Maud Gorbet | University of Waterloo (original) (raw)

Papers by Maud Gorbet

Research paper thumbnail of Ocular responses to biomaterials

The eye is a complex biological system, composed of different cell types, irrigated by several ph... more The eye is a complex biological system, composed of different cell types, irrigated by several physiological solutions, subject to various mechanical stresses and which environment can also be affected by diurnal variations. As a biomaterial is placed into this environment, protein adsorption, cell adhesion or activation may occur. The exact molecular mechanisms involved in biocompatibility of ocular biomaterials have not yet been determined and this chapter highlights some of the current knowledge related to biological mechanisms and material properties that affect the ocular response. The effects of aging and disease on ocular biocompatibility are also discussed.

Research paper thumbnail of The Effect of Closed-Eye Tear Film Conditions on Blood-Isolated Neutrophils, In Vitro

Ocular Immunology and Inflammation, 2017

Purpose: Eyelid closure results in influx of neutrophils onto the ocular surface, which are non-r... more Purpose: Eyelid closure results in influx of neutrophils onto the ocular surface, which are non-responsive to inflammatory stimuli. This investigation examined whether incubation of blood-isolated neutrophils in closedeye conditions induce a tear-film neutrophil phenotype. Methods: Blood-isolated neutrophils were incubated combining various conditions: hypoxia, corneal epithelial cells (HCEC), artificial tear solution (ATS). Results: A hypoxic environment induced no differential effect on membrane receptor expression. Incubation in the presence of HCEC resulted in membrane receptor upregulation and increase in caspase activation. Conclusions: Hypoxia, corneal epithelial cell exposure, or artificial tear fluid are insufficient to replicate a tear-film neutrophil phenotype using blood-isolated neutrophils.

Research paper thumbnail of The Impact of Silicone Hydrogel–Solution Combinations on Corneal Epithelial Cells

Eye & Contact Lens: Science & Clinical Practice, 2013

Silicone hydrogel (SiHy) contact lenses were introduced on the market over 10 years ago, and seve... more Silicone hydrogel (SiHy) contact lenses were introduced on the market over 10 years ago, and several multipurpose solutions (MPS) have since been developed for their cleaning and disinfection. Depending on the combination of lens and solution, clinical and retrospective studies have shown that different combinations may be more biocompatible than others. In vivo, sodium fluorescein is used to assess the corneal response, whereas in vitro studies typically investigate MPS toxicity by incubating diluted MPS with cells. This difference between in vivo and in vitro measurements makes it difficult to gain a better understanding of the biocompatibility of SiHy-solution combinations. This review discusses the recent progress in characterizing the interactions between sodium fluorescein and corneal epithelial cells and in vitro MPS cytotoxicity using solution on both monolayer and stratified epithelial models. As interactions between MPS and contact lens materials lead to uptake and release of various solution components, in vitro models that take into account the effect of lens material are also presented. With the improvement of advanced characterization methods and new in vitro models, we are moving in the right direction, but more effort is required to fully elucidate the interactions between contact lens, disinfecting solution, and the cornea.

Research paper thumbnail of Investigation of the response of tear-film neutrophils to interleukin 8 and their sensitivity to centrifugation, fixation, and incubation

Scientific Reports, 2020

During eye closure, a large number of neutrophils (polymorphonuclear neutrophils, PMNs) invade th... more During eye closure, a large number of neutrophils (polymorphonuclear neutrophils, PMNs) invade the ocular surface and are often referred to as tear-film PMNs. While immunophenotyping experiments have been performed on tear-film PMNs, the impact of commonly used experimental procedures on their phenotype as well as their response to interleukin-8 (IL-8), a physiological inflammatory mediator, have not yet been investigated. A gentle eye wash method was used to collect cells at home. In the morning upon awaking, participants washed their eyes with sterile phosphate buffer saline (PBS) and collected the runoff into a sterile polypropylene tube. The cell collection was then delivered to the lab within two hours. The effects of centrifugation, incubation and fixation with paraformaldehyde (PFA) before (pre-fixed staining) or after (post-fixed staining) incubation with antibodies were characterized. Tear-film PMNs as well as blood PMNs (used for comparison) were also stimulated with IL-8....

Research paper thumbnail of The Noninflammatory Phenotype of Neutrophils From the Closed-Eye Environment: A Flow Cytometry Analysis of Receptor Expression

Investigative Opthalmology & Visual Science, 2015

In the closed-eye environment (during sleep), there is an influx of neutrophils into the tear fil... more In the closed-eye environment (during sleep), there is an influx of neutrophils into the tear film, and the phenotype of these cells has yet to be characterized. This study was conducted to investigate the response of tear-film neutrophils to inflammatory stimuli. Immediately upon awakening, cells from healthy participants (n = 12) were collected using a gentle eye-wash with PBS. Tear-film neutrophils were counted and cell viability was determined. Neutrophils were also isolated from blood by density-gradient centrifugation. Tear-film and blood-isolated neutrophils were stimulated with phorbol myristate acetate (PMA), lipopolysaccharide (LPS), or N-Formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Changes in the expression of macrophage-1 antigen, intercellular adhesion molecule-1 (ICAM-1), CD66b (a degranulation membrane marker), C3aR (complement C3a receptor), CD45 (leukocyte common antigen) as well as reactive oxygen species (using dichlorodihydro-fluorescein diacetate) were characterized by flow cytometry. Hundreds of thousands of leukocytes were collected upon awakening. Tear-film neutrophils were alive as shown by trypan blue and propidium iodide (PI) exclusion. While tear-film neutrophils were able to mount an oxidative response, stimulation with LPS, PMA, or fMLP did not induce receptor upregulation. This lack of response to stimulus with tear-film neutrophils was significantly different from that of blood-isolated neutrophils. Incubation in the presence of tear film proteins did not affect the tear-film neutrophil response to stimuli. Our results indicate that while tear-film neutrophils are alive, they do not respond to inflammatory stimuli in the same manner as blood-isolated neutrophils. This refractory phenotype may be due to exposure to anti-inflammatory factors present in the tear film.

