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Purpose The purpose of this study was to characterize the central epithelial thickness (CET) of penetrating keratoplasty corneal specimens obtained from patients with keratoconus (KC) and correlate the histological patterns with their clinical history. Methods Ex vivo histological imaging was performed to measure CET and total corneal thickness (TCT) in 56 patients with KC. Microscopic slides from penetrating keratoplasty corneal specimens, stained with hematoxylin and eosin were evaluated using bright field microscopy. CET and TCT were measured, and morphological features were studied. Clinical history regarding duration of KC prior to surgery and length of and tolerance to contact lens wear were compared and analyzed. Results The microscopic slides of all patients available for follow up (n = 48) were analyzed and CET and TCT were measured. The histological evaluation revealed 3 distinctive epithelial patterns. Pattern 1 with central hypertrophic and hydropic changes (n = 19) measured 70.89 ± 25.88 μm in CET and 308.63 ± 100.74 μm in TCT; Pattern 2 (n = 14) had not changed, similar to normal epithelium CET and TCT measuring 36.5 ± 7.02 μm and 260.14 ± 87.93 μm respectively. Pattern 3 (n = 15) demonstrated thinner central epithelium characterized by atrophy and focal hydropic changes measuring 19.93 ± 4.60 μm and 268.00 ± 79.39 μm in CET and TCT respectively (all p

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. To assess the reproducibility of cell collection and variability in receptor expression over time, participants were also asked to collect cells three times over a period of a month. The change in expression of surface receptors, CD11b, CD16, CD55, CD66b, important inflammatory and activation markers, and CD45 (PAN leukocyte marker) was assessed by flow cytometry. Fixing tear-film PMNs prior to the staining with antibodies resulted in a significant (fivefold or more) reduction in the expression of CD11b, CD16 and CD45 when compared to unfixed samples, while CD16 was the only receptor to undergo significant downregulation upon post-staining fixation. Furthermore, additional centrifugation step prior to antibody incubation as well as long (4 h) incubation at 37 °C resulted in significant reductions in expression of CD11b, CD16 and CD55 when compared to control samples. As opposed to blood PMNs, stimulating tear-film PMNs with IL-8 did not induce any significant changes in expression of CD11b, CD16, CD55 and CD66b. When working with collected tear-film PMNs, our results suggest that any additional centrifugation and incubation step should be avoided, or at least limited, and post fixation staining is recommended in order to preserve cell phenotype and cell integrity of tear film PMNs. Our study also adds further information on the reproducibility of the gentle eye wash as well as the inability of tear-film PMNs to modulate their surface receptors upon stimulation with IL-8. The latter may be due to prior exposure to IL-8, activation in the closed-eye environment, or a reduced ability to respond to inflammatory stimulus. Further mechanistic studies will be needed to gain a better understanding of the tear-film neutrophil phenotype.

Cardiovascular diseases remain the leading cause of death worldwide. Patency rates of clinically-utilized small diameter synthetic vascular grafts such as Dacron® and expanded polytetrafluoroethylene (ePTFE) to treat cardiovascular disease are inadequate due to lack of endothelialization. Sodium trimetaphosphate (STMP) crosslinked PVA could be potentially employed as blood-compatible small diameter vascular graft for the treatment of cardiovascular disease. However, PVA severely lacks cell adhesion properties, and the efforts to endothelialize STMP-PVA have been insufficient to produce a functioning endothelium. To this end, we developed a one-pot method to conjugate cell-adhesive protein via hydroxyl-to-amine coupling using carbonyldiimidazole by targeting residual hydroxyl groups on crosslinked STMP-PVA hydrogel. Primary human umbilical vascular endothelial cells (HUVECs) demonstrated significantly improved cells adhesion, viability and spreading on modified PVA. Cells formed a confluent endothelial monolayer, and expressed vinculin focal adhesions, cell-cell junction protein zonula occludens 1 (ZO1), and vascular endothelial cadherin (VE-Cadherin). Extensive characterization of the blood-compatibility was performed on modified PVA hydrogel by examining platelet activation, platelet microparticle formation, platelet CD61 and CD62P expression, and thrombin generation, which showed that the modified PVA was blood-compatible. Additionally, grafts were tested under whole, flowing blood without any anticoagulants in a non-human primate, arteriovenous shunt model. No differences were seen in platelet or fibrin accumulation between the modified-PVA, unmodified PVA or clinical, ePTFE controls. This study presents a significant step in the modification of PVA for the development of next generation in situ endothelialized synthetic vascular grafts.

