EP2948155A1

EP2948155A1 - Biomaterial comprising poly(trimethylene carbonate-co-e-caprolactone) for promoting axonall growth and neuronal regeneration

Info

Publication number: EP2948155A1
Application number: EP14710658.7A
Authority: EP
Inventors: Ana Paula PÊGO; Daniela ROCHA
Original assignee: Ineb-instituto de Engenharia Biomedica
Current assignee: INEB-INSTITUTO NACIONAL DE ENGENHARIA BIOMEDICA
Priority date: 2013-01-28
Filing date: 2014-01-28
Publication date: 2015-12-02
Also published as: GB201301461D0; WO2014116132A1

Abstract

Mammalian central nervous system (CNS) neurons do not regenerate after injury due to the inhibitory environment formed by the glial scar, largely constituted by myelin debris. The use of biomaterials to bridge the lesion area and the creation of an environment favoring axonal regeneration is an appealing approach, currently under investigation. This work aimed at assessing the suitability of three candidate polymers - poly(?-caprolactone), poly(trimethylene carbonate-co- ?-caprolactone) (P(TMC-CL)) (11:89 mol%) and poly(trimethylene carbonate) - with the final goal of using these materials in medicine, namely in the development of conduits to promote spinal cord regeneration. Cortical neurons cultured on P(TMC-CL) in the presence of myelin were able to tame myelin inhibition in comparison with the control condition (glass substrate). This effect was found to be mediated by the glycogen synthase kinase 3p (GSK3p) signaling pathway with impact on the collapsin response mediator protein 4 (CRMP4), suggesting that nanomechanical properties were implicated in this process. The obtained results indicate P(TMC-CL) as a promising material for CNS regenerative applications as it promotes axonal growth, taming myelin inhibition.

Description

BIOM ATE RIAL COMPRISING POLY(TRIMETHYLENE CARBONATE-CO-E-CAPROLACTONE) FOR PROMOTING AXONAL GROWTH AND NEURONAL REGENERATION

Field of the Invention

The present invention relates to a biomaterial for use in medicine, in particular for promoting neuronal growth in the central nervous system, wherein the biomaterial comprises poly(trimethylene carbonate-co-8-caprolactone) (P(TMC-CL)). This biomaterial can be used in the regeneration of neurons in the central nervous system (CNS), such as the spinal cord.

Background to the Invention

When an injury is inflicted to the spinal cord, the blood-brain barrier (BBB) breaks down locally and a massive infiltration of immune cells is observed. After the initial mechanical trauma (primary damage), cell damage is triggered such that within hours the injury site and the surrounding haemorrhagic areas begin to undergo necrosis (secondary damage), a progressive process that can last for several days. Tissue necrosis is thought to result from a combination of processes, including free-radical induced injury, excitotoxic amino acids and the release of toxic cytokines by inflammatory cells. As the necrotic tissue is removed by macrophages, large fluid- filled cavities develop, which are bordered by areas of glial/connective tissue scarring. Even though this glial scar may provide several beneficial functions such as the restoration of the BBB, prevention of a devastating inflammatory response and limit action of cellular degeneration [1, 2], it also contributes to the establishment of a physical and chemical barrier to axonal regeneration [1]. Strategies aimed at preventing primary and delaying secondary damage need to be administered within minutes to hours after injury [3]. Furthermore, none of the clinical approaches currently available to control or minimize the impact of a SCI lead to neuronal regeneration [4], nor is there an efficient regenerative therapeutic strategy for SCI treatment [4]. Although injured axons show the ability to regenerate when in a peripheral nervous system environment [5], the major factor contributing to the failure of the central nervous system (CNS) regeneration is the lack of capacity of injured axons to spontaneously regenerate in the glial scar microenvironment [6].

The use of biocompatible biomaterials to bypass the glial scar is one of the promising approaches being investigated to promote spinal cord regeneration [3, 7-13]. These tissue-engineering approaches are usually based on the use of either cell-free bridges or of cellularized biomaterial- based matrices. There are some advantages in the use of a cell-free bridging material, as on one hand cell purification and expansion methods are laborious, time consuming and expensive, and on the other hand when the transplantation of allogenic cells is required, the use of immunosuppressants cannot be circumvented [13]. Therefore the idea of a cell-free bridging material that uses and controls endogenous cell population responses, by having the ability to promote axon regeneration and control inflammatory and glial reactions is appealing.

There are numerous polymeric materials under study for application in nerve repair strategies [3, 10, 14]. These can simultaneously provide a scaffold for tissue regeneration, as well as serve as a cell-delivery vehicle and a reservoir for sustained drug delivery [15]. Within this class of materials, biodegradable polymers are particularly advantageous for the preparation of these bridges, as polymer degradation can be tuned to match the neuronal cell growth. Besides the degradation rate, the mechanical properties of the selected material are also of extreme relevance, as they must be fitted to one's needs. While the implantable structures must be flexible but relatively strong, as well as easy to handle by surgeons, their mechanical properties have an influence on cell phenotype as well [ 16- 19] .

Poly(1ximethylene carbonate-co-s-caprolactone) (P(TMC-CL)) copolymers or the parental trimethylene carbonate (TMC) homopolymer are very flexible and tough materials that can be processed into highly porous three dimensional structures with degradation rates that can be modulated by adjusting the co-monomer content [20, 21]. These materials have been shown to be biocompatible [21-23] and have been previously explored for peripheral nerve regeneration conduits [20, 23-26]. Additionally, polymer degradation occurred with minimum swelling of the material [23], which is also an essential feature to prevent nerve compression that could compromise regeneration.

Although P(TMC-CL) copolymers have been previously explored for peripheral nerve regeneration, regeneration of nerves in the CNS is hampered by the inhibitory environment formed by the glial scar, largely constituted by myelin debris. For example, it has been shown that many materials, which have shown an effect in the periphery, do not promote nerve regeneration in the CNS. Furthermore, there is no material that has been found to be able to promote axonal elongation and neuronal polarization, even in the absence of myelin (an inhibitory substrate). Therefore, there would be no expectation that P(TMC-CL) copolymers would be able promote neuronal growth in the CNS, in particular, axonal elongation and neuronal polarization.

Summary of the Invention

In a first aspect, the present invention provides a biomaterial for use in medicine, namely for promoting neuronal regeneration in the CNS, wherein the biomaterial comprises poly(trimethylene carbonate-co-s-caprolactone) (P(TMC-CL)).

It has been surprisingly found that the copolymer poly(trimethylene carbonate-co-e- caprolactone) is able to promote neuronal regeneration, in particular, neuronal polarization and axonal elongation, in the CNS, even under inhibitory conditions. This is especially surprising as polymers of the two constituents, i.e. poly(trimethylene carbonate) and poly(8-caprolactone) do not promote this effect on their own.

The biomaterial can be used to treat conditions in which neuronal regeneration of the CNS is required. The CNS is defined as the brain and the spinal cord of a subject. The CNS does not include the sciatic nerves.

