Differential response of Staphylococci and osteoblasts to varying titanium surface roughness

The surface roughness of metallic orthopaedic implants has typically been used to influence osseointegration and spatially control load transfer to the surrounding bone. Because of the increasing recognition of biomaterials-associated infection as a leading implant failure mode, we are interested to know the relative importance of roughness not only on surface-osteoblast interactions but also on surface-bacteria interactions. This in vitro study thus compares the effects of surface topography on Staphylococcus epidermidis and human osteoblast behavior using four clinically relevant titanium surface finishes: polished, satin, grit-blasted and plasma-sprayed. Important differences between these surfaces are manifested not only by their vertical roughness parameters but also by the lateral length scales over which topographic fluctuations occur. We find that S. epidermidis adhesion and growth is substantially higher on the satin and grit-blasted surfaces than on the polished or plasma-sprayed surfaces. The former are both substantially rougher at length scales comparable to that of bacteria. In contrast, based on imaging and biochemical assays of proliferation, differentiation and matrix formation, we find that desirable osteoblast-surface interactions are maximized on plasma-sprayed surfaces and minimized on satin-finished surfaces. We attribute these differences to the fact that the plasma-sprayed surface is relatively smooth compared to the size of an individual osteoblast, while the satin surface is rough at this length scale. These findings indicate that both the vertical and lateral character of surface roughness can be modified to not only optimize implant-bone interactions but to simultaneously minimize implant-bacteria interactions.     

Adhesion of cultured human endothelial cells onto methacrylate polymers with varying surface wettability and charge

The adhesion of human endothelial cells (HEC) onto a series of well-characterized methacrylate polymer surfaces with varying wettabilities and surface charges was studied either in serum-containing (CMS) or in serum-free (CM) culture medium. HEC adhesion in CMS onto (co)polymers of hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA) was found to be optimal on the moderately wettable copolymer (mol ratio 25 HEMA/75 MMA). Positively-charged copolymers of HEMA or MMA with trimethylaminoethyl methacrylate-HCl salt (TMAEMA-Cl), both with mol ratios of 85/15 and a negatively-charged copolymer of MMA with methacrylic acid (MAA), mol ratio 85/15, showed high numbers of adhering HEC. In CM, HEC adhered onto the three charged copolymers mentioned above, but neither onto the copolymer of HEMA and MAA (mol ratio 85/15) nor onto the HEMA/MMA co- and homopolymers. Complete cell spreading in CM was only observed on the positively-charged copolymers.     

Novel transparent nano- to micro-heterogeneous substrates for in-situ cell migration study

Transparent substrates having heterogeneities ranging from nanometer to micrometer lateral length scale were fabricated to study cell migration. The surfaces were generated using thin films of block copolymers and homopolymer blends on ultra smooth transparent polyethylene terephthalate films. Results show that the lateral size scale of the surface heterogeneities affects fibroblast (NIH-3T3) adhesion, spreading and motility. More specifically, fibroblasts migrate faster on micron-sized than on nanometer-sized heterogeneities. Cell movements and morphology on the micron patterned surfaces resemble cells cultured in a 3D environment. These surfaces, therefore, can potentially be utilized as models to study cell behavior in physiologically relevant conditions which can add to our fundamental understanding of cell-substrate interactions and facilitate development of surfaces for medical devices.     

Cellular reactions of osteoblasts to micron- and submicron-scale porous structures of titanium surfaces

Osteoblast reactions to topographic structures of titanium play a key role in host tissue responses and the final osseointegration. Since it is difficult to fabricate micro- and nano-scale structures on titanium surfaces, little is known about the mechanism whereby the topography of titanium surfaces exerts its effects on cell behavior at the cellular level. In the present study, the titanium surface was structured in micron- and submicron-scale ranges by anodic oxidation in either 0.2 M H3PO4 or 0.03 M calcium glycerophosphate with 0.15 calcium acetate. The average dimensions of pores in the structured surface were about 0.5 and 2 microm in diameter, with roughness averaging at 0.2 and 0.4 microm, respectively. Enhanced attachment of cells (SaOS-2) was shown on micron- and submicron-scale structures. Initial cell reactions to different titanium surfaces, e.g. the development of the actin-containing structures, are determined by the different morphology of the surfaces. It is demonstrated that on either micron- or submicron-structured surfaces, many well-developed filopodia were observed to be primary adhesion structures in cell-substrate interactions, and some of them entered pores using their distinct tips or points along their length for initial attachment. Therefore, porous structures at either micro- or submicrometre scale supply positive guidance cues for anchorage-dependent cells to attach, leading to enhanced cell attachment. In contrast, the cells attached to a smooth titanium surface by focal contacts around their periphery as predominant adhesion structures, since repulsive signals from the environment led to retraction of the filopodia back to the cell bodies. These cells showed well-organized stress fibres, which exert tension across the cell body, resulting in flattened cells.     

