Cyclic straining of cell-seeded synthetic ligament scaffolds: development of apparatus and methodology

Cyclic tensile strains acting along a ligament implant are known to stimulate cells that colonize it to proliferate and to synthesize an extracellular matrix (ECM), which will then remodel and form a new ligament structure. However, this process of tissue induction is poorly understood. As a first step toward elucidating this process, we aimed to investigate the effect of cyclic tensile strain on the proliferation of, and possible ECM synthesis by, cells colonizing ligament scaffolds. Because there was no commercially available apparatus to undertake such investigation the objectives of this study were to develop an apparatus for the application of cyclic tensile strains on cell-seeded synthetic ligament scaffolds and to develop and validate (through preliminary data obtained using the apparatus) methodology for studying the effect of cyclic strain on cell proliferation. We designed a multi-station test apparatus that operated inside an incubator. It allowed the application of tensile cyclic strains of between 0.5% and 5% at a frequency of 1 Hz on cell-seeded polyester ligament scaffolds immersed in culture medium. Test stations with windows in their bases could be easily de-coupled from the apparatus. This allowed monitoring of cell proliferation and morphology, with inverted light microscopy, through the transparent glass bases of the culture wells. Preliminary experiments lasting for 1 day or 9 weeks examined the effect of selected aspects of the cyclic strain on proliferation of cells seeded onto ligament scaffolds. Tests lasting for 1 day showed that the application of cyclic tensile strain of 5% for 4 h increased cell proliferation 24% above that observed in unstrained controls (p < .05). Scanning electron microscopy data from tests lasting for 9 weeks demonstrated further the dependency of cell proliferation and possible ECM synthesis on the magnitude of the strain. The larger the amplitude, the greater was the coverage of the scaffold with cells and ECM. Transmission electron microscopy of the ECM observed at 9 weeks showed evidence of collagen fibrils aligned in the direction of load in strained scaffolds, whereas the tissue on the control scaffolds was random.