The biomaterial can be used to treat damage to the CNS. In this regard, the invention provides a biomaterial for use in treating damage to the CNS, wherein the biomaterial comprises poly(trimethylene carbonate-co-e-caprolactone) (P(TMC-CL)). This damage may occur as a result of a number of different causes. The damage may occur due to a disease, which causes damage to the CNS, or injury. For example, damage to the CNS may occur as a result of: trauma, e.g. a spinal cord injury (SCI); an infection such as poliomyelitis; neurodegeneration such as amyotrophic lateral sclerosis; a tumour; an autoimmune and/or inflammatory disease such as multiple sclerosis; or spinal stroke. Where the damage to the CNS is caused by disease, the biomaterial causes regeneration of nerves in the CNS rather than treating the underlying cause of the damage.

The biomaterial can be used to treat injury or trauma to the CNS. In particular, the biomaterial can be used to treat spinal cord injury. This typically happens as a result of damage to the spinal cord of a subject. Poly(trimethylene carbonate-co-e-caprolactone) is a copolymer formed from the monomers trimethylene carbonate and ε-caprolactone. This copolymer can be formed in any suitable way. For example, polymerisation may be carried out using ring-opening polymerization. This may be done in an inert atmosphere such as an argon atmosphere or in solution. In some embodiments, the polymerization reaction is carried out in an inert atmosphere. A catalyst may be used such as stannous octoate or yttrium isopropoxide. In some embodiments, stannous octoate is used as a catalyst. The polymerization reaction may be carried out for a period of between 1 and 40 days depending on the temperature of polymerisation and the size of polymer required. The polymerization reaction may be carried out for a period of between 1 and 5 days. In some embodiments, the polymerization reaction is carried out for between 2 and 4 days. In particular embodiments, the polymerization reaction is carried out for 3 days. The polymerization reaction may be carried out at between about 60°C and about 200°C or between about 100°C and about 160°C. In some embodiments, the polymerisation reaction is carried out at between about 110°C and about 150°C. In further embodiments, the polymerisation reaction may be carried out at between about 120°C and about 140°C. The polymerisation reaction may be carried out at about 130°C, e.g. 130°C ± 2°C.

In certain embodiments, the polymerization reaction is carried out for between 2 and 4 days at between about 100°C and about 160°C. In some embodiments, the polymerization reaction is carried out for 3 days at between about 120°C and about 140°C such as about 130°C, e.g. 130°C ± 2°C.

The poly(trimethylene carbonate-co-E-caprolactone) contains trimethylene carbonate and ε- caprolactone as monomers. The ratio of these monomers should be such that the polymer has the required physical properties to cause neuronal regeneration in the CNS. In some embodiments, the molar ratio of trimethylene carbonate to ε-caprolactone may be between about 1 :20 and about 1:3, respectively. In other embodiments, the molar ratio of trimethylene carbonate to ε- caprolactone may be between about 1:1 and about 1:3. In further embodiments, the molar ratio of trimethylene carbonate to ε-caprolactone may be between about 1:17 and about 1:3. The molar ratio of trimethylene carbonate to ε-caprolactone may be between about 1:15 and about 1:3. In particular embodiments, the molar ratio of trimethylene carbonate to ε-caprolactone may be between about 1:20 and about 1:4. In certain embodiments, the molar ratio of trimethylene carbonate to ε-caprolactone may be between about 1:15 and about 1:5. The molar ratio of trimethylene carbonate to ε-caprolactone may be between about 1:10 and about 1 :6. Further, the molar ratio of trimethylene carbonate to ε-caprolactone may be between about 1 :9 and about 1 :7. The molar ratio of trimethylene carbonate to ε-caprolactone may be about 1:8.

Alternatively, in some embodiments, the molar ratio of trimethylene carbonate to ε-caprolactone may be between about 5:95 and about 25:75, respectively. In other embodiments, the molar ratio of trimethylene carbonate to ε-caprolactone may be between about 5:95 and about 23:77. In further embodiments, the molar ratio of trimethylene carbonate to ε-caprolactone may be between about 5:95 and about 21:79. In particular embodiments, the molar ratio of trimethylene carbonate to ε-caprolactone may be between about 5:95 and about 19:81. In certain embodiments, the molar ratio of trimethylene carbonate to ε-caprolactone may be between about 5:95 and about 17:83. The molar ratio of trimethylene carbonate to ε-caprolactone may be between about 7:93 and about 15:85. Further, the molar ratio of trimethylene carbonate to ε- caprolactone may be between about 9:91 and about 13:87. Furthermore, the molar ratio of trimethylene carbonate to ε-caprolactone may be between about 10:90 and about 12:88. The molar ratio of trimethylene carbonate to ε-caprolactone may be about 11 :89.

The trimethylene carbonate and the ε-caprolactone should be polymerised to an appropriate degree to give a polymer with the required characteristics. The degree of polymerisation can be measured using the number average molecular weight (Mn).

The copolymer may have a number average molecular weight of at least about 0.2 x 10⁵. The copolymer may have a number average molecular weight of less than about 2 x 10⁵. In some embodiments, the copolymer has a number average molecular weight of less than about 1.5 x 10⁵. The copolymer may have a number average molecular weight of less than about 1 x 10⁵. In further embodiments, the copolymer may have a number average molecular weight of less than about 0.75 x 10^s. In other embodiments, the copolymer may have a number average molecular weight of less than about 0.5 x 10^s. In particular, embodiments, the copolymer has a number average molecular weight of at least about 0.2 x 10⁵ and less than about 1 x 10⁵. The copolymer may have a number average molecular weight of about 0.37 x 10⁵.

The copolymer may have a stiffness of between about 200 N/m and about 400 N/m. The copolymer may have a stiffness of about 312 N/m. The copolymer may have a hardness of between about 1 x 10⁶ N/m² and about 6 x 10⁶ N/m². In some embodiments, the copolymer has a hardness of between about 2 x 10⁶ N/m² and about 5 x 10⁶ N/m². In other embodiments, the copolymer has a hardness of between about 3 x 10⁶ N/m² and about 4 x 10⁶ N/m². The copolymer may have a hardness of about 3.32 x 10⁶ N/m².

The copolymer may have a roughness of between about 10 nm and about 50 nm. The copolymer may have a roughness of about 24 nm.

The poly(trimethylene carbonate-co-e-caprolactone) may be coated with a molecule that promotes cell adhesion to the polymer. This molecule may be any suitable molecule such as fibronectin, laminins (e.g. laminin-111), collagen (e.g. collagen type I), poly(ethylene imine) or synthetic oligopeptides or polypeptides (e.g. poly(L-lysine) (PLL)). In some embodiments the poly(trimethylene carbonate-co-e-caprolactone) is coated with laminin-111 or PLL. In some embodiments, the poly(trimethylene carbonate-co-e-caprolactone) is coated with PLL.