Novel adhesion prevention membrane based on a bioresorbable copoly(ester-ether) comprised of poly-L-lactide and Pluronic: in vitro and in vivo evaluations.

Block copolymers consisting of poly(L-lactide) (PLLA) and poly(oxyethylene-co-oxypropylene), with various compositions, were synthesized and characterized in vitro and in vivo for their application as postoperative adhesion prevention membranes. It was found that the flexibility and degradability of the cast films of the block copolymers grew with increasing Pluronic F68 [PN; poly(oxyethylene-co-oxypropylene] composition. The receding contact angle of the copolymer films against water became lower than that of the PLLA film, because the surface was predominantly covered with more hydrophilic PN segments in a wet state. This surface property significantly affects the cell attachment property of the copolymer films, and the fibroblasts cultured on the films exhibit a spheroid-like morphology. The copolymer films subcutaneously implanted in the back of rats induced milder tissue responses compared with PLLA homopolymers, because of the increased surface hydrophilicity in the former. In vivo evaluation using a uterus horn model in rats revealed that the performance of these copolymer films as an adhesion-prevention membrane is comparable to that of a conventionally utilized membrane of oxidized regenerated cellulose. These results indicate that the copolymer films are biocompatible materials with controllable mechanical properties and biodegradability as adhesion-prevention membranes.     

Interaction of Zap70 and CXCR4 receptor at lamellipodia that determines the directionality during Jurkat T cells chemotaxis

Directional migration of T-lymphocytes is a key process during immune activation and is tightly regulated both temporally and spatially. The initial cell membrane protrusion at a particular site is critical for determining the direction of cell migration. In this study, we found that ZAP-70 protein appeared not only at the margin of the spreading areas of polarized Jurkat T cells but also formed clusters near the center of the cell body on a fibronectin plate. Specifically, some pZAP-70 was located at the lamellipodia/filopodia and was closely associated with the most extended membrane contact. To visualize the dynamic distribution of ZAP-70 on migrating Jurkat T cells, we generated a fluorescent ZAP-70-EGFP fusion protein (hZAP70G). Expression of the hZAP70G in P116 cells, a ZAP-70 defective Jurkat derivative, restored its chemotactic migration toward SDF-1, adhesion to fibronectin matrix, and integrin activation. In addition, the distribution of hZAP70G protein is associated with changes in cell shape, specifically the membrane protrusion step, forming filopodia/lamellipodia and a retracting uropod. Furthermore, SDF-1 stimulated the formation of ZAP-70 and CXCR4 complex. CXCR4 was observed mainly at the leading edge of migrating cell. The localization of ZAP-70 at the very front edge of protruding lamellipodia was close to CXCR4 and a part of them were overlapped. Collectively, our data describe the critical early step of directional cell movement toward SDF-1 that ZAP-70 is recruited to the CXCR4 at the leading edge of membrane and consequently modulates lamellipodia/filopodia formation and integrin activation.     

Characterization of nano hydroxyapatite/collagen surfaces and cellular behaviors

Osseointegration at the bone-implant interface is a prerequisite for endosseous implants to succeed in achieving and maintaining their long-term stability in bone tissue. The achievement of osseointegration is significantly affected by surface nature of implants. To optimize osseointegration, this study presents the characterization of synthesized nanocrystalline hydroxyapatite (nano HA) and in vitro studies on nano HA, nano-HA/collagen, and titanium surfaces. Voids were found within the grain of nano HA, which consisted of the shell and the core. The finding assists the clarification of microstructures of nano HA. By low-temperature mixing nano-HA sol with collagen gel (nano-HA/collagen 80:20), nano HA, and nano-HA/collagen coated on pure titanium or porous anodic titanium oxides resulted in higher wettability and lower roughness. The in vitro studies showed that porous structures produced by anodic oxides on titanium served as positive anchorage sites for cell filopodia to connect, and nano HA decreased cell attachment of osteoblasts and induced well-developed long filopodia and broad lamellipodia, thereby enhancing cellular motility. Collagen involvement enhanced cell adhesion to nano HA. Cell reactions to nano HA, nano-HA/collagen, native, and porous titanium surfaces provide some guidance for an optimal osseointegration by their application in surface modifications for implants.     

