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

A lens-free 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 lens-free 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 lens-free 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 lens-free 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 lens-free 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.

In vitro methods of assessing ocular biocompatibility using THP-1-derived macrophages.

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.

Numerical investigation of fluid flow in a chandler loop.

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.

Impact of multipurpose solutions released from contact lenses on corneal cells.

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, ß1, and ß4 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.

One-Pot Covalent Grafting of Gelatin on Poly(Vinyl Alcohol) Hydrogel to Enhance Endothelialization and Hemocompatibility for Synthetic Vascular Graft Applications.

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.

Endotoxin: the uninvited guest.

In the laboratory environment where biomaterials are synthesized and their biocompatibility assessed, we find that endotoxin contamination is hard to avoid and must not be ignored. In those relatively few cases where endotoxin was known to be present, it has been clearly shown that endotoxin can significantly affect the biological response observed and hence confound any effect of the material. This short review explains what endotoxin is, how to test for it and remove it and what its effect on the biological response to biomaterials is. We advocate routine testing of endotoxin on biomaterials and of reagents used in experimental evaluation of biomaterials and this should be the responsibility of every scientist to ensure the validity of any biomaterial study.

Biomaterial-associated thrombosis: roles of coagulation factors, complement, platelets and leukocytes.

Our failure to produce truly non-thrombogenic materials may reflect a failure to fully understand the mechanisms of biomaterial-associated thrombosis. The community has focused on minimizing coagulation or minimizing platelet adhesion and activation. We have infrequently considered the interactions between the two although we are generally familiar with these interactions. However, we have rarely considered in the context of biomaterial-associated thrombosis the other major players in blood: complement and leukocytes. Biomaterials are known agonists of complement and leukocyte activation, but this is frequently studied only in the context of inflammation. For us, thrombosis is a special case of inflammation. Here we summarize current perspectives on all four of these components in thrombosis and with biomaterials and cardiovascular devices. We also briefly highlight a few features of biomaterial-associated thrombosis that are not often considered in the biomaterials literature: The importance of tissue factor and the extrinsic coagulation system. Complement activation as a prelude to platelet activation and its role in thrombosis. The role of leukocytes in thrombin formation. The differing time scales of these contributions.

Investigation of recombinant human elastin polypeptides as non-thrombogenic coatings.

We investigated the use of a recombinant human elastin polypeptide as a coating on synthetic materials with a view to determining if these polypeptides could improve the blood compatibility of cardiovascular devices such as vascular conduits and arterial/venous catheters. Platelet adhesion and activation were studied in vitro using three commercially available synthetic materials: polyethylene terephthalate (Mylar), a poly(tetrafluoroethylene/ethylene) copolymer (Tefzel) and a polycarbonate polyurethane (Corethane). Coated with adsorbed polypeptide, all three synthetic materials demonstrated reduced platelet activation and adhesion in platelet rich plasma in vitro. Compared to non-coated controls, there was a significant decrease (p=0.05) in both platelet microparticle release and P-selectin expression for the polypeptide-coated surfaces. Scanning electron microscopy indicated fewer adhering platelets on coated surfaces compared to non-coated controls. In vivo, in a rabbit model, evaluations of polyurethane catheters coated with the polypeptide showed a marked increase in catheter patency and a significant decrease in fibrin accretion and embolism when compared to uncoated controls. This polypeptide shows a strong potential for use as a non-thrombogenic coating for small diameter vascular grafts. In addition, the results of this study indicate that the elastin polypeptide would be a valuable component of a tissue engineered vascular conduit.

Development of a curved, stratified, in vitro model to assess ocular biocompatibility.

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.