U.S. patent application number 13/062234 was filed with the patent office on 2011-10-06 for medical device.
Invention is credited to Sandra Downes, Paul Kingham, Mingzhu Sun, Giorgio Terenghi.
Application Number | 20110245852 13/062234 |
Document ID | / |
Family ID | 39889145 |
Filed Date | 2011-10-06 |
United States Patent
Application |
20110245852 |
Kind Code |
A1 |
Downes; Sandra ; et
al. |
October 6, 2011 |
MEDICAL DEVICE
Abstract
The present invention provides a peripheral nerve growth conduit
for peripheral nerve repair, in particular conduits through which
peripheral nerves can grow. The conduit includes
poly-.epsilon.-caprolactone (PCL). Preferably, the inner (luminal)
surface of the conduit comprises pits having a depth of 1-4 .mu.m.
Suitably, the conduit may also include poly-lactic acid (PLA). The
inner surface of the conduit may have been treated with an alkaline
composition. The present invention also provides a method for
treating a peripheral nerve damage using a peripheral nerve growth
conduit including poly-.epsilon.-caprolactone (PCL). The present
invention also provides a kit for treating a peripheral nerve
damage having a peripheral nerve growth conduit including
poly-.epsilon.-caprolactone (PCL).
Inventors: |
Downes; Sandra; (Manchester,
GB) ; Terenghi; Giorgio; (Manchester, GB) ;
Sun; Mingzhu; (Bristol, GB) ; Kingham; Paul;
(Umea, SE) |
Family ID: |
39889145 |
Appl. No.: |
13/062234 |
Filed: |
March 4, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/GB2009/002161 |
Sep 9, 2009 |
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13062234 |
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Current U.S.
Class: |
606/152 ;
156/218; 427/2.24 |
Current CPC
Class: |
A61L 2430/32 20130101;
A61F 2/04 20130101; A61L 27/18 20130101; A61L 27/18 20130101; A61B
17/1128 20130101; Y10T 156/1038 20150115; A61F 2240/001 20130101;
C08L 67/04 20130101; A61L 27/56 20130101 |
Class at
Publication: |
606/152 ;
156/218; 427/2.24 |
International
Class: |
A61B 17/00 20060101
A61B017/00; B29C 53/08 20060101 B29C053/08; B05D 3/10 20060101
B05D003/10 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 10, 2008 |
GB |
0816574.8 |
Claims
1. A peripheral nerve growth conduit including
poly-.epsilon.-caprolactone (PCL) wherein the inner (luminal)
surface of the conduit comprises pits (blind holes).
2. A peripheral nerve growth conduit according to claim 1, wherein
the pits have an average diameter in the range 1-10 .mu.m.
3. A peripheral nerve growth conduit according to claim 1, wherein
the pits have an average depth in the range 1-4 .mu.m.
4. A peripheral nerve growth conduit according to claim 1, wherein
the % coverage of the pits is in the range 45% to 55%.
5. A peripheral nerve growth conduit according to claim 1, further
including poly-lactic acid (PLA), wherein the weight ratio of
PCL:PLA is in the range 20:1 to 2:1.
6. A peripheral nerve growth conduit according to claim 1 wherein
the conduit comprises at least 75 wt % PCL based on the total
weight of the conduit.
7. A peripheral nerve growth conduit according to claim 1, wherein
the PCL has a number average molecular weight in the range 60,000
to 100,000 g/mol.
8. A peripheral nerve growth conduit according to claim 1, wherein
the conduit is a tubular conduit and the thickness of the conduit
walls is in the range 20 .mu.m to 80 .mu.m.
9. A peripheral nerve growth conduit according to claim 1, wherein
the conduit has a length in the range 5 mm to 20 mm.
10. A peripheral nerve growth conduit according to claim 1, wherein
the conduit has a diameter in the range 1 to 5 mm.
11. A peripheral nerve growth conduit according to claim 1, wherein
the conduit is made from a film comprising PCL, preferably by
solvent evaporation and optionally opposite edges of the film are
joined together by heat sealing to form the conduit.
12. A peripheral nerve growth conduit according to claim 1, wherein
an inner (luminal) surface of the conduit has been treated with an
alkaline composition, wherein preferably the duration of the
treatment with alkaline composition is in the range 30 minutes to 3
hours, preferably the alkaline composition is aqueous NaOH, and
preferably the concentration of the aqueous NaOH is in the range of
8N to 12N.
13. A peripheral nerve growth conduit according to claim 1, wherein
the inner (luminal) surface of the conduit includes --COOH and/or
--OH terminated PCL chains, and preferably the conduit has nanopits
on the inner luminal surface of the conduit.
14. A peripheral nerve growth conduit according to claim 1, wherein
the inner surface of the conduit has an average surface roughness
(Ra) of at least 1 .mu.m.
15. A peripheral nerve growth conduit according to claim 1, wherein
the surface roughness of the outer surface of the conduit has a an
average surface roughness (Ra) of less than 1 .mu.m.
16. A peripheral nerve growth conduit according to claim 1, wherein
the difference in average surface roughness between the inner
(luminal) and outer surface is at least 1 .mu.m.
17. A peripheral nerve growth tubular conduit including
poly-.epsilon.-caprolactone (PCL), the inner (luminal) surface of
the conduit comprising pits (blind holes), the pits having an
average diameter in the range 1-10 .mu.m and an average depth in
the range 1-6 .mu.m, wherein the % surface coverage of the pits is
in the range 30% to 70% the conduit comprises at least 50 wt % PCL
based on the total weight of the conduit and the PCL has a number
average molecular weight in the range 60,000 to 100,000 g/mol, and
wherein the thickness of the conduit walls is in the range 10 .mu.m
to 80 .mu.m, the conduit has a length in the range 5 mm to 50 mm
and a diameter in the range 1 to 5 mm.
18. A kit for treating a peripheral nerve in a human or animal, the
kit including a peripheral nerve growth conduit according to claim
1.
19. A method of treating a damaged peripheral nerve using a
peripheral nerve growth conduit as defined in claim 1.
20. A method of treating the surface of a peripheral nerve growth
conduit according to claim 1 with hydroxide, comprising the step of
applying hydroxide to the surface.
21. A method of making a peripheral nerve growth'conduit according
to claim 1, the method comprising the step of: i) solvent casting a
film including PCL, and allowing the solvent to evaporate.
22. The method according to claim 21, further comprising the step
of: ii) joining opposite edges of the film together by heat sealing
to form the conduit.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention relates to scaffolds for peripheral
nerve repair, in particular to conduits through which peripheral
nerves can grow. The present invention is also concerned with
methods of making such scaffolds and of their use in the repair or
growth of peripheral nerves. Furthermore, the present invention
relates to methods of treating such scaffolds to enhance their
suitability for use in promoting peripheral nerve repair.
BACKGROUND
[0002] The peripheral nervous system (PNS) extends outside the
central nervous system (CNS) and provides the functions of, amongst
other things, bringing sensory information to the CNS and receiving
motor commands from the CNS, coordinating body movements and
controlling the involuntary muscles. Unlike the central nervous
system, the PNS is not protected by bone and is therefore
vulnerable to injuries.
[0003] Damage to nerves of the PNS can cause significant motor or
sensory impairment. In particular, patients with acute peripheral
nerve injury usually have nerve conduction defects that can
manifest as motor impairment or sensory dysfunction. Depending on
the severity of the injury and the nerve affected, a severed nerve
may cause paralysis, partial loss of mobility of the affected limb
and/or a loss of sensation. Nerve and muscle atrophy will follow if
no sufficient recovery occurs or no timely treatment is provided.
Similarly, crush damage to peripheral nerves can result in reduced
motor or sensory performance.
[0004] Surgical intervention is required if there is to be any
prospect of repairing severed peripheral nerves. One surgical
technique for attempting growth of a peripheral nerve involves
providing a scaffold, usually in the form of a conduit, at the site
of the nerve damage, to facilitate and encourage the extension of
regenerating axons. Specifically, the scaffold is selected to
provide an environment that will encourage nerve growth so that
nerve function can be returned. To date, success rates for
peripheral nerve growth have been low and it is presently not
possible to achieve the extent of peripheral nerve growth that
would be required in order to repair many of the injuries
experienced by peripheral nerves. It has been suggested [1] that
polyhydroxybutyrate (PHB) can be used to make peripheral nerve
growth conduits, but, again, only low levels of peripheral nerve
growth have been reported and the problem of repairing substantial
peripheral nerve damage remains.