The term "about" as used herein is defined as meaning that the values referred to can vary by plus or minus 5% (i.e. ±5%).

The biomaterial and/or the P(TMC-CL) may be in any suitable form for use in promoting neuronal regeneration. For example, the biomaterial and/or the P(TMC-CL) may be in the form of a scaffold, e.g. a three dimensional scaffold. The scaffold may be porous. The biomaterial may be in the form of a nerve guide, e.g. with a hollow tubular shape (cylindrical). Such a nerve guide will serve as a conduit for axonal growth. The conduit may be constituted by an assembly of smaller diameter nerve guides. The biomaterial can also be in the form of fibres, for example, prepared from extrusion, elecfrospinning or spun from solution. These fibres can be aligned in the longitudinal orientation of the nerve fibres. The nerve guides can also be filled with the fibres. The biomaterial may be in the form of films, beads or microspheres. The biomaterial may be in the form of a patch. These can be implanted into the brain or spinal cord for promoting neuronal regeneration. The films can have topographical features, like aligned channels or poles that can be obtained by microfabrication techniques. The biomaterial and/or the P(TMC-CL) may be plasma treated to alter surface morphology and/or chemistry. The biomaterial and/or the P(TMC-CL) may comprise one or more active pharmaceutical ingredients. The pharmaceutical ingredient may be an active agent such as ibuprofen, rolipram or diclofenac. A skilled person will appreciate that the various features described above can be combined together to form a biomaterial with certain characteristics. All the features can and are intended to be combined in any combination even if such combinations are not explicitly identified.

The present invention also provides the use of a biomaterial in the preparation of a composition for promoting neuronal regeneration in the CNS, wherein the biomaterial comprises poly(trimethylene carbonate-co-s-caprolactone) (P(TMC-CL)).

Further, the invention provides the use of a biomaterial in the preparation of a composition for treating damage to the CNS, wherein the biomaterial comprises poly(trimethylene carbonate-co- ε-caprolactone) (P(TMC-CL)).

In a further aspect, the present invention provides a method of promoting neuronal regeneration in the CNS of a subject, the method comprising administering a biomaterial comprising poly(rrimethylene carbonate-co-s-caprolactone) to a site of damage in the CNS of the subject.

The characteristics and features of the biomaterial are as described above.

The biomaterial may be administered in any suitable way. It may be implanted or injected. Generally, the biomaterial will be implanted by surgery.

The subject may be any suitable subject in which neuronal regeneration of the CNS is required. The subject may be any suitable subject with a CNS, which may require neuronal regeneration such as a mammal, for example, a cat, a dog, a horse or a human. Preferably, the subject is a human.

When the biomaterial is implanted at the site of damage in the CNS, it will promote neuronal regeneration to at least partially repair the damage. Over time, the P(TMC-CL) will be degraded and will be at least partially replaced by neural tissue.

Detailed Description of the Invention The invention will now be described in detail, by way of example only, with reference to the following figures:

Figure 1 - Cortical neuron culture on PLL coated films of P(T C-CL) and respective homopolymers. A. Number of cortical neurons with and without neurite extensions on polymeric surfaces coated with aqueous solutions at different concentrations of PLL. Glass coated with 24 g. Γ¹ of PLL for 30 minutes was used as control, (n = 3 independent studies, mean ± SD, p < 0.05) B. Percentage of PLL covered surface area as a function of the coating conditions. (n= 3, mean ± SD, p < 0.05) x = not tested condition, n.s. ~ non-signifieantly different from the control, a = total number of cells not significantly different from the control, β = number of cells with neurite extensions not significantly different from the control and χ = number of cells without extensions not significantly different from the control.

Figure 2 - Effect of the surface on neurite elongation and cellular polarization. A. Fluorescently labeled cortical neurons, immunostained for TAU (green); nuclei are counterstainned with Hoechst (blue); B. Number of neurites per cell; C. Total neurite length; D. Average neurite length and E. Length of the longest neurite. (n= 130 cells, mean ± SD, *** for p < 0.001)

Figure 3 - Morphology and mechanical properties of the tested polymeric surfaces. A. Root mean square (RMS) roughness of all polymeric surfaces; B. Representative photographs of the polymeric surfaces before and after nanoindentation; C. Representative nanoindentation force- displacement curves; D. Mean hardness values of all polymeric surfaces, calculated for the maximum load and E. Mean stiffness values for all polymeric surfaces, (n = 60 indentations, mean i SD, *** for p < 0.001)

Figure 4 - Effect of CNS myelin on neurite outgrowth of cortical neurons cultured for 4 days on PLL-P(TMC-CL) substrates coated with CNS myelin. A. Cortical neurons are immunostained for β-ΙΙΙ tubulin (green), myelin immunostained for MBP (red) and nuclei are counterstainned with Hoechst (blue); B. Effect of myelin on the ability of neurons to extend processes is presented as the % of cells with neurites in relation to the total number of cells, (n = 3 independent studies, mean ± SD; ** for p < 0.01) Figure 5 - Analysis of GSK3 in cortical neurons plated on P(TMC-CL) and effects of GSK3P inhibition on neurite extension. A. Schematic representation of the different phosphorylation forms of GSK3p and their activity status; B. Analysis of the phosphorylated forms of GSK3p by western blot. Representative blots are shown. Expression levels of GSK3P isoforms, βΐ and β2, are presented and quantified individually or together, (n = 3 independent studies, average ± SD); C. Morphology of neurons (immunostained for Tau in green and nuclei counterstained in blue) cultured for 24 hours in the presence of DMSO (Control) or in the presence of 6-bromoindirubin- 3'-acetoxime (BIO) at 30 and 300 nM. Quantifications of the longest neurite, average neurite length and the number of neurites per cell are shown (n = 130 cells, mean ± SD, * for p < 0.05, ** for p < 0.01 and *** for p < 0.001); D. Determination of CRMP4 phosphorylation levels in cortical neurons plated for 4 days on control or P(TMC-CL). Representative western blot is shown and below the quantification (n = 3 independent studies, average ± SD).

1. SUMMARY

Mammalian central nervous system (CNS) neurons do not regenerate after injury due to the inhibitory environment formed by the glial scar, largely constituted by myelin debris. The use of biomaterials to bridge the lesion area and the creation of an environment favouring axonal regeneration is an appealing approach, currently under investigation. This work aims to assess the suitability of three candidate polymers - poly(e-caprolactone), poly(trimethylene carbonate- co-s-caprolactone) (P(TMC-CL)) (11 :89 mol%) and poly(trimethylene carbonate) - with the final goal of using these materials in the development of conduits to promote spinal cord regeneration. Poly(L-lysine) coated polymeric films were tested for neuronal cell adhesion and neurite outgrowth, with P(TMC-CL) stimulating neuronal polarization and promoting axon elongation. Furthermore, cortical neurons cultured on P(TMC-CL) in the presence of myelin were able to overcome myelin inhibition in comparison with the control condition (glass substrate). This effect was found to be mediated by the glycogen synthase kinase 3β (GSK3p) signalling pathway with impact on the coUapsin response mediator protein 4 (CRMP4), suggesting that besides surface topography, mechanical properties were implicated in this process. The obtained results indicate P(TMC-CL) as a promising material for CNS regenerative applications as it promotes axonal growth, overcoming myelin inhibition.

