Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND DEVICES FOR IN SITU FORMED NERVE CAP WITH RAPID
RELEASE
INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS
[0001] This application is a continuation-in-part of Patent
Cooperation Treaty
Application No. PCT/US2019/040429, filed July 2, 2019 which claims the benefit
under
35 U.S.C. 119(e) as a nonprovisional application of U.S. Prov. App. No.
62/692,858 filed
on July 2, 2018 and U.S. Prov. App. No. 62/822,881 filed on March 24, 2019.
This
application also claims priority to U.S. Provisional Patent Application No.
62/960,564, filed
on January 13, 2020, all 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 and poor quality of life. 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
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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 fillers 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
at 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.
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[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) or a closed tube with a shelf (e.g.
AXOGEN Nerve
Cap ); 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 followed by securing the
nerve with
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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 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 due to the 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 due to the potential space, with resulting sensation
of pain.
Although these nerve caps degrade over a period of 3 months to 24 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 and
fragments
stimulating fibroblast infiltration and scar formation around the healing
nerve. As a result,
the efficacy of these pre-formed 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 ncuroma to the surrounding
environment and
degradation products triggering an adverse inflammatory response. Finally,
since these
methods require suturing using fine sutures (9-0 nylon or 8-0), the procedural
time and skill
required to secure these implants under surgical magnification (loupes) or a
surgical
microscope 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)
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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:
Hi stop ath o logic Evaluation. http://rnecting.-.h and surffm.
les/2018/e Po Rte rs/ HS EM 06 .pd ) 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] Other applications of the technology directed
towards wrapping or
coapting nerves are also described to prevent aberrant axon outgrowth and
encourage nerve
regeneration. In particular, methods to protect nerves by enveloping them in
in situ forming
hydrogels delivered inside a temporary Wrap form or methods to assist in nerve
regeneration
using growth supportive/permissive solutions or gels, rapidly resorbable thin
sheet wraps in
combination with in situ forming inhibitory hydrogels are described.
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
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of the form. The tip of 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 I 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. The method may alternatively
comprise the
step of performing axon fusion utilizing a 'PEG fusion' protocol, described
later, prior to
placing the nerve within the nerve form.
[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 a 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. Alternatively,
the form may
comprise a porous lyophilized biomateral that dissolves in minutes to hours
after being
exposed to physiological fluid
[0018] The identifying a severed nerve step may include the
step of severing a
target nerve, such as with scissors, a blade (e.g. No 10 or No 11), or a razor
blade. The step
may additionally comprise transecting the nerve cleanly at an oblique angle
prior to placing
the nerve within the form. Alternatively, nerves can be cute with a nerve
cutting or trimming
device.
[0019] The transformation step may comprise a crosslinking
reaction or a
polymerization and may use 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
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implementation, the transformation produces a synthetic crosslinked hydrogel
protective
barrier through which nerves cannot regenerate around the end of a transected
nerve stump.
By crosslinking the hydrogel at the distal tip of the transected nerve
containing severed
axons, the hydrogel provides a physical block to nerve regeneration or neuroma
formation
and, as it absorbs fluid and equilibrates, draws fluid from the axoplasm and
cellular/cytoplasmic debris away from the transected nerve, thereby improving
self-sealing
capability of the axonal membranes.
[0020] There is provided in accordance with a further
aspect of the present
invention a method of in situ formation of an implant with a rapid release
from a mold. The
method comprises the steps of identifying a nerve; positioning the nerve in a
cavity defined
by a form; introducing media into the cavity 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 surrounding the nerve; wherein a
hydrophilic
characteristic of the media cooperates with a hydrophobic characteristic of
the cavity to
facilitate a rapid release of the implant from the cavity following the
transformation.
[0021] The positioning step may comprise positioning a
severed end of a nerve
into the cavity and permitting the media to undergo a transformation to form a
protective
barrier surrounding the severed end of the nerve. The implant can be removed
from the
cavity by a pull force of no more than about 10 N, applied for no more than
about 5 seconds.
In some implementations, the implant can be removed from the cavity by a pull
force of no
more than about 5 N, or by a pull force of no more than about 2 N applied for
no more than
about 2 seconds. In general, the implant can be removed from the cavity within
10 seconds,
preferably within 5 seconds, preferably within about 2 seconds, without
disrupting
attachment between the implant and the nerve.
[0022] The introducing media step may include introducing a
first volume of
media, and following transformation of the first volume, introducing a second
volume of
media. In one embodiment, the first layer is delivered first to position the
nerve and a
subsequent second layer is delivered to completely cover the nerve.
[0023] A further method of in situ formation of an implant
comprises the steps of
identifying an implant formation site; positioning a form having an implant
cavity at the site;
introducing media into the cavity to form an implant precursor; and permitting
the media to
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undergo a transformation from a first, relatively flowable state to a second,
relatively non
flowable state to form the implant; wherein a hydrophilic characteristic of
the media
cooperates with a hydrophobic characteristic of the cavity to facilitate a
rapid release of the
implant from the cavity following the transformation.
[0024] The implant may be a nerve cap for inhibiting
neuroma formation around
a severed nerve end, a wrap for protecting the nerve from inflammation and
scar formation,
or may be a nerve conduit for guiding growth of a nerve. Alternatively, the
implant may be a
tissue bulking implant or a vascular occlusion device. Alternatively, the
implant may be a
serve a tendon protector or facilitate tendon repair.
[0025] A method of in situ formation of a nerve cap may
comprise 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 cavity 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 barrier surrounding the
severed nerve end;
wherein a hydrophilic characteristic of the media cooperates with a
hydrophobic
characteristic of the cavity to facilitate a rapid release of the nerve cap
from the cavity
following the transformation.
[0026] The method may additionally comprise the step of
removing the form.
The identifying step may comprise identifying a surgically severed nerve.
[0027] The form may comprise a nerve guide and said
positioning step may
comprise positioning the nerve such that the nerve guide maintains the severed
end within the
cavity spaced apart from a sidevvall of the form. The severed end may be
positioned at least
about 1 mm away from the sidewall. The nerve may be covered circumferentially
with a
layer of at least 0.05 mm, preferably at least 0.5 mm of hydrogel, more
preferably at least 1
mm of hydrogel although larger hydrogel thicknesses are also acceptable given
sufficient
space.
[0028] The transformation may occur within about 30 seconds
of the introducing
step, preferably within about 10 seconds of the introducing step, preferably
within about 5
seconds of the introducing step. The method may additionally comprise the step
of blotting a
volume of axoplasm from the severed nerve prior to the introducing step.
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[0029]
The form may comprise a first configuration in which the cavity is
exposed, and a second configuration in which the cavity is covered. The method
may further
comprise the step of advancing the form from the first configuration to the
second
configuration following the introducing media step. In another embodiment, the
nerve is
placed inside the cavity of the first configuration, the form is moved to a
second
configuration in which the nerve is enclosed in the form and the media is
subsequently
delivered through a port in the form. The form may have a clamshell lid and
may have an
entrance region for holding the nerve and preventing premature outflow of the
PEG from the
form. The form may comprise an open cell foam, and the cavity may comprise an
interstitial
volume within the foam. The identifying a severed nerve step includes the step
of severing a
target nerve.
[0030]
The transformation may comprise a crosslinking or polymerization. The
transformation may produce a synthetic crosslinked hydrogel protective
barrier. For the
prevention of neuroma formation, the protective barrier may have an in vivo
persistence of at
least about two months or at least about three months, more preferably 6
months or more.
The transformation may cause the media to swell in volume as it equilibrates
with the
surrounding tissue within the range of from about 2% to about 60%
volumetrically or within
the range of from about 5% to about 30%, preferably 10 to 20%.
[0031]
The method may further comprise the step of positioning a form at a
treatment site before the positioning the severed end step. The method may
further comprise
the step of forming the form in situ before the positioning the severed end
step. The method
may further comprise delivering the media around the nerve in two successive
steps. The
severing a target nerve and positioning a fat
_______________________________________ la at a treatment site steps may be
accomplished
using a single instrument.
[0032]
The viscosity of the flowable media may be less than 70,000 cps,
preferably less than 10,000 cps, more preferably less than 500 cps. In one
embodiment the
viscosity of the flowable media is similar to water (-1 cps). The density of
the flowable
media may be less than 1.1 g/cm3. The form may comprise a biocompatible
silicone. The
form may contain an integral silicone post for seating the nerve. The form may
contain a
biodegradable polymer post for seating the nerve that remains in situ after
the hydrogel
forms. In one embodiment, the polymer post is lyophilized in place and in
another
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embodiment the polymer post is adhered to the form with a biocompatible
adhesive. In one
embodiment, the biodegradable polymer post remains in situ with the hydrogel
cap and
detaches from the silicone form when the silicone form is removed. In another
embodiment
the he entire form may comprise PEG, with or without an integral PEG post for
seating the
nerve. In the latter embodiment, the form will remain in situ and degrade
within the
timeframe for degradation of the hydrogel.
[0033] Applications for supporting nerve survival or
regeneration. In some
embodiments, such as in situations where a nerve is in continuity that needs
protection during
healing after trauma or compression, an in situ forming hydrogel with
different
characteristics than the hydrogel developed for preventing neuroma formation
is desired. In
situations in which it is desirable to protect a nerve from scar tissue and
aberrant outgrowth
of damaged nerves, such as after neurolysis of a nerve, separating a nerve
from the
surrounding tissue, a hydrogel with a shorter in vivo degradation time is
preferable. Instead
of the degradation profile described earlier with no significant degradation
at 3 months to
prevent ncuroma formation during the regenerative period, nerve protection can
be
accomplished in a shorter period of time: hydrogels with degradation profiles
within 3
months, preferably substantially degraded within 6 weeks, more preferably
substantially
degraded within 4 weeks are desirable. Furthermore, unlike hydrogels developed
to prevent
a neuroma, the equilibrium swelling of hydrogels for protection must be
sufficient to
accommodate an edema on the compressesd nerve, swelling volumetrically to
between 20
and 50%, preferably about 25 to 40% volumetrically. As these hydrogels are
delivered
around an intact nerve, the cavity in which they are formed has an entrance
and an exit,
permitting the placement of the nerve through the continuity in an atraumatic
fashion. In yet
further embodiments such after partial or complete nerve transection in which
nerve fibers
are not in continuity, disclosed herein is a dual component in situ forming
biomaterial
composition comprising a nerve regeneration pernissive component and a nerve
growth
inhibitory component. In some embodiments, the growth permissive component is
comprised
of an in situ forming thermosensitive hydrogel and the growth inhibitory
component is
comprised of an in situ forming chemically crosslinked hydrogel. In other
embodiments, the
growth permissive component is comprised of a physically crosslinked hydrogel
or viscous
solution. Examples of growth permissive matrices are provided in Pabari et al
2011. Recent
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advances in artificial nerve conduit design: Strategies for the delivery of
luminal fillers. J.
Controlled Release, 156, 2-10, incorporated herein for reference.The in vivo
persistence of
the growth inhibitory component around partially or completely transected
nerves is longer
than the hydrogel delivered around nerves in continuity since the growth
inhibitory
component must carry the load of the nerve until the regenerating proximal
nerve has
traversed into the distal stump and can start to bear some of the load/tension
on the nerve
again. Preferably the hydrogel 'wrap' around the nerve will carry the load and
prevent
immune infiltration until the nerve is regenerated and then degrade thereafter
in order to
transfer the natural strain on nerve to the regenerated native tissue.
In yet other
embodiments, the hydrogels can be adapted for use to support the regeneration
and regain of
function after implantation of allografts and conduits. In one embodiment, the
hydrogel is
delivered around allograft to improve the handleability and assist with
coaptation with the
transected nerve. For example, after selecting the cables from the autograft
to match the
length and diameter of the transected nerve, the cables can be bound together
by using the in
situ forming hydrogel as an artificial cpincurium. The hydrogel may also
improve the
trimmability and overall handling of the allograft tissue. In a similar
approach, the in situ
forming hydrogel may be delivered around a conduit (solid, porous, grid/strut)
to adhere the
conduit to the proximal and distal transected stump either with or without
sutures.
[0034]
In some embodiments, the nerve growth permissive component is
delivered first and the nerve growth inhibitory component delivered second.
The nerve
growth permissive component is of sufficient mechanical integrity to prevent
the growth
inhibitory component from entering the desired growth regenerative zone.
[0035]
In some embodiments, the nerve growth permissive components conform
to the nerve and facilitate nerve ingrowth into, through and across the growth
permissive
biomaterial into the distal stump. In some embodiments, the nerve growth
permissive
component is injected between the proximal and distal tips of the transected
nerve. In other
embodiments, the nerve growth permissive component is delivered around the
nerve
circumferentially at the site of the compression, crush, partial transection,
full transection
without gap or full transection with gap. In one embodiment, the growth
permissive
component is a flowable composition and and may spread into the zone between
the
damaged or transected fibers. In some embodiments, the growth permissive
biomaterial is a
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temporarily filler that prevents the growth inhibitory biomaterial from
accessing the damaged
nerves and inhibiting regeneration.
[0036] In some embodiments, the nerve growth inhibitory
components prevent
nerve growth into the material. Preferably, the nerve growth inhibitory
component covers
the growth permissive material and the distal and proximal nerve stumps for a
distance of at
least 2 mm in each direction, more preferably a distance of 10 mm. In this
manner, immune
cell infiltration into the regenerating nerve fibers is limited and the
typical contraction of the
regenerating nerve cable is prevented.
[0037] In some embodiments, the nerve growth inhibitory
components act as a
guide upon or along which nerve regeneration can occur. In yet another
embodiment, the
nerve growth inhibitory components is additionally utilized as rods deployed
within the
growth permissive region and running in the same plane as the nerves to help
guide the
regenerating axons.
[0038] In one embodiment, the growth permissive biomaterial
is placed
temporarily as a barrier to the growth inhibitory biomaterial and is cleared
after the in situ
formation of the growth inhibitory hydrogel occurs. The clearance of the
growth permissive
biomaterial may be within 1 hour to 3 months after delivery of the growth
inhibitory
material, preferably 1 hour to 2 months, more preferably 1 hour to 3 days. In
the latter case,
rapid dissolution of the growth permissive biomaterial removes any physical
barriers to
regenerating neurons in the case of close apposition between two nerves
(proximal and distal
tips), either through suture or suturless placement.
[0039] In some embodiments, the biomaterials components
comprise in situ
forming crosslinked gel, in situ formed thermosensitive hydrogel, in situ
formed
thermoreversible hydrogel, viscous solutions of synthetic or natural polymers,
microparticles,
nanoparticles, foam, hydrogel microparticle slurry or micelles.
