Cell/surface interactions of human osteo-sarcoma (HOS) cells and micro-patterned polydimelthylsiloxane (PDMS) surfaces

This paper presents the results of an experimental study of the effects of three-dimensional micro-pattern geometry on cell/surface interactions and the adhesion between HOS cells and PDMS surfaces. Micro-grooves with well-controlled ridges and spacings were fabricated by curing poly-di-methy-siloxane (PDMS) in silicon molds produced by photolithography. HOS cells were then cultured onto these surfaces for durations of 6, 12 and 48 h. In cases, where the groove spacing was comparable to the spread cell size, the cells align well in the directions of microgrooves. However, as the ridge separation increases, the cell orientations become more random, and less dependent on ridge height and spacing. The actin cytoskeletal structure and the distribution of focal adhesions are also elucidated by immuno-fluorescence staining.     

Shear assay measurements of cell adhesion on biomaterials surfaces

The paper examines the adhesion of human osterosarcoma (HOS) cells to selected biomaterials surfaces that are relevant to implantable biomedical systems and bio-micro-electro-mechanical systems (BioMEMS). The four biomaterials that were explored include: silicon, silicon coated with a nanoscale layer of titanium, Ti–6Al–4V, and poly-di-methy-siloxane (PDMS). The interfacial strengths between the HOS cells and the biomaterials surfaces were determined using a shear assay technique. The adhesion forces were determined using a combination of confocal microscopy images of the three-dimensional cell structure, and computational fluid dynamics (CFD) simulations that coupled actual cell morphologies and non-Newtonian fluid properties in the computation of the adhesion forces. After cell detachment by the shear assay, immunofluorescence staining of the biomedical surfaces was used to reveal the proteins associated with cell detachment. These revealed that the nano-scale Ti coating increases the cell/surface adhesion strength. Silicon with Ti coating has the strongest adhesion strength, while the other surfaces had similar adhesion strength. The measured strengths are shown to be largely associated with the detachment of focal adhesion proteins from extra-cellular matrix (ECM) proteins.     

Monocyte, macrophage and foreign body giant cell interactions with molecularly engineered surfaces

To elucidate the mechanisms involved in monocyte/macrophage adhesion and fusion to form foreign body giant cells on molecularly engineered surfaces, we have utilized our in vitro culture system to examine surface chemistry effects, cytoskeletal reorganization and adhesive structure development, and cell receptor-ligand interactions in in vitro foreign body giant cell formation. Utilizing silane-modified surfaces, monocyte/macrophage adhesion was essentially unaffected by surface chemistry, however the density of foreign body giant cells (FBGCs) was correlated with surface carbon content. An exception to the surface-independent macrophage adhesion were the alkyl-silane modified surfaces which exhibited reduced adhesion and FBGC formation. Utilizing confocal immunofluorescent techniques, cytoskeletal reorganization and adhesive structure development in in vitro FBGC formation was studied. Podosomes were identified as the adhesive structures in macrophages and FBGCs based on the presence of characteristic cytoplasmic proteins and F-actin at the ventral cell surface. Focal adhesion kinase (FAK) and focal adhesions were not identified as the adhesive structures in macrophages and FBGCs. In studying the effect of preadsorbed proteins on FBGC formation, fibronectin or vitronectin do not play major roles in initial monocyte/macrophage adhesion, whereas polystyrene surfaces modified with RGD exhibited significant FBGC formation. These studies identify the potential importance of surface chemistry-dependent conformational alterations which may occur in proteins adsorbed to surfaces and their potential involvement in receptor-ligand interactions. Significantly, preadsorption of α₂-macroglobulin facilitated macrophage fusion and FBGC formation readily on the RGD surface in the absence of any additional serum proteins. As α₂-macroglobulin receptors are not found on blood monocytes but are expressed only with macrophage development, these results point to a potential interaction between adsorbed α₂-macroglobulin and its receptors on macrophages during macrophage development and fusion. These studies identify important surface independent and dependent effects in foreign body reaction development that may be important in the identification of biological design criteria for molecularly engineered surfaces and tissue engineered devices. © 1999 Kluwer Academic Publishers     

