Green synthesis of Pluronic stabilized reduced graphene oxide: Chemical and biological characterization.

The wonder material graphene has numerous potential applications in nanoelectronics, biomedicine, storage devices, etc. Synthesis of graphene is highly challenging due to the toxic chemicals used and its low yield. In the present study, a facile green route for synthesis of reduced graphene oxide (rGO) was carried out using ascorbic acid as reducing agent. rGO was stabilized using Pluronic P123 polymer to give Pluronic stabilized reduced graphene oxide (rGO-P) and gave superior yield (15 mg graphene oxide yielded ˜13 mg rGO-P). Despite the potential neuroscience applications of graphene, the impending toxicological outcome upon interaction with neurons is not well understood. Here, differentiated PC-12 neuron-like cells exposed to rGO-P showed a dose-dependent cytotoxicity. Membrane disruption and cytoskeletal integrity remained uncompromised after 24 h exposure. Oxidative stress in PC-12 was evident due to an increase in ROS generation in dose and time-dependent manner. In vivo acute toxicity was assessed in mice administered with 10 mg/kg body weight of rGO-P. There were no evident changes in behaviour, motor function or other morphological changes. In conclusion, rGO-P was successfully synthesized and provided superior yield. Even though in vitro toxicity testing showed dose-dependent toxicity, in vivo toxic effect was not apparent.

The use of an IL-1 receptor antagonist peptide to control inflammation in the treatment of corneal limbal epithelial stem cell deficiency.

Corneal limbal stem cell deficiency (LSCD) may be treated using ex vivo limbal epithelial stem cells (LESCs) derived from cadaveric donor tissue. However, continuing challenges exist around tissue availability, inflammation, and transplant rejection. Lipopolysaccharide (LPS) or recombinant human IL-1ß stimulated primary human keratocyte and LESC models were used to investigate the anti-inflammatory properties of a short chain, IL-1 receptor antagonist peptide for use in LESC sheet growth to control inflammation. The peptide was characterized using mass spectroscopy and high performance liquid chromatography. Peptide cytotoxicity, patterns of cell cytokine expression in response to LPS or IL-1ß stimulation, and peptide suppression of this response were investigated by MTS/LDH assays, ELISA, and q-PCR. Cell differences in LPS stimulated toll-like receptor 4 expression were investigated using immunocytochemistry. A significant reduction in rIL-1ß stimulated inflammatory cytokine production occurred following LESC and keratocyte incubation with anti-inflammatory peptide and in LPS stimulated IL-6 and IL-8 production following keratocyte incubation with peptide (1 mg/mL) (P < 0.05). LESCs produced no cytokine response to LPS stimulation and showed no TLR4 expression. The peptide supported LESC growth when adhered to a silicone hydrogel contact lens indicating potential use in improved LESC grafting through suppression of inflammation.

Hydroxyapatite promotes superior keratocyte adhesion and proliferation in comparison with current keratoprosthesis skirt materials.

AIM: Published clinical series suggest the osteoodontokeratoprosthesis (OOKP) may have a lower extrusion rate than current synthetic keratoprostheses. The OOKP is anchored in the eye wall by autologous tooth. The authors' aim was to compare adhesion, proliferation, and morphology for telomerase transformed keratocytes seeded on calcium hydroxyapatite (the principal mineral constituent of tooth) and materials used in the anchoring elements of commercially available synthetic keratoprostheses. METHODS: Test materials were hydroxyapatite, polytetrafluoroethylene (PTFE), polyhydroxyethyl methacrylate (HEMA), and glass (control). Cell adhesion and viability were quantified at 4 hours, 24 hours, and 1 week using a calcein-AM/EthD-1 viability/cytotoxicity assay. Focal contact expression and cytoskeletal organisation were studied at 24 hours by confocal microscopy with immunoflourescent labelling. Further studies of cell morphology were performed using light and scanning electron microscopy. RESULTS: Live cell counts were significantly greater on hydroxyapatite surfaces at each time point (p<0.04). Dead cell counts were significantly higher for PTFE at 7 days (p<0.002). ss(1) integrin expression was highest on hydroxyapatite. Adhesion structures were well expressed in flat, spread out keratocytes on both HA and glass. Keratocytes tended to be thinner and spindle shaped on PTFE. The relatively few keratocytes visible on HEMA test surfaces were rounded and poorly adherent. CONCLUSIONS: Keratocyte adhesion, spreading, and viability on hydroxyapatite test surfaces is superior to that seen on PTFE and HEMA. Improving the initial cell adhesion environment in the skirt element of keratoprostheses may enhance tissue integration and reduce device failure rates.