2. MATERIALS AND METHODS

2.1. Polymeric film preparation Poly(trimethylene carbonate) (P(TMC), poly(s-caprolactone) (P(CL)) and poly(trimethylene carbonate-co-e-caprolactone) (P(TMC-CL)) with 11 mol % of TMC were synthesized as previously described [20]. Briefly, prior to polymerization ε-caprolactone monomer (Fluka) was dried overnight over Ca¾ and distilled under reduced pressure. Trimethylene carbonate was obtained from Boehringer Ingelheim (Germany) and used as received. Polymerizations were conducted by ring-opening polymerization in an argon atmosphere using stannous octoate as a catalyst. All polymerizations were carried out for a period of 3 days at 130°C ± 2°C. The obtained polymers were purified by dissolution in chloroform and subsequent precipitation into a ten-fold volume of ethanol. The precipitated polymers were recovered, washed with fresh ethanol and dried under reduced pressure at room temperature (RT) until constant weight. The prepared polymers were characterized with respect to chemical composition by nuclear magnetic resonance (NMR). Four hundred MHz 1H-NMR (BRUKER AVANCE III 400) spectra were recorded using solutions of polymer in CDC1₃ (Sigma). Number average and weight average molecular weights (Mn and Mw, respectively), polydispersity indices (PDI) and intrinsic viscosities ([η]) of the (co)polymers were determined by gel permeation chromatography (GPC, GPCmax VE-2001, Viscotek, USA). The setup was equipped with ViscoGEL I-guard-0478, ViscoGEL I-MBHMW-3078, and ViscoGEL I-MBLMW-3078 columns placed in series and a TDA 302 Triple Detector Array with refractometer, viscometer, and light-scattering detectors, allowing the determination of absolute molecular weights. All measurements were performed at 30°C, using chloroform as the eluent at a flow rate of l.O ml.min^"1. The obtained results are compiled in Table 1.

Polymer films of 250 μπι in thickness were prepared by casting the polymer solution in chloroform onto glass Petri dishes. After drying the films under reduced pressure at RT, disks with a diameter of 14 mm were punched out. Prior to cell culture, disks were sterilized by two incubation steps in a 70% (v/v) ethanol solution for 15 min, followed by two rinsing steps of 15 min in autoclaved MilliQ water. After sterilization, polymer disks were placed in 24-well tissue polystyrene plates (BD Biosciences) and fixed with autoclaved silicon o-rings (EPIDOR).

2.2. Cortical neuron cell culture

Prior to cell seeding the air side surface of the polymeric disks was coated with 200 μΐ of a poly(L-lysine) (PLL, Sigma) solution in a concentration ranging from 24 to 73 μg.μ ¹, at 37°C for 30 min or overnight and, subsequently, rinsed with autoclaved MilliQ water. Coverglass (Menzel) coated with 24 μ μΓ¹, at 37°C for 30 min was used as control. Wistar Han rat embryos (embryonic day 17-18) were removed by caesarean section of the euthanized pregnant rats. The isolated cortices were dissociated for 30 min at 37°C in Hanks Balanced Salt Solution (HBSS) supplemented with 1.0 mM pyruvate, 2 mg.ml^"1 albumin, and 10% (v/v) trypsin (all from Gibco). Viable cells (trypan blue exclusion assay) were seeded at a density of 2.2 χ 10⁴ viable cells.cm^"2 onto PLL-coated polymeric discs or coverglasses in 24- well cell culture plates. Neural cells were seeded in 300 μΐ of Dubelcco's Modified Eagle Medium (DMEM) : Nutrient Mixture F-12 (F-12) (3:1) supplemented with 10% (v/v) inactivated fetal calf serum (FCS) (all from Gibco). Two hours later, medium and o-ring were removed and 1 ml of Neurobasal medium supplemented with 0.5 mM L-glutamine, 2% (v/v) B27 supplement, 1% (v/v) Penicillin-Streptomycin and 0.5% (v/v) Gentamicin (all from Gibco) was added and polymeric discs turned upside down. Cultures were maintained at 37 °C in a humidified atmosphere of 5 % C0₂. Culture purity was determined by immunocytochemistry as described further down. Half of the cell culture medium was changed on the third day of culture. After 4 days in culture, samples were treated for immunocytochemistry.

2.3. Poly(L-lysine) adsorption quantification

Polymeric disks were coated with PLL-FITC (fluorescein isothiocyanate) (Sigma) as described in the previous section. Coverglass coated with 24 μg.μ ¹ of PLL, at 37°C for 30 min, was used as control. Polymeric discs coated with PLL-FITC were further mounted on microscope slides using an aqueous mounting media (Sigma) and observed with an inverted fluorescence microscope (Axiovert 200M, Zeiss). Image analysis was performed with ImageJ 1.44 software.

2.4. Atomic force microscopy

2.4.1. Roughness analysis

The roughness of the polymer surfaces tested for cell culture was assessed by atomic force microscopy (AFM) using a PicoPlus scanning probe microscope interface with a PicoScan controller (Agilant Technologies, USA). A 10x10 μπι² piezoscanner was used in tapping mode, with a scan speed of 1 line.s^"1. A bar shaped silicon cantilever (ACT probe, from AppNano), with a spring constant of 25-75 N.m^"1 was used and roughness analysis was performed from scanned areas of 7x7 um² on five randomly chosen locations of each sample in air, at room temperature. The root-mean-square (RMS) roughness within the sampling area was determined using the WSxM scanning probe microscope software [25], according to where a represents the image height and N the total number of points.

2.4.2. Nanoindentation

These measurements were performed at CEMUP (Centro de Materials da Universidade do Porto), on a Veeco Metrology Multimode with Nanoscope IV controler (Veeco Instruments, Inc.) at RT conditions in Force-indent mode with a diamond tip, suitable for nanoindentation (DNISP Diamond-Tipped Probe from Veeco; spring constant 131 N.m^"1). Deflection sensitivity of the cantilever was calibrated by indenting a sapphire surface. Nanoindentations were made for 1 second and the peak load was confined up to 30 μΝ for P(TMC-CL) and P(CL) and 6.5 uN for P(TMC). Force-displacement curves were obtained during loading and unloading for each indentation, and further used to determine hardness and stiffness values according to the Oliver and Pharr method [26]. For each polymeric substrate type, 60 indents were done on the film side tested for cell culture, covering 3 randomly chosen regions of 4 different samples per material. In each region, a set of 16 indents was made at a distance of 2 um of each other. Stiffness was calculated as the slope of the tangent line to the unloading curve at the maximum loading point and hardness values were calculated for the maximum load and taking into consideration the shape of the indenter probe.