Laser-engineered topography: correlation between structure dimensions and cell control

Topographical cues have a significant impact on cell responses and by this means, on the fabrication of innovative implant materials. However, analysis of cell-topography interactions in dependence of the surface feature dimensions is still challenging due to limitations in the fabrication technology. Here, we introduce surface structuring via picosecond laser systems, which enable a fast production of micro-sized topologies. Changes in the processing parameters further control the feature sizes of so-called spikes. Using surfaces with big and small spike-to-spike-distances for comparisons, we focussed on cell adhesion via extracellular matrix adsorption and focal adhesion complexes, morphology, localisation and proliferation of fibroblasts. The observed cell control was dependent on a turnover point related to the structure dimensions: only big spike-to-spike-distances reduced cell behaviour. Therefore, this technology offers a platform to study cell and tissue interactions with a defined microenvironment.     

Various fates of neuronal progenitor cells observed on several different chemical functional groups

Neuronal progenitor cells cultured on gold-coated glass surfaces modified by different chemical functional groups, including hydroxyl (-OH), carboxyl (-COOH), amino (-NH₂), bromo (-Br), mercapto (-SH), -Phenyl and methyl (-CH₃), were studied here to investigate the influence of surface chemistry on the cells’ adhesion, morphology, proliferation and functional gene expression. Focal adhesion staining indicated in the initial culture stage cells exhibited morphological changes in response to different chemical functional groups. Cells cultured on -NH₂ grafted surface displayed focal adhesion plaque and flattened morphology and had the largest contact area. However, their counter parts on -CH₃ grafted surface displayed no focal adhesion and rounded morphology and had the smallest contact area. After 6 days culture, the proliferation trend was as follows: -NH₂>-SH>-COOH>-Phenyl>-Br>-OH>-CH₃. To determine the neural functional properties of the cells affected by surface chemistry, the expression of glutamate decarboxylase (GAD67), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were characterized. An increase of GAD67 expression was observed on -NH₂, -COOH and -SH grafted surfaces, while no increase in NGF and BDNF expression was observed on any chemical surfaces. These results highlight the importance of surface chemistry in the fate determination of neuronal progenitor cells, and suggest that surface chemistry must be considered in the design of biomaterials for neural tissue engineering.     

Morphology and adhesion of mesenchymal stem cells on PLLA,apatite and apatite/collagen surfaces

Biomimetic apatite/collagen composite coating, previously reported particularly with regard to its fabrication, characterization and interaction with osteoblast-like cells, has been investigated in this study to understand the response of human mesenchymal stem cells (hMSC) to such surface. PLLA films and PLLA films with apatite coating were compared with PLLA films with apatite/collagen composite coating. The hMSC morphology in response to such conditions was first observed using fluorescence microscopy. To further understand such cell-material interactions at a molecular level, integrin expression, actin assembly and vinculin-positive focal adhesion plaques were examined. Our results demonstrated that spreading of stem cells on the apatite/collagen composite surface was determined best among the three types of surfaces, followed by the apatite surface and then the PLLA control. Integrin expression on the apatite/collagen surface was higher than those on the apatite surface and PLLA surface. Immunostaining for vinculin and actin suggested that the composite coating on PLLA enhanced the formation of focal adhesion.     

Cell behaviour on phospholipids-coated surfaces

Effective integration of orthopedic biomaterials requires the rapid formation of the inorganic mineral phase during the first hours of implantation and the subsequent adhesion and proliferation of the osteoblasts. It has recently been demonstrated that phosphatidylserine-rich phospholipid coatings can induce a fast mineralisation of titanium implant surfaces on incubation in simulated body fluids. The aim of this work was to investigate the biocompatibility of these coatings in terms of cytotoxicity and ability to support osteoblast adhesion and activity. Cytotoxicity and cell adhesion to uncoated titanium, calcified phospholipid-coated titanium and HA-coated titanium was assessed using fibroblasts and osteoblast-like cells. The synthesis of type I collagen by osteoblast-like cells cultured on the calcified-phospholipid coatings was also comparable to that observed for osteoblast-like cells cultured on the titanium and HA-Ti surfaces. The results suggest that the fast mineralization of the phospholipid matrix, obtained in vitro by its pre-treatment in a SBF, exposes the cells to an environment similar to that present in the bone during its natural formation that allow cells to adhere, proliferate and produce proteins fundamental for bone growth. The biocompatibility of these phospholipid-based coatings, in combination with their ability to initiate rapid mineralisation, provides a promising material that could in vivo create bone cell interactions and bone integration.     

