Chan V, Drolle E, Phan CM, Hui A, Shi C, Subbaraman L, Wu J, Jones L. Evaluating the activity of lysozyme deposited on contemporary reusable silicone hydrogel contact lenses using an in vitro eye model The Association for Research in Vision and Ophthalmology, New Orleans, LA, USA, 2023 [ Show Abstract ][ PDF ]

Purpose: To evaluate lysozyme activity (LA) on five contemporary reusable silicone hydrogel contact lens (CL)materials over their proposed wear period using an advanced in vitro blink model.

Methods: Five CL materials (lotrafilcon B, samfilcon A, comfilcon A, senofilcon A, and serafilcon A) were cycled daily for 16h on an eye model, followed by 8h of soaking in OPTI-FREE PureMoist, to mimic a typical wear cycle. An artificial tear solution containing physiologically representative proteins and lipids was delivered to the model at a rate of 1.2-2.1μl/min. The model includes an artificial eyelid that blinks at a rate of 6blinks/min, which was kept at room temperature and humidity above 50%. Serafilcon A and senofilcon A were tested over 14 days, whereas the other CLs were evaluated for 30 days. At specified time intervals, including after 1, 7, 14 and 30 days, CLs were removed from the model and lysozyme extracted using a solvent containing acetonitrile and trifluoroacetic acid. The LA from the extracts were then evaluated using a micrococcal absorbance assay.

Results: Overall, LA decreased over time, reaching non-detectable levels by day 30 (p<0.05). Serafilcon A (13.9 ± 7.8μg/lens), and samfilcon A (9.6 ± 2.3μg/lens), had the highest LA after 1 day, followed by comfilcon A (4.7 ± 1.8μg/lens), lotrafilcon B (3.3 ± 1.6μg/lens), and senofilcon A (2.2 ± 3.7μg/lens). By day 7, LA for the weekly replacement lens, serafilcon A, decreased to 0.5 ± 0.6μg/lens. By day 14, LA for the biweekly replacement lens, senofilcon A, decreased to 0.6 ± 0.7μg/lens. Lotrafilcon B, samfilcon A, and comfilcon A, all monthly replacement lenses, decreased in activity by day 30 (0.1 ± 0.2μg/lens, 0.5 ± 0.7μg/lens, 0.0 ±0.0μg/lens respectively).

Conclusions: Deposition of biologically active lysozyme has been proposed to be an important factor for biocompatible CL wear. A decline in activity over time as the deposited protein becomes denatured may impact overall CL performance and has been linked to reduced comfort. LA decreases over time and reaches near zero for all lens types by the end of their proposed wearing period, confirming that they should be replaced within their specified replacement intervals. Compared to simple in vitro vial models, using an advanced blink model for CL deposition testing aims to provide more physiologically relevant results prior to clinical testing.

Garg P, Wulff D, Phan CM, Jones L. Evaluation of a biodegradable bioink for the fabrication of ophthalmic devices using 3D printing The Association for Research in Vision and Ophthalmology, New Orleans, LA, USA, April, 2023 [ Show Abstract ][ PDF ]

Purpose: To develop a degradable bioink for fabricating ophthalmic devices using 3D printing.

Methods: The bioink formulation consisted of 10% gelatin methacrylate (GelMA), 0.6% lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and 5% yellow dye as a light absorbing agent to improve print resolution. The bioink was used to 3D print square sheets (7x7x1 mm) using a commercial masked-stereolithography (mSLA) 3D printer at 95% humidity and 37°C temperature. The degradation of printed sheets was evaluated with different concentrations (0,25,50,100 μg/ml) of matrix metalloproteinase (MMP9) enzyme 37°C. MMP9s are naturally found in the tear film and elevated in various diseased states such as in corneal wounds and dry eye disease. The weights of the sheets were measured at t = 0,4,6,8,12,16,24 hrs. Another set of cubes (1x1x1 cm) was autoclaved and kept sealed in storage at different temperatures (4°C, 25°C, and 37°C) in phosphate buffered saline (PBS) and their weight was measured on day 10. An attempt was made to fabricate a contact lens using this bioink.

