Paclitaxel-coated stents to prevent hyperplastic proliferation of ureteral tissue: from in vitro to in vivo.

Ureteric stents have become an indispensable tool in the armamentarium of every urologist. However, they carry their own morbidity resulting mostly from infectious or abacterial fouling and biofilm formation, and/or urothelial hyperplastic reaction. All of these may interact and lead to clinical complications. Many different stent designs and coatings have been proposed. In this study, we focused on the effect of paclitaxel-coated stents on hyperplastic proliferation of ureteral tissue, using as example anastomotic strictures after ureteroureterostomy in a rat model. Human urothelial cells (SV-HUC-1) were used to determine paclitaxel dosages in vitro. Polyurethane stents were coated with a paclitaxel containing biodegradable polymer and studied in a ureteroureterostomy rat model. 48 male 9-week-old Sprague-Dawley rats underwent either sham surgery (n = 16) or ureteroureterostomy with sutured anastomosis, and consecutive stenting with either a paclitaxel-coated or an uncoated stent (16 per group), respectively. The animals received daily intraperitoneal injections of 5-bromo-2-deoxyuridine (20 mg/ml, 100 mg/kg body weight) during the first eight postoperative days, and were sacrificed on day 28. Healing of the ureteral anastomosis and proliferation of urothelial cells was examined histologically and immunohistochemically. In vitro, a concentration of 10 ng/mm2 paclitaxel can be considered as non-toxic, while still exerting an anti-proliferative effect on urothelial cells. Histologically, typical wound healing processes were seen at the site of the ureteral anastomosis in vivo. Proliferation of urothelial cells was significantly lower in animals with paclitaxel-coated stents compared to those with uncoated stents (LI 41.27 vs. 51.58, p < 0.001). Our results indicate that stenting of ureteral anastomoses with paclitaxel-coated stents can reduce hyperplastic proliferation of ureteral tissue. Paclitaxel-coated stents thus might be able to prevent not only scar-induced postoperative stenosis after reconstructive surgery, but also hyperplastic urothelial reaction in non-anastomotic stent patients as part of their inflammatory response to the foreign material.

Simulation of actin distribution of osteoblasts on titanium pillar arrays using a bio-chemo-mechanical model.

A numerical model for the adhesion of osteoblasts on titanium micropillar structures is suggested, and a function representing the concentration level of the adhesion on the pillars is constructed based on experimental observation. The introduction of this function helps a well-known bio-chemo-mechanical model to better predict the formation of actin in osteoblasts when they are laid on arrays of titanium micro-pillars of various size attached to silicon substrate. A parameter study suggests that each pillar is associated with a different pattern of adhesion. Our finding emphasises a capability of the bio-chemo-mechanical model that it can well explain the strong influence of the boundary condition on the formation of actin within the cells.

Aging effects of plasma polymerized ethylenediamine (PPEDA) thin films on cell-adhesive implant coatings.

Thin plasma polymer films from ethylenediamine were deposited on planar substrates placed on the powered electrode of a low pressure capacitively coupled 13.56 MHz discharge. The chemical composition of the plasma polymer films was analyzed by Fourier Transform Infrared Reflection Absorption Spectroscopy (FT-IRRAS) as well as by X-ray photoelectron spectroscopy (XPS) after derivatization of the primary amino groups. The PPEDA films undergo an alteration during the storage in ambient air, particularly, due to reactions with oxygen. The molecular changes in PPEDA films were studied over a long-time period of 360 days. Simultaneously, the adhesion of human osteoblast-like cells MG-63 (ATCC) was investigated on PPEDA coated corundum blasted titanium alloy (Ti-6Al-4V), which is applied as implant material in orthopedic surgery. The cell adhesion was determined by flow cytometry and the cell shape was analyzed by scanning electron microscopy. Compared to uncoated reference samples a significantly enhanced cell adhesion and proliferation were measured for PPEDA coated samples, which have been maintained after long-time storage in ambient air and additional sterilization by γ-irradiation.