[0040] In some embodiments, both the growth permissive and
growth inhibitory
components both contain polyethylene glycol (PEG).
[0041] In some embodiments, the PEG is a multi-arm PEG. In
some
embodiments, the PEG is a linear PEG. In some embodiments the growth
inhibitory PEG
comprises a multi-arm PEG and the growth regenerative PEG comprises a linear
PEG.
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[0042] In some embodiments, the PEG is comprised of a
urethane or amide
linkage.
[0043] In some embodiments, the PEG comprised of an ester
and/or amine
linkage.
[0044] In some embodiments, the PEG additionally comprises
a linear end-
capped PEG of 5,000 Daltons or less, such as PEG 3350.
[0045] In some embodiments, the crosslinking is performed
between a PEG-NHS
ester and a PEG-amine or a trilysine.
[0046] In some embodiments, the growth permissive gel
contains pores littni in
size or larger.
[0047] In some embodiments, the growth permissive gel
contains rods or
filaments.
[0048] In some embodiments, the growth permissive component
contains
chitosan, collagen, laminin, fibrin, fibronectin, HPMC, CMC or other natural
material. In
other embodiments, these components or regions of these components arc
covalently
conjugated to one another. In another embodiment, these components are blended
together.
[0049] 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%.
[0050] In some embodiments, the nerve growth permissive
components contains
between 0.001 and 20 % collagen, preferably between 3 and 6 wt%.
[0051] In some embodiments, the nerve growth permissive
component contains
fibronectin.
[0052] In some embodiments, the growth permissive component
contains poly-L-
ornithine.
[0053] In some embodiments, the growth permissive component
includes
laminin, preferably between 0 and 5 wt%, more preferably between 0 and 0.5%.
[0054] In some embodiments, the swelling of the growth
permissive component
is less than 20%, preferably between 0 and 20%. In some embodiments, the
viscosity of the
growth permissive component is over 5,000 Cps, preferably between 7,500 and
150,000 Cps,
more preferably between 10,000 and 20,000. In some embodiments, the Young's
modulus of
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the growth permissive component is less than 2 kPa, preferably less than 1
kPa, more
preferably less than 600 Pa. In one embodiment, the growth permissive
component is a soft
gel with a Young's modulus of between 100 and 300 Pa. In some embodiments, the
osmolarity of the growth permissive gel is between 275 and 320 mOsm,
preferably between
280 and 320 mOsm.
[0055] In some embodiments, the swelling of the growth
inhibitory component is
less than 50%, preferably between 0 and 30%, more preferably between 5 and
20%. In yet
another embodiment the swelling of the growth permissive component is the same
as the
growth inhibitory component.
[0056] In some embodiments, the swelling of the growth
permissive component
is less than or equal to the swelling of the growth inhibitory component.
[0057] In some embodiments, the compressive strength of the
growth inhibitory
component is greater than 10 kPa, preferably > 30 kPa.
[0058] In some embodiments, the growth permissive and
growth inhibitory
component are different colors.
[0059] In some embodiments, the growth permissive region
comprises agents that
support nerve survival, outgrowth, and regeneration.
[0060] In some embodiments, the growth permissive region
permits infiltration of
Schwann or other supporting cells. In some embodiments, the growth permissive
region may
be loaded with cells prior to placement in situ. The growth inhibitory region
may prevent the
migration of these cells away from the implantation site. In some embodiments,
the system
contains supporting cells such as glial cells, including Schwann cells,
oligodendrocytes, or
progenitor cells such as stems cells.
[0061] 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.
[0062] In some embodiments the growth permissive region can
be delivered with
a syringe accurately with preferably an 18 gauge or higher needle in volumes
of 10
microliters to 5 milliliters. Smaller volumes of less than 100 microliters can
be delivered to
smaller nerves or partially transected nerves and larger volumes can be
delivered to partial or
complete lesions in larger caliber nerves.
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[0063] In some embodiments, the system is delivered to
peripheral nerves, the
ventral or dorsal roots, the sympathetic ganglia, the cauda equina, the spinal
cord, or the
brain. In yet other embodiments, the system may be delivered to or around a
peripheral
neurovascular bundle or a tendon. In some embodiments, the system is delivered
to nerves
without utilizing the form, although using the form is preferable.
[0064] In some embodiments, the growth permissive and
growth inhibitory region
include a P2XR receptor antagonist.
[0065] In some embodiments, the P2XR receptor antagonist is
a P2X7 receptor
antagonist, including Brilliant Blue FCF (BB FCF) or Brilliant Blue G (BBG).
In some
embodiments, the P2XR antagonist is a P2X3 receptor antagonist, such as AF-219
or
gefapixant. In some embodiments, the concentration of the P2XR antagonist
concentration is
between 0.001 and 0.55 % the hydrogel.
[00661 In some embodiments, the hydrogel is delivered
circumferentially around
a nerve. In yet other embodiments, the hydrogel is delivered only partially on
the surface of
the nerve, for example around between 25% and 85%, preferably between 50 and
80% of the
nerve circumference. In yet other embodiments, multiple Wraps are delivered in
sequence to
cover longer lengths of nerve. For example, in some embodiments between one
and four
hydrogel wraps are delivered around the nerve in situations such as after
ulnar nerve
transposition when a long length of nerve has been released and moved. In yet
other
embodiments, if a nerve needs protection but a vascular or nerve bifurcates in
the region
where the wrap is desirable, the Wrap Form can be cut with surgical scissors
intraoperatively
to permit space for the bifurcating nerve to exit the Wrap Form outside of the
available exit
region of the Wrap Form.
[0067] In some embodiments, the length of the Cap Form or
Wrap Form is
between 10 mm and 60 mm, depending on the length of the nerve. For smaller
nerves less
than 4mm in diameter (Small Nerve Set), preferably the Cap Form or Wrap Form
cavities are
between 11 mm and 40 mm in length, preferably 15 mm to 20 mm in length. For
the Cap
Form, preferably at least 5 mm of nerve length needs to be covered with
hydrogel to secure
the nerve in the hydrogel, more preferably at least 10 mm of nerve length. As
a result, in
order to ensure preferably 1 mm of hydrogel surrounds the tip of the nerve,
the Cap Form
cavity length needs to be approximately 11 mm long, preferably 15 mm long for
small nerve
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coverage.
For the Cap Form and Wrap Form, longer cavities are preferable for
larger
nerves given the longer lengths of these nerves that are exposed, and so
cavity lengths on the
order of 15 mm to 60 mm, more preferably 20 mm to 50 mm in cavity length.
[0068]
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 one or more forms ¨ one foim for
receiving the
growth inhibitory hydrogel, the other for the growth permissive hydrogel. The
kit may
include a bioresorbable for
_________________________________________________________ 11 for receipt of
the growth permissive biomaterial followed by a
temporarily nondegradable form for the subsequent receipt of the growth
inhibitory hydrogel.
In yet another embodiment, disclosed is a kit including one in situ forming
hydrogel and one
low viscosity gel solution (viscosity between 5,000 and 30,000 Cps with a
modulus of less
than 1 kPa). The kit includes a dual applicator system clearly marked with
indicics as a
growth inhibitory applicator and a syringe/applicator system clearly marked as
a growth
permissive applicator. The kit may also include one or more forms ¨ one form
for receiving
the growth permissive gel solution that is biodegradable and one form for
receiving the
growth inhibitory hydrogel that is nondegradable. In one embodiment, the
biodegradable
form for receiving the growth permissive gel solution is a biodegradable
adherent sheet that
adheres to the nerve when wetted and is resorbed within several days after
application. In
another embodiment the biodegradable form degrades between one and two months
and is
positively charged to facilitate neurite extension.
[0069]
In some embodiments, disclosed herein is a method of delivering dual
gels, including an in situ forming hydrogel and a low viscosity gel to treat
conditions
involving nerves. The nerves can need repair, such as, for example, end-to-end
anastomoscs,
coaptation, repair with allograft or autograft or conduit or wrap, or gap
repair. The dual gel
system can be delivered to and around the site of anastomoses between proximal
nerve and
distal nerve stump, proximal nerve and allograft nerve or cable(s), or as a
connector-assisted
coaptation where the connector is provided by the in situ forming hydrogel.
The dual gel
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system can be delivered as an adjunct to suture repair, or preferably, without
the need for
sutures of the nerve tissue.
[0070] In some embodiments, a growth permissive region is
delivered between
the proximal and distal nerve stumps, between end-to-end anastomoscs sites,
between
proximal stump and allograft/autograft and between the allograft/autograft and
the distal
stump.
[0071] In some embodiments, a growth permissive region is
injected between the
proximal and graft and/or graft and distal stumps, with or without the
assistance of a form.
The growth permissive solution may comprise a volume of approximately 5 ul to
3 ml, more
preferably 10 ul to 1 cc. Sufficient volume must be delivered to fill the gap
between the
nerve and also cover 1 mm or more of length of the proximal and distal nerves,
preferably 2
to 5 mm of length of both the proximal and distal nerve.
[0072] In some embodiments, the growth permissive region is
delivered inside a
conduit or wrap. In some embodiments, the growth permissive region is
delivered inside a
lyophilized PEG conduit or wrap. The lyophilized PEG may comprise a
crosslinked multi-
arm PEG, a multi-arm PEG, a linear PEG solution or combination thereof that
provides
enough structural support during the procedure as a temporary form to prevent
the off-target
spread of the growth permissive material. Post-procedurally, the form is
cleared from the site
within 3 to 5 days, preferably less than 1 to 2 days.
[0073] 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. In yet
another embodiment, the growth permissive region is delivered into a porous
form permitting
adherence of the growth permissive gel to both the nerve and the form. The
growth
permissive region intercalates in the pores of the bioresorbable form to
provide good
adherence to the form.
[0074] In some embodiments, a growth inhibitory region is
delivered after the
growth permissive region. In some embodiment a growth inhibitory region is
delivered
around the growth permissive region, completely covering the growth permissive
material. In
another embodiment, a layer of the growth inhibitory region is delivered into
the form first
followed by placement of the nerves, delivery of the growth permissive region,
and
subsequent delivery of the final layer of growth inhibitory region.
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[0075] In some embodiments, a growth inhibitory region
covers the proximal and
distal nerves and growth permissive region. In some embodiments, the growth
inhibitory
region extends down the proximal and distal nerves from the anastomoses sight
at least 2 cm
in each direction, preferably 1 cm in each direction, preferably providing 5
mm coverage of
each nerve stump.
[0076] 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.
[0077] In some embodiments, a growth inhibitory region
covers the anastomoses
junction or site of direct nerve coaptation.
[0078] In some embodiments, a growth inhibitory region is
delivered to cover the
junction(s) between the nerve and the conduit or wrap.
[0079] In some embodiments, a growth inhibitory region
covers a healthy,
compressed, or contused nerve.
[0080] 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
facilitate 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 and adhere to the two nerve ends. In some embodiments, the
adhesion of the
growth inhibitory hydrogel to the nerves provides sufficient strength to
maintain the nerves
in proximity and support nerve regeneration without the need for sutures. In
some
embodiments, the nerves can be positioned within the biomaterials with an
angle or a flex in
the nerves to take the load off of the nerve ends and improve nerve repair.
[0081] 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. In some
embodiments,
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disclosed herein, is a method of supporting regeneration between a first nerve
end and a
second nerve end, comprising: placing the first nerve end and the second nerve
end in a
bioresorbable nerve cavity and introducing the growth permissive media into
the cavity and
into contact with the first nerve end and the second nerve end to form a
junction. The growth
permissive media/bioresorbable nerve cavity (e.g. wrap or conduit) can then be
handled with
forceps as a unit and then transferred into a second form cavity into which
the growth
inhibitory media is delivered.
[0082]
In some embodiments, disclosed herein, a dual component form is used to
complete the growth permissive and growth inhibitory biomaterial delivery
without the need
to handle or otherwise move the nerve unnecessarily. For example, the form
contains an
inner bioresorbable form (e.g. chitosan, HPMC, CMC film) that rests inside the
outer
nonresorbable form (e.g. silicone). The growth permissive material is
delivered directly into
the bioresorbable form and the growth inhibitory biomaterial is delivered in
between the
bioresorbable from and the non-resorbable fat
_______________________________________ la. In one embodiment, the
bioresorbable form
is a thin sheet that, when wet, becomes sticky and adhesive. The hydration of
the
biocompatible biodegradable sheet, which may occur through interaction with
the naturally
wet nerve and/or surrounding tissue fluids or through direct application of a
aqueous
solution, may either crosslink in situ to adhere to the tissue through
physical intercalation
with the tissue surface. After hydrogel formation, the non-resorbable form is
removed.
[0083]
Applications for preventing neuronal regeneration and neuroma
formation.
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.
[0084]
Applications for nerve protection and regeneration. 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
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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.
[0085] In some embodiments, disclosed herein is a Cap
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. In some embodiments,
disclosed herein
is a composition for an in situ forming growth inhibitory hydrogel with; a
compressive
strength greater than 20 kDa for over 6 months, in vivo persistence for at
least 6 months
comprising less than 15% mass loss, and/or in vitro swelling of less than 20%
for over 6
months. In a preferred embodiment, the growth inhibitory hydrogel for
preventing neuroma
formation has a compressive strength of greater than 30 kDa, in vivo
persistence for over 4
months comprising less than 15% mass loss, and/or swelling of less than 20%
for over 4
months. In the preferred embodiment, the growth inhibitory hydrogel swells
outwards
radially as it degrades, preventing compression of the nerve. Thus the
swelling of the
hydrogel occurs in concert with the loss of tensile strength of the hydrogel,
preventing
compression of the encapsulated nerve and of adjacent structures. In some
embodiments,
disclosed herein is a Wrap composition for an in situ forming growth
inhibitory hydrogel
with a compressive strength of greater than 10 k Da for over 2 weeks, with in
vivo
persistence of at least 4 weeks, and/or swelling of less than 60% over 3
months. In this
embodiment, swelling of at least 10% is desirable to accommodate any swelling
of the nerve
after trauma. In a preferred embodiment, the hydrogel swells at least 20%
after equilibrium,
more preferably greater than 30% but at no time point swells more than 60%
prior to
clearance. In this embodiment, the hydrogel swells sufficiently to permit any
nerve swelling
and then remains on the nerve to prevent immune cell infiltration and
secondarily, as
degradation and loss of tensile strength occurs, additional swelling in the
outward radial
direction permits gliding of the nerve within the hydrogel. In yet another
embodiment,
Wraps with an even shorter degradation time are desired to prevent nerve
compression from
occurring in the first week post-operatively. These Wrap hydrogels remain in
situ for at least
2 weeks in vivo but are rapidly cleared from the site and are substantially
cleared from the
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site within 4 weeks. These more rapidly degrading Wrap hydrogels permit
gliding during the
degradation phase of the hydrogel during which the hydrogel forms a viscous
solution around
the nerve through which the nerve can freely move.