SUMMARY OF THE INVENTION
[0005] The present inventors have noted that in order for a
peripheral nerve growth scaffold to effectively facilitate growth
or repair of damaged peripheral nerves, it is desirable for the
scaffold to exhibit a combination of properties.
[0006] Firstly, the material from which the scaffold is made must
be not only biocompatible but also subject to in vivo degradation
at a rate which is sufficiently slow to ensure adequate time for
the nerve to grow through the defect gap but fast enough to ensure
that the scaffold does not remain at the site of the injury such
that adequate healing can occur.
[0007] Secondly, the present inventors have found that the
mechanical properties of the scaffold must be such as to provide a
robust and durable connection between the portions of the damaged
peripheral nerve that is to be repaired (e.g. between proximal and
distal stumps of a severed peripheral nerve), for example without
breaking, swelling or collapsing once implanted. At the same time,
the scaffold must exhibit sufficient flexibility to withstand
handling and surgical implantation, as well as withstand movement
experienced when in situ.
[0008] Thirdly, the present inventors have found that sufficient
peripheral nerve growth is only likely to occur if the scaffold is
a biocompatible substrate for nerve cells and Schwann cells.
Suitably, the scaffold promotes or encourages the attachment and
proliferation of peripheral nerve cells and Schwann cells; and it
is desirable for the substrate to support the differentiation of
nerve cells. The scaffold must therefore be non-toxic and should
not release harmful break-down products. The scaffold should
preferably also possess surface properties that mimic the basal
lamina tissue in vivo.
[0009] Fourthly, the wall thickness of the nerve conduit should be
small enough to avoid neuroma formation, rigidity and tissue
compression associated with a thick wall. A thin wall, along with
small device size, means less allogenic biological material and
faster degradation rate. At its most general, the present invention
proposes that some or all of the above criteria can be achieved by
providing a peripheral nerve growth scaffold that comprises
poly-.epsilon.-caprolactone (PCL). This is based on the inventors'
experiments wherein sufficient peripheral nerve regeneration
occurred in a nerve conduit made from PCL.
[0010] In a first aspect, the present invention provides a
peripheral nerve growth scaffold including
poly-.epsilon.-caprolactone (PCL).
[0011] The present inventors have found that PCL, when provided as
a scaffold, for example a conduit, surprisingly exhibits excellent
mechanical properties and enhanced biocompatibility with peripheral
nerve cells and Schwann cells.
[0012] Suitably, the scaffold includes at least 50 wt % PCL, based
on the total weight of the scaffold. Preferably the scaffold
includes at least 60 wt % PCL, more preferably at least 70 wt %,
more preferably at least 75 wt % and most preferably about 80 wt %
PCL. In particularly preferred embodiments, the scaffold consists
essentially, preferably consists, of PCL.
[0013] The PCL as used herein can be PCL homopolymer or PCL
copolymer.
[0014] If the PCL is present as a PCL copolymer, it is preferred
that the PCL monomer comprises at least 50 wt % of the copolymer,
based on the total weight of the polymer. Preferably at least 60 wt
% of the copolymer is PCL monomer, more preferably at least 70 wt
%, more preferably at least 80 wt %, and most preferably at least
90 wt %.
[0015] The present inventors have found that the mechanical
properties and/or the peripheral nerve cell adhesion properties of
the scaffold can be further improved if the scaffold also includes
polylactic acid (PLA). Suitably, the PLA is provided as a mixture
with the PCL. Alternatively, the PLA may be provided as a copolymer
with PCL.
[0016] Suitably, if the PLA is provided as a copolymer, i.e. as
PCL-PLA copolymer, PCL and PLA are the only comonomers. However,
further comonomers can also be present.
[0017] It is preferred that the PLA is provided as a mixture
(blend) with the PCL.
[0018] Preferably no more than 50 wt % of the scaffold is PLA, more
preferably no more than 40 wt %, more preferably no more than 30 wt
% and more preferably no more than 25 wt %. A particularly
preferred concentration of PLA is about 20 wt %. This has been
found to provide a good balance of mechanical and cell adhesion
properties.
[0019] In this connection, if the content of PLA is greater than 50
wt %, it may be difficult or impossible to form a conduit by heat
sealing (discussed below). Furthermore, if the content of PLA is
greater than 50 wt %, the material is too quick to degrade in
vivo.
[0020] Conversely, the presence of some PLA can improve the
mechanical properties of the scaffold, in particular the
flexibility of the scaffold. In addition, incorporation of PLA in
combination with PCL provides improved peripheral nerve cell
viability and/or proliferation. Addition of PLA in the amounts
described herein also adjusts (typically increases) the rate of
biodegradation of the scaffold.
[0021] Preferably the weight ratio of PCL:PLA is in the range 20:1
to 1:1. More preferably the ratio is in the range 10:1 to 2:1, more
preferably 7:1 to 2:1, more preferably 6:1 to 2:1 and most
preferably 5:1 to 3:1. A particularly preferred ratio is about
4:1.
[0022] The term "PCLA" is used herein to denote a combination of
PCL and PLA. PCLA can be a mixture (blend) of PCL and PLA, or a
PCL-PLA copolymer.
[0023] Suitably the PCL has a number average molecular weight (Mn)
in the range 10,000 to 200,000. Preferably the Mn is in the range
20,000 to 140,000, more preferably 40,000 to 120,000 and most
preferably 60,000 to 100,000. A particularly preferred Mn is about
80,000.
[0024] Suitably the PLA, if present, has a number average molecular
weight (Mn) in the range 10,000 to 100,000. Preferably the Mn is in
the range 10,000 to 80,000, more preferably 10,000 to 50,000 and
most preferably 20,000 to 40,000. A particularly preferred Mn is
about 30,000.
[0025] Suitably the scaffold is a conduit. Suitably, the conduit
provides a luminal space in which peripheral nerve cells can grow
(e.g. regenerating nerve fibres can grow inside the conduit,
suitably in the lengthwise direction of the conduit). Typically a
conduit wall surrounds and defines the luminal space.
[0026] Suitably the conduit is tubular. Preferably the conduit has
tubular conduit walls. Suitably, the tubular conduit walls surround
and define a substantially cylindrical luminal space.
[0027] Suitably, the conduit has a circular cross section.
[0028] Preferably the conduit is substantially straight. However,
the conduit can also be bent or curved.
[0029] Preferably the thickness of the conduit walls is in the
range 10 .mu.m to 300 .mu.m. Preferably the conduit walls have a
thickness in the range 10 .mu.m to 200 .mu.m, more preferably 10
.mu.m to 100 .mu.m, more preferably 20 .mu.m to 100 .mu.m, more
preferably 20 .mu.m to 80 .mu.m and most preferably 55 .mu.m to 65
.mu.m. A particularly preferred thickness is about 60 .mu.m.
[0030] The present inventors have found that a conduit wall
thickness as described above provides a good balance between
degradation time, mechanical strength and flexibility.
[0031] Suitably the length of the scaffold, e.g. the conduit, is
selected to be appropriate to the nerve damage that is to be
repaired. For example, if the peripheral nerve damage comprises a
severed peripheral nerve with 10 mm of the peripheral nerve
missing, then the length of the scaffold will be chosen so as to be
sufficient to bridge the gap in the peripheral nerve. Typically,
the conduit will be longer (e.g. 10% to 50% longer) than the
gap.
[0032] Typically, the scaffold has a length in the range 5 mm to 50
mm, more preferably 5 mm to 30 mm, most preferably 5 mm to 20
mm.
[0033] As with the length of the scaffold, the width, e.g.
diameter, of the scaffold is selected so as to be appropriate to
the peripheral nerve damage that is to be repaired. Suitable
diameters are in the range 1 to 5 mm.
[0034] Preferably the scaffold is made from a film comprising PCL.
The present inventors have found that film formation can provide
control over mechanical and cell adhesion properties. Typically the
film is formed by solvent evaporation. That is, it is preferred
that a film comprising PCL is formed by dissolving or dispersing
the PCL in a solvent, casting the resultant solution or dispersion
onto a surface and allowing the solvent to evaporate.