Research paper thumbnail of Lens-free spectral light-field fusion microscopy for contrast- and resolution-enhanced imaging of biological specimens

Optics Letters, 2015

A lensfree spectral light-field fusion microscopy (LSLFM) system is presented for enabling contra... more A lensfree spectral light-field fusion microscopy (LSLFM) system is presented for enabling contrast-and resolution-enhanced imaging of biological specimens. LSLFM consists of a pulsed multispectral lensfree microscope for capturing interferometric light-field encodings at different wavelengths, and Bayesian-based fusion to reconstruct a fused object light-field from the encodings. By fusing unique object detail information captured at different wavelengths, LSLFM can achieve improved resolution, contrast, and signal-to-noise ratio (SNR) over a single-channel lensfree microscopy system. A five-channel LSLFM system was developed and quantitatively evaluated to validate the design. Experimental results demonstrated that the LSLFM system provided SNR improvements of 6.81-16.55 dB, as well as a six-fold improvement in the dispersion index (DI), over that achieved using a single-channel lensfree deconvolution microscopy system at individual wavelengths. Furthermore, the LSLFM system achieved an increase in numerical aperture (NA) of > 3 times over a single-channel lensfree deconvolution microscopy system at the highest-resolution wavelength used in the study. Samples of Staurastrum paradoxum, a waterborne algae, and human corneal epithelial cells were imaged using the system to illustrate its potential for enhanced imaging of biological specimens.

Research paper thumbnail of 006 Development of a Novel Matrix Metalloproteinase?Inhibiting Wound Dressing

Wound Repair and Regeneration, 2004

Research paper thumbnail of Leukocyte activation and leukocyte procoagulant activities after blood contact with polystyrene and polyethylene glycol–immobilized polystyrene beads

Journal of Laboratory and Clinical Medicine, 2001

Beads (45 μm) of polystyrene (PS) and polyethylene glycol modified PS (TentaGel) with an amino or... more Beads (45 μm) of polystyrene (PS) and polyethylene glycol modified PS (TentaGel) with an amino or hydroxyl terminal group were incubated with blood to assess the effect of surface area and material chemistry on leukocyte activation. After a 2-hour incubation, blood contact with beads activated leukocytes in the bulk (tissue factor expression, CD11b up-regulation, and association with platelets) independently of

Research paper thumbnail of The Effects of Methacrylic Acid Containing Beads on Angiogenesis in a Rodent Skin Graft Model

Journal of Burn Care & Rehabilitation, 2003

Research paper thumbnail of Does surface chemistry affect thrombogenicity of surface modified polymers?

Journal of Biomedical Materials Research, 2001

With some exceptions, surface chemistry had little effect on platelet and leukocyte activation, a... more With some exceptions, surface chemistry had little effect on platelet and leukocyte activation, and cell deposition, by scanning electron microscopy after blood exposure and clotting times among a group of 12 unmodified and plasma modified tubings. All materials activated platelets and leukocytes to detectable levels, although some materials increased the value of one activation parameter but not another. Unmodified materials [polyethylene (PE), Pellethane (PEU), latex, nylon, and Silastic] and modified materials (H(2)O plasma treated PE and PEU, CF(4) plasma treated PE, fluorinated PEU, NH(4) plasma treated PEU, polyethylene imine treated PEU, and heparin treated PEU) were characterised by XPS and contact angle. The objective of this project was to define a series of assays for the evaluation of hemocompatibility of cardiovascular devices with a view to clarify the specific requirements of ISO-10993-4, and to define an appropriate screening program for new blood contacting biomaterials. PE, PE--CF(4), PE--H(2)0, PEU--F, latex, and PEU-heparin were the exceptions to the general observations, although each behaved differently. PE proved to be least reactive, whereas PE-CF(4) was most reactive by several assays. Platelet microparticle formation (determined by flow cytometry), PTT, postblood exposure SEM, total SC5b-9, C3a, and platelet and leukocyte loss (cell counts) were able to distinguish differences among these materials, and often, but not always, showed expected correlations.

Research paper thumbnail of The effect of shear on in vitro platelet and leukocyte material-induced activation

Journal of Biomaterials Applications, 2013

The failure to understand the mechanisms of biomaterial-associated thrombosis prevents us from im... more The failure to understand the mechanisms of biomaterial-associated thrombosis prevents us from improving the blood compatibility of stents and mechanical heart valves. Blood-material interactions trigger a complex series of events and anticoagulant and anti-platelet therapies are needed to reduce the risks of thrombotic complications with most cardiovascular materials. While material interaction with platelets has been widely studied, little is currently known on material-induced leukocyte activation in the presence of shear. In vitro experiments were performed to assess the effect of flow on blood cell activation induced by medical grade metals, ST316L and TiAl6V4. Blood was circulated in flow chambers preloaded with or without metal wires at shear rates of 100, 500, and 1500 s⁻¹. Platelet and leukocyte activation, leukocyte-platelet aggregation, and tissue factor expression on monocytes were measured by flow cytometry. Metal surfaces were characterized by scanning electron microscopy. Under physiological shear rates, no significant platelet microparticle formation was observed. However, significant CD11b up-regulation, leukocyte-platelet aggregates, and tissue factor expression were observed at 100 s⁻¹. As shear rate increased to 1500 s⁻¹, leukocyte activation reduced to control values. TiAl6V4-induced leukocyte activation was generally lower than that of ST316L. Adhesion significantly decreased with increasing shear rate to 1500 s⁻¹. In blood, increase within physiological shear rates led to a significant reduction in in vitro material-induced leukocyte activation, suggesting that difference between material biocompatibility may be better identified at low shear rates or under pathological shear conditions.

