Different titanium surfaces modulate the bone phenotype of SaOS-2 osteoblast-like cells

Commercially pure titanium implants presenting a relatively smooth, machined surface or a roughened endosseous surface show a large percentage of clinical success. Surface properties of dental implants seem to affect bone cells response. Implant topography appears to modulate cell growth and differentiation of osteoblasts affecting the bone healing around the titanium implant. The aim of the present study was to examine the effects of 1cm diameter and 1mm thick titanium disks on cellular morphology, adhesion and bone phenotypic expression of human osteoblast-like cells, SaOS-2. SaOS-2 cells were cultured on commercially 1 cm pure titanium disks with three different surface roughness: smooth (S), sandblasted (SB) and titanium plasma sprayed (TPS). Differences in the cellular morphology were found when they were grown on the three different surfaces. An uniform monolayer of cells recovered the S surface, while clusters of multilayered irregularly shaped cells were distributed on the rough SB and TPS surfaces. The adhesion of SaOS-2 cells, as measured after 3h of culture, was not affected by surface roughness. ECM components such as Collagen I (CoI), Fibronectin (FN), Vitronectin (VN) and Tenascin (TN) were secreted and organized only on the SB and TPS surfaces while they remained into the cytoplasm on the S surfaces. Osteopontin and BSP-II were largely detected on the SB and TPS surfaces, while only minimal production was observed on the S ones. These data show that titanium surface roughness affects bone differentiation of osteoblast like-cells, SaOS-2, indicating that surface properties may be able to modulate the osteoblast phenotype. These observations also suggest that the bone healing response around dental implants can be affected by surface topography.     

Quantitative characterization of the influence of the nanoscale morphology of nanostructured surfaces on bacterial adhesion and biofilm formation

Bacterial infection of implants and prosthetic devices is one of the most common causes of implant failure. The nanostructured surface of biocompatible materials strongly influences the adhesion and proliferation of mammalian cells on solid substrates. The observation of this phenomenon has led to an increased effort to develop new strategies to prevent bacterial adhesion and biofilm formation, primarily through nanoengineering the topology of the materials used in implantable devices. While several studies have demonstrated the influence of nanoscale surface morphology on prokaryotic cell attachment, none have provided a quantitative understanding of this phenomenon. Using supersonic cluster beam deposition, we produced nanostructured titania thin films with controlled and reproducible nanoscale morphology respectively. We characterized the surface morphology; composition and wettability by means of atomic force microscopy, X-ray photoemission spectroscopy and contact angle measurements. We studied how protein adsorption is influenced by the physico-chemical surface parameters. Lastly, we characterized Escherichia coli and Staphylococcus aureus adhesion on nanostructured titania surfaces. Our results show that the increase in surface pore aspect ratio and volume, related to the increase of surface roughness, improves protein adsorption, which in turn downplays bacterial adhesion and biofilm formation. As roughness increases up to about 20 nm, bacterial adhesion and biofilm formation are enhanced; the further increase of roughness causes a significant decrease of bacterial adhesion and inhibits biofilm formation. We interpret the observed trend in bacterial adhesion as the combined effect of passivation and flattening effects induced by morphology-dependent protein adsorption. Our findings demonstrate that bacterial adhesion and biofilm formation on nanostructured titanium oxide surfaces are significantly influenced by nanoscale morphological features. The quantitative information, provided by this study about the relation between surface nanoscale morphology and bacterial adhesion points towards the rational design of implant surfaces that control or inhibit bacterial adhesion and biofilm formation.     

The relative importance of topography and RGD ligand density for endothelial cell adhesion

