Human corneal endothelial cell sheets for transplantation: thermo-responsive cell culture carriers to meet cell-specific requirements.

Corneal endothelial diseases lead to severe vision impairment, motivating the transplantation of donor corneae or corneal endothelial lamellae, which is, however, impeded by endothelial cell loss during processing. Therefore, one prioritized aim in corneal tissue engineering is the generation of transplantable human corneal endothelial cell (HCEC) layers. Thermo-responsive cell culture carriers are widely used for non-enzymatic harvest of cell sheets. The current study presents a novel thermo-responsive carrier based on simultaneous electron beam immobilization and cross-linking of poly(vinyl methyl ether) (PVME) on polymeric surfaces, which allows one to adjust layer thickness, stiffness, switching amplitude and functionalization with bioactive molecules to meet cell type specific requirements. The efficacy of this approach for HCEC, which require elaborate cell culture conditions and are strongly adherent to the substratum, is demonstrated. The developed method may pave the way to tissue engineering of corneal endothelium and significantly improve therapeutic options.

Biological relevance of ion energy in performance of human endothelial cells on ion-implanted flexible polyurethane surfaces.

To improve the biocompatibility of polyurethane (PUR), we modified the surface by irradiation with different ions (Carbon; C, Oxygen; O, Nitrogen; N, or Argon; Ar) at 0.3-50 keV energy and doses of 1,00E+13 - 1,00E+15 ions/cm(2). The effects of ion implantation using different ion energies and densities were observed on adhesion, proliferation, and viability of human umbilical vein endothelial cells (HUVECs). The long-term in vitro stability of ion-implanted PUR was also investigated. Ion irradiation moderately affected the surface roughness (R(a)), but strongly enhanced the work of adhesion (W(a)). Cell adhesion was markedly improved on O-, N-, and Ar-, but not on C-implanted PUR surfaces. Medium ion energies and lower ion doses produced the best HUVEC attachment and proliferation, indicating the importance of choosing the proper range of energy applied during ion irradiation. In addition, apoptosis rates were significantly reduced when compared with unmodified PUR (uPUR). N implantation significantly protected the surface, although C implantation led to stronger surface erosions than on uPUR. In total, ion implantation on flexible PUR surfaces strongly improved the material surface characteristics and biocompatibility. Electron beam ion implantation within an appropriate energy window is thus a key to improving flexible PUR surfaces for clinical use to support endothelial cell performance. Thus, it can contribute to designing small-diameter grafts, which are in great demand, towards vascular tissue engineering applications.

Physical vapor deposition of zirconium or titanium thin films on flexible polyurethane highly support adhesion and physiology of human endothelial cells.

The aim of this study was to develop and characterize novel metal-polymer constructs to improve the biocompatibility of flexible but hydrophobic polyurethane (PUR) implants. Using a physical vapor deposition (PVD) technique, thin films (< or =100 nm) of zirconium (Zr) or titanium (Ti) were deposited on the polyurethane surface. Both coatings displayed good stability when subjected to cross-cutting test and especially Zr showed only minor and superficial cracks in the scanning electron microscopy analysis. PVD coating resulted in significantly lowered contact angles and the standard surface free energy of wetting (Delta(wet)G degrees ) turned to more favorable negative values (Ti: -40; Zr: -30; untreated PUR (uPUR): +10.1 mN/m). This may lead to the highly enhanced adhesion and proliferation properties observed with human umbilical vein endothelial cells (HUVECs). In addition, the novel coatings had no toxic effect and even drastically reduced apoptosis rates of HUVECs. Cell morphology, nitric oxide production, and mitochondrial membrane potential--both at static and flow conditions--were superior compared with uPUR, thus demonstrating intact physiological functions. Therefore, we suggest that combining PUR as a flexible material with a thin coating of Zr or Ti as the improved biocompatible surface may have advantages for use, for example, vascular graft material.

Neurodegenerative diseases of the retina and potential for protection and recovery.

Recent advances in our understanding of the mechanisms in the cascade of events resulting in retinal cell death in ocular pathologies like glaucoma, diabetic retinopathy and age-related macular degeneration led to the common descriptive term of neurodegenerative diseases of the retina. The final common pathophysiologic pathway of these diseases includes a particular form of metabolic stress, resulting in an insufficient supply of nutrients to the respective target structures (optic nerve head, retina). During metabolic stress, glutamate is released initiating the death of neurones containing ionotropic glutamate (N-methyl-D-aspartat, NMDA) receptors present on ganglion cells and a specific type of amacrine cells. Experimental studies demonstrate that several drugs reduce or prevent the death of retinal neurones deficient of nutrients. These agents generally block NMDA receptors to prevent the action of glutamate or halt the subsequent pathophysiologic cycle resulting in cell death. The major causes for cell death following activation of NMDA receptors are the influx of calcium and sodium into cells, the generation of free radicals linked to the formation of advanced glycation endproducts (AGEs) and/or advanced lipoxidation endproducts (ALEs) as well as defects in the mitochondrial respiratory chain. Substances preventing these cytotoxic events are considered to be potentially neuroprotective.