Research paper thumbnail of The Impact of Silicone Hydrogel–Solution Combinations on Corneal Epithelial Cells

Eye & Contact Lens: Science & Clinical Practice, 2013

Silicone hydrogel (SiHy) contact lenses were introduced on the market over 10 years ago, and seve... more Silicone hydrogel (SiHy) contact lenses were introduced on the market over 10 years ago, and several multipurpose solutions (MPS) have since been developed for their cleaning and disinfection. Depending on the combination of lens and solution, clinical and retrospective studies have shown that different combinations may be more biocompatible than others. In vivo, sodium fluorescein is used to assess the corneal response, whereas in vitro studies typically investigate MPS toxicity by incubating diluted MPS with cells. This difference between in vivo and in vitro measurements makes it difficult to gain a better understanding of the biocompatibility of SiHy-solution combinations. This review discusses the recent progress in characterizing the interactions between sodium fluorescein and corneal epithelial cells and in vitro MPS cytotoxicity using solution on both monolayer and stratified epithelial models. As interactions between MPS and contact lens materials lead to uptake and release of various solution components, in vitro models that take into account the effect of lens material are also presented. With the improvement of advanced characterization methods and new in vitro models, we are moving in the right direction, but more effort is required to fully elucidate the interactions between contact lens, disinfecting solution, and the cornea.

Research paper thumbnail of In vitro methods of assessing ocular biocompatibility using THP-1-derived macrophages

Cutaneous and Ocular Toxicology, 2014

Abstract Macrophages play an important role in the elimination of infections, the removal of debr... more Abstract Macrophages play an important role in the elimination of infections, the removal of debris and in tissue repair after infection and trauma. In vitro models that assess ocular biomaterials for toxicity typically focus on the effects of these materials on epithelial or fibroblast cells. This investigation evaluated known ocular toxins deposited on model materials for their effects on the viability and activation of macrophages. THP-1-derived macrophages were cultured onto silicone films (used as a base biomaterial) deposited with chemical toxins (benzalkonium chloride (BAK), zinc diethyldithiocarbamate (ZDEC) and lipopolysaccharide (LPS)). Utilizing three fluorescent dyes calcein, ethidium homodimer-1 (EthD-1) and annexin V, the viability of macrophages attached to the biomaterial was determined using confocal microscopy. Propidium iodide (PI) staining and alamarBlue® (resazurin) reduction were used to assess cell death and metabolic activity. CD14, CD16, CD33, CD45, and CD54 expression of adherent macrophages, were also evaluated to detect LPS activation of macrophages using flow cytometry. The sensitivity of this test battery was demonstrated as significant toxicity from treated surfaces with ZDEC (0.001-0.01%), and BAK (0.001%-0.1%) was detected. Also, macrophage activation could be detected by measuring CD54 expression after exposure to adsorbed LPS. These in vitro methods will be helpful in determining the toxicity potential of new ocular biomaterials.

Research paper thumbnail of Human Corneal Epithelial Cell Shedding and Fluorescein Staining in Response to Silicone Hydrogel Lenses and Contact Lens Disinfecting Solutions

Current Eye Research, 2014

Purpose: A pilot study was conducted to evaluate human corneal epithelial cell shedding in respon... more Purpose: A pilot study was conducted to evaluate human corneal epithelial cell shedding in response to wearing a silicone hydrogel contact lens/solution combination inducing corneal staining. The nature of ex vivo collected cells staining with fluorescein was also examined. Methods: A contralateral eye study was conducted in which up to eight participants were unilaterally exposed to a multipurpose contact lens solution/silicone hydrogel lens combination previously shown to induce corneal staining (renu Õ fresh TM and balafilcon A; test eye), with the other eye using a combination of balafilcon A soaked in a hydrogen peroxide care system (Clear Care Õ ; control eye). Lenses were worn for 2, 4 or 6 hours. Corneal staining was graded after lens removal. The Ocular Surface Cell Collection Apparatus was used to collect cells from the cornea and the contact lens. Results: In the test eye, maximum solution-induced corneal staining (SICS) was observed after 2 hours of lens wear (reducing significantly by 4 hours; p50.001). There were significantly more cells collected from the test eye after 4 hours of lens wear when compared to the control eye and the collection from the test eye after 2 hours (for both; n = 5; p50.001). The total cell yield at 4 hours was 813 AE 333 and 455 AE 218 for the test and control eyes, respectively (N = 5, triplicate, p = 0.003). A number of cells were observed to have taken up the fluorescein dye from the initial fluorescein instillation. Confocal microscopy of fluorescein-stained cells revealed that fluorescein was present throughout the cell cytoplasm and was retained in the cells for many hours after recovery from the corneal surface. Conclusion: This pilot study indicates that increased epithelial cell shedding was associated with a lens-solution combination which induces SICS. Our data provides insight into the transient nature of the SICS reaction and the nature of fluorescein staining observed in SICS.

Research paper thumbnail of The response of tear film neutrophils to occasional overnight lens wear

Research paper thumbnail of Corneal epithelial cell biocompatibility to silicone hydrogel and conventional hydrogel contact lens packaging solutions