[0035] The present inventors have found that halogenated solvents
are particularly effective for film formation. Naturally, the
solvent should suitably be a liquid at room temperature. In
particular, halogenated hydrocarbon solvents have been found to
work well, especially halogenated alkanes (haloalkanes), alkenes,
benzene and toluene. Particularly preferred are halogenated
C.sub.1-10 alkanes and alkenes.
[0036] Chlorinated solvents are particularly preferred.
Chloro-substituted C.sub.1-4 alkanes especially chloro-substituted
methane, is especially preferred.
[0037] The most preferred solvents are dichloromethane (DCM) and
chloroform. DCM is particularly preferred. The present inventors
have found that DCM permits good control over the properties of the
film. In particular, the present inventors have found that the
surface morphology of the film is controllable with DCM such that
cell adhesion, for example, can be enhanced as compared to other
solvents. The surface morphology of the scaffold is discussed
below.
[0038] Suitably the solvent is heated, for example to a temperature
in the range 40-60.degree. C. This may assist in dissolving the
PCL.
[0039] Preferably the concentration of the PCL in the solvent is in
the range 1 to 10% (wt/vol), more preferably 1 to 5%, and most
preferably 2 to 4%. A particularly preferred concentration is about
3%.
[0040] Preferably the film is cast onto a smooth surface, for
example glass surface. The smooth surface can be provided by a
glass slide for example. Suitably the surface is degreased prior to
casting.
[0041] Suitably the film is allowed to dry in air. Optionally, air
flow is provided to facilitate evaporation of the solvent. The
present inventors have found that controlled evaporation of the
solvent produces the most desirable surface properties. Suitably,
the solvent is allowed to evaporate for at least 24 hours,
preferably at least 48 hours. Preferably, film drying/solvent
evaporation occurs at room temperature.
[0042] Typically, after solvent evaporation has been completed, the
film is washed. Suitable washing agents include water, preferably
distilled water.
[0043] Preferably the film is sterilised, for example sterilised
using UV radiation, .gamma. radiation or 70% ethanol. Indeed, any
suitable known technique for sterilising can be used.
[0044] The present inventors have found that the advantageous
properties of a scaffold comprising PCL can be further improved by
treating at least one surface of the scaffold with an alkaline
composition. Preferably this is achieved by treating the surface
prior to formation of the scaffold. In embodiments, a film is
treated with an alkaline composition prior to forming a conduit
from the film.
[0045] Preferably treatment with an alkaline composition includes
exposing the surface to an alkaline composition. A preferred
alkaline composition includes hydroxide. Suitably the alkaline
composition is an aqueous solution. A particularly preferred
composition is aqueous NaOH.
[0046] The strength (and hence alkalinity) of the alkaline
composition can be adjusted so as to provide the desired surface
modifying effect. In the case of NaOH, a concentration in the range
1N to 20N is preferred, with 5N to 15N being particularly
preferred, and 8N to 12N being yet more preferred. In embodiments,
a concentration of 10N is used.
[0047] The duration of the treatment can similarly be adjusted to
provide the desired surface modifying effect. However, a duration
of 30 minutes to 3 hours is preferred, with 30 minutes to 2 hours
being more preferred and 45 minutes to 90 minutes being even more
preferred. In embodiments, the treatment time is about 60
minutes.
[0048] Suitably, the surface of the scaffold that is treated is a
surface that in use is exposed to a peripheral nerve growing
volume. In other words, preferably the surface is a surface to
which it is desired that peripheral nerve cells adhere and/or
proliferate.
[0049] In the case of the scaffold being a conduit, the surface is
preferably a luminal surface of the conduit (i.e. an inward facing
surface).
[0050] Without wishing to be bound by theory, the present inventors
believe that alkali treatment of the scaffold causes ester
hydrolysis of the PCL. Suitably this causes formation of --COOH
and/or --OH terminated PCL chains. Thus, ester hydrolysis suitably
occurs as a result of alkali treatment. The present inventors
believe that the presence of the hydrolysed ester (and in
particular the --COOH and/or --OH moieties) may be, at least in
part, responsible for the observed enhancement of cell adhesion
and/or cell proliferation.
[0051] Furthermore, the present inventors have found that treatment
with an alkaline composition can increase the hydrophilicity of the
surface. Suitably this in turn enhances the attachment of
peripheral nerve cells. This increase in hydrophilicity is
demonstrated by an increase in the wettability of the surface.
[0052] In addition, the present inventors have observed that the
surface morphology of the surface can also change as a result of
treatment. For example, a change in the size of pits in the surface
may occur. Suitably, treatment with an alkaline composition reduces
the surface roughness (Ra) of the surface.
[0053] Furthermore, the present inventors have found that treatment
with an alkaline solution can also provide accelerated degradation
of the scaffold in vivo.
[0054] Preferably after treatment with an alkaline composition, the
surface is washed. Suitably the washing step removes residual
alkali. Suitably the washing step returns the pH of the surface to
neutral. Preferably water (especially distilled water) is used to
wash the surface.
[0055] Preferably the scaffold is provided as a conduit. Suitably
the conduit is formed from a film. Suitably this is achieved by
bringing two opposite edges of the film together, preferably by
rolling the film up. Typically the film is rolled around a conduit
forming member. Suitably this provides the desired dimensions (e.g.
diameter) of the conduit. The conduit forming member can be a
cannula or other suitably dimensioned structure (e.g. a
mandrel).
[0056] In embodiments, the conduit is formed from more than one
film. For example, a plurality of films may be rolled up to provide
a laminate structure (e.g. a conduit wall comprising a plurality of
layers of film).
[0057] Suitably the edges of the film are fixed together.
Preferably this is achieved by heat sealing the film in its rolled
up state. For example, the rolled up film (suitably on the conduit
forming member) is heat sealed. Preferably heat sealing is achieved
using a hot plate, but other heat sources could be used. Thus, the
conduit is suitably formed by rolling up a film and heat sealing
the edges of the film. Suitably heat sealing occurs at a
temperature in the range 50-100.degree. C., for example about
60.degree. C. In practice, the heat sealing temperature is selected
based on the melting temperature (Tm) of the material. Melting
temperature can be measured by DSC, for example. Other fixing
methods can also be used. However, heat sealing is preferred, not
least because the present inventors have found that the surface
morphology of the film is maintained after heat treatment. A
further advantage of this approach is that no other potentially
toxic materials (e.g. super glue) are introduced to this system by
using the heat sealing method.
[0058] Suitably the "air" side of the film (i.e. the side not in
contact with the glass surface) becomes the luminal or inner
surface of the conduit.
[0059] The surface of the scaffold that in use is exposed to a
peripheral nerve growth volume is referred to herein as the inner
surface of the scaffold (e.g. the inner or luminal surface of a
conduit).
[0060] Preferably the inner surface of the scaffold comprises
pits.
[0061] Preferably the pits have an average diameter in the range
1-20 .mu.m. Suitably the average diameter is in the range 1-15
.mu.m, preferably 1-10 .mu.m. Other preferred ranges are 2-15
.mu.m, more preferably 2-12 .mu.m and most preferably 8-10
.mu.m.
[0062] Pit dimensions are measured in accordance with the method
described herein.
[0063] Preferably the pits have an average depth in the range 0.5-8
.mu.m. Suitably the average depth is in the range 1-6 .mu.m,
preferably 1-5 .mu.m and most preferably 1-4 .mu.m.
[0064] Suitably the % coverage of the pits on the inner surface is
the range 20% to 80%, preferably 30% to 70%, more preferably 40% to
60% and most preferably 45% to 55%. A particularly preferred %
coverage is about 50%. Measurement of % coverage is discussed
below.
[0065] Preferably the surface is the luminal surface of a
conduit.
[0066] Suitably, the pits on the surface are formed by film
formation as described herein. In particular, the present inventors
have found that film formation using DCM provides a particularly
desirable distribution and/or size of pits.
[0067] Preferably the inner surface has an average surface
roughness (Ra) of at least 1 .mu.m, more preferably at least 2
.mu.m, more preferably at 2.5 .mu.m and most preferably at least
2.75 .mu.m. Suitably the average surface roughness is no more than
5 .mu.m. Measurement of average surface roughness is discussed
below.