An investigation of the initial attachment and orientation of osteoblast-like cells on laser grooved Ti-6Al-4V surfaces

This paper presents the results of an experimental study of the initial cell spreading and adhesion on longitudinally- and transversally-oriented micro-grooves produced by the laser irradiation of laser grooved Ti-6Al-4V surfaces. The initial spreading and orientations of human osteosarcoma (HOS) cells were observed and quantified after 15-min, 1-hour, 4-hour and 24-hour cell culture periods. Immuno-fluorescence staining of adhesion proteins (actin and vinculin) was then used to study the spreading and adhesion of HOS cells in 1 hour and 4 hour culture experiments. The initial cell adhesion was also quantified using enzymatic detachment tests. The results showed that cell spreading and adhesion were enhanced by longitudinally- and transversally-oriented micro-grooves. The effects, which increase with time, were not remarkable after 1 hour, but obvious after 4 hours. Contact guidance was found to promote cell adhesion due to the increase in interactions between the focal adhesions and the patterned extra-cellular matrix (ECM) proteins on the laser micro-grooved surfaces.     

Biological evaluation of ultrananocrystalline and nanocrystalline diamond coatings

Nanostructured biomaterials have been investigated for achieving desirable tissue-material interactions in medical implants. Ultrananocrystalline diamond (UNCD) and nanocrystalline diamond (NCD) coatings are the two most studied classes of synthetic diamond coatings; these materials are grown using chemical vapor deposition and are classified based on their nanostructure, grain size, and sp³ content. UNCD and NCD are mechanically robust, chemically inert, biocompatible, and wear resistant, making them ideal implant coatings. UNCD and NCD have been recently investigated for ophthalmic, cardiovascular, dental, and orthopaedic device applications. The aim of this study was (a) to evaluate the in vitro biocompatibility of UNCD and NCD coatings and (b) to determine if variations in surface topography and sp³ content affect cellular response. Diamond coatings with various nanoscale topographies (grain sizes 5–400?nm) were deposited on silicon substrates using microwave plasma chemical vapor deposition. Scanning electron microscopy and atomic force microscopy revealed uniform coatings with different scales of surface topography; Raman spectroscopy confirmed the presence of carbon bonding typical of diamond coatings. Cell viability, proliferation, and morphology responses of human bone marrow-derived mesenchymal stem cells (hBMSCs) to UNCD and NCD surfaces were evaluated. The hBMSCs on UNCD and NCD coatings exhibited similar cell viability, proliferation, and morphology as those on the control material, tissue culture polystyrene. No significant differences in cellular response were observed on UNCD and NCD coatings with different nanoscale topographies. Our data shows that both UNCD and NCD coatings demonstrate in vitro biocompatibility irrespective of surface topography.     

Influences of surface chemistry and swelling of salt-treated polyelectrolyte multilayers on migration of smooth muscle cells

The cell migration plays a crucial role in a variety of physiological and pathological processes and can be regulated by the cell–substrate interactions. We found previously that the poly(sodium 4-styrenesulphonate) (PSS)/poly(diallyldimethylammonium) chloride (PDADMAC) multilayers post-treated in 1–5 M NaCl solutions result in continuous changes of their physico-chemical properties such as thickness, chemical composition, surface charge, swelling ratio and wettability. In this study, the responses of human smooth muscle cells (SMCs) on these salt-treated multilayers, particularly the governing factors of cellular migration that offer principles for designing therapeutics and implants, were disclosed. The cell migration rate was slowest on the 3 M NaCl-treated multilayers, which was comparable with that on tissue culture plates, but it was highest on 5 M NaCl-treated multilayers. To elucidate the intrinsic mechanisms, cell adhesion, proliferation, adhesion and related gene expressions were further investigated. The SMCs preferred to attach, spread and proliferate on the PSS-dominated surfaces with well-organized focal adhesion and actin fibres, especially on the 3 M NaCl-treated multilayers, while were kept round and showed low viability on the PDADMAC-dominated surfaces. The relative mRNA expression levels of adhesion-related genes such as fibronectin, laminin and focal adhesion kinase, and migration-related genes such as myosin IIA and Cdc42 were compared to explain the different cellular behaviours. These results reveal that the surface chemistry and the swelling of the salt-treated multilayers govern the cell migration behaviours.     