2.5. Neurite outgrowth on myelin coated polymer films

2.5.1. Myelin isolation

Myelin was isolated from brains of C57BL/6 male mice, as previously described [27]. Briefly, the isolated brains were homogenized in 0.32 M sucrose and after centrifugation at 900g, the post-nuclear supernatant was collected. The post-nuclear supernatant was carefully overlaid on an ultracentrifuge tube containing a 0.85 M sucrose solution on top of a 50% (w/v) sucrose cushion. After centrifugation for 1 hour at 37000 g at 4 °C (Sorvall Pro80 centrifuge), the interphase between sucrose solutions was transferred to a new ultracentrifuge tube. Two rounds of osmotic shocks were performed by adding ice-cold water and centrifugation at 20000 g. The final myelin pellet was stored at -80°C until further use.

Myelin coating The polymeric disks and glass control were first coated overnight with PLL as described above and washed with 0.1M NaHC0₃. Myelin 1.25 μg myelin.cm^"2 aqueous solution was dried overnight onto the PLL coated disks as described by Cai et al. [28], and further used as substrates for cortical neuron culture.

2.6. Pharmacologic inhibition of glycogen synthase kinase 3

For neuronal outgrowth assays in the presence of a pharmacologic inhibitor of glycogen synthase kinase 3 (GSK3), a 30 or 300 nM solution of 6-bromoindirubin-3'-acetoxime (BIO) in dimethyl sulfoxide (DMSO) was added to cortical neuron cultures (DMSO final concentration 0.05% (v/v)) at two different time points: at seeding (t = 0) being in contact with cells for 4 days, and at the third day of culture (t = 3) being in contact with cells for 24h. Neurons seeded on polymer discs in the presence of 0.05% (v/v) DMSO were used as controls. After 4 days in culture samples were treated for immunocytochemistry.

2.7. Immunocytochemistry

Cells were fixed for immunocytochemistry staining with 2% (v/v) paraformaldehyde at RT and further permeabilized and blocked in phosphate buffered saline (PBS) containing 5% (v/v) Normal Goat Serum (NGS) (Biosource) and 0.2% (v/v) Triton X-100 (Sigma). Primary antibodies were diluted in PBS containing 1% (v/v) NGS and 0.15% (v/v) Triton X-100, and incubated overnight in a humid chamber at 4°C. Secondary antibodies were applied for lh at RT and subsequently treated for nuclear counterstaining at RT with Hoechst (Molecular Probes) at 2 μΙ.ιηΓ¹. Samples were mounted directly in aqueous mounting medium and observed with an inverted fluorescence microscope.

Culture purity was >99% in cortical neurons as determined by mouse anti-glial fibrillary acid protein (GFAP) (1:500, BD Biosciences)/ mouse anti-vimentin (1:100, Thermo Scientific)/ mouse anti-oligodendrocyte marker 4 (04) (1:100, Chemicon)/ rabbit anti-Tau (TAU protein) (1:100, Sigma)/ 2 ug-ml^"1 Hoechst fluorescent staining. Cells were counted from 18 radial fields and values were extrapolated to the total surface area of the sample (n=3). For axonal outgrowth assessment the length of the longest neurite and total neurite outgrowth per cell were determined using Axio Vision image analysis software. Neuronal processes were manually traced and quantified on 130 cells per condition. Three independent experiments were performed. For neuronal outgrowth analysis on myelin inhibition studies, neurons were stained with anti-βΙΙΙ tubulin (1:500, Abeam) and myelin with anti-myelin basic protein (MBP) SMI-94 (1:500, Abeam). The secondary antibodies used were anti-rabbit Alexa 488 (1:500, Invitrogen), anti- mouse 594 (1:1000, Invitrogen).

2.8. Western Blot

Cortical neuron lysates were prepared by washing cells with PBS and further lysed in buffer containing 20 mM acid (MOPS), 2 raM ethylene glycol tetraacetic acid (EGTA), 5 mM emylenediaminetetraacetic acid (EDTA), 30 mM NaF, 60 mM β- glycerophosphate, 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1% (v/v) Triton X-100, 1% (v/v) DL-dithiothreitol (DTT), 1 mM phenylmethanesulfonyl fluoride (PMSF) and protease inhibitor cocktail (Amersham). Protein lysates (25-100 μg/lane) were run on a 12% SDS-PAGE gel and then transferred to a nitrocellulose membrane (Amersham). For Western analysis, membranes were blocked with blocking buffer (5% (wt/v) non-fat dried milk in tris- buffered saline (TBS) 0.1% (v/v) Tween 20) and incubated overnight at 4°C in 5% (wt/v) bovine serum albumin (BSA) in TBS 0.1% Tween 20 with primary antibodies. The following primary antibodies were used: rabbit anti-phospho-glycogen synthase kinase 3 beta (GSK3P) Ser9 (1:1000, Cell Signaling), rabbit anti-phospho-GSK3p Tyr216 (1:2000, Santa Cruz Biotechnology), mouse anti-GSK3a/p (1:2000, Santa Cruz Biotech.), sheep anti-phospho- collapsin response mediator protein 4 (CRMP4) Thr 509 (1:1000, Kinasource) and mouse anti- total CRMP4 (1:500, Santa Cruz Biotech.). After washing, membranes were incubated with secondary antibodies for lh at RT. The secondary antibodies used were anti-rabbit HRP (1:10000, Jackson Immunoresearch), anti-mouse HRP (1:10000, Thermo Scientific) and anti- goat/sheep (1:10000, Binding Site). Proteins were detected using a chemiluminescent substrate Pierce ECL western blotting substrate (Thermo Scientific) according to the manufacturer's specifications. For each experiment representative western blots are shown. Phospho-protein expression was quantified by densitometry with QuantityOne software (BioRad) and levels were normalized to the total level of the same protein.

2.9. Statistical Analysis

For statistical analysis, one-way ANOVA followed by Tukey's post-hoc test were used. When Gaussian distribution was not confirmed non-parametric test Man- Whitney was applied, using the Graphpad Prism program. Data is expressed as the mean ± standard deviation (SD) and p values of < 0.05 were considered significant.

3. RESULTS

3.1. Cortical neurons adhere and extend neurites in a PLL dependent manner

As a first step in assessing P(CL), P(TMC-CL) and P(TMC) compatibility with the CNS and their potential application in devices for neuroregeneration, polymeric discs were tested as substrates for cortical neuron growth in vitro. Cortical neurons were seeded on PLL coated polymeric films and were found to adhere to the tested substrates in a PLL concentration dependent manner (Fig. 1A). Cell number and neurite outgrowth on the coated polymeric films were evaluated using coverglasses coated with a PLL concentration of 24 μ μ ¹ for 30 min as control. Cortical neurons adhered in comparable numbers to the control when these were coated overnight with 24 μg.μ ¹ and 48 μg.μ ¹ of PLL in the case of P(CL) films, and 72 μg.μ ¹ of PLL in the case of TMC containing films (see Fig. 1A). However, only on P(TMC-CL) the majority of adhered cells was able to extend neurites as in the control.