Results: Samples that were exposed to MMP9 enzymes showed a time-dependent degradation with increasing enzyme concentration. The samples incubated with 100 and 50 μg/ml of MMP9 were completely degraded by the end of 12 and 16 hrs, respectively. At the end of 24 hrs, the samples incubated at 25 μg/ml enzyme showed 72.8% degradation whereas the control samples did not show any signs of degradation. Interestingly, samples that were autoclaved and kept in storage also did not show any signs of degradation at all temperatures. A 3D-printed CL with overall diameter 14mm and thickness 1mm was printed without any support structures within 1 hour.

Conclusion: This study showed GelMA-based bioink can be used to fabricate biodegradable devices such as contact lenses. The biomaterials degrade in the presence of MMP-9 and future work will work on tuning the degradation kinetics of these materials, as well as incorporating ocular drugs.

Garg P, Wulff D, Phan CM, Jones L. Fabrication of a degradable ocular drug delivery system using 3D printing CBB 2023 Conference: Waterloo for Health, Technology and Society, March, 2023 [ PDF ]

Ho B, Phan CM, Ramasamy M, Hui A, Jones L. PDMS microfluidic devices fabricated from commercial 3D printers support growth of viable HCECs and enable cell biological assays for low-cost high-throughput screening The Association for Research in Vision and Ophthalmology, New Orleans, LA, USA, April, 2023 [ Show Abstract ]

Purpose: To integrate human corneal epithelial cells (HCECs) into a PDMS microfluidic chip fabricated from a novel 3D printing method to perform cell biological assays.
Introduction: The advent of microfluidic devices has enabled tight control over the physical and chemical cellular environment in vitro, while allowing for large-scale imaging and biochemical reactions at single-cell resolution. These devices are capable of miniaturizing assays to the microliter and nanoliter range, thereby increasing assay throughput with high sensitivity, a feature that is highly advantageous in high-throughput cell-based screens. Polydimethylsiloxane (PDMS) has been widely used in microfluidics devices due to its optical clarity and non-toxicity to cells, among other desirable features. However, the fabrication of PDMS devices traditionally requires specialized facilities and instruments. Additionally, PDMS itself is highly hydrophobic and does not support mammalian cellular viability and growth.

Methods: PDMS devices were cured in 3D-printed moulds generated using the FormLabs stereolithography (SLA) printer (FormLabs 3B+, FormLabs, Somerville, MA). These devices were sterilized by autoclaving, and coated with 0.01% polydopamine (PDA) and 20μg/mL collagen. HCECs were seeded onto the device, and allowed to grow for 18-36 hours in DMEM/F12 media at 37oC. HCECs were imaged by light microscopy, and viability was assessed by alamarBlue assays.

Results: Here, we present a novel and simple method of generating PDMS microfluidic devices suitable for mammalian cell biology assays using commercial 3D printing. We show that PDMS devices coated with polydopamine (PDA) support the growth of human corneal epithelial cells (HCECs) that are metabolically active (~60-90% viability) and are comparable to HCECs cultured in standard tissue culture plastic consumables. Finally, HCECs cultured in our devices are capable of growth with fluid flow rates of up to 1mm/s.

Conclusion: Our study shows that PDMS devices manufactured through the aid of a novel 3D printing pipeline support the growth of HCECs. We aim to utilize these microfluidic devices as a tool to screen different compounds and formulations while assessing cellular viability and acquiring high resolution microscopic and fluorescence images of HCECs.

Hui A. Commercialized Ocular Drug Delivery Devices XXV Biennial Meeting of the International Society for Eye Research, Feb 21, 2023 [ Show Abstract ]

The goal of creating a sustained drug delivery device for the anterior or posterior segment has been approached from numerous avenues, from inserts to contact lenses and punctal plugs. The utility of a device which can sustain drug release allows for the potential to increase patient compliance as well as improve clinical outcomes, as agents are able to be maintained at appropriate concentrations for desired periods with less administration. This presentation will discuss drug releasing devices for the eye which have reached commercialization. The indications for these devices, their reported effectiveness and known adverse reactions will be discussed. The experience on the use of the devices after they have been released on to the market will also be discussed, as well as their current clinical usage in contemporary eye care management.

