Bioactive surfaces for the delivery of nerve growth factor and brain derived neurotrophic factor and its effects in neurite outgrowth

Peripheral nerve injury is a severe condition that affects the quality of life of a patient as sensory and motor function are impaired. Current treatments include end-to-end suturing and end-to-side suturing of the injured nerve to restore function. The gold standard treatment for larger peripheral nerve injuries is the use of autografts, however, these are limited by donor site morbidity and size mismatches. Alternatively, allografts and xenografts have also been tried, but they might promote immunological responses that lead to rejection. To overcome these disadvantages, a hollow tube called nerve guide conduit (NGC) has been designed and fabricated, with natural and synthetic polymers, to aid peripheral nerve repair by connecting the proximal and distal stumps. Nonetheless, nerve guide conduits are limited to repair nerve gaps shorter than 20 mm. Research has focused on innovating the design of NGCs to improve their performance in a variety of ways. For example, topographical cues, such as channels and intraluminal fibres, have been included inside the lumen of the NGC. Even though promising results have been obtained, for both in vitro and in vivo evaluation, the regeneration potential of these NGCs is still not enough, as it has been recognized that peripheral regeneration is a complex process in which different cells and molecules play a significant role to achieve complete functional regeneration. For this reason, chemical cues have been added to NGCs. Chemical cues added to NGCs include mainly growth factors, peptides, and extracellular matrix components, such as collagen and laminin. These molecules have been incorporated into NGCs through different approaches, such as coatings on the inner wall, matrices filling the lumen, microspheres, adding the molecules within the wall of the NGC, and immobilisation, either on the wall or onto intraluminal fibres. Growth factors, such as nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3), and ciliary neurotrophic factor (CNTF), play an important role during peripheral nerve repair, therefore, these have been included in NGC to enhance neurite outgrowth and Schwann cell migration. Nevertheless, techniques to add these growth factors usually involve the use of solvents, which decrease the bioactivity of the growth factors. Thus, a method to introduce growth factors to NGCs that extends their half-life and preserves their bioactivity would be a promising approach to enhance neurite outgrowth and Schwann cell migration during nerve repair. The use of heparin has been reported to extend the half-life of growth factors as well as maintaining their bioactivity. Usually, heparin is bound covalently on different polymer surfaces, however, this technique could impair the bioactivity of heparin as covalent binding techniques also use solvents. Hence, binding heparin through electrostatic interactions to polymeric surfaces is an encouraging alternative. Heparin is a negatively charged glycosaminoglycan, thus, binding it to a positively charged surface would be the path to follow. Plasma deposition is a widely used technique to modify the surface of a material, making it hydrophilic or hydrophobic, depending on the application. Furthermore, plasma can be used to modify the charge on polymeric surfaces, either to encourage cell adhesion and proliferation of cells, or to bind other charged functional groups to the surface. Therefore, by using plasma deposition technique on a surface, it is possible to bind heparin, and then to bind growth factors, which would create a bioactive surface capable of acting as a local delivery system of growth factors. Specifically, growth factors such as NGF and BDNF can be used to aid neurite outgrowth and Schwann cell migration during nerve repair. This bioactive surface would deliver growth factors in a sustained manner, avoiding undesirable high burst release. Furthermore, taking advantage of the flexibility of the plasma deposition technique, this bioactive surface could be fabricated on the surface of any polymer of choice, regardless of the shape, which would make this bioactive surface an attractive delivery system to adapt to any polymeric scaffold. The work in this thesis consisted of, firstly, fabricating the bioactive surface on a commercially available amine (NH2+) coated plates (positively charged), to which heparin was bound (NH2+ + Heparin). Then, NGF, BDNF, or a combination of NGF plus BDNF, at different concentrations, were immobilised on heparin by electrostatic interactions (NH2+ + Heparin + Immobilised NGF, NH2+ + Heparin + Immobilised BDNF, and NH2+ + Heparin + Immobilised NGF plus BDNF). Contact angle and XPS analysis were used to characterise the bioactive surface and to confirm the presence of heparin. ELISA was performed to characterise the release of NGF and BDNF from the bioactive surface. The bioactive surface was then evaluated using NG108-15 neuronal cells and PC12 adh neuronal cells, finding that immobilised NGF and BDNF encouraged significant neurite outgrowth. Later, this bioactive surface was tested using dorsal root ganglia (DRG) and primary Schwann cells to evaluate neurite outgrowth and Schwann cell migration. The result of these experiments revealed that immobilised NGF at 1 ng/mL encouraged the growth of the longest neurites, in comparison to other test and control groups. Positive results were achieved so far, hence, it was important to implement this bioactive surface to an approach that, in the future, could be implemented in an NGC. Thus, this bioactive surface was added to polycaprolactone (PCL) electrospun fibres. To achieve this, plasma deposition was used to add positively charged amine (NH2+) functional groups on the surface of PCL electrospun scaffolds. Then, heparin was bound to the NH2+, and finally, NGF, BDNF, and a combination of NGF plus BDNF, at different concentrations, were immobilised onto the PCL + NH2+ + Heparin surface. XPS analysis was performed to confirm the successful addition of NH2+ and heparin. Moreover, ELISA showed that there was a sustained delivery of NGF and BDNF from the bioactive surface for up to21 days. Additionally, XPS analysis revealed that heparin was still present on the bioactive surface after 3 months of incubation at 4°C and 21°C. Furthermore, DRG were seeded on these PCL + NH2+ + Heparin + Immobilised NGF/BDNF/NGF plus BDNF scaffolds and neurite outgrowth and Schwann cell migration were evaluated. The results of this experiment revealed that PCL + NH2+ + Heparin + Immobilised NGF 1 ng/mL encouraged the longest neurite outgrowth as well as the furthest Schwann cell migration in comparison to other test groups, control groups, and importantly, to other results found in the literature. Even though PCL + NH2+ + Heparin + Immobilised BDNF did not encourage the longest neurite outgrowth, this surface stimulated the growth of longer neurites in comparison to when the bioactive surface was fabricated on flat, commercially available NH2+ coated plates, revealing that topological and chemical cues further improve the growth of neurites and migration of Schwann cells. Nevertheless, PCL + NH2+ + Heparin + Immobilised NGF plus BDNF did not stimulate an accumulative effect regarding neurite outgrowth and Schwann cell migration. In conclusion, the bioactive surface NH2+ + Heparin + Immobilised NGF/BDNF/NGF plus BDNF was successfully fabricated on PCL electrospun fibres. Then, it was found that NGF and BDNF were delivered in a sustained manner for 21 days. In addition, it was found that by immobilising a relatively low concentration of NGF (1 ng/mL), neurite length of 3 mm was achieved in vitro. Therefore, the use of this bioactive surface on PCL electrospun fibres makes this approach directly applicable and scalable for improving the function of NGCs.