Patent Application
20130184724
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.
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.
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.
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.
Furthermore, the direction in which axons grow along is an important issue and aberrant growth of axons results in neuromas.
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.
Firstly, the present inventors have found that ordered, aligned or directed growth and proliferation of nerve and Schwann cells are important in order to optimise repair of a peripheral nerve and so the nerve conduit should provide an environment in which such behaviour can be achieved.
Secondly, 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.
Thirdly, 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.
Fourthly, 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.
Fifthly, 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 the above criteria are addressed by providing a peripheral nerve growth scaffold that comprises poly-s-caprolactone (PCL) and a surface which comprises microgrooves adapted to encourage nerve and Schwann cell growth. This is based on the inventors' experiments wherein significant ordered nerve and Schwann cell growth occurred in a nerve conduit made from PCL having such microgrooves formed on the luminal (inner) surface of the conduit.
In a first aspect, the present invention provides a peripheral nerve growth scaffold including poly-ε-caprolactone (PCL) and microgrooves on an inner surface of the scaffold.
The present inventors have found that the combination of PCL and microgrooves, when provided as a scaffold, for example a conduit, surprisingly exhibits not only excellent mechanical properties and enhanced biocompatibility with peripheral nerve cells and Schwann cells but also provides a favourable environment for nerve and Schwann cell growth and proliferation. In particular the combination of PCL and microgrooves on an inner or luminal surface of the scaffold provides excellent bulk mechanical and biocompatible properties together with a surface environment adapted for directed growth and proliferation of nerve and Schwann cells. Such directed or aligned growth suitably reduces the rate of formation of neuromas.
Preferably the microgrooves form or are part of a pattern, for example an array of microgrooves. Thus, preferably the inner surface of the scaffold includes a pattern or array of microgrooves. Suitably the pattern or array comprises a plurality of microgrooves arranged side-by-side.
Suitably the microgrooves are aligned along the lengthwise or “long” direction of the scaffold. Thus, suitably, when the scaffold is inserted in vivo so as to bridge a gap in a peripheral nerve the microgrooves are aligned between the respective ends of the damaged nerve. In other words, it is preferred that the microgrooves are aligned in the direction of intended nerve growth. In the case of a tubular conduit scaffold, the microgrooves are preferably aligned substantially parallel to the longitudinal axis of the conduit.
Preferably the microgrooves extend to one or both ends of the scaffold, preferably both ends (the scaffold ends in use being associated with respective ends of the damaged nerve). For example this could be achieved with two sets of microgrooves, each set being associated with one of the scaffold ends, or a single set extending from end to end. Thus, suitably at least some microgrooves are substantially coterminous with the scaffold ends.
Preferably the microgrooves extend along at least 50% of the length of the scaffold, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90% and most preferably substantially all of the length of the scaffold.
Preferably the microgrooves are provided on at least 50% of the inner surface, by area, of the scaffold (e.g. conduit). That is, suitably the pattern comprising the microgrooves (i.e. the microgrooves themselves and the ridges or spaces between the microgrooves) is provided on at least 50% of the inner surface of the scaffold. More preferably the microgrooves are provided on at least 60% of the inner surface of the scaffold, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95% and more preferably substantially all of the inner surface.
The width of the microgrooves and the spacing of the microgrooves (i.e. the ridge between neighbouring microgrooves) can be selected independently. The width and the spacing can be the same or different. Preferably the width and the spacing are different. Suitably the width is larger than the spacing. Preferably the width is at least 1.5 times the spacing, more preferably at least 1.75 times the spacing, more preferably at least 1.9 times the spacing, more preferably at least 2 times the spacing, more preferably at least 2.5 times the spacing, more preferably at least 3 times the spacing, more preferably at least 3.5 times the spacing, more preferably at least 3.75 times the spacing and most preferably at least about 4 times the spacing.
Whilst there is no particular upper limit, a maximum ratio of 10:1, preferably 8:1, preferably about 6:1, more preferably about 5:1 and most preferably about 4.5:1 or 4:1 is preferred.
A particularly preferred range is about (width:spacing) 1.5:1 to 5:1, more preferably about 2:1 to 5:1, more preferably about 2.5:1 to 5:1, more preferably about 3:1 to 5:1, and most preferably about 3.5:1 to 4.5:1.
Preferably the microgrooves are substantially continuous, i.e. unbroken. Nevertheless, in embodiments some breaks or discontinuity may occur without a substantial adverse affect on the performance of the scaffold.
Suitably the width of the microgrooves is at least 2 μm, preferably at least 4 μm, more preferably at least about 5 μm, more preferably at least 8 μm, more preferably at least about 10 μm, more preferably at least 13 μm, more preferably at least 15 μm, more preferably at least 18 μm and most preferably at least about 20 μm. In embodiments, a groove width of about 20 μm was found to provide excellent results. But larger widths are possible, for example up to 50 μm.
Preferably the width of the microgrooves is 50 μm or less, preferably 30 μm or less, more preferably 25 μm or less and most preferably 22 μm or less.