Structural bone allografts are used to treat critically sized segmental bone defects (CSBDs) as such defects are too large to heal naturally. Development of biomaterials with competent mechanical properties that can also facilitate new bone formation is a major challenge for CSBD repair. 3D printed synthetic bone grafts are a possible alternative to structural allografts if engineered to provide appropriate structure with sufficient mechanical properties. In this work, we fabricated a set of novel nanocomposite biomaterials consisting of acrylated epoxidized soybean oil (AESO), polyethylene glycol diacrylate (PEGDA) and nanohydroxyapatite (nHA) by using masked stereolithography (mSLA)-based 3D printing. The nanocomposite inks possess suitable rheological properties and good printability to print complex, anatomically-precise, ‘by design’ grafts. The addition of nHA to the AESO/PEGDA resin improved the tensile strength and fracture toughness of the mSLA printed nanocomposites, presumably due to small-scale reinforcement. By adding 10 vol% nHA, tensile strength, modulus and fracture toughness (KIc) were increased to 30.8 ± 1.2 MPa (58% increase), 1984.4 ± 126.7 MPa (144% increase) and 0.6 ± 0.1 MPa·m1/2 (42% increase), respectively (relative to the pure resin). The nanocomposites did not demonstrate significant hydrolytic, enzymatic or oxidative degradation when incubated for 28 days, assuring chemical and mechanical stability at early stages of implantation. Apatite nucleated and covered the nanocomposite surfaces within 7 days of incubation in simulated body fluid. Good viability and proliferation of differentiated MC3T3-E1 osteoblasts were also observed on the nanocomposites. Taken all together, our nanocomposites demonstrate excellent bone-bioactivity and potential for bone defect repair.

From stents and large-diameter vascular grafts, to mechanical heart valves and blood pumps, blood-contacting devices are enjoying significant clinical success owing to the application of systemic antiplatelet and anticoagulation therapies. On the contrary, research into material and device hemocompatibility aimed at alleviating the need for systemic therapies has suffered a decline. This research area is undergoing a renaissance fueled by recent fundamental insights into coagulation and inflammation that are offering new avenues of investigation, the growing recognition of the limitations facing existing therapeutic approaches, and the severity of the cardiovascular disorders epidemic. This Opinion article discusses clinical needs for hemocompatible materials and the emerging research directions for fulfilling those needs. Based on the 2017 BloodSurf conference that brought together clinicians, scientists, and engineers from academia, industry, and regulatory bodies, its purpose is to draw the attention of the wider clinical and scientific community to stimulate further growth. STATEMENT OF SIGNIFICANCE: The article highlights recent fundamental insights into coagulation, inflammation, and blood-biomaterial interactions that are fueling a renaissance in the field of material hemocompatibility. It will be useful for clinicians, scientists, engineers, representatives of industry and regulatory bodies working on the problem of developing hemocompatible materials and devices for treating cardiovascular disorders.

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.

Lowering the thrombogenicity of materials has followed two broad paths – making materials inert or adding bioactive components. Heparin is the best example of the latter while the former has relied on hypotheses about the effect of surface properties (e.g., energy, chemistry) on proteins and platelets. Underlying this challenge is a limited understanding of the rate limiting mechanism of thrombosis on biomaterials.