The morphology and function of endothelial cells depends on the physical and chemical characteristics of the extracellular environment. Here, we designed silicon surfaces on which topographical features and surface densities of the integrin binding peptide arginine-glycine-aspartic acid (RGD) could be independently controlled. We used these surfaces to investigate the relative importance of the surface chemistry of ligand presentation versus surface topography in endothelial cell adhesion. We compared cell adhesion, spreading and migration on surfaces with nano- to micro-scaled pyramids and average densities of 6×10(2)-6×10(11) RGD/mm(2). We found that fewer cells adhered onto rough than flat surfaces and that the optimal average RGD density for cell adhesion was 6×10(5) RGD/mm(2) on flat surfaces and substrata with nano-scaled roughness. Only on surfaces with micro-scaled pyramids did the topography hinder cell migration and a lower average RGD density was optimal for adhesion. In contrast, cell spreading was greatest on surfaces with 6×10(8) RGD/mm(2) irrespectively of presence of feature and their size. In summary, our data suggest that the size of pyramids predominately control the number of endothelial cells that adhere to the substratum but the average RGD density governs the degree of cell spreading and length of focal adhesion within adherent cells. The data points towards a two-step model of cell adhesion: the initial contact of cells with a substratum may be guided by the topography while the engagement of cell surface receptors is predominately controlled by the surface chemistry.     

Use of cell contour analysis to evaluate the affinity between macrophages and glutaraldehyde-treated erythrocytes.

Recently, several authors evaluated the affinity between lipid bilayers or erythrocyte membranes by analyzing the deformation of cells or vesicles they brought into close contact using micromanipulators. In the present report, we extend this approach in a study of the adhesive properties of rough nucleated cells. Rat peritoneal macrophages were made to bind human red cells modified with glutaraldehyde or glutaraldehyde and polylysine. Conjugates were examined with electron microscopy, and photomicrographs were digitized for quantification of cell surface roughness in and out of adhesion areas. Also, macrophages were subjected to micropipette aspiration to find a relationship between apparent surface tension and area increase. Assuming that this increase was a direct consequence of a smoothing of the cell surface on the submicrometer scale, the actual affinity between macrophages and erythrocytes was estimated. The obtained values ranged between 8.4 X 10(-5) and 18.2 X 10(-5) J/m2. It is concluded that cell surface roughness may be an important parameter of cell adhesion and perhaps deformation. This is made amenable to experimental study by the present approach.     

Surface-enhanced nucleation of insulin amyloid fibrillation

Proteins can interact with biological surfaces such as cell membrane, chaperones, cornea, bone, arteries, veins, and heart cavities of the cardiovascular system and also with non-biological surfaces including dialysis membranes and tubing, catheters, invasive surgical instruments, needles, and artificial implants. Fibrillation of amyloid proteins is implicated in many human diseases, including Alzheimer’s, Parkinson’s, and type II diabetes. Here, we show that heterogeneous surfaces accelerate the human insulin nucleation process that is the rate-determining step during amyloid fibril formation. The observed shorter lag (nucleation) phase correlates both with surface wettability and surface roughness. Surfaces promote faster nucleation possibly by increasing the local concentration of protein molecules. A composite parameter combining both surface wettability and roughness suggests that the ideal surface for slower nucleation should be hydrophilic and smooth. These findings provide a basis for designing suitable biomaterials and biomedical devices, especially those to resist amyloidosis.     

Nanoparticles Covered Surfaces for Post-functionalization with Aromatic Groups to Obtain Parahydrophobic Surface with High Water Adhesion (Petal Effect)

Numerous exceptional properties can be observed in nature.Among these properties,parahydrophobic feature is of interest.This property describes material with high adhesion with water such as rose petals or gecko foot.Such kind of surface presents a real potential for applications in the field of water harvesting systems.In this work,we report a new synthetic strategy to mimic this property.Here,we combine three strategies in one.First,a monomer is electropolymerized in order to form the starting structured surface.Then,nanoparticles are grafted on the surface to increase the structuration and consequently to create the reactive surface.Finally,the grafted surface is post-functionalized (Huisgen reaction) with various aryl alkynes to control the surface chemistry and energy.This strategy allows to reach surfaces with both very high hydrophobic properties (θ=140°) and high water adhesion.This work also includes the surface wettability,roughness and morphology investigation in order to study the impact of the starting monomer structure and post-functionalization on the surface properties.     

