U.S. patent application number 17/547583 was filed with the patent office on 2022-03-31 for method of facilitating nerve growth.
The applicant listed for this patent is Incept, LLC, Tulavi Therapeutics, Inc.. Invention is credited to Corinne Bright, Farhad Khosravi, Kenneth M. Martin, Yong Ren, Amarpreet S. Sawhney.
Application Number | 20220096711 17/547583 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-31 |
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United States Patent
Application |
20220096711 |
Kind Code |
A1 |
Bright; Corinne ; et
al. |
March 31, 2022 |
METHOD OF FACILITATING NERVE GROWTH
Abstract
Disclosed are methods, devices and materials for the in situ
formation of a nerve cap and/or a nerve wrap to inhibit neuroma
formation following planned or traumatic nerve injury. The method
includes the steps of identifying a severed end of a nerve, and
positioning the severed end into a cavity defined by a form. A
transformable media is introduced into the form cavity to surround
the severed end. The media is permitted to undergo a transformation
from a first, relatively flowable state to a second, relatively non
flowable state to form a protective barrier surrounding the severed
end. The media may be a hydrogel, and the transformation may
produce a synthetic crosslinked hydrogel protective barrier. The
media may include at least one anti-regeneration agent to inhibit
nerve regrowth
Inventors: |
Bright; Corinne; (Mountain
View, CA) ; Ren; Yong; (Mountain View, CA) ;
Martin; Kenneth M.; (Mountain View, CA) ; Khosravi;
Farhad; (Sunnyvale, CA) ; Sawhney; Amarpreet S.;
(Lexington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Tulavi Therapeutics, Inc.
Incept, LLC |
Mountain View
Campbell |
CA
CA |
US
US |
|
|
Appl. No.: |
17/547583 |
Filed: |
December 10, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17138703 |
Dec 30, 2020 |
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17547583 |
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PCT/US2019/040429 |
Jul 2, 2019 |
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17138703 |
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62822881 |
Mar 24, 2019 |
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62692858 |
Jul 2, 2018 |
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International
Class: |
A61L 27/26 20060101
A61L027/26; A61B 17/11 20060101 A61B017/11; A61F 2/00 20060101
A61F002/00; A61F 2/02 20060101 A61F002/02; A61K 9/00 20060101
A61K009/00; A61L 27/18 20060101 A61L027/18; A61L 27/52 20060101
A61L027/52; A61L 31/14 20060101 A61L031/14 |
Claims
1. A method of facilitating nerve growth, the method comprising:
positioning a first nerve end of a first nerve into a form cavity;
positioning a second nerve end of a second nerve into the form
cavity; introducing an in situ forming media in contact with the
first nerve end; introducing the in situ forming media in contact
with the second nerve end, the in situ forming media being
photoresponsive such that the in situ forming media is configured
to transition from a flowable state to a nonflowable state upon
photopolymerization of the in situ forming media; and
photopolymerizing the in situ forming media to transition the in
situ forming media from the flowable state to the nonflowable
state.
2. The method of claim 1, wherein introducing the in situ forming
media in contact with the first nerve end comprises delivering the
in situ forming media at a junction between the first nerve and the
form cavity.
3. The method of claim 2, wherein introducing the in situ forming
media in contact with the second nerve end comprises delivering the
in situ forming media at a second junction between the second nerve
and the form cavity.
4. The method of claim 1 further comprising: placing a non-adhesive
sterile sheet along the first nerve and the second nerve prior to
introducing the in situ forming media; and removing the
non-adhesive sterile sheet after introducing the in situ forming
media.
5. The method of claim 4, wherein the non-adhesive sterile sheet is
configured to physically separate the in situ forming media from a
surrounding tissue during photopolymerization.
6. The method of claim 1, wherein the form cavity comprises
silicone.
7. The method of claim 1, wherein the form cavity comprises a nerve
wrap.
8. The method of claim 1, wherein the form cavity comprises a nerve
conduit.
9. The method of claim 1, wherein the in situ forming media
comprises a color additive configured aid in visual contrast
relative to background tissue.
10. The method of claim 1, wherein at least one of the first nerve
end or the second nerve end comprises a coaptation site.
11. The method of claim 1, wherein the in situ forming media
comprises PGSA.
12. The method of claim 1, wherein the in situ forming media
comprises a liquid when in the flowable state.
13. The method of claim 1, wherein the form cavity is
biodegradable.
14. The method of claim 1, wherein the in situ forming media is
biodegradable.
15. The method of claim 1, wherein introducing the in situ forming
media in contact with the first nerve end comprises delivering the
in situ forming media circumferentially along the first nerve.
16. The method of claim 1, wherein introducing the in situ forming
media in contact with the second nerve end comprises delivering the
in situ forming media circumferentially along the second nerve.
Description
INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 17/138,703, filed on Dec. 30, 2020, which claims the benefit
under 35 U.S.C. .sctn. 119(e) as a bypass continuation of PCT App.
No. PCT/US2019/040429, filed on Jul. 2, 2019, which in turn is a
nonprovisional application of U.S. Prov. App. No. 62/692,858, filed
on Jul. 2, 2018, and U.S. Prov. App. No. 62/822,881, filed on Mar.
24, 2019, each of which are hereby incorporated by reference in
their entireties.
BACKGROUND
[0002] Neuromas are benign tumors that arise from neural tissue and
are composed of abnormally sprouting axons, Schwann cells, and
connective tissue. Even though neuromas can appear following
various types of injuries, some of the most common and challenging
to treat are derived from trauma or surgical procedures in which
neural tissue was damaged or transected. Amputation surgeries
necessitate the transection of one or more sensory or mixed nerves.
Chronic neuropathic pain, attributed to neuroma formation, develops
in up to 30% of patient's post-surgery and results in downstream
challenges with wearing a prosthesis. In addition to traumatic and
amputation related neuromas, neuromas form across multiple clinical
indications such as in general surgery (hernia repair, mastectomy,
laparoscopic cholecystectomy), gynecologic surgery (C-section,
hysterectomy), and orthopedics (arthroscopy, amputation, knee
replacement).
[0003] Neuromas develop as a part of a normal reparative process
following peripheral nerve injury. They are formed when nerve
recovery towards the distal nerve end or target organ fails and
nerve fibers improperly and irregularly regenerate into the
surrounding scar tissue. Neuromas include a deranged architecture
of tangled axons, Schwann cells, endoneurial cells, and perineurial
cells in a dense collagenous matrix with surrounding fibroblasts
(Mackinnon S E et al. 1985. Alteration of neuroma formation by
manipulation of its microenvironment. Plast Reconstr Surg.
76:345-53). The up regulation of certain channels and receptors
during neuroma development can also cause 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). Haphazardly arranged nerve
fibers are known to produce abnormal activity that stimulates
central neurons (Wall P D and Gutnick M. 1974. Ongoing activity in
peripheral nerves; physiology and pharmacology of impulses
originating from neuroma. Exp Neurol. 43:580-593). This ongoing
abnormal activity can be enhanced by mechanical stimulation, for
example, from the constantly rebuilding scar at the injury site
(Nordin M et al. 1984. Ectopic sensory discharges and paresthesia
in patients with disorders of peripheral nerves, dorsal roots and
dorsal columns. Pain. 20:231-245; Scadding J W. 1981. Development
of ongoing activity, mechanosensitivity, and adrenaline sensitivity
in severed peripheral nerve axons. Exp Neurol. 73:345-364).
[0004] Neuromas of the nerve stump or neuromas-in-continuity are
unavoidable consequences of nerve injury when the nerve is not, or
cannot be, repaired and can result in debilitating pain. It has
been estimated that approximately 30% of neuromas become painful
and problematic. This is particularly likely if the neuroma is
present at or near the skin surface as physical stimulation induces
signaling in the nerve resulting in a sensation of pain.
[0005] The number of amputees in the world has risen significantly
in recent years, with war injuries and dysvascular diseases such as
diabetes accounting for approximately 90% of all amputee cases.
There are currently about 1.7 million amputees living in the United
States alone, and over 230,000 new amputee patients are discharged
annually from hospitals. Further, it has been estimated that there
will be a 20% increase in the number of new amputee cases per year
by 2050.
[0006] Unfortunately, due to persistent pain in limb remnants,
about 25% of amputees are not able to commence rehabilitation, much
less resume ordinary daily activities. The cause of such pain can
be a neuroma. One recent study reported that 78% of amputees
experienced mild to severe pain as a consequence of neuroma
formation over the 25-year study period, of which 63% described the
pain as constant aching pain. The pain is also frequently described
as sharp, shooting, or electrical-like phantom sensations that
persist for years after surgical amputation. In addition, patients
experience tenderness to palpation of the skin overlying the
neuroma, spontaneous burning pain, allodynia, and hyperalgesia.
[0007] While various methods have been used to prevent, minimize,
or shield neuromas in an attempt to minimize neuropathic pain, the
current clinical "gold standard" for treating neuromas is traction
neurectomy, in which the nerve is pulled forward under traction and
transected as far back as possible in the hope that, if a neuroma
forms, that it will be located deep in the tissue. Another well
recognized approach is to bury the proximal nerve end (that will
form the neuroma) into muscle or a hole drilled in bone. The nerve
is then sutured to the muscle or periosteum of the bone to maintain
its position. The rationale for this is that the surrounding tissue
cushions and isolates the neuroma to inhibit stimulation and the
resulting painful sensations. However, this procedure can greatly
complicate surgery, as significant additional dissection of
otherwise healthy tissue is required to place the nerve stump.
This, coupled with poor and variable efficacy, the lack of
appropriate/available tissue, and the additional procedural time
required, result in the procedure being rarely performed to prevent
neuroma formation.
[0008] Another method is to cut the nerve stump back to leave a
segment or sleeve of overhanging epineurium. This overhang can be
ligated to cover the face of the nerve stump. Alternatively, a
segment of epineurium can be acquired from other nerve tissue or a
corresponding nerve stump can be cut back to create an epineurium
sleeve that can be used to connect with and cover the other nerve
stump.
[0009] Yet another method that is commonly used is a suture
ligation, where a loop of suture is placed around the end of the
nerve and tightened. This pressure is believed to mechanically
block the exit of axons and causes the terminal end to eventually
form scar tissue over the site. Clinical and pre-clinical evidence
has shown, however, that this procedure can cause a painful neuroma
to form behind the ligation. Furthermore, the ligated nerve is
generally not positioned to minimize mechanical stimulation of the
neuroma, since it is anticipated that the scar tissue will provide
sufficient protection to the nerve end.
[0010] Other methods used clinically include placing the nerve
stump within a solid implantable silicone or biodegradable polymer
tube with an open, or more recently, a sealed end (e.g. Polyganics
NEUROCAP); wrapping the proximal nerve end with a harvested vein or
fat graft, again with the goal of providing a physical barrier to
aberrant nerve regeneration. The use of biomaterial implant devices
and methods necessitate insertion and securing the nerve with
sutures in the opening of the device, which can be difficult and
further damage the nerve end. For example, the current procedure
for securing the NEUROCAP requires a suture be placed in the
epineurium of the nerve and through the wall of the tube and
followed by pulling and stuffing the nerve into the lumen of the
tube using the suture and the placement of several sutures to
retain the nerve in the device. These methods and devices can also
result in mechanical stimulation of the neuroma tissue as a result
of 1) mismatch between the tissue compliance and the rigidity of
the conduit and 2) inability of the cap to prevent neuroma
formation within the cap, with resulting sensation of pain.
Although these nerve caps degrade over a period of 3 months to 18
months, substantial degradation-mediated mass loss occurs in the
first three to six months resulting in the exposure of a
temporarily protected neuroma to the surrounding environment. Thus,
the efficacy of these solid implantable caps is limited by the
ability of the cap to conform to the proximal end of the nerve and
prevent neuroma formation and secondarily their subsequent
degradation to expose the neuroma to the surrounding environment.
Finally, since these methods require suturing using fine sutures
(9-0 nylon) the procedural time and skill required to secure these
implants under surgical magnification (loupes) or harvested tissue
prohibits surgeons from more broadly adopting these procedures.
[0011] Unfortunately, current methods for addressing the formation
of and pain caused by neuromas have not been widely adopted. The
need therefore remains for an effective technology or therapy for
controlling or inhibiting neuroma formation following inadvertent
or planned surgical or traumatic nerve injury in addition to
reducing scar formation and perineural adhesions.
[0012] A variety of biomaterial conduits have been explored
preclinically to try to prevent neuroma formation, including other
solid implantable biodegradable polymeric conduits based on
polylactide/polycaprolactone (Onode et al (2019) Nerve capping with
a nerve conduit for the treatment of painful neuroma in the rat
sciatic nerve, J Neurosurg. p. 1-9; Yan et al (2014) Mechanisms of
Nerve Capping Technique in Prevention of Painful Neuroma Formation,
PLOS One, 9(4) p. 1-11; Yi et al (2018) Painful Terminal Neuroma
Prevention by Capping PGRD/PDLLA Conduit in Rat Sciatic Nerves Adv.
Sci, 1-11), atelocollagen (Sakai et al (2005) Prevention and
Treatment of Amputation Neuroma by an Atelocollagen Tube in Rat
Sciatic Nerves. J Biomed Mater Res Part B: Appl Biomater 73B:
355-360) or porcine small intestine submucosa (Tork et al (2018),
ePoster: Prevention of Neuromas with a Porcine SIS Nerve Cap:
Histopathologic Evaluation,
http://meeting.handsurgery.org/files/2018/ePosters/HSEP106.pdf) or
microcrystalline chitosan (Marcol et al (2011) Reduction of
Post-Traumatic Neuroma and Epineural Scar Formation in Rat Sciatic
Nerve by Application of Microcrystallic Chitosan. Microsurgery, 31:
642-649). These approaches have not been successful to date in
preventing the formation of neuromas either because, again, the
solid implants do not form in situ and create a potential space to
permit nerve outgrowth and varying degrees of neuroma formation or,
critically, because the in vivo persistence of the materials was
not sufficient to prevent neuroma formation.
[0013] Some embodiments as disclosed here have been demonstrated to
prevent neuroma formation preclinically and 1) eliminate the need
for suturing, dragging or stuffing of the nerve inside a conduit,
2) conform to the end of the nerve stump providing a physical
barrier to nerve regeneration, and 3) provide mechanical strength
to prevent nerve regeneration for a period of two months,
preferably three months or more necessary to prevent nerve
outgrowth during the growth regenerative phase after nerve injury.
In situ forming implants described herein can be compliant with the
surrounding tissue, adhere to but do not compress the underlying
nerve tissue, are flexible such that they can move over regions of
tissue involving joints or where nerves slide relative to other
tissues and prevent scar tissue and adhesions forming around
nerves. Finally, some of these in situ forming implants can be
delivered without advanced surgical training. In other situations,
there remains a need for a technology that prevent nerve outgrowth
into the surrounding tissue and direct the outgrowth of a
transected or compressed nerve into the distal nerve stump or
allograft/autograft. With this, in some aspects, a suture-free
technology that can direct nerve regeneration from a proximal nerve
stump directly (via direct coaptation/anastomoses with distal nerve
stump) or indirectly (through a nerve conduit, guidance channel,
allograft, autograft), or through a growth-permissive matrix into
the distal nerve stump is described. In addition, in some aspects,
a technology that allows detensioning of the anastomoses site is
described to promote better nerve regeneration. Lastly, in some
aspects, a technology that can be quickly and broadly applied to
nerves to prevent inadvertent damage to adjacent nerves during a
variety of surgical procedures is desirable.
SUMMARY
[0014] There is provided in accordance with one aspect of the
present invention a method of in situ formation of a conforming,
protective nerve cap to inhibit neuroma formation at a severed
nerve end. The method comprises the steps of identifying a severed
end of a nerve; positioning the severed end into a cavity defined
by a form; introducing media into the form to surround the severed
end; and permitting the media to undergo a transformation from a
first, relatively flowable state to a second, relatively non
flowable state to form a protective conforming barrier surrounding
the severed end. The method may further comprise the step of
removing the form, to leave behind a formed biocompatible in situ
protective nerve cap.
[0015] The identifying a severed end of a nerve step may comprise
identifying a nerve severed such as by cutting or ablation, or
severed traumatically. The form may comprise a nerve guide, and the
positioning step may comprise positioning the nerve such that the
nerve guide maintains the severed end within the cavity spaced
apart from a sidewall of the form. The severed end may be
positioned at least about 0.1 mm or 2 mm away from the sidewall or
more preferably about 1 mm away. Preferably the form is either
bioresorbable or is composed of a flexible nondegradable material
that can easily be removed from the surgical site after formation
of the in situ nerve cap.
[0016] The transformation from flowable to nonflowable state may
occur within about 1 minute, or within about 30 seconds or within
about 10 seconds of the introducing step. The method may
additionally comprise the step of blotting a volume of axoplasm
from the severed nerve prior to the introducing step.
[0017] In one implementation of the invention, the form may
comprise a first configuration in which the cavity is exposed, and
a second configuration in which the cavity is partially or
completely covered; and further comprising the step of advancing
the form from the first configuration to the second configuration
following the introducing nerve step. Alternatively, the step of
advancing the form from the first configuration to the second
configuration may occur prior to the introducing the nerve or media
steps. The form may alternatively comprise an open cell foam, and
the cavity comprises a tortuous, interconnected interstitial volume
within the foam. In the latter embodiment, the form would remain in
place in situ after integration with and formation of the nerve
cap.
[0018] The identifying a severed nerve step may include the step of
severing a target nerve. The step may additionally comprise
transecting the nerve cleanly at an oblique angle prior to placing
the nerve within the form. The transformation step may comprise a
crosslinking reaction or a polymerization and the use of an in situ
forming hydrogel that can intercalate with the host tissue to form
an adhesion between the hydrogel and the tissue. In the preferred
embodiment, the hydrogel is a neutral or negatively charged
material with submicron or smaller pores which permit nutrient and
protein exchange but not cellular infiltration. In one
implementation, the transformation produces a synthetic crosslinked
hydrogel protective barrier through which nerves cannot regenerate
around the end of a transected nerve stump.
[0019] The use of PEG as a biomaterial for delivery to nerves is
well known in the art. Increasingly, there is an appreciation that
biomaterials, including PEG hydrogels, need to be tuned for
specific applications. Properties, including molecular weight,
degradation kinetics, PEG shape (linear vs multi-arm vs dendritic),
degree of crosslinking, degree of substitution, crosslinking type
(electrophilic-nucleophilic or free radical), gelation time, arm
length (in the case of multi-arm PEGs), functional groups,
hydrolytic linkages, and other factors such as pH and buffer
selection are tailored for specific applications directed towards
nerves to prevent neuroma formation.
[0020] In some embodiments, disclosed herein is a dual component in
situ forming biomaterial composition comprising a nerve growth
permissive component and a nerve growth inhibitory component.
[0021] In some embodiments, the nerve growth permissive component
is delivered first and the nerve growth inhibitory component
delivered second.
[0022] In some embodiments, the nerve growth permissive components
conform to the nerve and facilitate nerve ingrowth into, through
and across the biomaterial into the distal stump.
[0023] In some embodiments, the nerve growth inhibitory components
prevents nerve growth into the material.
[0024] In some embodiments, the nerve growth inhibitory components
acts as a guide upon which nerve regeneration can occur.
[0025] In some embodiments, the biomaterial components comprise an
in situ forming gel.
[0026] In some embodiments, the biomaterial components comprise in
situ forming crosslinked gel, microparticles, nanoparticle, slurry
or micelles.
[0027] In some embodiments, both the growth permissive and growth
inhibitory components both contain polyethylene glycol (PEG).
[0028] In some embodiments, the PEG is a multi-arm PEG.
[0029] In some embodiments, the PEG is comprised of a urethane or
amide linkage.
[0030] In some embodiments, the PEG comprised of an ester
linkage.
[0031] In some embodiments, the PEG additionally comprises a linear
end-capped PEG of 5,000 Daltons or less.
[0032] In some embodiments, the crosslinking is performed between a
PEG-NHS ester and a PEG-amine or trilysine.
[0033] In some embodiments, the in situ forming gel contains pores
1 .mu.m in size or larger.
[0034] In some embodiments, the in situ forming gel contains rods
or filaments.
[0035] In some embodiments, the growth permissive component
contains chitosan.
[0036] In some embodiments, the growth permissive component
contains polylysine, preferably between 0.001 and 10 wt %, more
preferably between 0.01 and 0.1 wt %.
[0037] In some embodiments, the nerve growth permissive components
contains between 0.001 and 20% collagen, preferably between 3 and 6
wt %.
[0038] In some embodiments, the nerve growth permissive component
contains between fibronectin.
[0039] In some embodiments, the growth permissive component
contains poly-L-ornithine.
[0040] In some embodiments, the growth permissive component
includes laminin, preferably between 0 and 5 wt %, more preferably
between 0 and 0.5%.
[0041] In some embodiments, the swelling of the growth permissive
component is less than 20%, preferably between 5 and 20%.
[0042] In some embodiments, the swelling of the growth inhibitory
component is less than 30%, preferably between 0 and 10%.
[0043] In some embodiments, the swelling of the growth permissive
component is less than or equal to the swelling of the growth
inhibitory component.
[0044] In some embodiments, the compressive strength of the growth
inhibitory component is greater than 10 kPa, preferably >30
kPa.
[0045] In some embodiments, the growth permissive and growth
inhibitory component are different colors.
[0046] In some embodiments, the growth permissive region comprises
agents that support nerve survival, outgrowth, and
regeneration.
[0047] In some embodiments, the growth permissive region permits
infiltration of Schwann or glial cells.
[0048] In some embodiments, a composition includes agents which may
comprise one or more of growth factors, anti-inhibitory peptides or
antibodies, and/or axon guidance cues.
[0049] In some embodiments, the system contains supporting cells
such as glial cells, including Schwann cells, oligodendrocytes, or
progenitor cells such as stems cells.
[0050] In some embodiments, the system is delivered to peripheral
nerves or the spinal cord.
[0051] In some embodiments, the growth permissive and growth
inhibitory region include a P2XR receptor antagonist.
[0052] In some embodiments, the P2XR receptor antagonist is a P2X7
receptor antagonist, including Brilliant Blue FCF (BB FCF) or
Brilliant Blue G (BBG).
[0053] In some embodiments, the P2XR antagonist is a P2X3 receptor
antagonist, such as
[0054] In some embodiments, the concentration of the P2XR
antagonist is between 0.001 and 0.55% the hydrogel.
[0055] In some embodiments, disclosed is a kit including two or
more in situ forming hydrogels. The kit includes a dual applicator
system clearly marked with indicia as the growth permissive
applicator and a dual applicator system clearly marked as the
growth inhibitory applicator. Each component can be clearly color
coded and includes a powder vial, a reconstitution/diluent
solution, and an accelerator solution for use in the dual
applicator system. The kit also may include two or more forms--one
form for receiving the growth inhibitory hydrogel, the other for
the growth permissive hydrogel.
[0056] In some embodiments, disclosed herein is a method of
delivering dual in situ forming hydrogels to treat conditions
involving nerves. The nerves can need repair, such as, for example,
end-to-end anastomoses, coaptation, repair with allograft or
autograft or conduit or wrap, or gap repair.
[0057] In some embodiments, a growth permissive region is delivered
between the proximal and distal nerve stumps, between end-to-end
anastomoses sites, between proximal stump and
allograft/autograph.
