Production and in vitro evaluation of macroporous alginate hydrogel fibres for nerve tissue engineering

Abstract

The prospects for successful peripheral nerve repair using fibre guides are considered to be enhanced by use of a scaffold material which provides a good substrate for attachment and growth of glial cells and regenerating neurons. Alginate polymers exhibit a highly favourable balance of properties and exhibit the unique property of forming hydrogels under mild crosslinking conditions. As a result alginate has been widely applied for cell encapsulation. However the polysaccharide exhibits extremely poor cell adhesion properties, which limits its application for tissue regeneration. Alginate fibres were modified by gelatin a) to improve cell adhesion b) to investigate the potential for polypeptide growth factor incorporation in the fibres and c) to provide a method for macropore production by extraction of the gelatin particles from the fibres (Chapter 2). Incubation of hydrated alginate fibres in 0.5% w/w gelatin solution at 37 oC resulted in low protein loading (1.7% w/w) and rapid release of gelatin (90% of the initial load) over 24 h in distilled water at 37 oC. In comparison, incubation of hydrated alginate fibres in 5% w/v gelatin solution resulted in approximately 9% w/w protein loading and gradual protein release (80% of initial content) over 9 days. Alginate fibres incorporating gelatin particles were successfully produced by wet spinning suspensions of gelatin particles in 1.5% (w/v) alginate solution. Gelatin loading of the starting suspension of 40.0, 57.0 and 62.5% w/w resulted in gelatin loading of the hydrated alginate fibres of 16, 21 and 24% w/w respectively. Around 45 – 60% of the gelatin content of hydrated fibres was released in 1 h in distilled water at 37 oC, suggesting that a macromolecular structure for cell growth would be rapidly established in cell culture. Furthermore, the residual gelatin is expected to form a favourable surface for nerve cell adhesion and axonal extension. The production of macroporous alginate fibres for cell encapsulation (Chapter 3) was achieved by wet spinning suspensions of gelatin particles in alginate solution into CaCl2 crosslinking solution, followed by protein extraction in distilled water. Confocal laser scanning microscopy (CLSM) and image analysis provided detailed qualitative and quantitative information on pore size and size distribution. CLSM and image analysis also provided measurements of the connectivity of macropores within the hydrogel scaffold which is essential to permit a high level of cell ingrowth. The ability of plain alginate fibres and macroporous alginate fibres to maintain viability of encapsulated cells and to promote cell growth was assessed using fibroblasts in Chapter 4. Swiss 3T3 mouse fibroblasts were encapsulated in alginate fibres by wet spinning cell suspensions of alginate solutions. Increasing the cell concentration of the spinning solution from 1.4 × 104 to 1.5 × 106 cells/mL resulted in no significant improvement of fibroblast extension and growth. Human adult dermal fibroblasts (HDFa) were incorporated in macroporous alginate fibres by wet spinning alginate solutions containing both gelatin microparticles and suspended cells (cell concentration 3 × 105 cells/mL). Cell distribution was evaluated using CLSM (following staining with Calcein-AM) at an excitation wavelength of 488 nm. Fibroblast-loaded macroporous alginate fibres were characterised by a cell density of approximately 360 cells/mm3 which was significantly higher (*P<0.05) than that of non-macroporous fibres (approximately 140 cells/mm3). Cell viability was maintained for time periods of at least 12 days. This finding indicates a potential for repair of soft tissue by encapsulating cells or genetically modified, growth factor-secreting variants in alginate fibres. Encapsulation of nerve cells (primary DRG) in macroporous alginate fibres and cell development within the fibre are described in Chapter 5. Marked outgrowth of DRGs was evident within the fibre at day 11 in cell culture, indicating that macropores and channels created within the alginate hydrogel by gelatin extraction were providing a favourable environment for nerve cell development. Evidence of neurite contact was obtained at day 9. These findings indicate that macroporous alginate fibres encapsulating nerve cells may provide a useful strategy for nerve repair. Retinal degeneration including retinitis pigmentosa (RP) and age related macular degeneration (AMD) caused by progressive and eventual death of photoreceptor cells can eventually lead to blindness. Macroporous alginate fibres were produced incorporating cone photoreceptor-derived (661W) cells to investigate their potential as tissue engineering scaffolds for retinal repair (Chapter 6). Significantly higher cell proliferation (*P<0.05) was evident within macroporous fibres at day 14 in cell culture compared with non-macroporous fibres. Cell flattening and elongation with formation of cell extensions/projections was extensive in 2D culture on tissue culture plastic (TCP) but was not observed in 3D culture within the fibres, indicating that a suitable pore/channel structure for photoreceptor cell development was not created within the alginate hydrogel at 14 days in cell culture.