Collagen Self-Assembly on Orthopedic Magnesium Biomaterials Surface and Subsequent Bone Cell Attachment

Magnesium (Mg) biomaterials are a new generation of biodegradable materials and have promising potential for orthopedic applications. After implantation in bone tissues, these materials will directly interact with extracellular matrix (ECM) biomolecules and bone cells. Type I collagen, the major component of bone ECM, forms the architecture scaffold that provides physical support for bone cell attachment. However, it is still unknown how Mg substrate affects collagen assembly on top of it as well as subsequent cell attachment and growth. Here, we studied the effects of collagen monomer concentration, pH, assembly time, and surface roughness of two Mg materials (pure Mg and AZ31) on collagen fibril formation. Results showed that formation of fibrils would not initiate until the monomer concentration reached a certain level depending on the type of Mg material. The thickness of collagen fibril increased with the increase of assembly time. The structures of collagen fibrils formed on semi-rough surfaces of Mg materials have a high similarity to that of native bone collagen. Next, cell attachment and growth after collagen assembly were examined. Materials with rough surface showed higher collagen adsorption but compromised bone cell attachment. Interestingly, surface roughness and collagen structure did not affect cell growth on AZ31 for up to a week. Findings from this work provide some insightful information on Mg-tissue interaction at the interface and guidance for future surface modifications of Mg biomaterials.     

On the relationship between common amplitude surface roughness parameters and surface area: Implications for the study of cell–material interactions

In order to show that surface area is not always a quantity proportional to the surface roughness, we have constructed simple surfaces consisting of boxes of the same height equally spaced, and rms roughness and surface area have been computed. We have shown how we can get examples of surface configurations for which an increment in the surface roughness corresponds to a decrease in the surface area, although this is observed only for surfaces having similar rms roughness. We have also shown that even in the more intuitive situations where an increase in the surface roughness leads to an increase in the surface area, this increase is not necessarily equivalent. Analogous conclusions have been found when roughness was evaluated through the average roughness. These results could be interesting when analyzing interfacial phenomena such as cell adhesion, especially from a microscopic point of view, where the exact contact area between interacting phases governs these phenomena, and an exact-as-possible approximation to its real value is desirable. Also, the results of this paper could be of interest in various biomedical applications where the modulation of material surface-by-surface roughness may play a significant role. It can be concluded that care should be taken when using roughness parameters as estimators or indicators of the contact area between phases, since the relationship is not always simple.     

The effect of extracellular polymeric substances on the attachment of Pseudomonas NCIMB 2021 to AISI 304 and 316 stainless steel

Surfaces of AISI 304 and 316 stainless steels were pre-treated with three different types of extracellular polymeric substances, viz. (i) exopolymers released into the culture medium ("free"; or planktonic exopolymers), (ii) capsular exopolymers, and (iii) biofilm exopolymers, produced by continuous cultures of marine Pseudomonas NCIMB 2021. The initial attachment of Pseudomonas cells to exopolymer-conditioned steel surfaces varied with the exopolymer type and concentration. Results gained from wettability studies of exopolymer-treated steel using contact angle measurements, as well as from the surface roughness measurements conducted employing atomic force microscopy analysis, could not account for the observed, statistically significant differences (p < 0.1) in the level of bacterial surface colonisation. It is therefore proposed that neither surface hydrophobicity nor roughness play an important part in the early attachment of Pseudomonas NCIMB 2021 to the conditioned steel surfaces and that a difference in the chemistry of the exopolymers is most likely a key parameter influencing initial cell adhesion to pre-treated steel.     

Modulation of cell adhesion, proliferation and differentiation on materials designed for body implants