Antithrombogenicity and oxygen permeability of block and graft copolymers of polydimethylsiloxane and poly(alpha-amino acid)

A-B-A-type block copolymers consisting of poly(alpha-amino acid) as A component and polydimethylsiloxane as B component and graft copolymers consisting of polydimethylsiloxane as trunk polymer and poly(alpha-amino acid) as branch polymer were synthesized. gamma-Benzyl-L- or DL-glutamate, epsilon-benzyloxycarbonyl L-lysine, and sarcosine were used as alpha-amino acid. Different microphase-separated structures were found on the film surface according to the copolymer composition and the casting conditions. In vitro antithrombogenicity test showed higher antithrombogenicity of block or graft copolymers than homopolymers. The best antithrombogenicity was independent of the kind of alpha-amino acid and the degree of polymerization of copolymers. The best ratio was 65-75% in block copolymer and 40-50% in the case of graft copolymer. The oxygen permeability of block and graft copolymer film was intermediate between those of homopolymers and varied with changing the composition of the copolymer. These experiments showed that the microphase-separated structure on the film surface was most important both for the antithrombogenicity and oxygen permeability of these copolymer films.     

Sterically blocking adhesion of cells to biological surfaces with a surface-active copolymer containing poly(ethylene glycol) and phenylboronic acid.

Graft copolymers were designed that could spontaneously bind to biological surfaces and block subsequent recognition and adhesion at those surfaces. Phenylboronic acid (PBA) moieties in the polymer backbone provided binding to surfaces, forming reversible covalent complexes with cis-diols found in many biological molecules. Pendant poly(ethylene glycol) (PEG) side chains sterically protected those surfaces from subsequent interactions with other proteins and cells. The PEG and PBA grafting ratios on these poly-L-lysine-graft-(PEG;PBA) copolymers [PLL-g-(PEG;PBA)] were varied, and the polymers were tested in models relevant to undesirable wound-healing responses such as peritoneal adhesion formation and posterior capsule opacification. PLL-g-(PEG;PBA) polymers spontaneously coated tissue culture polystyrene and completely blocked rabbit lens epithelial cell adhesion to the surface over a wide range of PEG grafting ratios. PLL-g-(PEG;PBA)s with optimal grafting ratios were able to coat adsorbed serum proteins or extracellular matrices and block cell spreading on the surfaces at 4 h, although the effect was lost within 24 h. The polymer also enhanced the efficacy of surgical lysis of peritoneal adhesions in rats. The reversible covalent complexes formed by the PBA moieties on the copolymer backbone were more effective at binding biological surfaces than electrostatic interactions formed via a copolymer lacking the PBA moieties, that is, PLL-g-PEG.     

The in vitro blood compatibility of poly(ethylene oxide)-grafted polyurethane/polystyrene interpenetrating polymer networks

Poly(ethylene oxide) (PEO)-grafted polyurethane (PU)/polystyrene (PS) interpenetrating polymer networks (IPNs) were synthesized. The effects of the mobile pendant PEO chains with their microphase separated structure on blood-compatibility were investigated. The morphology of both the fracture surface as well as the top surface indicate that the size of the dispersed domains of the PS-rich phase decreased as the grafting with the PEO was increased. The swelling ratio also decreased as the grafting with the PEO was increased. However, the dynamic contact angle and the interfacial energy between IPN surface and water decreased, due to the structural reorganization of the pendant PEO chains. PU/PS IPNs have an excellent mechanical property as compared with PU homopolymers. The adsorption of bovine plasma fibrinogen (BPF) onto the PU/PS IPNs and PU homopolymers was effectively suppressed by the PEO-grafting. In the platelet adhesion test, the amount of platelets adsorbed, activated, and/or coagulated upon the PEO-grafted PU/PS IPNs were reduced when compared to the ungrafted PU homopolymers.     