[0058] In some embodiments, a growth permissive region is delivered
between the proximal and graft and/or graft and distal stumps.
[0059] In some embodiments, a growth permissive region is delivered
inside a conduit or wrap.
[0060] In some embodiments, a growth permissive region is delivered
into a form that permits adherence of the growth permissive gel to
the nerves but not the form.
[0061] In some embodiments, a growth inhibitory region is delivered
after the growth permissive region.
[0062] In some embodiments, a growth inhibitory region covers the
proximal and distal nerves and growth permissive region.
[0063] In some embodiments, a kit can include an in situ forming
hydrogel. The kit includes a dual applicator system and a powder
vial, a reconstitution/diluent solution, and an accelerator
solution for use in the dual applicator system. The kit also may
include a selection of forms in a range of sizes and lengths for
receiving the hydrogel.
[0064] In some embodiments, a growth inhibitory region covers the
anastomoses junction.
[0065] In some embodiments, a growth inhibitory region covers the
junction(s) between the nerve and the conduit or wrap.
[0066] In some embodiments, a growth inhibitory region covers a
healthy, compressed, or contused nerve.
[0067] In some embodiments, disclosed herein is a formed in place
nerve regeneration construct, comprising: a growth permissive
hydrogel bridge having first and second ends and configured to span
a space between two nerve ends and encourage nerve regrowth across
the bridge; and a growth inhibiting hydrogel jacket encapsulating
the growth permissive hydrogel bridge and configured to extend
beyond the first and second ends to directly contact the two nerve
ends.
[0068] In some embodiments, disclosed herein is a method of
encouraging 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 form cavity; introducing a growth permissive media
into the cavity and into contact with the first nerve end and the
second nerve end to form a junction; placing the junction into a
second form cavity; and introducing a growth inhibiting media into
the second form cavity to encapsulate the junction.
[0069] In some embodiments, disclosed herein is a form for creating
an in situ nerve cap to inhibit neuroma formation, 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 that is less than the area of a second plane
conforming to inside dimensions of the cavity and spaced apart into
the cavity and parallel to the first plane; and a concave nerve
guide carried by the wall and providing a side access to the
cavity.
[0070] In some embodiments, disclosed herein is a form for creating
an in situ wrap around a nerve to nerve junction, 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 that is less than the area of a second plane
conforming to inside dimensions 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 side access for
positioning a first nerve end in the cavity; and a second concave
nerve guide carried by the wall and providing a second side access
for positioning a second nerve end in the cavity.
[0071] In some embodiments, disclosed herein is a composition for
an in situ forming growth inhibitory hydrogel with: compressive
strength greater than 10 kDa for over 3 months, in vivo persistence
for at least 3 months comprising less than 15% mass loss, and/or
swelling of less than 30% for over 3 months.
[0072] In some embodiments, the composition includes one or more
of: poly(ethylene glycol) succinimidyl carbonate, a P2XR receptor
antagonist, and/or a P2X7 receptor antagonist.
[0073] In some embodiments, a P2X7 receptor antagonist is Brilliant
Blue FCF (BB FCF) or Brilliant Blue G (BBG).
[0074] In some embodiments, a method of in situ formation of a
nerve wrap, comprising identifying a section of a nerve;
positioning the nerve in a cavity defined by a form; introducing
media into the cavity of the form to surround the nerve; and
permitting the media to undergo a transformation from a first,
relatively flowable state to a second, relatively non flowable
state to form a protective barrier around the nerve.
[0075] In some embodiments, the nerve is healthy, compressed, or
contused.
[0076] In some embodiments, the nerve is repaired through direct
anastomoses, repair with allograft or autograft, or repair with a
conduit.
[0077] In some embodiments, the method includes removing the
form.
[0078] In some embodiments, the form 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
form.
[0079] In some embodiments, a method of in situ formation of a
nerve wrap includes where the nerve is covered circumferentially
with at least 0.5 mm of a protective barrier.
[0080] In some embodiments, the transformation occurs within about
10 seconds of the introducing step.
[0081] In some embodiments, the transformation comprises a
crosslinking or polymerizing.
[0082] In some embodiments, the transformation produces a synthetic
crosslinked hydrogel protective barrier.
[0083] In some embodiments, the protective barrier has an in vivo
persistence of at least about two months.
[0084] In some embodiments, the protective barrier has an in vivo
persistence of at least about three months.
[0085] In some embodiments, the transformation causes the media to
swell in volume within the range of from about 2% to about 60%.
[0086] In some embodiments, the transformation causes the media to
swell in volume within the range of from about 20% to 60%.
[0087] In some embodiments, the method includes forming a form in
situ before the positioning the severed end; and/or delivering the
media around the nerve in two successive steps.
[0088] In some embodiments, the severing a target nerve step and
the positioning a form at a treatment site step are accomplished by
a single instrument.
[0089] In some embodiments, the viscosity of the flowable media is
less than 70,000 cps.
[0090] In some embodiments, the density of the flowable media is
less than 1 g/cm.sup.3.
[0091] In some embodiments, the form is comprised of silicone.
[0092] In some embodiments, the form contains an integral posts for
seating longer lengths of the nerve.
[0093] In some embodiments, the wrap is comprised of PEG.
[0094] In some embodiments, the form has a clamshell lid.
[0095] In some embodiments, the growth permissive and growth
inhibitory region contain a P2XR receptor antagonist.
[0096] In some embodiments, the P2X7 receptor antagonist is a P2X7
receptor antagonist, including Brilliant Blue FCF or Brilliant Blue
G (BBG).
[0097] In some embodiments, the concentration of the P2XR
antagonist is between 0.001 to 0.55% in the hydrogel.
[0098] In some embodiments, disclosed herein are in situ forming
hydrogel(s) as a cap. In some embodiments, the nerve cap is not
pre-formed.
[0099] Some embodiments as disclosed herein include in situ forming
hydrogel scaffolds or ones that can be formed/wrapped in situ
around a nerve. In some embodiments, systems and methods do not
include a nerve guidance conduit (tube with two open ends) rather
than a cap. In some embodiments, disclosed herein are systems and
methods for delivering the hydrogel circumferentially around the
nerve in appropriately designed forms. In some embodiments, systems
and methods can include use of a form or the specific design of the
PEG hydrogel to prevent neuroma formation such as circumferential
delivery, in vivo persistence, minimal swelling etc. and delivering
them into a removable form.
BRIEF DESCRIPTION OF THE DRAWINGS
[0100] FIG. 1A is a perspective schematic view of a nerve end
positioned within a form cavity. An entrance region that permits
the nerve to guided into the form. The length of the form is
provided to provide a sufficient surface area over which the
hydrogel to form and adhere to the nerve tissue.
[0101] FIG. 1B is a side elevational cross section through the
construct of FIG. 1A.
[0102] FIG. 1C is a top view of the construct of FIG. 1A.
[0103] FIG. 1D is an end view of the construct of FIG. 1A.
[0104] FIG. 1E is a cross-sectional view taken along the line 1E-1E
in FIG. 1B.
[0105] FIG. 2 is a schematic illustration of a formed barrier
formed in accordance with some embodiments of the present
invention.
[0106] FIG. 3 is a perspective view of a form, having a stabilizing
feature.
[0107] FIG. 4 is a perspective view of a form for creating a wrap
around a nerve or a growth permissive region between a nerve. Thus,
depending on the application, the wrap form may contain a growth
permissive or growth inhibitory hydrogel.
[0108] FIGS. 5A-5E illustrate a series of steps for creating a
growth permissive hydrogel junction encapsulated by a growth
inhibitory hydrogel barrier.
[0109] FIG. 6 is a perspective view of a clamshell form.
[0110] FIGS. 7-10C illustrate embodiments of tools for transecting
nerves and/or creating a hydrogel junction.
[0111] FIGS. 11A-11E illustrate views of a form and methods of
use.
[0112] FIG. 12 is a perspective view of a form with a stabilizing
rod.
[0113] FIGS. 13A-13D is a perspective view of a cap form with a
partial cover and in internal rod to support the nerve.
[0114] FIGS. 14A-14C is a perspective view of a cap form with a
partial clamshell.
[0115] FIGS. 15A-15C is a perspective view of a tearable nerve cap
form.
[0116] FIGS. 16Ai-16E illustrate perspective views and photographs
of in situ formed hydrogels (growth inhibitory and growth
permissive) around nerves both in cap and wrap form.
[0117] FIGS. 17A-17B illustrate preclinical data demonstrating the
formation of neuroma after the delivery of hydrogels with adequate
initial mechanical strength but inadequate in vivo persistence
relative to hydrogels with longer duration mechanical strength and
persistence.
[0118] FIGS. 18A-18B illustrate a mixing element design to improve
the consistence of the hydrogel when delivering low volumes of
precursor solution.
DETAILED DESCRIPTION
[0119] Some aspects of the present invention involve in situ
formation of a protective barrier around an end of a nerve using
injectable or surgically introduced media which may be a
gel/hydrogel or gel precursors to block nerve regeneration and/or
neuroma formation and inflammation and adhesion etc. around/in
contact with nerves. Access may be by way of an open surgical
approach or percutaneous (needle, endovascular/transvascular). The
nerve end or stump may be formed by transection (cutting),
traumatic injury, or ablation through any of a variety of
modalities including RF, cryo, ultrasound, chemical, thermal,
microwave or others known in the art.
[0120] The hydrogels may `adhere` to the end of the nerves
providing a snug, conforming, cushioning barrier around the end of
a nerve as opposed to a cap with a void (inflammatory cells/fluid
cysts present causing neuroma formation). Hydrogels are transparent
for visualization, low-swelling, compliant, and are delivered into
a form to generate hydrogel caps with volumes 0.1 to 0.5 ml. The
barrier may inhibit neuroma formation purely through mechanical
blocking of nerve regrowth. The media may additionally comprise any
of a variety of drugs such as for inhibiting nerve regrowth as is
discussed in further detail herein.
[0121] Target nerves can vary widely in diameter or non circular
outside configuration, and the cut or severance angle and precision
can also vary. In accordance with some embodiments of the present
invention, capping is best accomplished by forming a soft,
cushioning and conformable protective barrier in situ. A flowable
media or media precursor(s) may be introduced to surround and
conform to the configuration of the nerve end, and then be
transformed into a non flowable state to form a protective plug in
close conformity with and bonded to the nerve end. To contain the
media before and during transformation (e.g., crosslinking), the
media may be introduced into a form into which the nerve end has
previously or will be placed. Filling the media into a form allows
the media to surround the nerve end and transform to a solid state
while contained in a predetermined volume and configuration to
consistently produce a protective, conforming nerve cap regardless
of the diameter and configuration of the nerve stump.
[0122] Referring to FIGS. 1A through 1D, there is illustrated a
nerve cap form 10. The form 10 extends between a proximal end 12, a
distal end 14 and includes a side wall 16 extending there between.
Sidewall 16 is concave to produce a form cavity 18 therein. The
form cavity 18 is exposed to the outside of the form by way of a
window 20.
[0123] The proximal end 12 of the form 10 is provided with a nerve
guide 22 to facilitate passage of the nerve 24 to position the
nerve end 26 within the form cavity 18. The nerve guide 22 may
comprise a window or opening in the proximal end wall 12 of the
form 10, and is configured to support the nerve at a level that
positions the nerve end 26 within the form cavity 18. In the
illustrated embodiment, the nerve guide 22 includes a support
surface 28 on an upwardly concave housing to produce a nerve guide
channel 30. See FIG. 1D. FIG. 1E is a cross-sectional view taken
along the line 1E-1E in FIG. 1B.
[0124] Referring to FIGS. 1B and 1C, the nerve end 26 is positioned
such that at least 1 mm and preferably 2 mm or more in any
direction separate the nerve end 26 from the interior surface of
the side wall of the form 10. This permits the media 27 to flow
into the form cavity and surround the nerve end 26 to provide a
protective barrier in all directions.
[0125] Following transformation of the media from a relatively
flowable state to a relatively non-flowable state, the form 10 may
be left in place, or may be peeled away to leave behind a formed
barrier 60 in the form of a plug as is schematically illustrated in
FIG. 2.
[0126] In order to stabilize the form 10 following placement and
during the filling and transformation stages, at least one
stabilizing feature 32 may be added. See FIG. 3. The stabilizing
feature 32 maybe at least one or two or four or more ridges,
flanges or feet which provide a transverse support surface 34 for
contacting adjacent tissue and stabilizing the form against motion.
The transverse support surface 34 may extend along or be parallel
to a tangent to the sidewall of the form 10.
[0127] In one implementation of the invention there is provided a
dual hydrogel construct with connectivity across the junction
between two nerve ends achieved by creating a growth permissive
hydrogel junction between the two opposing nerve ends, then
encapsulating that junction with a growth inhibitory hydrogel
capsule. The use of an in situ crosslinking hydrogel for the growth
permissive media produces a junction with sufficient mechanical
integrity and adhesiveness that it can be picked up as a unit as if
it were an intact nerve and then placed in a second form to form
the outer growth inhibitory hydrogel capsule.
[0128] Referring to FIG. 4, a form 10 includes a curved side wall
66 defining a form cavity 68. A first nerve guide 70 and a second
nerve guide 72 are in communication with the cavity 68 and
dimensioned and oriented to allow positioning first and second
nerve ends into the cavity 68 in a position where they will face
each other and become surrounded by flowable media introduced into
the cavity 68.
[0129] Referring to FIGS. 5A-5E, there is illustrated a sequence of
steps for forming a dual hydrogel conductive nerve junction between
two nerve ends. A first form 50 comprises an elongate side wall
curved to form a concavity such as in the form of a half of a
cylinder, having an inside diameter larger than the diameter of a
target nerve. The form 50 has a first end 52, a second end 54 and
an elongate channel 56 extending there between. The first nerve end
58 is positioned within the channel 56 from the first end 52. A
second nerve end 60 extends into the channel 56 from the second end
54. The result is a form cavity 62 formed between the first and
second nerve endings and the sidewall of the form 50.
[0130] A transformable growth permissive hydrogel precursor is
introduced into the form 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 second nerve end 60 as shown in FIG. 5B. Following
transformation of the gel to a less flowable state, the form 50 is
removed as shown leaving a junction comprising the nerve ends
connected by a conductive bridge 64 of polymerized growth
permissive gel 62. See FIG. 5C.
[0131] Thereafter the polymerized junction is placed within a
second form 66 having a central chamber 68 separating first and
second nerve supports 70, 72, such as that illustrated in FIG. 4. A
second growth inhibitory hydrogel precursor is introduced into the
central chamber 68 to surround and over form the conductive bridge
64 and nerve ends to produce a final construct in which the first
growth permissive polymer bridge 62 is encapsulated by second,
growth inhibitory polymer capsule 70. See FIG. 5E.
[0132] Either the nerve capping or nerve regeneration forms of some
embodiments of the present invention can be provided in a clam
shell configuration such as that illustrated in FIG. 6. A first
shell 80 defines a first cavity 82 and a second shell 84 defines a
second, complementary cavity 86. The first and second shells are
joined by a hinge 88 such as a flexible living hinge made from a
thin polymeric membrane. The first and second shells 80, 84 can be
rotated towards each other about the hinge 88 to form an enclosed
chambered form.
[0133] FIG. 7 illustrates a perspective view of a clamping tool 700
configured to cut nerve tissue, as well as to house a form for
forming a hydrogel nerve junction such as disclosed elsewhere
herein, e.g., after transecting a nerve. The tool 700 can include a
plurality of proximal movable grips 702, each connected to shafts
706 connected at pivot 704, and can have an unlocked configuration
as shown, movable to a locked configuration utilizing a locking
mechanism 705, such as a series of interlocking teeth. The distal
ends 707 of the shafts 706 can include end effectors 708 that can
include sidewalls 710 that can have a curved geometry as shown, and
complementary cutting elements 712 operably connected to the curved
sidewalls. In some embodiments, a form 10 can be connected to the
sidewall 710 after cutting the nerve. In other embodiments, a form
can include an integrally formed cutting element. In some
embodiments, the cutting element can be detached or otherwise
removed after cutting, leaving the form in place.
[0134] FIG. 8 is a close-up view of an end effector 708 of FIG. 7,
also illustrating that the end effector 708 can also carry a form
10. FIG. 9 is a side close-up view of the distal end of an
embodiment of the tool, illustrating that each of the end effectors
can include cutting elements and/or forms.
[0135] FIGS. 10A-10C illustrate various stages of a method of
transecting a nerve while removing axoplasm from the nerve tip, to
improve close apposition between the nerve end and the hydrogel. In
some embodiments, opposing end effectors 708 can include blades
712, which can be of equal or unequal length. Blades 712 on each
end effector 708 can be generally opposing, but offset from each
other as shown in some embodiments. Actuating the end effectors 708
can result in the blades transecting the nerve 24 creating a nerve
end 26. The blades can be within a form as previously described. An
absorbent material 780 such as a swab can be connected to one or
more end effectors 708 (such as within a form, for example) and be
proximate, such as directly adjacent one or more of the blades 712,
in order to absorb any axoplasm after nerve transection. The tip of
the swab can be, for example, less than 5 mm, more preferably less
than 2 mm in order that it may fit comfortably within the form and
hold the nerve while the hydrogel is delivered.
[0136] Referring to FIGS. 11A-11E, in some embodiments, a delivery
needle 1102 is advanced into an opening 1104 of the cap form 1100
to deliver the hydrogel precursor in and around the nerve 1124.
Also shown is nerve guide 1122 which can be as described elsewhere
herein. See FIG. 11A. Hydrogel may be delivered in to successive
applications, to half fill the form and for a hydrogel 1150 as
shown in FIG. 11B and then completely fill the form as shown in
FIG. 11C and form a hydrogel cap after which the form is removed.
Hydrogel is delivered in a small bolus 1152 to surround the tip of
the nerve as shown in FIG. 11D and then the remainder of the cap is
subsequently filled to form a hydrogel cap after which the form is
removed as shown in FIG. 11E.
[0137] Referring to FIG. 12, in some embodiments, a biodegradable
rod 1215 is placed adjacent to and in continuity with the length of
the nerve 1224. The rod 1215 provides additional strength to the
nerve 1224 and naturally adheres to the nerve 1224 such that,
irrespective of the rod's position, the nerve 1224 adheres to the
rod 1215. The hydrogel solution is then delivered on or around the
nerve 1224 and the biodegradable rod 1215 to form a nerve cap.
[0138] Referring to FIGS. 13A-13D, in some embodiments, one, two,
or more apertures 1310 are provided in the side of a cap or wrap
form 1300 to guide the needle to deliver the precursor solution in
the correct location. The hole 1310 may be in one of many locations
around the form as is needed to deliver the precursor solution 13A.
A post 1330 may be included in the bottom of a cap or wrap form to
provide additional support to the nerve. The nerve length is rested
on top of the post 1330 while taking care that the tip of the nerve
does not come in contact with the post 1330. The post 1330 may be
integral to the cap or wrap form and is subsequently removed when
the form is removed. Alternatively, the post 1330 may comprise a
biodegradable post that remains integral to the hydrogel cap. See
FIGS. 13B and 13C. In some embodiments, a cap form can include a
partial lid 1320, shown in FIG. 13A. The form is tilted such that
the precursor material will flow to and fill the distal cap first,
surrounding the proximal nerve stump end and then subsequently fill
the rest of the nerve cap. As shown in FIG. 13D, the cap or wrap
form can also include raised tabs 1333.
[0139] FIGS. 14A-14C illustrates various views of an embodiment of
a nerve cap form 1400 similar to that shown in FIGS. 13A-13D with a
partial lid 1420 connected via a hinge 1428 with an insert 1440 to
assist in centering the lid 1420 on the cap form 1400. Also shown
is nerve guide, which can be as described elsewhere herein.
[0140] FIGS. 15A-15C illustrate various views of a tearable cap
form 1500 that can include a peelable sheath 1560 including a
sidewall 1561, into which the nerve is placed (nerve channel 1562).
The precursor solution is delivered into and around a first nerve
channel 1562 and the peelable sheath 1560 is subsequently torn off
the nerve 1524, such as using a tearable tab 164 as shown in FIG.
15A. The nerve hydrogel 1570 is then rotated approximately 90
degrees and placed in a second larger diameter peelable cap form
1501. The precursor solution is then applied into the nerve channel
to surround the nerve and the first cap form. The peelable sheath
is then torn off the second tearable cap form 1501. The resultant
cylindrical cap form contains the centered nerve. The nerve 1524
can then be rotated back to the normal physiologic position, as
shown in FIG. 15B. FIG. 15C illustrates an alternate tearable cap
form design which can include a plurality of tabs.
[0141] FIG. 16Ai-6E illustrate hydrogel filling and surrounding a
nerve in a cap form. FIG. 16B illustrates a photograph of the
hydrogel formed inside a cap form. FIG. 16C illustrates a high
resolution image of a cap form. FIG. 16D illustrates an example of
a cap form and wrap forms around the pig sciatic nerve. Example of
a growth permissive hydrogel (pink) in a wrap form around a nerve
and then subsequently embedded in a second (blue) growth inhibitory
hydrogel wrap. Hydrogels are cut in cross section in order to see
the growth permissive (pink) hydrogel embedded within the growth
inhibitory (blue) hydrogel, as shown in FIG. 16E.
[0142] FIG. 17A illustrates neuroma formation after delivery of
DuraSeal in a cap form around a transected rat sciatic nerve. FIG.
17B illustrates the absence of neuroma formation after delivery of
the lead formulation around a transected rat sciatic nerve. Cap
form maintains mechanical strength and in vivo persistence of 3
months.
[0143] FIGS. 18A-18B schematically illustrates an embodiment of a
mixing element to mix a two-part hydrogel system. In some
embodiments, one static mixer 1800 delivers the hydrogel precursor
solution into a central chamber, permitting the backflow and
recirculation of the initial material coming out of the mixer. A
second static mixer captures the well mixed solution and delivers
it through the needle tip. The fluid entrance 1802 (from a dual
chamber applicator) and fluid exit 1804 (to a blunt needle) are
also shown.
[0144] The table to follow is related to specific non-limiting
embodiments and devices for delivering in situ forming
hydrogels.
TABLE-US-00001 Delivery of in situ forming Hydrogels to: Form Shape
Selected Hydrogel 1) Undamaged nerves Wrap Growth Inhibitory 2)
Nerves that have been compressed, Wrap Growth Inhibitory contused
or stretched 3) Stump neuroma or transected nerve that Cap Growth
Inhibitory can not be repaired 4) Nerves that have been transected
and Wrap Growth Permissive undergone direct suture repair and then
(coaptation, end-to-end anastamosed) Growth Inhibitory 5) Nerves
that have undergone suture repair Wrap(s)--protect Growth
Permissive and placement in a nerve conduit or wrap nerve-conduit
and then junction Growth Inhibitory 6) Nerves that have undergone
connector Wrap(s)--protect Growth Permissive assisted `suturelesss`
neuronaphy in nerve-conduit and then which sutures are placed
between the juncton Growth Inhibitory epineurium and the connector
but not to one another 7) Nerves that have been placed in a
Wrap(s)--protect Growth Permissive connector without suturing
anastamoses and then Growth Inhibitory 8) Nerves that are
undergoing conduit Wrap(s)--protect Growth Permissive detensioning
gap repair nerve-conduit and then junction Growth Inhibitory 9)
Nerves undergoing detensioning allograft Wraps(s)--protect Growth
Permissive interposition with connector assisted nerve-nerve and
then sutureless repair anastamoses Growth Inhibitory 10) Nerves
undergoing detensioning Wrap(s)--protect Growth Permissive
autologous nerve graft interposition suture nerve-nerve and then
repair Growth Inhibitory 11) Nerves that have non-union gaps (e.g.