A particularly preferred range for the width of the microgrooves is 2 μm to 30 μm, more preferably 4 μm to 25 μm, more preferably 8 μm to 25 μm and most preferably about 10 μm to about 20 μm.
In embodiments the width is selected from 4 to 6 μm, 8 to 12 μm, 13 to 17 μm and 18 to 22 μm. Widths selected from about 5 μm, about 10 μm, about 15 μm and about 20 μm are particularly preferred.
Suitably the width of each microgroove is substantially the same. Thus, suitably each microgroove in the pattern or array has substantially the same width.
Suitably the spacing between the microgrooves (e.g. the ridge width between the microgrooves, measured from respective ridge edges) is at least 1 μm, preferably at least 2 μm, more preferably at least 3 μm, more preferably at least 4 μm and most preferably at least about 5 μm.
In other embodiments a smaller spacing between the microgrooves is preferred. In embodiments the spacing between the microgrooves is less than 5 μm, optionally less than 4 μm, optionally less than 3 μm, optionally less than 2 μm, optionally less than 1 μm. In some embodiments the spacing between the microgrooves is 0 μm (i.e. the ridge is V-shaped).
Preferably the spacing between the microgrooves is 30 μm or less, more preferably 25 μm or less, more preferably 20 μm or less, more preferably 15 μm or less, more preferably 10 μm or less and most preferably 8 μm or less.
Particularly preferred ranges for the spacing of the microgrooves are 2 μm to 30 μm, 2 μm to 25 μm, 2 μm to 20 μm, 2 μm to 15 μm, 2 μm to 10 μm and 4 μm to 8 μm.
In embodiments, the spacing is selected from 4 μm to 6 μm, 8 μm to 12 μm, 13 to 17 μm and 18 μm to 22 μm. Widths selected from about 5 μm, about 10 μm, about 15 μm and about 20 μm are particularly preferred.
In embodiments, the groove spacing can be larger for deeper grooves. For example, a preferred spacing:depth ratio is 1:1 to 2:1, more preferably 1.2:1 to 1.8:1. In particularly preferred embodiments, the spacing and depths are as follows: spacing about 5 μm depth about 3 μm; spacing about 8 μm depth about 5 μm; spacing about 10 μm depth about 7 μm; and spacing about 15 μm depth about 10 μm.
Suitably the spacing between each pair of microgrooves is substantially the same. Thus, suitably each spacing (e.g. ridge between microgrooves) in the pattern or array is substantially the same.
The depth of the microgrooves can be selected independently of the width and/or spacing of the microgrooves. However, in embodiments, as discussed above, the depth can be related to the spacing and/or width. For example, where etching is used to create the grooves (e.g. by etching a template), groove width may increase as a function of increasing depth.
Suitably the depth (as measured at the deepest point in the case of a microgroove that has a contoured or profiled cross-section) is at least 1 μm, preferably at least 2 μm, more preferably at least 3 μm, more preferably at least 4 μm and most preferably at least about 5 μm.
A suitable upper limit for the depth is 10 μm, preferably 8 μm.
A particularly preferred range for the depth is 4 to 6 μm, with about 5 μm being especially preferred.
A preferred width:depth ratio is in the range 0.5:1 to 0.8:1, preferably 0.65:1 to 0.75:1 and preferably about 0.7:1.
Suitably, the depth of each microgroove is substantially the same. Thus, suitably each microgroove in the pattern or array has substantially the same depth.
A particularly preferred width:spacing:depth ratio is in the range 1.5:1:0.5 to 5:1:1.5, with 2.5:1:0.6 to 4.5:1:0.6 being particularly preferred.
A particularly preferred selection of width, spacing and depths is as follows: (width+spacing+depth) 5+10+3, 5+15+3, 5+20+3, 5+25+3, 5+30+3, 5+40+3 and 5+50+3; 8+10+5, 8+15+5, 8+20+5, 8+25+5, 8+30+5, 8+40+5 and 8+50+5; 10+15+7, 10+20+7, 10+25+7, 10+30+7, 10+40+7 and 10+50+7; and 15+20+10, 15+25+10, 15+30+10, 15+40+10 and 15+50+10.
The cross-section (taken perpendicular to the longitudinal axis of the grooves) or profile of the microgrooves can have any desired shape, for example rectangular (i.e. vertical sides and a flat bottom). Indeed, vertical side walls are preferred in some embodiments.
Nevertheless, the present inventors have found that a microgroove having sloping or non-vertical sides can give rise to particularly good cell growth performance, especially cell alignment. In particular, a V-shaped, especially a truncated V-shaped, cross-section is preferred. Another preferred topography is a V-shaped ridge, wherein the sloping sidewalls of adjacent grooves meant at a point, effectively giving a zero ridge width. Sloping sidewalls are referred to herein as SL-grooves, and the V-shaped ridge variant as SLV-grooves.
Preferably (e.g. in the case of V-shaped or truncated V-shaped configurations) one or both side walls has an angle to the vertical of at least 20°, more preferably at least 30°, more preferably at least 40°, more preferably at least 50° and most preferably about 55°. Preferably both side walls are at an angle selected from the above. Suitably both side walls have the same angle.