Purpose: Although all contact lenses (CLs) are applied initially to the eye directly from a packa... more Purpose: Although all contact lenses (CLs) are applied initially to the eye directly from a packaging solution, little is known about the effects of these solutions on human corneal epithelial cells (HCECs). Due to the porous nature of CL materials, they have the potential to sorb components of the packaging solution during storage, which could then be subsequently released upon insertion of the CL on the eye. The purpose of this study was to investigate the effect of various packaging solutions on HCECs, using an in vitro model. Methods: An in vitro assay was developed whereby various silicone hydrogels and conventional, poly-2hydroxyethylmethacrylate (polyHEMA)-based lens materials were removed directly from their packaging and then incubated for up to 24 h with HCECs. The effect of the retained and released packaging solution components on HCECs was assessed by measuring cell viability, adhesion phenotype, and apoptosis. Results: Incubation of HCECs with CLs stored in borate-buffered packaging solutions resulted in a significant reduction in cell viability. Adherent cells incubated with these CLs also exhibited reduced levels of β1 and α3 integrin. Soaking borate-buffered packaged CLs in PBS before cell incubation resolved viability and integrin expression in all cases, with the exception of galyfilcon A and balafilcon A, from which a 20% reduction in cell viability was still observed. In comparison, CLs stored in phosphate-buffered packaging solutions had cellular viability and expression of integrins similar to control cells (cells incubated in the absence of a lens). When incubated with cells at a 10% concentration in serum-free medium, borate-buffered packaging solutions and borate-containing saline (Unisol 4) significantly reduced cell viability and integrin expression. Neither caspase activation nor annexin V binding was observed on cells following exposure to borate buffer solution. However, a significant decrease in reactive oxygen species was observed at 24 h. These latter results suggest that in vitro exposure to low concentration of borate/boric acid results in cell dysfunction, leading to necrosis rather than apoptosis. Conclusions: Borate-buffered packaging solutions were shown to adversely affect the viability and integrin expression of HCECs in vitro. When used in ophthalmic packaging solutions, the antimicrobial properties of borate buffer may be outweighed by its relatively cytotoxic effects on cells. Molecular Vision 2010; 16:272-282 <http://www.molvis.org/molvis/v16/a33>

Research paper thumbnail of Impact of Multipurpose Solutions Released from Contact Lenses on Corneal Cells

Optometry and Vision Science, 2011

To assess, in vitro, the effect of the release of contact lens multipurpose solutions (MPS) from ... more To assess, in vitro, the effect of the release of contact lens multipurpose solutions (MPS) from two silicone hydrogel lenses on human corneal epithelial cells. A monolayer of immortalized human corneal epithelial cells was seeded in a 24-well plate in keratinocyte serum-free medium. Lotrafilcon A (LA) and balafilcon A (BA) lenses were placed on top of the adherent cells for 8 and 24 h, after being soaked in MPS, borate-buffered (Unisol) or phosphate-buffered saline overnight. Cells were assayed for viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay or for α3, β1, and β4 integrin expression and caspase activation by flow cytometry. After 8 h, LA lenses soaked in Unisol, Opti-Free Express (OFX), and ReNu MultiPlus (ReNu) showed decrease in cell viability. LA and BA soaked in Complete Moisture Plus (Complete) had similar viability at around 85% of control. After 24 h, a further decrease in viability was observed with all MPS-soaked lenses; LA soaked in OFX significantly reduced viability compared with Unisol-soaked lenses. In addition, reduced levels of integrin expression for lenses soaked in OFX and ReNu, and for BA soaked in Complete were observed. At 24 h, only LA soaked in OFX led to an increase in caspase activation. Our results indicate an increase in cytotoxicity with borate-based MPS solutions in vitro when compared with both phosphate-buffered saline and borate-exposed lenses, suggesting that biocides and/or additives play a role in the observed cell reaction. Moreover, the mechanism of in vitro solution-induced toxicity appeared to be mediated by lens type, suggesting differences in the preferential adsorption/release profile of certain compounds.

Research paper thumbnail of Bayesian-based deconvolution fluorescence microscopy using dynamically updated nonstationary expectation estimates

Scientific Reports, 2015

Fluorescence microscopy is widely used for the study of biological specimens. Deconvolution can s... more Fluorescence microscopy is widely used for the study of biological specimens. Deconvolution can significantly improve the resolution and contrast of images produced using fluorescence microscopy; in particular, Bayesian-based methods have become very popular in deconvolution fluorescence microscopy. An ongoing challenge with Bayesian-based methods is in dealing with the presence of noise in low SNR imaging conditions. In this study, we present a Bayesian-based method for performing deconvolution using dynamically updated nonparametric nonstationary expectation estimates that can improve the fluorescence microscopy image quality in the presence of noise, without explicit use of spatial regularization.

Research paper thumbnail of Bayesian-based deconvolution fluorescence microscopy using dynamically updated nonstationary expectation estimates

Scientific reports, 2015

Fluorescence microscopy is widely used for the study of biological specimens. Deconvolution can s... more Fluorescence microscopy is widely used for the study of biological specimens. Deconvolution can significantly improve the resolution and contrast of images produced using fluorescence microscopy; in particular, Bayesian-based methods have become very popular in deconvolution fluorescence microscopy. An ongoing challenge with Bayesian-based methods is in dealing with the presence of noise in low SNR imaging conditions. In this study, we present a Bayesian-based method for performing deconvolution using dynamically updated nonstationary expectation estimates that can improve the fluorescence microscopy image quality in the presence of noise, without explicit use of spatial regularization.

Research paper thumbnail of Effect of contact lens material on cytotoxicity potential of multipurpose solutions using human corneal epithelial cells

Molecular vision, 2011

Multipurpose solutions (MPS) are used daily to clean and disinfect silicone hydrogel (SiHy) conta... more Multipurpose solutions (MPS) are used daily to clean and disinfect silicone hydrogel (SiHy) contact lenses. This in vitro study was undertaken to identify the potential for interaction between MPS, SiHy surface treatments, and lens materials, which may lead to changes in the response of human corneal epithelial cells (HCEC) to MPS-soaked lenses. The MPS tested were renu fresh (formerly known as ReNu MultiPlus; ReNu), OptiFree Express (OFX), OptiFree RepleniSH, SoloCare Aqua, and Complete Moisture Plus. The SiHy materials evaluated were lotrafilcon A, lotrafilcon B, comfilcon A, galyfilcon A, and balafilcon A (BA). MPS-soaked lenses were placed on top of adherent HCEC. The effect of MPS dilutions (0.1 to 10% final concentration in medium) was also characterized. Cell viability, adhesion phenotype and caspase activation were studied after 24-h cell exposure. OFX released from lenses was determined using UV absorbance. A significant reduction in viability (between 30 to 50%) was observ...

