Elsevier

Biomaterials

Volume 28, Issue 3, January 2007, Pages 441-449
Biomaterials

Polylysine-functionalised thermoresponsive chitosan hydrogel for neural tissue engineering

https://doi.org/10.1016/j.biomaterials.2006.08.044Get rights and content

Abstract

Foetal mouse cortical cells were cultured on 2D films and within 3D thermally responsive chitosan/glycerophosphate salt (GP) hydrogels. The biocompatibility of chitosan/GP 2D films was assessed in terms of cell number and neurites per cell. Osmolarity of the hydrogel was a critical factor in promoting cell survival with isotonic GP concentrations providing optimal conditions. To improve cell adhesion and neurite outgrowth, poly-d-lysine (PDL) was immobilised onto chitosan via azidoaniline photocoupling. Increase in PDL concentrations did not alter cell survival in 2D cultures but neurite outgrowth was significantly inhibited. Neurons exhibited a star-like morphology typical of 2D culture systems.

The effects of PDL attachment on cell number, cell morphology and neurite outgrowth were more distinct in 3D culture conditions. Neurones exhibited larger cell bodies and sent out single neurites within the macroporous gel. Immobilised PDL improved cell survival up to an optimum concentration of 0.1%, however, further increases resulted in drops in cell number and neurite outgrowth. This was attributed to a higher cell interaction with PDL within a 3D hydrogel compared to the corresponding 2D surface. The results show that thermally responsive chitosan/GP hydrogels provide a suitable 3D scaffolding environment for neural tissue engineering.

Introduction

Most neurons in the adult mammalian central nervous system (CNS) do not proliferate or renew themselves and consequently interest has focussed upon cell replacement therapies to repair damage in the CNS. Growth of implanted cells must be controlled in order to guide differentiation and neurite outgrowth, and hence study of suitable scaffolding materials to support cells on implantation is required. The materials must therefore provide appropriate chemical and spatial microenvironment for cell proliferation, differentiation and axon extension.

Hydrogels have many advantages as cellular scaffolds, because they have similar mechanical properties to soft tissue, have low interfacial tension which allow cells to move across the tissue-implant boundary [1] and use only non-toxic aqueous solvents that promote diffusion of oxygen, nutrients and waste throughout the scaffold. Both synthetic and naturally occurring injectable hydrogels have been deployed in the nervous system, and many provide support to cells cultured within the material. Dorsal root ganglia (DRGs), PC12 cells or cortical cells grown within agarose will form neurites [2]. The ability for agarose to support neural cells was improved through optimisation of the gel in terms of porosity and gel stiffness [3], and was functionalised for in vitro culture of cortical or DRG cells [4], [5]. Cortical cells that die on hyaluronic acid hydrogels survive and differentiate when poly-d-lysine (PDL) was covalently bound [6]. Differentiation and neurite outgrowth of forebrain neurons increases as poly(ethylene glycol) (PEG) based hydrogels degrades to create porosity [7].

Chitosan is a well-known biodegradable polysaccharide used in biomedical and cosmetic applications. It is derived from chitin found in crustacean shell which, together with chitosan, is the second most abundant polysaccharide in nature, after cellulose. Tissue engineering applications include dehydrated sponges that absorb fluid [8], [9], material for encapsulating cells [10], [11] or as a gel [12], [13], [14]. The latter relies on the pH sensitivity of chitosan solution: chitosan is soluble in dilute aqueous conditions but precipitates into a gel at neutral pH.

Chitosan supports attachment and growth of a range of cell lines [11], [15], [16], [17], [18], [19] but does not offer the same support for neurons [17], [20]. Gong et al. [17] cultured both gliosarcoma cells (9 L) and foetal mouse cortical cells (FMCC) on chitosan films to determine biocompatibility and nerve cell affinity, respectively. They found that both could be improved to be equivalent with the polylysine control by either coating or blending with polylysine. A follow-on study by the same group determined the optimum concentration of polylysine for cell attachment to be 3 v/v% with chitosan, both in serum and serum-free conditions. This was significantly better than the collagen control surface [21], and three times greater than for chitosan.

Recently, a pH-neutral chitosan solution was developed by Chenite et al. [22] by the addition of a polyol salt (glycerophosphate salt (GP) is most commonly used). The solution forms a macroporous gel scaffold when the temperature is raised to 37 °C [23]. This quality allows the material to be injected and to form a scaffold [24] in situ with minimal surgical destruction. While chitosan/GP has been used successfully in vitro [22], [25], it has not been tested with nerve cells. However, since the physical properties of the fundamental material could be utilised in tissue engineering it would be of value to maximise the biocompatibility of chitosan/GP towards neurons. Currently, chitosan and chitosan/GP need improved neuronal compatibility if they are to be used with the nervous system. Rather than blending chitosan/GP with polylysine, leaving the polylysine free to diffuse away from the material, an examination of the effect of covalently binding polylysine to chitosan was undertaken with the intention of improving the biocompatibility and neuron affinity of the system. Polylysine was chosen for this purpose because its positive nature and high hydrophilicity is known to attract neurons and promote neurite outgrowth [26], [27], [28], [29].

This work explores cell–hydrogel interactions in different in vitro environments, in order to improve neuron affinity of chitosan/GP. Specifically, we wish to work towards developing a biodegradable and injectable scaffold that can be used in conjunction with cell replacement therapies to help repair damaged neural pathways within the brain.

Section snippets

Materials

The chitosan (Sigma) used was of degree of deacetylation (DD) 83% as determined by 13C cross-polarisation magic angle spinning nuclear magnetic resonance spectroscopy (CP/MAS NMR, data not shown) and molecular weight 9.8×104 Da determined by gel permeation chromatography. It was purified by dissolving in 0.1 m HCl (BDH), filtering through grade 3 filter paper (Whatman), heating, and then when cooled, stirring with granulated carbon and refiltering. The chitosan was precipitated by adding 100 mL

Mechanical properties of chitosan/GP

Initial gelation of chitosan/GP takes around half an hour before it becomes solid-like (Fig. 2), but because gel formation slows diffusion and mobility of the polymer chains, gel properties do not stabilise for another 9 h [30]. The strength of the gels varies non-linearly with chitosan concentration and ranges from 67 to 1572 Pa. Because the lower concentrations are of similar stiffness to brain tissue [31], [32], [33] they are suitable in vitro approximations of brain tissue when implanted into

Conclusion

Chitosan/GP has good cell adhesion properties and good neuron compatibility at low concentrations. It also supported neurons cultured three-dimensionally within the hydrogel, a better approximation to an ECM-like environment. Neuron survival was improved with the covalent attachment of polylysine to chitosan: cell survival doubled although neurite outgrowth did not change at an optimal concentration of 0.05% of chitosan mers with an attached polylysine molecule. Chitosan/GP functionalised with

Acknowledgements

This project was funded by an Australian Postgraduate Award, the CRC for Polymers and ARC project DP0450618. Thanks to X. Zhang for NMR characterisation, M. Dodla, J. Saul and R. Evans for discussions regarding chemistry and J. Nunan regarding cell culture.

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