Hui A, Heynen M, Chan V, Mirzapour P, Enstone D, Saad M, George M, Ngo W, Jones L. The impact of RGP care solutions on ISO measured lens parameters and the protein deposition on RGP lenses when managed with a hydrogen peroxide care solution Global Specialty Lens Symposium, Las Vegas, Jan 20, 2023 [ PDF ]

Jin Y, Jones L, Gorbet M. Investigation of the Circadian Recruitment of Tear Neutrophils to the Ocular Surface and Their Phenotypes  The Association for Research in Vision and Ophthalmology, New Orleans, LA, USA, April, 2023 [ Show Abstract ]

Purpose: Hundreds of thousands of leukocytes (with approximately 60% of the population being polymorphonuclear neutrophils (PMNs)) can be collected from the ocular surface immediately on waking, while only a few are harvested during the daytime. To better understand their role in ocular surface homeostasis and inflammation, this study aimed to investigate potential factors contributing to the circadian infiltration of tear PMNs and changes in phenotype across different time points in a 24-hour cycle.

Methods: Tear leukocytes were collected from 30 participants using a gentle eyewash after 2-hr and 7-hr of sleep at night, after 2-hr sleep during the day, and towards the end of the day (around 5 pm). Cell count and morphology were determined using a Moxi Z cell counter and May-Grunwald stain, respectively. Cells were stimulated by fMLP. Changes in the degranulation (lactoferrin, CD66b, CD63) and cell aging state (CD184) of PMNs were measured via flow cytometry. Neutrophil extracellular traps (NETs) were also identified by flow cytometry and microscopy following staining with myeloperoxidase, citrullinated histones, and CD15.

Results: Significantly more cells were collected from the nighttime compared to the daytime (p<0.001). There was a positive correlation between IL-8 concentrations and PMN numbers, but not with C5a, suggesting that the recruitment of tear PMNs to the ocular surface may be driven mainly by IL-8. 2hr-sleep-derived tear PMNs were less degranulated than 7hr-sleep-derived tear PMNs (p<0.03) and possessed a larger functional activation potential in response to stimulus (p<0.03). Furthermore, 7hr-sleep-derived tear PMNs exhibited hyper-segmented nuclei and were prone to aggregation, when compared to 2hr-night-derived tear PMNs, suggesting an aged and activated phenotype. A significantly increased number of NETs were present in 7hr-night-derived tear samples (p<0.05).

Conclusions: The diurnal-nocturnal recruitment pattern of tear PMNs may be driven by the increase in IL-8 in nighttime tears. Higher levels of degranulation and NETs indicate that tear PMNs become more activated on the ocular surface during prolonged eye closure at night. This PMN inflammatory response must then be balanced by other anti-inflammatory processes to prevent ocular surface damage.

Jones L. Osmolarity: Gold Standard for Dry Eye Disease Determination or Random Number Generator? XXV Biennial Meeting of the International Society for Eye Research, Feb 23, 2023 [ Show Abstract ]

The potential impact of osmolarity on dry eye symptoms dates back to the early 1970’s. The concept that tear film instability leads to hyperosmolarity and that this is a key factor in the development of dry eye disease (DED) is described in the 2017 TFOS Reports. Both aqueous deficient and evaporative DED can lead to tear hyperosmolarity, which stimulates ocular surface inflammation through increased expression and production of proinflammatory cytokines and chemokines, resulting in corneal epithelial cell death by apoptosis. Ocular surface disease and tear hyperosmolarity have also been associated with goblet cell loss, which can lead to altered mucin expression and tear film instability, further exacerbating inflammatory processes. These changes may stimulate sensory nerve fibers innervating the ocular surface, generating sensations of ocular discomfort. Although osmolarity has gained fairly widespread acceptance as a DED diagnostic, findings suggest that tear osmolarity measurements do not possess the very high sensitivity and specificity claimed by some studies and it may have limited value in the diagnosis of DED. A key factor in determining the relationship between DED and osmolarity relates to the ability to measure osmolarity, a process that is not without its challenges. The low volume of tears on the ocular surface necessitate using techniques that can estimate osmolarity using micolitre or nanolitre volumes of fluid. Clinical assessment methods typically occur through estimation of the osmolarity of the inferior tear meniscus. However, calculations using fluorescence assessments and hyperosmolar drops suggest that hyperosmolar levels in the tear film overlying the ocular surface may transiently spike during tear instability to levels far greater than that determined in the inferior tear meniscus, resulting in corneal inflammation and triggering sensory neurons. This may explain the relative disconnect between DED symptoms and osmolarity measurements.
Additonally, variability in results obtained between instruments, within-visit measures, differences between eyes and the impact
of treatments on osmolarity values over time require further examination.
In addition to DED, this review will examine other factors that may impact osmolarity values, including contact lens wear, cataract
and refractive surgery and systemic drugs. Finally, the osmolarity of ocular lubricants and contact lens blister packs and their
clinical relevance will be discussed.

