Analysis and measurement of Alignment of Human Corneal fibroblast in the presence of uniaxial mechanical load by Fast Fourier Transform

Abstract

Mechanical load has been shown to play a critical role on tissue formation, growth, maintenance, and disease in vertebrate animals. In this study, we measure the dynamics of human corneal fibroblast orientation from cell seeding through tissue formation on a collagen substrate. The experiments were run with or without applied uniaxial tensile mechanical load and the cell orientation was calculated using a 2D Fast Fourier Transform (FFT). FFT provides quantitative information on the relative anisotropy of the corneal fibroblasts which assume an elongated morphology during colonization of the substrate. The FFT analysis was performed on long-term, high-temporal resolution movies obtained with differential interference contrast (DIC) microscopy. The FFT results are converted in directional vectors and transformed to a polar diagram and histogram. The human corneal fibroblasts were cultured on thin isotropic collagen films (~40um thickness) and maintained in our mechanobioreactor on an inverted microscope for a week. Some collagen films (N=5) were strained to ~50KPa and a few (N=2) were totally unstrained. DIC images were captured every 6 minutes and then imported in a custom MATLAB programming to be analyzed by 2D FFT method. The dynamic FFT data (polar and histogram) show that single fibroblast cells have random orientation before cell confluence. After confluence there was consistent alignment of fibroblasts on the loaded collagen films at a relatively fixed angle (~ 45