[0086] In some embodiments, the composition includes one or
more of:
poly(ethylene glycol) succinimidyl carbonate, a P2XR receptor antagonist, such
as a P2X7
receptor antagonist.
[0087] In some embodiments, a P2X7 receptor antagonist is
Brilliant Blue FCF
(BB FCF) or Brilliant Blue G (BBG).
[0088] In some embodiments, a method of in situ
faimation 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 faun a protective barrier around the nerve.
[0089] In some embodiments, the nerve is healthy,
compressed, contused,
partially or completed transected. In some embodiments the nerve is transected
during the
procedure and in yet other embodiments the damage to the nerve, such as
formation of a
neuroma, has occurred 3 months to about 10 years previously and is excised
prior to
application of the hydrogel. In other embodiments, the nerve is repaired
within minutes to
three months after an injury, typically within a day to 14 days after injury.
In yet other
embodiments, such after trauma when the extent of nerve damage or capacity for
the nerve to
regenerate is unclear and the surgeon prefers to evaluate if the nerve
function is able to return
without surgical intervention, the surgeon may elect not to perform the
procedure until two
months to 6 months after the initial nerve trauma.
[0090] In some embodiments, the nerve is first repaired
through direct
anastomoses, repair with allograft or autograft, or repair with a conduit
prior to delivery of
the growth permissive and growth inhibitory biomaterials.
[0091] In some embodiments, the method includes removing
the form.
[0092] 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. In one embodiment, the hydrogel is formed around
a nerve
placed in a cylindrical tube which is subsequently removed after which the
nerve-hydrogel is
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rotated and placed in a second cylindrical tube and a second application of
the growth
inhibitory hydrogel is applied. By placing the successively larger diameter
tubes
appropriately, the nerve can be delivered in the center of the formed
hydrogel. This same
approach may be utilized for applications of growth permissive or growth
inhibitory gels.
[0093] In some embodiments, a method of in situ formation
of a nerve wrap
includes where the nerve is covered circumferentially with a biomaterial
thickness around the
nerve of at least 0.1 mm of a protective bather, preferably 0.5 mm to 10 mm,
more
preferably 0.5 to 5 mm thickness.
[0094] In some embodiments of delivering the in situ
forming hydrogel into the
cap or wrap form, the transformation occurs within about 10 seconds of the
introducing step,
preferably less than 5 seconds of the introducing step. In some embodiments of
delivering
the hydrogel into the wrap/cap form, the transformation preferably occurs
within 15 seconds
of the introducing step, or longer as needed for wrapping longer sections of
nerve. In order
to accommodate different gel times, kits will be designated for small nerves
(less than 4 mm
nerves) and large nerves (greater than 4 mm nerves). Kits may also contain
different needle
gauges to accommodate the delivery of different volumes and gel times to the
nerve
cap/wraps. For example, a small nerve kit may contain a 22 gauge needle and a
large nerve
kit may contain a 20 or 18 gauge needle.
[0095] In some embodiments, the transformation comprises a
crosslinking or
polymerizing. In some embodiments, the transformation comprises gelation after
a
temperature change from room temperature to body temperature.
[0096] In some embodiments, the transformation produces a
synthetic crosslinked
hydrogel protective barrier. In some embodiments, the transformation produces
a natural
crosslinked hydrogel protective barrier, such as can be obtained with fibrin
sealant (Tisseel,
Baxter).
[0097] In some embodiments, the protective barrier has an
in vivo persistence of
at least about two months.
[0098] In some embodiments, the protective barrier has an
in vivo persistence of
at least about three months.
[0099] In some embodiment, the protective barrier has an in
vivo persistence of at
least about 6 months.
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[0100] In some embodiments, the protective barrier does not
degrade in vivo.
[0101] In some embodiments, the transformation causes the
media to swell in
volume within the range of from about 2% to about 60%.
[0102] In some embodiments, the transformation causes the
media to swell in
volume within the range of from about 50% to 40%.
[0103] 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.
[0104] In some embodiments, the severing a target nerve
step and the positioning
a foul' at a treatment site step are accomplished by a single instrument.
[0105] In some embodiments, the viscosity of the flowable
hydrogel precursor
media is less than 70,000 cps, preferably less than 10,000 cps.
[0106] In some embodiments, the density of the flowable
media is less than about
1.2 g/cm3, or approximately that of water at 1 g/cm3.
[0107] In some embodiments, the form is comprised of
biocompatible medical
grade silicone.
[0108] In some embodiments, the form contains integral
posts for seating longer
lengths of the nerve.
[0109] In some embodiments, the cap or wrap form is
comprised of PEG.
[0110] In some embodiments, the form has a clamshell lid
and a separate port or
entrance for delivery the hydrogel
[0111] In some embodiments, the growth permissive and
growth inhibitory region
contain a P2XR receptor antagonist.
[0112] In some embodiments, the P2XR receptor antagonist is
a P2X7 receptor
antagonist, including Brilliant Blue FCF or Brilliant Blue G (BBG). In some
embodiments,
the concentration of the P2XR antagonist is between 0.001 to 0.55 % in the
hydrogel.
[0113] In other embodiments, the growth permissive and/or
growth inhibitory
region contain the antioxidant methylene blue. In other embodiments, the
growth permissive
and/or growth inhibitory contain FD&C No.1 alone or in combination with FD&C
No. 5 to
create blue, turquoise/teal, and varying shades of green hydrogels. In other
embodiments, the
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growth permissive and/or growth inhibitory region contain linear end-capped
PEG (3.35kDa,
or 5kDa or blends thereof) at solid contents of 1% to 50 wt%, more preferably
10-20 wt%.
[0114] In some embodiments, disclosed herein are in situ
forming hydrogel(s) as
a cap. In some embodiments, the nerve cap is not pre-formed.
[0115] 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,
these hydrogels are delivered without a pre-formed wrap or conduit.
[0116] In some embodiments, the nerve is prevented from
forming a neuroma by
delivering an in situ forming 'bridge to nowhere' conduit in the form of an in
situ formed
hydrogel with open lumens that permit the regeneration of nerves along and on
the hydrogel
until their ability to regenerate has aborted. In some embodiments, the
channel inside the
hydrogel is 1 cm or more in length, preferably 2 cm or more in length. In some
embodiments,
the channel is comprised of a rapidly resorbing biomaterial, such as low
molecular weight
PEG, collagen, hyaluronic acid or hydroxymethylcellulose or combinations
thereof. The
hydrogel is formed around the nerve and the rapidly absorbing biomaterial
channel. The
biomaterial cylinder maintains its three dimensional structure long enough to
provide a
scaffold over which the growth inhibitory cylinder can be formed. To achieve
this
configuration, the nerve is placed in a bioresorbable Wrap form and the form
filled with the
rapidly resorbing material. The Wrap form is wrapped circumferentially around
the nerve
and rapidly resorbing biomaterial, after which the growth inhibitory hydrogel
is delivered
around the nerve and wrap in a second, larger Wrap form. The larger Wrap form
may be
biodegradable or a removable nondegradable form.
[0117] 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 and
coverage of the tip of the nerve, sufficient in vivo persistence beyond the
time during which
nerves can regenerate (3 months or more), minimal swelling to prevent nerve
compression or
loss of adherence between the nerve and the hydrogel, sufficient tensile
strength to prevent
nerve outgrowth into the hydrogel and delivering them into a removable form.
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[0118] There is provided in accordance with a further
aspect of the invention, a
kit for in situ formation of an implant for guiding nerve regeneration between
two nerve
ends. The kit includes first components for producing a first, growth
permissive hydrogel;
second components for producing a second, growth inhibitory hydrogel; at least
one form
having a concavity; a first applicator for delivering the growth permissive
hydrogel into the
cavity; and a second applicator for delivering the growth inhibitory hydrogel
into the cavity.
[0119] The first components may include a powdered growth
permissive
hydrogel precursor, a reconstitution solution, and an accelerator solution.
The powdered
growth permissive hydrogel precursor may contain an agent to stimulate nerve
regeneration.
The second components may include a powdered growth inhibiting hydrogel
precursor, a
reconstitution solution, and an accelerator solution. The first components
include a
powdered growth permissive gel precursor and a reconstitution solution. The
second
components may include a pre filled syringe containing the growth permissive
gel.
[0120] The kit may further include a first form having a
first configuration for
receiving the growth inhibitory hydrogcl, and a second form having a second,
different
configuration for receiving the growth permissive hydrogel. The concavity may
have a
surface having a hydrophobic characteristic. At least one of the growth
permissive and
growth inhibitory hydrogel may have a hydrophilic characteristic.
[0121] The second form may comprise a biocompatible
biodegradable sheet,
which may have a thickness of less than about 60 microns or less than about 40
microns.
[0122] There is also provided a kit for in situ formation
of a hydrogel nerve cap.
The kit may include a dual applicator system; a vial with powdered hydrogel
precursor; a
reconstitution solution; an accelerator solution; and at least one nerve cap
form.
[0123] There is also provided a formed in place nerve
regeneration construct.
The construct comprises 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.
[0124] There is also provided a form for creating an in
situ nerve cap to inhibit
neuroma formation. The form comprises a concave wall defining a cavity, the
wall having a
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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; wherein the concave
wall has a
hydrophobic characteristic.
[0125] The form is configured to receive a second
biodegradable form containing
the nerve ends to be repaired. The form may facilitate nerve regrowth across a
nerve to nerve
junction. The form may facilitate the formation of a hydrogel to prevent nerve
compression
and scar tissue formation around the nerve.
[0126] There is also provided a form for creating an in
situ nerve conduit for
facilitating regrowth across 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
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.
[0127] There is also provided a composition for an in situ
forming growth
inhibitory hydrogel. The hydrogel exhibits 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 swelling of less than 30% for over 3 months. Degradation of the hydrogel
may result in
outward radial swelling of the hydrogel.
[0128] The composition may comprise one or more of a
poly(ethylene glycol)
with a biodegradable amide or urethane bond; a poly(ethylene glycol)
succinimicly1
carbonate; a P2XR receptor antagonist; a P2X7 receptor antagonist. The P2X7
receptor
antagonist may be Brilliant Blue FCF (BB FCF) or Brilliant Blue G (BBG).
[0129] The composition may also contain one or more of a
poly(ethylene glycol)
with a biodegradable ester bond; a poly(ethylene glycol) succinimidyl adipate;
a multi-arm
PEG with arm lengths of between 1 and 10 kDa; and at least one multi-arm PEG
with an arm
length of 5 kDa.
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[0130] There is also provided a composition for an in situ
forming growth
inhibitory hydrogel. The composition comprises a hydrogel which exhibits a
compressive
strength of greater than about 10 kPa; In vivo persistence for at least about
2 weeks; and
initial swelling of greater than about 20% but less than about 100%
[0131] Degradation of the hydrogel may result in outward
radial swelling of the
hydrogel with volumetric swelling less than about 160%. Degradation of the
hydrogel may
occur in less than about 16 weeks. The growth inhibitory hydrogel may be
configured to
encapsulate a growth permissive gel with a Young's modulus of less than about
10 kPa and a
viscosity greater than about 5,000 cP.
[0132] The composition may include one or more of a
poly(ethylene glycol) with
a biodegradable ester bond; a poly(ethylene glycol) succinimidyl adipate; a
multi-arm PEG
with arm lengths of between 1 and 10 kDa; or at least one multi-arm PEG with
an arm length
of 5 kDa.
[0133] There is also provided an absorbable, formed in situ
electrode anchor,
comprising a volume of hydrogel polymerized in situ around an electrode and
configured to
maintain the electrode in electrical communication with a nerve. The hydrogel
may be
electrically conductive.
[0134] There is also provided a formed in situ implant,
comprising a volume of
hydrogel transformed in contact with a form from a relatively flowable state
to a relatively
non flowable state and removed from the form by a pull force of no more than
about 5 N,
wherein removal was facilitated by a hydrophilic characteristic of the
hydrogel and a
hydrophobic characteristic of the form. The formed in situ implant may
comprise a nerve
cap, a nerve conduit for guiding regeneration of a nerve, or a nerve wrap for
preventing scar
formation and tether of a nerve.
[0135] 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
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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 he delivered without advanced surgical training. In other
situations, there
remains a need for a technology that prevents nerve outgrowth into the
surrounding tissue
and directs 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. By delivering the hydrogel
circumferentially
around the nerve, the tension can be distributed circumferentially and over a
distance over
the nerve to distribute the tension evenly across the nerve surface. In doing
so, the tension is
transferred to the hydrogel, not born at the focal points of the three or four
sutures at the
anastomoses site. As is done within conduits, by creating slack in the nerves
(placing with a
curve) prior to the delivery of the growth inhibitory hydrogel, additional
detensioning can be
achieved so that the tension at the anastomoses site is minimal. Lastly, in
some aspects, a
versatile 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.
[0136] There is further provided a kit for in situ
formation of an implant for
guiding nerve regeneration between two nerve stumps. The kit includes first
components for
producing a first, growth permissive hydrogel; second components for producing
a second,
growth inhibitory hydrogel; at least one form having a concavity; a first
applicator for
delivering the growth permissive hydrogel into the cavity; and a second
applicator for
delivering the growth inhibitory hydrogel into the cavity.
[0137] The first components may include a powdered growth
permissive
hydrogel precursor, a reconstitution solution, and an accelerator solution.
The second
components may include a powdered growth inhibiting hydrogel precursor, a
reconstitution
solution, and an accelerator solution. A first form having a first
configuration for receiving
the growth inhibitory hydrogel, and a second form having a second, different
configuration
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for receiving the growth permissive hydrogel may also be provided. The
concavity may have
a surface having a hydrophobic characteristic. At least the growth permissive
hydrogel may
have a hydrophilic characteristic.
[0138] A kit for in situ formation of a hydrogel nerve cap
is also provided. The
kit comprises a dual applicator system; a vial with powdered hydrogel
precursor; a
reconstitution solution; an accelerator solution; and at least one nerve cap
form.
[0139] 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 to directly contact the
two nerve ends.
[0140] There is also provided 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. At least the surface of
the concave wall
may have a hydrophobic characteristic.
[0141] There is also provided a form for creating an in
situ nerve conduit for
facilitating regrowth across 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.
[0142] There is also provided a composition for an in situ
forming growth
inhibitory hydrogel, having a compressive strength greater than 10 kDa for
over 3 months; an
in vivo persistence for at least 3 months comprising less than 15% mass loss;
and swelling of
less than 30% for over 3 months. The composition may comprise poly(ethylene
glycol)
succinimidyl carbonate. The hydrogel may contain contains a P2XR receptor
antagonist and
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/ or a P2X7 receptor antagonist. The P2X7 receptor antagonist may be Brilliant
Blue FCF
(BB FCF) or Brilliant Blue G (BBG).
