The present study has developed poly ε-caprolactone (PCL)/ poly lactic acid (PLA) films with specific internal structure suitable to prepare nerve guide conduit for peripheral nerve repair. The film preparation method has been carried out using an environmental chamber to prepare the solvent cast films with the specific surface structure. Different cellular behaviour of neuronal cell cultures was seen on the pitted films with different pits configurations (size and distribution). The consistent surface morphology provided a reliable surface structure for further in vitro and in vivo studies. The effect of a medical grade sterilisation process using gamma radiation at eight doses (0-45kGy) on PCL/PLA films was explored. It has been shown that material properties, including mechanical strength, were significantly affected, while cellular behaviour and responses (NG108-15) were improved. Grooved films with three groove shapes (Sloped, Square, and V shape) were prepared using patterned silicon substrates, photolithography and wet/dry etching. The groove patterns were successfully transferred and good mechanical strength was observed for grooved PCL/ PLA. Oriented growth of NG108-15 cells was observed on the patterned films with an improved alignment and organisation on SL and V shape grooved films. UV-ozone treatment was used to increase hydrophilicity of PCL/PLA films to improve Schwann cells behaviour. No negative effect was observed on cell growth and proliferation on the treated films however the mechanical properties were reduced. Schwann cells expressed typical long spindle-shape morphology with cell-to-cell interaction in longitudinal direction on the treated grooved films. Consistent to in vitro experiment with NG108-15, Schwann cells alignment was also improved on SL and V shape grooves. A three-week in vivo study was carried out to test grooved and non-grooved conduits in a rat sciatic nerve model. The grooved conduits showed better regeneration, with SL-grooved film showing a significant improvement of nerve regeneration. A separate in vivo study evaluated the effect of wall-thickness on nerve regeneration. However, it was shown that the wall thickness had no positive effect, and the conduit with improved mechanical strength adversely affected the nerve regeneration. In conclusion, a nerve guide conduit was developed with the optimised surface structure to support nerve regeneration. The promising in vitro and in vivo studies together with the suitable biomechanical properties and specific surface structure and morphology indicate that the grooved PCL/PLA conduit is a viable treatment for peripheral nerve repair.

Nerve injury is a very common trauma affecting 300,000 people in Europe every year. Although autografts are currently the gold standard in surgery, they can cause loss of sensation and scar tissue formation. Artificial nerve conduits are a valid alternative for peripheral nerve repair. They can provide a confined environment during the regeneration process, enabling axons sprouting from the proximal to the distal nerve segments as well as reducing scar tissue formation. Poly-e-caprolactone (PCL) is a biocompatible and biodegradable polymer suitable for the fabrication of nerve guidances. In particular, previous works demonstrated that neural cells are able to adhere and proliferate on micropitted PCL films obtained through solvent casting. Also, short term studies showed that axons were able to bridge 1cm injury gap. In this work a 18 weeks long term in vivo experiment using a rat model was performed to investigate the reinnervation of end organ skin and muscle. PCL conduits were compared to autografts, with no significant differences in terms of regeneration and reinnervation. However, Schwann cells (SCs), the most important glial cells in the peripheral nervous system, showed poor attachment in vitro on PCL scaffolds; hence, surface modification was carried out in order to improve the material biocompatibility. The effect of both hydrophilicity and functional groups on SCs was first investigated. PCL films were then hydrolysed and aminolysed to modify the surface with carboxylic and amino groups respectively. Hydrolysed films increased remarkably the surface hydrophilicity, although topography and mechanical properties were not affected. Conversely, the tensile modulus and strength were significantly reduced by aminolysis, but still suitable for the desired application. The two treatments influenced also the morphology of SCs. It was demonstrated that cell elongation was induced by hydrophilic surfaces, whilst cells preferred cell-cell interaction when cultured on aminolysed films. However, cell proliferation was remarkably increased on the latter surfaces, confirming previous results obtained on substrates characterised by amino groups. These results confirmed that a good balance between hydophilicity and surface chemistry is necessary to guarantee the best cell response. In order to enhance both proliferation and morphology of SCs, arg-gly-asp (RGD) sequences were immobilised on the PCL film surface using two different reaction mechanisms. Carbodiimide chemistry was compared to a new mechanism developed in the present study based on the Thiol chemistry. Biological tests performed on these modified films demonstrated the improvement of SC response after the peptide immobilisation using the novel approach. Cell attachment and proliferation were three times higher compared to untreated PCL films. It was also observed that the presence of peptides on the film surface induced the formation of focal adhesion plaques by SCs, important for the perception of cellular signals when in contact with a particular substrate. Hence, a good balance between focal adhesion and adhesion forces was achieved after peptide immobilisation. Overall the results of this study showed that material functionalisation is very important for SC response and it will be fundamental for the production of artificial nerve conduits.

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