Designing a CL surface to reduce corneal stress
Closing Date: 04/12/2020
Friction is a difficult parameter to compare across different contact lens brands. Even in the laboratory, the friction coefficient is sensitive to numerous factors, including the solution environment, contact geometry, pressure applied, sliding speeds, surface conditioning, temperature and surface roughness. To model how the surface of a contact lens rubs against the ocular surface, modern in vitro approaches have paired a corneal epithelial cell model surface against a sliding portion of a contact lens. The instrument that performs this type of direct contact sliding between these two surfaces is called a biotribometer, and it has been used to compare and contrast differences between five daily disposable contact lens materials: three were inherently wettable contact lens materials and two had soft high water content surface gel layers. The lenses with surface gel layers demonstrated statistically significant reductions in damage and trauma to the epithelial cells under direct contact sliding. These results correlate well with clinical findings and suggests that this approach could be used prior to initiating lengthy and often expensive clinical trials.
While it is seemingly easy for a contact lens wearer to report that their lenses are uncomfortable, determining what causes discomfort remains an ongoing challenge for clinicians and scientists. Numerous factors may be involved in contact lens discomfort, from properties of the lenses themselves to the environmental factors during wear. The reported discomfort may range from mild to an inability to continue wearing the lenses (figure 1).1 Contact lens solutions could interact with the lens, and the subsequent interactions between the lens and the ocular surfaces could impact comfort.2,3 Lubricity is a particular area of interest in studying how low-friction (good lubricity) between contact lens surfaces and ocular and conjunctival tissues during wear affects comfort.4 Intuitively, lenses with a lower coefficient of friction (COF) should be more comfortable.5
Figure 1: Contact lens discomfort can come from multiple sources, and progress through several stages (after the TFOS report, 20131)
A significant complication in laboratory friction studies is that the reported COF is a measurement of the interface between two surfaces (ie the surface of the contact lens and the countersurface upon which it slides) within a particular solution environment. For contact lenses, the selection of countersurfaces, surface preparation and the solution environments can differ widely, and the result is a wide variation in friction coefficient measurements even for the same materials.6 Contact lens biotribology (the science of friction and lubrication applied to biological systems) has demonstrated multiple factors that can affect friction measurements of contact lenses – the force applied, the contact area, the velocity across the surface, the contact time, the substrate and the composition of the lubricating fluid.7-11
A standardised in vitro method to allow comparison between lenses has not been agreed upon. However, a standard method may not reflect the actual performance of the lens in the eye, as tear composition is complex and varies from person to person. The addition of biological components such as proteins and lipids does not reflect the true composition of tears, and variations between the additives prevent credible inter-study comparison.12-15 One postulate presented and discussed in the literature is that frictional work will reduce comfort. This model suggests that both the coefficient of friction and the duration of sliding affect comfort, so that the longer the user wears a contact lens, the greater the degree of discomfort. However, in the clinical setting, replacement of a lens with a new one was not found to improve comfort,16 suggesting that conditioning of the ocular surfaces during contact lens wear is a more likely genesis of the discomfort.
It is difficult, if not impossible, to make comparisons of COF measurements between different laboratories, each potentially using different methods, contact conditions, countersurfaces and instruments. From this vantage point, we suggest that contact lens surfaces could be more fairly characterised using a method where the contact conditions are standardised to near physiological levels, and the countersurfaces are living epithelial cells of ocular origin.
Shear Stress as a predictor of cellular damage and contact lens discomfort
Designing experiments to match the physiological conditions of a contact lens in the eye is challenging and requires careful
attention to factors such as the contact pressure between the contact lens surface and a uniform layer of corneal epithelial cells (grown on a specially prepared smooth glass surface). The eye is in nearly constant movement while awake, and in addition to low forces and low contact pressure, these experiments were designed to perform 1,000 cycles of repeated sliding to study the progression of damage. Cells combine to form the epithelium, across which contact pressure and frictional forces interact with the surface. The spreading of these frictional forces is shear stress, τ (Greek letter tau), which is the product of the contact pressure, P, and the coefficient of friction, µ, and given by the equation:
Shear stress has been identified as a source of cellular disruption for corneal epithelial cells.17 Under repeated sliding the shear stress causes the release and accumulation of intracellular signals (such as cytokines), which may explain the observation of continued discomfort despite changing to fresh lenses. This biological model of shear stress activating damage-associated intracellular pathways is also consistent with in vitro production of pro-inflammatory cytokines and the start of apoptosis.18 Apoptosis is a programmed cell death process, which is different from necrosis and acute trauma.
