In vitro evaluation of bone cell response to novel 3D-printable nanocomposite biomaterials for bone reconstruction.

Critically-sized segmental bone defects represent significant challenges requiring grafts for reconstruction. 3D-printed synthetic bone grafts are viable alternatives to structural allografts if engineered to provide appropriate mechanical performance and osteoblast/osteoclast cell responses. Novel 3D-printable nanocomposites containing acrylated epoxidized soybean oil (AESO) or methacrylated AESO (mAESO), polyethylene glycol diacrylate, and nanohydroxyapatite (nHA) were produced using masked stereolithography. The effects of volume fraction of nHA and methacrylation of AESO on interactions of differentiated MC3T3-E1 osteoblast (dMC3T3-OB) and differentiated RAW264.7 osteoclast cells with 3D-printed nanocomposites were evaluated in vitro and compared with a control biomaterial, hydroxyapatite (HA). Higher nHA content and methacrylation significantly improved the mechanical properties. All nanocomposites supported dMC3T3-OB cells' adhesion and proliferation. Higher amounts of nHA enhanced cell adhesion and proliferation. mAESO in the nanocomposites resulted in greater adhesion, proliferation, and activity at day 7 compared with AESO nanocomposites. Excellent osteoclast-like cells survival, defined actin rings, and large multinucleated cells were only observed on the high nHA fraction (30%) mAESO nanocomposite and the HA control. Thus, mAESO-based nanocomposites containing higher amounts of nHA have better interactions with osteoblast-like and osteoclast-like cells, comparable with HA controls, making them a potential future alternative graft material for bone defect repair.

Investigation of the rhythmic recruitment of tear neutrophils to the ocular surface and their phenotypes.

Hundreds of thousands of polymorphonuclear neutrophils (PMNs) are collected from the ocular surface upon waking, while few are harvested during daytime. This study aimed to investigate potential factors contributing to the circadian infiltration of tear PMNs, including changes in IL-8 and C5a in tears, and their phenotypes across different time points in a 24-h cycle. Tear PMNs were collected using a gentle eyewash after 2-h and 7-h of sleep (eye closure, EC) at night, after 2-h EC during the day, and towards the end of the afternoon. Significantly fewer cells were collected after 2-h EC during the day compared to 2-h EC at night. A positive correlation between IL-8 and PMN numbers existed, but not with C5a. Tear PMNs collected after 2-h EC at night were less degranulated and possessed a larger activation potential compared to 7-h EC. Tear PMNs from 7-h EC at night exhibited hyper-segmented nuclei and more NETosis compared to 2 h EC night, indicating an aged and activated phenotype. The diurnal-nocturnal recruitment pattern of tear PMNs may be driven by increased IL-8 in nighttime tears. Higher degranulation and NETs point to the significant activation of tear PMNs on the ocular surface during prolonged eye closure at night.

The effect of triglycerol diacrylate on the printability and properties of UV curable, bio-based nanohydroxyapatite composites.

3D printable biopolymer nanocomposites composed of hydroxyapatite nanoparticles and functionalized plant-based monomers demonstrate potential as sustainable and structural biomaterials. To increase this potential, their printability and performance must be improved. For extrusion-based 3D printing, such as Direct Ink Writing (DIW), printability is important for print fidelity. In this work, triglycerol diacrylate (TGDA) was added to an acrylated epoxidized soybean oil:polyethylene glycol diacrylate resin to increase hydrogen bonding. Greater hydrogen bonding was hypothesized to improve printability by increasing the ink's shear yield strength, and therefore shape holding after deposition. The effects of this additive on material and mechanical properties were quantified. Increased hydrogen bonding due to TGDA content increased the ink's shear yield stress and viscosity by 916% and 27.6%, respectively. This resulted in improved printability, with best performance at 3 vol% TGDA. This composition achieved an ultimate tensile strength (UTS) of 32.4 ± 2.1 MPa and elastic modulus of 1.15 ± 0.21 GPa. These were increased from the 0 vol% TGDA composite, which had an UTS of 24.8 ± 1.8 MPa and a modulus of 0.88 ± 0.06 GPa. This study demonstrates the development of bio-based additive manufacturing feedstocks for potential uses in sustainable manufacturing, rapid prototyping, and biomaterial applications.

Chitosan Grafted with Thermoresponsive Poly(di(ethylene glycol) Methyl Ether Methacrylate) for Cell Culture Applications.

Chitosan is a polysaccharide extracted from animal sources such as crab and shrimp shells. In this work, chitosan films were modified by grafting them with a thermoresponsive polymer, poly(di(ethylene glycol) methyl ether methacrylate) (PMEO2MA). The films were modified to introduce functional groups useful as reversible addition-fragmentation chain transfer (RAFT) agents. PMEO2MA chains were then grown from the films via RAFT polymerization, making the chitosan films thermoresponsive. The degree of substitution of the chitosan-based RAFT agent and the amount of monomer added in the grafting reaction were varied to control the length of the grafted PMEO2MA chain segments. The chains were cleaved from the film substrates for characterization using 1H NMR and a gel permeation chromatography analysis. Temperature-dependent contact angle measurements were used to demonstrate that the hydrophilic-hydrophobic nature of the film surface varied with temperature. Due to the enhanced hydrophobic character of PMEO2MA above its lower critical solution temperature (LCST), the ability of PMEO2MA-grafted chitosan films to serve as a substrate for cell growth at 37 °C (incubation temperature) was tested. Interactions with cells (fibroblasts, macrophages, and corneal epithelial cells) were assessed. The modified chitosan films supported cell viability and proliferation. As the temperature is lowered to 4 °C (refrigeration temperature, below the LCST), the grafted chitosan films become less hydrophobic, and cell adhesion should decrease, facilitating their removal from the surface. Our results indicated that the cells were detached from the films following a short incubation period at 4 °C, were viable, and retained their ability to proliferate.