2 Wraps Growth Permissive cannot be repaired directly) and then
Growth Inhibitory 12) Nerves that have been repaired and one or
Wrap(s)--Protect Growth Permissive more wraps are placed around the
nerve-wrap and then anastomoses site Growth Inhibitory 13) Nerves
undergoing suture repair in Wrap--Protect Growth Permissive
targeted muscle reinnervation nerve-nerve and then interface Growth
Inhibitory
[0145] Peripheral Nerve Stimulation (PNS). As neurostimulators have
advanced from the spine to the periphery and the hardware has been
miniaturized, purpose built peripheral nerve stimulators are being
developed and advanced for blocking pain, stimulating muscle
contractions, and stimulating or blocking nerves to modulate
disease and/or symptomatology, and stimulate nerve regeneration. As
new applications and new neurostimulators have been developed, so
has an increased awareness of the need to be able to maintain the
stimulation electrodes and catheters in direct or close apposition
with the target nerve as 1) placing the electrodes next to the
nerve procedurally can be challenging and electrodes can migrate
procedurally even after ideal placement adjacent to a nerve and 2)
after placement, electrodes may drift through patient movement or
handling as muscles contract or the implant gets better seated
within the tissue. This can lead to loss of the therapy reaching
the target nerve and thus loss of efficacy.
[0146] Percutaneous delivery. With the advent of higher resolution
handheld ultrasound and better training amongst interventional pain
physicians, percutaneously delivered implantable neurostimulators
are increasingly being used as an alternate method to treat chronic
pain. In one embodiment once an electrode has been placed adjacent
to a nerve using a percutaneous delivery system, the position of
the electrode next to the nerve can be maintained by delivering
approximately 0.1 to 3 cc of an electroconductive hydrogel to form
around the electrode and maintain it in close apposition with the
nerve. The hydrogel media can be delivered through the lumen of the
catheter delivery system or the lumen of the electrode and will
form in situ. In some embodiments, the surface of the electrode can
be designed such that it interfaces is rougher, permitting stronger
intercalation between the hydrogel and the electrode to prevent
lead migration. In other embodiments, a coil or other screw like
design is placed on the end of the electrode to provide better
purchase between the electrode, the hydrogel, and the surrounding
tissue. For the percutaneous applications, delivery of a growth
inhibitory hydrogels or hydrogels with medium to long duration
mechanical strength are desirable. Again, longer-term the
maintenance of mechanical strength to maintain the position of the
electrode within the hydrogel is desirable until the chronic
foreign body response is sufficient to hold the electrode in place.
For example, to maintain longer term lead placement, the selection
of crosslinked PEG hydrogels containing more stable ester, urethane
or amide linkages is desirable, such as PEG-SG, PEG-SC, or
PEG-SGA.
[0147] In still other embodiments, the neurostimulators are
injectable wireless implants and takes the form of a pellet, rods,
beads, a wrap a sheet or a cuff that are held in place with a
hydrogel adjacent to a nerve, ganglia, or plexus. In one
embodiment, the hydrogel is delivered first to the target site and
the neurostimulator is delivered into the hydrogel. In another
embodiment, the neurostimulator implant(s) is delivered first,
adjusted to the desired location and then the hydrogel is then
delivered around it to secure it in the desired location.
Similarly, the neurostimulator implant location may be adjusted
using an external magnet to orient the implant adjacent to or in
contact with the nerve or neural tissue. In this embodiment, the
gelation time can be adjusted to provide sufficient time for the
appropriate alignment of the neurostimulator. In some embodiments,
a plurality of injectable microstimulator implants are injected
into a degradable or non degradable in situ forming hydrogel. In
yet another embodiment, microstimulators in the form of micro or
nanorods are implanted in the growth permissive hydrogel between
the two nerve stumps to promote neurite extension and accelerated
regeneration. These microstimulators may deliver magnetic,
chemical, or electric fields to stimulate nerve regeneration
through the gel and potentially along the microstimulator implants.
In one embodiment, the microstimulators are nanofibers and can be
injected through a low gauge needle or catheter to the nerve.
[0148] In another embodiment, short- or long-acting
microstimulators can be delivered with an injectable biocompatible
biomaterial such as a hydrogel to form a neurostimulator anisogel.
The microstimulators are magnetic, allowing directional control of
the microstimulator implant and, for example, parallel alignment of
the microimplants within the hydrogel prior to the gel converting
from a precursor solution to a gel. These hydrogels would be
injected around or in proximity to nerve bundles or tendrils and
then the microstimulators may physically provide regions across
which they can grow to and orient along as well as providing
chemical, electrical, or magnetic field stimulation to support
neurite outgrowth.
[0149] Open surgical. For open surgical applications, the hydrogel
may also be deposited in a similar manner around the electrode with
the electrode in direct contact and/or adjacent to the nerve under
direct visualization. Again, deposition of approximately 0.5 to 1
cc of hydrogel is sufficient to maintain the electrode position
relative to a nerve. The electrode can be inserted into a groove in
the silicone form adjacent to and with the nerve prior to the
delivery of the hydrogel. Forms can be envisioned that have a
second entrance region for the electrode. In this manner, for
example, the electrode can be aligned to run parallel to the nerve
or in direct apposition to the nerve when the gel is applied. For
applications where the neurostimulation therapy is only required
for a day to several weeks, pulling on the electrode will cause it
to be removed from the hydrogel with relative ease. Utilizing
combinations of growth inhibitory and growth permissive hydrogels
described above, may be selected depending on the application. For
examples in which electrodes placed next to the nerve only need to
stay in place for a matter of days or weeks, a shorter term
degradable hydrogel may be employed. This provides sufficient time
for the hydrogel to remain in place while the therapy is delivered
and then be rapidly cleared from the tissue. One example of this
would be the selection of crosslinked PEG hydrogels containing more
reactive ester linkages such as PEG-SS or PEG-SAZ. These hydrogels
are electrically conductive and thus suitable for applications
involving neurostimulators.
[0150] Generally, the selection of low swelling formulation is
critical to maintain apposition with the electrode; in one
embodiment, the hydrogel swelling is less than 30%, more preferably
less than 20% in order to maintain apposition with the nerve and
the electrode.
[0151] In yet another embodiment, the in situ forming hydrogel can
be used to secure a convection enhanced delivery system to the
site. Like the implantable neurostimulator, a drug delivery
catheter can be secured approximately 10 mm proximal to an injury
nerve site with the tip approximately 5 mm from the nerve injury.
Like the implantable neurostimulator, the silicone form can be
designed to include an entrance zone or cut out of the top edge of
the silicone form to permit the catheter or stimulator lead to rest
in the form in preparation for addition of the hydrogel. After
delivery of therapy (neurostimulation, convection enhanced drug
delivery), the catheter or neurostimulator can be removed from the
hydrogel without disrupting the protective barrier around the
hydrogel. For example U.S. Pat. No. 9,386,990 teaches the use of
DuraSeal to repair nerves with an in vivo persistence of two to
four weeks, the hydrogel does not provide the sustained mechanical
strength necessary to prevent neuroma formation or detension a
nerve during regeneration, such as at 3 and 4 months after surgical
repair. For example, crosslinked multi-arm PEGs containing rapidly
degrading ester linkages such as PEG-SS or PEG-SG are well-suited
suitable for applications to prevent the acute and subacute
adhesion formation. For another example, low molecular weight
linear PEGs have been demonstrated to act as a fusogen and promote
nerve repair and regeneration when injected around injured nerves
(but do not provide mechanical strength or persistence to prevent
neuroma formation. For example, PEG hydrogels, such as PEG
tetraacrylate hydrogels, have been used to rejoin nerves in
preclinical models (Hubbell 2004/0195710)
[0152] None of the previous examples contain the degradable
linkages necessary to support the required mechanical strength or
in vivo persistence required for applications to prevent aberrant
nerve outgrowth and neuroma formation. Commercially available PEG
hydrogels, particularly conventional PEGs with a hydrolytic ester
linkage, do not have the suitable mechanical strength or in vivo
persistence to prevent neuroma formation or detension a nerve for
three of four months until the nerve is repaired. These PEGs and
PEG gels may have sufficient mechanical strength initially to
temporarily assist in the repair of nerves across an anastomoses
and/or prevent adhesion formation, but the hydrogels do not have
sufficient mechanical strength at two months, or more preferably
three months post administration to prevent aberrant neuroma
formation (cap) and continue to provide mechanical offloading to
support to a regenerating nerve (wrap). FIG. 16 provides an example
of the lack of durability of the DuraSeal hydrogel in preventing
neuroma formation in a rat sciatic nerve transection model. The
hydrogels containing ester linkages have either degraded sufficient
that they no longer provide a barrier to nerve regeneration, have
fallen off the nerve, or have been cleared entirely. As a result,
the initial mechanical barrier was not sufficient to act as a
long-term barrier to prevent nerve outgrowth and neuroma
formation.
[0153] Furthermore, the other approaches teach delivering the an in
situ forming hydrogel around the nerves directly without protecting
the underlying muscle from adhesions or providing a method to
systematically circumferentially cover the proximal nerve tip with
hydrogel. In situ forming polymeric systems adhere to, albeit with
varying to degrees, to the surrounding tissues that they come into
contact with during crosslinking or polymerization. If the
non-target tissue (e.g. muscle or fascia) is not protected or
shielded from the reaction, the hydrogel also adheres to this
tissue. Since nerves glide freely within a fascial plane, typically
between muscles, limitation to their movement is not desirable and
may result in pain and or loss of efficacy. Some embodiments
described here provide forms that separate the in situ forming
hydrogel from the surrounding environment, preventing tethering
between the nerve and the surrounding tissue and permitting the
nerve to glide within the fascial tunnel.
[0154] Nerve blocking. In order to block nerve regeneration, the in
situ forming biomaterial needs to have the physical properties to
prevent nerves from migrating into the biomaterial including
negative or neutral charge, smaller pore size, hydrophilicity
and/or higher crosslinking density. Although most studies are
focused on materials through which nerves regenerate, several
studies have documented the biomaterials through which nerves will
not grow, including poly(ethylene glycol)-based hydrogels, agarose-
and alginate-based hydrogels, particularly at higher concentrations
of the polymers. Higher concentrations typically have higher
crosslinking density and thus smaller pore size. These hydrogels
can be employed for their ability to prevent neurite outgrowth in
vitro and in vivo by virtue of their charge, inert surface,
hydrophilicity, and pore size. One example of this agarose, in
which nerves will not extend across the biomaterial above
concentrations of 1.25% wt/vol. In another example, PEG hydrogels
can prevent neuroma formation at 4% w/v and higher. In other
embodiments, even positively charged or natural in situ forming
biomaterials can provide a barrier to nerve regeneration if the
solid content and crosslinking density are such that the pores are
too small for cellular ingrowth.
[0155] In order to prevent neuroma formation, the in situ forming
biomaterial needs to provide the requisite mechanical strength to
act as a barrier to nerve regeneration for two months, more
preferably three months or more. Many in situ forming gels,
including commercially in situ forming PEG hydrogels with
biodegradable ester linkages, may have sufficient mechanical
strength initially but hydrolyze at such a rate that their
crosslinks have broken sufficient that their mechanical strength at
1 to 2 months is not sufficient to prevent neuroma formation (See
Table 1). In vivo experiments in a rat sciatic nerve model
demonstrated the formation of bulbous neuromas at between one and
three months after delivery of these hydrogels around the end of a
transected nerve stump. Preclinical testing has demonstrated that a
mechanical strength of at least 10 kPa, more preferably 20 kPa or
more is necessary to prevent neuroma formation. At three months, in
vivo studies have demonstrated that these hydrogels have been full
degraded and cleared from the site or have lost their mechanical
integrity sufficient that the nerve has grown out into the soft,
collapsed and/or fractured gels and formed a neuroma. Thus,
although the prior art teaches the use of PEG hydrogels for the
purposes of nerve repair, not all hydrogels are suitable to support
the long-term mechanical strength and persistence requirements
necessitated to prevent neuroma formation and aberrant nerve
outgrowth. Preferably the barrier has an in vivo persistence of at
least about two month or at least about three months, preferably
four months or more depending upon the desired clinical response to
reduce chronic neuropathic pain after surgery. The mechanical
integrity of the hydrogels at various points in vitro and in vivo
can be assessed through compression testing, described further
below.
[0156] Persistence The in vivo persistence of biodegradable
hydrogels is related to the crosslinking density and thus the
mechanical integrity of the hydrogel. For applications to prevent
neuroma formation, the hydrogel degradation must be sufficiently
slow that the hydrogel does not lose significant structural
integrity during the weeks to months during which the nerves are
attempting to regenerate, which occurs over approximately 3 months
and may be 6 months or more in humans. In this manner, the
persistence of the hydrogel and the persistence of the mechanical
integrity of the hydrogel is critical to providing ongoing
protection and padding from neuroma and aberrant nerve outgrowth
preferably for 3 months or more, preferably 4 months or more. In
embodiments utilizing a degradable hydrogel, the mechanical
strength must be maintained for longer than 2 months, preferably 3
months and thus there must be no substantial degradation of the
hydrogel for this period of time, preferably 3 months or more.
Similarly, the persistence of the mechanical integrity and, in
turn, the hydrogel is critical to the ongoing offloading provided
by the hydrogel around the nerve-nerve or nerve-graft interface for
a period of preferably 2 months, more preferably 3 months as even
nerves that have been directly sutured to one another through
direct coaptation still have not regained their original strength
(nerves have approximately 60% of original strength at 3 months
after a transection).
[0157] The development of in situ forming polymers, and
particularly, in situ forming synthetic hydrogels, including
PEG-based hydrogels with longer in vivo mechanical strength and
longer persistence profiles beyond 2.5 months but less than 12
months is challenging. For example, there is a significant gap
between the in vivo persistence of PEG hydrogels with biodegradable
esters (weeks to less than 3 months) in and around the surgical
environment of the nerve and PEG hydrogels containing biodegradable
urethane or amide bonds, with degradation profiles in this
subcutaneous extramuscular location on the order of 9 months to 18
months or more. Some embodiments focus on in situ forming polymers,
preferably multi-arm PEGs, with the requisite mechanical strength
and persistence to prevent neuroma formation. In particular, the
swelling, mechanical strength and in vivo persistence of PEG
hydrogels are described to permit the long-term safety and efficacy
in applications requiring the long-term prevention of aberrant
nerve outgrowth and the ability to detension and offload nerves
over a period of months after the surgical repair.
[0158] In order to obtain a suitable in vivo mechanical strength
and persistence, conventional PEG hydrogels containing a degradable
ester linkers that are widely available commercially as dural and
lung sealants are not suitable for applications around nerves given
their loss of mechanical strength and/or clearance within a couple
months. Simply, degradation occurs at a rapid enough rate that
mechanical integrity can not be maintained for sufficient time,
making these hydrogels suitable for anti-adhesion prevention but
not the prevention of nerve outgrowth. In embodiments utilizing a
nondegradable PEG hydrogel, 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 testing
of a range of hydrogel with various molecular weights, degradable
linkages, crosslinking densities demonstrated that only hydrogels
with sufficient mechanical strength at 3 months (and with this, in
vivo persistence) were able to prevent neuroma formation. Examples
of hydrogels, degradation times, and formation of neuromas are
provided in the table below. FIGS. 16Ai-16Aiii illustrate the
formation of a neuroma after the delivery of DuraSeal.
[0159] Examples of Multi-Armed PEG Hydrogels with Various
Hydrolytically Labile Bonds
TABLE-US-00002 In Vivo Neuroma formation in Rat Sciatic PEG
Hydrogel Persistence Nerve Transection Model PEG-SS 2 weeks Large
bulbous neuroma observed (ester bond) at 1 month Duraseal 2 to 8
weeks Large bulbous neuroma formation (ester bond) observed at 2
and 3 months PEG-SG 4 to 8 weeks Large bulbous neuroma formation
(ester bond) observed at 3 months PEG-SAP 6 to 8 weeks Large
bulbous neuroma formation (ester bond) observed at 2 and 3 months
PEG-SAZ 2-3 weeks Large bulbous neuroma at 2 and 3 (ester bond)
months PEG-SGA 9 months No neuroma formation (amide bond) or more
PEG-SC 6 months No neuroma formation (urethane bond) or more
[0160] In vivo persistence refers to the absence of significant
absorption of the biomaterial, such as less than 25% resorption,
preferably less than 15% at a given time point. Depending on the
biomaterial, this can be assessed by mass loss, loss of
cross-linking density, or change in the form of the biomaterial.
Active bonds that have more extended degradation in vivo such as
the PEG-ureas (e.g. PEG isocyanate, PEG-NCO), PEG-urethanes
(PEG-succinimidyl carbonate) (PEG-SC) and PEG-carbamate. Hydrogels
comprised of polyethylene glycol succinimidyl carbonates (PEG-SCs)
with more than 2 arms, such as the 4-arm, 6-arm, or 8-arm PEGs with
molecular weights ranging from 1K to 50K, preferably 10K to 20K,
such as 10K, 15K or 20 kDa. In some embodiments, the 4-arm 10K
PEG-SC, 4-arm 20K PEG-SC, 8-arm 10K PEG-SC, 8-arm 15K PEG-SC, or
8-arm 20K PEG-SC are selected, more preferably 4-arm 10K PEG-SC or
8-arm 20K PEG-SC. The following patent is incorporated for
reference 20160331738A1.
[0161] Compressive Strength.
[0162] The desired compressive strength (elastic modulus, Young's
modulus) of the hydrogel is greater than 10 kPa, preferably greater
than 20 kPA, preferably greater than 30 kPa. In the preferred
embodiment, the compressive strength of the is greater than 10 kPa
after 3 months in vivo, more preferably 40 kPa at 3 months after
administration.
[0163] Compressive strength was measured benchtop after in vitro
equilibrium and also after harvesting implanted samples from the
subcutaneous space in rats, in which hydrogel cylinders (d=6 mm)
are cut to 100 mm long, pre-equilibrated (for 12 hours at
37.degree. C.) and evaluated for compressive strength. Compressive
properties of the hydrogel formulations were measured at a 1 mm/min
with the Instron. The modulus was calculated as the tangent slope
of the linear region between 0.05 and 0.17 of the stress-strain
curve.
[0164] Compressive Strength of Various Formulations
TABLE-US-00003 Compressive Compressive modulus modulus (t = 3
months, Polymer (t = 0) in vivo) Neuroma formation Formulation G 20
kPa 5 kPa Neuroma formation Formulation H 12 kPa 8 kPa Neuroma
formation Formulation I 1 kPa 1 kPa Neuroma formation Formulation I
25 kPa 17 kPa No neuroma formation Formulation J 72 kPa 55 kPa No
neuroma formation
[0165] Although in vitro mechanical strength and persistence of the
hydrogels (37.degree. C., PBS) typically does not correlate well
with in vivo persistence, the maintenance of mechanical strength of
the hydrogels at 3 months in vitro is a strong indicator of the
ability of the hydrogel to provide a sustained mechanical barrier
to nerve regeneration in vivo.
[0166] In some embodiments a cleavable carbamate, carbonate, or
amide linker in a biodegradable hydrogel permits a more stable
slowly degrading bond to maintain the requisite mechanical strength
to prevent nerve outgrowth for three months or more and, with this,
the in vivo persistence to provide the sustained mechanical barrier
to nerve regeneration.
[0167] Generally, the structure of multi-armed PEGs are
C-[(PEG).sub.n-M-L-F].sub.m
[0168] where
[0169] C=core structure of the multi-arm PEG
[0170] n=repeating units of PEG on each arm (25 to 60 units)
[0171] M=Modifier
[0172] L=cleavable or noncleavable linker (ester, urethane, amide,
urea, carbamate, carbonate, thiourea, thioester, disulfide,
hydrazone, oxime, imine, amidine, triazole and
thiol/maleimide).
[0173] F=reactive functional group for covalent crosslinking, e.g.
maleimide, thiol or protected thiol, alcohols, acrylates,
acrylamides, amines, protected amines, carboxylic acids or
protected carboxylic acids, azides, alkynes, 1,3-dienes, furans,
alpha-halocarbonyls, and N-hydroxysuccinimidyl,
N-hydroxysulfosuccinimidyl, or nitrophenyl esters or carbonates
[0174] m=number of PEG arms (e.g. 2, 3, 4, 6, 8, 10)
[0175] In some embodiments, hydrolysis modifiers (M) can be
incorporated into the backbone of the hydrogels to slow the
hydrolytic degradation of the ester bonds (L) in the hydrogel. This
can be accomplished with electron donating groups which regard the
reaction or by increasing the length of the carbon chain adjacent
to the ester bond in order to increase the hydrophobicity and
shield the bond from hydrolysis. For example, PEG-SAP, PEG-SAZ are
examples of PEG-ester bonds with 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 a PEG-aromatic carboxyl ester,
including a benzoic acid ester or a substituted benzoic acid
ester.
[0176] In some embodiments, a more stable or slowly degrading bond
such as a urethane bond or amide bond may be selected to provide
the requisite mechanical strength and in vivo persistence to
prevent the neuroma from forming.
[0177] In other embodiments, hydrolysis modifiers (M) can be
designed in the backbone of the hydrogels to increase the
hydrolytic degradation of urethane in the hydrogel. This can be
accomplished with the addition of electron withdrawing groups which
accelerate the reactions.
##STR00001##
[0178] In one embodiment, the hydrolysis rate of carbamate bond can
be modulated by the adjacent groups, thus modulating the
persistence of hydrogel in vivo. R1 and R3 can be any aliphatic
hydrocarbon group (--CH.sub.2--, --CHR--, --CRR'--), substituted
aliphatic hydrocarbon group, aromatic groups and substituted
aromatic group in any arranged forms. The aromatic group includes
but not limit to phenyl, biphenyl, polycyclic aryls and
heterocyclic aryls. The substitution moiety for aliphatic and
aromatic group include but not limit to halogen, alkyl, aryl,
substituted alkyl, substituted aryl, substituted heteroaryl,
alkenylalkyl, alkoxy, hydroxy, amine, phenol ester, amide,
carboalkoxy, carboxamide, aldehyde, carboxyl, nitro and cyanide. R2
can be H and any group in R1 and R3. In addition, R1 can include
isocyanate, aromatic isocyanate, diisocyanate (e.g. LDI). In one
embodiment, R3 can be Anilide and in another embodiment R1 can be
phenyl.