To explain this PLL-dependent behaviour, the amount of PLL adsorbed to the polymeric films surface was evaluated by fluorescence quantification of PLL-FITC coated samples. As one can observe in Fig. IB, the surface area covered by PLL (in %) was only comparable to the control conditions when the polymeric films were treated with a PLL solution of at least 48 μ μΓ¹ and 72 μg.μΓ¹ in the case of the CL containing materials and P(TMC), respectively. Consequently, cell adhesion can be directly correlated with the PLL adsorption profile to the polymeric films. Taking into consideration the obtained results, both in terms of cell adhesion and neurite extension, the coating conditions used in the subsequent studies were established to be polymer surface treatment overnight with 48 μg.μl^Λ for P(CL) and 72 μg.μ ¹ for P(TMC-CL) and P(TMC).

3.2. P(TMC-CL) stimulates axonal elongation

In order to evaluate neurite outgrowth on the different PLL-coated polymeric surfaces, the number of neurites per cell, as well as the neurite length were determined. As seen in Fig. 2A, neurons behave differently on each surface. More than 80% of the cells seeded on polymeric films sho one or two neurites, while more than 80% of the cells seeded on glass (control) present between 3 to 5 neurites (Fig. 2B). Furthermore, as shown in Fig. 2A neurons seeded on the polymeric surfaces exhibit a lower degree of branching than those seeded on glass. However, 0006

16

on P(CL) and P(TMC) the adhered cells show smaller neurites than on P(TMC-CL) and the control (Fig. 2C-E). Despite the fact that for P(TMC-CL) the total neurite length was similar to the one observed on glass, given the lower number of neurites per cell in this condition, the average neurite length was higher (Fig. 2D). More remarkably, the length of the longest neurite was increased relatively to the control (Fig. 2E).

3.3. P(TMC-CL)'s mechanical properties stimulate axonal outgrowth

As shown in Fig. 3 A, AFM analysis indicated that the RMS roughness was similar for P(CL) and P(TMC-CL), with mean values of 21.8 ± 11.5 nm and 24.4 ± 12.1 nm respectively, while significantly lower for P(TMC) with a mean value of 1.6 nm ± 1.0.

Nanoindentation is one of the most versatile techniques and particularly suited for the measurement of localized mechanical properties on the surface of materials [29]. Representative photos of the nanoindentations and force/displacement curves are represented in Fig. 3B and C, respectively. These show that P(CL) poses a greater resistance to deformation in relation to the other two materials tested, as the force needed to achieve the same displacement is higher than for P(TMC-CL) or P(TMC). As shown in Fig. 3 D-E stiffness and hardness values are significantly different between the three different substrates. A stiffness value of 312 ± 56.4 N.m^" ¹ and a hardness of 3.32 xlO⁶ ± 0.373 x 10⁶ N.m^-2 was found for P(TMC-CL), while P(CL) shows the highest values with a stiffness of 435 ± 40.4 N.m^"1 and a hardness value of 6.60 xlO⁶ ± 2.11 x 10⁶ N.m-². As seen in the photos before and after nanoindentation, P(TMC) samples recover almost completely from the indentations and, consequently, show stiffness and hardness values close to zero.

3.4. P(TMC-CL) promotes overcome of myelin inhibition

Myelin-associated inhibitors (MAIs) are present at a spinal cord lesion site and are known to be among the major impediments of the spontaneous axonal regeneration after SCI. Cortical neurons were seeded on myelin coated surfaces. P(TMC-CL) was chosen from the three surfaces tested as it showed the best results for neuronal adhesion and neurite extension, presenting a positive influence on axonal elongation. As seen in Fig. 4, when comparing surfaces coated and uncoated with myelin, the number of cells with neurites is smaller in the first case. Nevertheless, this decrease is not significant on P(TMC-CL) seeded neurons in contrast to the control where this reduction is statistically significant (p < 0.01). 3.5. GS 3 signalling pathway mediates neuronal sensing of P(TMC-CL) surface nanomechanical properties

GSK3 is implicated in many processes in the nervous system and is known to play a critical role in the regulation of neuron physiology. It is highly expressed in neurons and crucial for the establishment of neuronal polarity, as well as for the establishment of the branching-elongation equilibrium [30-32]. In view of this knowledge, the involvement of GSK3 as a mediator of P(TMC-CL) effect on neurite formation and axonal outgrowth was examined. GSK3P is regulated by phosphorylation and its activity can be reduced by phosphorylation at Ser-9. Contrarily, tyrosine phosphorylation at Tyr-216 increases the enzyme's activity (Fig. 5A) [33, 34]. As shown in Fig. 5B cortical neurons seeded on P(TMC-CL) present lower levels of GSK3p₂ Ser-9 phosphorylation and higher level of Tyr-216 phosphorylation, in comparison to neurons cultured on glass. This indicates that neurons seeded on P(TMC-CL) display more kinase activity than those on glass. It is also perceptible from Fig. 5B that the GSK3P isoform that is differently expressed is GSK3p2, which is known to be expressed exclusively in the nervous system [35]. To further confirm the involvement of GSK3p as a mediator of the P(TMC- CL) effect on axonal outgrowth and number of neurites per cell, cortical neurons were cultured in the presence of a GSK3 pharmacologic inhibitor - BIO. It is expected that inhibiting GSK3 activity should inhibit the polymeric surface's effect on cortical neurons. In fact, as shown in Fig. 5C, when BIO is added to the culture medium one can observe a decrease in the length of the longest neurite and in the average neurite length, as well as an increase on the number of neurites per cell. These effects occur in a dose-dependent manner, with the highest concentration of BIO tested (300 nM) leading to statistically significant differences. Alabed et al. [36] have established that GSK3P phosphorylation and consequent inactivation, regulates the interaction of CRMP4 and RhoA through CRMP4 de-phosphorylation. If this mechanism is active in our setup, phospho-CRMP4 levels should be higher for neurons seeded on P(TMC-CL). To test this hypothesis, the levels of CRMP4 phosphorylation in cortical neurons seeded on P(TMC-CL) and glass surfaces were assessed. As expected, phospho-CRMP4 levels were increased for neurons cultured on P(TMC-CL) as shown in Fig. 5D.