Vascular-adrenal niche--endothelial cell-mediated sensitization of human adrenocortical cells to angiotensin II.

Alterations in both vasculature and renin-angiotensin-aldosterone system are a consistent finding in the metabolic syndrome. Adrenal tissue is highly vascularized and encounters blood flow, exceeding by far the volume expected for its size. Endothelial cells in the adrenal vasculature are therefore a major cellular component of adrenocortical tissue. The aim of the study was to analyze the cellular interaction between endothelial and steroid producing cells, focusing on endothelial cell-factor-mediated activation of aldosterone synthesis. The interaction between human endothelial (HUVECs) cell-conditioned medium and human adrenocortical (NCI-H295R) cells IN VITRO induced a significant surge in aldosterone secretion. The endothelial cell-conditioned medium together with angiotensin II and forskolin also potentiated aldosterone release by 1.5-fold and 2.6-fold, respectively, while preincubation of NCI-H295R cells for 24 h with endothelial cell-conditioned medium enhanced and sensitized the response of NCI-H295R to subsequent angiotensin II and forskolin stimuli by 2.5-fold and 2.2-fold, respectively. The increase in aldosterone release after preincubation with endothelial cell-conditioned medium was sensitive to cycloheximide and KN-93. Cellular conditioning with endothelial-cell factors exerts a hitherto unknown paracrine regulation of aldosterone production in human adrenocortical cells. This interaction may contribute to altered basal aldosterone release and have a role in patients with hypertension.

Effects of different titanium alloys and nanosize surface patterning on adhesion, differentiation, and orientation of osteoblast-like cells.

To test nanosize surface patterning for application as implant material, a suitable titanium composition has to be found first. Therefore we investigated the effect of surface chemistry on attachment and differentiation of osteoblast-like cells on pure titanium prepared by pulsed laser deposition (TiPLD) and different Ti alloys (Ti6Al4V, TiNb30 and TiNb13Zr13). Early attachment (30 min) and alkaline phosphatase (ALP) activity (day 5) was found to be fastest and highest, respectively, in cells grown on TiPLD and Ti6Al4V. Osteoblasts seeded on TiPLD produced most osteopontin (day 10), whereas expression of this extracellular matrix protein was an order of magnitude lower on the TiNb30 surface. In contrast, expression of the corresponding receptor, CD44, was not influenced by surface chemistry. Thus, TiPLD was used for further experiments to explore the influence of surface nanostructures on osteoblast adhesion, differentiation and orientation. By laser-induced oxidation, we produced patterns of parallel Ti oxide lines with different widths (0.2-10 microm) and distances (2-20 and 1,000 microm), but a common height of only 12 nm. These structures did not influence ALP activity (days 5-9), but had a positive effect on cell alignment. Two days after plating, the majority of the focal contacts were placed on the oxide lines. The portion of larger focal adhesions bridging two lines was inversely related to the line distance (2-20 microm). In contrast, the portion of aligned cells did not depend on the line distance. On average, 43% of the cells orientated parallel towards the lines, whereas 34% orientated vertically. In the control pattern (1,000 microm line distance), cell distribution was completely at random. Because a significant surplus of the cells preferred a parallel alignment, the nanosize difference in height between Ti surface and oxide lines may be sufficient to orientate the cells by contact guiding. However, gradients in electrostatic potential and surface charge density at the Ti/Ti oxide interface may additionally influence focal contact formation and cell guidance.

[Characteristic features of optic nerve ganglion cells and approaches for neuroprotection. From intracellular to capillary processes and therapeutic considerations]. / Besonderheiten der Optikusganglienzellen und Ansätze zur Neuroprotektion. Von intrazellulären Vorgängen zur Kapillare bis zu therapeutischen Uberlegungen.

In many diseases associated with deterioration of the visual field and eyesight, optic nerve ganglion cells are at the highest risk. The clinical course of primary chronic open-angle glaucoma (PCOAG) is also determined by the degree of damage to these cells. Due to their anatomy, they are subject to extreme stress exerted by metabolic and microcirculatory forces. The interaction between hypoxia and metabolic stress leads to damage of the retinal ganglion cells. This is compounded by oxidative stress and age-dependent increase of advanced glycation end products. The following contribution gives consideration to approaches for delaying ganglion cell death in PCOAG, e.g., with neuroprotective agents. Furthermore, agents that reduce calcium influx into the cells could prevent cell destruction. Likewise, NMDA receptor antagonists could be effective; however, considerable side effects are to be feared. Antioxidants are also attributed with theoretical impact in combating PCOAG by preventing apoptosis. Finally, the ideal glaucoma medication should be well tolerated when taken orally, prevent destruction of retinal ganglion cells, and possess a low side effect profile.

Collagen type I prevents glyoxal-induced apoptosis in osteoblastic cells cultured on titanium alloy.