Luensmann D, Tucker AW, Voltz K, Guthrie S, Woods J, Vega J. Orthokeratology Lens Fit Success Using a New Software Global Specialty Lens Symposium, Las Vegas, Jan 20, 2023 [ Show Abstract ][ PDF ]

Purpose: To determine orthokeratology (Ortho-K) lens parameters in as few steps possible is beneficial for the eye care professionals (ECPs) and patient and this study investigated how the new Visavy software can help to inform the initial lens parameters.

Methods: This prospective study recruited participants aged 6-35 years and fit them with Paragon CRT or Paragon CRT Dual Axis Ortho-K lenses (CooperVision Specialty EyeCare), worn every night for 1 month. Topography images (Oculus Keratograph 5) were uploaded in the software and together with additional entries for subjective refraction and white-to-white corneal diameter, the software populated the initial lens parameters. Lens modifications for fit and/or vision were permitted at any of the following three timepoints: the dispense visit, after the first night or after one week of wear. Visual acuity (LogMAR) was determined with subjective refraction at baseline and unaided after one month of Ortho-K wear. Subjective comfort was collected after the first lens application and after one month (0-10 scale, 10=very comfortable). Subjective vision clarity was collected via home ratings just after lens application on the first night and after one month (0-10 scale, 10=Sharp, clear/ very good vision).

Rresults: Sixteen participants (12F:4M), mean age 11.3±3.2 years [7 to 18 years] were included in the analysis. The mean refraction of all 32 eyes was Sph -2.80±1.38DS [-1.00 to -5.75DS] and Cyl -0.56±0.46DC [0.00 to -1.25DC]. Best-corrected visual acuity prior to lens insertion was 0.00±0.01 logMAR and unaided vision after 1 month was +0.09±0.11 logMAR. Remaining correction after 1 month was Sph -0.12±0.46DS [+0.75 to -1.25DS]. Almost all lens designs predicted by the software were considered acceptable (fit and vision) by the investigator with just one lens requiring a modification after the first night due to corneal staining. Comfort ratings were significantly better after 1 month (7.8 ± 1.4) compared to the dispense visit (5.2 ± 2.2)(p<0.01). Vision clarity ratings were also better after 1 month (8.4 ± 1.5) compared to after the first night (7.0 ± 3.2)(p=0.02).

Conclusions: The Visavy software could help determine acceptable lens parameters for the Paragon CRT or Paragon CRT dual axis Ortho-K lenses in 97% of eyes (31 of 32 eyes). This high initial success rate has the potential to reduce chair time and assist ECPs to confidentially fit these lenses to their patients.

Phan C-M, Chan V, Walther H, Pereira da Mota A, Lorenzo CA, Jones L. Developing a High-throughput in vitro Eye Model for Evaluating Ocular Drug Delivery with Contact Lenses XXV Biennial Meeting of the International Society for Eye Research, Feb 21, 2023 [ Show Abstract ]