The University of Florida (UF) quantified apoptosis of corneal epithelial cells in contact with contact lens surfaces during sliding by combining in situ staining, molecular biology techniques, and scanning fluorescence confocal microscopy. In baseline experiments, a spherically capped polyacrylamide gel probe was fabricated, loaded against an epithelial cell layer to a contact pressure of 30Pa and slid continuously for over 16 hours resulting in 10,000 cycles over the same area. This baseline study showed no measurable cell damage19 at this level of pressure. However, by increasing the shear stress through increases in contact pressure, coefficient of friction, or both, cellular damage and pro-inflammatory cytokine release was observed.20
Shear stress has emerged as a clear biological predictor of epithelial cell damage, whose impact on the eye may be particularly important for contact lenses. From a design perspective, stiffer contact lens surfaces will focus loads over smaller areas of the ocular tissues, increasing localised contact pressure and shear stress. In contrast softer surfaces will spread the load over a wider area, reducing both pressure and shear. The surface softness of a contact lens is not necessarily related to the published bulk modulus. To test this hypothesis, two silicone hydrogel daily disposable contact lenses with soft surface gel layers, delefilcon A (Dailies Total 1) and verofilcon A (Precision 1), were compared to three other silicone daily disposable contact lenses without soft surface gels (ie inherently wettable surfaces). The higher surface water content of the lenses with the surface gel layers results in a lower elastic modulus and stiffness at the surface, a reduced coefficient of friction (improved lubricity) and, under the same applied force, should reduce the activation of damaging intracellular pathways.
Direct contact experiments between the surfaces of commercially available daily disposable contact lenses and a layer of immortalised human corneal epithelial cells were performed using a biotribometer with in situ optical microscopy. The set of lenses tested included two lenses with high water outer surface layers (delefilcon A and verofilcon A) and three other commonly available contact lenses, one hydrogel (etafilcon A) and two silicone hydrogels (stenfilcon A and somofilcon A). Vital stains were used to determine cellular health by observing cellular structures, such as actin filaments, the nucleus and various mucins. The biotribometer used an applied normal force of 200µN, a stroke length of 3mm and sliding velocity of 1mm/sec while sliding a probe carrying the contact lenses across the surface of the epithelial cells for 1,000 cycles at a temperature of 37 ± 1°C (figure 2). As a control, a matched set of cells were isolated from sliding, but otherwise experienced the same culture and handling conditions.
Figure 2: a) A biotribometer can prescribe normal force and measure frictional forces on living cell monolayers. b) A pHEMA contact lens holder is integrated with the cantilever to perform experiments on different lens materials. c) The membrane probe design allows for independent control of contact pressure to physiological levels. d) The contact area and corresponding pressures are spread across a large cell area. e) A two well design allows for direct differential analysis of cell responses to shear against contacting materials
Periodically during testing, the entire contact zone was imaged with a low concentration of propidium iodide (PI) within the liquid media. Fluorescence microscopy visualises how the PI molecule is preferentially taken up by the nuclei of damaged cells (figure 3). The cells that stain positive for PI are identified, outlined, and counted across the entire sliding path. At discrete imaging time points, based on the number of reciprocating cycles, the cell damage density is computed by dividing the number of cells that stain positive for PI by the overall contact area (ie damage density is the number of cells damaged per mm2). All experiments were performed using biological replicates in triplicate, and each contact lens experiment had measurements of damage at cycles 0, 1, 10, 100, and 1,000. Additionally, cells outside of the contact zone were measured to compute baseline (normal) apoptotic cell turnover rates. Figure 3 shows the approach taken to create the cell damage maps from cell identification, tracing, and final construction.