The Differential Reactive Oxygen Species Production of Tear Neutrophils in Response to Various Stimuli In Vitro.

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.

mSLA-based 3D printing of acrylated epoxidized soybean oil - nano-hydroxyapatite composites for bone repair.

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.

Impact of contact lens wear on epithelial alterations in keratoconus

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 Mum in CET and 308.63 ± 100.74 Mum in TCT; Pattern 2 (n = 14) had not changed, similar to normal epithelium CET and TCT measuring 36.5 ± 7.02 Mum and 260.14 ± 87.93 Mum respectively. Pattern 3 (n = 15) demonstrated thinner central epithelium characterized by atrophy and focal hydropic changes measuring 19.93 ± 4.60 Mum and 268.00 ± 79.39 Mum in CET and TCT respectively (all p < 0.0001). The presence of Pattern 2 characterized by similar to normal CET was correlated with the duration of the condition (R = 0.600, p = 0.030). There was a significant difference in the length of CL wear comparing those with patterns 1 and 2 versus 3 (least no. of CL years) (p = 0.05 and p = 0.33 respectivelly). CONCLUSIONS: Patients with advanced disease have various central corneal epithelial changes detected with histology. Although each central epithelial pattern type was distinctive comparing the 3 patterns, there was no correlation with years of CL wear but only with the duration of the condition

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Impact of contact lens wear on epithelial alterations in keratoconus.

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±25.88µm in CET and 308.63±100.74µm in TCT; Pattern 2 (n=14) had not changed, similar to normal epithelium CET and TCT measuring 36.5±7.02µm and 260.14±87.93µm respectively. Pattern 3 (n=15) demonstrated thinner central epithelium characterized by atrophy and focal hydropic changes measuring 19.93±4.60µm and 268.00±79.39µm in CET and TCT respectively (all p<0.0001). The presence of Pattern 2 characterized by similar to normal CET was correlated with the duration of the condition (R=0.600, p=0.030). There was a significant difference in the length of CL wear comparing those with patterns 1 and 2 versus 3 (least no. of CL years) (p=0.05 and p=0.33 respectivelly). CONCLUSIONS: Patients with advanced disease have various central corneal epithelial changes detected with histology. Although each central epithelial pattern type was distinctive comparing the 3 patterns, there was no correlation with years of CL wear but only with the duration of the condition.

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

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.

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.

The blood compatibility challenge. Part 3: Material associated activation of blood cascades and cells.

Following protein adsorption/activation which is the first step after the contact of material surfaces and whole blood (part 2), fibrinogen is converted to fibrin and platelets become activated and assembled in the form of a thrombus. This thrombus formation is the key feature that needs to be minimized in the creation of materials with low thrombogenicity. Further aspects of blood compatibility that are important on their own are complement and leukocyte activation which are also important drivers of thrombus formation. Hence this review summarizes the state of knowledge on all of these cascades and cells and their interactions. For each cascade or cell type, the chapter distinguishes statements which are in widespread agreement from statements where there is less of a consensus. STATEMENT OF SIGNIFICANCE: This paper is part 3 of a series of 4 reviews discussing the problem of biomaterial associated thrombogenicity. The objective was to highlight features of broad agreement and provide commentary on those aspects of the problem that were subject to dispute. We hope that future investigators will update these reviews as new scholarship resolves the uncertainties of today.

BloodSurf 2017: News from the blood-biomaterial frontier.

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 Effect of Closed-Eye Tear Film Conditions on Blood-Isolated Neutrophils, In Vitro.

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.

Corneal epithelial cells exposed to shear stress show altered cytoskeleton and migratory behaviour.

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 ß1 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.

Stirred, shaken, or stagnant: What goes on at the blood-biomaterial interface.

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.

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.

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

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Bayesian-based deconvolution fluorescence microscopy using dynamically updated nonstationary expectation estimates.

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.

Human corneal epithelial cell response to substrate stiffness.

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-α3ß1 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.

Coculture with intraocular lens material-activated macrophages induces an inflammatory phenotype in lens epithelial cells.

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 < 0.03) versus control (no intraocular lens), while the polymethylmethacrylate intraocular lens enabled adhesion and multinucleated fusion, but induced no significant activation. Coculture with either intraocular lens increased reactive oxygen species production in macrophages after one day (p < 0.03) and increased expression of α-smooth muscle actin in HLE B-3 after six days, although only poly(2-hydroxyethyl methacrylate) induced a significant difference versus control (p < 0.01). Our results imply that-contrary to prior uveal biocompatibility understanding-macrophage adherence is not necessary for a strong inflammatory response to an intraocular lens, with hydrophilic surfaces inducing higher activation than hydrophobic surfaces. These findings provide a new method of inquiry into uveal biocompatibility, specifically through the quantification of cell-surface markers of leukocyte activation.