[0143] There is also provided an absorbable, formed in situ
electrode anchor,
comprising a volume of hydrogel polymerized in situ around an electrode and
configured to
maintain the electrode in electrical communication with a nerve. The hydrogel
may be
electrically conductive.
[0144] There is also provided a formed in situ implant,
comprising a volume of
hydrogel transformed within a form cavity from a relatively flowable state to
a relatively non
flowable state and removed from the cavity by a pull force of no more than
about 5 N,
wherein removal was facilitated by a hydrophilic characteristic of the
hydrogel and a
hydrophobic characteristic of the cavity. The implant may be a nerve cap or a
nerve conduit
for guiding regeneration of a nerve.
[0145] 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 prevents nerve outgrowth into the
surrounding tissue
and directs 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. By delivering the hydrogel
circumferentially
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around the nerve, the tension can be distributed circumferentially and over a
distance over
the nerve to distribute the tension evenly across the nerve surface. In doing
so. the tension is
transferred to the hydrogel, not born at the focal points of the three or four
sutures at the
anastomoses site. As is done within conduits, by creating slack in the nerves
(placing with a
curve) prior to the delivery of the growth inhibitory hydrogel, additional
detensioning can be
achieved so that the tension at the anastomoses site is minimal. Lastly, in
some aspects, a
versatile 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0146] Figure 1A is a perspective schematic view of a nerve
end positioned
within a form cavity. An entrance region that permits the nerve to be guided
into the form.
The length of the form provides a sufficient surface area over which the
hydrogel to form and
adhere to the nerve tissue.
[0147] Figure 1B is a side elevational cross section
through the construct of
Figure 1A.
[0148] Figure 1C is a top view of the construct of Figure
1A.
[0149] Figure 1D is an end view of the construct of Figure
1A.
[0150] Figure lE is a cross-sectional view taken along the
line lE ¨ lE in Figure
1B .
[0151] Figure IF is a top view of another embodiment of the
construct of Figure
1A.
[0152] Figure 2 is a schematic illustration of a formed
barrier formed in
accordance with some embodiments of the present invention.
[0153] Figure 3A is a perspective view of a form, having a
stabilizing feature.
[0154] Figure 3B is a perspective view of a form for
creating a wrap around a
nerve or a growth permissive region in a gap between nerve ends. Thus,
depending on the
application, the wrap form may contain a growth permissive or growth
inhibitory hydrogel.
[0155] Figure 4A shows electrical stimulation of nerve ends
across a growth
permissive media in an open surgical procedure..
[0156] Figure 4B shows anchoring of an electrode adjacent a
nerve to deliver pain
management stimulation in a percutaneous procedure.
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[0157] Figures 5A ¨ 5M illustrate a series of steps for
creating a growth
permissive hydrogel junction encapsulated by a growth inhibitory hydrogel
barrier.
[0158] Figure 6 is a perspective view of a clamshell form.
[0159] Figures 7-10C illustrate embodiments of tools for
transecting nerves
and/or creating a hydrogel junction.
[0160] Figures 11A-11E illustrate views of a form and
methods of use.
[0161] Figure 12 is a perspective view of a form with a
stabilizing rod.
[0162] Figures 13A-13D are perspective views of a cap form
with a partial cover
and an internal rod to support the nerve.
[0163] Figures 14A-14C are perspective views of a cap form
with a partial
clamshell.
[0164] Figures 15A-15C are perspective views of a tearable
nerve cap form.
[0165] Figures 16A-16E illustrate perspective views and
photographs of in situ
formed hydrogels (growth inhibitory and growth permissive) around nerves both
in cap and
wrap form.
[0166] Figures 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.
[0167] Figures 18A-18B illustrate a mixing element design
to improve the
consistency of the hydrogel when delivering low volumes of precursor solution.
[0168] Figures 18 C and 18 D illustrate distal mixing tip
configurations.
[0169] Figure 18 E is a cross-section taken along the lines
18E-18E of Figure 18
D.
[0170] Figure 19 schematically illustrates a dual chamber
syringe system.
[0171] Figure 20 schematically illustrates a dual chamber
syringe and mixer
system.
DETAILED DESCRIPTION
[0172] 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
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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)
approach. The nerve end or stump may be formed by transection (cutting),
traumatic injury,
or ablation through any of a variety of modalities including radiofrequency
(RF),
cryotherapy, ultrasound, chemical, thermal, microwave or others known in the
art.
[0173] 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 supporting neuroma formation).
Hydrogels are
transparent for visualization, low-swelling, compliant, and are delivered into
a form to
generate hydrogel caps with volumes 0.05 to 10 ml, preferably 0.1 to 5 ml,
more preferably
0.1 to 2.8 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.
[0174] Target nerves can vary widely in diameter with a
spherical or non-
spherical outside configuration, and the cut or severance angle and precision
can also vary.
While it can be appreciated that all nerves would benefit from this hydrogel
technology,
nerves approximately 0.2 mm to 15 mm, preferably 1 mm to 5 mm, more preferably
1 mm to
2 mm may be treated with this approach. 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. The form prevents the media
from contacting
the surgical site and creates a smooth shape around the nerve, permitting
gliding of the nerve
and the form in the surrounding tissue.
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[0175] Referring to Figures lA 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.
[0176] 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
Figure 1D.
[0177] Figure lE is a cross-sectional view taken along the
line lE ¨ lE in Figure
1B. The width of the window 20 in a circumferential direction is less than the
inside
diameter of the cavity. This results in a reentrant wall segment 29 on either
side of the
window 20, having an edge that lies on a plane 33 that is parallel to a
central vertical (not
shown) through the center of the window 20. The edge plane 33 is parallel to
and spaced
apart from a tangent 31 to the inside surface of the sidewall 16. The distance
between the
edge plane 33 and the tangent 31 is within the range of from about 2% to about
30%,
generally within the range of from about 5% and about 20% of the inside
diameter of the
cavity.
[0178] Referring to Figures 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.
[0179] In general, a sufficient volume of hydrogel
precursor will be introduced
into the cavity to produce a continuous protective coating around the nerve
end and at least
about 1 mm or 2 mm or 5 mm or more of the nerve leading up to the end. In some
embodiments, the axial length of the cavity will be as much as 10 mm or 12 mm
up to about
2 cm or less, and the ID width may be less than about 2.5 mm or 2 mm or less.
The volume
of flowable precursor is generally at least about 100 microliters or 200
microliters or more
but no more than about 500 microliters for small nerves (e.g., nerves up to
about a 4 mm
diameter). Form cavities intended for larger nerves (e.g., nerves with
diameters from about 4
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mm to about 10 mm) may receive at least about 1 ml or 1.5 ml but generally
less than about 4
ml or 3 ml of flowable precursor. As further discussed elsewhere herein, the
precursor may
be introduced into the cavity as two or three or more separate layers that
adhere together to
form the final cap. For example, a first, base layer may be introduced into
the cavity either
before or after positioning the nerve in the cavity. During or following
transformation of the
first layer, a second layer may be introduced, to bond to the first layer and
encapsulate the
nerve and form the protective nerve cap. The first layer preferably will
contact at least the
lower surface of the nerve, and may partially enclose the nerve, with at least
an upper portion
of the surface of the nerve exposed. Within about 5 minutes, preferably within
about 1
minute or within about 30 seconds from completion of delivery of the first
layer, the second,
top layer is applied in contact with the exposed portion of the nerve and the
exposed surface
of the top layer to encase the nerve and form the final cap construct. In
another preferred
embodiment, the nerve is completely or partially embedded in the precursor
solution until it
forms a gel. The mixer/blunt needle on the tip of the applicator is removed
and a new
mixer/blunt needle tip is attached so that additional material can be
delivered in a second
layer to cover the nerve.
[0180] Referring to Figure 1F, the inside surface of the
cavity may be provided
with one or more smface structures 35 for facilitating mixing and / or filling
of the cavity. In
the illustrated embodiment, the surface structure 35 comprises a flow guide in
the form of a
radially inwardly extending projection for facilitating flow, such as a
helical thread. The
pitch and depth of the thread may be optimized with the viscosity of the
flowable media, to
facilitate filling and complete circumferential coverage of the nerve root as
the flowable
hydrogel precursor media is injected into the cavity.
[0181] 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 Figure
2.
[0182] 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 Figure 3A.
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
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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.
[0183] 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
cros slinking 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.
[0184] In another implementation, an in situ forming
thermosensitive hydrogel
(such as a PEG-PCL-PEG triblock copolymer) is selected as a growth permissive
hydrogel.
The hydrogel formed in the junction is soft enough that nerves can grow
through the
hydrogel without hindrance but viscous enough to prevent the egress of the
inhibitory
hydrogal into the junction between the two nerves. In another implementation,
the growth
permissive biomaterial provides a temporary barrier to the egress of the
growth inhibitory
hydrogel, such as through the use of a viscous hyaluronic acid, pluronic, PEG,
fibrin or
collagen solution.
[0185] In another implementation, the injectable growth
permissive biomaterial is
delivered into a bioabsorbable wrap form, such a wrap comprised of PEG-,
pullulan-,
pullulan-collagen, or HPMC-based dried sheet. These films can be cast into a
cap or wrap
shape and dried by solvent casting with organic or aqeous solvents and the
films dried
through evaporation at room temperature or in a lyophilizer. For example,
these material can
be formed into cap-like shapes similar to the manufacturing process for
pullulan or gelatin
capsules (Capsugel Plantcaps) in which the biomaterial is melted, compressed
and formed
into the desired form. Plasticizers include sorbitol and glycerin. General
instructions on the
formation of soft gelatin capsules can be found on the SaintyCo website
(haps ://ww w.sainty tec.comisoft-gelatin-capsules-inallufacturing-processt)
utilizing small or
large scale automatic encapsulation equipment with forms adapted to be the
appropriate size
and shape for use as caps and wraps around nerves.
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[0186] Other sheets of interest include fibrinogen and
thrombin sheets (US
10,485,894), hydroxypropylcellulose (JP2009/183649A) or hydrophobic polymers
(US2012/0095418). The films have sufficient thickness to support the delivery
of the
hydrogel precursor solution and are ideally 10 to 100 microns, preferably 50
to 200 microns,
more preferably 10 to 150 microns in thickness. The films preferably swell
minimally, less
than 50% in thickness, after hydration. In one embodiment, the thin-film
bioerodible cap and
wrap forms dissolve after 5 minutes into growth permissive molecules or
polymers. In
another embodiment the films remain in place and are cleared over one day to 6
months
preferably one day to three months.
[0187] In another embodiment, the injectable growth
permissive biomaterial is
delivered in a more traditional wrap sheet form similar in size and form to
that available
commercially (Axoguard Nerve Protector), approximately 1 to 4 cm lengthwise
and 0.5 cm
to 4 cm wide (similar in thickness and size to the oral sheets Listerine
POCKETPAKs).
When wrapped around nerves and the growth permissive biomaterial, these form
wraps
approximately 2 mm in diameter by 40 mm long (2 mm, 3.5 nana, 5 mm, 7 mm. 10
mm and
20 mm to 40 mm long).The Wrap forms or Conduit forms containing a
biodegradable
biocompatible material may or may not be pre-assembled in the second larger
wrap form. If
the latter is the case, after the growth permissive biomaterial is placed in
the wrap form
around a nerve or nerve stumps, for example, the biomaterial-wrap do not need
to be handled
and the physician can directly deliver the growth inhibitory biomaterial
around the growth
permissive material. By delivering the growth permissive biomaterial in a
biocompatible
biodegradable wrap, softer or lower viscosity solutions, gels, or slurries can
be delivered in
close apposition to the nerve without dripping away from the site. Last, the
growth
inhibitory biomaterial is delivered into the second larger diameter Wrap form
around the
nerve (typically 1-4 mm in diameter larger than the wrap form diameter and, if
the second
Wrap form is not biodegradable, it can be removed and discarded.
[0188] Referring to Figure 3B, 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.
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[0189] Referring to Figures 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.
[0190] 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
Figure 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 Figure Sc.
[0191] 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 Figure 3B. 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 Figure 5E.
[0192] 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 Figure 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.
[0193] Figure 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
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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.
[0194] Figure 8 is a close-up view of an end effector 708
of Figure 7, also
illustrating that the end effector 708 can also carry a form 10. Figure 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.
[0195] Figures 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.
[0196] Referring to Figures 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. Opening 1104 may communicate with the
cavity 18
through the sidewall at about the level of the support surface 28 or below, to
facilitate
introduction of media below the nerve to form a first layer on the underside
and partially
encapsulating the nerve. Also shown is nerve guide 1122 which can be as
described
elsewhere herein. See Figure 11A.
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[0197]
Hydrogel may be delivered in two or more successive applications, to
partially (e.g. half) fill the form and form a hydrogel layer 1150 as shown in
Figure 11B.
Then a second volume of precursor can be introduced to completely fill the
form as shown in
Figure 11C and form a hydrogel cap encasing the nerve end after which the form
is removed.
Hydrogel may be delivered in a small bolus 1152 to surround the tip of the
nerve as shown in
Figure 11D and then the remainder of the cap is subsequently filled to form a
hydrogel cap as
shown in Figure 11E after which the form is removed. Thus, a multilayer (two
or three or
four or more) hydrogel cap may be formed to encapsulate the nerve end.
[0198]
Referring to Figure 12, in some embodiments, a support rod 1215 is
placed adjacent to and in contact with a section 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 reinforced nerve cap. The rod 1215 may be biodegradable.
[0199]
Referring to Figures 13A-13D, in some embodiments, one, two, or more
apertures 1310 are provided in the side of a cap or wrap fat
________________________ la 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. 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 may be subsequently removed when the form is removed. Alternatively,
the post
1330 may comprise a biodegradable post that remains integral to the hydrogel
cap. See
Figures 13B and 13C. Alternatively, a first layer of hydrogel can be formed in
the bottom of
the cavity before introducing the nerve end into the cavity. The nerve end may
then be
positioned on top of the first, base layer. Precursor may then be introduced
to encase the
nerve and bond to the first, base layer. The first, base layer may be formed
during the
clinical procedure, or previously at the point of manufacture of the form.
[0200]
In some embodiments, a cap form can include a partial lid 1320, shown
in
Figure 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
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nerve cap. As shown in Figure 13D, the cap or wrap form can also include
raised tabs 1333
to incline the longitudinal axis of the form. By slightly tilting the cap
form, the spill of the
precursor material from the nerve entrance zone can be minimized.