The influence of surface chemistry and topography on the contact guidance of MG63 osteoblast cells

The purpose of the present study was to determine in vitro the effects of different surface topographies and chemistries of commercially pure titanium (cpTi) and diamond-like carbon (DLC) surfaces on osteoblast growth and attachment. Microgrooves (widths of 2, 4, 8 and 10 μm and a depth of 1.5–2 μm) were patterned onto silicon (Si) substrates using microlithography and reactive ion etching. The Si substrates were subsequently vapor coated with either cpTi or DLC coatings. All surfaces were characterized using atomic force microscopy (AFM), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and contact angle measurements. Using the MG63 Osteoblast-Like cell line, we determined cell viability, adhesion, and morphology on different substrates over a 3 day culture period. The results showed cpTi surfaces to be significantly more hydrophilic than DLC for groove sizes larger than 2 μm. Cell contact guidance was observed for all grooved samples in comparison to the unpatterned controls. The cell viability tests indicated a significantly greater cell number for 8 and 10 μm grooves on cpTi surfaces compared to other groove sizes. The cell adhesion study showed that the smaller groove sizes, as well as the unpatterned control groups, displayed better cell adhesion to the substrate.     

Effect of substrate surface conditions and impact velocity of sprayed particles on coatings produced by plasma spraying

In high temperature plasma spraying, surface conditions of the substrate and the impact velocity of sprayed particles are particularly important in the consideration of factors influencing the resultant coatings. In this study an induction plasma torch was used to spray aluminum, tungsten and nickel particles onto flat (111) single-crystal silicon substrates with different surface preparations. In addition to experiments with silicon substrates with clean surfaces and substrates with previously evaporated aluminum over oxide coatings, oxide films and water vapor, hydraulic oil and grease contaminant films were used to investigate the influence of these surface conditions on adhesion formation. The impact velocity of the sprayed particles was controlled by controlling the speed of a centrifugal disc on which the substrate was installed to traverse the plasma effluent.Scanning electron microscopy was used extensively to characterize features of the particle-substrate interaction and to compare the individual behaviors of the metal particles used. Microcavitations and dislocations resulting from the plastic deformation of the substrate were revealed by the successive use of a metal and a special dislocation etchant.     

Korrosionsinhibierende Plasmapolymerschichten auf Stahluntergründen

Plasma polymeric coatings with inhibitory effect on steel surfaces The plasma based surface technique facilitates scientifically and technically, economically and ecologically interesting alternatives to traditional treatment procedures. From that the evolution of a new procedure for the improvement of the corrosion resistance of low‐alloy steel surfaces through ultrathin plasma polymeric coatings was a main topic at the iLF in the last years. After plasma based super cleaning process under oxidized conditions should be produced plasma polymeric coatings with a corrosion inhibiting / passivation effect. For this purpose corrosion inhibitor molecules are implanted into the normally electrochemically inactive coating during the process of the plasma based polymerization of silicon‐organic monomers. Through that it has been proved that the long‐term corrosion protection improves. Besides the surface of the produced plasma polymer shift can be modified by plasma‐technical procedures so that it shows also optimal adhesion promoter characteristics.     

