Susan Sandeman

Aging and the cornea.

Aging, the persistent decline in age specific fitness of an organism as a result of internal physiological deterioration, is a common process among multicellular organisms.1 In humans, aging is usually monitored in relation to time, which renders it difficult to differentiate between time dependent biological changes and damage from environmental insults. There are essentially three types of aging at work in any adult tissue; the aging of long lived proteins, the aging of dividing cells, and the aging of non-dividing cells.2 Dividing cells may be derived from renewing populations in which the rate of cell loss and division is great. An example is the corneal epithelium in which complete turnover occurs within 5–7 days after terminal differentiation.34Conditional renewal populations, which normally have an extremely low proliferation rate, can also produce dividing cells in response to extrinsic stimuli. Stromal keratocytes are a prime example of a conditional renewing population.5 Corneal endothelial cells retain the capacity to undergo mitosis and conditional renewal in humans although they very seldom do so.6-9 Non-dividing cells are those from static cell populations (exemplified by cerebral neurons) which never divide during adult life.3 Corneal aging produces both structural and functional changes. These changes in turn can affect the ability of the organ to refract light, to repair itself, and to protect itself and the internal structures of the eye.10 A variety of corneal aging changes have been reported. However, as it is difficult to distinguish age specific deterioration from degenerations modified by environmental and genetic factors, we think it is helpful to consider these alterations within the broader framework of the aging process. The study over the past 30 years of isolated cells in culture as a model system for aging changes has greatly advanced our understanding of …

Adsorption of anionic and cationic dyes by activated carbons, PVA hydrogels, and PVA/AC composite

The textural and adsorption characteristics of a series of activated carbons (ACs), porous poly(vinyl alcohol) (PVA) gels, and PVA/AC composites were studied using scanning electron microscopy, mercury porosimetry, adsorption of nitrogen (at 77.4 K), cationic methylene blue (MB), anionic methyl orange (MO), and Congo red (CR) from the aqueous solutions. Dye-PVA-AC-water interactions were modeled using the semiempirical quantum chemical method PM6. The percentage of dye removed (C(rem)) by the ACs was close to 100% at an equilibrium concentration (C(eq)) of less than 0.1 mM but decreased with increasing dye concentration. This decrease was stronger at C(eq) of less than 1 mM, and C(rem) was less than 50% at a C(eq) of 10-20 mM. For PVA and the PVA/AC composite containing C-7, the C(rem) values were minimal (<75%). The free energy distribution functions (f(ΔG)) for dye adsorption include one to three peaks in the -ΔG range of 1-60 kJ/mol, depending on the dye concentration range used and the spatial, charge symmetry of the hydrated dye ions and the structural characteristics of the adsorbents. The f(ΔG) shape is most complex for MO with the most asymmetrical geometry and charge distribution and adsorbed at concentrations over a large C(eq) range. For symmetrical CR ions, adsorbed over a narrow C(eq) range, the f(ΔG) plot includes mainly one narrow peak. MB has a minimal molecular size at a planar geometry (especially important for effective adsorption in slit-shaped pores) which explains its greater adsorptive capacity over that of MO or CR. Dye adsorption was greatest for ACs with the largest surface area but as molecular size increases adsorption depends to a greater extent on the pore size distribution in addition to total and nanopore surface areas and pore volume.

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 Descemets membrane folds. The central corneal thickness, 620 (SD 71) μm, in affected eyes was significantly higher (p<0.005, two tailed paired t test) than that of the contralateral eyes, 563 (SD 48) μm. The endothelial cell density was significantly lower (p<0.0001, two tailed paired t test) in affected eyes: 830 (SD 280) cells/mm2v 2017 (SD 446) cells/mm2. The mean average cell area was significantly higher in the BAC treated eyes: 1317 (SD 385) μm2v 521 (SD 132) μm2. 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.

Mesoporous carbide-derived carbon for cytokine removal from blood plasma

Porous carbons can be used for purification of bio-fluids due to their excellent biocompatibility with blood. Since the ability to adsorb a range of inflammatory cytokines within the shortest possible time is crucial to stop the progression of sepsis, the improvement of the adsorption rate is a key factor to achieving efficient removal of cytokines. Here, we demonstrate the effect of synthesis temperatures (from 600 degrees C to 1200 degrees C), carbon particle sizes (from below 35 microm to 300 microm), and annealing conditions (Ar, NH(3), H(2), Cl(2), and vacuum annealing) that determine the surface chemistry, on the ability of carbide-derived carbons (CDCs) to remove cytokines TNF-alpha, IL-6, and IL-1 beta from blood plasma. Optimization of CDC processing and structure leads to up to two orders of magnitude increase in the adsorption rate. Mesoporous CDCs that were produced at 800 degrees C from Ti(2)AlC with the precursor particle size of <35 microm and annealed in NH(3), displayed complete removal of large molecules of TNF-alpha in less than an hour, with >85% and >95% TNF-alpha removal in 5 and 30 min, respectively. This is a very significant improvement compared to the previously published results for CDC (90% TNF-alpha removal after 1h) and activated carbons. Smaller interleukin IL-6 and IL-1 beta molecules can be completely removed within 5 min. These differences in adsorption rates show that carbons with controlled porosity can also be used for separation of protein molecules.