The role of the interplay between polymer architecture and bacterial surface properties on the microbial adhesion to polyoxazoline-based ultrathin films

Surface platforms were engineered from poly(L-lysine)-graft-poly(2-methyl-2-oxazoline) (PLL-g-PMOXA) copolymers to study the mechanisms involved in the non-specific adhesion of Escherichia coli (E. coli) bacteria. Copolymers with three different grafting densities α (PMOXA chains/Lysine residue of 0.09, 0.33 and 0.56) were synthesized and assembled on niobia (Nb?O?) surfaces. PLL-modified and bare niobia surfaces served as controls. To evaluate the impact of fimbriae expression on the bacterial adhesion, the surfaces were exposed to genetically engineered E. coli strains either lacking, or constitutively expressing type 1 fimbriae. The bacterial adhesion was strongly influenced by the presence of bacterial fimbriae. Non-fimbriated bacteria behaved like hard, charged particles whose adhesion was dependent on surface charge and ionic strength of the media. In contrast, bacteria expressing type 1 fimbriae adhered to the substrates independent of surface charge and ionic strength, and adhesion was mediated by non-specific van der Waals and hydrophobic interactions of the proteins at the fimbrial tip. Adsorbed polymer mass, average surface density of the PMOXA chains, and thickness of the copolymer films were quantified by optical waveguide lightmode spectroscopy (OWLS) and variable-angle spectroscopic ellipsometry (VASE), whereas the lateral homogeneity was probed by time-of-flight secondary ion mass spectrometry (ToF-SIMS). Streaming current measurements provided information on the charge formation of the polymer-coated and the bare niobia surfaces. The adhesion of both bacterial strains could be efficiently inhibited by the copolymer film only with a grafting density of 0.33 characterized by the highest PMOXA chain surface density and a surface potential close to zero.     

The in vitro blood compatibility of poly(ethylene oxide)-grafted polyurethane/polystyrene interpenetrating polymer networks

Poly(ethylene oxide) (PEO)-grafted polyurethane (PU)/polystyrene (PS) interpenetrating polymer networks (IPNs) were synthesized. The effects of the mobile pendant PEO chains with their microphase separated structure on blood-compatibility were investigated. The morphology of both the fracture surface as well as the top surface indicate that the size of the dispersed domains of the PS-rich phase decreased as the grafting with the PEO was increased. The swelling ratio also decreased as the grafting with the PEO was increased. However, the dynamic contact angle and the interfacial energy between IPN surface and water decreased, due to the structural reorganization of the pendant PEO chains. PU/PS IPNs have an excellent mechanical property as compared with PU homopolymers. The adsorption of bovine plasma fibrinogen (BPF) onto the PU/PS IPNs and PU homopolymers was effectively suppressed by the PEO-grafting. In the platelet adhesion test, the amount of platelets adsorbed, activated, and/or coagulated upon the PEO-grafted PU/PS IPNs were reduced when compared to the ungrafted PU homopolymers.     

Stem cell attachment to layer-by-layer assembled TiO2 nanoparticle thin films

Surface topography is one of the most important factors influencing the attachment and spreading of cells. In the present study, layer-by-layer assembled titanium dioxide (TiO2) nanoparticle thin films were chosen for attachment, proliferation and spreading studies on mouse mesenchymal stem cells (MSC). Increasing surface roughness was observed with increasing number of layer-by-layer assembled TiO2 thin films. Four layer TiO2 thin film showed higher number of attached cells than a one layer thin film and control surfaces. MSCs experienced no cytotoxic effects after culture on the TiO2 coated substrates as observed from the cytotoxicity tests. Cell spreading, visualized with scanning electron microscopy, showed a faster rate of spreading on a rougher surface. Cells on a four-layer substrate, at 12 h showed complete spreading, where as most of the cells on a control surface and a one-layer surface, at 24 h, retained a rounded morphology. In conclusion, TiO2 nanoparticle thin films were successfully assembled in alternation with polyelectrolytes and in-vitro studies with MSC showed an increase in the attachment and faster spreading of cells on rougher surfaces.     