In embodiments, one or both side walls has an angle to the vertical in the range of 30° to 50°. Suitably both side walls have an angle to the vertical in the range of 30° to 50°.
In the case of microgrooves having non-vertical side walls, the width of the microgroove as referred to herein is the width at the top of the microgrooves, i.e. generally at the widest point. In such cases, the depth as referred to herein is the deepest point of the microgroove cross-section.
Suitably the microgrooves are straight or linear microgrooves. As noted above, preferably the microgrooves are aligned parallel to the longitudinal axis of the conduit. Suitably there is substantially no branching of the microgrooves. Suitably the microgrooves are discrete microgrooves in the sense that they do not merge or intersect with each other.
Preferably the microgrooves are parallel to each other.
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.
Suitably the inner surface on which the microgrooves are formed comprises PCL, preferably consists essentially, most preferably consists, of PCL.
The PCL as used herein can be PCL homopolymer or PCL copolymer.
If the PCL is present as a PCL copolymer, it is preferred that the PCL monomer is present in an amount of 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 %.
The present inventors have found that the degradation rate 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.
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.
It is preferred that the PLA is provided as a mixture (blend) with the PCL.
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.
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 may be too quick to degrade in vivo. Furthermore, phase separation may occur.
Incorporation of PLA in combination with PCL may provide 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.
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.
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.
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.
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.
Suitably the scaffold is a conduit. Suitably, the conduit provides a luminal space (i.e. the lumen, cavity or channel within the conduit) 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.
Preferably the conduit wall consists essentially, preferably consists, of PCL or a PCL-PLA mixture (blend).
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.
Suitably, the conduit has a circular cross section.
Preferably the conduit is substantially straight. However, the conduit can also be bent or curved.
Preferably the thickness of the conduit walls is in the range 10 μm to 300 μm. Preferably the conduit walls have a thickness in the range 10 μm to 200 μm, more preferably 10 μm to 100 μm, more preferably 20 μm to 100 μm, more preferably 20 μm to 80 μm and most preferably 55 μm to 65μ. A particularly preferred thickness is about 60 μm.
In other embodiments the preferred thickness of the conduit walls is in the range 20 μm to 200 μm, and more preferably 60 μm to 180 μm.
The present inventors have found that a conduit wall thickness as described above provides a good balance between degradation time, mechanical strength and flexibility and it also surprisingly permits microgrooves of a depth as described herein to be formed on the inner surface of the conduit, for example by film casting as discussed herein, without damage to the wall (e.g. film) structure and without significantly changing the mechanical properties of the conduit.
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.
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. As discussed above, preferably the microgrooves extend along at least 50%, preferably substantially all of this length.
As with the length of the scaffold, the width (measured from the outer surface of the scaffold), e.g. the 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.
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. In particular, as discussed below, film formation on a (patterned) template permits efficient and low cost formation of microgrooves whilst retaining good mechanical properties. Surprisingly, in embodiments films can be cast directly on to suitable templates and removed without damage to the film. This is a valuable advantage because it permits rapid and efficient formation of patterned films and provides consistent microgroove patterns.
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. Suitably the surface is the surface of a template as described herein.
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 C1-10 alkanes and alkenes.
Chlorinated solvents are particularly preferred. Chloro-substituted C1-4 alkanes, especially chloro-substituted methane, are especially preferred.
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.
These solvents have been found to be a good solvent for casting on a template: the pattern is accurately and reliably transferred to the film and the film can be readily removed from the template. The surface morphology of the scaffold is discussed below.
Suitably the solvent is heated, for example to a temperature in the range 40-60° C. This may assist in dissolving the PCL.
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%.
Preferably the film is cast onto a template, for example a silicon and/or silica (silicon dioxide) template. The template can be a patterned silicon wafer for example. Suitably the template is cleaned to remove impurities (e.g. degreased) prior to casting.
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.
Typically, after solvent evaporation has been completed, the film is washed. Suitable washing agents include water, preferably distilled water.
Preferably the film is sterilised, for example sterilised using UV radiation, γ radiation or 70% ethanol. Indeed, any suitable known technique for sterilising can be used. UV radiation is preferred.
The present inventors have found that the advantageous properties of a scaffold comprising PCL and microgrooves can be further improved by treating at least one surface comprising the microgrooves 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.
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.
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.
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.
Suitably, the surface of the scaffold that is treated is the 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. 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).
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.
Furthermore, the present inventors have found that treatment with an alkaline composition can increase the hydrophilicity of the surface without significant adverse affect on the microgrooves. 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. SEM studies of the microgroove pattern shows that the microgrooves retain their desired shape and dimensions after alkaline treatment.
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.
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). The present inventors have found that the microgrooves are not adversely affected by this fabrication technique.
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). In such embodiments, only the innermost film need comprise the microgrooves.
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° C., for example about 60° 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, including the microgrooves 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.
Suitably the microgrooved side of the film becomes the luminal or inner surface of the conduit.
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).
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).
Suitably the outer surface is not provided with microgrooves. Indeed, suitably the outer surface is substantially smooth.
