Abstract
Abstract: :
Purpose: Experiments have shown that outflow facility is strongly affected by acto–myosin "tone" in outflow pathway cells, indicating a role for cellular biomechanical properties in determining outflow resistance. No data about the biomechanical properties of trabecular meshwork (TM) cells are available. Here we use paramagnetic microbeads to directly measure the stiffness of cultured TM cells. Methods: TM cells were harvested from human donor eyes and maintained in DMEM containing 10% fetal bovine serum. Cells in passages 5–8 were grown to a sub–confluent monolayer and serum starved for 24 hours before being used for experiments. 4.5um diameter tosyl–activated paramagnetic beads (Dynal Biotech) were coated with a synthetic RGD peptide (Integra LifeSciences) and bound to integrins on TM cells. Beads were added in a quantity that ensured attachment of 1 to 2 beads per cell. A magnetic microneedle was then used to pull the beads as described elsewhere (Matthews et al., BBRC, 313:758–764, 2004) and the resulting bead motion was captured by real–time videomicroscopy and analyzed. An effective elastic modulus (stiffness) for each cell was obtained by fitting the bead displacements to a lumped parameter model for the cell as described by Bausch et al. (Biophysical Journal, 75:2038–2049, 1998). The stiffnesses of cultured human umbilical vein endothelial cells (HUVEC) (Cambrex Biosciences) were also determined by the same method, for comparison to the TM cell stiffnesses. Results: TM cells showed viscoelastic behaviour, with an initial elastic displacement and a subsequent viscous "creep". The mean stiffness of cultured TM cells from passage 5 was 0.023 ± 0.006 Pa–m (mean ± SEM; n=22 measurements from 10 cells). The mean stiffness of cultured HUVEC cells from passage 6 was 0.024 ± 0.006 Pa–m (n=12 measurements from 4 cells), which was not significantly different from TM cells (p<0.05). Conclusions: These results indicate that it is feasible to directly measure the biomechanical properties of outflow pathway cells. TM cells grown under static culture conditions have similar mechanical properties to HUVEC cells, but are less stiff than previously reported values for fibroblasts. These experiments will be extended to measure the elastic modulus of Schlemm's Canal (SC) endothelial cells as well as TM and SC cells treated with agents known to affect actin architecture, such as Dexamethasone and Latrunculin B.
Keywords: trabecular meshwork • cytoskeleton • intraocular pressure