A model for the preliminary biological screening of potential keratoprosthetic biomaterials.

A series of in vitro screening assays for the preliminary selection of biomaterials for use in the fabrication of artificial corneas (keratoprostheses) (KPros) have been investigated. These screening assays assessed the initial binding of inflammatory and cell adhesive proteins, activation of inflammatory proteins, adhesion of keratocytes, epithelial cells and macrophages and the production of inflammatory cytokines by keratocytes contacting biomaterials. Central optic biomaterials were selected on the basis of low-inflammatory and cell adhesion potential. Peripheral skirt materials were selected on the basis of low-inflammatory potential but good cell adhesion to anchor the implant within the host cornea. Green fluorescent protein (GFP) gene transfer was used in a novel context to investigate cell invasion in the absence of external staining techniques. Confocal laser scanning microscopy and scanning electron microscopy were used to investigate GFP positive keratocyte invasion of porous materials. The results of in vitro assays were compared to a corneal organ culture system in which the biomaterials were assessed within a stromal environment. A range of polyurethane-based interpenetrating polymers with a range of water contents were screened. All materials showed low-inflammatory potential. A reduction in biomaterial water content induced an increase in complement C3 and fibronectin binding and in cell adhesion to materials, whilst differences in co-monomer formulation had little impact. The screening methods used in the current study provide a suitable preliminary assessment regime for the in vitro evaluation of potential KPro materials.

Corneal toxicity secondary to inadvertent use of benzalkonium chloride preserved viscoelastic material in cataract surgery.

AIMS: To study the long term toxic effects of intraocular benzalkonium chloride (BAC). METHODS: 19 patients exposed to intraocular BAC preserved viscoelastic during cataract surgery in February 1999 developed severe striate keratopathy immediately postoperatively. 16 patients, including two who underwent penetrating keratoplasty, were studied in the period April to June 2000. Ocular symptoms, visual acuity, biomicroscopy, intraocular pressure, dilated funduscopy, specular endothelial microscopy, and corneal pachymetry findings were recorded. The corneal and iris specimens of the two patients who underwent keratoplasty were studied by light, transmission, and scanning electron microscopy. RESULTS: Six males and 10 females, aged 64-98 years, were studied 14-16 months postoperatively. All patients were symptomatic. 12 patients had best corrected visual acuity of 6/12 or better and four patients of between 6/18 and 6/60. Five patients had corneal epithelial oedema and 11 had Descemet's membrane folds. The central corneal thickness, 620 (SD 71) microm, in affected eyes was significantly higher (p<0.005, two tailed paired t test) than that of the contralateral eyes, 563 (SD 48) microm. The endothelial cell density was significantly lower (p<0.0001, two tailed paired t test) in affected eyes: 830 (SD 280) cells/mm2 v 2017 (SD 446) cells/mm2. The mean average cell area was significantly higher in the BAC treated eyes: 1317 (SD 385) microm2 v 521 (SD 132) microm2. There was no significant difference in the coefficient of variation of cell size between the two eyes (p=0.3, two tailed paired t test). Two corneal specimens displayed morphological features of bullous keratopathy and other non-specific abnormalities. Extracellular melanosomes were present in a portion of the iris of one case. CONCLUSION: BAC is toxic to the corneal endothelium when used intraocularly, leading to severe striate keratopathy. This cleared in most cases but left varying degrees of residual stromal thickening in all eyes. If penetrating keratoplasty is required the results are excellent.

Does MMP-2 expression and secretion change with increasing serial passage of keratocytes in culture?