[0068] Preferably the scaffold comprises a surface which in use is
not exposed to a peripheral nerve growth volume. This is referred
to herein as the outer surface of the scaffold (e.g. the outer
surface of a conduit). Preferably, in the case where the scaffold
is formed from a film, the outer surface is the surface of the film
that was in contact with the surface on which the film was cast
(e.g. a glass surface).
[0069] Suitably the outer surface is substantially free of pits.
However, if such pits are present, preferably they have an average
diameter in the range 1-5 .mu.m. Suitably the average diameter is
in the range 1-3 .mu.m.
[0070] Suitably, if pits are present on the outer surface, they
should have a small depth, preferably an average depth of less than
1 .mu.m, more preferably less than 0.75 .mu.m, more preferably less
than 0.5 .mu.m.
[0071] The present inventors have found that it is desirable for
the outer surface to be smoother than the inner (luminal) surface.
Thus, suitably, the surface roughness of the inner surface of the
scaffold (e.g. conduit) is greater than the surface roughness of
the outer surface.
[0072] In particular, the present inventors have found that a low
density of pits on the outer surface is desirable.
[0073] Preferably the inner surface of the scaffold (e.g. a
conduit) includes nanopits, suitably the nanopits have a depth in
the range 50-800 nm, preferably 50-500 nm. Suitably these nanopits
are in addition to the pits discussed above.
[0074] Suitably the scaffold is made using a film and the nanopits
are formed during film formation. In particular, the nanopits may
be formed during solvent evaporation from the film. Alternatively
or additionally, the nanopits may be formed by alkaline treatment
(e.g. NaOH treatment).
[0075] Preferably the outer surface has an average surface
roughness (Ra) of less than 2 .mu.m, more preferably less than 1.5
.mu.m, more preferably less than 1 .mu.m and most preferably less
than 0.5 .mu.m.
[0076] Preferably the surface roughness of the outer surface is
less than the surface roughness of the inner surface. Suitably, the
difference between the surface roughness of the inner surface and
the outer surface is at least 0.5 .mu.m, preferably at least 1
.mu.m, and most preferably at least 1.5 .mu.m.
[0077] When the scaffold is a conduit, it is preferred that the
conduit wall does not include any pores extending through the
thickness of the wall (through holes). This arrangement has been
found to provide advantages because it prevents the escape of the
regenerating axons from the conduit. It may also prevent ingrowth
of fibrous tissues which can lead to unwanted scarring. This may
assist in providing a controlled environment within the conduit for
nerve repair.
[0078] However, a small number of such pores can be present, for
example no more than 5% of the surface area comprises such pores.
Preferably no more than 2% and more preferably no more than 1% of
the surface area comprises such pores. Suitably, if such pores are
present, they have a diameter not larger than 15 .mu.m. Preferably
they have a diameter in the range 1-10 .mu.m. If present, these
pores can assist in avoiding the building up of pressure resulting
from fluid retention.
[0079] Preferably the film used to form the scaffold has a tensile
strength of at least 5 MPa, more preferably at least 8 MPa, more
preferably at least 10 MPa and most preferably at least 15 MPa.
[0080] Preferably the film used to form the scaffold has a Young's
modulus of at least 80 MPa, more preferably at least 100 MPa, more
preferably at least 110 MPa and most preferably at least 120 MPa.
Preferably the Young's modulus is no more than 200 MPa and more
preferably no more than 180 MPa.
[0081] Preferably the film used to form the scaffold has a maximum
strain of at least 1 mm/mm.
[0082] Preferably the scaffold is flexible. In embodiments, the
present inventors have found that the PCL scaffold is highly
flexible. This flexibility reduces or avoids irritation to
surrounding tissues.
[0083] In particular, the present inventors have found that a
scaffold comprising PCL provides an excellent combination of
mechanical properties, making it suitable for handling by a
surgeon, whilst providing a surprisingly effective surface
environment for peripheral nerve growth.
[0084] Preferably the scaffold is used to treat peripheral nerve
damage.
[0085] Peripheral nerve damage can be a gap in a peripheral nerve,
i.e. a severed peripheral nerve. Alternatively or additionally,
peripheral nerve damage can be a partially severed peripheral
nerve. Alternatively or additionally, peripheral nerve damage can
be a crushed peripheral nerve.
[0086] Suitably the scaffold provides a microenvironment at the
injured site with protecting and promoting effects for the
regenerating peripheral nerve. For example, it can prevent the
infiltration of fibroblasts and the escape of regenerating
neurites; at the same time it can contain endogenous growth factors
in situ. Therefore, the scaffold is suitable for treating
crushed/damaged peripheral nerves as well as severed peripheral
nerves.
[0087] In particular, the scaffold of the present invention can be
used to treat neurapraxia (nerve nonfunction), axonotmesis (axon
cutting), and neurotmesis (nerve cutting).
[0088] It is envisaged that the scaffold of the present invention
is used to treat some or all of these types of peripheral nerve
damage.
[0089] It particular, the scaffold is preferably used to treat
acute peripheral nerve injury.
[0090] The peripheral nerve damages can occur as a result of
accidental injury, disease or surgical procedures. For example,
peripheral nerve damage can occur as a result of a cut to the hands
or feet, crush injuries, organ transplant, tumour removal,
congenital birth defects or previous attempts to repair peripheral
nerves.
[0091] The scaffold of the present invention can be used to repair
peripheral nerve damage wherever it occurs in the body. Examples of
peripheral nerves that are most frequently damaged include: palmar
digital nerves, median nerves, the ulnar nerve and the radial
nerve. Further examples include the brachial plexus and
musculocutaneous nerves. Yet further examples (in the lower limbs)
include plantar digital nerve, peroneal and the sciatic nerve.
[0092] In embodiments, the scaffold is used to enclose the affected
part of the peripheral nerve (i.e. the damaged portion).
[0093] In other embodiments wherein the peripheral nerve damage
includes a severed peripheral nerve such that there is a gap in the
peripheral nerve, the scaffold is positioned so as to bridge the
gap between the respective proximal and distal ends of the severed
nerve. In preferred embodiments wherein the scaffold is a conduit,
the conduit is positioned so as to provide a guide for peripheral
nerve growth between the axial and distal ends of the severed
nerve.
[0094] The scaffold can be attached to the peripheral nerve by any
means known to the skilled reader. Suitably the scaffold is
attached using a suture. Suitably the suture provides attachment
between the epineurium and the scaffold. Bioglue can also be
used.
[0095] The scaffold can be used to treat peripheral nerve damage in
an animal, including humans and non-humans. Treatment of humans is
particularly preferred.
[0096] To assist in the treatment of peripheral nerve damage, the
scaffold may be used in conjunction with a peripheral nerve cell
growth medium (e.g. a gel matrix). Suitably the peripheral nerve
cell growth medium includes one or both of growth factors and
Schwann cells/differentiated stem cells. Suitably the peripheral
nerve cell growth medium is a transport media for cells and/or
growth factors (i.e. the peripheral nerve cell growth medium is, or
comprises, a transport matrix). Typically the peripheral nerve cell
growth medium is a hydrogel.
[0097] In use, the peripheral nerve cell growth medium (e.g. a gel
matrix) is preferably introduced into the scaffold in situ.
Typically the scaffold is positioned at the site of the peripheral
nerve damage (e.g. after suturing) and then the growth medium (gel
matrix) may be delivered to the scaffold. In preferred embodiments
wherein the scaffold is a conduit, suitably the growth medium is
delivered into the luminal volume of the conduit, optionally
together with cells and/or growth factors. Suitably this is
achieved by injecting the growth medium, for example through the
end of the conduit after suturing.
[0098] While the invention has been discussed above in relation to
a scaffold, the present invention also provides methods and uses
relating to the scaffold.
[0099] In a further aspect, the present invention provides a
peripheral nerve growth conduit, wherein the conduit includes
poly-.epsilon.-caprolactone.
[0100] In a further aspect, the present invention provides a
peripheral nerve growth conduit, wherein the conduit includes
poly-.epsilon.-caprolactone and polylactic acid.
[0101] In a further aspect, the present invention provides a
peripheral nerve growth conduit, wherein the conduit is prepared
using solvent evaporation method wherein the solvent comprises a
halogenated solvent, preferably dichloromethane or chloroform.
[0102] In a further aspect, the present invention provides a
peripheral nerve growth scaffold, wherein at least part of the
surface of the scaffold has been treated with alkali.