Characterization of proteins and fibroblasts on thin inorganic films

The ability of biomaterial surfaces to regulate cell behavior requires control over surface chemistry and material microstructure. One of the goals in the development of silicon-based biomedical devices such as biosensors or drug delivery systems is improved biocompatibility which may be achieved through the deposition or adsorption of thin films. In this study, films of single crystal silicon, stoichiometric and low stress silicon nitride, doped and undoped polysilicon, as well as Arg-Gly-Asp (RGD) peptide adsorbed surfaces characterized in terms of protein adsorption or cellular adhesion for a period of four days. Protein adsorption studies using fibrinogen and albumin, two proteins implicated in cellular adhesion and surface activity, reveal that low stress silicon nitride surfaces have a 223%±2.50% greater protein adsorption compared to undoped polysilicon surfaces, followed by silicon nitride, unmodified silicon, and doped polysilicon surfaces, respectively. The thickness of the adsorbed albumin and fibrinogen layer on various thin films was measured by ellipsometry and compared to contact angle measurements. The greatest cellular adhesion was observed on undoped polysilicon, followed by unmodified (control) silicon, low stress silicon nitride, silicon nitride, and doped polysilicon surfaces. Cellular binding supports the differential protein adsorption found on modified and unmodified silicon surfaces. Understanding the biological response to thin films will allow us to design more appropriate interfaces for implantable diagnostic and therapeutic silicon-based microdevices.     

Effects of hydroxyapatite/Zr and bioglass/Zr coatings on morphology and corrosion behaviour of Rex-734 alloy

To improve corrosion resistance of metallic implant surfaces, Rex-734 alloy was coated with two different bio-ceramics; single-Hydroxyapatite (HA), double-HA/Zirconia(Zr) and double-Bioglass (BG)/Zr by using sol–gel method. Porous surface morphologies at low crack density were obtained after coating and sintering processes. Corrosion characteristics of coatings were determined by Open circuit potential and Potentiodynamic polarization measurements during corrosion tests. Hardness and adhesion strength of coating layers were measured and their surface morphologies before and after corrosion were characterized by scanning electron microscope (SEM), XRD and EDX. Through the SEM analysis, it was observed that corrosion caused degradation and sphere-like formations appeared with dimples on the coated surfaces. The coated substrates that exhibit high crack density, the corrosion was more effective by disturbing and transmitting through the coating layer, produced CrO₃ and Cr₃O₈ oxide formation. It was found that the addition of Zr provided an increase in adhesion strength and corrosion resistance of the coatings. However, BG/Zr coatings had lower adhesion strength than the HA/Zr coatings, but showed higher corrosion resistance.     

Induced tissue integration of bone implants by coating with bone selective RGD-peptides in vitro and in vivo studies

The optimal function of medical implant materials used in tissue substitution is often limited due to its healing properties. This effect is linked to reduced interactions of the implants with the surrounding tissue. Implant surfaces biologically functionalized with arginine-glycine-aspartic acid (RGD) peptides, a class of cellular adhesion factors, are described in this paper. The RGD-peptides are either bound via bovine serum albumin linking on culture plastic dishes as a model surface or via acrylic acid coupling on PMMA surface as a potential implant material. Resulting functionalized surfaces aquire the capability to bind cultured osteoblasts in high levels and show high proliferation rates in vitro. These results are observed for osteoblast cultures as well as from different species with different preparation procedures. A critical minimum distance between the bioactive portion of the RGD-peptides and the implant surface of 3.0–3.5 nm is crucial for the induction of an optimum cell binding process. In vivo animal studies in the rabbit show that newly formed bone tissue generated a direct contact with the RGD-peptide coated implants. In contrast uncoated implants are separated from newly formed bone tissue by a fibrous tissue layer thereby preventing the formation of a direct implant–bone bonding.     