Evaluation of antimicrobial effects of novel implant materials by testing the prevention of biofilm formation using a simple small scale medium-throughput growth inhibition assay.

Staphylococcal colonization of implants is a serious complication of orthopaedic surgery. Anti-infectious modification of implant surfaces may serve to prevent bacterial colonization. The authors set out to develop an in vitro test system for the analysis of prevention of biofilm formation by Staphylococcus epidermidis and Staphylococcus aureus on implant materials. Biofilm growth was monitored over 10 days on titanium disks in order to develop appropriate test parameters. Bacterial cell counts following ultrasonic treatment of the colonized samples were compared with scanning electron microscope images of the specimens. Copper ion containing surfaces (ie copper [Cu] and inter-metallic Ti-Cu films) were used for growth inhibition assays: copper ion releasing specimens led to reduced bacterial numbers in biofilms and decreased bacterial persistence in the model used. The assay used represents an inexpensive and quick in vitro screen for the antibacterial effects of novel implant surface materials.

In vivo investigation of the inflammatory response against allylamine plasma polymer coated titanium implants in a rat model.

Titanium (Ti) is an established biomaterial for bone replacement. However, facilitation of osteoblast attachment by surface modification with chemical groups could improve the implant performance. Therefore, this study aimed to evaluate the effect of a plasma polymerized allylamine (PPAAm) layer on the local inflammation in a rat model. Three series (RM76AB, RM78AB, RM77AB) of PPAAm-treated Ti plates were prepared using different plasma conditions. Twelve male LEW.1A rats received one plate of each series and one uncoated control plate implanted into the back musculature. After 7, 14 and 56 days, four rats were euthanized to remove the implants with surrounding tissue. Total monocytes/macrophages, tissue macrophages, T-cells and MHC-class-II-positive cells were morphometrically counted. On day 14, the macrophage/monocyte number was significantly higher for the controls than for the PPAAm samples. On day 56, the RM76AB and RM78AB samples had significantly lower numbers than RM77AB and the controls. The same was found for the tissue macrophages. No change over time and no differences between the implants were found for the T-cells. For the number of MHC-class-II-positive cells, a significant decrease was found only for the RM78AB implants between day 14 and day 56. Physico-chemical analysis of the PPAAm implants revealed that the RM77AB implants had the lowest water absorption, the highest nitrogen loss and the lowest oxygen uptake after sonication. These results demonstrate that the PPAAm samples and the controls were comparable regarding local inflammation, and that different plasma conditions lead to variations in the material properties which influence the tissue reaction.

Mathematical correlation between biomaterial and cellular parameters--critical reflection of statistics.

For mathematical modelling of the biomaterial-cell contact, it is necessary to find both parameters characterizing physical and chemical properties of the material surface and also such describing the reaction of the adhering cells. Only those material and cell parameters that correlate with each other are applicable to model this contact mathematically. Only few papers are dealing with this special problem. The aim of this paper is to present results of physical/chemical and biological investigations made on differently modified rough titanium implant surfaces in order to find out only the correlating parameters. Furthermore we discuss several ways to apply statistical methods to the correlation problem. Only few ones of all investigated parameters both on material and on cellular side were applicable for correlation. For example we found in our studies that fractal structure parameter topothesy has influence on the spreading behaviour of the osteoblastic cells. However the value of the correlation coefficient and its statistical significance heavily depend on the method of averaging the available data. Especially the biological data (spreading area) were afflicted with relatively high error up to 30%. Averaging of this data masks the true facts. That is why the correlation coefficient considerably decreases if the biological parameters are not averaged. On the other hand, the statistical reliability increases due to the higher number of investigated cases. Critical error discussion is necessary in statistical correlation between material and biological parameters. Often the results are heavily influenced by the statistical handling of data, especially if only few data are available. May be that new unconventional methods like bootstrap method can show a way out of this dilemma.

Spark plasma sintering synthesis of porous nanocrystalline titanium alloys for biomedical applications.

