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Objectives: A tight seal between the epithelium and the dental implant surface is required to prevent bacterial inflammation and soft tissue recession and therefore to demonstrate a long-term success. Surface hydrophilicity was recently shown to promote osseointegration. The aim of this study was to investigate the influence of surface hydrophilicity in combination with surface topography of Ti implant surfaces on the behavior and activation/differentiation of epithelial cells using a set of in vitro experiments mimicking the implant-soft tissue contact., Methods: Hydrophobic acid-etched (A) and coarse-grit-blasted, acid-etched (SLA) surfaces and hydrophilic acid-etched (modA) and modSLA surfaces were produced. The behavior of an oral squamous cell carcinoma cell line (HSC-2) grown on all surfaces was compared through determination of cell attachment and proliferation/viability (CCK-8 and MTT assay), time-lapse microscopy of fluorescence labeled cells and determination of gene expression by real time polymerase chain reaction., Results: Within the surfaces with similar wettability cell spreading and cell movements observed by time-lapse microscopy after one day of incubation were most pronounced on smoother (A and modA) surfaces compared to rougher (SLA and modSLA) surfaces. Within the surfaces with similar roughness the hydrophilic surfaces (modA and modSLA) showed more cell spreading and cell activity compared to the hydrophobic surfaces (A and SLA). The relative gene expressions of cytokeratin14, integrin α6, integrin β4, vinculin, transforming growth factor (TGF)-β, TGF-β1, and TGF-β3 were decreased in HSC-2 on all four types of Ti surfaces compared to control surfaces (tissue culture polystyrene; p<0.01) and there was no significant difference of gene expression on the four different implant-surfaces., Significance: We have demonstrated that for proliferation and spreading of HSC-2 cells the smoother and hydrophilic surface is optimal (modA). These results suggest that surface hydrophilicity might positively influence the epithelial seal around dental implants. All tested titanium surfaces downregulate cell attachment, cell proliferation, expression of adhesion promoters, and cytokines involved in wound healing in HSC-2 cells compared to control surfaces., (Copyright © 2012. Published by Elsevier Ltd.)

The crucial factor of metal implant ingrowth in the bone is the rapid cellular acceptance. Therefore, the knowledge about additionally used adhesion mechanisms of osteoblasts, like their negatively charged hyaluronan coat, generates new surface functionalization strategies. Here, titanium was coated with a very thin, adherent, cross-linked, pinhole- and additive-free allylamine plasma polymer layer (PPAAm) resistant to hydrolysis and delamination and equipped with a high density of positively charged amino groups. This plasma polymer-functionalization of titanium is advantageous concerning osteoblastic focal adhesion formation as vinculin and paxillin, actin cytoskeleton development and, in consequence in differentiated cell functions, compared to a pure titanium surface-but similar such as the collagen I bonded surface via a polyethylenglycol-diacid (PEG DA)-spacer.

Stress fibers (SFs), a contractile bundle of actin filaments, play a critical role in mechanotransduction in adherent cells; yet, the mechanical properties of SFs are poorly understood. Here, we measured tensile properties of single SFs by in vitro manipulation with cantilevers. SFs were isolated from cultured vascular smooth muscle cells with a combination of low ionic-strength extraction and detergent extraction and were stretched until breaking. The breaking force and the Young's modulus (assuming that SFs were isotropic) were, on average, 377 nN and 1.45 MPa, which were approximately 600-fold greater and three orders of magnitude lower, respectively, than those of actin filaments reported previously. Strain-induced stiffening was observed in the force-strain curve. We also found that the extracted SFs shortened to approximately 80% of the original length in an ATP-independent manner after they were dislodged from the substrate, suggesting that SFs had preexisting strain in the cytoplasm. The force required for stretching the single SFs from the zero-stress length back to the original length was approximately 10 nN, which was comparable with the traction force level applied by adherent cells at single adhesion sites to maintain cell integrity. These results suggest that SFs can bear intracellular stresses that may affect overall cell mechanical properties and will impact interpretation of intracellular stress distribution and force-transmission mechanism in adherent cells.

Topographically patterned surfaces are known to influence cellular behavior in a controllable manner. However, the relatively large surface areas (several cm2) required for many biomaterial applications are beyond the practical limits of traditional lithography. Langmuir-Blodgett lithography, a recently developed method, was used to fabricate regularly spaced grooves of different depths (50 and 150 nm) with a periodicity of 500 nm over several square centimeter on silicon surfaces. These topographies were transferred into polystyrene surfaces by means of nanoimprinting. Primary osteoblasts were cultured on the patterned polymer surfaces. They were observed to align, elongate and migrate parallel to the grooves. The combination of Langmuir-Blodgett lithography with nanoimprinting enables the fabrication of large, nanostructured surface areas on a wide spectrum of different biomaterials. Osteoblasts show a significant anisotropic behavior to these surfaces, which can enhance cell settlement on the surface or be used to direct tissue generation on the biomaterial interface.