Interactions of Fibroblasts with Different Morphologies Made of an Engineered Spider Silk Protein

Spider silk has been investigated for decades due to the intriguing mechanical and also biomedical properties of the silk fibers. Previously, it has been shown that recombinant silk proteins can also be processed into other morphologies. Here, we characterized scaffolds made of the recombinant spider silk protein eADF4(C16) concerning their surface interactions with fibroblasts. Studies of BALB/3T3 cells on hydrogels and films made of eADF4(C16) showed low cell adhesion without observable duplication. Electro‐spun non‐woven scaffolds made of eADF4(C16), however, enabled both their adhesion and proliferation. Since eADF4(C16) lacks specific motifs for cell attachment, fibroblasts cannot generate focal adhesions with the material's surface, and, therefore, other cell–interface interactions such as topographical anchorage or cell attachment mediated by adhesion of extracellular matrix proteins are discussed in this paper. On non‐woven meshes protrusion of filopodia and/or lamellipodia between individual fibers increase the surface contact area, which depends on the diameter of the fibers of the non‐woven meshes. In contrast, at flat (film) or microstructured surfaces (hydrogels) such interactions seem to be precluded.     

Human osteoblast adhesion on titanium alloy, stainless steel, glass and plastic substrates with same surface topography

Osteoblast adhesion on materials will depend on the surface aspects of materials which may be described according to their surface chemistry, surface topography or surface energy. To separate the effects of roughness and composition of materials on osteoblast response, we chose to compare substrates with various surface composition but with the same smooth surface. Ti6Al4V alloy, stainless steel, glass and standard tissue culture polystyrene were tested. Adhesion was evaluated using specific antibodies against adhesion proteins and by a quantitative cell detachment assay. After 1, 7 and 14 days, cells expressed extracellularly fibronectin fibers, and intracellularly type I collagen and osteopontin. Vinculin-labeled focal contacts were visible on all materials but were more frequent on glass and stainless steel surfaces. ₁-integrin subunit-labeled patches were visible on all surfaces at each delay. The quantitative cell detachment assay showed few differences between materials. Adhesion was higher on metallic substrates although cell proliferation was higher on glass and stainless steel compared to tissue culture polystyrene and Ti6Al4V alloy. Substrates with various surface composition but with the same surface topography did not induce significant differences of adhesion although cell proliferation was variable.     

Contractility-dependent modulation of cell proliferation and adhesion by microscale topographical cues

Engineering of cellular assembly on biomaterial scaffolds by utilizing microscale topographical cues has emerged as a powerful strategy in cardiovascular tissue engineering and regenerative medicine. However, the mechanisms through which these cues are processed to yield changes in canonical cell behaviors remain unclear. Previously, we showed that when mixtures of cardiomyocytes and fibroblasts were cultured on polydimethylsiloxane surfaces studded with microscale pillars (micropegs), fibroblast proliferation was dramatically suppressed, which suggests that the micropegs could be exploited to minimize fibrosis and scar formation. Here, we demonstrate that this effect relies on altered adhesive and micromechanical interactions between individual cells and micropegs. First, we show that the proliferation of a cell physically attached to a micropeg is significantly lower than that of a cell cultured on a featureless region of the substrate. Micropeg adhesion is accompanied by a marked elongation in cell and nuclear shape. When fibroblast contractility is pharmacologically attenuated through low-dose inhibition of either Rho-associated kinase or myosin light chain kinase, the potency with which micropeg adhesion suppresses cell proliferation is significantly reduced. Together, our results support a model in which cell fate decisions may be directly manipulated within tissue engineering scaffolds by the inclusion of microtopographical structures that alter cellular mechanics.     

Adhesion and friction properties of micro/nano-engineered superhydrophobic/hydrophobic surfaces

Hydrophobic micro/nano-engineered surfaces (MNESs) with good adhesion and frictional performances were fabricated by the combination of aluminum-induced crystallization (AIC) of amorphous silicon (a-Si) and octadecyltrichlorosilane (OTS) coating. The AIC of a-Si technique was used to produce silicon micro/nano-textured surfaces, while an OTS self-assembled monolayer was used to lower the surface energies of the textured surfaces. The wetting properties of the MNESs were studied using a video-based contact angle measurement system. The adhesion and friction properties of the MNESs were investigated using a TriboIndenter. This study shows that the adhesion and frictional performances of all MNESs are significantly improved compared to untreated silicon substrate surfaces, and the adhesion and frictional performances of the OTS-modified textured surfaces strongly correlate to their surface wetting property, i.e., the larger the water contact angle, the better the adhesion and frictional performances of the OTS-modified textured surfaces.