A large number of polymorphonuclear neutrophils (PMNs) invade the ocular surface during prolonged eye closure (sleep); these leukocytes are commonly referred as tear PMNs. PMNs contribute to homeostasis and possess an arsenal of inflammatory mediators to protect against pathogens and foreign materials. This study examined the ability of tear PMNs to generate reactive oxygen species (ROS), an essential killing mechanism for PMNs which can lead to oxidative stress and imbalance. Cells were collected after sleep from healthy participants using a gentle eye wash. ROS production in stimulated (phorbol-12-myristate-13-acetate (PMA), lipopolysaccharides (LPS) or N-Formylmethionyl-leucyl-phenylalanine (fMLP)) and unstimulated tear PMNs was measured using luminol-enhanced chemiluminescence for 60 min. A high level of constitutive/spontaneous ROS production was observed in tear PMNs in the absence of any stimulus. While tear PMNs were able to produce ROS in response to PMA, they failed to appropriately respond to LPS and fMLP, although fMLP-stimulated tear PMNs generated ROS extracellularly in the first three minutes. Higher ROS generation was observed in isolated tear PMNs which may be due to priming from the magnetic bead cell separation system. The differential responses of tear PMNs in ROS generation provide further evidence of their potential inflammatory roles in ocular complications involving oxidative stress.

Engineering design is a critical skill that all engineering students are expected to learn and is often the focus of final year capstone projects and first-year cornerstone projects. In the Systems Design Engineering Department at the University of Waterloo, engineering design is introduced to the students during an intense two-day Design Days Boot Camp. Design Days was originally conceived of and run in Fall 2016. The Fall 2018 version, Design Days 2.0, included substantial improvements focused on adding two additional design activities and a writing activity, strengthening the connection with first year content, and providing a greater variety of team experiences. The methods of achieving the nine intended learning outcomes of Design Days 2.0 are discussed and connected to CEAB graduate attributes. This demonstrates that meaningful learning can be achieved during a two-day boot camp that will starts students on the path towards professional engineering. Other departments are encouraged to use the presented intended learning outcomes, graduate attributes connections, and Design Days 2.0 descriptions as a template for their own design boot camp. Finally, Design Days 2.0 inspired ideas for further improvements including the incorporation of a software-focused design activity, adding budgetary constraints, and providing an opportunity for student reflection.

– Engineering design is a core aspect of engineering education. Students might not appreciate the importance of engineering design early on, especially in the first term of their academic study. In this paper, we propose an approach for motivating students to think early about engineering design, namely at the start of their first academic term. The approach entails organizing an immersive design boot camp –“Design Days” – that replaces lectures for the first two days of the term. During the two days, students are divided into randomly assigned teams, and provided specifications to work on a design challenge. As part of the challenge, students are incentivized to fully understand the problem before attempting to solve it, instructed to follow a design process and apply iterative design, and asked to document their design and its rationale. The proposed method was successfully applied in Fall 2016. The approach will be applied again in Fall 2017 with minor modifications based on student feedback.

It has been reported that mechanical stimulus can affect cellular behavior. While induced differentiation in stem cells and proliferation and directional migration in fibroblasts are reported as responses to mechanical stimuli, little is known about the response of cells from the cornea. In the present study, we investigated whether changes in substrate stiffness (measured by elastic modulus) affected the behavior of human corneal epithelial cells (HCECs). Polyacrylamide substrates with different elastic moduli (compliant, medium and stiff) were prepared and HCECs were cultured on them. HCECs responses, including cell viability, apoptosis, intercellular adhesion molecule-1 (ICAM-1) expression, integrin-α3β1 expression and changes in cytoskeleton structure (actin fibers) and migratory behavior, were studied. No statistically significant cell activation, as measured by ICAM-1 expression, was observed. However, on compliant substrates, a higher number of cells were found to be apoptotic and disrupted actin fibers were observed. Furthermore, cells displayed a statistically significant lower migration speed on compliant substrates when compared with the stiffer substrates. Thus, corneal epithelial cells respond to changes in substrate stiffness, which may have implications in the understanding and perhaps treatment of corneal diseases, such as keratoconus.