##STR00002##
[0179] In another embodiment, the hydrolysis rate of carbamate bond
can be modulated by the modulator at the beta position. The
modulator can be CF.sub.3PhSO.sub.2--, ClPhSO.sub.2--,
PhSO.sub.2--, Me.sub.nPhSO.sub.2--, MeOPhSO.sub.2--, MeSO.sub.2--,
O(CH.sub.2CH.sub.2)NSO.sub.2--, CN--, (Et).sub.2NSO.sub.2--. In yet
other embodiments, these modifiers can be adapted for use in PEG
hydrogels containing amide, carbonate and urea linkages. Additional
modifiers that affect the hydrolysis rate of the carbamate linkage
are described in U.S. Pat. No. 7,060,259, incorporated for
reference herein. Additional cleavable crosslinks are described in
Henise et al (2019) In vitro-in vivo correlation for the
degradation of Tetra-PEG-hydrogel-microspheres with tunable
b-eliminative crosslink cleavage rates. International Journal of
Polymer Science, incorporated in entirety. These modifier groups,
M, can be on the backbone itself or a nearby side chain such as
with a beta-eliminative linker as described by D. V. Santi et al
(2012) Predictable and tunable half-life extension of therapeutics
agents by controlled chemical release from macromolecular
conjugates. PNAS, 109(6) 6211-6216 and US20170312368A1 incorporated
herein for reference. In some embodiments, a soft chain extender is
added, such as an amino acid-peptide based chain extender with
ester linkages. For example poly (phosphoester urethanes) with
chain extenders containing phosphoester linkages. For example poly
(DL, lactide) is a chain extender or poly(caprolactone) to extend
the PEG chain and add a soft segment. Preferably the molecular
weight of the chain extender may be 0.5 kDa to 5 kDa, preferably 1
to 2 kDa, more preferably 2 kDa. The soft segments can provide
additional properties to enhance the physical properties of the
hydrogel including the thermosensitivity, crystallinity, potential
resulting in physical in addition to chemical crosslinking. These
hydrogels may be comprised of, for example, PEGs with molecular
weight between 1,000 Da and 50 kDa including multi-arm
PEG-succinimidyl carbonate (4-arm or 8-arm) with molecular weights
of 5 to 40 kDa and arm lengths between 1 and 3 kDa, and PEG-amine
(4-arm or 8-arm) with molecular weights between 5 to 40 kDa,
preferably 10 or 20 kDa. In one embodiment, PEG-SC (4-arm 10K) is
crosslinked with PEG-amine (8-arm 20 k). In another embodiment,
PEG-SC (8-arm 15K) is crosslinked with trilysine amine. In another
embodiment, PEG-SC (4-arm 20K) is crosslinked with trilysine amine.
Examples of other in situ forming PEG-SC formulations are described
in U.S. Pat. No. 6,413,507, incorporated herein for reference. In
another embodiment, a 4-arm PEG succinimidyl glutaramide 4-arm 10K
(PEG-SGA) may used in combination with 8-arm PEG-amine 20K at 8%
solid content.
[0180] Alternatively, the functionalized PEG urethanes and esters
may be covalently crosslinked with another reactive polymer or
small molecule (e.g. trilysine) containing amines or protected
amines, maleimides, thiols or protected thiols, acrylates,
acrylamides, carboxylic acids or protected carboxylic acids,
azides, alkynes including cycloalkynes, 1-3 dienes and furans,
alpha-hydroxycarbonyls, and N-hydroxysuccinimidyl,
N-hydroxysulfosuccinimidyl, or nitrophenyl esters or
carbonates.
[0181] In yet other embodiments, blends of faster degrading
PEG-esters and slower degrading PEG-SGA or PEG-SC with ratios of
10:1 or 5:1 may permit slowing of the in vivo degradation profile.
Similarly, blends of multi-arm PEG-SC and PEG-amine that crosslink
to form carbamate bonds and PEG-carbonate ester bonds (delayed
reaction of PEG-SC and with hydroxyl functional groups) to form
blended 60:40 hydrogels (Kelmansky et al (2017) In Situ Dual
Cross-Linking of Neat Biogel With Controlled Mechanical and
Delivery Properties. Molecular Pharmaceutics, 14(10) 3609-3616.
[0182] In yet other embodiments, multi-arm PEGs can be combined
with blocks of other hydrolytically degradable polymers that can be
used to tailor the degradation time of the PEG hydrogels. For
example, soft segments with diblock with polyester or triblocks can
be synthesized with low molecular weight polyester regions to
permit the hydrogel to be formed in an aqueous environment
(polycaprolactone, polylactic acid, polyglycolic acid,
polyurethane, polyhydroxyalkanoates (PHA), poly(ethylene adipate)
(PEA), alipathic diisocyanates such as isophorone diisocyanate
(IPDI) or L-lysine ethyl ester diisocyanate (LDI)). These blocks
can be comprised of lactide, glycolide, or caprolactone regions
can, depending on the degree of crystallinity (D,L or L,L) be used
to provide additional mechanical strength to the hydrogels permit
tuning of the degradation profile. For example, a block of
caprolactone can be added to a multi-arm PEG which each arm
comprising a PEG-PCL-NHS ester. In this embodiment, the PCL domain
may extend the degradation of a previously poor in vivo persistent
multi-arm PEG with hydrolytic ester linkages. In the preferred
embodiment, a PCL block of between 1 and 5 kDa, preferably 1 to 2
kDa is added on the PEG arm. For example, a 4-arm 28K PEG-PCL-NHS
ester may react with an 4-arm 10K PEG-amine to form a crosslinked
hydrogel in situ, where the PEG is a 2K block. The addition of the
block renders the hydrogel in situ forming both through chemical
and physical crosslinking. Amino acids can also be incorporated as
chain extenders in the PEG-SC to improve the degradation of the
PEG-urethane. In some embodiments low molecular weight
trifunctional polyester polyols are selected for incorporation.
Please refer to FIG. 1--common monomers used for synthesis of
biostable and biodegradable polyurethanes, incorporated herein for
reference (Chapter: Degradation of Polyurethanes for Cardiovascular
Applications, Book: Advances in Biomaterials Science and Biomedical
Applications).
[0183] In some embodiments heterobifunctional crosslinkers are used
to enable polyesters to be conjugated to some arms and NHS esters
or other functional group with other arms.
[0184] In yet other embodiments, excipients may be incorporated
into the hydrogels to modify the mechanical strength, density,
surface tension, flowability, and in vivo persistence of the
hydrogels. These modifiers are encapsulated in the hydrogel when
the hydrogel is formed. Modifiers may include amphiphilic
excipients such as vitamin E TPGS, low molecular weight polyesters
such a caprolactone or solvents such as ethanol. In one embodiment,
ethanol is incorporated in the diluent or accelerator solutions to
yield a 5% to 70% v/v ethanol loaded hydrogel. The ethanol improves
the elasticity of the hydrogel and reduces the density of the
hydrogel precursor solution relative to the nerve density. Further
more, low concentrations of ethanol may be incorporated in the
hydrogel to improve the pot or functional life of the PEG/diluent
solution after PEG powder suspension. In another embodiment,
Pluronic may be incorporated in diluent or accelerator solution to
yield a 5 to 15% w/v to yield a PEG-SG hydrogel with improved
elasticity and in vivo persistence. In yet another embodiment, low
molecular caprolactone is incorporated into diluent solutions to
yield a 1 to 5% w/v PEG/caprolactone blended hydrogel. In another
embodiment, vitamin TPGS can be incorporated into the diluent
solution to yield a 5 to 20% w/v of PEG/vitamin E TPGS blend.
[0185] Swelling Another critical element of these hydrogels is the
swelling of the hydrogels for applications around nerves. The
hydrogels, when delivered circumferentially around an object such
as a nerve, undergo positive swelling in an outward radial
direction. Initially, the hydrogels undergo equilibrium-mediated
swelling as they equilibrate with the fluids in the surrounding
environment, and, later, when a critical number of hydrolytic bonds
have broken, the hydrogels swell as a result of loss of mechanical
strength. This latter phase of degradation-mediated swelling
results in the progressive loss of mechanical strength and hydrogel
softening that the hydrogel collapses and is ultimately cleared
from the site. In vivo experiments in which transected rat sciatic
nerves were surrounded within hydrogels that swelled 5%, 10%, 20%,
30% and 60%, demonstrated that hydrogels that swell more than 30%
were significantly more likely to fall off of the nerve as a result
of the creation of a gap between the nerve and the hydrogel. Of
note, PEG hydrogels that swell at or less than 0%, shrink when
equilibrating in vitro or in vivo and the resultant compression may
result in persistent hydrogel-mediated local nerve pain. For
example, the DuraSeal hydrogel swells significantly and have a
tendency to fall of the proximal nerve stump when delivered in
situ.
[0186] Equilibrium swelling. For applications in which hydrogels
are delivered to nerves to prevent nerve regeneration, maintaining
close adherence and apposition between the nerve and the
conformable hydrogel is desirable. As a result, minimizing the
equilibrium swelling post-hydrogel delivery is desirable. The
equilibrium swelling occurs during in the minutes to days as the
hydrogel equilibrates with the fluids in the in situ environment.
In one embodiment, the hydrogel swells greater than 0% but less
than 40%, preferably greater than 5% and less than 30%, more
preferably greater than 5% and less than 25%.
[0187] Furthermore, in some embodiments, it is desirable to avoid
hydrogels that shrink as these hydrogels may compress the nerve and
result in aberrant nerve firing and therefore it is preferable to
use hydrogels that swell greater than 0%. In addition, the nerve
may swell after injury, and so some swelling is desirable to permit
some space for the nerve to swell.
[0188] Equilibrium swelling may be preferably assessed in vitro at
body temperature conditions (37.degree. C. in PBS). Hydrogel
samples were prepared in cylindrical silicone tubing (6 mm) and cut
to dimensions of 6 mm diameter by 12 mm length. Samples were
weighed and merged into PBS at 37.degree. C. After swelling in PBS
for 12 to 24 hours at 37.degree. C., samples were taken out and
weighted again. The swelling is calculated by the percentage of
mass increase.
[0189] Degradation swelling. A secondary characteristic in
biodegradable or bioerodible hydrogels, after the initial
equilibrium swelling, is an appreciation for a second ongoing phase
of swelling that occurs as a result of the degradation of the
hydrogel. The swelling may occur through the hydrolytic, enzymatic,
or oxidatively-sensitive bonds in the hydrogel. This is an equally
important characteristic because 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 an animal model,
the period of time is shorter and in the clinical setting this
period is longer. In some instances, if the degradation rate is too
rapid, the hydrogel may fracture and fall off the nerve or be
cleared before the hydrogel can serve the function to prevent nerve
outgrowth and/or neuroma prevention. In other instances, if the
hydrogel appears intact on the nerves, there may be a substantial
loss of the mechanical integrity within the hydrogel as a result of
degradation that the nerve may extend out into the softened or
fractured hydrogel and form a neuroma formation. As a result, it is
preferable that a biodegradable system have no more than 50% of the
hydrolytically labile linkages cleaved at 3 months, more preferably
no more than 30% of the linkages, and even more preferably no more
than 20% of the linkages. After the period of time in which the
hydrogel provides a mechanical barrier to nerve regeneration, the
crosslinking density can drop and the degradation can continue
until the hydrogel is entirely cleared. The loss of bonds can be
evaluated in part through the reduction in the mechanical integrity
of the hydrogel. The loss of bonds can be evaluated in part through
the reduction in the mechanical integrity of the hydrogel. Thus, it
is desirable that the hydrogel maintain a compression modulus of 40
kPa at 3 months post delivery, this hydrogel is sufficiently stiff
that nerves will not grow through it.
[0190] Pressure. In addition to ensuring that the swelling is not
so great that the hydrogel falls off of the nerve, confirmation
that the swelling (or low swelling, shrinkage) will not result in
nerve compression is also desirable. In one experiment, a pressure
transducer catheter was placed next to a nerve and an in situ
forming hydrogel was delivered to form around the nerve/pressure
transducer (Millar Mikro-Tip pressure catheter, 3.5 F, single
straight, AD Instruments). The hydrogel was then placed at
37.degree. C. in PBS and measurements of the pressure as a function
of time were taken. Hydrogels with approximately 0% or negative
swelling resulted in high and sustained increases in the pressure
(>80 mmHg) exerted on the embedded nerve whereas hydrogels that
swelled 10% or more did not result in any significant increases in
pressure (.about.20 mmHg). In the preferred embodiment, the
pressure reading after equilibrium swelling is around 5 mmHg In
preclinical and clinical models, pressure at the site of nerve
damage may be between 5 and 15 mmHg (Khaing et al 2015--Injectable
Hydrogels for Spinal Cord Repair). For example, although a variety
of materials have been evaluated for modulating nerve regeneration
in a spinal cord model, the majority do not have the requisite
linear compressive modulus (G) to prevent neuroma formation (Table
1, Khaing et al, 2015).
[0191] Stiffness. Stiffness of the hydrogel can measured/inferred
either by rheology (G'=storage modulus, G*=shear modulus, or the
linear compressive modulus (G). Preferably the stiffness of the
hydrogels, as measured through the linear compressive modulus (G)
is greater than 10 kPa, preferably greater than 30 kPa, more
preferably greater than 50 kPa. The stiffness prevents nerve
outgrowth into the surrounding hydrogel.
[0192] Compression and rebound. In addition to injectable gels that
have minimal swelling, gels that are compressible are desirable. In
this manner, even if the hydrogel implant is pressed, it will not
fracture. Compression and rebound testing is performed on
cylindrical samples (6 mm diameter, 10 mm long) that have been
incubated for at least one hour at 37 oC until equilibrated. The
samples are loaded into the Instron and a displacement
perpendicular to the longitudinal axis of the cylinder will be
applied at a crosshead speed of 1 mm/min to a final displacement of
60% of the diameter of the conduit. Verification that the hydrogels
can withstand compressive forces of greater than 0.25N and that no
changes in the shape and diameter have occurred after removal of
the compressive forces.
[0193] Flexibility. Another critical parameter of these in situ
forming polymers is the ability of the hydrogels to bend and flex
at physiologically relevant angles in the body. To evaluate the
flexibility of the hydrogels, the hydrogels were formed inside 0.1
to 0.25'' inner diameter silicone tubing to form 12 to 24'' long
cylindrical hydrogel cables. Preferably, the hydrogels have
sufficient flexibility to bend greater than 90.degree. and more
preferably cylindrical strands of hydrogel can be readily be tied
into a knot. Since the flexibility and elasticity is determined, in
part, by the distance between the core of one multi-arm PEG to the
core of the adjacent multi-arm PEG, PEG hydrogels with core-to-core
distances of 3 kDa, more preferably 5 kDa or more. Flexible robust
hydrogels that will not fracture in the highly mobile and
compressive environment of the body. As a result, more flexible
hydrogels are desired such as combinations of the 4-arm 10K or 20 K
PEGs with 4-arm or 8-arm 20K PEG-amines may be desirable.
[0194] Viscosity. Low and medium viscosity precursor solutions may
be selected to encapsulate the hydrogel with generally better
adhesion in the low viscosity solution and improved handling of the
nerve in the medium viscosity precursor solutions. In one
embodiment of the invention, the flowable media is a low viscosity
hydrogel precursor solution, having a viscosity of no more than
about 100 cP and in some embodiments no more than about 20 cP or no
more than about 5 cP. In yet another embodiment, the flowable media
is a medium viscosity hydrogel precursor solution, having a
viscosity preferably 300 to 10 kcP, more preferably 300 to 900 cP.
In one embodiment, a viscosity enhancer/modifier or thickening
agent can be added to the gel precursor to modify the fluidic
properties and help to positioning the nerve in the cap before
gelling. The viscosity modifier can be natural hydrocolloids,
semisynthetic hydrocolloids, synthetic hydrocolloids and clays.
Natural hydrocolloids include but not limited to Acacia,
Tragacanth, Alginic acid, Alginate, Karaya, Guar gum, Locust bean
gum, Carrageenan, Gelatin, Collagen, Hyaluronic acid, Dextran,
Starch, Xanthan gum, Galactomannans, Konjac Mannan, Gum tragacanth,
Chitosan, Gellan gum, Methoxyl pectin, Agar, Gum arabic, Dammar
gum. Semisynthetic hydrocolloids include but not limited to
Methylcellulose, Carboxymethylcellulose, Ethyl cellulose, Hydroxy
ethyl cellulose, Hydroxy propyl methyl cellulose (HPMC, 0.3%),
Modified starches, Propylene glycol alginate. Synthetic
hydrocolloids include but not limited to Polyethylene glycol,
Polyacrylic acid, polyvinyl alcohol, polyvinyl pyrrolidone,
polyglycerol, Polyglycerol polyricinoleate. Clays include but not
limited to Magnesium aluminum silicate (Veegum), Bentonite,
Attapulgite.
[0195] In another embodiment, viscosity can be modified by
pre-crosslinking PEG-amine and PEG-urethane. PEG-amine and
PEG-urethane can pre-crosslinked at a ratio from 10000:1 to 1:10000
at total PEG concentration of 0.01% to 100%. The pre-crosslinking
can be further cross-linked by itself, or with PEG-amine, or with
PEG-urethane, or with PEG-amine and PEG-urethane to form a higher
viscosity precursor solution.
[0196] Density of precursor solution. Nerve tissue, as a result of
the myelin and adipose tissue, is hydrophobic and thus has a
tendency to float in solutions with density approximating that of
water (.about.1 g/cm.sup.3). By adjusting the density of the
flowable media, the nerve position can be adjusted to reduce the
propensity of smaller diameter nerves to float up in the precursor
solution without sacrificing adhesion strength that comes with
increasing precursor viscosity. In some embodiments, the density of
the precursor solution or media solution is decreased so that the
nerve is relatively more dense than the solution to <1
g/cm.sup.3, preferably <0.9 g/cm3. In still other embodiments,
the density of the precursor solution is adjusted to be
approximately equivalent to that of the nerve. Polar and less dense
solvents can be added including ethanol (10 to 70%), toluene (10%),
ethyl acetate or chlorobenzene to reduce the density of the
precursor solution. In another embodiment, vitamin E TPGS (1-2 kDa,
10-20%) can be added to reduce the propensity of the nerve to float
up. Some of these solvents also reduce the surface tension of the
precursor solution, causing the nerve to sink.
[0197] Open Surgical Neuroma Procedure. After openly exposing the
surgical site and isolating the target nerve, fresh transection of
the nerve is desirable to clean up the nerve end. In some
embodiments, the clinician may elect to transect the nerve at a 90
degree or, alternatively, a 45 degree angle. In some embodiments,
the clinician may elect to use other methods such as electrocautery
of the nerve end, ligation of the nerve stump, apply low molecular
weight end-capped PEG (e.g. 1-5 kDa, 50 w/v % hypotonic solution)
or other approaches that they have developed to seal or ablate the
end of the nerve.
[0198] Axoplasm. As axoplasm, a viscous and sticky material that
oozes from the end of the cut nerve after transection, may reduce
close apposition between the nerve end and the hydrogel, it may be
desirable to remove it from the nerve tip. This can be accomplished
through the contact between the nerve and an absorbent material,
such as a swab. An absorbable swab may be provided in order to
absorb any of the axoplasm after nerve transection. The tip of the
swab is preferably less than 5 mm, more preferably less than 2 mm
in order that it may fit comfortably within the form and hold the
nerve while the hydrogel is delivered. The swab may be provided as
part of the kit. Alternatively, this can be accomplished by
contacting the nerve tip with the surrounding tissue, which results
in the rapid formation of an adhesion between the nerve and the
tissue that must then be secondarily severed.
[0199] Coverage of the proximal nerve stump. The hydrogel itself
preferably extends at least 0.5 mm, preferably 1 mm to 20 mm,
preferably 2 mm to 10 mm beyond the end of the proximal nerve
stump.
[0200] Length coverage. It is preferable that a minimum of 10 mm of
nerve be embedded/encapsulated in the hydrogel although, in some
instances, 5 mm may be sufficient. A greater length of nerve
embedded in the hydrogel accomplishes several things a) increased
surface area of apposition between the nerve and the hydrogel and
b) decreased likelihood of proximal sprouting from nodes of Ranvier
proximal to the transection as these proximal nodes are embedded in
hydrogel. Again, the greater the length of nerve encapsulated, the
higher the likelihood that even the regions that were damaged
through handling with forceps, previous trauma, etc. are embedded
in the hydrogel, thereby preventing sprouting of nerve fibers.
[0201] Once an approximately 10 mm section of nerve is isolated,
whether the nerve is adherent to the swab, the side of a forcep or
rod, or is gently held by a pair of forceps, the nerve is elevated
slightly to allow a form to be placed underneath the nerve. In one
embodiment, the nerve is then gently lowered into a channel or
entry zone to align the nerve in the center of the form. See, e.g.
FIGS. 1A and 1B. The form, once the desirable position is reached,
is left in place.
[0202] While holding the nerve tip in one hand in the center of the
form, the clinician then delivers the in situ forming hydrogel
using the other hand to fill the form and retain the nerve in the
center of the form. The top of the silicone form serves as a guide
for when to stop filling the form. As the hydrogel fills past the
top of the nerve, the swab/forcep is removed so that the nerve is
retained within the hydrogel and not the tool. In this manner,
there is no direct path for a nerve to regenerate through the
surrounding tissue and the nerve is completely surrounded by the
hydrogel.
[0203] Gel thickness. Preventing nerve regeneration requires
providing a conformable barrier at the proximal end of a transected
nerve. The hydrogel also preferably surrounds the nerve with a
thickness of 100 .mu.m to 5 mm radially. In one embodiment, the
hydrogel precursor solution is dripped over the nerve to form a
thin protective coating approximately 100 microns to 2 mm in
thickness circumferentially around the proximal nerve stump and
remain in the place to block neurite outgrowth. A thin coating is
sufficient to provide a barrier to nerve regeneration and thus in
some embodiments, the nerve is dip-coated in the flowable media and
the hydrogel subsequently forms in a thin layer around the nerve.
In this embodiment, the hydrogel gelation time is adjusted to 10 to
20 seconds to permit the removal of the coated nerve prior to the
conversion to a nonflowable form with gel formation. Thin coatings
providing adhesion to and coverage of the circumference and tip of
the nerve stump on the order of 50 microns to 500 microns to cover
the end of the nerve are desirable.
[0204] In yet another embodiment, it is desirable to form the
hydrogel around the nerve in an implant or bolus, providing a
robust adhesive layer of hydrogel around the nerve, approximately
0.5 to 5 mm, more preferably 1-2 mm in thickness around the
circumference of the nerve and 1 to 5 mm beyond the tip of the
nerve stump tip.
[0205] Pore size. In order to prevent nerve regeneration into the
biomaterial, the pore size of the hydrogel needs to be sufficiently
small to prevent nerves and other supporting cells from
infiltrating the biomaterial. Nerve axon diameters are between
approximately 0.5 and 30 .mu.m. Preferably, the growth inhibitory
hydrogel is microporous or mesoporous, with pores less than 1
.mu.m, preferably less than 0.5 microns, more preferably less than
500 nm in diameter.
[0206] Charge Neutrally or negatively charged biomaterials are
preferred as growth inhibitory gels as neurites prefer to grow into
positively charged biomaterials. Similarly, hydrophilic materials
or amphiphilic are preferable to hydrophobic materials.
[0207] Nondegradable hydrogels. If a nondegradable hydrogel system
is used, the same equilibrium swelling characteristics apply but,
since the hydrogel is nondegradable or biostable, degradation
swelling is not relevant. For example, the nondegradable in situ
forming thermoresponsive copolymer, Pluronic (PEO-PPO-PEO),
polyvinyl alcohol, or PEO may be utilized to form hydrogels.