Objectives: To study the presence of intercellular adhesion molecule-1 (ICAM-1), P-selectin, and E-selectin, as well as the cytoskeletal components talin and vinculin that bind to cellular adhesion molecules (CAMs), in bladder biopsies from patients with interstitial cystitis (IC) and controls. IC is a sterile, bladder disorder characterized by urinary frequency and pelvic floor pain. The pathologic bladder findings include defective urothelium, activated mast cells, and variable inflammation. Mast cells can induce the expression of CAMs necessary for initiation of inflammation., Methods: Fresh frozen biopsies were analyzed immunocytochemically from 2 female normal bladders, 10 female IC bladders, 1 clear margin of transitional cell carcinoma of female bladder, 1 normal foreskin, 1 transitional cell carcinoma of foreskin, and 1 inflamed male finger., Results: Of the 10 IC samples, 9 were positive for ICAM-1, 6 for P-selectin, 6 for vinculin, 5 for talin, and 4 for E-selectin, all exclusively perivascular. Both normal bladders were negative for ICAM-1 and P-selectin and faintly positive for E-selectin, and one was weakly positive for talin and vinculin; the normal foreskin was negative. The "control" samples from the transitional cell carcinoma of the bladder and foreskin, as well as the inflamed finger skin, were positive only for ICAM-1. An increased number of activated mast cells associated with ICAM-1 was noted in IC., Conclusions: These results showed that ICAM-1 is expressed in IC, with variable expression of the other markers studied, supporting the different degrees of bladder inflammation noted in patients with IC.

Objectives: To characterize endothelial cell-to-cell junctions in the sinusoids and microvasculature of the corpus cavernosum., Methods: Corporal tissue was obtained from 6 potent human subjects, cut into 5-microm cryosections, and double-labeled with consecutive applications of primary and secondary antibodies. Laser scanning confocal microscopy identified subcellular localization of endothelial anchoring and adhesion molecules. Fluorescence intensity was rated as strong, weak, or absent by two observers., Results: The cavernosal endothelial adherens junction was composed of vascular endothelial cadherin, alpha-catenin, plakoglobin, vinculin, and the regulatory proteins beta-catenin and ZO-1. Adherens junctions in sinusoids were elongated, redundant, and narrow versus short, dense, linear cell-to-cell contacts in small arterioles and venules. Vinculin expression along the basal interface of the endothelium and stroma was weak in the sinusoids and strong in the arterioles. Definite sinusoidal expression of CD31 and CD34 was noted. P-selectin was only expressed within the cavernosal microvessels., Conclusions: The regulatory and structural proteins extending from vascular endothelial cadherin provide immunohistochemical evidence of a role for adherens junctions in cavernosal endothelial barrier function and cellular homeostasis. The sinusoidal endothelium has a unique junctional phenotype consistent with its blood trapping function. Differential expression of functional proteins in sinusoidal and microvascular endothelium may reflect segmental variation in hemodynamic exposure to pressure, stretch, or flow.

The interactions between an implant material and the surrounding tissue are of a complicated nature, and the initial attachment of cells to the surface is important in determining the implant success. HAPEX has been developed as a second-generation orthopaedic biomaterial, with both mechanical and biological characteristics that make it suitable for bone augmentation. Further optimisation of the material is being continued to increase the attachment of osteoblasts coupled with improving mechanical characteristics, so it may be used in load bearing applications. It has been previously observed that polishing followed by roughening the surface of HAPEX enhances osteoblast proliferation and phenotype. This article discusses the recruitment of primary human osteoblast cells onto the optimised surface, by examining morphology and cytoskeletal changes using scanning electron microscopy and confocal laser scanning microscopy. The results show that the cells attach in greater numbers to the optimised surface, and develop notably faster, than cells on machined HAPEX.