Cells that form the corneal epithelium, the outermost layer of the cornea, are exposed to shear stress through blinking during waking hours. In this in vitro study, the effect of fluid shear stress on human corneal epithelial cells (HCECs) was investigated. Following exposure to shear stresses of 4 and 8 dyn/cm2, HCECs showed cytoskeletal rearrangement with more prominent, organized and elongated filamentous actin. Cytoskeletal changes were time-dependent, and were most significant after 24 hours of shear stress. Higher rates of migration and proliferation, as evaluated by a scratch assay, were also observed following 24 hours of low shear stress exposure (4 dyn/cm2). This result contrasted the poor migration observed in samples scratched before shear exposure, indicating that shear-induced cytoskeletal changes played a key role in improved wound healing and must therefore precede any damage to the cell layer. HCEC cytoskeletal changes were accompanied by an upregulation in integrin β1 and downregulation of ICAM-1. These results demonstrate that HCECs respond favourably to flow-induced shear stress, impacting their proliferation and migration properties as well as phenotype.

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 closed-eye 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.

The development of medical-device technology is constrained by the financial and ethical considerations of animal testing and clinical trials. Organ-on-a-chip systems are being developed to speed up drug development, yet there has been little recognition or support for the use of this technology in the development and testing of medical devices. In this Perspective, we introduce the concept of medical-device-on-a-chip (MDoC), highlight possible applications and discuss the potential of microfluidic high-throughput technologies for achieving significant time and cost savings over conventional testing. Rather than testing an entire macroscale device, a MDoC has the ability to recapitulate biological function in a physiome associated with medical-device use and to test interactions with device components. In the clinic, MDoCs could also incorporate individual human samples for personalized diagnostics. We also review research towards the integration of key elements of organ-on-a-chip technologies with medical-device testing. This Perspective puts forward the concept of medical-device-on-a-chip, that is, a microphysiological system that leverages organ-on-a-chip technology for the development and testing of medical devices.

Cataracts are the leading cause of blindness worldwide, requiring surgical implantation of an intraocular lens. Despite evidence of leukocyte ingress into the postoperative lens, few studies have investigated the leukocyte response to intraocular lens materials. A novel coculture model was developed to examine macrophage activation by hydrophilic acrylic (poly(2-hydroxyethyl methacrylate)) and hydrophobic acrylic (polymethylmethacrylate) commercial intraocular lens. The human monocytic cell line THP-1 was differentiated into macrophages and cocultured with human lens epithelial cell line (HLE-B3) with or without an intraocular lens for one, two, four, or six days. Using flow cytometry and confocal microscopy, expression of the macrophage activation marker CD54 (intercellular adhesion molecule-1) and production of reactive oxygen species via the fluorogenic probe 2′,7′-dichlorodihydrofluorescein diacetate were examined in macrophages. α-Smooth muscle actin, a transdifferentiation marker, was characterized in lens epithelial cells. The poly(2-hydroxyethyl methacrylate) intraocular lens prevented adhesion but induced significant macrophage activation ( p

Understanding the cellular and molecular mechanisms of the corneal tissue and translating them into effective therapies requires organotypic culture systems that can better model the physiological conditions of the front of the eye. Human corneal in vitro models currently exist, however, the lack of tear replenishment limits corneal in vitro models' ability to accurately simulate the physiological environment of the human cornea. The tear replenishment system (TRS), a micro-fluidic device, was developed to mimic the in vivo tear replenishment in the human eye in an in vitro corneal model. The TRS is capable of generating adjustable intermittent flow from 0.1 µL in every cycle. The TRS is a sterilizable device that is designed to fit standard 6-well cell culture plates. Experiments with the corneal models demonstrated that exposure to the TRS did not damage the integrity of the stratified cell culture. Contact lenses "worn" by the in vitro corneal model also remained moist at all times and the cytotoxicity of BAK could also be verified using this model. These in vitro results confirmed that the TRS presents novel avenues to assess lens-solution biocompatibility and drug delivery systems in a physiologically relevant milieu.