[0103] In a further aspect, the present invention provides a
peripheral nerve growth scaffold, wherein at least one surface of
the scaffold includes --COOH and --OH groups.
[0104] In a further aspect, the present invention provides a
peripheral nerve growth scaffold, wherein one surface of the
scaffold includes pits having an average diameter in the range 1-15
.mu.m.
[0105] In a further aspect, the present invention provides a
peripheral nerve growth scaffold, wherein one surface of the
scaffold includes pits having an average depth in the range 1-5
.mu.m.
[0106] In a further aspect, the present invention provides a
peripheral nerve growth conduit, wherein the surface roughness of
the inner surface of the conduit is greater than the surface
roughness of the outer surface.
[0107] In a further aspect, the present invention provides a
peripheral nerve growth conduit, wherein the thickness of the wall
of the conduit is in the range 20-100 .mu.m.
[0108] In a further aspect, the present invention provides a kit
for treating a peripheral nerve in a human or animal, the kit
including a peripheral nerve growth scaffold according to any one
of the preceding claims.
[0109] Preferably, the kit includes the peripheral nerve growth
scaffold in a sterilised package.
[0110] Preferably, the kit includes a plurality of peripheral nerve
growth scaffolds as described herein. More preferably, the
peripheral nerve growth scaffolds vary in size according to one or
more of the following dimensions: scaffold length, scaffold
internal diameter and scaffold wall thickness. A user may then
select the correct size of nerve repair scaffold from the kit to
suit the requirements of a particular nerve repair treatment.
Suitably, each peripheral nerve growth scaffold in the kit is in an
individual sterilised package.
[0111] In a further aspect, the present invention provides use of
poly-.epsilon.-caprolactone (PCL) in a peripheral nerve growth
scaffold.
[0112] In a further aspect, the present invention provides use of
hydroxide to treat the surface of a peripheral nerve growth
scaffold including poly-.epsilon.-caprolactone (PCL). Suitably the
action of the hydroxide encourages growth of peripheral nerves on
said surface.
[0113] In a further aspect, the present invention provides use of
PCL for the manufacture of a peripheral nerve growth scaffold for
treatment of a damaged peripheral nerve.
[0114] In a further aspect, the present invention provides PCL for
use in treating a damaged peripheral nerve.
[0115] In a further aspect, the present invention provides PCLA for
use in a method of treatment of the human or animal body.
[0116] In a further aspect, the present invention provides PCLA for
use in treating a damaged peripheral nerve.
[0117] In a further aspect, the present invention provides a method
of treating a damaged peripheral nerve using PCLA.
[0118] In a further aspect, the present invention provides a method
of treating a damaged peripheral nerve using a peripheral nerve
growth scaffold including PCL.
[0119] In a further aspect, the present invention provides a method
of treating a severed peripheral nerve, the method including the
steps of [0120] (i) providing a peripheral nerve growth scaffold
including PCL, [0121] (ii) coupling a first severed end of the
nerve to a first portion of the scaffold, and [0122] (iii) coupling
a second severed end of the nerve to a second portion of the
scaffold, [0123] wherein the first and second portions of the
scaffold are separated by a growth portion of the scaffold having a
growth surface on which at least one of the first and second
severed ends of the nerve is able to grow in a direction towards
the respective other severed end.
[0124] Suitably the first severed end is the proximal end of the
nerve and the second severed end is the distal end of the severed
nerve.
[0125] Any one or more of the aspects of the present invention may
be combined with any one or more of the other aspects of the
present invention. Similarly, any one or more of the features and
optional features of any of the aspects may be applied to any one
of the other aspects. Thus, the discussion herein of optional and
preferred features may apply to some or all of the aspects. In
particular, optional and preferred features relating to the
scaffold, methods of making the scaffold and methods of using the
scaffold, etc apply to all of the other aspects. Furthermore,
optional and preferred features associated with a method or use may
also apply to a product (e.g. scaffold) and vice versa.
BRIEF DESCRIPTION OF THE DRAWINGS
[0126] Embodiments of the invention and experiments illustrating
the advantages and/or implementation of the invention are described
below, by way of example only, with respect to the accompanying
drawings, in which:
[0127] FIG. 1 shows schematically the heat sealing method preferred
for forming the conduit of the present invention;
[0128] FIG. 2 shows SEM images of the air (inner) (2A) and glass
(outer) (2B) surfaces of a PCL film;
[0129] FIG. 3 shows 3-D AFM images of the air (inner) (3A) surfaces
and glass surface (outer) (3B) of a PCL film;
[0130] FIG. 4 shows SEM images of a cast PCLA film before (4A) and
after (4B) NaOH treatment;
[0131] FIG. 5 shows XPS spectra for dichloromethane cast PCL films
before (5A) and after (5B) NaOH treatment;
[0132] FIG. 6 shows a graph of wettability data for a number of
different materials formed as films;
[0133] FIG. 7 shows a graph of MTS cell attachment data for
NG108-15 cells on different materials;
[0134] FIG. 8 shows a graph of DNA cell attachment data for
NG108-15 cells on different materials;
[0135] FIG. 9 shows a graph of proliferation data for NG108-15
cells on untreated PCL films; NaOH treated PCL films, and PLA;
[0136] FIG. 10 shows the results of Schwann cell proliferation on
treated and untreated PCL films;
[0137] FIG. 11 shows (A) an SEM image of differentiated NG108-15
cells on NaOH treated PCL film; (B) SEM image of (A) at higher
resolution; (C) Confocal microscope image of phalloidin stained
cells; (D) Confocal microscope image of anti-neurofilament antibody
stained cells. Bar=100 .mu.m in (C) and (D).
[0138] FIG. 12 shows (A) an SEM image of Schwann cells growing on
NaOH treated PCL film; (B) Immunohistochemical-stained cells, using
antibody against marker protein S100; (C) Toluidine Blue O stained
Schwann cells.
[0139] FIG. 13 shows images of haematoxylin stained nuclei of
NG108-15 cells on different materials;
[0140] FIG. 14 shows a photograph of a PCL conduit sutured in place
to bridge a 10 mm nerve gap;
[0141] FIG. 15 shows a photograph of the healed wound of a rat 14
days/2 weeks after surgery;
[0142] FIG. 16 shows a photograph of a PCL conduit 14 days/2 weeks
after implantation;
[0143] FIG. 17 shows photographs of a harvested PCL nerve conduit
after 14 days/2 weeks of in vivo testing (A) and regenerated nerve
tissue after the removal of the PCL conduit (B);
[0144] FIG. 18 shows photographs of peripheral nerve regrowth in
the conduit of FIG. 17, being anti-PGP9.5 antibody stained
regenerated nerve fibres (18A) and anti-S100 antibody stained
Schwann cells (18B); and
[0145] FIG. 19 shows an SEM image of the inner surface of a PCL
conduit after 14 days/2 weeks in vivo;
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0146] The term "scaffold" as used herein is well known to the
skilled reader. In particular, a scaffold in the context of the
present invention is a structure adapted for peripheral nerve
growth. Suitably the scaffold promotes or enhances peripheral nerve
growth.
[0147] The term "pit" as used herein means a closed-end pore or
"blind" hole. In short, a "pit" as used herein does not extend all
of the way through the wall of the scaffold.
[0148] The term "nanopit" as used herein means a pit having at
least one dimension on the nano- or sub-.mu.m scale.
Film Formation
[0149] PCL pellets (Sigma-Aldrich) were dissolved in
dichloromethane (3.0%, wt/v) and gentle heating at a temperature of
approximately 50.degree. C. could be used to assist dissolving. PCL
solution was evenly applied onto borosilicate glass slides
(75.times.25 mm.sup.2), which had been degreased with
acetone/ethanol (1:1, v/v).
[0150] Complete solvent evaporation was allowed in a fume cupboard
for at least 48 hours, to provide films with a thickness of 60.+-.5
.mu.m.
[0151] The polymer films were washed in distilled H.sub.2O and
sterilized by UV irradiation for 1 hour prior to in vitro and in
vivo testing.
[0152] Complete solvent evaporation was confirmed by FTIR (Thermo
Nicolet Nexus.TM. FTIR (Cambridge, UK) controlled by OMNIC Software
Version 6.1a), which ensured that no solvent toxic effect would
occur in the subsequent cell growth and in vivo testing.