[0208] Clarity. In the preferred embodiment, the hydrogel is clear
and transparent to confirm the location of the nerve after hydrogel
formation. A visualization agent may be incorporated in the
hydrogel to aid in contrast relative to the background tissues. The
color additive or color additive blend may be included in a the
polymer powder solution. In the case of the use of multiple
hydrogels (described below), the use of one or more different
visualization agents is desirable to provide visual confirmation,
for example, that the growth permissive hydrogel was correctly
delivered between the nerves and the growth inhibitory hydrogel was
delivered around the growth permissive hydrogel.
[0209] Elasticity. In some embodiments, the elasticity of hydrogel
can be modulated by incorporating hydrophobic domain into the
hydrogel. The hydrophobic domain can be incorporating by
crosslinking or mixing of molecules, particles, fibers and
micelles. The molecules incorporated can be amphiphilic molecules
including pluronic, polysorbate and tocopherol polyethylene glycol
succinate. The particles, fibers and micelles can be made from
amphiphilic molecules described in the above section. In addition,
many low molecular weight hydrophobic drugs that are incorporated
into the hydrogel (described below) improve the elasticity of the
hydrogel.
[0210] Kit. The in situ forming hydrogel is delivered through a
dual applicator system comprising a dual channel applicator, a dual
adapter, 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 the dual
applicator system. The kit may include one or more forms into which
the hydrogel is delivered. The kit may also include on or more
mixer-blunt syringes. The Mixer syringes may be conventional single
lumen mixers with a static mixing element or the mixers may be
mixers in which there is recirculation and turbulent flow of the
contents to improve the mixing of the precursor solution.
[0211] Chemical and physical. Preferably, the hydrogel networks are
predominantly hydrophilic with high water content and are formed
through the physical or chemical crosslinking or synthetic or
natural polymers, copolymers, block copolymers or oligomers.
Examples of synthetic hydrogels that are not growth permissive
include agarose, PEG, or alginate, PVA hydrogels with 2% w/v solid
content or higher, preferably 6% w/v solid content, more preferably
8% w/v solid content or higher. PEGs. Multi-arm PEGs are described
above but may be selected according to the desired properties from
PEG-amine, PEGarboxyl, PEG-SCM, PEG-SGA, PEG-Nitrophenyl carbonate
(carbonate linker), PEG-Maleimide, PEG-Acrylate, PEG-Thiol,
PEG-Vinysulfone, PEG-Succinimidyl Succinate (SS), PEG-Succinimidyl
Glutarate (SG), PEG-Isocianate, PEG-Norbornene or PEG-Azide.
Alginate. Viscous injectable alginate sol (1 to 5%) may be
delivered around the nerve. Similarly, agarose gel at
concentrations of 1% wt/vol or more prevent nerve extension.
[0212] Hypotonic solutions. In one embodiment, a hypotonic solution
(Ca2+ free, slightly hypotonic, saline solution containing 1-2 mM
EGTA) is delivered to the cut nerve to assist in the sealing of
crushed or transected axonal ends prior to repair with the in situ
forming biomaterial.
[0213] PEG Fusion Combined with an In Situ Nerve Cap or Wrap. As
described in the many publications outlining methods for PEG fusion
of nerves (3.35-5 kDa, 30-50% w/v solution), a PEG solution can be
delivered to the nerves to first fuse the nerves, alone or in
combination with methylene blue. After sealing the membranes, the
growth permissive hydrogel is delivered in between and around the
compressed or severed nerves.
[0214] Cross-linked Particles. In some embodiments, the hydrogel
can be made with cross-linkable particles, fibers, or micelles.
These particles, fibers or micelles are functionalized with
reactive groups, including but not limited to active ester, amine,
carboxyl, aldehyde, isocyanate, isothiocyanate, thiol, azide and
alkyne, which can be cross-linked with small molecules, polymers,
particles fibers or micelles with reactive groups to form bonds
including amide, carbamate, carbonate, urea, thiourea, thioester,
disulfide, hydrazone, oxime, imine, amidine and triazole. In some
embodiments, the micelles, fibers and particles can be formed from
amphiphilic macromolecules with hydrophilic and hydrophobic
segments. The hydrophilic segments can be natural or synthetic
polymers, including polyethylene glycol, polyacid, polyvinyl
alcohol, polyamino acid, polyvinyl pyrrolidone, polyglycerol,
polyoxazolines, and polysaccharides. Hydrophobic segments can be
fatty acids, lipids, PLA, PGA, PLGA, PCL and the polymer ester
copolymer at different ratio. In another embodiment, functionalized
microparticles form physical crosslinks with one another after a pH
change, and then, when placed in situ, the functionalized particles
crosslink to form an interconnected network of microparticles.
[0215] Sealants. Some of the in situ forming gels developed for
adhesion prevention and sealants may also be adapted for this
application to preventing neuromas and aberrant nerve outgrowth
into scar tissue, such as low molecular weight polyanhydrides of
acids like sebacic acid, including poly(glycerol-co-sebacate)
(PGSA) based sealants (U.S. Pat. No. 9,724,447, US20190071537,
Pellenc et al (2019) Preclinical and clinical evaluation of a novel
synthetic bioresorbable, on demand light activated sealant in
vascular reconstruction, incorporated herein and adapted for use
around nerves, for reference). Another sealant that may be adapted
for delivery around peripheral nerves is the Adherus Dural Sealant,
which comprises a PEG-polyethylenimine (PEI) copolymer that forms
in situ, as it exhibits low swelling and degrades in about 90 days
(U.S. Pat. No. 9,878,066, incorporated herein). Other sealants
include BioGlue Surgical Adhesive (Cryolife), composed of bovine
serum albumin and glutaraldehyde, Omnex (Ethicon), ArterX (Baxter),
Coseal (Baxter) and TissuGlu, composed of lysine based urethane
(Cohera Medical).
[0216] Photoresponsive. In some embodiments, photoresponsive,
photopolymerizing or photocrosslinked biomaterials are envisioned
that could be delivered in a liquid (low to medium viscosity) state
into the form (cap or wrap form) around the nerve, and then, when
the proper positioning of the nerve is obtained within the form,
the photopolymerization is initiated with either ultraviolet or
visible light. In one embodiment, the light source can be attached
directly or via fiber optic cable that interfaces directly with an
opening in the cap or wrap form. The cap form diffracts the light
such that it ensures that the entire form is sufficiently
illuminated to achieve consistent homogenous crosslinking across
the gel. In the preferred embodiment, the light source housing is
coupled directly with the form at the distal end of the cap facing
the nerve stump face to ensure that the light directly penetrates.
Alternatively, the form may be designed with embedded
light-emitting elements that permit the light to be delivered
circumferentially around the nerve. Hydrogels include PNIPAAM
hydrogels modified with a chromophore such as trisodium salt of
chlorophyllin.
[0217] Other forms. In addition to a cross-linked network,
hydrogels may be comprised of dendrimers, self-assembling
hydrogels, or low molecular weight synthetic polymer liquids.
[0218] In one embodiment, lower molecular weight hydrogels (2 kDa,
liquid) can be formed in situ without water as a solvent as
described in Kelmansky et al (2017) In Situ Dual Cross-Linking of
Neat Biogel with Controlled Mechanical and Delivery Properties),
Molecular Pharmaceutics, 14(10) 3609-3616, incorporated herein. In
yet other embodiments, hydrogels can be photocrosslinked to form
hydrogels, as extensively described in the literature. Crosslinking
agents include eosin,) In yet other embodiments, electroconductive
hydrogels are used including poly(3,4-ethylenedioxythiophene)
(PEDOT), poly(pyrrole), polyaniline, polyacetylene, polythiophene,
ester derivative, 3,4-propylenedioxythiophene (ProDOT), natural or
synthetic melanin, derivatives, and combinations thereof.
[0219] Addition of sulfated proteoglycans. In some embodiments, it
may be desirable to deliver inhibitory environmental cues to the
nerves in addition to the mechanical barrier provided by the
hydrogel. This can be accomplished by the addition of inhibitory
molecules and/or extracellular matrix to the hydrogel either
through physical blending or chemical crosslinking. Sulfated
proteoglycans, such as side chains with a negative charge such as
glycosaminoglycans are of interest. Of particular interest is
dermatan sulfate (DS).
[0220] Blends. In some embodiments, it may be desirable to create
blends of two PEGs to improve the degradability of the system. In
one embodiment, the PEG-SC is combined with PEG-SG prior to
crosslinking with trilysine amine to create a hydrogel that has
sufficient mechanical support to prevent nerve outgrowth but then
degrades more rapidly than PEG-SC. The persistent time of gel in
vivo is fine-tuned by the ratio of PEG-SG and PEG-SC. With the
increase of PEG-SC content, the persistent time of gel in vivo
increases. In another embodiment, the PEG-carbamates are blended
with the PEG-carbonates. Other hydrogels include PEG hydrogels
comprised of carbamate derivatives (U.S. Pat. No. 7,060,259).
[0221] Incorporation of agents. In some embodiments, agents can be
dissolved or suspended in the diluent or accelerator solution and
surfactants or ethanol can be added to stabilize the suspension.
The drug can be also encapsulated in microparticles, nanoparticles
or micelles and then suspended in diluent or accelerator. In some
embodiments, the hydrogel can be made with cross-linkable particles
and micelles. These particles or micelles have reactive groups such
as the active ester, amine, carboxyl, thiol and those described in
U.S. Pat. No. 7,347,850 B2 and can be crosslinked with small
molecules, polymers, particles or micelles with reactive groups
which reacts with the former particles or micelles and forming
bonds including amide, carbamate, carbonate, urea, thiourea,
thioester, disulfide, hydrazone, oxime, imine, amidine and
triazole. In other embodiments, gel can be form by the swelling of
particles. The large volume of swelling can increase the particle
contact and lock them into their location to form gel.
[0222] Solid content. The solid content can be adjusted to fine
tune the swelling and tensile properties of the hydrogel. For
example, the solid content can be adjusted above the critical
gelation concentration, such as between 6 to 15% loading, more
preferably 7-9% loading, more preferably 8-8.5% solid content.
[0223] Crosslinking. Hydrogels may be formed in situ through
electrophilic-nucleophilic, free radical, or
photo-polymerization.
[0224] In vivo Persistence. In some embodiments a longer in vivo
persistence may be preferred, in which the hydrogel remains in situ
for between 3 months and 3 years, more preferably 6 months and 18
months, more preferably 6 months and 12 months.
[0225] Adhesion. Adhesive strength is an important criterion for
maintaining the hydrogel in close apposition to the nerve. Adhesion
may occur through crosslinking reactions between the hydrogel and
the primary and secondary amines on the tissue surface e.g. the
epineurium or the amine groups found on the surface of nerves,
glia, and associated cells. The adhesion strength should be greater
than 10 kPa, preferably greater than 50 kPa, more preferably
greater than 100 kPa. Adhesive strength on nerves can be estimated
by embedding the sciatic nerve in the hydrogels. The ends of the
nerves are embedded in superglue between sandpaper and placed in
titanium clamps in a Bose ElectroForce 3200-ES. Nerves are pulsed
at a rate of 0.08 mm/s until failure. Care was taken to ensure that
the nerves were used shortly after harvest and that the hydrogel
and nerve were equilibrated in PBS at 37.degree. C. prior to
testing.
[0226] Other hydrogels. In situ forming polyanhydrides are also of
interest for developing applications directed towards nerves. In
one embodiment, polyanhydride polymers can be acrylated so that
they can form in situ through free radical polymerization.
Alternatively, they can form through photocrosslinking. At lower
concentrations, the polymers are water soluble e.g. 10%. The
prevention of nerve regeneration is conferred in part through their
hydrophobicity. Incorporated for reference are US20180177913A1,
U.S. 62/181,270, and US201562181270P.
[0227] Applicator. Dual channel applicators used to deliver the in
situ forming hydrogels are commercially available (Nordson Medical
Fibrijet Biomaterial Applicators, Medmix Double Syringe Biomaterial
Delivery System, K-System). However, these mixers, delivering
between 2.75 and up to 10 ml of hydrogel, include a single lumen
with a static mixing element and are designed for the adequate
mixing and delivery of large volumes of hydrogel solution and are
not ideal for delivering small volumes (<1 ml) of hydrogel
solution to a site. As a result, there is a need for a mixer that
provides mixing of small volumes of two component systems as
inevitably, one of the two solutions is typically advanced slightly
ahead of the other solution, leading to a small volume of partially
or inadequately mixed gel existing the needle first. In one
embodiment, a custom mixer is designed to fit onto the Nordson
Medical or K-System applicator, through either a luer or a snap
fit, as the dual chamber applicator system necessitates, to
recirculate some of the initial solutions that enter the mixer in
order to ensure better mixing of the hydrogel, including the
initial components. FIG. 18 illustrates the design (transparent) of
the center portion of the mixer containing an entrance port with at
least one static mixer, a larger container through which the
contents are delivered and recirculated and a second port which
captures the mixed recirculated fluid and delivers it to the exit
port of the mixer. The second port may or may not contain
additional static mixers.
EXAMPLE 1
[0228] In some embodiments the 4-arm-PEG 10K-SC is crosslinked with
an 8-arm PEG 20K amine. The PEG-SC and PEG-amine were dissolved in
an acidic diluent at a ratio of 1:1. The suspension was mixed with
accelerator buffer and delivered through a static mixer to form a
hydrogel. This formulation gelled in 4 seconds.
EXAMPLE 2
[0229] In other example, 8-arm 15K PEG-SC is crosslinked with
trilysine. The PEG-SC was suspended in buffered trilysine solution
and then mixed with accelerator buffer through a static mixer. This
formulation gelled in 2 seconds and the gel provided compression
strength up to 200 kPa.
EXAMPLE 3
[0230] In other example, 8-arm 20 K PEG-thioisocyanate is
crosslinked with trilysine at a ratio of 1:1. The formulation
gelled in 3 seconds and has a compression strength of 120 kPa and
5% swelling.
[0231] Cap Form. The method may comprise the step of positioning a
form at a treatment site before the positioning the severed end
step. The form is provided as part of the kit containing the
delivery system and is composed of an inert, biocompatible,
flexible and nonadhesive material to provide the desired shape to
the in situ formed material. The form is designed to be filled with
the flowable media such that it flows around the proximal nerve
stump end where it transforms to a non flowable composition,
conforming to the nerve stump and preventing neuroma formation. In
the preferred embodiment, the form creates a low profile nerve cap
with a smooth transition between the nerve and the cap and
approximately cylindrical shape around the nerve.
[0232] Shape. A form is desirable, not only because it reduces off
target spread of the in situ forming material but because it
provides a low-profile circumferential lubricious shape that cannot
be achieved with the application of the hydrogel alone. The profile
and transitions of the form design reduce the friction and
interference with the surrounding tissue, allowing the hydrogel to
glide and rotate relative to the surrounding tissue. The cap is
designed to be able to cover at least 5 mm, preferably a 10 mm
length or more of nerve.
[0233] In accordance with another aspect of the present invention,
there is provided a form for creating a formed in situ nerve cap to
inhibit neuroma formation. The form comprises a concave wall
defining a cavity, the wall having a top opening for accessing the
cavity. The top opening lies on a first plane and has an area that
is less than the area of a second plane conforming to inside
dimensions of the cavity and spaced apart into the cavity and
parallel to the first plane. A concave nerve guide is carried by
the wall and provides a side access to the cavity for receiving a
nerve end. The wall is flexible so that it can be removed from a
crosslinked nerve cap formed within the cavity, and may comprise
silicone, preferably with a durometer of 30-50, most preferably a
durometer of 40. The wall design of the form has a slight undercut
such that the material, when filling the form to the top edge of
the form, forms a convex surface due, in part, to surface tension
of the media, that completes the cylindrical shape of the
hydrogel.
[0234] Silicone. In one embodiment, the form is comprised of a
nonadherent nondegradable material. In the preferred embodiment,
the material is medical-grade silicone, sufficiently flexible to be
peeled or popped off of the in situ formed material (e.g. durometer
40). After the transition of the media to a substantially
non-flowable state, the silicone form is removed and discarded. In
one embodiment, the silicone form is colored to provide contrast
against the surrounding tissue so that the nondegradable polymer is
not accidentally left in situ. Darker colors are preferable to
enhance the light that enters the cap and illuminate the nerve,
such as dark blue, dark purple or dark green.
[0235] Biodegradable. The method may alternatively comprise the
step of placing a biodegradable form before the positioning the
severed end step. The biodegradable form may be comprised of a
non-crosslinked lyophilized (or dried) synthetic biomaterial that
remains in place for approximately 5 to 10 minutes during the time
that the in situ forming hydrogel is delivered, and then is rapidly
dissolved and cleared from the site in less than, for example, one
or two days. In one embodiment, the bioerodible form is comprised
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 comprise the step
of forming a biodegradable form in situ before the positioning the
severed end step. In alternate embodiments, the form is composed of
materials typically used for nerve conduits and wraps, such as
polyvinyl alcohol, chitosan, polylactic acid, polyglycolic acid,
polycaprolactone.
[0236] In yet another embodiment, the ex vivo implantable form is
comprised of the same material as the in situ formed material that
is delivered into the form. In this manner, the properties of the
equilibrated form are comparable and match well with the
equilibrated in situ formed hydrogel. In these embodiments, the
biodegradable form for remains in place after delivery of the
hydrogel and is not removed but is cleared from the implant site at
approximately the same rate as the in situ formed material. In yet
another embodiment, the form is comprised of lyophilized
non-crosslinked PEG into which the in situ formed hydrogel media is
delivered. The non-crosslinked PEG is readily solubilized and
cleared from the site, making the form a rapidly bioerodible form.
In yet another embodiment, the form is comprised of a crosslinked
PEG matrix that will be cleared rapidly from the site as a result
of a rapidly cleaved hydrolytic bond, such as can be obtained with
the ester linkage in a PEG succinimidyl succinate (PEG-SS).
[0237] Forms can be synthesized by injection molding, crosslinking
or polymerizing in a cavity, solvent casting, or 3D printing. A
range of synthetic and natural materials can be selected for the
implantable form, including collagen, PEG-PEI, alginate, chitosan,
or agarose.
[0238] Forms may be rapidly dissolving forms, that, upon wetting,
dissolve and are cleared within an hour so after the procedure,
leaving the in situ formed biomaterial in place. Alternatively,
forms may be more slowly degrading forms that swell to a similar or
greater extent that the in situ formed material that is delivered
inside them. The swelling prevents scenarios in which the hydrogel
swells during equilibrium swelling and compresses the nerve if the
form into which it is delivered is shrinking or has minimal
compliance.
[0239] In some embodiments, the nerve positioned is held in the
desired location or orientation with forceps in one hand and the
media is delivered with the other hand into the form. As the media
fills the form, the nerve is released and subsequently the media
changes to a non-flowable state. Alternatively, a supporting
physician or nurse may assist with the procedure. In another
embodiment, the growth inhibitory hydrogel is formed around the
nerve in a two-step process. In the first step, the hydrogel is
delivered to the nerve tip to encapsulate the nerve end. In the
second step, the hydrogel is applied to fill the entire form,
including the nerve tip. In another embodiment, the growth
inhibitory hydrogel is formed around the nerve in a first layer and
then a second layer of hydrogel is subsequently applied, in a
two-step process.
[0240] Conformability. Unlike wraps, which still have a gap between
the wrap and the nerve, the hydrogel conforms directly to the nerve
itself providing a barrier to inflammatory and pro-scar forming
cells to the site while allowing nutrients through. Because the
hydrogel adheres 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 scar and adhesion formation around the nerve in
the initial healing phase.
[0241] Centering. Nerves, by virtue of their low density and high
fat content and flexibility, particularly smaller nerves, have a
tendency may flow upwards in a low viscosity solution. The
following embodiments are designed to assure that the nerve does
not float up after delivery of the media to the top of that
surface.
[0242] Viscosity. As described above, the viscosity of the flowable
solution can be increased to minimize the nerve floating up within
the solution.
[0243] Flow. In another approach, the needle that delivers the
flowable media is directed in such a way that the flow of the media
permits the circumferential spread of the solution around the nerve
prior to the gel forming. The cap form can also be designed to
improve the flow dynamics of the media and improve nerve alignment.
In one implementation of the invention the severing a target nerve
step and the positioning a form at a treatment site step are
accomplished by a single instrument. In another implementation of
the invention, the nerve cap form is designed such that the
delivery system and the form are integrated. In the preferred
embodiment, the delivery system is connected to the form via a
catheter. The catheter entrance in the cap resides at the same
entrance where the nerve is entering the form. The catheter permits
the flow of material down the shaft and circumferentially around
the nerve such that the media acts to self-center the nerve within
the form.
[0244] Stabilizer. In another embodiment, a stabilization rod or
piece is aligned directly under the nerve or against the nerve such
that it provides sufficient adhesive forces that the hydrogel can
be delivered around the centered nerve into the form.
[0245] Peelable conduits In yet another embodiment, the nerve is
positioned in the center of the in situ biomaterial through the
consecutive placement within two peelable conduits. In brief, the
nerve is placed inside the first peelable conduit and the in situ
forming material is delivered to surround the top and distal end of
the nerve. The peelable conduit may be an open ended or close ended
conduit. After the hydrogel forms, the sheath is pulled apart along
weaker peel lines in the material an discarded. The resultant
nerve-hydrogel is then placed in a second larger peelable conduit.
By rotating the nerve slightly, the hydrogel surface can be placed
on the bottom of the second sheath so that the nerve is centered in
approximately the middle of the second sheath. A second application
of the in situ forming hydrogel results in the cumulative formation
of a circumferential hydrogel around the nerve which protects and
centers the nerve within the nerve cap. In one embodiment, the
sheath is composed of an extruded splittable PTFE tube with a
vertical tab to assist in tearing the piece apart in the surgical
setting, similar to vascular introducers.
[0246] In another embodiment, the nerve is placed so that the
proximal stump rests at a ninety degree angle downward in a
cup-shaped form and the hydrogel delivered into the cup-shape to
form around the nerve. The cup-shaped form is subsequently removed
and discarded and the proximal stump is adjusted back to its
resting position in the tissue.
[0247] In yet another embodiment, the nerve can be delivered in an
amphiphilic or hydrophobic solution to prevent the nerve from
floating to the surface of the medial. In yet another embodiment,
the in situ forming material may be more viscous, to prevent the
nerve from migrating within the form.
[0248] Tilted. Alternatively, the form may have a tilt in the form,
to bias the nerve filling from the distal to the proximal end. In
this manner, the nerve can be positioned in such a way that the
hydrogel forms circumferentially around the proximal nerve tip
first and then, either through a second application or a
continuation of the first application, fills the rest of the
form.
[0249] Entrance centering. In one embodiment, the forms are
designed in such a way that the nerve enters at a lower level
relative to the top of the form to permit the material to be
delivered circumferentially around the nerve. In another
embodiment, the entrance region of the form is sloped so that the
nerve enters the form at a downward angle, biasing the proximal
nerve tip location downward.
[0250] Ribs. Tabs or ribs are provided on the external face of
nondegradable temporary forms, such as silicone forms, to assist
with the removal of the form after formation of the gel. These tabs
are placed in such a place to provide additional stability for the
cap form on irregular surfaces or to provide a surface to grip with
forceps or other surgical instruments. In yet another embodiment,
the form is designed to be self centering. In other words, the form
may naturally seat itself so that the top surface of the form is
level in preparation for delivery of the in situ forming
hydrogel.