4. DISCUSSION

In the aftermath of a SCI, a glial scar is formed. Despite its key role in constraining the damaging effects caused by the lesion, the glial scar also prevents axon regeneration. The astroglial scar not only contains secreted and transmembrane molecular inhibitors of axon growth but also constitutes an almost impenetrable physical barrier to regeneration [4], Consequently, it was hypothethized that by creating a favourable environment at the lesion site, one will be able to enhance axonal regeneration and ultimately promote some gain of function. Therefore, the use of an implantable scaffold to bypass the glial scar area is one of the promising approaches being investigated to promote spinal cord regeneration. A prerequisite in the design of such biomaterial is its biocompatibility, which in this context means that it must support neuronal survival and axonal growth. The aim of this study was, therefore, to investigate the suitability of P(CL), P(TMC-CL) and P(TMC) as substrates for spinal cord regeneration purposes.

One of the most commonly used strategies to assess neuronal behaviour in vitro when testing biomaterials for nerve regeneration applications is to evaluate axonal growth [28, 36-39]. In the present work rat cortical neurons were firstly seeded on the PLL-coated polymeric substrates to assess adhesion and neurite outgrowth ability. PLL is a synthetic homo-poly-(amino acid), characterized by an isopeptide bond between the ε-amino and the a-carboxyl groups of L-lysine, commonly used to coat cell culture substrates [40]. Initially, the polymer surface coating conditions were optimized - PLL concentration and time of contact - in order to achieve a comparable surface area covered by PLL and, consequently, similar cell adhesion in all tested surfaces. The observed PLL dependent behaviour can be explained by the different adsorption capacity of PLL on polymeric and glass surfaces. Differences that can be attributable to the surface properties of the tested materials, as these polymers present a more hydrophobic surface than glass [20], although after this process one could obtain comparable numbers of cortical neurons after 4 days of culture on the tested materials, significant morphological differences were found between neurons cultured on polymeric surfaces, particularly P(TMC-CL), and the control. Firstly, only on P(TMC-CL) the majority of neurons is able to extend neurites. Furthermore, our results show that among all the tested surfaces, including glass, seeding cortical neurons on P(TMC-CL) stimulates neuronal polarization and promotes axon elongation, as neurons on P(TMC-CL) show significantly enhanced neurite outgrowth and significantly lower numbers of neurites per cell. This switch to polarized and elongated morphology is noteworthy as successful regeneration requires that neurons survive and initiate rapid and directed neurite outgrowth [41]. A decreased number of neurites per cell were also found on P(CL) and P(TMC) but on these materials axonal outgrowth was significantly impaired. Moreover, while control neurons have, on average, twice the number of neurites of neurons seeded on P(TMC-CL), when one sums the length of all neurites of each cell (total neurite length) no significant differences are found. Altogether, cortical neurons seeded on P(TMC-CL) were found not only to be polarized but also to extend significantly longer neurites. To the best of our knowledge, no previous reports have shown this neuronal behaviour on any studied material.

The potential of materials to trigger specific cellular responses is getting to be a well established phenomenon mediated by a number of factors that range from the properties of the surface that contacts with the cell, to the mechanical properties of the material [16, 17, 42-44]. We have previously characterized the family of these copolymers and when varying the monomer ratio mainly the thermal and, consequently, the mechanical properties of these materials are drastically affected [21]. P(TMC) and P(TMC-CL) copolymers with high CL content are flexible and though materials that range from amorphous to semi-crystaline elastomers when the CL content increases. Therefore, we hypothesise that surface topography and the nanomechanical properties of the tested materials play a key role in influencing cell behaviour. The local characterization of roughness, hardness and elastic properties of a wide range of materials has been reported including for thin films and biomolecules [45-48] but so far the characterization of TMC-CL copolymers has not been performed. The roughness of the three tested polymeric surfaces was first investigated. Values of 22 nm and 24 nm were found for P(CL) and P(TMC- CL) respectively, while for the P(TMC) the roughness values were found to be significantly lower. In 2002, Fan et al. [49, 50] showed that neuronal cells adherence and survival is optimum on surfaces with a RMS roughness ranging from 10 to 50 nm. Taking this data in consideration, both P(CL) and P(TMC-CL) show not only similar but also optimum roughness values for neural adhesion and survival, while P(TMC) is outside this optimum roughness range. Therefore, the different neuronal behaviour on these surfaces cannot be explained simply by topography. Aiming to measure localized mechanical properties on the surface of the polymeric films, nanoindentations were performed and force-displacement curves obtained for each indentation. Mean hardness and stiffness values were calculated and significant differences were found between all polymeric surfaces, with P(TMC-CL) being significantly less resistant to deformation than P(CL) and significantly more resistant to deformation than P(TMC). Although roughness values were similar between P(CL) and P(TMC-CL) and within the optimum range, P(CL) was two times harder than P(TMC-CL), which could explain the different cellular behaviour on these surfaces, indicating that changes in stiffness and hardness values may have caused changes in cell morphology, specifically in axonal elongation.

Having observed the ability of P(TMC-CL) surfaces in promoting neuronal polarization and axonal elongation under normal cell culture conditions, the capacity of P(TMC-CL) to positively affect cortical neurons in a typical CNS inhibitory environment was tested, envisaging its application in the design of an axonal regeneration promoting strategy. While axons in the context of a mature mammalian CNS do not regenerate if damaged, the immature mammalian CNS is able to regenerate after injury [51, 52]. Probably the most notable difference between the mature and the immature nervous system is the presence of myelin [28]. Indeed, the limited regenerative capacity of the mammalian CNS is known to be partially due to myelin inhibition. So far, no biomaterial has shown the ability to overcome myelin inhibition unless blockers of myelin protein receptors were used [53]. Recently, Mohammad and co-workers have shown that a nano-textured self-assembled aligned collagen hydrogel was able to promote directional neurite guidance and overcome inhibition by a recombinant myelin-associated glycoprotein of dorsal root ganglia cultures [54]. To assess P(TMC-CL)'s neuronal effect under adverse, and more biologically relevant conditions, cortical neurons were seeded on P(TMC-CL) films in the presence of myelin. As expected, in the glass control surface we observed a statistically significant reduction of the number of cells extending neurites when cultured in the presence of myelin. In contrast, when P(TMC-CL) was used as a substrate, this reduction was not statistically significant (Fig. 4 B), suggesting that P(TMC-CL) is, to some extent, contributing to the promotion of the overcome of myelin inhibition. This is of extreme relevance as it has been already demonstrated that some degree of functional recovery can be obtained simply by counteracting the activity of myelin inhibition [55, 56]. The existence of a biomaterial that has the capacity to overcome this inhibition per se, without the need for the administration of antibodies or chemical inhibitors, can prove to be of great importance for therapeutic purposes.