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.
However, in embodiments 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 μm. Preferably they have a diameter in the range 1-10 μm. If present, these pores can assist in avoiding the building up of pressure resulting from fluid retention.
Preferably the film used to form the scaffold has a tensile strength of at least 1 MPa.
Preferably the film used to form the scaffold has a Young's modulus of at least 20 MPa.
Preferably the film used to form the scaffold has a maximum strain of at least 1 mm/mm.
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.
In particular, the present inventors have found that a scaffold comprising PCL and having microgrooves on an inner surface provides an excellent combination of mechanical properties, making it suitable for handling by a surgeon, whilst providing a surface environment adapted for nerve and Schwann cell growth and proliferation.
Preferably the scaffold is used to treat peripheral nerve damage.
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.
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.
In particular, the scaffold of the present invention can be used to treat neurapraxia (nerve nonfunction), axonotmesis (axon cutting), and neurotmesis (nerve cutting).
It is envisaged that the scaffold of the present invention is used to treat some or all of these types of peripheral nerve damage.
It particular, the scaffold is preferably used to treat acute peripheral nerve injury.
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.
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.
In embodiments, the scaffold is used to enclose the affected part of the peripheral nerve (i.e. the damaged portion).
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 proximal and distal ends of the severed nerve.
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.
The scaffold can be used to treat peripheral nerve damage in an animal, including humans and non-humans. Treatment of humans is particularly preferred.
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). For example, the peripheral nerve cell growth medium includes growth factors and Schwann 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.
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.
While the invention has been discussed above in relation to a scaffold, the present invention also provides methods and uses relating to the scaffold.
In a further aspect, the present invention provides a peripheral nerve growth conduit, wherein the conduit includes poly-ε-caprolactone and microgrooves on a luminal (inner) surface of the conduit.
In a further aspect, the present invention provides a peripheral nerve growth conduit, wherein the conduit includes poly-ε-caprolactone and polylactic acid and microgrooves on a luminal (inner) surface of the conduit.
In a further aspect, the present invention provides a peripheral nerve growth conduit as described herein, wherein the conduit is prepared by solvent evaporation wherein the solvent comprises a halogenated solvent, preferably dichloromethane or chloroform.
In a further aspect, the present invention provides a peripheral nerve growth scaffold as described herein, wherein at least part of the surface of the scaffold has been treated with alkali.
In a further aspect, the present invention provides a peripheral nerve growth scaffold as described herein, wherein at least one surface of the scaffold includes —COOH and —OH groups.
In a further aspect, the present invention provides a peripheral nerve growth conduit as described herein, wherein the thickness of the wall of the conduit is in the range 20-100 μm.
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 as described herein.
Preferably, the kit includes the peripheral nerve growth scaffold in a sterilised package.
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.
In a further aspect, the present invention provides use of poly-ε-caprolactone (PCL) in a peripheral nerve growth scaffold as described herein.
In a further aspect, the present invention provides use of hydroxide to treat the surface of a peripheral nerve growth scaffold as described herein. Suitably the action of the hydroxide encourages growth of peripheral nerves on said surface.
In a further aspect, the present invention provides use of PCL for the manufacture of a peripheral nerve growth scaffold as described herein for treatment of a damaged peripheral nerve.
In a further aspect, the present invention provides PCL for use in treating a damaged peripheral nerve using a peripheral nerve growth scaffold as described herein.
In a further aspect, the present invention provides a method of treating a damaged peripheral nerve using a peripheral nerve growth scaffold as described herein.
In a further aspect, the present invention provides a method of treating a severed peripheral nerve, the method including the steps of
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.
In a further aspect, the present invention provides a method of making a biocompatible polymer material having a patterned surface, the method comprising the steps of
Suitably the pattern is a pattern of microgrooves as described herein.
Suitably the biocompatible polymer material is a biocompatible polymer, preferably a biodegradable polymer, preferably PCL. Suitable PCL-containing polymers are described herein. Preferably it consists essentially of and most preferably consists of PCL.
Suitably the method comprises the step of (C) removing the biocompatible material from the template. Preferably the biocompatible polymer material is treated with an alkaline composition, suitably as described herein.
Suitably the method comprises the step of (D) forming a nerve repair scaffold from the patterned biocompatible polymer material. Suitably the scaffold is formed so that the patterned surface is an inner surface of the scaffold, e.g. a luminal or inner surface of a conduit. Step (D) may comprise rolling up the patterned biocompatible polymer material. In embodiments the heat sealing method described herein is used to form the scaffold. Suitably the scaffold is sterilised.
Thus, preferably, the method is a method of making a nerve repair scaffold, suitably a peripheral nerve repair scaffold, more suitably a peripheral nerve repair scaffold as described herein.
Suitably step (B) includes forming a film of the biocompatible polymer material on the template. Suitable film thicknesses are as disclosed herein.
Preferably step (B) comprises (i) apply a solution or dispersion of the biocompatible polymer material (e.g. PCL) in a solvent to the template, and (ii) removing solvent from the solution or dispersion. In other words, a casting method to form a film is preferred.
