The use of reflection anisotropy spectroscopy to assess the alignment of collagen

The alignment of collagen fibres in tissue has a major influence on their mechanical properties. This study investigated the ability of reflection anisotropy spectroscopy (RAS) to determine the degree of alignment of collagen fibres deposited onto surfaces and secreted by mouse fibroblast cells in vitro. Aligned nanofibres of polytetrafluoroethylene were deposited on glass coverslips using a simple friction transfer method. These linear parallel nanofibres were used as topographical cues to orientate and align L929 fibroblasts and their deposited collagen. The strength of the RAS signal was demonstrated to correlate with the degree of collagen alignment. Immunochemical staining and atomic force microscopy were used to visualize the topography of the fibres and confirm that the RAS signal was as a result of collagen fibres. Collagen deposited onto glass coverslips from a solution that had been subjected to dialysis that caused 'nanofibrillar' collagen to form also resulted in a strong RAS signal whereas collagen adsorbed from a simple solution of collagen in which collagen fibres are not formed resulted in no RAS signal. It was concluded that the RAS signal could be used to determine the degree of alignment of collagen and that this could have a potential application in the assessment of collagen orientation in tissue repair.