Cell Type-Specific Adhesion and Migration on Laser-Structured Opaque Surfaces.

Cytocompatibility is essential for implant approval. However, initial in vitro screenings mainly include the quantity of adherent immortalized cells and cytotoxicity. Other vital parameters, such as cell migration and an in-depth understanding of the interaction between native tissue cells and implant surfaces, are rarely considered. We investigated different laser-fabricated spike structures using primary and immortalized cell lines of fibroblasts and osteoblasts and included quantification of the cell area, aspect ratio, and focal adhesions. Furthermore, we examined the three-dimensional cell interactions with spike topographies and developed a tailored migration assay for long-term monitoring on opaque materials. While fibroblasts and osteoblasts on small spikes retained their normal morphology, cells on medium and large spikes sank into the structures, affecting the composition of the cytoskeleton and thereby changing cell shape. Up to 14 days, migration appeared stronger on small spikes, probably as a consequence of adequate focal adhesion formation and an intact cytoskeleton, whereas human primary cells revealed differences in comparison to immortalized cell lines. The use of primary cells, analysis of the cell-implant structure interaction as well as cell migration might strengthen the evaluation of cytocompatibility and thereby improve the validity regarding the putative in vivo performance of implant material.

Liquid-Infused Structured Titanium Surfaces: Antiadhesive Mechanism to Repel Streptococcus oralis Biofilms.

To combat implant-associated infections, there is a need for novel materials which effectively inhibit bacterial biofilm formation. In the present study, the antiadhesive properties of titanium surface functionalization based on the "slippery liquid-infused porous surfaces" (SLIPS) principle were demonstrated and the underlying mechanism was analyzed. The immobilized liquid layer was stable over 13 days of continuous flow in an oral flow chamber system. With increasing flow rates, the surface exhibited a significant reduction in attached biofilm of both the oral initial colonizer  Streptococcus oralis and an oral multispecies biofilm composed of S. oralis, Actinomyces naeslundii, Veillonella dispar, and Porphyromonas gingivalis. Using single cell force spectroscopy, reduced S. oralis adhesion forces on the lubricant layer could be measured. Gene expression patterns in biofilms on SLIPS, on control surfaces, and expression patterns of planktonic cultures were also compared. For this purpose, the genome of S. oralis strain ATCC 9811 was sequenced using PacBio Sequel technology. Even though biofilm cells showed clear changes in gene expression compared to planktonic cells, no differences could be detected between bacteria on SLIPS and on control surfaces. Therefore, it can be concluded that the ability of liquid-infused titanium to repel S. oralis biofilms is mainly due to weakened bacterial adhesion to the underlying liquid interface.

Development of Laser-Structured Liquid-Infused Titanium with Strong Biofilm-Repellent Properties.

Medical implants are commonly used in modern medicine but still harbor the risk of microbial infections caused by bacterial biofilms. As their retrospective treatment is difficult, there is a need for biomedical materials that inhibit bacterial colonization from the start without using antibacterial agents, as these can promote resistance development. The promising concept of slippery liquid-infused porous surfaces (SLIPS) possesses enormous potential for this purpose. In the present study, this principle was applied to titanium, a common material in implantology, and its biofilm-repellent properties were demonstrated. To simplify prospective approval of the medical device and to avoid chemical contamination, surface structuring was performed by ultrashort pulsed laser ablation. Four different structures (hierarchical micro- and nanosized spikes, microsized grooves, nanosized ripples, and unstructured surfaces) and five infusing perfluoropolyethers of different viscosities were screened; the best results were obtained with the biomimetic, hierarchical spike structure combined with lubricants of medium viscosities (20-60 cSt at 37 °C, 143 AZ, and GPL 104). The surfaces exhibited extremely low contact angle hysteresis, as is typical for liquid-infused materials and a reliable 100-fold reduction of human oral pathogen Streptococcus oralis biofilms. This characteristic was maintained after exposure to shear forces and gravity. The titanium SLIPS also inhibited adherence of human fibroblasts and osteoblasts. Toxicity tests supported the explanation that solely the surface's repellent properties are responsible for the vigorous prevention of the adhesion of bacteria and cells. This use of physically structured and liquid-infused titanium to avoid bioadhesion should support the prevention of bacterial implant-associated infections without the use of antibacterial agents.

Dry friction of microstructured polymer surfaces inspired by snake skin.

The microstructure investigated in this study was inspired by the anisotropic microornamentation of scales from the ventral body side of the California King Snake (Lampropeltis getula californiae). Frictional properties of snake-inspired microstructured polymer surface (SIMPS) made of epoxy resin were characterised in contact with a smooth glass ball by a microtribometer in two perpendicular directions. The SIMPS exhibited a considerable frictional anisotropy: Frictional coefficients measured along the microstructure were about 33% lower than those measured in the opposite direction. Frictional coefficients were compared to those obtained on other types of surface microstructure: (i) smooth ones, (ii) rough ones, and (iii) ones with periodic groove-like microstructures of different dimensions. The results demonstrate the existence of a common pattern of interaction between two general effects that influence friction: (1) molecular interaction depending on real contact area and (2) the mechanical interlocking of both contacting surfaces. The strongest reduction of the frictional coefficient, compared to the smooth reference surface, was observed at a medium range of surface structure dimensions suggesting a trade-off between these two effects.

