Cellular biomechanics of the sclera: role of strain on cell-mediated extracellular matrix contraction and cellular differentiation

© 2010 Nethrajeith SrinivasaluMyopia is a refractive error characterised by an abnormal increase in eye size. The sclera, a structure that maintains ocular integrity, is responsible for controlling eye size and hence is a potential target for myopia control. Active remodelling of the scleral extracellular matrix during myopia has been believed to alter the tissue’s biomechanical properties and result in ocular elongation. However, studies carried out in other connective tissues have demonstrated a possible role for the α-smooth muscle actin (α-SMA) expressing myofibroblasts in contributing to the tissue biomechanics. The effect of myofibroblasts at contributing to tissue biomechanics is further mediated by the stresses affecting the surrounding matrix. As the sclera contains a resident myofibroblast population and is under constant yet varying strain during eye growth and myopia, this study aimed to identify the effect of matrix strain in mediating matrix contraction and scleral myofibroblast differentiation. The first experiment demonstrated the role of cellular strain (intrinsic and applied) in mediating contraction of the in vitro scleral cell-populated matrix. Increasing densities of scleral fibroblasts (2.5 x 10 squared – 3.75 x 10 cubed/mm cubed) were seeded into three-dimensional collagen matrices, which were formulated to approximate the in vivo scleral matrix, demonstrated a cell density-dependent increase in matrix contraction when no external strain was applied. When these matrices were exposed to an external strain of 8.5% and 11.6%, which was calculated to reflect the in vivo strain under normal and myopic conditions, a rapid increase in contraction was observed within 6 hours, while contraction plateaued after 24 hours. The matrix contraction achieved at 24 hours after application of external strain was significantly higher than that achieved by the matrices maintained under intrinsic strain at that time point. The role of myofibroblasts in mediating the contraction of the cell-populated matrices observed in experiment one was investigated in the second experiment using immunocytochemical studies. The results of the second experiment demonstrated a cell density-dependent increase in the population of myofibroblasts in matrices maintained under intrinsic strain for 5 days. However, the α-SMA stained cells in matrices exposed to external strains appeared pyknotic at the end of 5 days. Specifically, these cells were smaller in volume and circular with even smaller nuclei. In addition, these cells lacked a-SMA containing stress fibres apparent in the matrices maintained under intrinsic strain. Since a rapid increase in matrix contraction was observed within 6 to 24 hours after the application of strain, the early expression of myofibroblasts (until 24 hours) was investigated. In these matrices there was an increase in α-SMA staining at various early time points (≤ 6 hours), while at 24 hours these cells became pyknotic and lacked stress fibres. These changes are consistent with the plateauing of matrix contraction from 24 hours to 120 hours after the application of strain. Such a finding suggests that rapid initial increase matrix contraction when exposed to 8.5% strain is mediated by the rapid myofibroblast differentiation, while possible apoptosis underpins the lack of significant contraction after 24 hours. This apoptosis is likely to result from removal of strain on scleral cells due to remodelling of the surrounding extracellular matrix. The sclera is under constant yet varying strain during eye growth due to fluctuations in IOP from events such as rapid eye movements. Thus, the sclera must alter its contractile properties within few seconds to maintain ocular integrity. This study demonstrated that scleral cells are capable of rapid (