Purpose: To develop a high-throughput in vitro eye model for evaluating ocular drug delivery with contact lenses (CLs).
Method: The eye model was designed using CAD software and manufactured using a combination of fabrication methods, including moulding, CNC machining, laser cutting, and 3D printing. The model consists of an eyeball, an upper and lower eyelid, and a collection tray to collect flow-through fluid. The portion of the upper eyelid that comes into contact with the eyeball is moulded with a highly wettable and durable polyvinyl alcohol hydrogel. The centre of the eyeball is designed with a 300 µm thick, 15 mm diameter cut-out that allows for a contact lens to be mounted. Simulated tear fluids can be delivered through an inlet located on the upper eyelid using a pump. During each blink cycle, the eyelid slides and flexes across the eyeball to create an artificial tear film layer. The blink distance, speed and rate are actuated using a motor controlled by an Arduino board and software. The release of a red dye from two CLs (etafilcon A and senofilcon A) and the release of two drugs (resveratrol and pravastatin sodium) from drug-loaded CLs were evaluated using the model and compared to the traditional vial testing method. Phosphate buffered saline (PBS) was used as the simulated tear fluid and infused into the model at 5 µL/min, at a blink rate of 1 blink/10 s.
Results: The fluid flows from the inlet, spreads across the eye, accumulates in the lower eyelid and then flows into the collection tray via gravity. During this process, approximately 25% of the fluid originally injected into the model was lost due to evaporation, nonspecific absorption, and residual dead volume. Overall, the release of the dye and drugs from the CLs was higher in a vial compared with the eye model. Interestingly, the drug release profiles from the drug-loaded CLs on the eye model were similar to in vivo results previously collected from a rabbit study, although the total amount of drugs released was significantly less. 9 or 24 CLs can be tested with one syringe or peristaltic pump, respectively.
Conclusion: The current eye model developed from this study could be used to measure the release of ophthalmic drugs or comfort agents from CLs in a high-throughput manner. However, further work is required to fine-tune the parameters of the model, such as the composition of the tear fluid, blink rate, tear flow rates, and temperature, to better simulate in vivo conditions.

Phan CM, Ramasamy M, Ho B, Hui A, Jones L. Fabrication of a microfluidic chip using 3D printing for evaluating ocular cytotoxicity The Association for Research in Vision and Ophthalmology, New Orleans, LA, USA, April, 2023 [ Show Abstract ][ PDF ]

Purpose: To develop a PDMS (polydimethyl siloxane) microfluidic chip to evaluate ocular cytotoxicity with ophthalmic formulations and materials.

Methods: The microfluidic chip was designed using CAD software (FreeCAD), and the moulds of the chips were printed using (1) a stereolithography (SLA) and (2) digital light processing (DLP) 3D printer. The printed moulds were washed with isopropyl alcohol (IPA), UV-cured for 1-hour at 60oC, followed by heating in an oven at 120oC for 2 hours to remove any unreacted polymers. The surface of the chips was smoothed with sandpaper with increasing grit, followed by an application of nail polish. The moulds were then cast with PDMS, a gas-permeable and clear polymer commonly used for the fabrication of microfluidic chips. The moulds and chips were imaged using SEM (scanning electron microscopy). The light transmittance of the chips was also measured. The PDMS top half of the chip was adhered to a microscope slide using medical-grade double-sided tape. For a pilot study, the PDMS chips were sterilized via autoclaving, coated with 0.1% polydopamine to improve their surface wettability, and then seeded with immortalized human corneal epithelial cells (HCEC). After 2 days of incubation in a nutrient media broth (no flow), cell adhesion and growth were evaluated using light microscopy.

Results: Both 3D printers were able to print moulds with high resolution, with channel dimensions as low as 50 µm, and with faster print times for the DLP printer. SEM images revealed that moulds that were both sanded and had a nail coating were significantly smoother than the original 3D-printed moulds. The chips cast from the polished moulds were transparent, with >85% transmittance from 450-700 nm, and could be used to image cells through a microscope. The microfluidic chips were able to handle flow rates up to 1 mL/min for 24 hours without any signs of leakage. HCEC cells were able to adhere and grow on the coated PDMS microfluidic chip after 2 days.

Conclusion: This study showed that SLA and DLP printers could be used to fabricate PDMS microfluidic chips as a low-cost rapid prototyping approach. The fabricated chips were clear and could be used to incorporate HCEC cells. Future work will examine the viability of cells under different flow rates and shear stress conditions on these chips.

Ramasamy M, Ho B, Phan CM, Jones L. Fabrication of a microfluidic chip for ophthalmic drug delivery studies using 3D printing The Association for Research in Vision and Ophthalmology, New Orleans, LA, USA, April, 2023 [ Show Abstract ]

Purpose: To develop a microfluidic chip for testing the release of ocular drugs from soft contact lenses using 3D printing.