Although the Chandler loop model has been used in various in vitro flow studies, there is a lack of guidance on the selection of the appropriate sample volume. The questions of how to determine the appropriate sample volume and its effect on blood activation have not been fully addressed. This study proposes a new criterion for determining sample volume and defines a time-averaged wall shear stress equation for this model. In vitro experiments were performed to investigate the implications of sample volume on blood cell activation in the presence of model stent. Experimental results indicated that in the absence of a stent and for shear stress up to about 56 dyn/cm2, platelet activation was independent of volume and shear. On the other hand, the formation of platelet–leukocyte aggregates was affected by volume as well as the presence of a stent. Doubling blood volume for the same stent resulted in a twofold decrease in platelet microparticle formation and platelet–leukocyte aggregation. These results demonstrate the importance of selecting appropriate sample volume for the Chandler loop model, since it influences blood activation parameters, especially platelet–leukocyte aggregation formation, which can play an important role in material-induced thrombosis. These results have significance for in vitro screening of materials for biocompatibility.

Two thermally induced phase separation (TIPS) methods have been used to fabricate biodegradable poly(L-lactic acid) (PLLA) tissue engineering scaffolds each with fibrous (F-TIPS) and porous (P-TIPS) microstructures. Three levels of PLLA concentration (3, 5 and 7 wt%) were employed in each fabrication method and both wet and dry specimens were studied. Simple compression testing revealed that an elastic-plastic representation of the mechanical behavior was possible for all specimens. Both elastic and plastic moduli were higher for the P-TIPS, for higher polymer concentration, and might be somewhat higher for dry as opposed to wet specimens. For F-TIPS specimens, permanent deformation occurred successively during cyclic deformation but a "memory effect" simplified the behavior. Although F-TIPS microstructure better resembled the natural extracellular matrix, human osteosarcoma fibroblast cells showed more consistent viability in the P-TIPS scaffolds under our unloaded test protocols. Biodegradation in cell culture medium resulted in a decreased elastic moduli for F-TIPS specimens. Information presented regarding the microstructure, mechanical properties and cell viability of these PLLA scaffolds that should help reduce the number of iterations involved in developing tissue engineering products.

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.

There is a widely recognized need to improve the performance of vascular implants and external medical devices that come into contact with blood by reducing adverse reactions they cause, such as thrombosis and inflammation. These reactions lead to major adverse cardiovascular events such as heart attacks and strokes. Currently, they are managed therapeutically. This need remains unmet by the biomaterials research community. Recognized stagnation of the blood-biomaterial interface research translates into waning interest from clinicians, funding agencies, and practitioners of adjacent fields. The purpose of this contribution is to stir things up. It follows the 2014 BloodSurf meeting (74th International IUVSTA Workshop on Blood-Biomaterial Interactions), offers reflections on the situation in the field, and a three-pronged strategy integrating different perspectives on the biological mechanisms underlying blood-biomaterial interactions. The success of this strategy depends on reengaging clinicians and on the renewed cooperation of the funding agencies to support long-term efforts.

Purpose. To report the development of a new apparatus for non-invasive collection of human corneal epithelial cells. Methods. Previous methods of non-invasive, irrigative corneal cell collection resulted in low cell yields limiting potential analysis. A new ocular surface cell collection apparatus (OSCCA) was designed to collect more epithelial cells from direct irrigation of the corneal surface to allow for clinical comparisons. Forty-five samples were obtained (unilateral or bilateral over seven visits) from five human participants. Cell yield, size, phenotype, and corneal staining (prior and post eye wash) were examined. Results. On average 364 ± 230 epithelial cells were collected from the cornea per eye. Epithelial cell sizes ranged from 8.21 to 51.69 μm in diameter, and 67.30 to 2098.85 μm2 area. The proportion of corneal specific cells collected per sample was 75 ± 14% as determined by positive K3 expression with AE5. On average, 77 ± 0.2% of epithelial cells harvested were nucleated, the remainder were non-nucleated ghost cells. Corneal staining was reduced in the OSCCA-washed vs. contralateral non-washed eyes (p = 0.02). Conclusions. The OSCCA allows collection of human corneal epithelial cells with significantly higher yields, and greater specificity than previously reported. Reduced corneal staining observed post eye-wash demonstrated the safety of the technique, and its ability to remove cells directly from the corneal surface. The OSCCA could provide an objective non-invasive method of investigating pathological changes, effects of topical therapeutics, and impact of contact lenses and care-solutions of the cells of the ocular surface.

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.