The effects of ageing on matrix metalloprotease degradation of the extracellular matrix during corneal wound healing are largely unknown. The following study used an in vitro model of ageing to assess changes in MMP-2 RNA expression and protein secretion. Early passage (EP) EK1.BR keratocyte cultures from 14 to 18 cumulative population doublings (cpds) and late passage (LP) cultures from 40 to 47 cpds were used to isolate protein and mRNA samples. Total protein from EP and LP cultures was measured using the Bradford protein assay. Zymographic analysis of EP and LP samples was carried out to compare MMP-2 activity. Northern blot analysis was used to assess changes in MMP-2 mRNA expression by EP and LP cultures, using a digoxigenin (DIG) based chemiluminescent detection system. LP cultures secreted more total protein per cell. MMP-2 but not MMP-9 activity was detected in keratocyte cultures. Densitometric analysis of zymograms and calculation of MMP-2 activity indicated a significant increase in MMP-2 activity per cell (P<0.05, n=11). No difference was observed in the levels of MMP-2 mRNA expressed by EP and LP cultures. An increase in MMP-2 activity per cell by LP cultures suggests that senescent keratocytes increase their degradative capacity. Similar changes in the keratocyte phenotype within the ageing cornea may alter the balanced response necessary for adequate wound healing and may have implications for the therapeutic use of MMP inhibitors in the eye.

Human keratocyte migration into collagen gels declines with in vitro ageing.

Although senescence in various cell types has been shown to have detrimental effects on wound repair, the effect of this phenomenon on corneal function with increasing age has yet to be elucidated. This study investigated the effect of in vitro ageing on keratocyte migration into a collagen gel matrix. The keratocyte cell strain EK1. BR was cultured to late passage and a comparison of early passage migration with that of late passage migration was carried out. Early or late passage keratocytes were seeded onto 6 collagen gels (1.75 mg ml(-1)) for each experiment. Gels were incubated at 37 degrees C for 72 h, stained with calcein AM (0.5 mg ml(-1)) and assayed for cell migration using fluorescent microscopy. Changes in the effect of EGF on keratocyte migration with age were assessed by the addition of EGF (20 ng ml(-1)) to 3 of the 6 gels in each experiment. Proliferative lifespan was measured by immunocytochemical detection of Ki67 activity. This study shows for the first time that keratocyte migration, and migration in response to EGF stimulation, significantly declines with increasing age of keratocytes in culture (P<0.001). As keratocyte migration in response to cytokine stimulation is vital for corneal repair, the accumulation of senescent keratocytes with age may impair corneal wound healing.

Novel materials to enhance keratoprosthesis integration.

BACKGROUND: The successful integration of keratoprostheses (KPros) within the cornea depends in part on peripheral host keratocyte adhesion to anchor the implant in place and prevent epithelial downgrowth. The following study incorporated different acrylate co-monomers with poly(hydroxyethyl methacrylate) (p(HEMA)) and measured the suitability of these materials as potential skirt materials in terms of their ability to enhance keratocyte adhesion to p(HEMA). METHODS: p(HEMA) hydrogels incorporating varying amounts of the acrylate co-monomers methacrylic acid (MA), 2-(dimethylamino)ethyl methacrylate (DEM), or phenoxyethyl methacrylate (PEM) were formed by free radical polymerisation. Keratocytes were seeded onto discs of each material and incubated at 37 degrees C for 72 hours. Assays for viable cell adhesion were carried out. A viability/cytotoxicity assay using solutions of calcein-AM (0.5 mM) and ethidium homodimer-1 (EthD-1) (0.5 microM) were used to measure viable and non-viable cell adhesion, respectively. An ATP assay was also used to quantify cell adhesion in terms of the amount of ATP present following lysis of adherent cells. RESULTS: The viability/cytotoxicity assays indicated that the incorporation of 15 mol% of the co-monomer PEM or of 20 mol% DEM increased cell adhesion to p(HEMA) by at least four times. The ATP assays confirmed the results for PEM but absorption of ATP to the DEM containing hydrogel indicated that the assay was not a suitable measure of cell adhesion to this material. CONCLUSIONS: The properties of p(HEMA) may be moderated to enhance keratocyte adhesion by the incorporation of PEM or DEM suggesting that these may be suitable materials for use in the further development of a novel KPro skirt material.

A standard strain of human ocular keratocytes.

The ability of an injured cornea to regenerate from deep tissue trauma is largely due to wound healing processes mediated by the surviving stromal keratocytes. Despite the importance of the wound healing process, and the ease with which keratocytes can be grown in tissue culture, a standardised strain of the cells has never been made available. Accordingly, this study reports a strain of human embryonic keratocytes, designated EK1.BR as a research tool for the ophthalmic community. EK1.BR has been characterised with respect to life-span, fraction of dividing cells and maintenance of a keratocyte phenotype in culture. It is hoped that these cells will prove useful in the in vitro study of stromal wound healing and the characterisation of keratocyte gene expression.