Plasma Treatment of PDMS for Microcontact Printing (µCP) of Lectins Decreases Silicone Transfer and Increases the Adhesion of Bladder Cancer Cells.

The present study investigates silicone transfer occurring during microcontact printing (µCP) of lectins with polydimethylsiloxane (PDMS) stamps and its impact on the adhesion of cells. Static adhesion assays and single-cell force spectroscopy (SCFS) are used to compare adhesion of nonmalignant (HCV29) and cancer (HT1376) bladder cells, respectively, to high-affinity lectin layers (PHA-L and WGA, respectively) prepared by physical adsorption and µCP. The chemical composition of the µCP lectin patterns was monitored by time-of-flight secondary ion mass spectrometry (ToF-SIMS). We show that the amount of transferred silicone in the µCP process depends on the preprocessing of the PDMS stamps. It is revealed that silicone contamination within the patterned lectin layers inhibits the adhesion of bladder cells, and the work of adhesion is lower for µCP lectins than for drop-cast lectins. The binding capacity of microcontact printed lectins was larger when the PDMS stamps were treated with UV ozone plasma as compared to sonication in ethanol and deionized water. ToF-SIMS data show that ozone-based treatment of PDMS stamps used for µCP of lectin reduces the silicone contamination in the imprinting protocol regardless of stamp geometry (flat vs microstructured). The role of other possible contributors, such as the lectin conformation and organization of lectin layers, is also discussed.

The Effect of the Topmost Layer and the Type of Bone Morphogenetic Protein-2 Immobilization on the Mesenchymal Stem Cell Response.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) plays a key role in the stem cell response, not only via its influence on osteogenesis, but also on cellular adhesion, migration, and proliferation. However, when applied clinically, its supra-physiological levels cause many adverse effects. Therefore, there is a need to concomitantly retain the biological activity of BMP-2 and reduce its doses. Currently, the most promising strategies involve site-specific and site-directed immobilization of rhBMP-2. This work investigated the covalent and electrostatic binding of rhBMP-2 to ultrathin-multilayers with chondroitin sulfate (CS) or diazoresin (DR) as the topmost layer. Angle-resolved X-ray photoelectron spectroscopy was used to study the exposed chemical groups. The rhBMP-2 binding efficiency and protein state were studied with time-of-flight secondary ion mass spectrometry. Quartz crystal microbalance, atomic force microscopy, and enzyme-linked immunosorbent assay were used to analyze protein-substrate interactions. The effect of the topmost layer was tested on initial cell adhesion and short-term osteogenesis marker expression. The results show the highest expression of selected osteomarkers in cells cultured on the DR-ended layer, while the cellular flattening was rather poor compared to the CS-ended system. rhBMP-2 adhesion was observed only on negatively charged layers. Cell flattening became more prominent in the presence of the protein, even though the osteogenic gene expression decreased.

Effect of poly(tert-butyl methacrylate) stereoregularity on polymer film interactions with peptides, proteins, and bacteria.

The impact of polymer stereoregularity on its interactions with peptides, proteins and bacteria strains was studied for three stereoregular forms of poly(tert-butyl methacrylate) (PtBMA): isotactic (iso), atactic (at) and syndiotactic (syn) PtBMA. Principal component analysis of the time-of-flight secondary ion mass spectrometry data recorded for thin polymer films indicated a different orientation of ester groups, which in the case of iso-PtBMA are exposed away from the surface whereas for at-PtBMA and syn-PtBMA these are located deeper within the film. This arrangement of chemical groups modified the interactions of iso-PtBMA with biomolecules when compared to at-PtBMA and syn-PtBMA. For peptides, the affected interactions were explained by the preferential hydrogen bonding and electrostatic interaction between the exposed polar ester groups of iso-PtBMA and positively charged peptides. In turn, for protein adsorption no impact on the amount of adsorbed proteins was observed. However, the polymer stereoregularity influenced the orientation of immunoglobulin G and induced conformational changes in bovine serum albumin structure. Moreover, the impact of polymer stereoregularity occurred equally for their interactions with Gram-positive bacteria (S. aureus), which absorbed preferentially onto iso-PtBMA films as compared to two other stereoregularities.

Type of Inflammation Differentially Affects Expression of Interleukin 1ß and 6, Tumor Necrosis Factor-α and Toll-Like Receptors in Subclinical Endometritis in Mares.

Mares that fail to conceive or lose their embryos, without showing typical signs of clinical endometritis, should be suspected of subclinical endometritis (SE). In this study, the question was addressed: does SE fully activate selected mechanisms of innate immunity in mares? For this aim, expression of mRNAs for Toll-like Receptor 2 and 4 (TLR 2/4), interleukin 1ß (IL-1ß), interleukin 6 (IL-6) and tumor necrosis factor α (TNF) was examined in control mares versus either mares suffering from chronic endometritis (ChE) or subacute suppurative endometritis (SSE). The concentrations of IL-1ß, IL-6 and TNF-α in supernatants from endometrial tissue cultures after 4 h incubation were measured using the enzyme immunoassay (EIA) method. Eighty-two warmblood mares, of known breeding history, were enrolled in this study. Based on histopathological assessment, mares were classified as suffering from ChE, SSE or as being healthy. In addition, immuno-localization of both TLR2 and TLR4 as well as TNF-α was investigated in the equine endometria. The mRNA expression of TLR2 (P < 0.01), IL-1ß (P < 0.0001), IL-6 (P < 0.0001) and TLR4 and TNF (P < 0.05) was up-regulated in endometria of mares suffering from SSE compared with unaffected mares. Concentrations of IL-6 and TNF-α were increased only in mares exhibiting SSE, compared with unaffected (P < 0.01 for both) and ChE mares (P < 0.05 for both). Immuno-localization of TNF-α and TLRs was confirmed, both in unaffected and SE-affected endometria, and was present in the luminal and glandular epithelia and stromal cells. The severity of inflammation impacts the immune response and fosters activation of innate immunity mechanisms, as observed in the endometria of mares. The intracellular localization of TLRs and TNF-α in the endometria indicates a key role of endometrial epithelial and stromal cells in the immune response and inflammation.