A METHODOLOGY FOR THE SYSTEMATIC AND QUANTITATIVE STUDY OF CELL CONTACT GUIDANCE IN ORIENTED COLLAGEN GELS - CORRELATION OF FIBROBLAST ORIENTATION AND GEL BIREFRINGENCE

Despite the likely role of contact guidance in every physiological pro cess involving cell migration, its study in a three-dimensional tissue -equivalent environment has been precluded, heretofore, by inherent di fficulties in systematically preparing well-defined contact guidance f ields and quantifying the resultant contact guidance. Here, we describ e a novel use of a magnetic field to orient collagen fibrils during fi brillogenesis, entrapping cells dispersed in the collagen solution. Us ing computer-controlled staging and image analysis, we show from autom ated birefringence measurements of the resultant slab of cell-populate d gel contained in a specially designed observation chamber that the f ibril orientation is biased along the long axis of the chamber uniform ly throughout the chamber. Further, we show that the degree of fibril orientation, and consequently the elicited contact guidance, can be co ntrolled by independently varying the magnetic field strength or tempe rature during fibrillogenesis. We characterize the contact guidance re sponse to the imposed contact guidance field by measuring cell orienta tion relative to the axis of fibril orientation from still images obta ined in time-lapse via automated image analysis. We present the first quantitative correlation of contact guidance (based on cell orientatio n) with collagen fibril orientation (based on birefringence) for human foreskin fibroblasts cultured in a collagen gel, by using gels of var ying orientation resulting from different magnetic field strengths and temperatures during fibrillogenesis, and by using sufficiently low ce ll concentrations and early observation times.