Understanding the cellular basis of osteoblastic cell-biomaterial interaction is crucial to the analysis of the mechanism of osseointegration, a requirement of long-term orthopedic implant stability. Clinically, the amount of bone ingrowth is variable, and cellular parameters that influence ingrowth have yet to be clearly determined. In this study, two clinically relevant orthopedic alloys, titanium Ti6A14V (Ti) and cobalt-chrome-molybdenum (CC), were used for a comparative analysis of primary human osteoblastic cell adhesion and spreading, where cell adhesion represents the initial interaction between cellular elements and the biomaterial surface. The kinetic profile of adhesion revealed enhanced cell attachment upon rough Ti surfaces relative to rough CC. Using confocal laser scanning microscopy (CLSM), we observed that, during the first 12 h of contact with the substratum, osteoblastic cells were relatively less spread on rough Ti, whereas cells appeared elongated with multiple cellular extensions on rough CC. Focal adhesion contacts, as indicated by vinculin immunostaining, were distributed throughout the cells adhering to Ti, but were relatively sparse and localized to cellular processes on CC. Furthermore, three-dimensional CLSM reconstruction analysis indicated the presence of vinculin at all membrane-to-surface contact points on both Ti and CC. On Ti, these contact points closely followed the surface contour, whereas, on CC, they were restricted to relative topographic peaks only. Actin cytoskeletal reorganization was prominent in cells cultured on Ti, with stress fibers arranged throughout the cell body, whereas, on CC, actin filaments were sparse and localized primarily to cellular extensions. Because cell attachment mechanisms are likely to influence signal transduction and regulation of gene expression, these early differential responses of osteoblastic cells on Ti and CC may have functional implications on subsequent extracellular matrix mineralization and bone ingrowth at the cell-biomaterial interface.

12(S)-HETE, a lipoxygenase metabolite of arachidonic acid, has been demonstrated to induce a reversible retraction of vascular endothelial cells (EC). 12(S)-HETE-induced microvascular EC retraction was blocked by a selective protein kinase C inhibitor, calphostin C, but not by the protein kinase A inhibitor, H8. EC exposed to 12(S)-HETE demonstrated a gradual dissolution of actin microfilaments and a decrease of vinculin-containing focal adhesions. The intermediate filaments, vimentin, also underwent extensive reorganization (i.e., filament bundling and enrichment to the cell filapodia) following 12(S)-HETE treatment. In vivo phosphorylation studies revealed that 12(S)-HETE induced a hyperphosphorylation of several major cytoskeletal proteins including myosin light chain, actin, and vimentin. The increased phosphorylation of these cytoskeletal proteins following 12(S)-HETE stimulation was abolished by calphostin C but not by H8. Confluent EC express alpha v beta 3 in focal adhesions at both the cell body and the cell-cell borders. 12(S)-HETE induced a sequential rearrangement of the alpha v beta 3-containing focal adhesions, resulting in a general decrease in alpha v beta 3 integrin receptors, especially in those retracted EC. 12(S)-HETE-induced rearrangement of alpha v beta 3 was inhibited by calphostin C but not by H8. In contrast to alpha v beta 3, confluent EC enrich alpha 5 beta 1 integrin receptors primarily at the cell-cell borders, colocalizing with extracellular fibronectin and cell cortical microfilaments. 12(S)-HETE treatment also disrupted the cell-border distribution pattern of alpha 5 beta 1 as EC retracted, but no distinct alterations (such as time-related redistribution and quantitative differences) in alpha 5 beta 1 were observed.

Cell detachment by laminar shear stresses was used to characterize cellular interactions with hydrophilic glass and hydrophobic silane. In this study, we examined whether smaller, rounder cells were preferentially detached by laminar flow, and whether cell detachment occurred by dissociation of adhesion proteins and their membrane receptors or rupture of the membrane. Shear-induced detachment from glass and silane were similar after 0.5 h static attachment to the surfaces, even though 3T3 cells had a greater projected area on silane. No particular cell size was preferentially detached by fluid shear stresses. After 2 h attachment and spreading, 3T3 cells were more easily detached from the silane surface even though the cells were more spread than on glass. On glass, smaller cells were preferentially detached below 30 dyne/cm2, increasing the mean projected area of the population. Above 30 dyne/cm2, larger cells also detached from the surface. Cell detachment from the silane surfaces did not show any size preference. The strength of adhesion and projected areas on both surfaces increased significantly when the surfaces were preincubated with fibronectin. Simple geometric models of spreading cells were used to estimate the forces exerted on cells. The hydrodynamic forces exerted on spreading cells were similar, but the bond density needed to resist detachment declined as the projected area increased. Analysis of Dil-C18(3) membrane fragments indicated that cell detachment by membrane rupture was a significant mechanism of cell detachment from glass for shear stresses above 40 dyne/cm2, but was unimportant for cell detachment from the silane surfaces. The results indicate that differences in the strength of 3T3 cell adhesion were probably due to differences in bond strength and the numbers of receptor-ligand bonds formed on the two surfaces and, on glass, cell detachment due to membrane failure at higher shear stresses.