[0201]
Figures 14A-14C illustrates various views of an embodiment of a nerve
cap form 1400 similar to that shown in Figures 13A-13D with a partial lid 1420
connected
via a hinge 1428 with an insert 1440 to assist in centering the lid 1420 over
the window on
the cap form 1400. Also shown is nerve guide 1405, which can be as described
elsewhere
herein.
[0202]
Figures 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 sheeth 1560 is subsequently torn off the nerve
1524, such as
using a tearable tab 1564 as shown in Figure 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 Figure 15B.
Figure 15C
illustrates an alternate tearable cap form design which can include a
plurality of tabs.
[0203]
Figure 16A-16E illustrate hydrogel filling and surrounding a nerve in a
cap form. Figure 16B illustrates a photograph of the hydrogel fat
___________________ lied inside a cap form.
Figure 16C illustrates a high resolution image of a cap. Figure 16D
illustrates an example of
a cap and wrap 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. See also Figures 5A-5E. 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 Figure 16E.
[0204]
Figure 17A illustrates neuroma formation after delivery of DuraSeal in
a
cap form around a transected rat sciatic nerve. Figure 17B illustrates the
absence of neuroma
formation after delivery of a formulation of the present invention around a
transected rat
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sciatic nerve. The hydrogel cap maintains mechanical strength and in vivo
persistence of at
least about 3 months, more preferably about 6 months
[0205] Figures 18A-18B schematically illustrate 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.
[0206] Referring to Figures 18A and 18B, there is
illustrated a mixer 1800 for
mixing the two part precursor components of the gel of the present invention.
The mixer at
1800 comprises a housing 1802 having an influent port 1804 and an effluent
port 1806 in
fluid communication via a flow path 1808. The influent port 1804 and effluent
port 1806 may
comprise luer connectors or other standard connection structures. Media
introduced through
the influent port 1804 follows flow path 1808 through at least a first static
primary mixing
column 1810. The mixing column 1810 includes a tubular housing 1812 and an
internal
column of mixing elements in the form of baffles 1814.
[0207] Media exiting the first static mixing column 1810
enters a secondary
mixing chamber 1816. In the illustrated embodiment, the secondary mixing
chamber 1816
causes media following the flow path 1808 to enter an optional second static
mixing column
1818. Media exiting the second mixing column 1818 is directed out of the
effluent port 1806.
[0208] The total volume of delivered, mixed media will
generally be less than
about 5 ml, typically no more than about 2 ml and in some applications less
than 1 ml. The
first component to enter the primary mixing column 1810 will generally be the
first to exit
the primary mixing column 1810. The secondary mixing chamber 1816 functions to
accomplish a different type of folding mixing, to blend the effluent from the
primary mixing
column 1810 with itself and achieve superior uniformity. Addition of a third
mixing function
by addition of the optional second static mixing column 1818 further ensures
uniformity in
the mixing of the hydrogel components for the first 0.5 ml. or 1 ml of
hydrogel to exit the
effluent port 1806.
[0209] The mixing column 1810 preferably includes at least
4 mixing elements
1814 and generally includes between about 6 and 12 baffles 1814 and generally
no more than
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about 32 baffles 1814. The baffles may have an outside diameter of no more
than about 1/8
of an inch and in some implementations no more than about 1/16 or 1/32 of an
inch. The
length of the static primary mixing column 1010 is generally less than about 4
inches and
typically less than about 2 inches or less than about 1.5 inches. In one
implementation, the
length is within the range of from about 0.4 to 1.0 inches, more particularly
about 0.5 to 0.7
inches.
[0210] Referring to Figures 18 C-E, there is illustrated a
combined dual chamber
dispenser and mixing assembly 1830. Referring to Figure 18C, the dispensing
and mixing
assembly 1830 comprises a housing 1832 enclosing a first chamber 1834 and a
second
chamber 1836 for containing and maintaining separate first and second
components. The
components begin to mix at amerger1838 of flow paths from the chambers, such
as in
response to advancing plungers (not illustrated) into the proximal ends of the
first and second
chambers. The combined media streams then advance through a primary mixing
column
[0211] In Figure 18D, a combined dual chamber dispenser and
mixing assembly
1830 is shown, having a single primary mixing column 1810 and a secondary
mixing
chamber 1816. Media exiting the static mixing column 1810 follows a flow path
1808
through a secondary mixing chamber 1816, and eventually through an aperture
1820 and into
an exit chamber 1822. A baffle 1824 may be provided to direct effluent from
the static
mixing column 1810 into the secondary mixing chamber 1816 which will
substantially fill
before exiting via the aperture 1820.
[0212] Referring to Figure 19, there is illustrated a dual
component syringe for
use with the mixer of Figure 18 A. The syringe comprises a housing 1842
enclosing a first
chamber 1844 having a first plunger 1846, and a second chamber 1848 enclosing
a second
plunger 1850. The first and second plungers are connected via a bridge 1852,
to prevent
dispensing from either chamber ahead of the other.
[0213] An adapter 1854 may be provided for removable
coupling to the housing
1842 and to a first chamber 1858 and a second chamber 1864 removably coupled
to the
adapter 1854. The adapter comprises a first connector 1856 for removable
connection to the
first chamber 1858 which contains a first media 1860. A second connector 1862
may be
removably coupled to a second container 1864 which may contain a second media
1866.
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[0214] Proximal retraction of the first and second plungers
such as by manually
retracting the bridge 1852 will draw first media 1860 and second media 1866
into their
respective chambers on the syringe. Adapter 1854 be there after be
disconnected from the
housing 1842 and the dual chamber syringe then coupled to a mixer such as one
of those
disclosed herein.
[0215] Referring to Figure 20, the dual component syringe
1840 is illustrated as
filled with the first media 1860 and second media 1866. A connector 1805 may
be provided
on a distal end of the syringe 1840 for connection to the influent port 1804
on a mixer such
as mixer 1800. Media expressed from the syringe follows the flow path 1808 as
has been
described, and eventually fully mixed first and second media blend may be
expressed in via a
needle 1868 into a mold as is described elsewhere herein.
[0216] The table to follow is related to specific non-
limiting embodiments and
devices for delivering in situ forming hydrogels.
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 partially or Wrap Growth Permissive and
completely transected and undergone then
Growth Inhibitory
direct suture repair (coaptation, end-to-
end anastomosis)
5) Nerves that have undergone suture repair Wrap(s) ¨protect Growth
Permissive and
and placement in a nerve conduit or nerve-conduit junction
then Growth Inhibitory
wrap
6) Nerves
that have undergone connector Wrap(s) ¨ protect Growth Permissive and
assisted `suturelesss' neurorraphy in nerve-conduit junction
then Growth Inhibitory
which sutures are placed between the or
Growth Inhibitory
epineurium and the connector but not to only
one another
7) Nerves
that have been placed in a Wrap(s) ¨ protect Growth Permissive and
connector without suturing anastomosis then
Growth Inhibitory
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or Growth Inhibitory
Only
8)
Nerves that are undergoing conduit Wrap(s) ¨ protect Growth
Permissive and
detensioning gap repair nerve-conduit junction
then Growth Inhibitory
or Growth Inhibitory
Only
9)
Nerves undergoing detensioning Wraps(s) ¨ protect Growth
Permissive and
allograft interposition with connector nerve-nerve
then Growth Inhibitory
assisted sutureless repair anastomosis or
Growth Inhibitory
Only
10) Nerves undergoing detensioning
Wrap(s) ¨protect Growth Permissive and
autologous nerve graft interposition nerve- nerve
then Growth Inhibitory
suture repair
11) Nerves that have non-union gaps (e.g.
Wraps Growth Permissive
cannot be repaired directly) and
then Growth
Inhibitory
12) Nerves that have been repaired and one
Wrap(s) ¨ Protect Growth Permissive
or more wraps are placed around the nerve-wrap and
then Growth
anastomoses site
Inhibitory
13) Nerves undergoing suture repair in
Wrap ¨ Protect nerve- Growth Permissive
targeted muscle reinnervation nerve interface and
then Growth
Inhibitory
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[0182] Peripheral Nerve Stimulation (PNS). As
neurostimulators have advanced
from the spine to the periphery and hardware and batteries 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 (e.g. pain), 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.
[0217] Percutaneous delivery. With the advent of higher
resolution handheld
ultrasound and better training amongst interventional pain and orthopedic
physicians,
percutaneously delivered implantable neurostimulators arc 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. In
this embodiment, the electrode is placed at the desired location and then the
in situ forming
hydrogel is delivered to anchor its location. 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 the
interface 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, for the treatment of
nerve
regeneration, more rapidly degradable PEG hydrogels are desirable, maintaining
mechanical
strength for a week or two prior to eroding and clearing from the site. While
these in situ
formed hydrogels have sufficient adhesiveness to hold the catheter or
electrode at the site of
delivery, should the electrode or catheter need to be removed, a strong pull
on the electrode
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of catheter will permit percutaneous removal from the delivery site. Suitable
PEG hydrogels
for these applications are based on multi-arm PEGS with faster degradation
such as PEG-SS
(PEG-succinimidyl succinate ¨ NHS ester) or PEG-SG (PEG-succinimidyl glutarate
¨ NHS
ester). For treatment applications for chronic pain in which an indwelling
peripheral
electrode is desirable, delivery of a growth inhibitory hydrogels or hydrogels
with medium to
long duration mechanical strength are desirable such as multi-arm PEGs based
on more
slowly degrading bonds is 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 -
succinimidyl glutarate¨ NHS ester), PEG-SAP (PEG - succinimidyl adipate¨ NHS
ester),
PEG-SC (succinimidyl carbonate¨ NHS ester), or PEG-SGA (succinimidyl
glutaramide¨
NHS ester). Preferably these PEG-NHS esters are blended and subsequently
crosslinked
with PEG-amines for improved flexibility over small molecule crosslinking
systems, such as
trilyine, for example.
[0218] 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 a
hydrogel slurry. 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. for example, 15 seconds to one
minute
gelation. In some embodiments, a plurality of injectable microstimulator
implants are
injected into a degradable or non-degradable in situ forming hydrogel.
[0219] 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
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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.
[0220] In another embodiment, short- or long-acting
microstimulators can he
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 forming from a precursor solution. 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.
[0221] Referring to Figure 4A, there is schematically
illustrated a construct in
accordance with the present invention for electrically stimulating nerve
regrowth. A proximal
nerve stump 100 and distal nerve stump 102 are positioned within a temporary
form 104 such
as a silicone wrap in a manner previously disclosed herein. A growth
permissive gel 106 is
introduced into the form, to span the gap between the proximal nerve stump 100
and distal
nerve stump 102. An electrode assembly 108 having a probe or support 110 with
at least one
conductive surface 112 and, in a bipolar system, a second conductive surface
114, is
positioned within the temporary form 104. An electrically conductive hydrogel
116 is
introduced into the form 104 and solidified to support the nerve stumps and
growth
permissive gel, and retain the position of the electrode 108 with respect to
the growth
permissive gel 106.
[0222] RF stimulation may be accomplished using any of a
variety of
microneedle electrodes, such as a stainless steel needle electrode (0.35 mm
outer diameter,
12 mm length) connected to the negative wick (cathode) of a stimulator (Trio
300; Ito,
Tokyo, Japan). Operating parameters may include low frequency stimulation,
generally less
than about 200 Hz and preferably in the range of from about 2 Hz to about.
Current may be
in the range of from about 1 to about 10 ma or more. Voltage may be about 3 V.
with a
square waveform having about 0.1 ms pulse intermittently. Duration may be from
about one
hour to 2 weeks, depending upon the desired clinical performance.
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[0223] 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 0.1 to 5
cc, preferably 0.2 to 2 cc, more preferably 0.5 to 1 cc of hydrogel is
sufficient to maintain the
electrode position relative to a nerve. In one embodiment,he 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 erosslinked 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 neuro stimulators. In other embodiments, non
electrically conductive
polymers may also be employed to isolate the electrical signal from the
surrounding tissue.
[0224] 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.
[0225] Referring to Figure 4B, there is illustrated a
formed in situ hydrogel
anchor, for securing an electrode in electrical communication with a nerve. A
support 110
carries conductors in electrical communication with at least a first
conductive surface 112
and preferably at least a second conductive surface 114 for delivery of RF
energy from an
external energy source. Second or third or more pairs of electrically
conductive surfaces may
be provided. A volume of electrically conductive hydrogel 116 may be
introduced into a
form and solidified in situ in a manner previously discussed. The conductive
hydrogel 116
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encases the electrode and stabilizes the electrode with respect to an adjacent
nerve 120 such
that the electrode is in electrical communication with the nerve 120 through
the conductive
hydrogel 116. Alternatively, the electrode may be pinned between an in situ
formed
hydrogel anchor and the nerve 120, or by forming the conductive hydrogel
anchor around
both the nerve 120 and the adjacent electrode. Electrode may be configured to
be
proximately withdrawn from the electrically conductive hydrogel.
Alternatively, the
electrode may be withdrawn from the patient following absorption of the
hydrogel. In one
embodiment, the electrical conductivity of PEG hydrogels can be enhanced by
incorporating
PSS in a PEG hydrogel matrix, resulting in the in situ formation of PEDOT to
form a
PEDOT:PSS loaded PEG hydrogel (Kim et al 2016. Highly conductive and hydrated
PEG-
based hydrogels for the potential application of a tissue engineering
scaffold. Reactive and
Functional Polymers, DOT: 10. 1016/j .re actfunctpolym.2016.09.003) In another
embodiment,
metal nanoparticles and carbon-based materials can be delivered in the
hydrogel, including
gold, silver, platinum, iron oxide, zinc oxide, or polypyrrole (PPy),
polyaniline (PANi),
polythiophonc (PT), PEDOT (above), or poly(p-phenylene vinylenc) (PPV) as
described in
Min et al 2018. Incorporation of Conductive Materials into Hydrogels for
Tissue Engineering
Applications, Polymers, 10, 1078; doi: 10.3390/polym10101078, incorporated
herein.
[0226]
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 nana 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
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suitable for applications to prevent the acute and subacute adhesion formation
around nerve.
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).
[0227] Generally, PEGs with ester bonds susceptible to
hydrolysis do not contain
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 for three of four months until the nerve is repaired
or neuroma
prevention is achieved. 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 and therefore may not be suitable for a hydrogelcap. Figure 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 a
neuroma was
formed.
[0228] In another embodiment mechanical offloading of the
nerves is desirable.
By designing the nerves to be covered by at least 8 mm, preferably a 10, 15 or
20 mm length
of nerve, there is sufficient adhesion circumferentially that the tension is
partially offloaded
into the gel and better distributed. By providing mechanical offloading the
hydrogel can
support a regenerating nerve (wrap). Given a 10 mm or longer length of nerve
stump (one or
more nerve stumps), the nerve can be embedded in the hydrogel is a bend or an
'S' turn, such
that the tenion on the nerve anastamoses is minimized.