[0153] Using the same method, a mixture of PCL and PLA was formed
as a film (the "PCLA film"). The weight ratio of PCL to PLA was
4:1.
Alkaline (Hydroxide) Treatment
[0154] PCL films were soaked in 10N NaOH for 1 hour with horizontal
shaking at 150 rpm at room temperature and then rinsed thoroughly
with distilled H.sub.2O to return the pH to neutral (pH 7.2-7.4).
Subsequent XPS analysis (discussed below) confirmed the cleavage of
the ester bond (ester hydrolysis) as follows:
##STR00001##
Molecular Structure of Poly(.epsilon.-Caprolactone)
##STR00002##
[0155] After Alkaline Hydrolysis
[0156] For comparison, a film of PHB was treated with NaOH.
However, the PHB film did not withstand NaOH treatment; it was too
brittle and shattered into pieces.
Conduit Formation
[0157] FIG. 1 illustrates schematically the methodology used to
form the PCL and PCLA conduits. The films 2 were wrapped around a
16G cannula 4, to form a tubular conduit. Sealing of the
overlapping edges of the film was carried out by briefly (several
seconds) pressing the edges on to a hot plate 6 at 60.degree. C. A
thin layer of tin foil was provided (at location 8) between the
outer surface of the conduit and the hot plate. This provided a
durable seal and the resultant tubular conduit was self
supporting.
[0158] The inner (luminal) surface of the PCL and PCLA conduits was
unchanged as a result of the heating step.
Surface Analysis--AFM & SEM
[0159] PCL and PCLA films prepared as described above were imaged
using Atomic Force Microscopy (AFM, Veeco CP II) and Philips XL30
Field Emission Gun Scanning Electron Microscopy (SEM) techniques.
3-D images were created, and dimension of individual pores measured
using IP Image Analysis 2.1 software (Image Processing and Data
Analysis version 2.1.15. TM Microscopes, copyright .COPYRGT.
1998-2001).
[0160] FIG. 2A shows an SEM image of the PCL film, being the "air"
surface of the film that is destined to become the inner (luminal)
surface of the conduit.
[0161] FIG. 2B shows an SEM image of the "glass" surface of the
film, which when formed as the conduit will be the outer
surface.
[0162] It is clear from FIGS. 2A and 2B that the outer surface is
considerably smoother (i.e. has a lower surface roughness) than the
inner surface. In particular, FIG. 2A shows that the inner surface
is pitted and that the plurality of pits have diameters in the
range 1 to 10 .mu.m. FIG. 2B shows that the outer surface has
smaller and shallower pits.
[0163] Indeed, SEM imaging revealed that PCL films comprised pits
on the air surface in the range of 1-10 .mu.m in diameter; the
depth of these pits was between 1-5 .mu.m. The glass (outer)
surface was also pitted, with pores in the diameter of 1-5 .mu.m.
However, the depth of pits on this side of the films was down to
100 nm-800 nm.
[0164] The diameter and depth of the pits for the inner surface of
both PCL and PCLA films are set out in Table 1. Also included is
diameter and depth data for the same surfaces after treatment with
NaOH.
TABLE-US-00001 TABLE 3 Inner surface pit size of PCL and PCLA
films. Samples Pore diameter (.mu.m) Pore depth (.mu.m) PCL 1-10
1-5 PCL (NaOH treated) 1-10 1-5 PCLA 1-8 1-3 PCLA (NaOH treated)
1-8 1-3
[0165] The results in Table 1 show that NaOH treatment didn't
affect the overall morphology of the materials but that some
reduction in the surface roughness was observed. In addition, the
results show that PCLA films have smaller pit size than PCL
films.
[0166] The % coverage of pits on the inner surface is 51%, measured
using SEM image and data and Image J software [2].
[0167] The 3-D image generated from AFM data of the PCL film inner
("air") and outer ("glass") surfaces are shown in FIG. 3. The
scanned area of 3A is 30.times.30 .mu.m.sup.2; for 3B it is
10.times.10 .mu.m.sup.2. The pits ("closed end" holes) can be seen
clearly.
[0168] The average surface roughness (Ra) of the untreated inner
surface is 3.883 .mu.m, and of the NaOH treated surface is 3.041
.mu.m.
[0169] The average surface roughness (Ra) of the outer surface is
0.569 .mu.m and 0.576 .mu.m respectively before and after NaOH
treatment.
[0170] The average surface roughness (Ra) and pit size were
measured using AFM images and IP Image Analysis 2.1 software.
[0171] FIG. 4, being SEM images of a PCLA film before (4A) and
after (4B) treatment with NaOH, shows that the pitted morphology is
maintained after treatment. Nanoscale structure (nanopits) can also
be seen in 4B indicating that NaOH treatment causes formation of
nanopits.
[0172] Measurement of % coverage of pits using Image J software [2]
and SEM image data showed that the % coverage of pits for the PCL
film is 51%, and for the PCLA film it is 35.8%. In addition, the
size of the pits on the PCLA film is smaller than that for the PCL
film.
Surface Analysis--XPS
[0173] X-ray Photoelectron Spectroscopy (XPS, AXIS Ultra) was used
to analyse the chemical and electronic state of the carbon and
oxygen elements existing in the PCL film before and after treatment
with NaOH.
[0174] FIG. 5 shows XPS spectra for a PCL film before (5A) and
after (5B) NaOH treatment. The reduced peak of C--O group confirms
that alkaline hydrolysis has cleaved the ester bond.
Wettability
[0175] The hydrophilicity of the PCL and PCLA films before and
after NaOH treatment was compared by measuring the static contact
angles using Kruss DSA 100 prop Size Analyser. Ten treated or
untreated films were tested and five randomly selected areas were
measured on each film. A glass coverslip was tested for comparison.
The results are reported in Table 2 below, where "--OH" denotes
NaOH treatment.
TABLE-US-00002 TABLE 2 Water contact angle for PCL and PCLA films
("S" designates smooth outer surface; "P" pitted inner (luminal)
surface) Samples Water Content Angle (%) Standard Deviation (%)
PCL-OH-S 36.7 4.65 PCL-OH-P 52.79 10.8 PCL-S 43.81 6.3 PCL-P 64.58
2.8 PCLA-OH-S 61.49 4.6 PCLA-OH-P 74.45 9.2 PCLA-S 69.36 4.2 PCLA-P
76.63 6.5 PLA-P 71.33 3.3 Glass Coverslips 31.25 9.87
[0176] The results are graphed in FIG. 6.
[0177] The results show that the smooth outer surface is more
hydrophilic than the porous inner surface and that NaOH treated
materials are more hydrophilic than the untreated counterparts.
Also that PCL is more hydrophilic than the PCLA composite either
before or after the NaOH treatment.
[0178] For comparison, the wettability of Poly (3-hydroxybutyrate)
(PHB) was tested. PHB (Astra Tech, Sweden) was dissolved into
chloroform at 70.degree. C. and then applied evenly onto the
surface of glass slides. PHB (1% wt/v) film had a contact angle of
80.03.degree..
Mechanical Testing
[0179] The tensile strength, Young's modulus and maximum strain of
PCL and PCLA films were measured, before and after NaOH
treatment.
[0180] Tensile strength is defined as the maximum amount of tensile
stress that a material can be subjected to before failure. Young's
modulus is a measurement of stiffness.
[0181] Maximum strain is measured as the total elongation per unit
length of material subject to same applied stress.
[0182] Tensile strength, Young's modulus and maximum strain were
measured on a mechanical tensile tester (Instron 1122) at
23.+-.1.degree. C., 50%.+-.2% relative humidity. The cross
sectional area was (3.8.times.0.06) mm.sup.2; grip distance was 35
mm; strain rate was set at 50 mm/min and the full scale load 0.005
KN.
[0183] Young's modulus was measured from the initial slopes in the
elastic region and the tensile strength was the average of ultimate
stress at the breaking point of the films.
[0184] The results are set out in Table 3.