The interaction of cells and tissues with artificial materials designed for applications in biotechnologies and in medicine is governed by the physical and chemical properties of the material surface. There is optimal cell adhesion to moderately hydrophilic and positively charged substrates, due to the adsorption of cell adhesion-mediating molecules (e.g. vitronectin, fibronectin) in an advantageous geometrical conformation, which makes specific sites on these molecules (e.g. specific amino acid sequences) accessible to cell adhesion receptors (e.g. integrins). Highly hydrophilic surfaces prevent the adsorption of proteins, or these molecules are bound very weakly. On highly hydrophobic materials, however, proteins are adsorbed in rigid and denatured forms, hampering cell adhesion. The wettability of the material surface, particularly in synthetic polymers, can be effectively regulated by physical treatments, e.g. by irradiation with ions, plasma or UV light. The irradiation-activated material surface can be functionalized by various biomolecules and nanoparticles, and this further enhances its attractiveness for cells and its effectiveness in regulating cell functions. Another important factor for cell-material interaction is surface roughness and surface topography. Nanostructured substrates (i.e. substrates with irregularities smaller than 100nm), are generally considered to be beneficial for cell adhesion and growth, while microstructured substrates behave more controversially (e.g. they can hamper cell spreading and proliferation but they enhance cell differentiation, particularly in osteogenic cells). A factor which has been relatively less investigated, but which is essential for cell-material interaction, is material deformability. Highly soft and deformable substrates cannot resist the tractional forces generated by cells during cell adhesion, and cells are not able to attach, spread and survive on such materials. Local variation in the physical and chemical properties of the material surface can be advantageously used for constructing patterned surfaces. Micropatterned surfaces enable regionally selective cell adhesion and directed growth, which can be utilized in tissue engineering, in constructing microarrays and in biosensorics. Nanopatterned surfaces are an effective tool for manipulating the type, number, spacing and distribution of ligands for cell adhesion receptors on the material surface. As a consequence, these surfaces are able to control the size, shape, distribution and maturity of focal adhesion plaques on cells, and thus cell adhesion, proliferation, differentiation and other cell functions.     

Substrata mechanical stiffness can regulate adhesion of viable bacteria

The competing mechanisms that regulate adhesion of bacteria to surfaces and subsequent biofilm formation remain unclear, though nearly all studies have focused on the role of physical and chemical properties of the material surface. Given the large monetary and health costs of medical-device colonization and hospital-acquired infections due to bacteria, there is considerable interest in better understanding of material properties that can limit bacterial adhesion and viability. Here we employ weak polyelectrolyte multilayer (PEM) thin films comprised of poly(allylamine) hydrochloride (PAH) and poly(acrylic acid) (PAA), assembled over a range of conditions, to explore the physicochemical and mechanical characteristics of material surfaces controlling adhesion of Staphylococcus epidermidis bacteria and subsequent colony growth. Although it is increasingly appreciated that eukaryotic cells possess subcellular structures and biomolecular pathways to sense and respond to local chemomechanical environments, much less is known about mechanoselective adhesion of prokaryotes such as these bacteria. We find that adhesion of viable S. epidermidis correlates positively with the stiffness of these polymeric substrata, independently of the roughness, interaction energy, and charge density of these materials. Quantitatively similar trends observed for wild-type and actin analogue mutant Escherichia coli suggest that these results are not confined to only specific bacterial strains, shapes, or cell envelope types. These results indicate the plausibility of mechanoselective adhesion mechanisms in prokaryotes and suggest that mechanical stiffness of substrata materials represents an additional parameter that can regulate adhesion of and subsequent colonization by viable bacteria.     

A short-time scale colloidal system reveals early bacterial adhesion dynamics

The development of bacteria on abiotic surfaces has important public health and sanitary consequences. However, despite several decades of study of bacterial adhesion to inert surfaces, the biophysical mechanisms governing this process remain poorly understood, due, in particular, to the lack of methodologies covering the appropriate time scale. Using micrometric colloidal surface particles and flow cytometry analysis, we developed a rapid multiparametric approach to studying early events in adhesion of the bacterium Escherichia coli. This approach simultaneously describes the kinetics and amplitude of early steps in adhesion, changes in physicochemical surface properties within the first few seconds of adhesion, and the self-association state of attached and free-floating cells. Examination of the role of three well-characterized E. coli surface adhesion factors upon attachment to colloidal surfaces--curli fimbriae, F-conjugative pilus, and Ag43 adhesin--showed clear-cut differences in the very initial phases of surface colonization for cell-bearing surface structures, all known to promote biofilm development. Our multiparametric analysis revealed a correlation in the adhesion phase with cell-to-cell aggregation properties and demonstrated that this phenomenon amplified surface colonization once initial cell-surface attachment was achieved. Monitoring of real-time physico-chemical particle surface properties showed that surface-active molecules of bacterial origin quickly modified surface properties, providing new insight into the intricate relations connecting abiotic surface physicochemical properties and bacterial adhesion. Hence, the biophysical analytical method described here provides a new and relevant approach to quantitatively and kinetically investigating bacterial adhesion and biofilm development.     