The Chandler loop is an artificial circulatory platform for in vitro hemodynamic experiments. In most experiments, the working fluid is subjected to a strain rate field via rotation of the Chandler loop, which, in turn, induces biochemical responses of the suspended cells. For low rotation rates, the strain rate field can be approximated using laminar flow in a straight tube. However, as the rotation rate increases, the effect of the tube curvature causes significant deviation from the laminar straight tube approximation. In this manuscript, we investigate the flow and associated strain rate field of an incompressible Newtonian fluid in a Chandler loop as a function of the governing nondimensional parameters. Analytical estimates of the strain rate from a perturbation solution for pressure driven steady flow in a curved tube suggest that the strain rate should increase with Dean number, which is proportional to the tangential velocity of the rotating tube, and the radius to radius of curvature ratio of the loop. Parametrically varying the rotation rate, tube geometry, and fill ratio of the loop show that strain rate can actually decrease with Dean number. We show that this is due to the nonlinear relationship between the tube rotation rate and height difference between the two menisci in the rotating tube, which provides the driving pressure gradient. An alternative Dean number is presented to naturally incorporate the fill ratio and collapse the numerical data. Using this modified Dean number, we propose an empirical formula for predicting the average fluid strain rate magnitude that is valid over a much wider parameter range than the more restrictive straight tube-based prediction.

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.

Purpose To further improve in vitro models of the cornea, this study focused on the creation of a three-dimensional, stratified, curved epithelium; and the subsequent characterization and evaluation of its suitability as a model for biocompatibility testing. Methods Immortalized human corneal epithelial cells were grown to confluency on curved cellulose filters for seven days, and were then differentiated and stratified using an air-liquid interface for seven days before testing. Varying concentrations of a commercial ophthalmic solution containing benzalkonium chloride (BAK), a known cytotoxic agent, and two relevant ocular surfactants were tested on the model. A whole balafilcon A lens soaked in phosphate buffered saline (BA PBS) was also used to assess biocompatibility and verify the validity of the model. Viability assays as well as flow cytometry were performed on the cells to investigate changes in cell death and integrin expression. Results The reconstructed curved corneal epithelium was composed of 3–5 layers of cells. Increasing concentrations of BAK showed dose-dependent decreased cell viability and increased integrin expression and cell death. No significant change in viability was observed in the presence of the surfactants. As expected, the BA PBS combination appeared to be very biocompatible with no adverse change in cell viability or integrin expression. Conclusions The stratified, curved, epithelial model proved to be sensitive to distinct changes in cytotoxicity and is suitable for continued assessment for biocompatibility testing of contact lenses. Our results showed that flow cytometry can provide a quantitative measure of the cell response to biomaterials or cytotoxic compounds for both the supernatant and adherent cell populations. As a specifically designed in vitro model of the corneal epithelium, this quantitative model for biocompatibility at the ocular surface may help improve our understanding of cell-material interactions and reduce the use of animal testing.

The Chandler loop is an artificial circulatory platform for in vitro hemodynamic experiments. In most experiments, the working fluid is subjected to a stress field via rotation of the Chandler loop, which, in turn, induces biochemical responses of the suspended cells. For very low rotation rates, the stress field can be approximated using laminar flow in a straight tube as a model. However, as the rotation rate increases, while still maintaining laminar flow, the effect of the tube curvature causes the stress field to deviate considerably from the straight tube approximation. In this manuscript, we investigate the flow and associated strain rate field of an incompressible Newtonian fluid in a Chandler loop as a function of the governing non-dimensional fluid dynamic parameters. We find that the Dean number, which is proportional to the rotation rate, is the dominant parameter in determining the fluid strain rate. We propose an empirical formula for predicting the average fluid strain rate magnitude in the working fluid that is valid over a wide parameter space to be used in lieu of the common, yet restrictive, straight tube-based prediction.

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 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; 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 � 333 and 455 � 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.