Suitable solvents are described herein.
In embodiments, the solution is a solution of PCL. Suitably the solvent is a halogenated solvent as described herein, preferably DCM. The solution may contain other polymers, as discussed herein.
Preferably the biocompatible polymer solution (e.g. PCL solution) is applied directly to the template, i.e. without an intervening layer.
Typically the template comprises a substrate. Suitably the substrate is an inorganic material, preferably silicon. In embodiments the substrate is a silicon wafer.
Suitably the method comprises a photolithography process. Preferably the method comprises an etching process, preferably photolithography and etching processes.
Preferably step (A) includes forming a barrier layer on the surface of the substrate (e.g. a silicon wafer). Preferably the barrier layer comprises silicon dioxide. The barrier layer, e.g. silicon dioxide, can be thermally deposited onto the surface of the substrate, for example by heating the silicon substrate, for example at 1100 to 1200° C. in a furnace.
Suitably a photoresist is formed on the substrate or a barrier layer, preferably on a barrier layer. Suitable photoresists are known to the skilled reader. Typically the photoresist is applied by spin coating. In embodiments the photoresist-coated substrate is heated, for example at about 110° C., to remove solvent.
A photomask is preferably used to demarcate the desired pattern. Typically the photoresist is exposed to radiation, typically UV radiation, through the photomask.
Suitably the solubilised areas of photoresist are removed, typically with a developer solution.
Preferably a post-developed heating step is carried out, for example to improve durability of the remaining photoresist.
Suitably the barrier layer (e.g. silicon dioxide) is etched, the pattern of etching being dictated by the pattern of the remaining photoresist. A preferred etching solution is HF (aq), preferably buffered, for example with NH4F.
In embodiments, etching comprises plasma etching, for example Deep Reactive Ion Etching (DRIE).
After etching of the barrier layer preferably the remaining photoresist is removed by solvent washing, for example using acetone.
In embodiments the resultant material, comprising the substrate with etched (patterned) barrier layer, is then etched. That is, the substrate is selectively etched in areas where it is not covered by the barrier layer. A suitably etching material is KOH.
Suitably etching of the substrate is carried out for sufficient time to achieve the desired pattern depth in the substrate. Preferred dimensions for the microgrooves are discussed above.
The etched substrate is then typically washed with a solvent, suitably isopropanol.
Thus, embodiments of the present invention apply template-forming techniques from semi-conductor chip manufacturing to biocompatible materials such as PCL to form microgroove patterns.
In a further aspect, the present invention provides a method of making a peripheral nerve repair conduit comprising PCL and having microgrooves on a luminal surface of the conduit, the method comprising the steps of
Preferably the PCL solution is applied directly to the template, i.e. without an intervening layer.
In embodiments of the solvent casting method described herein, biocompatible polymers can be simply dissolved and poured onto a master (template) cured, and removed. Many replicates (copies) can be fabricated from a single patterned master; as such the cost and processing time can be minimized.
In a further aspect, the present invention provides a peripheral nerve growth scaffold including a biocompatible material and microgrooves on an inner surface of the scaffold, wherein the microgrooves have a width:spacing ratio in the range 1.5:1 to 8:1.
Preferably the biocompatible material is a biocompatible polymer, preferably a bioresorbable polymer. Particularly preferred is PCL.
Preferably the width:spacing ratio is in the range 2:1 to 5:1.
Preferably the width is in the range 10 μm to 30 μm, more preferably 18 μm to 22 μm, for example about 20 μm.
Preferably the spacing is in the range 5 μm to 15 μm, more preferably 5 μm to 10 μm, more preferably 3 μm to 7 μm, for example about 5 μm, about 8 μm or about 10 μm.
Suitably the depth of the microgrooves is in the range 3 μm to 10 μm, more preferably 3 μm to 7 μm, for example about 5 μm.
Suitably the microgrooves are as described herein.
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.
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:
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.
The term “microgroove” as used herein means a groove having a width of at least 1 μm. In this connection, the skilled reader will understand “groove” to include elongate channels and trenches formed on or in the surface of the scaffold material.
PCL pellets (Sigma-Aldrich average Mn ˜80,000 g/mol) were dissolved in dichloromethane (3.0%, wt/v) and gentle heating at a temperature of approximately 50° C. could be used to assist dissolving. PCL solution was evenly applied onto the desired template, which had been cleaned in Decon, dH2O, and then isopropanol, followed by drying with an N2 gun. If appropriate, the template can be vacuum dried before fabrication of polymer films.
Complete solvent evaporation was allowed in a fume cupboard for at least 48 hours, to provide films with a thickness of 60±5 μm.
The polymer films were peeled away from the template, washed in distilled H2O and sterilized by UV irradiation for 1 hour prior to testing.
Complete solvent evaporation was confirmed by FTIR (Thermo Nicolet Nexus™ FTIR (Cambridge, UK) controlled by OMNIC Software Version 6.1a), which ensured that no solvent toxic effect would occur in the subsequent cell growth testing.
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.
Chrome photo mask was manufactured by Photronics UK Ltd.