Methods: The microfluidic chips were designed using CAD (computer-aided design) software consisting of a top and bottom portion. The top portion comprised of inlet, outlet, and channels for fluid flow. The lower portion contained a dome-shaped mount to mount a contact lens. The chips were printed using clear resin on a commercial stereolithography (SLA) 3D-printer. The printed chips were washed in isopropyl alcohol (IPA) for 30 minutes, air dried and UV cured for 30 minutes. The top and bottom portions of the chip were fused by applying a thin layer of resin, followed by UV-curing for 10 minutes. In another design iteration, moulds for the chips were 3D printed and casted with polydimethylsiloxane (PDMS). The two halves of the PDMS chips were fused using double-sided adhesive tape. In a preliminary study, two commercial contact lenses, etafilcon A and senofilcon A, were soaked in 2 ml of red food dye for 2 hours. The release of the dye was measured using the PDMS chip with phosphate-buffered saline at a flow rate of 1.5 L/min over 24 hours via absorbance at 520 nm. The dye extraction from both lenses was
performed by incubating the dye-soaked lenses in 1:1 acetonitrile/water solution for 24 hours with gentle shaking.

Results: Both the chip and moulds were printed in less than 5 hours, with a minimum resolution of 50 μm. The resulting resin and PDMS chips can also be sterilized by autoclaving. The top and bottom parts of the chips were completely sealed such that no leakage was detected at a flow rate of up to 100 μL/min for 24 hours. The release kinetics of the dye was linear throughout the 24 h period for both lens types under the current parameters. The total amount of dye released after 24 h was higher for etafilcon A (26.26 mg/lens) than senofilcon A (18.41 mg/lens), which corresponded to approximately 83.1% and 40.01% release, respectively. Both the lens types were still visibly red after 24 hours. The output of the microfluidic chip could be used as an input for subsequent analyses.

Conclusions: This study showed a cost- and time-efficient approach to fabricate a microfluidic chip for evaluating drug release from contact lenses. Future work will examine the release profile of various ocular drugs from contact lenses using different flow conditions.

Wulff D, Phan C-M, Jones L. Development of a 3D-printed hydrogel eye model for evaluating ocular drug delivery The Association for Research in Vision and Ophthalmology, New Orleans, LA, USA, April, 2023 [ Show Abstract ][ PDF ]

Purpose: To 3D-print a soft hydrogel eye model using a novel bioink for evaluating ocular drug delivery.

Methods: The eye model was designed using CAD software. It includes several key components made from a hydrogel, including an upper and lower eyelid, a frontal surface to mimic the cornea and sclera, and an internal chamber to mimic the interior of the eye. The components were designed to fit with an existing blink model that was developed previously in our laboratory that allows for automated blinking and tear collection. The eyeball and the lower eyelid were 3D bioprinted using a modified commercial mSLA printer (Photon Mono X, AnyCubic, Shenzhen, China). Various bioinks were tested, consisting of 5-15% gelatin methacryloyl, 1-5% polyvinyl alcohol, 5-30% polyethylene glycol diacrylate, 0.4-0.6% lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and 2-3% of a yellow food-grade dye in phosphate buffer solution. Different formulations were evaluated to create prints that were desirable in terms of print quality, stiffness, and flexibility. Printing was undertaken at 40˚C to ensure the ink remained a liquid and at 90% humidity to protect the parts and ink from desiccation.

Results: Both the eye and the lower eyelid were successfully printed in high resolution using 100 µm layer heights without any support structures within 3 hours. The prints are hydrophilic with a 60-80% water content, are soft and flexible, and are fabricated with biocompatible biomaterials. Both components were able to be incorporated into an in vitro blink model which will allow for improved testing that more closely mimics a human ocular system, in particular, drug absorption through the cornea. A port on the eye allows for the sampling of fluid from the interior eye model for testing the diffusion of drugs to the posterior chamber when released by topical ophthalmic formulations or anterior segment devices such as contact lenses.

Conclusion: This study demonstrated that a modified mSLA 3D printer can be used to fabricate soft, hydrophilic ocular model components using a novel biocompatible bioink.