Research paper thumbnail of Ocular responses to biomaterials

The eye is a complex biological system, composed of different cell types, irrigated by several ph... more The eye is a complex biological system, composed of different cell types, irrigated by several physiological solutions, subject to various mechanical stresses and which environment can also be affected by diurnal variations. As a biomaterial is placed into this environment, protein adsorption, cell adhesion or activation may occur. The exact molecular mechanisms involved in biocompatibility of ocular biomaterials have not yet been determined and this chapter highlights some of the current knowledge related to biological mechanisms and material properties that affect the ocular response. The effects of aging and disease on ocular biocompatibility are also discussed.

Research paper thumbnail of The Effect of Closed-Eye Tear Film Conditions on Blood-Isolated Neutrophils, In Vitro

Ocular Immunology and Inflammation, 2017

Purpose: Eyelid closure results in influx of neutrophils onto the ocular surface, which are non-r... more Purpose: Eyelid closure results in influx of neutrophils onto the ocular surface, which are non-responsive to inflammatory stimuli. This investigation examined whether incubation of blood-isolated neutrophils in closedeye conditions induce a tear-film neutrophil phenotype. Methods: Blood-isolated neutrophils were incubated combining various conditions: hypoxia, corneal epithelial cells (HCEC), artificial tear solution (ATS). Results: A hypoxic environment induced no differential effect on membrane receptor expression. Incubation in the presence of HCEC resulted in membrane receptor upregulation and increase in caspase activation. Conclusions: Hypoxia, corneal epithelial cell exposure, or artificial tear fluid are insufficient to replicate a tear-film neutrophil phenotype using blood-isolated neutrophils.

Research paper thumbnail of The Impact of Silicone Hydrogel–Solution Combinations on Corneal Epithelial Cells

Eye & Contact Lens: Science & Clinical Practice, 2013

Silicone hydrogel (SiHy) contact lenses were introduced on the market over 10 years ago, and seve... more Silicone hydrogel (SiHy) contact lenses were introduced on the market over 10 years ago, and several multipurpose solutions (MPS) have since been developed for their cleaning and disinfection. Depending on the combination of lens and solution, clinical and retrospective studies have shown that different combinations may be more biocompatible than others. In vivo, sodium fluorescein is used to assess the corneal response, whereas in vitro studies typically investigate MPS toxicity by incubating diluted MPS with cells. This difference between in vivo and in vitro measurements makes it difficult to gain a better understanding of the biocompatibility of SiHy-solution combinations. This review discusses the recent progress in characterizing the interactions between sodium fluorescein and corneal epithelial cells and in vitro MPS cytotoxicity using solution on both monolayer and stratified epithelial models. As interactions between MPS and contact lens materials lead to uptake and release of various solution components, in vitro models that take into account the effect of lens material are also presented. With the improvement of advanced characterization methods and new in vitro models, we are moving in the right direction, but more effort is required to fully elucidate the interactions between contact lens, disinfecting solution, and the cornea.

Research paper thumbnail of Investigation of the response of tear-film neutrophils to interleukin 8 and their sensitivity to centrifugation, fixation, and incubation

Scientific Reports, 2020

During eye closure, a large number of neutrophils (polymorphonuclear neutrophils, PMNs) invade th... more During eye closure, a large number of neutrophils (polymorphonuclear neutrophils, PMNs) invade the ocular surface and are often referred to as tear-film PMNs. While immunophenotyping experiments have been performed on tear-film PMNs, the impact of commonly used experimental procedures on their phenotype as well as their response to interleukin-8 (IL-8), a physiological inflammatory mediator, have not yet been investigated. A gentle eye wash method was used to collect cells at home. In the morning upon awaking, participants washed their eyes with sterile phosphate buffer saline (PBS) and collected the runoff into a sterile polypropylene tube. The cell collection was then delivered to the lab within two hours. The effects of centrifugation, incubation and fixation with paraformaldehyde (PFA) before (pre-fixed staining) or after (post-fixed staining) incubation with antibodies were characterized. Tear-film PMNs as well as blood PMNs (used for comparison) were also stimulated with IL-8....

Research paper thumbnail of The Noninflammatory Phenotype of Neutrophils From the Closed-Eye Environment: A Flow Cytometry Analysis of Receptor Expression

Investigative Opthalmology & Visual Science, 2015

In the closed-eye environment (during sleep), there is an influx of neutrophils into the tear fil... more In the closed-eye environment (during sleep), there is an influx of neutrophils into the tear film, and the phenotype of these cells has yet to be characterized. This study was conducted to investigate the response of tear-film neutrophils to inflammatory stimuli. Immediately upon awakening, cells from healthy participants (n = 12) were collected using a gentle eye-wash with PBS. Tear-film neutrophils were counted and cell viability was determined. Neutrophils were also isolated from blood by density-gradient centrifugation. Tear-film and blood-isolated neutrophils were stimulated with phorbol myristate acetate (PMA), lipopolysaccharide (LPS), or N-Formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Changes in the expression of macrophage-1 antigen, intercellular adhesion molecule-1 (ICAM-1), CD66b (a degranulation membrane marker), C3aR (complement C3a receptor), CD45 (leukocyte common antigen) as well as reactive oxygen species (using dichlorodihydro-fluorescein diacetate) were characterized by flow cytometry. Hundreds of thousands of leukocytes were collected upon awakening. Tear-film neutrophils were alive as shown by trypan blue and propidium iodide (PI) exclusion. While tear-film neutrophils were able to mount an oxidative response, stimulation with LPS, PMA, or fMLP did not induce receptor upregulation. This lack of response to stimulus with tear-film neutrophils was significantly different from that of blood-isolated neutrophils. Incubation in the presence of tear film proteins did not affect the tear-film neutrophil response to stimuli. Our results indicate that while tear-film neutrophils are alive, they do not respond to inflammatory stimuli in the same manner as blood-isolated neutrophils. This refractory phenotype may be due to exposure to anti-inflammatory factors present in the tear film.