Mask 1: A chrome photo mask was designed with 16 different areas, each comprising a microgroove pattern with different groove and spacing sizes (
Mask 2: Based on the results obtained from Mask 1, a new mask with 4 selected patterns (5+5; 10 ; 20+20, and 5+20) and enlarged pattern area (2.7×2.7 cm2) was produced for fabricating implantable nerve conduits.
To make the desired patterns, n-type silicon wafers (100) (4-inch in diameter with a <100> crystallographic orientation) were firstly cleaned to remove particulate matter from the surface as well as any other traces of impurities. The cleaning procedure used a mixture of sulphuric acid and hydrogen peroxide. Following cleaning, a barrier layer (approximately 500 nm in thickness) of silicon dioxide (102) was thermally deposited onto the surface of the substrate by placing the silicon wafer in an 1100-1200° C. furnace for 40 mins (step (1) of
Masked by photoresist, a solution referred to as buffered HF containing concentrated HF (49%) and a buffering salt (NH4F) in the ratio of 1:4 was used to etch through the layer of SiO2 (step (5) of
The resulting template was washed in Decon, dH2O and isopropanol and dried using a nitrogen gun. To form films, PCL solution (3% wt/v) was directly cast onto the surface of the patterned wafer (step (8) of
DRIE method was utilised to produce grooved silicon template with upright walls (i.e. substantially non-sloping sides). The desired pattern was produced on a silicon wafer by photolithography then the samples were etched using the DRIE process. In detail, silicon wafer (100) with silicon nitride barrier layer was cut in required dimension (2.7 cm*2.7 cm). Then the sample was cleaned with acetone, IPA and oxygen plasma cleaning process. The photoresist, S1805, was then applied to the silicon substrate by spin coating. The photoresist-coated silicon was then heated to remove the remaining solvent. The substrate was exposed to UV radiation through the photomask. The developer, MF319, removed the exposed photo resist. Then the hard baking step was carried out to bake the photoresist on the substrate.
DRIE is a dry etching process to fabricate deep, steep-sided trenches in wafers, which uses reactive plasma to etch. Reactive particles diffuse to the silicon wafer and react and adsorb at the surface then the volatile reaction particles diffuse away from the surface. The main technique in DRIE is the Bosch process.
The Bosch process consists of passivation and etching cycles. At the passivation step, C4F8 based plasma is deposited to a few monolayers thickness across the silicon surface. Then in the etching step, SF6 is introduced to the system to isotopically etch the silicon. At the beginning of the etching step the ions bombard the surface and remove fluorocarbon from the surfaces parallel to the wafer surface. After selective etching, the unprotected silicon surface is exposed to reactive fluorine-based species and isotropically etched, whilst the vertical walls protected from etching by fluorocarbon polymer. By controlling the passivation and etch time during the process the degree of lateral etch is reduced. Furthermore, this process can lead to a smooth vertical wall in the wafer.
The resulting template was washed in Decon, dH2O and isopropanol and dried using a nitrogen gun. To form films, PCL solution (3% wt/v) was directly cast onto the surface of the patterned wafer (step (8) of
PCL films were soaked in 10/V NaOH for 1 hour with horizontal shaking at 150 rpm at room temperature and then rinsed thoroughly with distilled H2O 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:
The inner (luminal) surface of the PCL conduits including the microgrooves was unchanged as a result of the heating step.
PCL 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.
In the case of analysis of films formed using the patterning process, a scanning frequency of approximately 0.4 kHz was used in tapping mode for the AFM imaging. The scanned area was 80×80 μm2. Samples were gold coated in a sputter coater (Edwards Ltd.) and mounted onto Aluminium stubs (Agar Scientific Ltd.) prior to SEM imaging and the microscope was operated at 5 kV with a 20 mm working distance.
The photolithography and subsequent HF and KOH etching steps were found to be efficient approaches to produce the silicon master template for the surface patterning of PCL films (
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.
XPS spectra for a PCL film before and after NaOH treatment showed a reduced peak for the C—O group, which confirms that alkaline hydrolysis has cleaved the ester bond.
The tensile strength, Young's modulus and maximum strain of PCL were measured, before and after NaOH treatment.
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. Maximum strain is measured as the total elongation per unit length of material subject to same applied stress.
The mechanical property of films was measured by Instron 1122 at 23±1° C. temperature, 50%±2% relative humidity. The grip distance and crosshead speed were set on 10 mm and 50 mm/min, respectively. The full load of 0.02KN was used for PCL films and 0.01 KN for PCL/PLA films.
The films were cast on silicon template and were approximately 25×25 mm2 in size. The groove on silicon substrates has five different dimensions which illustrated as width-space-depth (μm); 10-10-5, 10-20-5, 20-20-3 and 20-20-5. 10 specimens were prepared for each pattern and their width and thickness were measured by digital micrometer.
The results are set out in the following tables.
The results show that microgrooved PCL and PCL/PLA films can be fabricated at micro-thickness and at the same time retain mechanical strength and flexibility.
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 μM forskolin mitogen supplemented media.