[0251] Holes. In some embodiments, a hole or guidance sheath is
provided to direct the needle to deliver the media into the form in
a particular direction. The direction of the media flow can be
designed to better position the nerve in the channel. In one
embodiment, the hole is provided adjacent to or on top of where the
nerve enters the form to guide the solution from proximal to distal
in the conduit and encourage laminar flow within the form.
[0252] Lids. In some embodiments, the form contains a partial or
complete hinged lid to permit the centering of the nerve according
to the direction of flow within the form.
[0253] Volumes delivered. As with the cap form, the volume of media
delivered may range from 0.1 cc to 10 cc, typically 0.2 to 5 cc,
more typically 0.3 to 1 cc.
[0254] Needle size. The kits contain a 21 gauge or 23 gauge needle
for delivering smaller volumes into smaller size wrap (or cap)
forms and 18 gauge needles for filling larger forms. These needles
provide additional control to the speed of delivery of the in situ
forming material, permitting, the deposition of a bead of the
hydrogel to the rapid filling of a larger conduit.
[0255] Gelation time. Similarly, the gelation time can be adjusted
depending on the fill volume of the wrap or cap forms, providing
longer gelation times of 10 to 20 seconds for larger wraps and a
shorter gelation time of around 5 seconds for smaller wraps or
caps.
[0256] Form sizes and hydrogel thickness. The range of form sizes
are designed with an entrance region to accommodate a nerve
diameter .+-.1 to 3 mm, or more preferably .+-.1 mm. The diameter
of the form determines the thickness of the gel that forms around
the nerve. The thickness of the hydrogel that forms around the
nerves may be 0.05 mm to 10 mm, more preferably 1 mm to 5 mm, more
preferably 1 to 3 mm depending on the size of the nerve.
[0257] Kit design. Instead of each kit containing one form for only
one size of nerve, as is the case with implantable nerve conduits
and wraps, kits will contain one to ten, more typically one to cap
forms (or wrap forms, or combinations thereof), allowing the
physician to select the appropriate size for the procedure as well
as the ability to switch the form out without having to request an
additional kit. Kits may be labelled according to forms
selected--for example forms for a ranges of nerve sizes, forms for
a type of surgical procedure (nerve protector for inguinal repair),
or forms appropriate for a specific location of the procedures
(nerve cap for hand surgery, nerve protector for upper limb, nerve
form for brachial plexus).
[0258] Sheets. In some embodiments, the in situ forming material
may be delivered to a nerve that is placed on a temporary
nonadherent biocompatible sheet such as an Esmarch bandage or other
biocompatible sheet or background (Mercian Surgical Visibility
Background Material) routinely used to isolate the nerve from the
surrounding tissue. The gelation time can be shortened to limit the
spread of the hydrogel around the nerve, for example to 10 seconds
or less, preferably 5 seconds or less. Any excess hydrogel can then
be removed from the surgical site and discarded.
[0259] Liquid Cap Form. In another embodiment, the form is not a
physical form but created by the injection of a soluble hydrophobic
solution, preferably a viscous solution, such as glycerol. For
example, while securing the nerve out of the way, a viscous oil can
be delivered to the surrounding tissue to coat it and prevent the
hydrogel from adhering the surrounding tissue. The solution, if
viscous enough, can create a rapidly bioerodible form for
delivering the in situ forming hydrogel. In the preferred
embodiment, Solution A is delivered first to block the amine and
tissue binding sites and to create a space or region into which
Solution B can be delivered. In the next step, Solution B is
delivered in the space created by Solution A, or it is delivered in
the center of Solution B, displacing Solution B away from the
site.
[0260] No Form. In some embodiments, the space or access does not
permit the use of a form. In some cases, such as in brachial plexus
injuries, the surgical window is so small or the concern of
damaging adjacent tissues is so small that placing a form in the
site into which to deliver the hydrogel is not possible. In these
instances, the hydrogel may be delivered directly into a surgical
pocket or region in or around the nerve. If the region around the
nerve is utilized as a natural form, the cap has an irregular shape
that is defined by the boundaries of the tissue on the bottom and
sides of the nerve. In one embodiment, since the hydrogel is
adherent to both the nerve and the tissue, the in situ formed
material should be carefully peeled off of the muscle and fascia so
that it forms a free-floating bolus in contact with the nerve. This
will permit the nerve to continue to move within the region without
being tethered to the surrounding tissue.
[0261] In still further embodiments, it is desirable for the
hydrogel to take the shape of the surrounding tissue around the
nerve. For example, in embodiments where the nerve is to be ablated
and the hydrogel needs to fill the potential space where the nerve
is/was and the surrounding area to prevent regeneration.
Alternatively, when the hydrogel is delivered around visceral
nerves where there is frequently a loose and fine network of tiny
nerve fibers and the space around these nerve fibers needs to be
filled. In another embodiment, the hydrogel fills around
irregularly shaped nerves or bundles/clusters of nerve fibers
and/or cell bodies. In this way, the hydrogel can most effectively
deliver therapeutic agents to the region.
[0262] Hydrogel placed in a controlled manner in situ around a
nerve. In another embodiment, however, particularly in dynamic
environments in which nerves are sliding during motion between
muscles, joints, bones, or tendons, such as between muscles in the
periphery, it is not desirable for the nerve to be tethered via the
hydrogel to the surrounding tissue. Instead, it is desirable to
develop solutions in which the nerve can glide freely within the
channel. In these embodiments, the hydrogel can be physically
separated from the surrounding tissue during in situ crosslinking
or in situ polymerization. This can be achieved with something as
simple as a nonadhesive sterile sheet which can be placed at the
site and then removed after gel formation. This can also be
achieved through the placement of a form in/around the nerve. The
form may take several forms depending on the size and location of
the nerve, the presence or absence of a sheath, the goal of the
therapy that is delivered to the site (nerve stimulation, nerve
blocking, nerve ablation, or a barrier to nerve regeneration). In
one embodiment, the form is a cap that can be gently placed around
the end of a nerve and the in situ forming gel injected into this
form in order that it assume the shape of the form. The material in
the cap can then be pushed out and the cap form removed and
discarded. In yet other embodiments, the cap is biocompatible and
thus is not removed. In another embodiment, a half-cylinder (halved
longitudinally) can be placed underneath the nerve and the in situ
forming material delivered into the half cylinder. In this same
manner, it is possible to deliver a gel circumferentially around
the nerve without the gel developing attachments to the surrounding
tissue.
[0263] Alternate cap forms. In order to reduce the chances of
adhesions forming, the 3 to 10 mm sections of nerve can be placed
inside a syringe barrel (with the luer lock end portion removed)
and the plunger pulled back to the appropriate gel distance that is
desired on the end of the nerve. The hydrogel is delivered into and
around the nerve inside the plunger where the gel sets. After the
gel forms, the plunger is gently depressed to extrude the nerve
encased in the hydrogel. Using this approach again, minimal or no
sutures are required to avoid additional damage or over handling of
the nerve. Preferably the syringe barrel has a lubricious
coating.
[0264] Laprascopic or endoscopic surgery. The forms may be advanced
down a channel during laprascopic or endoscopic surgery and placed
under a nerve, similar to the approach in open surgery. The form
may be folded to permit transit through smaller conduits and then
released at the site of the procedure. Alternatively, instruments
can be designed with the nerve form (cap or wrap) build into the
tip of one instrument with the in situ forming biomaterial
delivered through the lumen of another instrument. Gelation time of
the in situ forming biomaterial needs to be adjusted to 20 to 30
seconds or more to allow for travel time of the hydrogel down the
instrument lumen.
[0265] In a needleoscopic approach to the procedure, a first
material can be injected that coats the outside tissues to prevent
direct adhesion between the gel and the surrounding tissue. After
this, the hydrogel can be delivered into the same site, forming a
depot around the nerves and displacing the first material to the
periphery of the injection site. This can be achieved with a
hydrophobic substance, such as an oil or a viscous substance such
as glycerol. This may also be achieved with a low molecular weight
PEG solution which has the added benefit of helping seal the
membranes of the nerve prior to the hydrogel forming the nerve
block/cap around the nerve.
[0266] In another embodiment, the nerve is dipped in the flowable
material solution prior to it crosslinking to form a thin
protective surface on the hydrogel. In some embodiments, only a
thin coating of the biodegradable polymer is necessary around the
nerve. The coating may be only 100 microns to 500 microns thick. In
other embodiments, a coating is not sufficient to prevent the
inflammatory infiltration and or prevent early degradation--in
these cases it is desirable to use a coating of between 0.5 mm to
10 mm thick.
[0267] Attempts at developing nerve caps to date have been focused
on solid physical caps that are sutured in place around the end of
a transected nerve. These caps have, by necessity, had a gap around
the end of the nerve at the end of the proximal stump as well as
circumferentially. As a result, neuroma formation occurs into the
end of the cap. Examples include resorbable poly(D,L
lactide-co-caprolactone) implant, aligned silk fibroin (SF) blended
with poly(L-lactic acid-co-e-caprolactone) (SF/P(LLA-CL)) nanofiber
scaffolds, poly(lactic acid)-co-(glycolic
acid)/arginylglyclaspartic acid) modified poly(lactic
acid-co-glycolic acid-alt-L-lysine) (PRGD-PDLLA) implant with pores
on the order of 10 microns in diameter [), Yi et al 2018, Adv
Sci],. The PRGD/PDLLA conduit was 10 mm long with an inner diameter
of 2 mm and a tube wall thickness of 200 microns, the SF/P(LLA-CL)
conduit is 1.5 cm long with an internal diameter of 1.5 mm. These
caps require suture placement. Another approach, called
Neurocap.RTM., is a synthetic nerve capping device including of a
solid tube with a closed end that is placed over the nerve bundle
and then has to be both sutured to the nerve to keep the nerve
within the cap and sutured to the surrounding tissue to hold the
cap in position, published as WO2016144166A1. Another approach also
utilizes a solid implant, published as US20140094932A1. In
contrast, the injectable gel approach can flow around nerves of any
size from tiny fibers to large nerve bundles, does not require
cutting or suturing, and provide a reduction in pain and neuralgia.
In short, an injectable flowable system is not limited to nerve
stumps but also can prevent aberrant nerve outgrowth in fibers too
small to be picked up.
[0268] NERVE PROTECTOR/WRAP. In accordance with a further aspect of
the invention, there are provided methods and devices to protect
intact or compressed nerves. In some instances, it may be desirable
to protect nerves that are surgically exposed as a result of a
procedure or adjacent nerves or tissues, such as in the instance
where these nerve would otherwise dry out. In some instances, it
may be desirable to protect and mark the nerves that are exposed as
part of another surgery so that additional handling, stretching,
contusion and/or compression can be reduced or avoided. In one
embodiment, an in situ forming material is delivered around the
nerve to provide a protective layer and prevent the nerve from
damage from forceps and other surgical equipment in the region.
Additionally, a dye in the hydrogel can provide sufficient contrast
from the surrounding tissue that the physician can also visually
stay away from the nerve during the procedure. This may
dramatically reduce the incidence of iatrogenic nerve injury during
surgical procedures.
[0269] In accordance with a further aspect of the invention, there
is provided a form for creating a formed in situ capsule around a
nerve to nerve junction. The form comprises a concave wall defining
a cavity, the wall having a top opening for accessing the cavity.
The top opening lies on a first plane and has an area that is less
than the area of a second plane conforming to inside dimensions of
the cavity and spaced apart into the cavity and parallel to the
first plane. A first concave nerve guide is carried by the wall and
provides a first side access for positioning a first nerve end in
the cavity; and a second concave nerve guide is carried by the wall
and provides a second side access for positioning a second nerve
end in the cavity.
[0270] One example of this is the prophylactic treatment of the
ilioinguial and iliohypogastric nerves during procedures to repair
hernias, particularly inguinal hernia repair. These nerves may be
partially or completely exposed during the repair of the hernia,
resulting in compression, contusion, and partial or complete
transection. Post-surgically, the damaged nerve may send aberrant
nerve sprouts out into the post-surgical scar tissue which may
result in neuroma formation and nerve entrapment resulting in a
high rate of post-operative and chronic pain. In addition, these
nerves may be incidentally or purposefully transected surgically in
an attempt to prevent the nerves from being entrapped surgically or
tangled in the mesh used to repair the hernia. In one embodiment, a
kit containing the in situ forming growth inhibitory hydrogel and
appropriate forms permit the surgeon to select a form to provide
either a `cap` or `wrap` shaped form cavity. Depending on the
surgery, the physician may then select a wrap if the nerve is not
transected and the physician wishes to protect the nerve from
further damage or a cap if the nerve is transected and the
physician wishes to prevent the formation of distal neuroma at the
end of the transected nerve.
[0271] Another example of this is the exposure of the sciatic nerve
during hip procedures. Although the nerve is not the target of
these procedures, the nerve is often exposed and placed in traction
such that it runs a risk that it be damaged and/or dry out during
the procedure. In one embodiment, a wrap-shaped form is provided to
deliver hydrogel around the region of sciatic nerve at risk. For
larger nerves, these region may be 5 to 50 cm or more. Wrap-shaped
forms may be provided to span this entire length or alternately
multiple cap forms can be placed in series along the nerve to
provide protection. In another embodiment, an anti-inflammatory
agent is delivered in the hydrogel in order to reduce the
inflammation around the nerve that may result as a result of
positioning or moving the nerve during the surgery. In another
embodiment, a local anesthetic is delivered into the hydrogel that
is placed around the nerve.
[0272] In another embodiment, the hydrogel is delivered around the
nerve to reduce inflammatory neuropathies that may result after
surgery, particularly peripheral neuropathy that may result in
slowly developing severe pain and/or weakness in the affected limb.
The in situ forming hydrogel may be delivered after open surgery or
through a percutaneous image-guided procedure. For percutaneous
ultrasound guided or fluoroscopic delivery, echogenic needles are
desirable to confirm not only depth but location of the needle
relative to relevant structures.
[0273] In another embodiment, an in situ forming hydrogel is
delivered around the nerve in a `wrap` form cavity to form a
protective compliant wrap around the nerve. The wrap form cavity is
left place, providing additional support and protection during the
surgery, and then the wrap form is removed and discarded after
completion of the procedure prior to closing the site. The hydrogel
remains in place is to protect the nerve, prevent aberrant nerve
outgrowth, and any scar tissue from infiltrating the nerve.
[0274] Coaptation aid. In some embodiments, a nerve that has
undergone direct coaptation with sutures can be placed into a wrap
form cavity. The direct coaptation site may be filled with an
injected growth permissive hydrogel or temporary spacer material
(e.g. fibrin glue) that can spread into the interstices of the site
and the growth inhibitory hydrogel delivered directly around the
anastomoses site using a wrap form cavity.
[0275] Wrap or protector form. The wrap form cavity comprises a
form with two entrance zones for the nerve with a variable cavity
length around the region of the nerve that needs protection. In
shorter wrap forms, the cavity is designed such that the nerve
rests on the entrance zones and the nerve is `floating` between
this region and does not make contact with the walls of the form.
The needle that delivers the in situ forming hydrogel delivers the
flowable hydrogel solution into the form, surrounding the nerve,
where it forms a protective hydrogel around the nerve. The form
prevents the off target spread of the hydrogel to adjacent tissues
and maintains a consistent thickness of hydrogel around the
nerve.
[0276] Longer lengths. In situations where there is a long length
of nerve to protect, a longer wrap form cavity may be utilized with
small posts designed in the bottom of the form to prop and provide
stability to the nerve over longer distances. These posts are then
removed when the form is removed, leaving only a small exposure
between the nerve and the surrounding environment at a non-critical
location away from the proximal nerve stump tip (if one exists). In
yet another embodiment, in which it is desirable to protect longer
lengths of nerve, the in situ forming hydrogel solution may be
delivered in multiple layers or regions. In one embodiment, the
form is filled in multiple sections in order to maintain the nerve
within the center of the form. In another embodiment, a first layer
of in situ forming hydrogel is delivered to the bottom of the form,
either with the nerve or without the nerve completely or partially
embedded, and then a second layer of hydrogel is delivered on top
of the first layer in order to completely cover and protect the
nerve.
[0277] In one embodiment, kits are provided which contain the
appropriate volume of in situ forming hydrogel to fill the wrap
form as well as multiple mixers and needle components to allow the
physician to switch the mixer-needle tip and continue to deliver
more of the media in second or third applications, as needed.
[0278] Protecting anastomoses sites. With the increased use of
allograft in addition to autograft in larger nerve gap repairs,
there is an increased recognition that aberrant nerve outgrowth
from the peripheral nerve stump into the surrounding tissue at the
nerve-allograft, nerve-autograft, or nerve-conduit junction can
result in local pain and reduce the effectiveness of the nerve
repair. In addition, the compliance mismatch between a solid
implantable conduit used to secure two nerve stumps and the nerves
themselves may cause friction at the interface between the nerve
and the conduit resulting in additional aberrant nerve tethering
into surrounding tissue. In one embodiment, the delivery of an in
situ forming hydrogel at the interface between the proximal nerve
and the allograft or autograft anastomoses, or the delivery of the
hydrogel between the allograft or autograft and distal nerve stump,
or similarly at the junction between where the nerve stump enters
and exists the conduit, is anticipated. A smaller volume of
hydrogel delivered either directly or in a shorter wrap form
segment provides protection of neurite outgrowth and reduction in
scar formation and immune cell infiltration into the graft and
conduit. Alternatively, a wrap form may span the entire length of
the anastomoses to cover the allograft/autograft/conduit in
addition to the nerve.
[0279] Nerve gliding. Some peripheral nerves undergo a considerable
amount of motion in the fascial plane in which they reside and thus
scarring and tethering of these nerves is particularly painful. For
example, the median nerve in the carpal tunnel or the ulnar nerve
location relative to the elbow, are locations where a provision for
gliding is important. With implantable conduits, wraps and
protectors, the form of the implant is such that the gliding of the
nerves is not enhanced with the biomaterial and may be further
inhibited. In one embodiment, the in situ forming hydrogel is
delivered as a protector around these nerves to permit the nerves
to continue to glide within their fascial plane. One way this can
be accomplished is by delivering a higher swelling in situ forming
material that swells significantly, e.g. greater than 30%,
preferably greater than 60% outward radially after delivery around
a nerve such that the nerve can glide within the channel created
after the hydrogel reaches equilibrium. In this manner, even though
the hydrogel eventually becomes encased in a thin capsular layer,
the nerve itself, within the hydrogel, is free to glide within the
channel and is free from significant scar tissue, nerve outgrowth
into surrounding tissue and also is not compressed in these
critical locations.
[0280] Indications. The in situ forming hydrogels can also be
introduced intraoperatively to aid in the maintenance of a
successful microsurgical anastomosis of donor and recipient nerves
using a wrap form. They hydrogels can be injected in a wrap form at
the junction of the anastomoses to protect the aberrant
inflammatory response, the formation of scar tissue, and aid in the
coaption of the donor and recipient nerves. The transfer of a
noncritical nerve to reinnervate a more important sensory or motor
nerve is known as neurotization. In one example, a patient
undergoing breast reconstruction after mastectomy can select
autologous flap reconstruction to connect the nerves with the chest
wall. Wrap forms can be placed at the junction between the proximal
nerve stump and distal nerve tissue to which it is sutured and the
hydrogel delivered to protect the anastomoses sites. This repair
may lead to restoration of sensory function and an improvement in
physical and quality of life advancement for women.
[0281] Compression repair. In another embodiment, the in situ
forming hydrogels can be delivered in a wrap form around the nerve
as a barrier to the attachment of surrounding tissue while the
nerve repairs. This approach allows the hydrogel to infiltrate and
conform to the nerve and act as a replacement for vein wrapping, in
which autologous vein in wrapped around the nerve in a spiral
wrapping technique, providing a barrier to attachment of surround
tissue. This also provides an alternative to the AxoGuard Nerve
Protector which has to be wrapped around the nerve potentially
stretching and damaging it further. The solid nerve protector
requires extensive handling of the nerve with forceps, stretching
open the solid wrap to hold it open, and then suturing the nerve to
the wrap. Using a soft, conformal, hydrogel based approach, a
liquid or viscous liquid may be delivered directly around the nerve
in a form with minimal nerve handling. Soft tissue attachments are
minimized, swelling is minimized and mechanical support provided by
the gel reduces the tension and stress on the coaption site.
Nutrients can diffuse through the hydrogel network. Also, the
hydrogel may reduce the physician's procedure time.
[0282] In one embodiment, the solution in based on hyaluronic acid.
In another embodiment, the solution is based on a hydrogel slurry
(TraceIT, Boston Scientific).
[0283] Distance applied. The hydrogel can be delivered
circumferentially around the nerve using a syringe or applicator
tip, in this manner, the nerve has protection over the length of
the damage. Ideally, the hydrogel would be applied between e.g. 5
and 15 mm on each site of the damaged or transected nerve. Volumes
administered may be between 100 microliters and 10 ml, more
preferably 0.2 to 3 ml. The syringe contains the hydrogel (or the
two precursor components to the hydrogel) can be designed with the
exact volume to be delivered to allow for controlled automated
delivery of the in situ forming hydrogel. Alternatively, an excess
volume can be provided to allow the individual to use his/her
judgment on how much to deliver around the site. At minimum, the
hydrogel should form a cellular barrier approximately 200 microns
thick around the outside of the nerve, although the hydrogel may
also be delivered to fill a site and thus form a circumferential
bolus with a 2 cm radius around the hydrogel site.
[0284] End-to-side Repair. A window in the outer nerve sheath is
made and a nerve transfer is attached to the side of the nerve.
After the suturing, the in situ forming hydrogel is delivered
around this to keep these in close apposition with one another.
[0285] Internal neurolysis. After a nerve is stretched or
chronically compressed, internal scarring and swelling my occur.
The outer sheath of these nerves may be opened to relieve pressure
and assist with blood flow.
[0286] External neurolysis. If nerves have become scarred or
develop neuromas, stretching or moving may result in additional
nerve damage, pain, and additional nerve scarring. Neurolysis can
be used to remove the scar tissue around the nerve without entering
into the nerve itself. The in situ forming hydrogel can be
delivered around the nerve after the external neurolysis to prevent
additional nerve scarring and reduce pain.
[0287] Neurotization. In one embodiment a percutaneous nerve
protector is delivered around the damaged or crushed nerve. In this
embodiment, if applied within a day to several days after injury,
the local inflammatory response can be reduced. In situ forming
hydrogels that are 1) biocompatible, 2) biodegradable or
bioerodible, 3) permit diffusion of nutrients and oxygen into and
out of the tissue while preventing inflammation, 4) flexible and
compliant so that the axons are protected without being compressed,
5) non or minimally swelling, and 6) prevent fibrous ingrowth to
the injury site.
[0288] Location. An injectable conformable hydrogel also permits
the same product to be delivered to multiple nerve diameters and
multiple locations (between bones, fascia, ligaments, muscle) as
the material will flow in the region around the nerve.
[0289] Delivery location. In some embodiments, the needle location
impacts the delivery of the hydrogel. In some embodiments, the
needle delivers the hydrogel directly on top of the target nerve or
region. In another embodiment, the needle is run distally to
proximally to first fill the end of the form and the distal nerve
stump and later to fill the rest of the conduit.
