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Fibroblast Shape and Organization Changes within Contracting Load Bearing Collagen Gels.   B. P. Johnson-Wint and B. Coon  Dept. of Biological Sciences, Northern Illinois University, DeKalb IL.

   How do specialized dense connective tissue and bone cells work to create structurally and mechanically sound tissue?   We are interested in the physical organization of the collagen fibers by fibroblasts and osteocytes during this process and the role of tissue load on cell function.   In the present study we examined fibroblast filopodia formation and cell-cell interaction during the reorganization of type-I collagen in weight bearing cell populated collagen gels (CPCGs).

   To track cell bodies and filopodia, isolated rat tendon fibroblasts were stained in vitro with 25 µM Cell Tracker Green or Cell Tracker Orange (Molecular Probes).  The 2 types of stained cells were then mixed and seeded into type I collagen gels.  The CPCGs were placed under load and imaged live with a Zeiss laser scanning confocal microscope at multiple times up to 55 hours.  Time point data stacks were converted into 3D images and movies for structural analysis using Image J.

    An extensive interconnected network of filopodia formed between fibroblasts in contracting CPCGs.   By 4 hours cells had extended multiple, frequently branched, thin filopodia of 1 cell diameter length.  By 16 hours these thin filopodia were 3 to 5 times longer and often in point or lateral contact with the filopodia of neighboring cells.  By 36 hours of collagen gel contraction the cells were closer together and their filopodia were shorter and fatter with a larger surface of contact between the ends of the filopodia.  Finally, by 55 hours the cells had drawn next to each other forming long branching strings of side-by-side adherent cells within the now highly compact CPCG.

   In conclusion, we have found that early in collagen gel contraction fibroblasts extend multiple thin filopodia that attach to each other forming a tight filopodial network in the 3D collagen gel.  Later, cells adhere side-by-side forming a network of branching cell strings.  These two types of network appear to be under tension and may reflect two mechanisms whereby cells squeeze fluid out of collagen gels and compact the collagen fibers creating a denser stronger tissue of a particular shape.  Supported by NIU and the NIU Foundation.