TABLE-US-00003 TABLE 3 Mechanical strength of PCL and PCL + PLA
(=PCLA) films, before and after NaOH treatment. Young's Samples (3%
Thickness Max. STR. Max. STN. Modulus weight/volume) (.mu.m) (MPa)
(mm/mm) (MPA) PCL 0.057 16.3 7.67 115.48 PCL (NaOH 0.054 14.98 7.14
118.89 treated) PCL + PLA 0.053 11.59 2.86 175.52 PCl + PLA (NaOH
0.053 10.73 2.44 156.48 treated)
[0185] The results show that mechanical strength of the PCLA film
is lower than that of the PCL film. It is expected that the PCLA
film will have a faster degradation rate than the PCL film. Thus,
the inclusion of 20 wt % PLA has modified the mechanical properties
of PCL and provides a favourable balance in terms of handling ex
vivo (e.g. by a surgeon) and performance in vivo.
[0186] The results also show that PCL films (with or without a PLA
component) can be fabricated at micro-thickness and at the same
time retain mechanical strength and flexibility.
Cell Compatibility Analysis
Cell Source
[0187] The NG108-15 cell line was purchased from ECACC (Porton
Down, UK). Schwann cells were isolated from neonate rats as
previously described [3] and maintained with 63 ng/ml glial growth
factor (GGF) and 10 .mu.M forskolin mitogen supplemented media.
Cell Culture
[0188] NG108-15 cells were maintained in DMEM (Dulbecco's Modified
Eagle's Medium), containing 4.5 g/L glucose; 5% foetal bovine
serum; 1% antibiotics, and supplemented with 1.times.HAT (a liquid
mixture of sodium hypoxanthine, aminopterin and thymidine)
solution, at 37.degree. C. in a 5% CO.sub.2 humidified
atmosphere.
[0189] Schwann cells were cultured in DMEM containing 10% serum and
antibiotics (penicillin 100 IU/ml and streptomycin 100
.mu.g/ml).
Cell Attachment Analysis
[0190] 1 ml of NG108-15 cells (10.sup.5/ml) were seeded onto PCL
and/or PCLA films (3.14 cm.sup.2) and cultured for 3 hours at
37.degree. C. in a 5% CO.sub.2 humidified atmosphere.
[0191] For the MTS assay, films were transferred into fresh cell
culture plates and washed gently twice in 37.degree. C. cell
culture medium to ensure that only attached cells were tested. The
CellTiter 96.RTM.Aqueous One Solution Cell Proliferation Assay
(MTS) (Promega UK) is a colorimetric method for determining the
number of viable cells. The active component is a tetrazolium
compound called MTS which is reduced by cells to a colored formazan
product. The amount of formazan product is directly proportional to
the number of living cells; therefore, cell proliferation or death
can be quantified by reading the plate at 490 nm.
[0192] DNA assay for the attachment of NG108-15 cells was conducted
using the Hoechst stain reagent (Hoechst 33258 from Sigma-Aldrich),
which specifically binds onto DNA and as such can be used to detect
the contents of a sample DNA by plotting a standard
emission-to-content curve. After 3 hours of culturing films were
washed twice in PBS followed by three freeze and thaw cycles in
dH.sub.2O to release the DNA from cells. FLUOstar OPTIMA
fluorescence microplate reader was used to measure the
fluorescence.
[0193] The results of the MTS analysis are shown in FIG. 7. FIG. 7A
shows measured absorbance for the inner surfaces of PCL and PCLA
films, with and without NaOH treatment. FIG. 7B shows cell number
for the inner surfaces of PCL and PCLA films, with and without NaOH
treatment.
[0194] The results show that NaOH treated materials are more
compatible with NG108-15 cells than untreated materials. This is
quantified in Table 4, which provides the ratio (as a %) of the
cell attachment achieved with untreated material compared to
treated material.
TABLE-US-00004 TABLE 4 Comparison of NG108-15 cell attachment on
NaOH treated and untreated PCL and PCLA films (inner surface).
Samples 2 hour 3 hour 4 hour PCL/NaOH treated PCL 39.6% 45.7% 47.5%
PCLA/NaOH treated PCLA 66.9% 77.5% 84.9%
[0195] The data obtained is in keeping with the results from DNA
attachment analysis, discussed below.
[0196] The results of the DNA (Hoechst) analysis are presented in
FIG. 8. The results show that NaOH treated materials are more
compatible with NG108-15 cells than untreated ones and the NaOH
treated pitted surface of PCLA showed the best result. These
results confirmed those of the MTS assay.
Cell Proliferation Analysis
[0197] The proliferation rate of NG108-15 cells on PCL and PCLA
films (both NaOH treated and untreated) was also analyzed using the
MTS method. NG108-15 cells (5000/cm.sup.2) were seeded onto films
in each well of the 12-well plate and cultured as described above.
Another resorbable biomaterial, poly (D,L-lactic acid) (PLA) was
included as a comparison.
[0198] The results are provided in FIG. 9. The results show that in
six days cell number increased approximately 9 fold on PLA films
and NaOH treated PCL films. The effect of NaOH treatment on the PCL
film is remarkable and demonstrates that NaOH treatment of PCL
provides a surface having a significantly enhanced compatibility
for peripheral nerve cells and provides an "active" environment
that encourages peripheral nerve cell proliferation.
[0199] Schwann cell proliferation was also studied using the MTS
method. Schwann cells were grown on NaOH treated and untreated PCL
films cast from DCM. 6000/cm.sup.2 cells were seeded onto the
surface of PCL and NaOH-treated PCL films. Cells were cultured in
DMEM containing 10% serum and antibiotics (penicillin 100 IU/ml and
streptomycin 100 .mu.g/ml). Cell culturing was conducted for 8
days; readings were taken on every second day (antibody staining
was carried out after 7 days of culturing; see below). The results
were graphed in FIG. 10. The results show that Schwann cells
proliferate on PCL regardless of whether or not there has been
hydroxide treatment.
[0200] In vitro testing showed that the PCL and PCLA films, with
and without hydroxide treatment, supported the attachment and
proliferation of both NG108-15 cells and Schwann cells, which are
involved in maintenance of axons and are crucial for neuronal
survival and regeneration. Importantly, NG108-15 cells could also
be induced into differentiated phenotype with long branched
neurites extending across the surface of the material. FIGS. 11A
and 11B show the differentiated NG108-15 cells branching and
extending over the pitted PCL surface. FIG. 11C shows phalloidin
stained cells and FIG. 11D shows anti-neurofilament antibody
stained cells, which confirms proper differentiation. The excellent
neurite elongation and branching indicates good cell-material
compatability.
[0201] FIGS. 12A to C show Schwann cell growth on the NaOH treated
PCL film. FIG. 12A shows a typical bipolar spindle-shaped
phenotype. The immunohistochemical-stained cells shown in FIG. 12B
confirms expression of marker protein and this together with the
Toluidine Blue O stained cells of FIG. 12C indicates excellent
cell-material compatibility.
Haematoxylin Staining
[0202] Images of haematoxylin stained nuclei of NG108-15 cells on
PCL films, NaOH treated PCL films and PLA films (reference) were
obtained after 5 days in culture. The images are shown in FIG. 13.
For each material, experiments were carried out in triplicates and
repeated three times.
[0203] As can be seen from FIG. 13, there is excellent
reproducibility between each of the 3 films for each material.
Furthermore, good levels of cell proliferation are seen for PCL.
Most impressive is the result provided by NaOH treated PCL where
surprisingly high levels of cell proliferation were observed.
Furthermore, the cells are distributed evenly on the surface.
Nerve Re-Growth--In Vivo
[0204] NaOH treated PCL films were cut into rectangular sheets and
rolled around a 16G intravenous cannula (16G Abbocath.RTM.-T,
Abbott Ireland, Sligo, Republic of Ireland). The standardised
internal diameter of the conduits is 1.6 mm, more than 1.5 times
the diameter of rat sciatic nerve, thus allowing space for
post-injury swelling. Conduits were sealed by controlled heating at
60.degree. C. while still mounted on the cannula. Prior to surgical
implantation, the conduits were sterilised using UV radiation.
[0205] All work was conducted in keeping with the terms of the
Animals (Scientific Procedures) Act 1986, and the experimental
design recognised the need to optimise animal welfare.
[0206] Eight-week-old female adult Sprague-Dawley rats (Harlan,
Inc. USA) (weighing between 180-220 g) were anesthetised with
isofluorane (Abbott Laboratories Ltd.). The site for implantation
was shaved and sterilised with surgical alcohol. The left sciatic
nerve of the rat was exposed through a gluteal muscle-splitting
incision at the mid-thigh level after a dorsolateral skin incision
and splitting of the fascia between the gluteus and biceps femoris
muscle. The surrounding tissues were separated and a piece of 8 mm
in length was removed from the sciatic nerve, leaving a 10-mm nerve
gap after retraction of both ends.