Figure 3: Propidium iodide stains the nuclei of damaged epithelial cells, enabling them to be counted and the damage quantified. a) cell identification (circle around PI positive nucleus), b) tracing of the cell nucleus and representative cell border, c) expanded example showing the tracing over a single microscope image and d) construction of the cell damage map
Results and Discussion
Baseline measurements of PI staining for the corneal epithelial cells under culture and without contact with lenses had a damage score of 7±4 cells/mm2, indicating that less than 0.1% of the cells were undergoing normal apoptosis and were healthy.
The measurements of cell damage after 1,000 cycles of sliding for a single representative sample are shown for the different lenses in figure 4a, which is ordered based upon the average cell damage density. The averages and standard deviations for the replicates (error bars) are shown for each lens type at all time points in figure 4b. The model was able to differentiate the lenses without high water content surface gel layers (somofilcon A, stenfilcon A, and etafilcon A) to a high degree of statistical significance. The two lenses with surface gel layers (verofilcon A and delefilcon A) demonstrated cell damage less than one-tenth that of the lenses without. The average value for the lenses with the surface gel layers were found not to be statistically different from one another and the mean of measured cell damage after 1,000 cycles was within the measurement variance of baseline cell damage.
Figure 4 a) Representative cell damage maps from a single experiment after 1,000 cycles of sliding for each of the materials examined. b) Graph of the measured levels of damage for three replicates at cycles 1, 10, 100 and 1,000 (error bars are standard deviations). c) Individual measurements of damage after 1,000 cycle of sliding for each material examined. The arrows indicate the measurements shown in (a) and the mean values as shown by the horizontal dashed line are plotted in (b)
These findings of increased cell damage associated with frictional shear are consistent with earlier studies on corneal epithelial cell models that measured the release of friction induced inflammation18 and apoptosis (pre-inflammatory) markers.20 In these experiments, which were performed against polyacrylamide probes (as opposed to contact lens specimens), increased shear stress increased the expression of pro-inflammatory genes and damage associated apoptotic genes. These findings are consistent with our understanding of epithelial cells, which are known to sense and respond to mechanical strains and deformations through a process called mechanotransduction.
The lenses with high-water content surface gel layers to reduce the contact pressure, friction coefficient, and shear stress demonstrated a reduction in damage to the ocular epithelial cell models in vitro. Biotribological cell model systems have repeatedly shown that shear stress drives the production of pro-inflammatory cytokines, cell damage and apoptosis. These results are consistent with the symptomatology of dry eye disease, suggesting that underlying mechanisms of contact discomfort may be a result of contact lens-induced shear stress and the cellular responses to shear. Clinical study findings are consistent with this hypothesis. For example, delefilcon A lenses often show better comfort and less dryness sensation when compared with other daily disposable contact lenses.21-24 Additionally, patients with symptomatic end of day dryness also report improved comfort with delefilcon A compared to other silicone hydrogel daily disposable lenses.25 Recently introduced verofilcon A lenses, which also have a high water content surface gel layer, have also shown good clinical results.26
The development of lenses which have softer and more lubricious high water content surface gel layers results in significantly lower in vitro damage to epithelial cells from shear stresses induced during rubbing. The lenses have been shown to have superior comfort in clinical tests. This in vitro technique can help scientist measure lens-cellular interaction before full clinical trials are needed.
Table 1: Upregulation of genes associated with the production of pro-inflammatory cytokines and damage associated apoptosis were studied after sliding under contact pressures of 1.3 and 2.1 kPa and shear stresses of 30 and 60 Pa (18). Increased gene expression was observed following 5.5 h of tribological testing for a pro-inflammatory genes (IL-1β, IL-6, and MMP9) and damage associated pro-apoptotic genes (DDIT3 and FAS)
Many thanks to all of the students and faculty at the University of Florida for their expertise and facilities in performing these experiments.
Bob Tucker and John Pruitt are employed by Alcon Laboratories LLC, Fort Worth, TX, USA. Greg Sawyer is an Ebaugh Professor at University of Florida.
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