A series of plasma-modified tubes (along with an unmodified control and other commercially available tubing) were prepared, surface characterized, and exposed to heparinized whole blood (1 U/mL heparin) for one hour at 37°C. The surface modifications included several different plasma vapors (H 2 O, CF 4 , and fluorine). The 1.5 mm ID tubing was incubated with whole blood on a rocking platform to gently agitate the blood and keep the cells from overtly settling. This system does not probe the effect of shear on cell activation; rather the agitation and long incubation time are thought sufficient to create “well-mixed” conditions. One of the conclusions from this study is that the materials with the lowest levels of platelet and leukocyte activation (microparticle formation, CD11b upregulation) were the unmodified materials (polyethylene, Pellethane™, a polyurethane), and that the surface modifications tested here had either no effect or only made things worse from the perspective of platelet and leukocyte activation.

Background/Aims: Recent advances in nanotechnology have made it possible to mass-produce ultrathin silicon membranes with pore sizes in the range of nanometers. In this study, we investigate the possibility of employing ultrathin nanoporous silicon membranes with pore diameters of 5 and 20 nm for dialysis of human whole blood by performing in vitro clearance and hemocompatibility assessments. Methods: A mini blood dialyzer is fabricated by mounting nanoporous silicon membranes on a Teflon structure. Clearance is calculated based on the concentration of sodium, chloride, ionized calcium, total CO2, glucose, creatinine and hematocrit measured before and after dialysis. Blood activation is assessed by flow cytometry. Results: Blood contact with the nanoporous membranes induces considerable leukocyte activation. Coating of the membranes with polyethylene glycol significantly improves hemocompatibility without blocking the nanopores. Conclusion: Silicon nanoporous membranes are potential candidates for fabrication of miniaturized blood dialyzers. Their mechanical strength and hemocompatibility can be further improved.

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.

A novel automated cell counting technique for cell sample images used to study the side-effects of lens cleaning solutions on human corneal epithelial cells is developed. The proposed multi-step approach integrates non-maximum suppression, seeded region growing, connected component analysis, and adaptive thresholding to produce segmentation and classification results that are robust to background illumination variation and clustering of cells. The proposed algorithm is computationally efficient, and experimental results show that the average detection rate of nucleated cells is greater than 90% with the proposed technique as opposed to the state-of-the-art level set method which gives an accuracy of less than 65%.

Purpose. 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. Methods. 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. Results. 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. Conclusions. 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.

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 various 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 6-12 dB, as well as a six-fold improvement in the dispersion index (DI), over that achieved using a single-channel, resolution-enhancing lensfree deconvolution microscopy system or its multi-wavelength counterpart. Furthermore, the LSLFM system achieved an increase in numerical aperture (NA) of ~16% over a single-channel resolution-enhancing 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., Comment: 5 pages

Rimon Therapeutics develops synthetic therapeutic polymers (Theramers™) that combine bioactivity with a broad range of desirable mechanical and physical properties. The MI Theramer™ is a polymer that modulates the activity of matrix metalloproteinases (MMPs).. Excessive proteolytic cleavage of extracellular matrix and growth factors resulting from elevated levels of MMPs is believed to contribute to the impaired wound healing associated with chronic, non-healing wounds. MI Theramer™ beads suspended in ThermaGel™(a thermoreversible gel) make up MI-Gel™ Dressing. The dressing may be applied as a liquid that is poured into a wound, where it will gel on contact. The dressing can be reliquified and removed easily without re-injuring the wound by cooling it. In vitro inhibition of collagenase type IV (equivalent to MMP-2) from clostridium histolyticum (Molecular Probes) was measured. A dose-dependent inhibition of MMP-2 activity was measured for MI Theramer™ beads alone and suspended in ThermaGel™ indicating that MMP-2 is able to diffuse into the gel and bind to the beads suspended therein. In vivo efficacy was demonstrated by observing the degradation of glutaraldehyde cross-linked gelatin tubes using a mouse subcutaneous pouch model. Gelatin zymography on extracts collected from the gelatin tubes co-implanted with MI Theramer™ beads also showed reduced MMP-2 and MMP-9 activity in comparison to controls. The insoluble MI Theramer™ beads were able to modulate MMP activity both in vitro and in vivo. The beads retained their inhibitory activity when they were suspended in a novel thermoreversible gel (ThermaGel™). The combined MI-Gel™ Dressing may have promise as a novel chronic wound healing product.