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[0229] Other approaches teach delivering 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 it is preferable that 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 formed hydrogel from the surrounding environment,
preventing
tethering between the nerve and the surrounding tissue and permitting the
nerve to glide
within the fascial tunnel. Gliding can be achieved through two mechanisms: 1)
the
lubriciousness and streamlined outside form of the hydrogel after formation in
the Cap or
Wrap form, or 2) by selecting a formulation with minimal equilibrium swelling
for two to
three weeks, utilizing a faster degrading PEG hydrogel, the hydrolysis of the
hydrogel
supports swelling of betwee 20 to 80% swelling, more preferably 40 to 70%. The
second
phase of swelling, the degradation swelling, permits the gliding of the
damaged nerve
through the inner lumen of the hydrogel (lumen expands as the swelling of the
hydrogel
translates to an outward swell) after negligible equilibrium swelling to
prevent inflammatory
response at the time of the surgery and acutely post-operatively.
[0230] 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,
hydrophillicity 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(ethylcne glycol)-bascd hydrogcls, agarosc- 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. In one embodiment
agarose, at a
concentration of example 1.25% wt/vol, can be selected to prevent nerve
regeneration. In
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another example, PEG hydrogels can prevent neuroma formation at 4% w/v and
higher,
preferably 6 to 9 % w/v, more preferably 8 % w/v or 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 arc such that the
pores are too small
for cellular ingrowth.
[0231]
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
commercial in situ forming PEG hydrogels with biodegradable ester linkages,
may have
sufficient mechanical strength initially but hydrolyze at such a rate that
their cros slinking
density is lost 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,
comparable to nerve
transection alone. Preclinical testing has demonstrated that a mechanical
strength of at least 5
kPa, preferably 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 PEG hydrogels are suitable to support the long-tat
____________ -11 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, more preferably 6 months or
to reduce or
prevent neuroma formation and 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.
[0232]
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
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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).
[0233] The development of in situ forming polymers, and
particularly, in situ
forming synthetic hydrogels, including PEG-based hydrogcls with longer in vivo
mechanical
strength and longer persistence profiles beyond 2.5 to 3 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 including combinations of PEG-NHS-esters and PEG-
amines
with biodegradable linkages, 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.
[0234] 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,
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degradation occurs at a rapid enough rate that mechanical integrity cannot 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.
Figure 16A
illustrates the formation of a neuroma after the delivery of DuraS eal.
[0235]
Examples of Multi-Armed PEG Hydrogels with Various Hydrolytically
labile bonds
PEG Hydrogel In Vivo Persistence
Neuroma formation in Rat Sciatic
Nerve Transection Model
PEG-SS (ester bond) 2 weeks
Large bulbous neuroma observed
at 1 month
Duraseal (ester bond) 2 to 8 weeks
Large bulbous neuroma formation
observed at 2 and 3 months
PEG-SG (ester bond) 4 to 8 weeks
Large bulbous neuroma formation
observed at 3 months
PEG-SAP (ester bond) 6 to 8 weeks
Large bulbous neuroma formation
observed at 2 and 3 months
PEG-SAZ (ester bond) 2-3 weeks
Large bulbous neuroma at 2 and 3
months
PEG-SGA (amide bond) 9 months or more No neuroma
formation
PEG-SC (urethane bond) 6 months or more No neuroma
formation
[0236]
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
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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 are preferable for the Cap or nerve
repairs in which
a longer in vivo persistence of the hydrogel is preferable. 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 to
blend
with 8-arm 20K amine or 4-arm 10K amine. The following patent is incorporated
for
reference 20160331738A1. In other embodiments, such as for wrapping a nerve,
the 8-arm
20K PEG-SG or 8-arm 15K PEG-SAP combined with blends of 8-arm 40K amine or 8-
arm
20 K amine are preferable for providing structural support while the nerve
regenerates and/or
to prevent nerve compression and scar tissue formation in the acute and
subchronic period
and then subsequently degrading and being cleared from the site. For
applications to prevent
nerve compression or support nerve regeneration after nerve injury, growth
inhibitory PEG
hydrogels with shorter in vivo degradation profiles are preferable. For these
applications, the
hydrogel should provide sufficient mechanical strength to prevent aberrant
nerve outgrowth
and prevent immune infiltration into the healing nerve. PEGs suitable for
these
[0237] Compressive strength. The desired compressive
strength (elastic modulus,
Young's modulus) of the growth inhibitory 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 20 kPa after 3 months in vivo,
more preferably 40
kPa at 3 months after administration.
[0238] Compressive strength was measured benchtop after in
vitro equilibrium
and also after harvesting implanted samples from the subcutaneous space in
rats, in which
hydrogel cyclinders (d=6 mm) are cut to 100 mm long, pre-equilibrated (for 12
hours at
37 C) and evaluated for compressive strength. Compressive properties of the
hydrogel
formulations were measured at a 1 mm/min with the lnstron. The modulus was
calculated as
the tangent slope of the linear region between 0.05 and 0.17 of the stress-
strain curve.
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[0200] Compressive Strength of Various Formulations
Polymer Compressive Compressive modulus Neuroma
formation
modulus (t=0) (t= 3 months, in vivo)
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
Formation K 80 kPa 75 kPa No neuroma
formation
[0239] Although in vitro mechanical strength and persistence of the
hydrogels
(37 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.
[0240] 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.
[0241] Generally, the structure of multi-armed PEGs are
[0242] C ¨
[0243] where
[0244] C = core structure of the multi-arm PEG
[0245] n = repeating units of PEG on each arm (25 to 60 units)
[0246] M = Modifier
[0247] L = cleavable or noncleavable linker (ester, urethane, amide, urea,
carbamate, carbonate, thiourea, thioester, disulfide, hydrazone, oxime, imine,
amidine,
triazole and thiol/maleimide).
[0248] 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-
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halocarbonyls, and N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, or
nitrophenyl
esters or carbonates
[0249] m= number of PEG arms (e.g. 2, 3, 4, 6, 8, 10)
[0250] In some embodiments, hydrolysis modifiers (M) can he
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.
[0251] 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.
[0252] 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.
0
~eat 'R1
N-
[0253] 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 (-CH2-, -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 hut not
limit to halogen, alkyl, aryl, substituted alkyl, substituted aryl,
substituted heteroaryl,
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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). Jr
one embodiment,
R3 can he Anilide and in another embodiment R1 can be phenyl.
Mod H 0
R ---3."Crjt" NH
[0254] In another embodiment, the hydrolysis rate of
carbamate bond can be
modulated by the modulator at the beta position. The modulator can he CF3PhS02-
,
ClPhS02-, PhS02-, Me11PhS02-, Me0PhS02-, MeS02-, 0(CH2CH2)NS02-, CN-, (E02NS02-
.
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 carbarnate linkage are described in 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. S anti et al
(2012) Predictable and
tunable half-life extension of therapeutics agents by controlled chemical
release from
macromolccular 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
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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 20k).
Preferably
the solid content of PEG is between 6 and 10 wt%. more preferably 8 wt%. 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 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.
[0255] 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.
[0256] 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 without appreciable loss of mechanical strength
during the initial
period of nerve regeneration. 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.
[0257] 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,
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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 figure 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 Biomatcrials Science and Biomedical
Applications).
[0258] 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.
[0259] 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
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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.
[0260]
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.
[0261]
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%.
[0262]
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.
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[0263] Equilibrium swelling may be preferably assessed in
vitro at body
temperature conditions (37 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 C. After swelling in PBS for 12 to 24
hours at
37 C. samples were taken out and weighted again. The swelling is calculated by
the
percentage of mass increase.
[0264] 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.
[0265] 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
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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, 3.5F,
single straight, AD Instruments). The hydrogel was then placed at 37 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 (- 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).
[0266] Stiffness. Stiffness of the hydrogel can
measured/inferred either by
rhcology (G' = storage modulus, G*=shcar 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.
[0267] 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 37oC 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.
[0268] 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 and more
preferably
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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.
[0269] 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 100cP and in some
embodiments no more
than about 20cP 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
10kcP, 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.
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-cros slinking can
be further
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cross-linked by itself, or with PEG-amine, or with PEG-urethane, or with PEG-
amine and
PEG-urehtane to form a higher viscosity precursor solution.
[0270] 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 (-1g/cm3). By adjusting the density of the
flowable media, the
nerve position can he 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/cm3,
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.
[0271] 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.
[0272] In yet another embodiment, the clinician may elect
to employ a PEG
fusion protocol as described in US 10,398,438 or 10,136,894 to the nerve prior
to application
of the in situ forming hydrogel. These PEG fusion approaches can be used to
either fuse
nerves, as is desrible to seal nerve ends after transection for prevention of
neuroma. They
may also be employed to seal nerves together, such as the proximal and distal
stump in close
apposition for improving outcomes in nerve regeneration. By applying this
sequence of
solutions beforehand (Ca2+ free, optional antioxidant, fusogen. Ca2+
solution), the nerve
ends may be optimally sealed to improve neuronal survival, or, in the case of
nerve
regeneration, nerve outgrowth. Kits containing both the PEG fusion component
solutions
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described in the above patents and incorporated herein for reference in their
entirety may be
combined with the in situ forming hydrogel kits.
[0273] 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. Alternatively, for applications for the prevention of
neuroma, the tip of
the nerve can be washed with a Ca2+ free solution to wash away excess axoplasm
and
growth factors prior to delivery of the in situ forming hydrogel around the
nerve.
Alternatively or in addition to this, the low molecular weight biomembrane
fusion agent may
be delivered in the in situ forming hydrogel to the nerve to seal the
membranes in concert
with the delivery of PEG hydrogel. As above, concentrations between 40 and
70%,
preferably 50% and molecular weights of PEG (< 5kDa, 10-50 mM) have been
widely
demonstrated to seal damaged plasmalemma. Several protocols for PEG fusion
(for use in
neuroma prevention applications by sealing cut nerves) or PEG sealing (for use
in nerve
repair procedures) are widely publicly available in the preclinical literature
(PMID:
30586569, 22302626, 30737092, 30909624. Although Colton Riley et al 2017
(PMID: 29053522) have illustrated the delivery of the PEG fusion solution and
associated
solutions inside the wrap form, this could be better achieved by delivering
the PEG fusion
and associated solutions into the wrap or cap form.
[0274] Coverage of the proximal nerve stump (tip). 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.
[0275] Length coverage. It is preferable that a minimum of
10 mm of nerve be
embedded/encapsulated in the hydrogel although, in some instances, 5 nun may
be sufficient.
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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.
[0276] 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. Figures lA and 1B. The form, once
the desirable
position is reached, is left in place.
[0277] While holding the nerve tip in one hand in the
center of the form, the
clinician then delivers the in situ forming hydrogcl 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. In another embodiment, a post is
added in cap
form to adhere the nerve to the cap form prior to the application of the
hydrogel. The post is
flexible and can be removed after hydrogel formation. In one embodiment, the
post is
constructed of a bioresorbable polymer that is left in place after deliver of
the hydrogel and
detaches from the cap form after formation of the hydrogel inside the cap
form. Thus, this
post remains embedded in the hydrogel cap that remains in situ. In one
embodiment, the post
is comprised of the same material that the hydrogel cap is made out of. This
permits the
similar or identical swelling behavior as the hydrogel cap which has formed
around the post
and the nerve. In another embodiment, the post is comprised of a low molecular
weight
PEG, permitting the post to be cleared more quickly than the hydrogel cap. The
post may be
formed from a PEG solution lyophilized in place inside a cap form. Preferably,
the post is
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set back 2-3 mm from the tip of the transected nerve so that regenerating
nerves do now grow
back retrogradely and exit through the void created by the post.
[0278] 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 pm 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.
[0279] For applications in which it is desirable to protect
long lengths of nerve, a
wrap form can be designed that is 1 cm to 100 cm long, preferably 1 cm to 50
cm long, more
preferably 1 cm to 10 cm long. For longer nerve wraps, the gelation time of
the in situ
formed hydrogel can be extended so permit coverage of the entire length of the
nerve.
Alternatively, the mixer/needle on the end of the hydrogel applicator can be
exchanged so
that the longer wraps can be filled through successive regions of gel
formation. Alternatively,
multiple wraps approximately 1.5 cm to 2 cm long can be placed in sequence.
These wraps
can be filled with one applicator each.
[0280] 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.
[0281] 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 iLtm. Preferably, the growth inhibitory hydrogel is
microporous or
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mesoporous, with pores less than 1 um, preferably less than 0.5 microns, more
preferably
less than 500 nm in diameter.
[0282] Charge. Neutrally or negatively charged biomaterials
are preferred as
growth inhibitory gels as neurites prefer to grow into positively charged
biomatcrials.
Similarly, hydrophilic materials or amphiphilic are preferable to hydrophobic
materials.
[0283] 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.
[0284] Clarity. In the preferred embodiment, the hydrogel
is clear and transparent
to confirm the location of the nerve after hydrogel formation. Of particular
relevance to nerve
repair cases, the transparency permits confirmation that the nerve repair was
optimal and that
no fascicles are building from the repair site or that the optimal distance
between the two
nerve stumps has been maintained. 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.
[0285] The cap of the present invention can be formed from
a hydrogel having
sufficient optical clarity that the nerve can be visually observed through the
material of the
cap following cap formation. Referring to Figures 1B and 1E, the cap once
released from
the mold will have a first (lower) convex sidewall surface that conformed to
the concave
surface of the mold, and a second (upper) surface that aligned with the window
20 and has a
relatively flat, meniscus conformation. At least a portion of the first
surface has a curvature
that may substantially conform to the surface of a cylinder. This along with
the optically
transparent characteristic of the hydrogel functions as a lens, magnifying the
appearance of
the nerve when viewed through the first surface. The cap may thus function in
the manner of
a convexo-concave lens (sometimes called negative meniscus), where the concave
interface
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at the surface of the nerve has a tighter radius of curvature than the radius
of the outer first
surface to produce the magnification. The radius of the hydrogel lens is
typically between
about 1 mm and about 12 mm from the center of the nerve and the thickness of
the hydrogel
lens through the first surface may he between about 0.5 mm and about 5 mm
thick,
preferably about 1 mm to about 1.5 mm thick. The refractive index of the
hydrogel convex
lens is similar to objects embedded in glass, at about 1.45 to 2.00,
effectively bending the
light rays inward to make the nerve inside appear larger than it is. The
hydrogel is
transparent and is over 90% water and thus is well suited
[0286] Elasticity. In some embodiments, the elasticity of
hydrogel can be
modulated 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.