Inflammatory cytokine removal by an activated carbon device in a flowing system

A prototype in-line filtration/adsorption device has been developed using novel synthetic pyrolysed carbon monoliths with controlled mesoporous domains of 2-50nm. Porosity was characterized by SEM and porosimetry. Removal of inflammatory cytokines TNF, IL-6, IL-1beta and IL-8 was assessed by filtering cytokine spiked human plasma through the walls of the carbon modules under pressure. The effect of carbon filtration on plasma clotting response and total plasma protein concentration was also assessed. Significant removal of the cytokines IL-6, IL-1beta and IL-8 was observed. Initially marked TNF removal diminished over time. The coagulation studies indicated that the carbon device does not exacerbate the propensity of blood plasma to clot. The total plasma protein concentration remained constant. The device offers a broader approach to the treatment of systemic inflammatory response syndrome (SIRS) by the removal of inflammatory mediators central to its progression.

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). ß1 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.

Nano carriers for drug transport across the blood–brain barrier

Abstract Effective therapy lies in achieving a therapeutic amount of drug to the proper site in the body and then maintaining the desired drug concentration for a sufficient time interval to be clinically effective for treatment. The blood–brain barrier (BBB) hinders most drugs from entering the central nervous system (CNS) from the blood stream, leading to the difficulty of delivering drugs to the brain via the circulatory system for the treatment, diagnosis and prevention of brain diseases. Several brain drug delivery approaches have been developed, such as intracerebral and intracerebroventricular administration, intranasal delivery and blood-to-brain delivery, as a result of transient BBB disruption induced by biological, chemical or physical stimuli such as zonula occludens toxin, mannitol, magnetic heating and ultrasound, but these approaches showed disadvantages of being dangerous, high cost and unsuitability for most brain diseases and drugs. The strategy of vector-mediated blood-to-brain delivery, which involves improving BBB permeability of the drug–carrier conjugate, can minimize side effects, such as being submicrometre objects that behave as a whole unit in terms of their transport and properties, nanomaterials, are promising carrier vehicles for direct drug transport across the intact BBB as a result of their potential to enter the brain capillary endothelial cells by means of normal endocytosis and transcytosis due to their small size, as well as their possibility of being functionalized with multiple copies of the drug molecule of interest. This review provids a concise discussion of nano carriers for drug transport across the intact BBB, various forms of nanomaterials including inorganic/solid lipid/polymeric nanoparticles, nanoemulsions, quantum dots, nanogels, liposomes, micelles, dendrimers, polymersomes and exosomes are critically evaluated, their mechanisms for drug transport across the BBB are reviewed, and the future directions of this area are fully discussed.

Hierarchical porous carbide-derived carbons for the removal of cytokines from blood plasma.

Series of silicon carbonitride ceramics are utilized to obtain hierarchically porous carbide-derived carbons (CDCs) for cytokine removal. The removal rate of TNF-α and IL-6, as two examples of pro- and anti-inflammatory cytokines, is proportional to the surface area of pores larger than the size of the protein molecule

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.

The in vitro corneal biocompatibility of hydroxyapatite coated carbon mesh

The purpose of this study was to consider the use of a hydroxyapatite (HA) coated porous carbon matrix as a synthetic dental laminate substitute in osteo-odonto-keratoprosthetic (OOKP) design. 3 types of carbon meshes were coated with HA by sonoelectrochemical deposition. The materials were characterised by scanning electron microscopy (SEM) and HA deposition was characterised by elemental analysis and X-ray diffractometry (XRD). In vitro assays were carried out to quantify the effects of HA coating on human keratocyte adhesion. Cellular cytokine production was used to assess inflammatory potential. HA coating significantly increased keratocyte adhesion to the carbon matrix (p<0.01). The materials did not induce excessive cytokine production by the adherent keratocytes. In addition, the matrices themselves adsorbed significant levels of the cytokine IL-8 (p<0.05). The results indicate that HA coated carbon matrices provide a suitable environment to enhance in-growth of corneal cells without inducing further inflammation. The materials may also suppress excessive inflammation by adsorption of the cytokine IL-8 into the porous, internal carbon structure.