A cell-binding, immunoglobulin-like protein from human plasma. II. Cell-binding activity

Cell adhesion to plastic surfaces coated with a new high-molecular-mass immunoglobulin-like protein from normal human plasma was studied. Mouse subdermal fibroblasts, hamster kidney cells, human umbilical vein endothelial cells, and human skin fibroblasts were found to become attached to the surface, but cancer cells derived from human stomach cancer and human breast cancer did not. The appearance of the attached cells differed from that of cells attached to surfaces coated with fibronectin or concanavalin A. The cell adhesion to the surfaces coated with the protein was inhibited by goat anti-human IgM. Furthermore, the binding of the protein to the cell surfaces was demonstrated by the indirect immunofluorescence method. It is concluded that this protein is a new cell-binding protein.     

Estimation of the solubility parameters of model plant surfaces and agrochemicals: a valuable tool for understanding plant surface interactions

Background

Most aerial plant parts are covered with a hydrophobic lipid-rich cuticle, which is the interface between the plant organs and the surrounding environment. Plant surfaces may have a high degree of hydrophobicity because of the combined effects of surface chemistry and roughness. The physical and chemical complexity of the plant cuticle limits the development of models that explain its internal structure and interactions with surface-applied agrochemicals. In this article we introduce a thermodynamic method for estimating the solubilities of model plant surface constituents and relating them to the effects of agrochemicals.

Results

Following the van Krevelen and Hoftyzer method, we calculated the solubility parameters of three model plant species and eight compounds that differ in hydrophobicity and polarity. In addition, intact tissues were examined by scanning electron microscopy and the surface free energy, polarity, solubility parameter and work of adhesion of each were calculated from contact angle measurements of three liquids with different polarities. By comparing the affinities between plant surface constituents and agrochemicals derived from (a) theoretical calculations and (b) contact angle measurements we were able to distinguish the physical effect of surface roughness from the effect of the chemical nature of the epicuticular waxes. A solubility parameter model for plant surfaces is proposed on the basis of an increasing gradient from the cuticular surface towards the underlying cell wall.

Conclusions

The procedure enabled us to predict the interactions among agrochemicals, plant surfaces, and cuticular and cell wall components, and promises to be a useful tool for improving our understanding of biological surface interactions.

     

Response of MG63 Osteoblast Cells to Surface Modification of Ti-6Al-4V Implant Alloy by Laser Interference Lithography

The response of human osteoblast-like osteosarcoma cells (MG63) to surface modification of Ti-6Al-4V implant alloy was investigated by Laser Interference Lithography (LIL).In this work,laser interference lithography was employed to fabricate the microstructures of grooves,dots and dimples onto the surfaces of Ti-6Al-4V samples.Two and three beam LIL systems were developed to carry out the experiments.The laser treatment resulted in the increases of the roughness and the contact angle of water on the implant alloy surfaces.The proliferation of osteoblasts was analyzed by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay for the time periods of 4 hours,2 days,3 days,and 6 days.The MTT test results demonstrated that the laser treatment surfaces had a positive impact on the proliferation of osteoblast cells after 24 hours.The alloy surface morphology and the morphological changes of MG63 cells cultured on the laser textured Ti-6Al-4V surface were observed by Scanning Electron Microscope (SEM).The SEM results indicated that the osteoblast cells were aligned on grooved surfaces and they were prolonged with the structures.Enzymatic detachment results showed that the 20 μm grooved structures provided the better cell adhesion to the textured Ti-6Al-4V surfaces.     

Analysis of adhesion of large vesicles to surfaces.

An experimental procedure that can be used to measure the interfacial free energy density for the adhesion of membranes of large vesicles to other surfaces is outlined and analyzed. The approach can be used for both large phospholipid bilayer vesicles and red blood cells when the membrane force resultants are dominated by isotropic tension. The large vesicle or red cell is aspirated by a micropipet with sufficient suction pressure to form a spherical segment outside the pipet. The vesicle is then brought into close proximity of the surface to be tested and, the suction pressure reduced to permit adhesion, and the new equilibrium configuration is established. The mechanical analysis of the equilibrium shape provides the interfacial free energy density for the surface affinity. With this approach, the measurable range of membrane surface affinity is 10(-4)-3 erg/cm2 for large phospholipid bilayer vesicles and 10(-2)-10 erg/cm2 for red blood cells.     