Research paper thumbnail of Lens-free spectral light-field fusion microscopy for contrast- and resolution-enhanced imaging of biological specimens

Optics Letters, 2015

A lensfree spectral light-field fusion microscopy (LSLFM) system is presented for enabling contra... more A lensfree spectral light-field fusion microscopy (LSLFM) system is presented for enabling contrast-and resolution-enhanced imaging of biological specimens. LSLFM consists of a pulsed multispectral lensfree microscope for capturing interferometric light-field encodings at different wavelengths, and Bayesian-based fusion to reconstruct a fused object light-field from the encodings. By fusing unique object detail information captured at different wavelengths, LSLFM can achieve improved resolution, contrast, and signal-to-noise ratio (SNR) over a single-channel lensfree microscopy system. A five-channel LSLFM system was developed and quantitatively evaluated to validate the design. Experimental results demonstrated that the LSLFM system provided SNR improvements of 6.81-16.55 dB, as well as a six-fold improvement in the dispersion index (DI), over that achieved using a single-channel lensfree deconvolution microscopy system at individual wavelengths. Furthermore, the LSLFM system achieved an increase in numerical aperture (NA) of > 3 times over a single-channel lensfree deconvolution microscopy system at the highest-resolution wavelength used in the study. Samples of Staurastrum paradoxum, a waterborne algae, and human corneal epithelial cells were imaged using the system to illustrate its potential for enhanced imaging of biological specimens.

Research paper thumbnail of 006 Development of a Novel Matrix Metalloproteinase?Inhibiting Wound Dressing

Wound Repair and Regeneration, 2004

Research paper thumbnail of Leukocyte activation and leukocyte procoagulant activities after blood contact with polystyrene and polyethylene glycol–immobilized polystyrene beads

Journal of Laboratory and Clinical Medicine, 2001

Beads (45 μm) of polystyrene (PS) and polyethylene glycol modified PS (TentaGel) with an amino or... more Beads (45 μm) of polystyrene (PS) and polyethylene glycol modified PS (TentaGel) with an amino or hydroxyl terminal group were incubated with blood to assess the effect of surface area and material chemistry on leukocyte activation. After a 2-hour incubation, blood contact with beads activated leukocytes in the bulk (tissue factor expression, CD11b up-regulation, and association with platelets) independently of

Research paper thumbnail of The Effects of Methacrylic Acid Containing Beads on Angiogenesis in a Rodent Skin Graft Model

Journal of Burn Care & Rehabilitation, 2003

Research paper thumbnail of Does surface chemistry affect thrombogenicity of surface modified polymers?

Journal of Biomedical Materials Research, 2001

With some exceptions, surface chemistry had little effect on platelet and leukocyte activation, a... more With some exceptions, surface chemistry had little effect on platelet and leukocyte activation, and cell deposition, by scanning electron microscopy after blood exposure and clotting times among a group of 12 unmodified and plasma modified tubings. All materials activated platelets and leukocytes to detectable levels, although some materials increased the value of one activation parameter but not another. Unmodified materials [polyethylene (PE), Pellethane (PEU), latex, nylon, and Silastic] and modified materials (H(2)O plasma treated PE and PEU, CF(4) plasma treated PE, fluorinated PEU, NH(4) plasma treated PEU, polyethylene imine treated PEU, and heparin treated PEU) were characterised by XPS and contact angle. The objective of this project was to define a series of assays for the evaluation of hemocompatibility of cardiovascular devices with a view to clarify the specific requirements of ISO-10993-4, and to define an appropriate screening program for new blood contacting biomaterials. PE, PE--CF(4), PE--H(2)0, PEU--F, latex, and PEU-heparin were the exceptions to the general observations, although each behaved differently. PE proved to be least reactive, whereas PE-CF(4) was most reactive by several assays. Platelet microparticle formation (determined by flow cytometry), PTT, postblood exposure SEM, total SC5b-9, C3a, and platelet and leukocyte loss (cell counts) were able to distinguish differences among these materials, and often, but not always, showed expected correlations.

Research paper thumbnail of The effect of shear on in vitro platelet and leukocyte material-induced activation

Journal of Biomaterials Applications, 2013

The failure to understand the mechanisms of biomaterial-associated thrombosis prevents us from im... more The failure to understand the mechanisms of biomaterial-associated thrombosis prevents us from improving the blood compatibility of stents and mechanical heart valves. Blood-material interactions trigger a complex series of events and anticoagulant and anti-platelet therapies are needed to reduce the risks of thrombotic complications with most cardiovascular materials. While material interaction with platelets has been widely studied, little is currently known on material-induced leukocyte activation in the presence of shear. In vitro experiments were performed to assess the effect of flow on blood cell activation induced by medical grade metals, ST316L and TiAl6V4. Blood was circulated in flow chambers preloaded with or without metal wires at shear rates of 100, 500, and 1500 s⁻¹. Platelet and leukocyte activation, leukocyte-platelet aggregation, and tissue factor expression on monocytes were measured by flow cytometry. Metal surfaces were characterized by scanning electron microscopy. Under physiological shear rates, no significant platelet microparticle formation was observed. However, significant CD11b up-regulation, leukocyte-platelet aggregates, and tissue factor expression were observed at 100 s⁻¹. As shear rate increased to 1500 s⁻¹, leukocyte activation reduced to control values. TiAl6V4-induced leukocyte activation was generally lower than that of ST316L. Adhesion significantly decreased with increasing shear rate to 1500 s⁻¹. In blood, increase within physiological shear rates led to a significant reduction in in vitro material-induced leukocyte activation, suggesting that difference between material biocompatibility may be better identified at low shear rates or under pathological shear conditions.