[0290] TMR. In another embodiment, the hydrogels can be delivered
around nerve that are reconnected as part of targeted muscle
reinnervation (TMR) procedures. Due to frequent the size mismatch
between the transected donor nerve and the typically smaller
denervated recipient nerve, it may be desirable to apply a hydrogel
at the junction to help direct the regenerating fibers into the
target recipient motor nerve. For these indications the hydrogel
may be applied directly or within a form. Typically this is
performed between a mixed motor and sensory nerve.
[0291] Inhibitory drugs to caps and wraps. Depending upon the
desired clinical performance, the mechanical barrier may be
assisted or enhanced by any of a variety of chemical agents that
inhibit or prevent nerve regrowth (sometimes referred to as
"anti-regeneration agents"). These agents include inorganic and
organic chemical agents, including small molecule organic chemical
agents, biochemical agents, which may be derived from the patient
and/or from an external source such as an animal source and/or a
synthetic biochemical source, and cell-based therapies.
Anti-regeneration agents may be applied directly to target tissue
prior to or following forming the nerve end. Alternatively, the
anti-regeneration agents may be carried within the media where they
become trapped in the media and are then released over time in the
vicinity of the nerve end.
[0292] Some specific examples of anti-regeneration agents that may
be used in conjunction with some embodiments of the present
invention include, among others: (a) capsaicin, resiniferatoxin and
other capsaicinoids (see, e.g., J. Szolcsanyi et al.,
"Resiniferatoxin: an ultrapotent selective modulator of
capsaicin-sensitive primary afferent neurons", J Pharmacol Exp
Ther. 1990 November; 255(2):923-8); (b) taxols including paclitaxel
and docetaxel (i.e., at concentrations are sufficiently elevated to
slow or cease nerve regeneration, as lower concentrations of
paclitaxel may facilitate nerve regeneration; see, e.g., W. B.
Derry, et al., "Substoichiometric binding of taxol suppresses
microtubule dynamics," Biochemistry 1995 Feb. 21; 34(7):2203-11),
botox, purine analogs (see, e.g., L A Greene et al., "Purine
analogs inhibit nerve growth factor-promoted neurite outgrowth by
sympathetic and sensory neurons," The Journal of Neuroscience, 1
May 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 anti-microtubule agents such as nocodazole and colchicine;
(e) platinum-based antineoplastic drugs (platins) such as
cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin,
nedaplatin and triplatin; (f) ZnSO.sub.4 (i.e., neurodegenerative
factor); (g) latarcins (short linear antimicrobial and cytolytic
peptides, which may be derived from the venom of the spider
Lachesana tarabaevi); (h) chondroitin sulfate proteoglycans (CSPGs)
such as aggrecan (CSPG1), versican (CSPG2), neurocan (CSPG3),
melanoma-associated chondroitin sulfate proteoglycan or NG2
(CSPG4), CSPGS, SMC3 (CSPG6), brevican (CSPG7), CD44 (CSPG8) and
phosphacan (see, e.g., Shen Y et al. "PTPsigma is a receptor for
chondroitin sulfate proteoglycan, an inhibitor of neural
regeneration", Science, 2009 Oct. 23; 326(5952):592-6); (i)
myelin-associated glycoprotein (MAG); (j) oligodendrocytes; (k)
oligodendrocyte-myelin glycoprotein; and (I) Reticulon-4, also
known as Neurite outgrowth inhibitor or Nogo, which is a protein
that in humans is encoded by the RTN4 gene (see, e.g., Lynda J.-S.
Yang et al., "Axon regeneration inhibitors, Neurological Research,
1 Dec. 2008, Volume 30, Issue 10, pp. 1047-1052) (m) ethanol or
glycerol.
[0293] Further examples of anti-regeneration agents include agents
that induce the formation of the inhibitory scar tissue, which may
be selected from the following, among others: (a) laminin,
fibronectin, tenascin C, and proteoglycans, which have been shown
in inhibit axon regeneration (see, e.g., Stephen J. A. Davies et
al., "Regeneration of adult axons in white matter tracts of the
central nervous system," Nature 390, 680-683 (18 Dec. 1997); (b)
reactive astrocyte cells, which are the main cellular component of
the glial scar, which form dense web of plasma membrane extensions
and which modify extracellular matrix by secreting many molecules
including laminin, fibronectin, tenascin C, and proteoglycans; (c)
molecular mediators known to induce glial scar formation including
transforming growth factor .beta. (TGF .beta.) such as TGF.beta.-1
and TGF.beta.-2, interleukins, cytokines such as interferon-.gamma.
(IFN.gamma.), fibroblast growth factor 2 (FGF2), and ciliary
neurotrophic factor; (d) glycoproteins and proteoglycans that
promote basal membrane growth (see, e.g., C C Stichel et al., "The
CNS lesion scar: new vistas on an old regeneration barrier", Cell
Tissue Res. (October 1998) 294 (1): 1-9); and (e) substances that
deactivate Schwann cells. Still other examples of anti-regeneration
agents include Semaphorin-3A protein (SEMA3A) (which may be used to
induce the collapse and paralysis of neuronal growth cones) to
block regeneration is incorporated into the hydrogels to release
approximately 1 .mu.g per day for a total of 2 .mu.g over a couple
weeks, calcium (which may lead to turning of nerve growth cones
induced by localized increases in intracellular calcium ions), f)
inhibitory dyes such as methylene blue, and g) radioactive
particles. Yet other inhibitory drugs include ciguatoxins,
anandamide, HA-1004, phenamil, MnTBAB, AM580, PGD2, topoisomerase I
inhibitor (10-HCT), anti-NGF, and anti-BDNF.
[0294] Pain and inflammation. The media may additionally include
one or more agents intended to relieve pain in the short-term
post-procedure timeframe where increased pain over baseline may be
experienced due to local tissue reaction depending upon the
ablation procedure. Examples of suitable anesthetic agents that can
be incorporated into the hydrogel for this purpose include, for
instance, bupivicaine, ropivicane, lidocaine, or the like, which
can be released to provide short-term local pain relief
post-procedure around the treatment region. Inflammation and scar
tissue in the surrounding tissue can also be minimized with the
incorporation of methylprednisolone into the hydrogel.
[0295] Growth permissive form. Regarding the examples of FIGS.
5A-5E in some instances, it is desirable to provide a
growth-permissive substance between the proximal and distal stump
of the nerve to encourage nerve regeneration rather than growth
inhibition. In some embodiments, the growth permissive substance
simply provides a temporary barrier to the growth inhibitory gel
leaking into the anastomoses site or damaged nerve tissue and
inhibiting regeneration. In other embodiments, the growth
permissive substance provides a medium through which nerves can
regenerate without the need for autograft/allograft or
conduit/wrap.
[0296] In accordance with a further aspect of the invention, there
are provided methods and devices to encourage guided nerve growth,
such as to span a gap between two opposing nerve stumps and restore
nerve function or to fill a small gap between nerves that have been
directly coapted with sutures. The method may comprise the steps of
placing a first nerve end and a second nerve end in a first form
cavity; Introducing an in situ forming growth permissive media into
the cavity and into contact with the first nerve end and the second
nerve end to form a junction; the media changing from a flowable to
a nonflowable state. The nerves, coupled together by the in situ
formed media, are then removed from the first form cavity and
placed inside a second larger form cavity; and Introducing a growth
inhibitory media into the second form cavity to encapsulate the
junction. The growth inhibitory media changes from a flowable to a
nonflowable state, covering the nerves and the growth permissive
media; The second form is then removed and discarded. In another
embodiment, the first and/or second forms remain in place.
[0297] There is also provided a formed-in-place nerve regeneration
construct, comprising a growth permissive hydrogel bridge having
first and second ends and configured to span a space between two
nerve ends and encourage nerve regrowth across the bridge; and a
growth inhibiting hydrogel jacket encapsulating the growth
permissive hydrogel bridge and configured to extend beyond the
first and second ends of the growth permissive region to directly
contact the proximal and distal nerves, respectively. In yet
another embodiment, growth permissive media is delivered into an
inhibitory form cavity where it undergoes a change from a
substantially flowable to a nonflowable state. The form remains in
place and provides acts as a growth inhibitory substrate through
which nerves cannot regenerate.
[0298] Preferably, the growth permissive media is comprised of an
in situ forming gel, such as a hydrogel and the growth inhibitory
media is comprised of an in situ forming gel, such as a hydrogel.
However, the growth permissive media may be comprised of an in situ
forming gel and the form into which it is delivered is comprised of
an ex vivo crosslinked gel.
[0299] In some embodiments it is desirable that the growth
permissive hydrogel adheres to the nerve tissue, providing a method
to anastamose the tissue without the need for sutures. In doing so,
the nerve-growth permissive gel-nerve unit can be picked up and
handled as one continuous nerve unit, permitting their later
placement of the unit in a growth inhibitory hydrogel. In other
embodiments, the growth permissive hydrogel can provide a temporary
glue lasting approximately half an hour. The glue is strong enough
to lightly adhere the two nerves but has comparable mechanical
strength to e.g. fibrin glue.
[0300] Tensionless repair. Another advantage of in situ forming
hydrogels is that they can be designed to provide tensionless
repair of nerves. In one embodiment, the wrap form of the conduit
is deep enough such that the directly repaired/anastamosed nerve
ends are placed in the form with the repaired region detensioned
inside the form. When the hydrogel is formed around the detensioned
nerve, the nerve-nerve repair is not under tension; any tension is
carried by the hydrogel around it. In this manner, the nerves are
not under tension and the hydrogel carries the load in a more
evenly distributed way than a suture repair can.
[0301] In another embodiment, tensionless repair is additionally
provided by the growth inhibitory hydrogel. In this embodiment, the
proximal and distal nerves are placed in the conduit and the growth
inhibitory hydrogel is delivered at the nerve-conduit interface to
prevent nerves from escaping out of the conduit and tethering with
the surrounding scar tissue. In another embodiment, the nerves are
purposefully detensioned within the growth permissive hydrogel, by
creating slack in the nerves within the form. In cases where the
nerves are directly reanastamosed, care is taken to make sure that
the tension, if any, is at the interface between the nerve and the
entrance to the form on either side of the wrap form, and that the
nerve inside the wrap is slack or free of tension before applying
the growth permissive hydrogel precursor solution into the wrap. In
this manner, the nerve anastomoses, nerve-gel-nerve or nerve-nerve
interface is without tension. In the preferred embodiment, the
nerve-growth-permissive hydrogel-nerve unit sits entirely within
the cavity of the second nerve form. The delivery of the inhibitory
hydrogel provides additional protection and detensioning, providing
approximately 3 to 10 mm of circumferential coverage around the
nerve on either side of the injury.
[0302] Coverage. In one embodiment, the growth permissive media is
located substantially in between the two nerve ends and does not
appreciably cover the outer surface of the nerves. Thus, the
diameter of the growth permissive media closely approximates that
of the diameter of the nerve. As a result of the location of the
growth permissive media, the growth inhibitory media is delivered
around the external or epineurial surface of the proximal and
distal nerves as well as the growth permissive media, covering
preferably 10 mm or 5 mm of more of healthy nerve on either side.
This permits the guidance of nerves directly from the proximal
nerve stump into the distal nerve stump. The additional coverage
provides adhesion strength and protection from aberrant nerve
outgrowth at the junction of the proximal nerve-gel.
[0303] Color. In one embodiment, the growth permissive hydrogel is
one color, such as blue, and the growth inhibitory hydrogel has no
color. In another embodiment the growth permissive hydrogel is blue
and the growth inhibitory hydrogel is green or turquoise.
[0304] Preferably, the growth permissive substance is an in situ
forming hydrogel. Preferably, the growth permissive substance
contains growth inhibitory and growth permissive microdomains.
Nerves will naturally pathfind along the growth inhibitory domains
and within the growth permissive domains. Growth permissive
hydrogels leveraging the in situ forming PEG platform are
desirable. These hydrogels may be crosslinked chemically or using
photo-crosslinkable approaches as with the non-growth permissive
hydrogels described above. The in situ growth permissive hydrogels
are preferably more rapidly degrading than the growth inhibitory
hydrogels, encouraging cellular ingrowth and replacement of the
synthetic matrix with natural extracellular matrix. As a result,
preferred PEG hydrogels for these applications are formed through
the crosslinking of PEG-NHS esters with hydrolytically labile ester
bonds (PEG-SS, PEG-SG, PEG-SAZ, PEG-SAP), preferably PEG-SS. These
PEGs can be crosslinked with PEG-amines or trilysines, for
example.
[0305] Other hydrogels may be selected to provide non-growth
permissive zones of the growth permissive hydrogel including
PEG-PPO-PEG, PEG-polyesters (triblocks, deblocks), alginate, agar,
and agarose. Other synthetic hydrogels include PEG-poly(amido
amine) hydrogels, PEO, PVA, PPF, PNIPAAm, PEG-PPO-PEO,
PLGA-PEG-PLGA, poly(aldehyde guluronate), or polyanhydrides. An
extensive list of hydrogel matrices that may be adapted for in situ
formation is found in Hoffman (2012) Hydrogels for biomedical
applications. Advanced Drug Delivery Reviews, 64: 18-24,
incorporated herein for reference. Another soft hydrogel that may
be suitable includes the InnoCore Liquid Polymer (LQP)
(PCLA-PEG-PCLA) which is a liquid polymer which forms a soft
macroscopic depot after delivery in vivo and degrades slowly over a
period of two to three moths. Another potentially suitable hydrogel
includes a six-armed star-shaped poly(ethylene oxide-stat-propylene
oxide) with acrylate end groups (star-PEG-A) can be photocured.
Other star-shaped PEGs include a 6-arm or 8-arm NHS ester PEGs
include mPEG-SCM (PEG-NHS: Succinimidyl Carboxyl Methyl ester) and
mPEG-SG (PEG-NHS: Succinimidyl Glutarate ester), 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. PEG concentration. If PEG is used in the growth permissive
matrix, preferably the PEG concentration in these hydrogels is
preferably between 3 and 8%, more preferably 3 to 5 wt % for the
applications to support nerve extension.
[0306] In some embodiments, the growth permissive region is
directly conjugated or chemically linked to the non-growth
permissive hydrogel region. For example, chitosan may be coupled to
the inhibitory region. The chitosan may be a 100 kDa to 350 kDa
molecular weight, more preferably 130 kDa to 160 kDa with a 0.85
degree of deacetylation. In another embodiment, an interpenetrating
network of gelatin methacrylamide polymerized with a PEG
framework.
[0307] Alternative growth permissive matrices. In addition to
incorporating positively charged matrix components that encourage
glial invasion, cellular division, and three-dimensional cellular
organization, the growth permissive components can also support
nerve ingrowth with or without the presence of supporting cells.
These growth promoting substances are applied at a concentration
sufficient to support growth but not at such a high concentration
to impact the mechanical properties of the hydrogel. Growth
permissive hydrogels contain combinations of natural growth
promoting biomaterials such as natural polymers collagen type I
(0.01 to 5 wt %, preferably 0.3-0.5 wt %, 1.28 mg/ml), laminin (4
mg/ml), hyaluronic acid, fibrin (9 to 50 mg/ml, strength 2.1 kPa)
or synthetic/semi synthetic polymers such as poly-L-arginine or
poly-L-lysine (0.001-10 wt %). By combining the growth inhibitory
domains above with the growth permissive matrices, growth
supportive matrices can be formed. These blends support the 1)
creation of a path through which regenerating nerves can path find,
2) provide a substrate to which neurites can adhere and Schwann
cells can migrate in. In one embodiment the hydrogel is 8-arm 15K
PEG-succinimidyl succinate (PEG-SS) crosslinked with trilysine,
containing 5 wt % collagen. In another embodiment, the hydrogel
comprises an 4% 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 crosslinked PEG solution relative to the
growth inhibitory PEGs used in neuroma blocking applications and
increasing the concentration of the positively charged growth
permissive biomaterial, an in situ forming hydrogel can be created
with both inhibitory and permissive domains to encourage nerve
outgrowth.
[0308] In another example, a non-growth permissive hydrogel (e.g.
crosslinked PEG hydrogel, alginate, methacryloyl-substituted
tropoelastin MeTro hydrogel) may be blended with a growth
permissive hydrogel (e.g. fibrin gelatin-methacryloyl GelM,
GelM/PEG or GelMA/MeTro composites) Soucy et al (2018)
Photocrosslinkable Gelatin-Tropoelastin Hydrogel Adhesives for
Peripheral Nerve Repair, Tissue Engineering, PMID: 29580168.
Incorporation of polylysine. Polylysine--either the D,L, or L
forms, can be incorporated into the growth permissive hydrogel
region. For example Epsiliseen (Siveele, Epsilon-poly-L-lysine).
The growth permissive hydrogel may be an in situ forming hydrogel
comprising chitosan and poly-lysine
(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/bm201763n).
[0309] PEG+Collagen in Backbone. Alternatively, natural polymers,
such as type I collagen, can be crosslinked with PEG hydrogel (e.g.
8-arm 15K SG) with collagen concentrations ranging from 30 to 60
mg/ml and PEG concentration at 50 or 100 mg/ml ((Sargeant et al
2012. An in situ forming collagen-PEG hydrogel for tissue
regeneration. Acta Biomaterialia 8, 124-132 and Chan et al (2012)
Robust and semi-interpenetrating hydrogels from PEG and Collagen
for Elastomeric Tissue Scaffolds. 12(11) 1490-1501.
[0310] Other gels. In yet another embodiment, the first growth
permissive material may comprise a viscous solution, a
nanoparticle- or microparticle-based gel, a slurry, or a macrogel.
In one embodiment a fibrin glue can be delivered around nerves. In
another embodiment, the solution is a slurry of biocompatible
nanoparticles or microparticles through which nerves can
regenerate. In another embodiment, a microgel or modugel is
delivered to the site. Microgels are created for stable dispersions
with uniform size, large surface area through precipitation
polymerization. Modugels, scaffolds formed from microgels,
properties can be varied through the degree of crosslinking and
scaffold stiffness (Preparation of the gels, including PEG-based
hydrogels, can be found in Scott et al (2011) Modular Poly
(ethylene glycol) scaffolds provide the ability to decouple the
effects of stiffness and protein concentration on PC12 cells. Acta
Biomater 7(11) 3841-3849, incorporated herein for reference.) In
addition, the use of electrically conductive hydrogels, such as
piezoelectric polymers like polyvinylidene fluoride (PVDF) which
generates transient surface charges under mechanical strain, may be
beneficial in supporting the growth of nerves through the hydrogel.
For example, dendrimers comprised of metabolites such as succinic
acid, glycerol, and beta-alanine may be incorporated into the
hydrogels to encourage extracellular matrix infiltration
(Degoricija et al (2008) Hydrogels for Osteochondral Repair Based
on Photocrosslinkable Carbamate Dendrimers, Biomacromolecules,
9(10) 2863.
[0311] Plain natural hydrogels. In another embodiment, an entirely
growth permissive hydrogel is provided without growth inhibitory
microdomains. In one embodiment, a fibrin hydrogel (such as those
crosslinked with thrombin) with a lower linear compressive modulus
is selected. Numerous other biomaterials have also been
demonstrated to support nerve regeneration in 2D and 3D scaffolds
and include chitosan, chitosan-coupled alginate hydrogels, viscous
fibronectin, collagen type I (.about.1.2 mg/ml), assist in
regeneration, fibrin (9 to 50 mg/ml), fibronectin, laminin
(https://www.ncbi.nlm.nih.gov/pubmed/15978668)), Puramatrix,
heparin 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
blends of these materials. Additional growth permissive hydrogels
include thermosensitive hydrogels like
chitosan-beta-glycerophosphate hydrogels (C/GP) mixtures. Other
thermosensitive hydrogels include poly (N-isopropylacrylamide)
(PNIPAAM). In one embodiment, a poly(propylene fumarate) PPF can be
injected as a liquid and chemically, thermally, or photo
cross-linked in situ to form a gel to provide a growth supportive
hydrogel. In another embodiment, an interpenetrating network of HA
and photocrosslinkable glycidyl methacrylate hyaluronic acid (GMHA)
provides a growth permissive substrate. Other growth permissive
hydrogels include: crosslinked hyaluronic acid gel (Hyaloglide gel)
or ADCON-T/N gel (Gliatech). These materials may be physically or
covalently crosslinked. Other scaffold materials may be anticipated
for the growth permissive region
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5899851/).
[0312] Charge. It is well known in the art that nerves prefer to
grow on or through positive charged surfaces. In some embodiments,
the positive charges are incorporated into the polymer backbone. In
other embodiments, these charges are incorporated in other
components, such as extracellular matrix proteins that become
trapped in the hydrogel when it forms in situ.
[0313] Incorporation of Adjuvants. In some embodiments,
anti-inhibitory molecules can 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 the polymer powder, diluent, or accelerator
depending on the stability requirements of the adjuvant.
[0314] Incorporation of Lipid Domains. Lipid domains may be added
to the backbone or side chains or these polymers to encourage nerve
outgrowth. Hydrophobic domains may also be incorporated into the
backbone of the hydrogel to support nerve ingrowth through soft and
hard regions of the hydrogel. In one embodiment, lipids are added
to diffuse between polymer chains and act as plasticizers for the
polymeric material that facilitates chain moving and improves
elasticity.
[0315] Adhesion strength. The growth permissive media and the
growth inhibiting media may transform into hydrogels and have
sufficient adherence that, once formed, the nerve ends can be
picked up and handled with ease. The adhesive strength of the
subsequently formed nonflowable growth permissive media, though,
permits the nerve-gel-nerve unit to be picked up with forceps. The
unit can be gently placed within a second form to permit
circumferential delivery of the inhibitory hydrogel. The adhesion
strength also permits good coupling between the nerve end and the
hydrogel.
[0316] Stiffness. As matrix stiffness and compressive strength of
the hydrogel play a significant role in promoting or inhibiting
nerve regeneration, the mechanical properties of the growth
inhibitory and growth permissive hydrogels differ substantially.
The growth permissive hydrogel is significantly softer and less
stiff to support and encourage the regeneration of nerve growth
cones into the media. Gel stiffness (G*, dynes/sq cm) of the growth
permissive hydrogel is preferably softer and more elastic in
character with G* less than 800 dynes/sqcm, more preferably less
than 200 dynes/sqcm. In some embodiments, regions of soft substrate
(100-500 Pa) are placed adjacent to regions of stiffer substrate
(1,000 to 10,000 Pa). The elasticity of this growth permissive
substrate should preferably be less than 0.1-0.2 MPa, preferably
less than 1.5 KPa. On the other hand, the growth inhibitory
hydrogel provides the necessary mechanical strength to maintain the
coupling and relationship between the proximal and distal nerve
stumps, reduce or eliminate the need for suturing, and potentially
permit tensionless repair. Nerve extension into the growth
permissive matrix is strongly dependent on matrix stiffness, and
pore interconnectivity of pores, charge. As the gel degrades, the
nerve extension may also be impacted by the hydrolytic, oxidative,
or enzymatic degradation of the matrix. For the growth permissive
hydrogel, the stiffness of the gel should more closely approximate
the elastic modulus of the nerve tissue, at or below 1 kPa,
preferably 200-300 Pa. Swelling. Given the placement of the growth
inhibitory hydrogel around the growth permissive hydrogel, the
growth permissive hydrogel swelling must be less than or equal to
the swelling of the growth inhibitory hydrogel. Alternatively, the
growth permissive hydrogel must be sufficiently soft that it does
not have the strength to push on the growth inhibitory hydrogel.