Impact of laser-structured biomaterial interfaces on guided cell responses.

To achieve a perfect integration of biomaterials into the body, tissue formation in contact with the interface has to be controlled. In this connection, a selective cell control is required: fibrotic encapsulation has to be inhibited, while tissue guidance has to be stimulated. As conventional biomaterials do not fulfil this specification, functionalization of the biointerface is under development to mimic the natural environment of the cells. One approach focuses on the fabrication of defined surface topographies. Thereby, ultrashort pulse laser ablation is very beneficial, owing to a large variety of fabricated structures, reduced heat-affected zones, high precision and reproducibility. We demonstrate that nanostructures in platinum and microstructures in silicon selectively control cell behaviour: inhibiting fibroblasts, while stimulating neuronal attachment and differentiation. However, the control of fibroblasts strongly correlates with the created size dimensions of the surface structures. These findings suggest favourable biomaterial interfaces for electronic devices. The mechanisms which are responsible for selective cell control are poorly understood. To give an insight, cell behaviour in dependence of biomaterial interfaces is discussed-including basic research on the role of the extracellular matrix. This knowledge is essential to understand such specific cell responses and to optimize biomaterial interfaces for future biomedical applications.

Two-photon polymerization microstructuring in regenerative medicine.

Two-photon polymerization has developed as a powerful tool for making micro- and nanoscale structures for regenerative medicine applications. This review discusses micro- and nanoscale aspects of tissue engineering, which are followed by a brief description of the two-photon polymerization process and how it has been used thus far in tissue engineering and other regenerative medicine applications. Lastly, potential future applications of two-photon polymerization in regenerative medicine are presented. This review provides a comprehensive summary of the uses of two-photon polymerization thus far in regenerative medicine and a look into how this technique will be used in the future.

Topography and coating of platinum improve the electrochemical properties and neuronal guidance.

To improve neuronal-electrode interfaces, we analyzed the influence of surface topographies combined with coating on the electrochemistry of platinum and neuronal differentiation of PC-12 cells. Surface structuring on nanoscale was realized by femtosecond laser ablation. Additional coating with laminin (LA), collagen type I (COL) or poly-d-lysine (PDL) did not change the produced topography. We further demonstrated that impedance could be improved in all cases. The pre-requisites of differentiation - viability and attachment - were fulfilled on the topography. Cell attachment of non-differentiated and differentiated cells and their formation of focal adhesion complexes were even enhanced compared to unstructured platinum. However, without the nerve growth factor (NGF) no cellular outgrowth and differentiation were possible. The topography enabled cell elongation and reduced the amount of rounded cells, but less effective than coating. Differentiation was either comparable or increased on the structures when compared with unstructured coatings. For instance, microtubule associated protein (MAP2) was detected most on the topography alone. But a combination of surface structuring and coating had the strongest impact on differentiation: the usage of COL provoked best cell elongation and beta III tubulin expression, PDL best synaptophysin. LA-coating had no noteworthy effect. These findings point out that innovative electronic devices like cochlear implants include two aspects: (a) nanotopography to improve the transmission of electrical signals and neuronal attachment; and (b) an additional coating to stimulate neuronal differentiation.

Evaluation of single-cell force spectroscopy and fluorescence microscopy to determine cell interactions with femtosecond-laser microstructured titanium surfaces.

One goal in biomaterials research is to limit the formation of connective tissue around the implant. Antiwetting surfaces are known to reduce ability of cells to adhere. Such surfaces can be achieved by special surface structures (lotus effect). Aim of the study was to investigate the feasibility for creating antiwetting surface structures on titanium and to characterize their effect on initial cell adhesion and proliferation. Titanium microstructures were generated using femtosecond- (fs-) laser pulses. Murine fibroblasts served as a model for connective tissue cells. Quantitative investigation of initial cell adhesion was performed using atomic force microscopy. Fluorescence microscopy was used for the characterization of cell-adhesion pattern, cell morphology, and proliferation. Water contact angle (WCA) measurements evinced antiwetting properties of laser-structured surfaces. However, the WCA was decreased in serum-containing medium. Initial cell adhesion to microstructured titanium was significantly promoted when compared with polished titanium. Microstructures did not influence cell proliferation on titanium surfaces. However, on titanium microstructures, cells showed a flattened morphology, and the cell orientation was biased according to the surface topography. In conclusion, antiwetting properties of surfaces were absent in the presence of serum and did not hinder adhesion and proliferation of NIH 3T3 fibroblasts.