NG108-15 cells were maintained in high glucose (4.5 g/ml) DMEM (Dulbecco's Modified Eagle's Medium) medium, containing 5% fetal bovine serum, 1% Penicillin/Streptomycin, and supplemented with 1×HAT solution at 37° C. in a 5% CO2 humidified atmosphere. To induce differentiation, NG108-15 cells (1000/cm2) were cultured onto patterned PCL films in medium containing 10% serum and 1% antibiotics for 2 days. On day 3, cell culture medium was replaced with medium containing 1.5% serum, 1% antibiotics and 1 mM dibutyryl-cAMP. Cells were cultured for another 5 days. Schwann cells were cultured at the density of 104/cm2 for 6 days before SEM and antibody staining (see below) were conducted.
Cells growing on patterned PCL films were prepared for SEM imaging using hexamethyldisilazane (HMDS) chemical drying method [4]. Cells were rinsed twice in PBS and then fixed with 1.5% glutaraldehyde (24.8% stock from TAAB Laboratories) in 0.1M phosphate buffer (containing NaH2PO4.2H2O 15.6 g and Na2HPO4 14.2 g in 1000 ml dH2O; pH=7.3) for 30 mins at room temperature. After fixation, cells were rinsed twice with phosphate buffer and dehydrated through a series of increasing concentrations of ethanol solutions (50%, 70%, 90%, and 100%) with 2×5 mins in each solution. Films were then dried in HMDS for 2×5 mins.
Finally, HMDS was removed and the films left overnight in fume cupboard for the fully evaporation of HMDS. Prior to SEM imaging, films were mounted onto an aluminium stub and sputter coated with gold.
For immunocytochemical analysis, NG108-15 cells or Schwann cells growing on PCL films were washed gently in PBS and fixed with 4% paraformaldehyde for 30 mins. Following fixation, cells were washed twice in PBS and then permeabilised with 0.2% Triton X-100 for 20 mins. After washing in PBS, a blocking solution of 5% normal goat serum was incubated with the cells for 1 hour at room temperature. The blocking solution was removed and mouse monoclonal anti-neurofilament antibody was used as primary antibody for NG108-15 cells (1:500 dilution; Abeam plc) or rabbit polyclonal S100 for Schwann cells (1:500 dilution; Dako cytomation). Incubation was conducted at 4° C. overnight.
Following thorough washing in PBS (3×10 mins) to remove non-specifically bound antibodies, goat anti-mouse CY3 secondary antibody for NG108-15 cells (1:200 dilution; Amersham plc) or goat anti-rabbit FITC-conjugated secondary antibody for Schwann cells (1:100 dilution; Vector Labs) was incubated with the cells for 1 hour in dark. Films were then washed again (3×10 mins) before they were mounted onto microscope slides.
ProLong® Gold antifade reagent with DAPI (Invitrogen/Gibco) was used to prevent the samples from bleaching and to counter-stain the nuclei of the cells. Pictures were taken using Nikon Eclipse 50i fluorescence microscope.
Resazurin fluorometric assay was carried out to compare the proliferation rate of NG108-15 cells on micro-grooved polymer films and the porous/non-grooved samples. Briefly, 105 cells (in 100 μl suspension) were seeded onto each sample film (25×25 mm2) and incubated (37° C., 5% CO2, humidified incubator) for 2 hours before the wells were topped up with 2 mls of cell culture medium. To avoid the attachment of cells onto the cell culture plates, ultra low cell attachment plates (Corning) were used in this experiment. On day 1, day 3, day 5 and day 7, cell culture medium was removed and 200 μl of Resazurin solution (Resazurin Sodium Salt (Sigma 199303-5G; 0.125% wt/v) in PBS), was added into each well of the 6-well plates together with 2 ml of fresh cell culture medium. Samples were incubated (37° C., 5% CO2, humidified incubator) for 2 hours before the absorbance reading was taken at 540 nm and 630 nm with a standard spectrophotometer. Cell culture medium was used as control.
The results are set out in the table below. Results on day 3, day 5 and day 7 showed that NG108-15 cells had a significantly higher proliferation rate on micro-grooved polymer films (p<0.05), including both micro-grooved PCL homopolymer films and the PCL/PLA blend films, than that on the micro-porous scaffolds. In addition, cell proliferation rate was higher on PCL/PLA blend films than that on PCL homopolymer films, regardless of the surface patterning.
The fraction of cells within ±10° of the patterning direction is the definition adopted to quantify cell alignment.
Although no cell alignment was observed on PCL films with a groove depth of 500 nm, both Schwann cells and NG108-15 cells were found to be successfully aligned along the 5 μm deep grooves.
