Architectural, mechanical and biochemical control of collagen films and scaffolds to guide neural- and fibroblast cell behaviour

Abstract

Collagen-based scaffolds offer the potential to bridge gaps in damaged nerves via infiltration of cells along oriented pore structures which can guide sprouting axons and direct Schwann cell migration. Neural cells have been shown to be highly mechanosensitive and hence substrate stiffness is an important factor in the repair process. However, the competition between neural cell attachment and fibrotic scar tissue formation also needs to be addressed. The goal of this thesis was to investigate the effects of mechanically and biochemically tailored collagen scaffolds on the attachment, migration and proliferation of neural- and fibroblast cells. By assessing cell behaviour in contact with scaffolds and thin films (as a surrogate for scaffold struts) it was possible to deconvolute the effects of two- and three dimensional architectures.

Chemical cross linking and incorporation of elastin in collagen films were investigated by atomic force- and optical microscopy, and tensile mechanical testing. 10% elastin incorporation was shown to increase the surface roughness of the films three-fold and decrease the stiffness from 10s of MPa to 100s of kPa. Carbodiimide cross linking using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in the presence of an N-hydroxy-succinimide (NHS) catalyst resulted in dose-dependent stiffness. Cross linking was varied from 0% to 100% (defined as molar ratio EDC:NHS:COOH[from collagen] of 5:2:1), resulting in an increase in modulus from 7 MPa to 65 MPa for collagen films and from 340 kPa to 740 kPa for collagen-10% elastin films. It was concluded that control of composition and degree of cross linking can be used to tailor film stiffness and surface roughness.

Three dimensional collagen scaffolds with aligned pore structures were produced by a unidirectional freezing process. The pore morphology, including pore size and percolation diameter was characterised by micro computed tomography and scanning electron microscopy, with median pore sizes of approximately 50 μm. Carbodiimide cross linking and elastin incorporation were investigated to create structures with physiologically relevant stiffness.

The biological response to 2D films was assessed using rat Schwann cells (RSC96), model neuronal (PC-12) cells and human dermal fibroblasts (HDF). Fluorescence imaging and lactate dehydrogenase assays revealed that elastin incorporation and lower levels of cross linking led to an increase in the attachment of RSC96 and PC-12 cells. Elastin incorporation alone led to a doubling of PC-12 attachment. It was concluded that this was due to changes in film mechanical properties as the cells were found to bind non-specifically to the films. HDFs showed minimal sensitivity to elastin incorporation, but binding was reduced by up to 45% at higher cross linking densities due to specific integrin binding motifs being consumed by the cross linking process.

Schwann cell infiltration and proliferation in 3D scaffolds was investigated with fluorescence microscopy and a PrestoBlue assay. The cells appeared to be insensitive to the degree of cross linking and elastin incorporation and full penetration of the scaffolds occurred by Day 5 with a relatively high degree of proliferation. The results suggested that the scaffolds provided sufficient guidance to Schwann cells to allow their migration and ultimately help to support tissue regeneration.