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.

Directing neuronal cell growth on implant material surfaces by microstructuring.

For best hearing sensation, electrodes of auditory prosthesis must have an optimal electrical contact to the respective neuronal cells. To improve the electrode-nerve interface, microstructuring of implant surfaces could guide neuronal cells toward the electrode contact. To this end, femtosecond laser ablation was used to generate linear microgrooves on the two currently relevant cochlear implant materials, silicone elastomer and platinum. Silicone surfaces were structured by two different methods, either directly, by laser ablation or indirectly, by imprinting using laser-microstructured molds. The influence of surface structuring on neurite outgrowth was investigated utilizing a neuronal-like cell line and primary auditory neurons. The pheochromocytoma cell line PC-12 and primary spiral ganglion cells were cultured on microstructured auditory implant materials. The orientation of neurite outgrowth relative to the microgrooves was determined. Both cell types showed a preferred orientation in parallel to the microstructures on both, platinum and on molded silicone elastomer. Interestingly, microstructures generated by direct laser ablation of silicone did not influence the orientation of either cell type. This shows that differences in the manufacturing procedures can affect the ability of microstructured implant surfaces to guide the growth of neurites. This is of particular importance for clinical applications, since the molding technique represents a reproducible, economic, and commercially feasible manufacturing procedure for the microstructured silicone surfaces of medical implants.

Bacterial retention on superhydrophobic titanium surfaces fabricated by femtosecond laser ablation.

Two-tier micro- and nanoscale quasi-periodic self-organized structures, mimicking the surface of a lotus Nelumbo nucifera leaf, were fabricated on titanium surfaces using femtosecond laser ablation. The first tier consisted of large grainlike convex features between 10 and 20 µm in size. The second tier existed on the surface of these grains, where 200 nm (or less) wide irregular undulations were present. The introduction of the biomimetic surface patterns significantly transformed the surface wettabilty of the titanium surface. The original surface possessed a water contact angle of θ(W) 73 ± 3°, whereas the laser-treated titanium surface became superhydrophobic, with a water contact angle of θ(W) 166 ± 4°. Investigations of the interaction of S. aureus and P. aeruginosa with these superhydrophobic surfaces at the surface-liquid interface revealed a highly selective retention pattern for two pathogenic bacteria. While S. aureus cells were able to successfully colonize the superhydrophobic titanium surfaces, no P. aeruginosa cells were able to attach to the surface (i.e., any attached bacterial cells were below the estimated lower detection limit).

Femtosecond laser fabricated spike structures for selective control of cellular behavior.

In this study we investigate the potential of femtosecond laser generated micrometer sized spike structures as functional surfaces for selective cell controlling. The spike dimensions as well as the average spike to spike distance can be easily tuned by varying the process parameters. Moreover, negative replications in soft materials such as silicone elastomer can be produced. This allows tailoring of wetting properties of the spike structures and their negative replicas representing a reduced surface contact area. Furthermore, we investigated material effects on cellular behavior. By comparing human fibroblasts and SH-SY5Y neuroblastoma cells we found that the influence of the material was cell specific. The cells not only changed their morphology, but also the cell growth was affected. Whereas, neuroblastoma cells proliferated at the same rate on the spike structures as on the control surfaces, the proliferation of fibroblasts was reduced by the spike structures. These effects can result from the cell specific adhesion patterns as shown in this work. These findings show a possibility to design defined surface microstructures, which could control cellular behavior in a cell specific manner.

Differential fine-tuning of cochlear implant material-cell interactions by femtosecond laser microstructuring.

Cochlear implants (CIs) can restore hearing in deaf patients by electrical stimulation of the auditory nerve. To optimize the electrical stimulation, the number of independent channels must be increased by reduction of connective tissue growth on the electrode surface and selective neuronal cell contact. The femtosecond laser microstructuring of the electrode surfaces was performed to investigate the effect of fibroblast growth on the implant material. A cell culture model system was established to evaluate cell-material interactions on these microstructured CI-electrode materials. Fibroblasts were used as a cell culture model for connective tissue formation, and differentiating neuronal-like cells were employed to represent neuronal cells. For nondestructive microscopic examination of living cells on the structured surfaces, the cells were genetically modified to express green fluorescent protein. To investigate the special interaction between the electrode material and the tissue we used electrode material which is originally used for manufacturing CI for human applications, namely platinum (contact material) and silicone carrier material (LSR 30, HCRP 50). Microstructures of various dimensions (groove width 1-10 microm) were generated by using femtosecond laser ablation. The highest fibroblast growth rate was observed on platinum, but cell growth rates on the silicone carrier material were lower. Microstructuring reduced fibroblast cell growth on platinum significantly. On the microstructured silicone, a trend to lower cell growth rates was observed. In addition, microgrooves on platinum surfaces can direct neurite outgrowth parallel to the grooves. The implications of the results are discussed with respect to the design of a microstructured CI surface.