The reason for the extended use of titanium and its alloys as implant biomaterials stems from their lower elastic modulus, their superior biocompatibility and improved corrosion resistance compared to the more conventional stainless steel and cobalt-based alloys [Niinomi, M., Hattori, T., Niwa, S., 2004. Material characteristics and biocompatibility of low rigidity titanium alloys for biomedical applications. In: Jaszemski, M.J., Trantolo, D.J., Lewandrowski, K.U., Hasirci, V., Altobelli, D.E., Wise, D.L. (Eds.), Biomaterials in Orthopedics. Marcel Dekker Inc., New York, pp. 41-62]. Nanostructured titanium-based biomaterials with tailored porosity are important for cell-adhesion, viability, differentiation and growth. Newer technologies like foaming or low-density core processing were recently used for the surface modification of titanium alloy implant bodies to stimulate bone in-growth and improve osseointegration and cell-adhesion, which in turn play a key role in the acceptance of the implants. We here report preliminary results concerning the synthesis of mesoporous titanium alloy bodies by spark plasma sintering. Nanocrystalline cp Ti, Ti-6Al-4V, Ti-Al-V-Cr and Ti-Mn-V-Cr-Al alloy powders were prepared by high-energy wet-milling and sintered to either full-density (cp Ti, Ti-Al-V) or uniform porous (Ti-Al-V-Cr, Ti-Mn-V-Cr-Al) bulk specimens by field-assisted spark plasma sintering (FAST/SPS). Cellular interactions with the porous titanium alloy surfaces were tested with osteoblast-like human MG-63 cells. Cell morphology was investigated by scanning electron microscopy (SEM). The SEM analysis results were correlated with the alloy chemistry and the topographic features of the surface, namely porosity and roughness.

The in vitro effects of H-89, a specific inhibitor of protein kinase A, in the human colonic carcinoma cell line Caco-2.

SUMMARY: H-89 is a compound characterized in vitro as a potent and selective inhibitor of protein kinase A. In the present study, we observed that H-89 induced morphological transformation and caused growth inhibition of the human colon cancer cell line Caco-2 in a dose-dependent manner. However, another protein kinase A inhibitor, H-8, had no effect on Caco-2 cells. To evaluate the possible molecular mechanism of H-89-evoked effects in Caco-2 cells, we analysed the capacity of H-89 to regulate the protein kinase B (Akt/PKB) signalling pathway. H-89 treatment led to an activation of Akt/PKB in Caco-2 cells. This activation was phosphatidylinositol 3 (PI3)-kinase-dependent and promoted survival of Caco-2 cells because the PI3 kinase inhibitor LY294002 inhibited the Akt/PKB activation and induced apoptosis of Caco-2 cells. To test whether Akt/PKB activity promoted resistance to H-89-induced effects, LY294002 was added in combination with H-89. LY294002 greatly potentiated the H-89-induced growth inhibition and apoptosis of Caco-2 cells. These results suggest that the H-89-induced growth inhibition of Caco-2 cells is associated with phosphorylation of Akt/PKB protein and that the cells become more sensitive to H-89 and die by apoptosis upon inhibition of the PI3K/Akt pathway.

[Fibronectin-chemotaxis and collagen-gel contraction of the palmar aponeurosis in morbus dupuytren]. / Fibronektin-Chemotaxis und Kollagengelkontraktion von Fibroblasten aus der Palmaraponeurose beim Morbus Dupuytren.

Causes for Morbus Dupuytren (MD) on the cellular level are largely unknown. We have studied chemotaxis and collagen-gel contraction of cultivated cells from nodules and cords of Morbus Dupuytren patients and normal palmar aponeurosis. The cells of the seized tissue showed an increased chemotaxis in gradients of the chemo-attractant fibronectin. Furthermore embedded into collagen-gels cells from MD patients especially from the region of cords to the skin had an enhanced ability to contract a three-dimensional collagen network compared to those originating from the center of the nodules and control palmar aponeurosis. One reason for the increase of chemotactic motion and the ability to contract gels could be a surplus of receptors for extracellular matrix proteins (integrins) on the surface of cells from seized tissues. Flow cytometry of cells fluorimetrically stained for beta 1 -, alpha 2 - and alpha 3 -integrins displayed no differences in the quantity of these main cell surface receptors.