Adhesion and proliferation of OCT-1 osteoblast-like cells on micro- and nano-scale topography structured poly(L-lactide)

The impact of the surface topography of polylactone-type polymer on cell adhesion was to be concerned because the micro-scale texture of a surface can provide a significant effect on the adhesion behavior of cells on the surface. Especially for the application of tissue engineering scaffold, the pore size could have an influence on cell in-growth and subsequent proliferation. Micro-fabrication technology was used to generate specific topography to investigate the relationship between the cells and surface. In this study the pits-patterned surfaces of polystyrene (PS) film with diameters 2.2 and 0.45 microm were prepared by phase-separation, and the corresponding scale islands-patterned PLLA surface was prepared by a molding technique using the pits-patterned PS as a template. The adhesion and proliferation behavior of OCT-1 osteoblast-like cells morphology on the pits- and islands-patterned surface were characterized by SEM observation, cell attachment efficiency measurement and MTT assay. The results showed that the cell adhesion could be enhanced on PLLA and PS surface with nano-scale and micro-scale roughness compared to the smooth surfaces of the PLLA and PS. The OCT-1 osteoblast-like cells could grow along the surface with two different size islands of PLLA and grow inside the micro-scale pits of the PS. However, the proliferation of cells on the micro- and nano-scale patterned surface has not been enhanced compared with the controlled smooth surface.     

Self-assembling polystyrene-block-poly(ethylene oxide) copolymer surface coatings: Resistance to protein and cell adhesion

In this paper we report a method for biomaterial surface modification that utilizes the self-assembly of block copolymers of poly(styrene-block-ethylene oxide) (PS–PEO) to generate micro-phase separated surfaces with varying density PEO domains. These PS–PEO self-assembled surfaces showed a significant reduction in protein adsorption compared to control polystyrene surfaces. The adhesion of NIH-3T3 fibroblast cells was shown to be significantly affected by the surface coverage of PEO nano-domains formed by copolymer self-assembly. These nano-domains, when presented at high number density (almost 1000 domains per square micron), were shown to completely prevent cellular attachment, even though small amounts of protein were able to bind to the surface.     

Suppression of platelet activity on microdomain surfaces of 2-hydroxyethyl methacrylate-polyether block copolymers

Block copolymers constructed from chains of poly(2-hydroxyethyl methacrylate) (PHEMA) and either poly-ethyleneoxide (PEO) or poly-propyleneoxide (PPO) were synthesized. These block copolymers exhibited microdomain structure. Platelet adhesion on their surfaces was investigated by a column elution method to examine the effect of microdomain structure. The number of platelets adhered from whole blood was smaller for the block copolymer systems than for the homopolymers. Minimum points of platelet adhesion appeared at approximately 0.38 mol fraction of HEMA in the HEMA-PO system. Both block copolymer surfaces showed microdomains of alternate lamellar structure. Furthermore, the percent of platelets released from the column after incubation was investigated using PRP. In the case of homopolymers, released platelet percentages decreased with an increase of incubation time. Released platelet percentages from the block copolymers, however, were nearly constant with changing incubation time. These results show that HEMA-EO and HEMA-PO block copolymers had the ability to suppress both reversible and irreversible adhesion of platelets to their respective microdomain surfaces.     

Conditions of lateral surface confinement that promote tissue-cell integration and inhibit biofilm growth

Surfaces with cell adhesiveness modulated at micro length scales can exploit differences between tissue/bacterial cell size, membrane/wall plasticity, and adhesion mechanisms to differentially control tissue-cell/material and bacteria/material interactions. This study explores the short-term interactions of Staphylococcus aureus and osteoblast-like cells with surfaces consisting of cell-adhesive circular patches (1–5 μm diameter) separated by non-adhesive electron-beam patterned poly(ethylene glycol) hydrogel thin films at inter-patch distances of 0.5–10 μm. Osteoblast-like U2OS cells both bind to and spread on the modulated surfaces, in some cases when the cell-adhesive area comprises only 9% of the total surface and in several cases at least as well as on the continuously adhesive control surfaces. In contrast, S. aureus adhesion rates are 7–20 times less on the modulated surfaces than on the control surfaces. Furthermore, the proliferation of those bacteria that do adhere is inhibited by the lateral confinement imposed by the non-adhesive boundaries surrounding each patch. These findings suggest a new approach to create biomaterial surfaces that may promote healing while simultaneously reducing the probability of infection.     