It was observed that both NG108-15 cells and Schwann cells had a tendency to settle in the grooves in all fourteen different patterns, with the average percentage of neural cells in grooves being over 58.2% and Schwann cells 69%. In the case of NG108-15, when the widths of the grooves were larger, more cells were found in the grooves. This effect was clearly seen in patterns of 5 μm+20 μm, 10 μm+20 μm, 10 μm+15 μm and 5 μm+15 μm; however, when the widths of the spacing were also large (e.g. 20 μm+20 μm and 20 μm+10 μm), this type of cell response diminished (
The design of the pattern with small spacings (5 μm) and large grooves (20 μm) in mask 2 was based on the observation that NG108-15 cells tended to align in the grooves and the chance for the regenerating nerve fibers to grow across the grooves was therefore reduced. This is believed to be unique in our design because other researchers previously adopted the same sizes for both the grooves and the spacings. However, no such effect was found for Schwann cells. This was speculated to be due to the smaller size of Schwann cells, which could align the cells in smaller grooves i.e. 10 μm and 5 μm.
To make implantable conduits, PCL films with enlarged pattern area (2.7×2.7 cm2) were produced from Mask 2. Data obtained from these substrates are shown in the Table below. Differentiated NG108-15 cells were used in this experiment and it was shown that patterns 5 μm+20 μm and 10 μm+10 μm were comparable with each other in performance and 20 μm+20 μm was significantly worse (p<0.05) both in terms of the average neurite length and the alignment rate. Statistics was conducted using Student's T-test: two-samples assuming equal variance.
The results of the present investigation with differentiated NG108-15 cells, showed that both the ridge width and the groove width played important roles in guiding and deciding the orientation of regenerating nerve fibers. In particular the present study, small ridge width (5 μm) combined with big groove width (10 μm, 15 μm and 20 μm) were found to produce neurites that were considerably longer as compared to the ridge and groove widths being the same and the cross-growth of nerve fibres across the substrata was also reduced.
Embodiments of the present invention provide a simple and efficient approach to producing micro-grooves on the surface of biodegradable ultra-thin polymer films, which can be easily rolled up to form implantable nerve conduits. The use of grooves that are wider than the ridges provides enhanced contact guidance of regenerating axons. With the added advantages of the ultra-thin polymer scaffolds, such as excellent handle-ability, low cost and easy availability, these patterned conduits are adapted for peripheral nerve repair.
In order to further test cell attachment and alignment to the smooth, pitted and grooved (square grooves with vertical walls (SQ); grooves with sloping sidewalls (SL); and grooves with sloping sidewalls and zero ridge width, i.e. V-shaped “ridges” (SLV)) PCL/PLA (4:1) films, differentiated adipose stem cells (dASCs) were seeded and allowed to adhere for 24 hours. Adherent cells were fixed with 4% PFA/PBS and stained with Alexa Fluor 488® phalloidin (Invitrogen) and mounted with vectashield containing DAPI (Vector). Cells were imaged using a fluorescent microscope and image-pro plus software. For quantitative analysis of cell attachment 5 separate fields of view were captured for each sample and average cell counts calculated. In addition, cell proliferation/cytotoxicity experiments were carried out using the AlamarBlue® assay (abD Serotec) to test cell proliferation of dASCs on grooved and non-grooved PCL/PLA (4:1) films. Absorbance readings were taken at 570 nm and 600 nm using a spectrophotometer and calculated as percentage reduction of AlamarBlue®. All experiments were performed in triplicate and the results plotted using GraphPad Prism software. Statistical analysis was carried out using one-way ANOVA followed by Bonferroni's Multiple Comparison Test.
The grooved PCL/PLA (4:1) surfaces were prepared as described above. A silicon substrate having grooves with width-space-depth dimensions of 10 μm-10 μm-5 μm was used to form the PCL/PLA (4:1) surfaces. This silicon substrate used to form the SQ-grooved PCL/PLA (4:1) surface had vertical side walls, resulting in a SQ-grooved PCL/PLA (4:1) surface having grooves with width-space-depth dimensions of 10 μm-10 μm-5 μm. The silicon substrate used to form the SL-grooved PCL/PLA (4:1) surface had side walls at an angle of 30° to 50° to the vertical, resulting in a SL-grooved PCL/PLA (4:1) surface having grooves of width 15 μm to 20 μm, spacing of 3 μm to 6 μm and depth of 5 μm. The silicon substrate used to form the SLV-grooved PCL/PLA (4:1) surface had side walls at an angle of 30° to 50° to the vertical, resulting in a SLV-grooved PCL/PLA (4:1) surface having grooves of width 15 μm to 20 μm, spacing of 0 μm (V-shaped ridges) and depth of 5 μm. Of course, other width-space-depth dimensions may be used, as discussed above.
A first observation is that statistically significant increases in cell attachment are achieved for the pitted PCL/PLA (4:1) surface when compared to the smooth PCL/PLA (4:1) surface (
Taken together these results demonstrate that topographical modifications to PCL/PLA surfaces, especially the sloping wall variants SL and SLV, significantly enhance dASC attachment and proliferation. Furthermore these modifications result in the highly ordered alignment of dASCs to a spindle shaped Schwann cell-like morphology that resemble longitudinally aligned bands of Bungner found in damaged nerves following wallerian degeneration that provide essential pathways for guided axonal growth. Thus these topographical features will improve axonal guidance and enhance nerve regeneration in PCL/PLA nerve conduits.
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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1015828.5 | Sep 2010 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB2011/001358 | 9/16/2011 | WO | 00 | 3/18/2013 |