The potential of materials to trigger specific cellular responses, such as interference and/or activation of defined pathways is extremely promising for tissue engineering. Stiffness and hardness sensing probably involves transduction into biological signals [17]. GSK3p is known to regulate axonal growth through the modification of the phosphorylation status of several microtubule-binding proteins and consequently the assembly of microtubules [34, 57]. Moreover, Alabed et al. [36] showed that the overexpression of active GSK3P attenuates MAI- dependent neurite outgrowth inhibition. For these reasons, GS 3 was studied as a possible mediator of P(TMC-CL)'s effect. Mammalian GSK3 is generated from two genes, GSK3a and GSK3p. GSK3 expression in neurons is further characterized by an alternative splicing of GSK3P originating two main variants: GSK3pi and GSK3P2. GSK3P2 is specifically expressed in the nervous system [34]. GSK3p is regulated by phosphorylation and its activity is dependent on the balance between tyrosine (Tyr-216) and serine (Ser-9) phosphorylation as shown in Fig. 5A, with a reduction of activity if phosphorylated at Ser-9, and its increase if phosphorylated at Tyr-216 [33, 34]. Our results show that GSK3P is differently regulated in neurons seeded on glass and P(TMC-CL), with the later showing lower levels of Ser9 phosphorylation, a site of GSK3P inactivation, and higher levels of Tyr216 phosphorylation, which facilitates the activity of GSK3p by promoting substrate accessibility [34]. Neurite elongation and neuronal polarization on P(TMC-CL) may be promoted by an increase GSK3p activity in vitro. The relationship between axonal elongation and GSK3P activity was further confirmed through pharmacological inhibition of GS 3 in vitro. As expected, inhibition of GSK3P blocked P(TMC-CL) effect, as there was a decrease in neurite length and an increase on the numbers of neurites per cell. Cells seeded on P(TMC-CL) and treated with BIO acquired a morphology that resembles more closely the neurons seeded on glass Fig. 2A.

Activation of GSK3P activity occurs in cortical neurons when these are cultured on P(TMC-CL), resulting in an increase in neurite outgrowth and decrease on the number of neurites per cell. Increased axonal outgrowth in the presence of higher GSK3P activity has also been shown in prior reports, for cerebellar, dorsal root ganglia and hippocampal neurons [31, 36, 58].

The Rho signalling pathway is known to play an important role in neuronal growth regulation and it has been shown that inhibitors of RhoA, and/or its downstream effector Rho kinase, facilitate growth on myelin substrates [59, 60]. Wozniak et al. [16] have studied the effects of stiffness on cell shape and shown that ROCK mediated contractility is essential for breast epithelial cells to sense the biophysical properties of the surrounding environment. Alabed et al.

[61] have identified CRMP4 as a protein that functionally interacts with RhoA to mediate neurite outgrowth. Later on, this team has found that CRMP4-RhoA interaction is regulated by dephosphorylation of CRMP4 as a direct consequence of GSK3P inactivation by phosphorylation at Ser-9 [36]. This observation indicates that overexpression of GSK3P and consequent inhibition of CRMP4-RhoA complex formation may be protective in the context of myelin inhibition. Our findings are consistent with Alabed et al. [36] as for neurons seeded on P(TMC-CL), which show higher levels of GSK3 activity and longer neurites the levels of phospho-CRMP4 are higher than in glass seeded neurons. Overall these results suggest that the activation of GSK3P activity, and consequent neurite elongation, is mediated by the surface mechanical properties of P(TMC-CL).

5. CONCLUSIONS

This work shows that P(TMC-CL) with a high CL content can promote axonal regeneration, prompting neurons into a regeneration mode, even under inhibitory conditions. This effect is mediated by the GSK3 signalling pathway, which is triggered by P(TMC-CL)'s surface mechanical properties.

These findings establish P(TMC-CL) as a promising material in the design of devices to promote neural regeneration, serving as a protective barrier for de novo scar tissue formation while supporting axonal growth and rendering neurons insensitive to myelin dependent neurite outgrowth inhibition without the administration of any therapeutic drug.

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Claims

1. A biomaterial for use in medicine, wherein the biomaterial comprises poly(trimethylene carbonate-co-e-caprolactone) (P(TMC-CL)).

2. A biomaterial for use in promoting neuronal regeneration in the central nervous system, wherein the biomaterial comprises poly(trimethylene carbonate-co-s-caprolactone) (P(TMC-CL)).

3. The biomaterial for use according to claim 2, wherein the biomaterial is for use in treating damage to the central nervous system.

4. The biomaterial for use according to claim 3, wherein the damage is a traumatic injury.

5. The biomaterial for use according to claim 4, wherein the damage is a spinal cord injury (SCI).

6. The biomaterial for use according to claim 3, wherein the damage is as a result of a disease which causes damage to the CNS such as the spinal cord.

7. The biomaterial for use according to claim 3 or claim 6, wherein the damage is caused by: an infection such as poliomyelitis; neurodegeneration such as amyotrophic lateral sclerosis; a tumour; an autoimmune and/or inflammatory disease such as multiple sclerosis; or spinal stroke.

8. The biomaterial for use according to any preceding claim, wherein the molar ratio of trimethylene carbonate to ε-caprolactone in the P(TMC-CL) is between about 1 :20 and about 1:3.

9. The biomaterial for use according to any preceding claim, wherein the molar ratio of trimethylene carbonate to ε-caprolactone in the P(TMC-CL) is between about 1:10 and about 1:6.

10. The biomaterial for use according to any preceding claim, wherein the molar ratio of trimethylene carbonate to ε-caprolactone in the P(TMC-CL) is about 11:89.

11. The biomaterial for use according to any preceding claim, wherein the P(TMC-CL) is coated with a molecule that promotes cell adhesion.

12. The biomaterial for use according to claim 10, wherein the molecule that promotes cell adhesion is poly(L-lysine) (PLL).

13. The biomaterial for use according to any preceding claim, wherein the P(TMC-CL) has a number average molecular weight of at least about 0.2 x 10⁵.

14. The biomaterial for use according to any preceding claim, wherein the P(TMC-CL) has a number average molecular weight of less than about 2 x 10^s.

15. The biomaterial for use according to any preceding claim, wherein the P(TMC-CL) has a stiffness of between about 200 N/m and about 400 N/m.

16. The biomaterial for use according to any preceding claim, wherein the P(TMC-CL) has a hardness of between about 1 x 10⁶ N/m2 and about 6 x 10⁶ N/m2.

17. The biomaterial for use according to any preceding claim, wherein the P(TMC-CL) has a roughness of between about 10 nm and about 50 nm.

18. The biomaterial for use according to any preceding claim, wherein the biomaterial is in the form of a scaffold, a nerve guide, a fibre, a film, a bead, a microsphere, or a patch.

19. The biomaterial for use according to any preceding claim, wherein the biomaterial is in the form of a scaffold or a nerve guide.

20. The biomaterial for use according to any one of claims 1 to 17, wherein the biomaterial is in the form of a patch. 27

21. The biomaterial for use according to any preceding claim, wherein the biomaterial comprises one or more active pharmaceutical ingredients.

22. A method of promoting neuronal regeneration in the central nervous system of a subject, the method comprising administering a biomaterial comprising poly(trimethyIene carbonate-co-e-caprolactone) to a site of damage in the central nervous system of the subject.