[0287] 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.
[0288] 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,
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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-Norbomene 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.
[0289]
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.
[0290]
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.
[0291]
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 fat
_____________________ ii 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 microp articles.
[0292]
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
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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
(9,724,447, US20190071537, PeIlene 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 he 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 (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).
[0293] 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, infrared, visible
light. In one
embodiment, the light source can be attached directly or via a fiber optic
cable to an opening
in the cap or wrap form. By designing the light to shine over the entire cap
or wrap form, a
consistency in cros slinking density can be obtained. 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-emmitting 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. Other biomaterials that form in situ
include
elastomers that can be crosslinked in situ including US 10,035,871 (and PMID
31089086),
incorporated in their entirety either via a chemical or photocrosslinking
process. For these
biomaterials, the transparency and color of the cap and the wrap form can be
adjusted to
reflect ultraviolet light back into the form, such as an opaque white form.
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[0294] 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.
[0295] In one embodiment, lower molecular weight hydrogels
(2kDa, 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 photocrosslined 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.
[0296] 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).
[0297] 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
(7,060,259).
[0298] Growth permissive gels. In some embodiments, the
growth permissive
solution comprises a low viscosity solution of collagen at 1.5 nag/m1 or less,
more preferably
0.6 mg/ml or 0.8 mg/ml. In another embodiment. A laininin solution at a
concentration of
0.4 mg/ml is preferable. In another embodiment, an HPMC or CMC formulation at
a
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concentration of 2% provide a low viscosity solution through which nerves can
pathfind
without appreciable mechanical barriers.
[0299] 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 patent
US 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.
[0300] 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.
[0301] Crosslinking. Hydrogels may be formed in situ
through electrophilic-
nucleophilic, free radical, or photo- polymerization.
[0302] 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.
[0303] 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.
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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 C prior to testing.
[0304] Other hydrogels. In situ forming polyanhydrides are
also of interest for
developing applications directed towards nerves. In one embodiment,
polyanyhydride
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,
US62/181,270, and US201562181270P.
[0305] Applicator. Dual channel applicators used to deliver
the in situ forming
hydrogels are commercially available (Nordson Medical Fibrijet Biomateral
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 arc 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 arc 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.
[0306] Example 1.
[0307] 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
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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 and provides
compressive
strength between 50 and 70 kPa. In addition swelling is between 10 and 30 wt%.
[0308] Ex amp le 2
[0309] 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.
[0310] Example 3
[0311] 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.
[0312] 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.
[0313] 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.
[0314] 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 or a
nerve wrap to prevent nerve compression or facilitate nerve regeneration. The
form
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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 20 to 40, preferably 20 to 30, most preferably
a durometer of
20. 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. This
durometer is
considerably softer than the FDA cleared silicone nerve tubes which are so
rigid, with
durometers of 50 or 60 that they result in constriction and chronic
neuropathic pain.
[0315] 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
20 or 30). 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 sun-ounding 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.
[0316] While select natural rubbers may be selected for the
nerve forms, they are
not desirable due to their lack of biocompatibility and poor characterization
for applications
involving direct tissue contact. As a result, medical grade silicones, such as
those sold by
NuSil/Avantor, Elkem, Dow and Momentive are preferable as the majority have
undergone
class VI USP biocompatibility testing. The vast majority of medical grade
liquid silicone
rubbers (LSRs) are designed for high tensile strength applications with psi
greater than 1200
psi, not designed for working with relatively more fragile biomaterials. While
these
properties are desirable for some medical devices (implantable ICDs, for
example), their high
tensile strength makes them a poor choice for applications requiring
flexibility in order to
facilitate the release of a biomaterial from the mold. As a result, a range of
LSRs were
evaluated to determine which material best suited the application for
releasing a hydrogel
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from a form. Quickly, higher tensile strength silicones were eliminated, and
the focus was on
a smaller set of two-part silicone systems for injection molding that are
cured by heat. For
these applications in which direct contact with tissue is brief, materials
with established
hiocompatibility for human implantation for a period less than 29 days arc
sufficient
although materials with established biocompatibility for longer implantation
period may be
desirable.
[0317] However, for this application, silicones with lower
durometer and lower
tensile strength are preferable in order to facilitate the removal of the
hydrogel from the
form. Therefore, silicones with a durometer less than 45, preferably less than
30, more
preferably around a durometer of 20 are desirable. Although the majority of
commercially
available liquid silicone rubbers (LSRs) have tensile strengths greater than
1000 psi, for this
applications, LSRs with lower tensile strengths are preferable. For example,
tensile strengths
of less than 900 psi, preferably less than 800 psi are desirable. Similarly,
elongation is
another factor that determines the ease with which the biodegradable
biomaterial can be
removed from the mold. Materials with a percent elongation greater than 400%,
preferably
between 400 and 2000%, more preferably between 400 and 1200%, more preferably
between
400 and 800% are desirable. Examples of these materials include MED-4920
(NuSil,
Durometer Type A 20, 700% elongation, tensile strength 750 psi), MED-4930
(Durometer
Type A 30, 450% elongation, tensile strength of 800 psi), LIM-6010 (Durometer
15,
elongation 440%, tensile strength 3 Mpa), Silopren LSR 4020 (durometer 22,
elongation
1000%, tensile strength 7 MPa) or MED50-5338 (durometer 30, elongation 350%,
tensile
strength 650 psi), and SIL-5940 (durometer 40, elongation 680%, tensile
strength 1,350 psi),
Silbione LSR 4340 (durometer 40, elongation 605%, tensile strength 1250 psi),
Silibione
4325 (durometer 26, elongation 1035%, and tensile strength of 1198 psi), MED-
5840
(durometer 40, elongation 680%, tensile strength 1,350 psi), and MED-4840
(durometer 43,
elongation 590%. and tensile strength 1180 psi), S1LASTIC Biomedical Grade LSR
7-6840
(durometer 42, elongation 700%, tensile strength 1430) or S1LASTIC BioMedical
Grade
LSR (Q7-4840 (durometer 44, elongation 540%, tensile strength 1370 psi).
Preferably the
biocompatibility of these raw materials would be established to pass USP Class
VI standards.
[0318] Alternatively but less preferably, high consistency
rubbers (HCRs) such as
the peroxide or platinum cured MED-4035 (durometer 35, elongation 1,055%
elongation,
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tensile strength 1565 psi), MED-4025 (durometer 30, elongation 890%, tensile
strength of
1,285 psi), MED-4020 (durometer 25, elongation 1,245%, tensile strength
1,400), or
preferably MED-4014 (durometer 15, elongation 1330%, tensile strength 700
psi), more
preferably MED-4920 (durometer 20, elongation 700%, tensile strength 750 psi).
Class VT
high consistency rubbers may also be carefully selected, although these tend
to have
significantly higher tensile strength on the higher end of acceptable over
1,000 psi such as
1,300 to 1.600 psi. Lastly, room temperature vulcanizing silicone (e.g. RTV-
2), such as P-44
(durometer 42, elongation 250%, tensile strength 600 psi, Silicones Inc. High
Point, NC) are
also suitable. In some embodiments, the LSR is white as opposed to translucent
(e.g. MED-
4942) or colored for contrast with tissue using Nusil's healthcare color
masterbatches in
purple or blue. In some embodiments, in which longer or larger forms are
desirable, a
silicone with a higher durometer within this range (e.g. Shore A hardness 40)
may be
desirable to help maintain the shape for, for example, wrap forms with a
length of 3 cm.
Examples of materials include MED-4940 (Nusil).
[0319] LSRs with durometers above 50 and tensile strengths
higher than 1300 psi
are less desirable (e.g. Silbione LSR 4370, Silbione LSR 4365). Similarly,
some silicones
are designed to be adhesive, such as for adhesive wound dressings, such as the
Silbione HC2
2031 A&B. These silicones are less desirable because they are designed to
adhere to tissue
as opposed to being designed to be lubricious and lift off the tissue easily.
[0320] Demolding. Easy demolding can be achieved by
decreasing the adhesion
of nerve cap/wrap form to the mold. If the nerve cap/wrap precursor spreads
well on the
mold, it will adhere to the surface after gelling. To achieve easy demolding,
the wettability of
the nerve cap/wrap precursor to the mold can be decreased. Interfacial
tension, determined
largely by polarity, plays a decisive role in wettability. The worst
wettability condition for
adhesion is conditions in which the liquid polarity is far from the polarity
of the surface of
the cap/wrap form. In one embodiment, hydrophobic materials, include but not
limited to
biomedical-grade silicone and their blends at different ratios, can be used to
make the mold.
The interfacial tension of a liquid with particular solid can also be
calculated from the contact
angle of the various solids with the liquid.
[0321] In another embodiment, polar interactions and non-
polar interaction
between the hydrogel nerve cap/wrap and the nerve cap/wrap faint will make the
nerve
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cap/wrap challenging to demold. Polar interactions include but not limited to
dipole-dipole,
dipole-induced dipole, and hydrogen bonding. Materials with less polar
interactions with
nerve cap/wrap, especially hydrogen bond, can be used to make the mold.
[0322]
In another embodiment, increase the surface tension of the gel
precursor
will decrease the wettability of gel precursor on surface. High polarity
materials, include but
not limited to salts, can be added to the gel formulation.
[0323]
In another embodiment, materials without chemical reaction to nerve
cap/wrap can be used as mold materials. Chemical reaction between nerve
cap/wrap and
mold materials increase the adhesiveness.
[0324]
In another embodiment, the surface smoothness of the mold affects the
adhesiveness of nerve cap/wrap and the demolding. Generally, the smoother of
the mold
surface, the easier the demolding. For example, the mold material can be
hydrophobic and
rough at the microscopic scale. It can trap air at its surface, causing the
unsolidified nerve
cap/wrap held up by its own surface tension. Such surfaces are called
superhydrophobic.
Superhydrophobic surfaces can be designed by forming microscalc roughness or
patterns on
a hydrophobic material.
[0325]
In one embodiment, the hydrophobicity of the cap/wrap forms generates
sufficient surface tension that, when hydrogel precursor solution is delivered
to fill the
cap/wrap form, the hydrogel precursor solution forms a convex cap that rises
above the form
itself, providing a cylindrical or oblong cross-section to the wrap or cap
form.
[0326]
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 (3.35kDa) or crosslinked multi-arm PEG (e.g. 8-arm 15kDa). 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
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used for nerve conduits and wraps, such as polyvinyl alcohol, chitosan,
polylactic acid,
polyglycolic acid, polycaprolactone.
[0327] 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).
[0328] 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 agaro se.
[0329] Forms may be rapidly dissolving forms, that, upon
wetting, dissolve and
are cleared within an hour so after the procedure, leaving the in situ foimed
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.
[0330] 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,
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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.
[0331] 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.
[0332] 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.
[0333] Viscosity. As described above, the viscosity of the
flowable solution can
be increased to minimize the nerve floating up within the solution.
[0334] 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. Similarly, but utilizing shorter gelation times, flow and thus the
movement of the nerve
is limited.
[0335] 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.
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[0336] 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 settng, similar to vascular introducers.
[0337] 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.
[0338] 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.
[0339] 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.
[0340] 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.
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[0341] 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.
[0342] 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.
[0343] 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.
[0344] 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.
[0345] 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.
[0346] 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 no more than about 10 seconds
or no more
than about 5 seconds but generally at least about 2 or 3 seconds for smaller
wraps or caps.
[0347] 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
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mm, more preferably 1 mm to 5 mm, more preferably 1 to 3 mm depending on the
size of the
nerve.
[0348] 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).
[0349] 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.
[0350] 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.
[0351] 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
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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.
[0352] 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 hydrogcl 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.
[0353] 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.
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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.
[0354] 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.
[0355] Laprascopic or endoscopic surgery. The fat
________________ ins 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 if a catheter is employed and the
gel mixing
occurs at a distance from the gel formation. Treatment of hernias is
particularly appropriate
for block nerve regeneration in nerves transected in the course of performing,
for example,
inguinal hernia repair through an open, robotic, or laprascopic approach
[0356] In a ncedleoscopic 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
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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.
[0357] 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.
[0358] 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 Neurocap0, 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 W02016144166A 1.
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.
[0359] 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
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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.
[0360] 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.
[0361] 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
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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.
[0362] 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.
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.
[0363] 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.
[0364] 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.
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[0365] 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.
[0366] 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.
[0367] 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.
[0368] 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
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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.
[0369] 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 biomatcrial 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
enchased 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.
[0370] The hydrogel thus forms a hollow cylindrical sleeve
having a central
lumen within which a nerve or tendon can glide. The gliding may occur in one
of two ways:
the gliding may occur over the outer surface of the formed hydrogel, with the
hydrogel and
the nerve or tendon moving together. Alternatively, and preferably, the nerve
or tendon
glides within the lumen of the hydrogel form while the hydrogel is anchored in
place with a
minimal capsule. In this manner, the nerve or tendon is free to move in a
soft, compliant,
scar-free, manner through the hydrogel.
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[0371] 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
wrarp 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.
[0372] Tendon repair. In another embodiment, the hydrogels
may be used to
assist in the repair of tendons and prevent adhesion/scar tissue from forming
around tendons.
In some embodiments, the hydrogel is placed around the tendon using a similar
form
permitting delivery of solution to form a gel circumferentially around the
tendon. In this
manner, the gel permits gliding of the tendon in the same way that this can he
achieved with
the gels around nerves.
[0373] 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
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tension and stress on the coaption site. Nutrients can diffuse through the
hydrogel network.
Also, the hydrogel may reduce the physician's procedure time.
[0374] In one embodiment, the solution in based on
hyaluronic acid. In another
embodiment, the solution is based on a hydrogel slurry (TraceIT, Boston
Scientific).
[0375] 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.
[0376] 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.
[0377] 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.
[0378] 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.
[0379] 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
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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.
[0380] 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.
[0381] 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.
[0382] 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 receipient 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.
[0383] 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.
[0384] 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.,
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"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 February 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</sub> (i.e., neurodegenerative factor); (g) latarcins
(short linear
antimicrobial and cytolytic peptides, which may be derived from the venom of
the spider
Lachcsana tarabacvi); (h) chondroitin sulfate protcoglycans (CSPGs) such as
aggrccan
(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
October 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.
[0385] 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
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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., CC 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 [tg per day for a total of 2 pg 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.
[0386] 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.
[0387] Growth permissive form. Regarding the examples of
Figures 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.
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[03881 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 coaptcd 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 foim 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.
[0389] 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 peimissive media is delivered into an inhibitory foim 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.
[0390] 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
cros slinked gel.
[0391] 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
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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.
[0392] 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.