Adhesion and differentiation of adipose-derived stem cells on a substrate with immobilized fibroblast growth factor

Control of cell-matrix interactions plays a role in the regulation of stem cell function. In this study basic fibroblast growth factor (bFGF) linked to maltose-binding protein (MBP) was designed as a matrix for cell adhesion. MBP-FGF was immobilized on polystyrene (PS) surfaces by spontaneous adsorption. The amount of MBP-bFGF immobilized on the PS surface increased with increasing protein concentration, being 158 ng cm(-2) at 10 μg ml(-1) protein. Human adipose-derived stem cell (hASC) adhesion to MBP-bFGF immobilized on a PS surface (PS-MBP-bFGF) was inhibited by heparin. Integrin signaling and cell spreading of hASC on PS-MBP-bFGF were down-regulated compared with those on fibronectin-coated surfaces or tissue culture polystyrene (TCP). hASC differentiated into adipocytes, which stained positive for lipid vacuoles with Oil Red, more readily on PS-MBP-bFGF than on TCP. In contrast, hASC hardly differentiated into osteoblast on PS-MBP-bFGF or on TCP. These results suggest that the mechanism of hASC adhesion to MBP-bFGF immobilized on a PS substrate is mediated by a specific interaction between bFGF and heparin, and that the adhesion mechanism might provide an insight into the design of biomaterials to control the fate of stem cells.     

Two different types of nonthrombogenic surfaces: PEG suppresses platelet adhesion ATP-independently but HEMA-St block copolymer requires ATP consumption of platelets to prevent adhesion

Poly(ethylene glycol) (PEG) and a hydrophobic-hydrophilic microdomain structured block copolymer comprising poly(2-hydroxyethyl methacrylate) and polystyrene (HEMA-St) have been reported to show good blood compatibility owing to inhibition of platelet activation. By using a computer-assisted novel technique to analyze platelet behavior on the surfaces, we found two different mechanisms to prevent platelet adhesion. Platelets were prevented from adhesion and spreading on the microdomain surface and retained cell movement for a long time. The platelet movement velocity was not significantly different between PEG-grafted surfaces and HEMA-St block copolymer-cast surfaces. However, platelet motion was qualitatively different. Platelets on HEMA-St block copolymer-cast surfaces moved with rolling, spinning, and vibrating, whereas platelet movement was limited to oscillatory vibration on PEG-grafted surfaces. When platelets were treated with NaN(3), an adenosine triphosphate (ATP) synthesis inhibitor, before contacting the surfaces, platelets movement velocity was decreased only on HEMA-St block copolymer-cast surfaces. Such an inhibitory effect was hardly observed with platelets on PEG-grafted surfaces. We propose two different mechanisms to prevent platelet adhesion onto surfaces. One is ATP-independent as observed with PEG, and the other is ATP-dependent for HEMA-St block copolymer, where platelets consume ATP to prevent adhesion.     

Block copolymer nanotemplating of tobacco mosaic and tobacco necrosis viruses

This paper examines the interaction between a block copolymer and a virus. A poly(styrene-b-4-vinylpyridine) block copolymer was loaded with nickel, and cast from a selective solvent mixture to form a cylindrical microstructure (PS/P4VP–Ni). The nickel ions were confined within the P4VP block of the copolymer. The binding of tobacco mosaic virus (TMV) and tobacco necrosis virus on microphase-separated PS/P4VP–Ni was examined. A staining technique was developed to simultaneously visualize virus and block copolymer structure by transmission electron microscopy. Electron microscopy revealed virus particles associated with block copolymer microphase-separated domains, even after extensive washes with Tween. In contrast, virus associated with PS/P4VP block copolymers lacking Ni were readily removed by Tween. The cylinder long axis of the microstructure was oriented using a hot press and a cooled channel die for quenching, resulting in PS/P4VP cylinders that had a strong anisotropic directional preference. When exposed to flowing solutions of TMV, the PS/P4VP–Ni surface exhibited an ability to retain TMV in a partially aligned state, when the direction of flow coincided with the long axis of the PS/P4VP–Ni cylinders. These results suggest that Coulombic interactions provide a robust means for the binding of virus particles to block copolymer surfaces.