Cell-extracellular matrix interaction and physico-chemical characteristics of titanium surfaces depend on the roughness of the material.

The interaction of cells with the extracellular matrix at the interface of an implant determines the biology of cells and tissues. We analysed components of cell adhesion and measured physico-chemical characteristics of structural modifications of titanium surfaces: polished, machined, glass particle-blasted, corundum-blasted, vacuum plasma-sprayed. Scanning electron microscopy and profilometry revealed a differentiated topography from smooth to rough surfaces, respectively. Osteoblastic MG-63 cells showed an increased spreading on surfaces with low roughness, although without a straight correlation with the surface topography. Integrin expression was increased on structured surfaces compared with polished material, and the organization of the actin cytoskeleton and fibronectin was impaired on extremely rough surfaces. Electrochemical methods, especially the electrochemical impedance spectroscopy (EIS) was used to evaluate physico-chemical characteristics, and the impedance curves revealed a dependence on the roughness of the material surfaces. Further analyses of the EIS results were performed using equivalent circuits which model the electrical flow through the interface. First indications for a correlation between parameters from the equivalent circuits with surface properties were obtained which promise a relevance for the biological response of the cells.

Effects of lectins on CCK-8-stimulated enzyme secretion and differentiation of the rat pancreatic cell line AR42J.

INTRODUCTION: The peptide hormone cholecystokinin (CCK) plays an important role in the gastrointestinal tract. The rat pancreatic CCK receptor is a highly glycosylated membrane receptor that is able to bind to plant lectins such as wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA-I). AIMS AND METHODOLOGY: We used both lectins to block this receptor for studying the pathophysiologic relevance of its oligosaccharide side chains. In the present study we investigated the influence of WGA and UEA-I on CCK-8-induced alpha-amylase secretion of the rat pancreatic tumor cell line AR42J, which expresses both CCK-A and CCK-B receptors. RESULTS: Under the influence of WGA (25 microg/mL), the alpha-amylase release was reduced by 25% after 30 minutes compared with the hormone-stimulated controls. UEA-I (25 microg/mL) caused a reduction of 20%. The simultaneous application of the lectins with CCK antagonists L 364,718 or L 365,260 led to a reduction of secretion, but the assignment to CCK-A or CCK-B receptors was not possible. CONCLUSION: In long-term studies, both lectins revealed no toxic or apoptosis-inducing effects. On the contrary, WGA showed an inhibitory effect on cell proliferation and led to improved differentiation of cells.

Inhibition of lens epithelial cell adhesion by the calcium antagonist Mibefradil correlates with impaired integrin distribution and organization of the cytoskeleton.

BACKGROUND: Posterior capsule opacification is the most common complication of primary cataract surgery and is caused by migration and proliferation of residual lens epithelial cells onto the posterior capsule. Interfering with the mechanisms involved in cell adhesion is a suitable approach to prevent posterior capsule opacification. METHODS: Mibefradil, a T-type calcium-channel blocker, was used to examine the influence on adhesion-mediating mechanisms in human lens epithelial cells derived from cataract surgery. Adhesion was evaluated by light microscopy on the anterior capsules. Expression of integrin receptors was studied by flow cytometry. The influence on the distribution of integrin receptors on the cell surface and the organization of the cytoskeleton was examined by immunofluorescence using a confocal microscope. RESULTS: The calcium-channel blocker Mibefradil inhibited cell adhesion on the anterior capsule wall at concentrations between 10 and 100 pNM. The cells expressed the integrin subunits beta1 and alpha3. Mibefradil distinctly impaired the distribution of these integrins on the cell surface in culture. The cells express the cytoskeletal components actin, vimentin and, very weakly, cytokeratin. The structural organization of the actin filaments and vimentin was strongly disrupted with pronounced fragmentation of the actin filaments in the presence of the calcium-channel blocker. CONCLUSION: The results suggest that the inhibition of cell adhesion by the calcium-channel blocker Mibefradil involves the impairment of integrin-mediated mechanisms. The use of this calcium antagonist appears to be a suitable therapeutic approach to prevent posterior capsule opacification.