Osteoclastogenic differentiation of human precursor cells over micro- and nanostructured hydroxyapatite topography

BackgroundSurface topography is a key parameter in bone cells–biomaterials interactions. This study analyzed the behavior of human osteoclast precursor cells cultured over three hydroxyapatite (HA) surfaces ranging from a micro- to nanoscale topography.MethodsHA surfaces were prepared with microsized HA particles, at 1300 °C (HA1), and with nanosized HA particles at 1000 °C (HA2) and 830 °C (HA3). Human osteoclast precursors were cultured in the absence or presence of M-SCF and RANKL.ResultsHA surfaces had similar chemical composition, however, HA1 and HA3 presented typical micro- and nanostructured topographies, respectively, and HA2 profile was between those of HA1 and HA3. The decrease on the average grain diameter to the nanoscale range (HA3) was accompanied by an increase in surface area, porosity and hydrophilicity and a decrease in roughness. Compared to HA1 surface, HA3 allowed a lower osteoclastic adhesion, differentiation and function. Differences in the cell response appeared to be associated with the modulation of relevant intracellular signaling pathways.ConclusionsThe decrease in HA grain size to a biomimetic nanoscale range, appears less attractive to osteoclastic differentiation and function, compared to the HA microsized topography.General significanceThis observation emphasizes the role of surface topography in designing advanced biomaterials for tailored bone cells response in regenerative strategies.     

Impact of surface energy and roughness on cell distribution and viability

The aim of this study was to assess the respective impacts of the surface energy and surface roughness of bare and coated steels on biofouling and sanitisation. Bioadhesion of Staphylococcus aureus CIP 53.154 was studied on two stainless steel surfaces with smooth or specific micro-topography. Two coatings were also studied: silicon oxide (hydrophilic) and polysiloxane (hydrophobic). On smooth surfaces, adhesion was reduced on an apolar coating and cell viability increased with the surface polarity. A specific micro-topography decreased the level of bacterial adhesion on bare surfaces by a factor ten. On this surface, only single adherent cells were observed, contrasting with cells in clusters on smoother surfaces. As a consequence, cell repartition influenced bacterial viability. Most isolated adherent cells were dead whereas cells in clusters were still alive. In addition, the quaternary ammonium chloride used in sanitisation, acted at once both as a tensio-active molecule and a biocide. It only displaced adherent cells but did not remove them.     

Observation of optimal gecko's adhesion on nanorough surfaces

In this letter we report experimental observations on the times of adhesion of living Tokay geckos (Gekko geckos) on polymethylmethacrylate (PMMA) inverted surfaces. Two different geckos (male and female) and three surfaces with different root mean square (RMS) roughness (RMS=42, 618 and 931 nm) have been considered, for a total of 72 observations. The measured data are proved to be statistically significant, following the Weibull Statistics with coefficients of correlation between 0.781 and 0.955. The unexpected result is the observation of a maximal gecko adhesion on the surface with intermediate roughness of RMS=618 nm, that we note has waviness comparable to the seta size.     

A thermodynamic model to predict Phanerochaete chrysosporium INA-12 adhesion to various solid carriers in relation to lignin peroxidase production

A thermodynamic model was used in this study to predict the adhesion of Phanerochaete chrysosporium INA-12 as conidiospores or mycelium to various solid carriers. Theoretical predictions were closely reflected by experimental results. Amount of immobilized mycelium was higher for hydrophobic (polypropylene and polyurethane) than for hydrophilic carrier (stainless steel and grey). Lignin peroxidase production was stimulated in the same way. However, better results were obtained with polyurethane than with polypropylene and with grey than with stainless steel. These results were attributed to roughness effects of solid surfaces. Surface morphology characterization showed that the surface roughness parameter R(A) was higher for polyurethane and grey as compared to polypropylene and stainless steel, respectively. On the other hand, polyurethane is not simply rugous; it has an intraparticle porosity as well as a higher total surface area as compared to polypropylene.