Advanced glycation end products (AGEs) irreversibly cross-link proteins with sugars and accumulate at a higher age and in diabetes, processes which can interfere with the integration of implants into the tissue. Glyoxal is a highly reactive glycating agent involved in the formation of AGEs and is known to induce apoptosis, as revealed by the upregulation of caspase-3 and fractin (caspase-3 being a key enzyme activated during the late stage of apoptosis and fractin being a caspase-cleaved actin fragment). In this study, we investigated the influence of collagen type I coating on the cytotoxic effect of glyoxal on rat calvarial osteoblastic cells and on human osteosarcoma cells (Saos-2) grown on titanium alloy, Ti6Al4V. Activation of caspase-3 and fractin was measured by counting immunohistochemically stained cells and by flow cytometry with propidium iodide (detection of the apoptosis indicating a sub-G1 peak). Our results showed an increased number of apoptotic osteoblasts after incubation with glyoxal on Ti6Al4V discs. However, the number of apoptotic cells on collagen-coated titanium was significantly smaller than on uncoated titanium after the same treatment. The present findings demonstrate that osteoblasts treated with glyoxal undergo apoptosis, whereas collagen type I coating of titanium alloys (used for implants) has an antiapoptotic function.

Coenzyme Q10 reduces the toxicity of rotenone in neuronal cultures by preserving the mitochondrial membrane potential.

Defects in mitochondrial energy metabolism due to respiratory chain disorders lead to a decrease in mitochondrial membrane potential (DeltaPsim) and induce apoptosis. Since coenzyme Q10 (CoQ10) plays a dual role as an antioxidant and bioenergetic agent in the respiratory chain, it has attracted increasing attention concerning the prevention of apoptosis in mitochondrial diseases. In this study the potential of CoQ10 to antagonize the apoptosis-inducing effects of the respiratory chain inhibitor rotenone was explored by video-enhanced microscopy in SH-SY5Y neuroblastoma cells. The cationic fluorescent dye JC-1 which exhibits potential-dependent accumulation in mitochondria was used as an indicator to monitor changes in DeltaPsim. The relative changes in fluorescence intensity after incubation with rotenone for 15 minutes were calculated. Pre-treatment with CoQ10 (10 or 100 microM) for 48 h led to a significant reduction of rotenone-induced loss of DeltaPsim. These results suggest, that cytoprotection by CoQ10 may be mediated by raising cellular resistance against the initiating steps of apoptosis, namely the decrease of DeltaPsim. Whether these data may provide new directions for the development of neuroprotective strategies has to be investigated in future studies.

Intracellular changes in astrocytes and NG 108-15 neuroblastoma X glioma cells induced by advanced glycation end products.

INTRODUCTION: The extracellular effects caused by advanced glycation end products (AGEs) themselves formed by the Maillard-reaction from proteins and sugars, are well known. Less is known about the effects of AGEs on cellular function. Cellular dysfunctions accumulate with increasing age and can be found as a pathogenetic principle of different diseases where AGEs are involved. The causal mechanisms of these cellular dysfunctions are not fully understood. MATERIAL AND METHODS: AGEs effects were examined in cell culture using video microscopy, immunohistochemistry, flow cytometry and biochemical methods in neural (NG 108-15) and glial (primary culture of astrocytes) cells. RESULTS: AGEs can bind to the cell surface of neural and glial cells. AGEs induce an altered distribution of vesicles and a statistically significant reduction in the velocity of microvesicles. The proteins dynamin 2 and clathrin, which are an important part of the transport apparatus, show changes in intracellular distribution similar to that of microvesicles. The immunoreactivity of dynamin 2 in AGE-treated cells is increased after AGE-incubation. After incubation durations of 2 h, 6 h and 12 h we found an increase (maximum +34.6%) of the relative fluorescence intensities of dynamin 2 in the AGE-BSA treated cells in comparison to the control cells (BSA). Using flow cytometry we found a minimal increase (14.4%) in clathrin immunoreactivity after 2 h incubation with 500 nM AGE-BSA. The non-quantitative western blot showed no difference of the electrophoretic behaviour of both dynamin 2 and clathrin in AGE-BSA-incubated cells (2 h, 500 nM) and in control cells (2 h, 500 nM BSA-incubation). The incubation with AGE-BSA up to a concentration of 500 nM in culture was not followed by apoptotic or necrotic cell death. DISCUSSION: We could show defined effects on the cells induced by incubation with AGEs. One of the effects is a significant reduction of the velocity of microvesicles, the reason for this is unclear. It could be due to disturbed calcium metabolism or caused by microtubuli-changes. The aggregation of intracellular microvesicles could be the result of an altered binding-behaviour of the vesicles or the destruction of the transport apparatus. The proteins dynamin 2 and clathrin, which are involved in transport apparatus, show changes in intracellular distribution indicating a breakdown of the normal cellular distribution system. The current results may contribute to the understanding of the pathologic processes in the cell in Mb. Alzheimer and other neurodegenerative diseases, where AGEs-accumulation can be found in tissues.