Porosity. In some embodiments, the growth permissive media
comprises a growth inhibitory hydrogel filled with highly
interconnected macroscopic growth permissive pores that provide a
channel through which regenerating nerves can pathfind. Pores may
created in the hydrogels through porogen leaching (solid, liquid),
gas foaming, emulsion-templating to generate macroporosity. The
pores may be created by a growth permissive porogen 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 therefore are
encompassed in a term called tortuosity. Preferably, the hydrogel
is a macroporous hydrogel with pores greater than 1 .mu.m, more
preferably greater than 10 .mu.m, preferably between >100 .mu.m
more preferably >150 .mu.m, with an average pore radius of 0.5
to 5%. The density of the pores should be greater than 60%, or
preferably greater than 90% pore volume, of sufficient density that
the pores are interconnected. In this manner, the remaining PEG
hydrogel provides a non-growth permissive scaffold through which
neurons can grow. In one embodiment, porosity is created by
creating air or nitrogen bubbles in the hydrogel through shaking,
pushing the plunger in the applicator back and forth, or
introducing the air through another port in the applicator. In
another embodiment, a surfactant is used as an air-trapping agent
to create porosity in the hydrogel, such as sodium dodecyl sulfate
(SDS). In situ gas foaming with up to 60% porosity and 50 to 500
micron pores and a compressive modulus of 20-40 Mpa, described in
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3842433/. In another
embodiment, the creation of a foaming agent which generates
macroscale pores to permit cellular migration and proliferation. In
some embodiments, the porogen is a degradation enhancer.
Preferably, the concentration of pores is sufficient that the pores
are interconnected with one another. Preferably, >70% of the
pores are interconnected, more preferably 80% or more. The pores
create and define growth permissive zones and preferably the
interconnectivity is sufficiently high that the tortuosity is low
that nerves will extend out into them. In addition, if the walls of
the pores are formed of PEG, nerves can pathfind along the walls of
the hydrogel. Pores may be created by low molecular weight
end-capped PEGs, such as PEG 3350, which can be delivered in up to
50 wt % solution. The growth permissive regions or pores may
contain natural biomaterials such as collagen/gelatin, chitosan,
hyaluronic acid, laminin (Matrigel), fibrin that provide a growth
permissive substrate for nerve outgrowth.
[0317] Channels. In another embodiment, channels are created in the
hydrogel in situ to permit nerve guidance. In one embodiment,
channels are approximately 150 .mu.m, 300 .mu.m in diameter, more
preferably 500 .mu.m in diameter to 1 mm. Preferably, the channels
are filled with saline in situ.
[0318] Fibers and other structural elements. Adding fibers or
structural elements (e.g. beads, macrospheres, gel particle slurry,
microspheres, rods, nanoparticles, liposomes, rods, filaments,
sponges) to reinforce the structural integrity of the hydrogel,
improve the hydrogels in vivo persistence and/or to provide a
substrate along which neurites can extend and growth for guidance,
is desirable. The nanofibers can be flexible or rigid and can range
in size from nanometers to micrometers in diameter, and can be
linear or irregularly shaped. In the preferred embodiment, the
fiber deposition through the needle containing the media into the
form permits the generally longitudinal parallel alignment of the
fibers within the conduit. The fiber-loaded media is laid down in
the conduit by first filling the distal end and advancing the
needle to proximal end of the form in a smooth continuous motion
while depositing the hydrogel. Rapid gelation (less than 20
seconds, preferably less than 10 seconds, more preferably less than
7 seconds) permits the fibers to be captured in the desired
orientation as the media changes to a nonflowable form. In another
embodiment, the media solutions is more viscous, between 10 and 20
cP, permitting the suspension of these fibers within the growth
permissive media. In another embodiment, the fibers are provided in
the kit and are placed in the lumen with forceps.
[0319] Fibers, rods, filaments, sponges. In another embodiment, the
fibers are added to the form either immediately before or after the
delivery of the growth permissive hydrogel solution in the wrap
form to provide a surface along which nerves can grow en route to
the distal stump. The fibers may be added using forceps, another
syringe, or sprinkling them within the conduit. The gelation time
of the hydrogel media is sufficiently delayed that the fibers can
be embedded within the media prior to the media change to a
substantially nonflowable form.
[0320] Injecting nanorods Similarly, shorter nanorods may be
incorporated into the polymer solution, polymer powder, diluent, or
accelerator and then injected in situ. By injecting smoothly and in
one direction and utilizing a fast gelling hydrogel, the alignment
of these fibers may be improved. The fibers may be constructed from
nondegradable or biodegradable materials. In some embodiments the
fibers are made of chitosan, polycaprolactone, polylactic or
glycolic acid, or combinations thereof. The fibers may be inert of
functionalized with a positive charge or addition of a coating such
as laminin. https://www.ncbi.nlm.nih.gov/pubmed/24083073. In
another embodiment the fibers undergo molecular self-assembly to
form a fiber or cable.
[0321] In one embodiment, fibers will be incorporated either
randomly or in an aligned fashion in order to provide the support
for nerve regeneration across a gel. Filaments and sponges can be
formed out of collagen. Rods can be formed out of collagen-gag,
fibrin, hyaluronic acid, polyamide,
polyarylonitrile-co-methacrylate, PAN-MA, PGA, PHBV/PLGA blends,
PLLA, PLGA or PP. The filaments may be between 0.5 and 500 .mu.m in
diameter, more preferably 15 to 250 .mu.m in diameter. In one
embodiment, the rods, fibers, and filaments may be coated with
laminin.
[0322] Nanofibers can be incorporated into the hydrogel to provide
structural support. Fibers may be composed of PEG, PGA, PLA, PCL,
PCL mixed with gelatin, PCL with a laminin coating, chitosan,
hyaluronic acid, gels, hyaluronan, fibrin, or fibrinogen (10
mg/ml). In one embodiment, a fibrillar fibrin hydrogel (AFG), or
P(D,L, LA) fibers, fabricated through electrospinning is are
incorporated into in situ forming gels. (Electrospinning methods
are described in McMurtrey (2014) Patterned and functionalized
nanofiber scaffolds in three-dimensional hydrogel constructs
enhance neurite outgrowth and functional control. J. Neural Eng 11,
1-15, incorporated herein.) In another embodiment, polyethylene
glycol is incorporated as a porogen and nanofibers, such as
cellulose nanofibers, are employed to provide structural integrity
to the soft porous hydrogel (Naseri et al (2016) 3-Dimensional
Porous Nanocomposite Scaffolds Based on Cellulose Nanofibers for
Cartilage Tissue Engineering: Tailoring of Porosity and Mechanical
Performance. RSC Advances, 6, 5999-6007, incorporated for reference
herein.)
[0323] Microparticles. In yet another embodiment, microparticles,
nanoparticles or micelles can be introduced into the growth
permissive media to deliver drugs to the nerve tissue. In one
embodiment, microparticles are composed of PEG hydrogels (e.g.
8-arm 15K SG, 10%), poly(D,lysine) microparticles. For example,
cross-linked PEG particles formed ex vivo can be formulated into a
slurry lubricated by low MW PEG (1-6%, 12 kDa). Alternatively, the
particles can be suspended in a collagen or hyaluronic acid
solution to provide a growth permissive matrix through which the
nerves can regenerate. Similarly, hydrophobic particles and oils
may be incorporated to create growth permissive voids in the
hydrogel to encourage nerve outgrowth.
[0324] Compressive modulus of growth promoting hydrogels. Matching
the compressive modulus of the nervous tissue to the growth
permissive hydrogel may also be advantageous--approximately 2.6 to
up to 9.2 kPa (Seidlits et al (2010) The effects of hyaluronic acid
hydrogels with tunable mechanical properties on neural progenitor
cell differentiation are promising (Biomaterials 31, 3930-3940).
Similarly, the linear compressive modulus is less than 20 kPa,
preferably less than 10 kPa, more preferably less than 1 kPa to
encourage nerve and Schwann cells ingrowth into the gel.
[0325] In situ forming growth-permissive hydrogels that can be
delivered in a wrap form or thin layer around partially transected,
compressed, or completely transected nerve ends are desirable. The
use of an in situ forming gel eliminates the need to transect
otherwise largely intact nerves and provides a mechanism to support
nerve regeneration through a substrate and into the distal tissue.
Coupling the growth-permissive hydrogel with a growth-inhibitory
hydrogel assists in guiding and directing these neurites within the
growth permissive region. In one embodiment, the in situ forming
hydrogel has sufficient adhesive strength and stiffness that it can
be delivered between the nerve stumps into an appropriate form and
then be picked up and removed from the first wrap form and placed
into a second larger wrap form into which the growth inhibitory in
situ forming hydrogel is delivered.
[0326] Hydrogel thickness. Growth permissive gel. The thickness or
diameter of the growth permissive gel should roughly approximate
the diameter of the nerves to which it is being delivered. In the
case where only a small defect exists in the nerves, the growth
permissive gel can be dropped directly on the injured tissue to
form a thin layer. Growth inhibitory gel. Given the often rigorous
environment in which a nerve is located, often in a fascial plain
between or along muscles, in some embodiments it is desirable that
a minimum thickness of growth inhibitory hydrogel be maintained
around the nerve, preferably 1mm circumferentially, more preferably
2-3 mm. For example, for a form that would be place around the
common digital nerve, approximately 2-2.5 mm in diameter, a conduit
of approximately 3 to 4 mm in diameter is used, providing a 0.5 to
2 mm hydrogel layer around the nerve. For the digital nerve,
approximately 1 to 1.5 mm, a conduit approximately 2 to 2.5 mm in
diameter is selected. For larger nerves embedded in the arm or
thigh, between 2 and 10 mm, preferably the gel thickness is 2 to 6
mm, preferably 2 to 3 mm around the nerve circumferentially.
[0327] Gelation time. After 30 seconds or less, preferably 20
seconds or less, more preferably 10 seconds or less, more
preferably 3 to 7 seconds, the hydrogel forms around the nerve. The
hydrogel is transparent so the location of the nerve can be
visually confirmed in the hydrogel. The clinician visually or
mechanically confirms the hydrogel formation and the silicone form
is slipped off of the hydrogel cap and discarded. See FIG. 2. The
surrounding tissues (muscles, skin) are then sutured up again per
standard surgical technique.
[0328] In Vivo Persistence of Growth Permissive Gel or Slurry. The
in vivo persistence may be considerably less in the growth
permissive hydrogel than the growth inhibitory gel, permitting the
progressive invasion by Schwann cells and regenerating nerve
fibers. For growth permissive hydrogels, more rapidly degrading
hydrogel networks are desirable to permit cellular infiltration and
subsequent nerve regeneration. Preferably, the hydrogels should
degrade in between 2 months and 6 months, more preferably 3 months.
Degradation of the inhibitory region. The inhibitory guide
preferably remains in place for 1 month or more, more preferably 3
months or more, to provide support to the regenerating nerve. In
some embodiments, the degradation of the growth permissive hydrogel
is days to months, preferably days to weeks, permitting the
clearance of the material as the cellular tissue replaces and
regenerates.
[0329] Charge. Preferably, the growth permissive hydrogels are
positive charged or contain positively charged domains. Addition of
PEG fusogens. In some embodiments, it may be desirable to add a
nonreactive fusogen to the hydrogel formulation. Thus, in addition
to the mechanical blocking properties of the hydrogel, the damaged
proximal surviving nerves may be protected from excitotoxic damage
and their membranes resealed. Furthermore, the hyperexcitability of
the cell bodies is reduced, such as the dorsal root ganglia,
reducing neuropathic paresthesia and dysthesia accompanying nerve
injury. In one embodiment, low molecular weight end-capped or
nonreactive PEG (methoxy-PEG) to the formulation. For example, the
trilysine buffer may contain nonreactive low molecular weight
linear PEG (0.2 kDa, 2 kDa, 3.35 kDa, 4 kDa, or 5 kDa). When mixed
with the 8-arm 15K star-shaped PEG, the resulting hydrogel will
have low molecular weight PEG (2 kDa, 10-50% w/v) which may help to
seal up the damaged nerve endings and thus further reduce the
influx and efflux of ions. In this manner, lysosome formation,
demyelination, and 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 the survival of the ablated
axons.
[0330] In another embodiment, six-armed star-shaped end-capped PEG
(poly(ethylene-oxide-stat-propylene oxide) or star-PEG-OH) can be
added as the fusogen. The linear PEG that is mixed in the polymer
blend can diffuse out of the crosslinked network, creating
micropores up to one micron in diameter, facilitating diffusion of
nutrients but not neurite extension. The linear PEG based hydrogels
are more stiff than the star-PEG based fusogen addition. The
addition of the linear PEG is based on findings which had been
demonstrated 2 kDa PEG to be beneficial in rapidly restoring axonal
integrity, called `PEG-fusion` between nerves of cut- and
crushed-axons (Britt et al 2010, J. Neurophysiol, 104: 695-703).
The theory is that this is in part because of sealing of the
plasmalemmal and axolemma at the lesion site.
[0331] Reapplying or repositioning. Should the clinician not be
happy with the location of the nerve, the hydrogel `cap` can be
removed with forceps and the procedure repeated. Nerve sparing. In
yet another embodiment, it may be desirable to deliver the in situ
forming hydrogel around a nerve in order to reduce the handling of
the nerve during a procedure. By delivering it in and around the
nerve bundle, the hydrogel can set and prevent forceps or any other
micromanipulators from crushing it during the procedure. In
additional to protecting the nerve from mechanical damage, the
hydrogel may also protect from thermal damage such as through
cauterizing or RF ablation, cryoablation.
[0332] There are several embodiments where existing nerve wraps
(e.g. conduits with a top slit in them that allow the nerve to be
pushed into the semi-rigid wrap) and/or conduits are still desired
but the physician would like to provide additional support for
regeneration either in the form of the application of a growth
permissive hydrogel, a growth inhibitory hydrogel, or the
combination.
[0333] The form for the growth permissive hydrogel is designed to
be substantially the same size as the nerves into which they are
placed. In one embodiment, a silicon form which is a hemi-tube with
an inner diameter approximately equivalent to the outer diameter of
the nerves is selected. The nerve are placed in the form either in
direct apposition, within close approximation, or, with a gap in
order to prevent tension, so that they rest within the form without
any tension. The nerves rest directly on the surface of the form
itself for delivery of the growth permissive hydrogel.
[0334] Drugs to Promote Nerve Regeneration. Drugs may be delivered
to the nerve directly prior to the placement of the form. For
example, local anesthetic, anti-inflammatory, growth factors agents
may be delivered directly to the nerve prior to encapsulation with
the hydrogel. Alternatively, drugs may be incorporated directly
into the hydrogel or incorporated through encapsulation in
drug-loaded microspheres, micelles, liposomes, or free-base to
achieve an improved sustained release profile.
[0335] Drugs for pain relief. In some embodiments, drugs used for
the treatment of chronic neuropathic pain may be delivered in the
hydrogels including tricyclic antidepressants, selective serotonin
and noradrenaline reuptake inhibitors, antiepileptics, and opioids.
For example, pregabalin and gabapentin may be selected for their
analgesic properties. Similarly, duloxetine, vennlafaxine, the SNRI
inhibitor and combinations thereof to provide more comprehensive
pain relief. Anti-inflammatories such as diclofenac may also be
promising. Other potential targets include ligands for the
FK506-binding protein family, neuroimmunophilin ligands, which are
neuritotrophic, neuroprotective and neuroregenerative agents.
[0336] The local delivery of taxol and cetuximab have also shown
promise for improving the survival and regeneration of neurons and
may be suitable for stimulating nerve regeneration when delivered
locally in an in situ forming hydrogel. In another embodiment,
cyclic AMP (cAMP) or cAMP analogue dibutyryl cAMP promotes
regeneration of nerves and may be incorporated into an in situ
forming hydrogel to promote nerve regeneration after injury. In
another embodiment, Kindlin-1 and Kindlin-2 (fermitin family) and
drugs which bind to the integrin superfamily of cell surface
receptors, allow nerves to extend across inhibitory matrix and can
be incorporated into the hydrogels to enhance regeneration across
inhibitory extracellular matrix.
[0337] In another embodiment, tacrolimus (FK506), an
immunosuppressant, may be incorporated into the hydrogel to enhance
axon generation and speed. The final concentration of FK506 in the
formed hydrogel is 100 ug/ml to 10 mg/ml, more preferably 0.1
mg/ml. The drug is released for weeks to months, preferably at
least a month, more preferably at least 3 months to aid in
immunosuppression and enhance nerve outgrowth. Drugs include FK506,
drugs selective for selective inhibition of FKBP12 or FKBP51.
[0338] Drugs that are P2X receptor antagonists (P2XR), P2X3
receptor antagonists (e.g. AF-219 Gefapivant, AF-130), P2X4 and
P2X7 receptor antagonists that are implicated in visceral and
neuropathic pain (as well as migraine and cancer pain), are of
interest. P2X7 Receptor Antagonists. The purinergic receptor
antagonist Brilliant Blue G (BBG) and the structurally similar
analogue, Brilliant Blue FCF (BB FCF), are of particularly interest
for their ability to modulate the nerve environment after injury
(Wang et al. 2013. The food dye FD&C Blue No. 1 is a selective
inhibitor of the ATP release channel Panx1. J. Gen. Physiol. 141(5)
649-656)). Other dyes of interest include the FD&C Green No. 3
dye which, like BBG and BB FCF, inhibit the ATP release Pannexin1
channel with an IC50 between 0.2 and 3 uM. A structurally similar
analogue, Brilliant Blue FCF (BB FCF) otherwise known as FD&C
#1 (https://pubchem.ncbi.nlm.nih.gov/compound/Acid_Blue_9), has
also been demonstrated to improve nerve survival and regeneration
after injury when used in combination with a low molecular weight
end capped PEG 3350 Da
(https://www.ncbi.nlm.nih.gov/pubmed/23731685). Similar efficacy
has been demonstrated using BBG in rat models of sciatic nerve
crush (Ribeiro et al 2017) and ischemia in the myenteric plexus
(Palombit et al 2019). In addition, BBG is thought to have
anti-inflammatory and anti-nociceptive effects through reducing
high extracellular ATP concentrations and high calcium influxes
after nerve damage. In one embodiment, Brilliant Blue FCF is
incorporated into the in situ forming hydrogel. The dye can be
incorporated into the polymer vial, the diluent, or the accelerator
solutions to yield 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 approximately 1 to 1000 ppm, preferably 10 to 100 ppm. On a per
site anatomic basis, local doses of between 5 .mu.g to as high as
25 mg of dye may be delivered in a hydrogel locally. For example,
the FD&C #1 dye may be delivered at 0.01% concentration in the
hydrogels to reduce neuronal injury after stroke (Arbeloa et al
2012--Referenced in Palmobit et al 2019). By incorporating the dye
into the hydrogel, the dye may help improve the survival of the
transected axons, reduce the local inflammation while the hydrogel
provides a barrier to regeneration.
[0339] In another embodiment, TRPV1 agonists, such as capsaicin are
delivered to the nerve to deliver a preconditioning injury to the
nerve that in term results in a neuroregenerative response
downstream to enhance nerve regeneration (PMID:29854941). In one
embodiment, capsaicin loaded hydrogels (1 to 8 wt % drug loading)
are delivered percutaneously to intact nerves to reduce painful
diabetic neuropathy). In another embodiment, pifithrin-u or
acetyl-L-carnitine is delivered in the hydrogel to reduce and treat
chemotherapy-induced peripheral neuropathy (CIPN) by reducing
neuronal mitochondrial damage.
[0340] In another embodiment, drugs that block the deregulated long
non-coding RNAs may also be incorporated into the hydrogels, such
as targets of endogenous Kcna2 antisense RNA. In one embodiment,
mitomycin C is incorporated into the in situ forming hydrogel in
order to inhibit Schwann cell proliferation and stimulate apoptosis
in fibroblasts. In one embodiment, 0.1 to 5 mg mitomycin C are
loaded into the polymer powder and utilized to form an in situ
formed gel with 0.01-0.5 wt % mitomycin C releasing between 0.1 and
0.5 mg/ml are released per day, preferably for 7 days or more. In
still another embodiment, Rho Kinase (ROCK) inhibitors or ROK
antagonists or Rac1 antagonists may be incorporated, such as
ripasudil hydrochloride.
[0341] Additional drugs include the anti-inflammatory curcurmin,
rapamycin, paclitaxel, cyclosporin A, pyrimidine derivatives (RG2
and RG5) to stimulate remyelination, Axon guidance molecule Slit 3,
triptolide, KMUP-1. calcium modulating agents including calcitonin,
calcium antagonist 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--used as a neuroprotective and
neurotrophic drug.
[0342] Example 1. Growth inhibitory hydrogel. Into the vial
containing 80 mg PEG with NHS ester reactive group, 80 .mu.g of BB
FCF is added to yield a 0.1% dye concentration in the PEG
hydrogel.
[0343] Example 2. Growth inhibitor hydrogel with a fusogen. Into
the vial containing 80 PEG with NHS ester reactive group, 80 .mu.g
of BB FCF and 500 mg PEG 3350.
[0344] Example 3. Phospholipids are incorporated into the PEG
hydrogel, such as cephalin, to improve fusion. Phospholipids are
surface active amphiphilic molecules and can be incorporated as an
emulsifier, wetting agent, solubilizer, and membrane fusogen. These
may include phosphatidylcholine, phosphatidylethanolamine or
phosphatidylglycerol
(https://www.ncbi.nlm.nib.gov/pmc/articles/PMC4207189/,
incorporated for reference herein).
[0345] Example 4. In some embodiments, hydrogels are loaded with
amiodarone with or without the addition of ethanol. For example,
0.1 to 5 wt % loading of amiodarone or more can be achieved. This
can also be accomplished and improved with the incorporation of
ethanol into the solution. For example, 50 to 75% ethanol can be
incorporated with 0.25 wt % amiodarone to achieve burst release of
amiodarone between 3 to 5 days. Similarly, 1% amiodarone can be
delivered from the hydrogels for a period of 30-60 days.
[0346] Various other modifications, adaptations, and alternative
designs are of course possible in light of the above teachings.
Therefore, it should be understood at this time 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. Further, the
disclosure herein of any particular feature, aspect, method,
property, characteristic, quality, attribute, element, or the like
in connection with an embodiment can be used in all other
embodiments set forth herein. Accordingly, 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. Thus, it is intended
that the scope of the present inventions herein disclosed should
not be limited by the particular disclosed embodiments described
above. Moreover, 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 described and the appended claims. Any methods
disclosed herein need not be performed in the order recited. The
methods disclosed herein include certain actions taken by a
practitioner; however, they can also include any third-party
instruction of those actions, either expressly or by implication.
The ranges disclosed herein also encompass any and all overlap,
sub-ranges, and combinations thereof. Language such as "up to," "at
least," "greater than," "less than," "between," and the like
includes the number recited. Numbers preceded by a term such as
"approximately", "about", and "substantially" as used herein
include the recited numbers (e.g., about 10%=10%), and also
represent an amount close to the stated amount that still performs
a desired function or achieves a desired result. For example, the
terms "approximately", "about", and "substantially" may refer to an
amount that is within less than 10% of, within less than 5% of,
within less than 1% of, within less than 0.1% of, and within less
than 0.01% of the stated amount. Furthermore, various theories and
possible mechanisms of actions are discussed herein but are not
intended to be limiting.
* * * * *
References