Structural alterations of adhesion mediating components in cells cultured on poly-beta-hydroxy butyric acid.

Polymers may serve as a biodegradable material in tissue engineering. To assess the biocompatibility of poly-beta-hydroxy butyric acid (PHB), we studied the structural organization of cellular molecules involved in adhesion using osteoblastic and epithelial cell lines. On PHB, both cell lines revealed a rounded cell shape due to reduced spreading. The filamentous organization of the actin cytoskeleton was impaired. In double immunofluorescence analyses we demostrated that the colocalization of the fibronectin fibrils with the actin filaments was lost in cultures on PHB. Similarly, collagen II distribution was altered, whereas the organization of collagen I was not obviously affected. Further evidence for impaired structural organization was obtained for the beta1-integrin receptor and vinculin which mediate the interaction of the cytoskeleton with the extracellular matrix. In confluent epithelial cells, the tight junction protein ZO-1 showed a larger lateral extension in the cell-cell contacts when cells were grown on PHB. Because structural organization of components which mediate cell-matrix and cell-cell adhesion controls cell physiology these parameters could be a sensitive indicator for the biocompatibility of implant materials.

Use of human preconditioned phagocytes for extracorporeal immune support: introduction of a concept.

Neutrophils are critical effector cells in humoral and innate immunity and play a vital role in phagocytosis and bacterial killing. If they and/or their specific functions are lacking, then immunoparalysis may occur, and severe diseases like systemic inflammatory response syndrome (SIRS) or sepsis can take a fatal course. In this paper, we discuss the possibility of using preconditioned cells in an extracorporeal biohybrid immune support system. A human promyelocytic cell line was stimulated for different times with all-trans retinoic acid. The resulting cells displayed major signs and functions of mature neutrophilic granulocytes including oxygen radical production, phagocytosis of living and dead Escherichia coli, Staphylococcus aureus, Candida albicans, intracellular killing, and interleukin production. The cells can be expanded to yield a sufficient cell mass, and subsequent prestimulation results in an expression of specific neutrophil functions. Extracorporeal bioreactor experiments seem to be feasible to test the benefit in immunoparalysis-associated diseases like SIRS or sepsis.

Dielectric changes in membrane properties and cell interiors of human mesothelial cells in vitro after crocidolite asbestos exposure.

Asbestos induces cytogenetic and genotoxic effects in cultured cell lines in vitro. For further investigations of the fiber-induced cellular changes, electrorotation (ROT) measurements can be used to determine early changes of surface properties and dielectric cellular changes. In the present study, human mesothelial cells (HMC) were exposed to nontoxic concentrations of crocidolite asbestos (1 microg/cm(2)) for 12, 24, 30, 50, and 72 hr, and were investigated for changes in dielectric properties, morphologic and biochemical changes using ROT measurements, electron microscopy, and flow cytometry, respectively. The results of ROT measurements revealed slightly increased internal conductivity and decreased membrane conductance of HMC during the first 12 hr of exposure to crocidolite. This may be due to functional changes of ion channels of the cellular membrane. However, after exposures of >= 30 hr, reduced internal conductivity and increased membrane conductance of HMC occurred. These effects may be caused by permeabilization of the cell membrane and the leakage of ions into the surrounding medium. The membrane capacitance of HMC is always decreased during exposure of cells to crocidolite fibers. This decreased membrane capacitance may result from the observed reduction in the number of microvilli and from the shrinkage of cells as observed by electron microscopy and flow cytometry. Changes in composition of the plasma membrane were also observed after the labeling of phosphatidylserines (PS) on the cell surface. These observed changes can be related to apoptotic events. Whereas during the first 50 hr of exposure only a small number of HMC with increased exposure of PS on the cell surface was detected by flow cytometry, the dielectric properties of HMC showed marked changes during this time. Our results show that surface property changes of the cellular membrane of HMC as well as interior dielectric changes occur after the exposure of cells to crocidolite fibers. The observed changes are discussed in terms of complex combined cellular effects after amphibole asbestos exposure.