Research paper thumbnail of The Impact of Silicone Hydrogel–Solution Combinations on Corneal Epithelial Cells

Eye & Contact Lens: Science & Clinical Practice, 2013

Silicone hydrogel (SiHy) contact lenses were introduced on the market over 10 years ago, and seve... more Silicone hydrogel (SiHy) contact lenses were introduced on the market over 10 years ago, and several multipurpose solutions (MPS) have since been developed for their cleaning and disinfection. Depending on the combination of lens and solution, clinical and retrospective studies have shown that different combinations may be more biocompatible than others. In vivo, sodium fluorescein is used to assess the corneal response, whereas in vitro studies typically investigate MPS toxicity by incubating diluted MPS with cells. This difference between in vivo and in vitro measurements makes it difficult to gain a better understanding of the biocompatibility of SiHy-solution combinations. This review discusses the recent progress in characterizing the interactions between sodium fluorescein and corneal epithelial cells and in vitro MPS cytotoxicity using solution on both monolayer and stratified epithelial models. As interactions between MPS and contact lens materials lead to uptake and release of various solution components, in vitro models that take into account the effect of lens material are also presented. With the improvement of advanced characterization methods and new in vitro models, we are moving in the right direction, but more effort is required to fully elucidate the interactions between contact lens, disinfecting solution, and the cornea.

Research paper thumbnail of In vitro methods of assessing ocular biocompatibility using THP-1-derived macrophages

Cutaneous and Ocular Toxicology, 2014

Abstract Macrophages play an important role in the elimination of infections, the removal of debr... more Abstract Macrophages play an important role in the elimination of infections, the removal of debris and in tissue repair after infection and trauma. In vitro models that assess ocular biomaterials for toxicity typically focus on the effects of these materials on epithelial or fibroblast cells. This investigation evaluated known ocular toxins deposited on model materials for their effects on the viability and activation of macrophages. THP-1-derived macrophages were cultured onto silicone films (used as a base biomaterial) deposited with chemical toxins (benzalkonium chloride (BAK), zinc diethyldithiocarbamate (ZDEC) and lipopolysaccharide (LPS)). Utilizing three fluorescent dyes calcein, ethidium homodimer-1 (EthD-1) and annexin V, the viability of macrophages attached to the biomaterial was determined using confocal microscopy. Propidium iodide (PI) staining and alamarBlue® (resazurin) reduction were used to assess cell death and metabolic activity. CD14, CD16, CD33, CD45, and CD54 expression of adherent macrophages, were also evaluated to detect LPS activation of macrophages using flow cytometry. The sensitivity of this test battery was demonstrated as significant toxicity from treated surfaces with ZDEC (0.001-0.01%), and BAK (0.001%-0.1%) was detected. Also, macrophage activation could be detected by measuring CD54 expression after exposure to adsorbed LPS. These in vitro methods will be helpful in determining the toxicity potential of new ocular biomaterials.

Research paper thumbnail of Human Corneal Epithelial Cell Shedding and Fluorescein Staining in Response to Silicone Hydrogel Lenses and Contact Lens Disinfecting Solutions

Current Eye Research, 2014

Purpose: A pilot study was conducted to evaluate human corneal epithelial cell shedding in respon... more Purpose: A pilot study was conducted to evaluate human corneal epithelial cell shedding in response to wearing a silicone hydrogel contact lens/solution combination inducing corneal staining. The nature of ex vivo collected cells staining with fluorescein was also examined. Methods: A contralateral eye study was conducted in which up to eight participants were unilaterally exposed to a multipurpose contact lens solution/silicone hydrogel lens combination previously shown to induce corneal staining (renu Õ fresh TM and balafilcon A; test eye), with the other eye using a combination of balafilcon A soaked in a hydrogen peroxide care system (Clear Care Õ ; control eye). Lenses were worn for 2, 4 or 6 hours. Corneal staining was graded after lens removal. The Ocular Surface Cell Collection Apparatus was used to collect cells from the cornea and the contact lens. Results: In the test eye, maximum solution-induced corneal staining (SICS) was observed after 2 hours of lens wear (reducing significantly by 4 hours; p50.001). There were significantly more cells collected from the test eye after 4 hours of lens wear when compared to the control eye and the collection from the test eye after 2 hours (for both; n = 5; p50.001). The total cell yield at 4 hours was 813 AE 333 and 455 AE 218 for the test and control eyes, respectively (N = 5, triplicate, p = 0.003). A number of cells were observed to have taken up the fluorescein dye from the initial fluorescein instillation. Confocal microscopy of fluorescein-stained cells revealed that fluorescein was present throughout the cell cytoplasm and was retained in the cells for many hours after recovery from the corneal surface. Conclusion: This pilot study indicates that increased epithelial cell shedding was associated with a lens-solution combination which induces SICS. Our data provides insight into the transient nature of the SICS reaction and the nature of fluorescein staining observed in SICS.

Research paper thumbnail of The response of tear film neutrophils to occasional overnight lens wear

Research paper thumbnail of Corneal epithelial cell biocompatibility to silicone hydrogel and conventional hydrogel contact lens packaging solutions