[0393] 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 prescursor 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 nun of
circumferential
coverage around the nerve on either side of the injury.
[0394] 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
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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.
[0395] 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.
[0396] Preferably, the growth peimissive 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 arc
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.
[0397] 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
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potentially suitable hydrogel includes a six-armed shar-shaped poly(ethylene
oxide-stat-
propylene oxide) with acrylate end groups (star-PEG-A) can be photocured.
Other start-
shaped PEGs include a 6-arm or 8-arm NHS ester PEGs include mPEG-SCM (PEG-NHS:
Succinimidly Carboxyl Mcthy elstcr) and mPEG-SG (PEG-NHS: Succinimidly
Glutaratc
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.
[0398] 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 famework.
[0399] 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-argininc or poly-L-lysine (0.001-10 wt%). 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.
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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.
[0400]
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) Photocrosslinable
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 chito s an and poly-lysine ( tittps://pu bs.
acs,orgidoi110.1021/acs. biomac..5b01550).
The growth permissive hydrogel may be an in situ forming hydrogel comprising
PEG and
polylsyinc (https://pu bs.acs.orialdoila bsil 0. 1021/bm2.01763n) .
[0401]
The growth permissive component of the present invention may
alternatively comprise a decellularized peripheral nerve specific scaffold,
formulated into an
injectable hydrogel form. It is based, at least in part, on the use of a
decellularized tissue
which substantially lacks immunogenic cellular components but retains
sufficient amounts of
nerve specific components to be effective at supporting nerve regrowth and
reducing or
preventing muscular atrophy. In certain non-limiting embodiments, the
decellularized tissue
scaffold may be formulated into a hydrogel through the use of enzymatic
degradation. These
hydrogels may be non-cytotoxic to neurons and also support neuronal outgrowth
of cultured
cells. Thus one aspect of the methods of the present invention includes
applying or injecting
a growth permissive media including a hydrogel comprising a decellularized
peripheral nerve
scaffold that has been enzymatically degraded, so that it bridges a gap in the
nerve. The
growth permissive media may be encased within a growth inhibiting media in any
of the
manners discussed elsewhere herein. The peripheral nerve scaffold may
originate from an
organism that is non-autologous to an intended recipient of the hydrogel, from
an organism
that is the same species as the intended recipient, or from an organism that
is not the same
species as the intended recipient. Harvested nerves may include, but are not
limited to, the
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sciatic nerve, femoral nerve, median nerve, ulnar nerve, peroneal, or other
motor/sensory
nerve. Additional details of the decellularized scaffold may be found in US
patent No.
9,737,635 to Brown, et al., entitled Injectable Peripheral Nerve Specific
Hydrogel, issued
August 22, 2017, the disclosure of which is hereby expressly incorporated in
its entirety
herein by reference.
[0402] In addition to nerve-derived or tissue specific
materials, other growth
permissive components produced from human (autologous) or animal ( non-
autologouse
bovine, porcine) sources may be employed in whole or in part into the growth
permissive
solution that is delivered between the proximal and distal stump. These
include non-viable
human umbilical cord allograft, amniotic membrane allograft, amnion and/or
chorion
membrane graft (submucosa of human placenta), autologous skin graft,
unprocessed
allogeneic cadaver/pig skin graft, autologous connective tissue, autologous or
allograft
tendon tissue, bovine collagen, fibrillar collagen, fibronectin, laminin,
proteoglycans. These
components may take the form of a micronized or dehydrated or lyophilized
powder, a gel, a
sheet or rolled sheet, or a dehydrated powder that can be rehydrated prior to
use. Dehydrated
products may be rehydrated prior to use in aqueous solutions, such as saline,
or solutions
with 6 to 9% low molecular weight poly(ethylene glycol)s, such as PEG 3350 or
PEG 5000.
In yet another embodiment, the growth permissive environment may contain
cells.
[0403] The growth permissive solutions may be used to
support nerve
regeneration in direct anastomoses repair, small grap repair (< 7 mm) and
larger gap repair
(>7 mm). The growth permissive and/or growth inhibitor solutions may be
delivered acutely
after injury, subacutely or several days after injury when the injury has had
a chance to
declare itself, or for chronic nerve repair.
[0404] 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.
[0405] Other gels. In yet another embodiment, the first
growth permissive
material may comprise a viscous solution, a nanoparticle- or microparticle-
based gel, a
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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 arc created for stable dispersions with
uniform size, large
surface area through precipitation polymerization. Modugels, scaffolds foimed
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 polyvinvylidene 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 (Dcgoricij a et al (2008) Hydrogels for
Ostcochondral Repair
Based on Photocrosslinkable Carbamate Dendrimers, Biomacromolecules, 9(10)
2863.
[0406]
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 (-1.2 mg/m1), assist
in regeneration,
fibrin (9 to 50 mg/m1),fibronectin, laminin
(fittps1/www,nobi.nlimnikgov/pubmes.1/15978668)),
Puramatrix, heparin sulfate proteoglycans, hyaluronic acid (1% sodium
hyaluronate viscous
solution), polylysine (poly (D, or L, or D,L) lysine), xyloglucan,
polyomithine, 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
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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 cross linked. Other scaffold materials may he anticipated for the
growth permissive
region (11 ttp s ://w ww ,nchi .nlm.n e.o v/vmclarti cles/P MC5899851/).
[0407] 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.
[0408] 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). 1-ittps://www. TIC bi .11i al, .govipubmed/2062020 These may be
incorporated with
the polymer powder, diluent, or accelerator depending on the stability
requirements of the
adjuvant.
[0409] 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.
[0410] 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, penults 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.
[0411] Stiffness. As matrix stiffness and compressive
strength of the hydrogel
play a significant role in promoting or inhibiting nerve regeneration, the
mechanical
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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 0.1-0.2MPa, preferably less than 1.5KPa. 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 Itm,
more preferably greater than 10pm, preferably between >100 pm more preferably
>150 pm,
with an average pore radius of 0.5 to 5%. The density of the pores should be
greater than
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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 hubbies 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
b
s :thy ww .nrbi .11ifIl, nth .tgp yip mclartici es/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 tortuoisity is low that neves 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.
[0412]
Channels. In another embodiment, channels are created in the hydrogel
in situ to permit nerve guidance. In one embodiment, channels are
approximately 150 lam,
300 pm in diameter, more preferably 500 lam in diameter to 1 mm. Preferably,
the channels
are filled with saline in situ.
[0413]
Fibers and other structural elements. Adding fibers or structural
elements
(e.g. beads, macrospheres, gel particle slurry, microspheres, rods,
nanoparticles, liposomcs,
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
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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) pet
___________________________________ nits 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.
[0414]
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.
[0415]
Injecting nanorods. Similarly, shorter nanorods may he 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.
fat.ps://www.ncbi,rilm.nih.govimihmt-A/2408:3073. In another embodiment the
fibers undergo
molecular self-assembly to form a fiber or cable.
[0416]
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 lam in
diameter,
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more preferably 15 to 250 pm in diameter. In one embodiment, the rods, fibers,
and
filaments may be coated with laminin.
[0417]
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 (AFC), 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.)
[0418]
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.
[0419]
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.
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[0420] 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.
[0421] 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 1 mm
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
mm, preferably the gel thickness is 2 to 6 mm, preferably 2 to 3 mm around the
nerve
circumferentially.
[0422] 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 Figure
2. The surrounding tissues (muscles, skin) are then sutured up again per
standard surgical
technique.
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[0423] 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.
[0424] 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 tray sine 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.
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
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addition. The addition of the linear PEG is based on findings which had been
demonstrated
2kDa 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 scaling of the plasmalcmmal and
axolcmma at the
lesion site.
[0425] 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.
[0426] 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.
[0427] 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.
[0428] 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 micro spheres,
micelles,
liposomes, or free-base to achieve an improved sustained release profile.
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[0429] 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.
[0430] 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 extracelluku- matrix.
[0431] 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 .
[0432] Drugs that are P2X receptor antagonists (P2XR), P2X3
receptor
antagonists (e.g. A14-219 (Jefapivant, A14-130), 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
(BB G) 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
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Panxl. 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 Pannexinl
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.nchi.nlm.nih.govicompound/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
(lattps://wwv.i.nebi stInt.ilill.fd.ovipubmer1/23731 685).
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 pg to as high as 25 mg of dye may be delivered in a
hydrogel locally. For
example, the FD&C #1 dye may he 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.
[0433]
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
pet-cutaneously 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.
[0434]
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
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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 Racl
antagonists may be
incorporated, such as ripasudil hydrochloride.
[0435] 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.
[0436] Example 1. Growth inhibitory hydrogel. Into the vial
containing 80 mg
PEG with NHS ester reactive group, 80 pg of BB FCF is added to yield a 0.1%
dye
concentration in the PEG hydrogel.
[0437] Example 2. Growth inhibitor hydrogel with a fusogen.
Into the vial
containing 80 PEG with NHS ester reactive group, 80 pg of BB FCF and 500 mg
PEG 3350.
[0438] 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
(Ittl,ps /iv/ ww,nebi , nJ . nilLgo v ipmciarti cles/PMC42071891, incorporated
for reference
herein).
[0439] 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
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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.
[0440]
The following examples are of in situ forming growth inhibitory wrap
formulations suitable for preventing aberrant nerve outgrowth, scar tissue
formation, and
supporting nerve gliding within the hydrogel.
[0441]
Example 5. In some embodiments the 8-arm20K PEG-SAP is crosslinked
with an 4-arm 10K PEG amine with an excess of PEG-SAP to PEG-amine. For
example, the
PEG-SAP and PEG-amine are dissolved in an acidic diluent at a ratio of 1.2:1.
The
suspension is mixed with accelerator buffer and delivered through a static
mixer to form a
hydrogel. The formulation gels in 3 seconds, provides compressive strength
between 70 and
100 kPa and swelling is between 10 and 30 wt%.
[0442]
Example 6. In some embodiments the 8-arm 15K PEG-SAP is crosslinked
with an 8-arm 40K PEG amine. The PEG-SAP and PEG-amine are dissolved in an
acidic
diluent at a ratio of 1.6:1. The suspension is mixed with accelerator buffer
and delivered
through a static mixer to form a hydrogel. This formulation gels in 4 seconds,
provides
compressive strength between 30 and 80 kPa and equilibrium swells between 20
and 60 wt%.
[0443]
Example 7. in some embodiments the 8-arm 20K PEG-SG is crosslinked
with an 4-arm 20K PEG-amine. The PEG-SG and PEG-amine are dissolved in an
acidic
diluent at a ratio of 1.0:1. The suspension is mixed with accelerator buffer
and delivered
through a static mixer to form a hydrogel. This formulation gels in 5 seconds,
provides
compressive strength between 20 and 70 kPa and undergoes equilibrium swelling
between 40
and 80 wt%.
[0444]
The following examples support in situ forming growth inhibitory wrap
formulations suitable for preventing aberrant nerve outgrowth, scar tissue fat
______ -nation, and
supporting nerve gliding within the hydrogel as it degrades and progressively
softens while
preventing cell infiltration.
[0445]
Example 8. In some embodiments the 8-arm 40K PEG-SG is crosslinked
with an 8-arm 40K PEG amine. The PEG-SG and PEG-amine are dissolved in an
acidic
diluent at a ratio of 1.2:1. The suspension is mixed with accelerator buffer
and delivered
through a static mixer to form a hydrogel. This formulation gels in 4 seconds,
provides
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compressive strength between 30 and 60 kPa and undergoes equilibrium swelling
between 40
and 80 wt%.
[0446] Example 9. In some embodiments the 8-arm 20K PEG-SAZ
(PEG-
Succinimidyl Azelate) is crosslinked with a 4-arm 40K PEG amine. The PEG-SAZ
and PEG-
amine are dissolved in an acidic diluent at a ratio of 1.2:1. The suspension
was mixed with
accelerator buffer and delivered through a static mixer to form a hydrogel.
This formulation
gelled in 4 seconds and provides compressive strength between 20 and 50 kPa.
In addition,
equilibrium swelling is between 50 and 100 wt%.
[0447] Example 10. In some embodiments the 4-aim 15K PEG-
SAZ is
crosslinked with a 4-arm 40K PEG amine. The PEG-SAZ and PEG-amine were
dissolved in
an acidic diluent at a ratio of 1.2:1. The suspension was mixed with
accelerator buffer and
delivered through a static mixer to form a hydrogel. This formulation gelled
in 8 seconds and
provides compressive strength between 10 and 40 kPa. In addition, equilibrium
swelling is
between 70 and 130 wt%.
[0448] Example 11. In another example the 3-arm 15K PEG-SS
(succinimidyl
succinate) is crosslinked with a 4-arm 40K PEG amine.
[0449] The following are examples of materials that can be
utilized in a
biodegradable form or sheet that is delivered around nerve ends needing
repair.
[0450] Example 12. In some embodiments the material of
biodegradable form is
crosslinked or uncrosslinked chitosan. The deacetylation of the chitosan is
ranging from 70%
to 100% and the thickness of the form is between 10 gm to100 gm, preferably
around 30 gm.
[0451] Example 13. In some embodiments the material of the
biodegradable form
is composed of crosslinked or uncrosslinked chitosan blended with HPMC
(Hydroxypropyl
methylcellulose), CMC (Carboxymethyl cellulose) or HA (Hyaluronic acid). The
deacetylation of the chitosan is ranging from 70% to 100%. The thickness of
the chitosan
layer is ranging from 10 gm to 100 gm. The layer of the other component (HPMC,
CMC or
HA) is from 5 pm to 50 pm.
[0452] Example 14. In some embodiments the material of
biodegradable form is
crosslinked or uncrosslinked Gelatin. The thickness of the conduit is ranging
from 10 gm to
100 id M.
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[0453] Example 15. In some embodiments the material of
conduit is crosslinked
or uncrosslinked CMC/HA blends. The thickness of the conduit is ranging from
10 gm to
100 gm, preferably around 40 gm.
[0454] Example 16. In some embodiments, the growth
permissive solution is
comprised of 3 to 5 wt% PEG-diacrylate, preferably 3 wt% PEG-DA, with a
compressive
modulus of about 70 Pa.
[0455] Example 17. In one example, the growth permissive
solution is comprised
of a 800 .is/m1 collagen solution.
[0456] Example 18. In one example, the growth permissive
solution is comprised
of an aqueous solution of 2% hydroxypropyl methylcellulose (HPMC) with a
viscosity
between 7500 and 14000 mPa-s .
[0457] Example 19. In one embodiment the growth permissive
solution is
comprised of fibrin solution.
[0458] Example 20. In another embodiment, the growth
permissive solution is
comprised of a solution of collagen-chrondoitin-6-sulfatc protcoglycan.
[0459] 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
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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.
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