Analysis of spatial distributions of cellular molecules during mechanical stressing of cell surface receptors using confocal microscopy.

Confocal laser scanning microscopy represents a suitable technique to study the localization of cellular components in three dimension. The authors used this technique to analyse cellular events related to mechanical stimulation of integrin receptors on the cell surface. By performing optical sections the distribution of integrin receptors on the apical surface of an osteoblastic cell was determined. Concerning intracellular compartimentalization of signal transduction events, it was demonstrated that mechanical stimulation of integrins induced their linkage to the cytoskeleton. Cytoskeletally associated proteins like vinculin and talin accumulated in the vicinity of the site where the mechanical stress was applied to integrins on the cell surface. Optical sections revealed that clustering of these proteins proceeded to the base of the cell with gradually decreasing extent. In summary, it was demonstrated that the local distribution of cellular components is an important factor in mechanically induced signal transduction.

Hepatocyte growth factor enables enhanced integrin-cytoskeleton linkage by affecting integrin expression in subconfluent epithelial cells.

Hepatocyte growth factor (HGF) exerts mitogenic and motogenic effects in different cell types. In the epithelial cell line mHepR1 we found that HGF induced pronounced alterations in cell morphology and promoted cell adhesion and spreading. To analyze the mechanisms how HGF affects these integrin mediated functions we studied the physical linkage of integrins with the cytoskeleton. First we found that HGF increased the expression of different integrin subunits in subconfluent cells and influenced the distribution of integrins on the cell surface. To address the physical association of integrins with the cytoskeleton we analyzed Triton X-100-extracted cell fractions using flow cytometry. Here we show that cultivation of the cells with HGF for 24 h prior to integrin cross-linking significantly enhanced the cytoskeletal anchorage of integrins. To further find out whether HGF directly induces an integrin-cytoskeleton link without subsequent cross-linking we added HGF to suspended cells but failed to detect cytoskeletally immobilized integrins in the detergent-insoluble cell fraction which could be related to the absence of a calcium response induced by HGF. Overall, the results indicate that HGF promotes the physical linkage of integrins to the cytoskeleton which requires additional stimulation of integrins.

Increased cytosolic Ca2+ amplifies oxygen radical-induced alterations of the ultrastructure and the energy metabolism of isolated rat pancreatic acinar cells.

BACKGROUND: Oxygen radicals have been implicated as important mediators in the early pathogenesis of acute pancreatitis, but the mechanism by which they produce pancreatic tissue injury remains unclear. We have, therefore, investigated the effects of oxygen radicals on isolated rat pancreatic acinar cells as to the ultrastructure, cytosolic Ca2+ concentration and energy metabolism. METHODS: Acinar cells were exposed to an oxygen radical-generating system consisting of xanthine oxidase, hypoxanthine and chelated iron ions. Cell injury was assessed by LDH release and electron microscopy. Cytosolic Ca2+ levels and mitochondrial membrane potential were determined by flow cytometry; adenine nucleotide concentrations by HPLC. Mitochondrial dehydrogenase activity was measured by spectrophotometric assay. RESULTS: Oxygen radicals damaged the plasma membrane as shown by a 6-fold LDH increase in the incubation medium within 180 min. At the ultrastructural level, mitochondria were the most susceptible to oxidative stress. In correlation to the pronounced mitochondrial damage, the mitochondrial dehydrogenase activity declined by 70%, whereas the mitochondrial membrane potential was enhanced by 27% after 120 min. Together this may cause the 85% decrease in the ATP concentration and the corresponding increase in ADP/AMP observed in parallel. In addition, an immediate 26% increase in cytosolic Ca2+ was found, a change which could be inhibited by BAPTA, reducing cellular damage. CONCLUSION: Cytosolic Ca2+ synergizes with oxygen radicals causing alterations of the ultrastructure and energy metabolism of acinar cells which might contribute to the cellular changes found in early stages of acute pancreatitis.