Purpose: Although all contact lenses (CLs) are applied initially to the eye directly from a packa... more Purpose: Although all contact lenses (CLs) are applied initially to the eye directly from a packaging solution, little is known about the effects of these solutions on human corneal epithelial cells (HCECs). Due to the porous nature of CL materials, they have the potential to sorb components of the packaging solution during storage, which could then be subsequently released upon insertion of the CL on the eye. The purpose of this study was to investigate the effect of various packaging solutions on HCECs, using an in vitro model. Methods: An in vitro assay was developed whereby various silicone hydrogels and conventional, poly-2hydroxyethylmethacrylate (polyHEMA)-based lens materials were removed directly from their packaging and then incubated for up to 24 h with HCECs. The effect of the retained and released packaging solution components on HCECs was assessed by measuring cell viability, adhesion phenotype, and apoptosis. Results: Incubation of HCECs with CLs stored in borate-buffered packaging solutions resulted in a significant reduction in cell viability. Adherent cells incubated with these CLs also exhibited reduced levels of β1 and α3 integrin. Soaking borate-buffered packaged CLs in PBS before cell incubation resolved viability and integrin expression in all cases, with the exception of galyfilcon A and balafilcon A, from which a 20% reduction in cell viability was still observed. In comparison, CLs stored in phosphate-buffered packaging solutions had cellular viability and expression of integrins similar to control cells (cells incubated in the absence of a lens). When incubated with cells at a 10% concentration in serum-free medium, borate-buffered packaging solutions and borate-containing saline (Unisol 4) significantly reduced cell viability and integrin expression. Neither caspase activation nor annexin V binding was observed on cells following exposure to borate buffer solution. However, a significant decrease in reactive oxygen species was observed at 24 h. These latter results suggest that in vitro exposure to low concentration of borate/boric acid results in cell dysfunction, leading to necrosis rather than apoptosis. Conclusions: Borate-buffered packaging solutions were shown to adversely affect the viability and integrin expression of HCECs in vitro. When used in ophthalmic packaging solutions, the antimicrobial properties of borate buffer may be outweighed by its relatively cytotoxic effects on cells. Molecular Vision 2010; 16:272-282 <http://www.molvis.org/molvis/v16/a33>

Research paper thumbnail of Impact of Multipurpose Solutions Released from Contact Lenses on Corneal Cells

Optometry and Vision Science, 2011

To assess, in vitro, the effect of the release of contact lens multipurpose solutions (MPS) from ... more To assess, in vitro, the effect of the release of contact lens multipurpose solutions (MPS) from two silicone hydrogel lenses on human corneal epithelial cells. A monolayer of immortalized human corneal epithelial cells was seeded in a 24-well plate in keratinocyte serum-free medium. Lotrafilcon A (LA) and balafilcon A (BA) lenses were placed on top of the adherent cells for 8 and 24 h, after being soaked in MPS, borate-buffered (Unisol) or phosphate-buffered saline overnight. Cells were assayed for viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay or for α3, β1, and β4 integrin expression and caspase activation by flow cytometry. After 8 h, LA lenses soaked in Unisol, Opti-Free Express (OFX), and ReNu MultiPlus (ReNu) showed decrease in cell viability. LA and BA soaked in Complete Moisture Plus (Complete) had similar viability at around 85% of control. After 24 h, a further decrease in viability was observed with all MPS-soaked lenses; LA soaked in OFX significantly reduced viability compared with Unisol-soaked lenses. In addition, reduced levels of integrin expression for lenses soaked in OFX and ReNu, and for BA soaked in Complete were observed. At 24 h, only LA soaked in OFX led to an increase in caspase activation. Our results indicate an increase in cytotoxicity with borate-based MPS solutions in vitro when compared with both phosphate-buffered saline and borate-exposed lenses, suggesting that biocides and/or additives play a role in the observed cell reaction. Moreover, the mechanism of in vitro solution-induced toxicity appeared to be mediated by lens type, suggesting differences in the preferential adsorption/release profile of certain compounds.

Research paper thumbnail of Bayesian-based deconvolution fluorescence microscopy using dynamically updated nonstationary expectation estimates

Scientific Reports, 2015

Fluorescence microscopy is widely used for the study of biological specimens. Deconvolution can s... more Fluorescence microscopy is widely used for the study of biological specimens. Deconvolution can significantly improve the resolution and contrast of images produced using fluorescence microscopy; in particular, Bayesian-based methods have become very popular in deconvolution fluorescence microscopy. An ongoing challenge with Bayesian-based methods is in dealing with the presence of noise in low SNR imaging conditions. In this study, we present a Bayesian-based method for performing deconvolution using dynamically updated nonparametric nonstationary expectation estimates that can improve the fluorescence microscopy image quality in the presence of noise, without explicit use of spatial regularization.

Research paper thumbnail of Bayesian-based deconvolution fluorescence microscopy using dynamically updated nonstationary expectation estimates

Scientific reports, 2015

Fluorescence microscopy is widely used for the study of biological specimens. Deconvolution can s... more Fluorescence microscopy is widely used for the study of biological specimens. Deconvolution can significantly improve the resolution and contrast of images produced using fluorescence microscopy; in particular, Bayesian-based methods have become very popular in deconvolution fluorescence microscopy. An ongoing challenge with Bayesian-based methods is in dealing with the presence of noise in low SNR imaging conditions. In this study, we present a Bayesian-based method for performing deconvolution using dynamically updated nonstationary expectation estimates that can improve the fluorescence microscopy image quality in the presence of noise, without explicit use of spatial regularization.

Research paper thumbnail of Effect of contact lens material on cytotoxicity potential of multipurpose solutions using human corneal epithelial cells

Molecular vision, 2011

Multipurpose solutions (MPS) are used daily to clean and disinfect silicone hydrogel (SiHy) conta... more Multipurpose solutions (MPS) are used daily to clean and disinfect silicone hydrogel (SiHy) contact lenses. This in vitro study was undertaken to identify the potential for interaction between MPS, SiHy surface treatments, and lens materials, which may lead to changes in the response of human corneal epithelial cells (HCEC) to MPS-soaked lenses. The MPS tested were renu fresh (formerly known as ReNu MultiPlus; ReNu), OptiFree Express (OFX), OptiFree RepleniSH, SoloCare Aqua, and Complete Moisture Plus. The SiHy materials evaluated were lotrafilcon A, lotrafilcon B, comfilcon A, galyfilcon A, and balafilcon A (BA). MPS-soaked lenses were placed on top of adherent HCEC. The effect of MPS dilutions (0.1 to 10% final concentration in medium) was also characterized. Cell viability, adhesion phenotype and caspase activation were studied after 24-h cell exposure. OFX released from lenses was determined using UV absorbance. A significant reduction in viability (between 30 to 50%) was observ...