[0207] Under an operating microscope (Zeiss.RTM., Germany), the
proximal and distal nerve stumps of the transected nerve were
secured epineurially within the 14 mm long guidance conduit using a
9-0 ETHILON suture. Both the nerve ends were positioned 2 mm from
the conduit ends to ensure the proximal and distal nerve stumps
were separated by a 10 mm gap (20, FIG. 14). A single 4-0 coated
VICRYL was used to suture the muscle and skin. After the operation,
4 .mu.g of buprenorphine (20 .mu.g/kg) was injected into the rats
as an analgesic intramuscularly. The depth of anaesthesia, heart
rate and breathing were checked periodically to ensure the rat was
in a good surgical condition. A total of 9 animals were implanted
in the same manner. The animals were caged in a temperature- and
humidity-controlled room with a 12-hour light/dark cycle. Food and
water was provided immediately.
[0208] 14 days/2 weeks post-operation, the site was well-healed
without any sign of swelling and inflammation (22, FIG. 15). The
animals were killed using Schedule I method.
[0209] FIG. 16 shows that the conduit 20 was integrated with both
proximal 24 and distal 26 stumps of the natural nerve. No severe
inflammatory response was found in all nine animals. The conduits
didn't open or collapse in all samples (n=9). (FIG. 16, Bar=10
mm).
[0210] FIG. 17A shows the harvested PCL peripheral nerve conduit
after 14 days of in vivo testing. FIG. 17B shows the regenerated
nerve tissue after the removal of PCL conduit.
[0211] For immunohistochemical studies, the entire implants with a
2 mm length of proximal and distal nerve were harvested en bloc,
pinned onto a plastic card to avoid shrinkage and marked at the
proximal end. Fixation was carried out in 4% (wt/v)
paraformaldehyde solution for 24 h at 4.degree. C. and then washed
three times with phosphate buffered saline (PBS) containing 15%
sucrose and 0.1% sodium azide.
[0212] Blocks for cryostat sectioning were prepared by rapid
freezing of samples into OCT.TM. mounting medium in liquid
nitrogen. Systematic longitudinal 15 .mu.m transversal sections
were cut using Bright (Model OTE) cryostat instrument at
-23.degree. C. and collected onto glass slides coated with
Vectabond (Vector Laboratories). Samples were dried overnight in
37.degree. C. oven. Immunostaining was performed by using
polyclonal rabbit antibodies directed against protein gene product
(PGP9.5) (Dako, dilution 1:200) in order to identify neurites.
Schwann cells were identified using polyclonal rabbit anti-protein
S100 (Dako, dilution 1:500). Secondary antibody used in the
staining was FITC conjugated anti-rabbit IgG (Vector labs, F1-1000;
1:100).
Schwann Cell Detection (Immunostaining)
[0213] FIG. 18 shows the results of immuno-staining of
neurofilament and Schwann cells in the PCL conduit used in the in
vivo testing discussed above.
[0214] FIG. 18A shows anti-PGP9.5 antibody stained neurofilaments
and FIG. 18B shows anti-S100 antibody stained Schwann cells.
[0215] The results of the preclinical testing show that the
regenerating neurites have grown through the whole length (i.e. 10
mm) of the conduit together with the infiltrated Schwann cells.
[0216] In contrast, the results reported in [1] (using the same
preclinical testing method) show that only a much smaller extent of
nerve re-growth was achieved when a PHB conduit is used. The effect
of fibrin matrix (Tisseel.RTM.) and Schwann cells
(SC)/differentiated mesenchymal stem cells (dMSC) on the
regeneration of peripheral nerves in PHB conduits is shown in Table
5. PHB conduits were used to bridge a 10 mm gap in the left sciatic
nerve of adult Sprague-Dawley rats (Harlan Inc. USA). Regeneration
was analysed by immunohistochemical staining to identify PGP9.5 for
neurofilament and S100 for Schwann cells two weeks
post-implantation.
[0217] The results from [1] are set out in Table 5 below.
TABLE-US-00005 TABLE 5 PHB conduits filled with fibrin gel matrix
and/or cells were used to bridge a 10 mm gap in the left sciatic
nerve of adult Sprague-Dawley rats. PHB PHB Antibodies used for
Empty with fibrin with fibrin immunohistochemical PHB PHB with
matrix- matrix- staining conduit fibrin matrix dMSC SC PGP9.5 1.91
mm* 2.28 mm* 3.16 mm* 3.17 mm* S100 Proximal 2.2 mm 2.4 mm 3.30 mm*
3.40 mm* Distal 1.7 mm 2.1 mm 2.80 mm* 2.91 mm* *Numbers in bold
were accurate data from the original work in [1]. The other four
measurements were extracted from the figure in [1].
[0218] It is clear from the above results that the PCL scaffold of
the present invention is an "active" scaffold in that it encourages
and promotes peripheral nerve growth.
[0219] FIG. 19 shows an SEM image obtained for the inner (luminal)
surface of a PCL conduit after 14 days/2 weeks in vivo. The arrows
are pointing at the regenerated nerve fibres. The SEM image also
serves to show that the pitted surface morphology was not affected
by the heat sealing method used to the form the conduit.
18 Week In Vivo Study
[0220] A 1 cm sciatic nerve gap in adult Sprague-Dawley rats was
created and repaired with either NaOH treated PCL conduits or a
nerve autograft (9 subjects in each group).
[0221] In both groups, 3 rats were prematurely culled due to
autotomy, a commonly reported phenomenon occurring as a result of
the surgical procedure.
[0222] The remaining 6 rats in each group adopted a normal living
style without any visible difference in behaviour. Before
sacrifice, the rats treated with PCL conduits were observed to
support themselves on both hind-limbs, indicative of significant
distal regeneration. This was supported by electrophysiological
measurements.
[0223] Briefly, after induction of anaesthesia (week 18), the
sciatic nerves were exposed from the sciatic notch to the distal
branches emanating from the popliteal fossa. A stimulating
electrode was placed in the proximal nerve segment and a recording
electrode distal to the repair site. In response to the electrical
stimulation, we were able to record action potentials (nerve
conduction) in the sural, medial gastrocnemius and tibial nerve
branches indicating significant regeneration across the nerve
conduit and distal towards the end organs.
[0224] Reinnervation of hind-limb muscles was indicated by recovery
of gastrocnemius muscle weight. In previous studies of nerve
repair, we have shown that peak muscle atrophy (loss of weight)
occurs at 7 weeks post-injury. At this time point, muscle weight on
the operated side was 27.87.+-.3.04% of the contra-lateral
side.
[0225] However, 18 weeks after repair with the PCL conduits, the
muscle weight was significantly (P<0.05) increased to
44.64.+-.4.67% and to 61.37.+-.2.37% (P<0.01) with
autografts.
[0226] These results indicate the capacity of the PCL nerve conduit
to support nerve regeneration and reinnervation comparable to the
gold standard nerve autograft.
[0227] It is to be understood that variants of the above described
examples of the invention in its various aspects, such as would be
readily apparent to the skilled person, may be made without
departing from the scope of the invention in any of its
aspects.
REFERENCES
[0228] A number of publications are cited herein in order to more
fully describe and disclose the invention and the state of the art
to which the invention pertains. Full citations for these
references are provided below. Each of these references is
incorporated herein by reference in its entirety into the present
disclosure, to the same extent as if each individual reference was
specifically and individually indicated to be incorporated by
reference. [0229] [1] Kalbermatten, D. F. et al., "Fibrin matrix
for suspension regenerative cells in an artificial nerve conduit",
Journal of Plastic, Reconstructive & Aesthetic Surgery (2008),
Volume 61, Issue 6, Pages 669-675. [0230] [2] Rasband, W. S., Image
J, U.S. National Institutes of Health, Bethesda, Md., USA,
http://rsb.info.nih.gov/ij/, 1997-2008. [0231] [3] Caddick, J. et
al., "Phenotypic and functional characteristics of mesenchymal stem
cells differentiated along a Schwann cell lineage", Glia 54 (2006),
pp. 840-849.
* * * * *
References