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.

Hyaluronic acid based materials for scaffolding via two-photon polymerization.

Hydrogels are able to mimic the basic three-dimensional (3D) biological, chemical, and mechanical properties of native tissues. Since hyaluronic acid (HA) is a chief component of human extracellular matrix (ECM), it represents an extremely attractive starting material for the fabrication of scaffolds for tissue engineering. Due to poor mechanical properties of hydrogels, structure fabrication of this material class remains a major challenge. Two-photon polymerization (2PP) is a promising technique for biomedical applications, which allows the fabrication of complex 3D microstructures by moving the laser focus in the volume of a photosensitive material. Chemical modification of hyaluronan allows application of the 2PP technique to this natural material and, thus, precise fabrication of 3D hydrogel constructs. To create materials with tailor-made mechanochemical properties, HA was combined and covalently cross-linked with poly(ethylene glycol) diacrylate (PEGDA) in situ. 2PP was applied for the fabrication of well elaborated 3D HA and HA-PEGDA microstructures. For enhanced biological adaption, HA was functionalized with human epidermal growth factor.

3D fabrication of all-polymer conductive microstructures by two photon polymerization.

A technique to fabricate electrically conductive all-polymer 3D microstructures is reported. Superior conductivity, high spatial resolution and three-dimensionality are achieved by successive application of two-photon polymerization and in situ oxidative polymerization to a bi-component formulation, containing a photosensitive host matrix and an intrinsically conductive polymer precursor. By using polyethylene glycol diacrylate (PEG-DA) and 3,4-ethylenedioxythiophene (EDOT), the conductivity of 0.04 S/cm is reached, which is the highest value for the two-photon polymerized all-polymer microstructures to date. The measured electrical conductivity dependency on the EDOT concentration indicates percolation phenomenon and a three-dimensional nature of the conductive pathways. Tunable conductivity, biocompatibility, and environmental stability are the characteristics offered by PEG-DA/EDOT blends which can be employed in biomedicine, MEMS, microfluidics, and sensorics.

Fabrication of microneedles using two photon polymerization for transdermal delivery of nanomaterials.

Microneedle devices for transdermal delivery of nanoscale pharmacologic agents were fabricated out of organically-modified ceramic (Ormocer) materials using two photon polymerization. Out-of-plane hollow microneedle arrays with various aspect ratios were fabricated using this rapid prototyping process. Human epidermal keratinocyte (HEK) viability on Ormocer surfaces fabricated using two photon polymerization was similar to that on control surfaces. Nanoindentation studies were performed to determine hardness and Young's modulus values for Ormocer materials. Microneedies were shown to enable more rapid distribution of the PEG-amine quantum dot solution to the deep epidermis and dermis layers of porcine skin than topical administration. Our results suggest that two photon polymerization may be used to create microneedle arrays for transdermal delivery of nanoscale pharmacologic agents.

Shrinkage of microstructures produced by two-photon polymerization of Zr-based hybrid photosensitive materials.

An investigation of the shrinking behaviour of a zirconium-based sol-gel composite micro-structured by two-photon polymerization is presented and a simple, straightforward methodology allowing the evaluation of shrinkage is suggested. It is shown that volume reduction is directly related to the average laser power (irradiation dose) used for the microfabrication and becomes a critical issue near the polymerization threshold. It is demonstrated that this shrinkage can be employed beneficially to improve the structural resolution. This is demonstrated by the presence of stopbands in the photonic crystal nanostructures fabricated with controlled volume reduction. Well above the polymerization threshold, the studied material exhibits remarkably low shrinkage. Therefore, no additional effort for the pre-compensation of distortion and for the improvement of structural stability is required.

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.

Three-dimensional cell growth on structures fabricated from ORMOCER by two-photon polymerization technique.

Two-photon polymerization technique was applied to generate three-dimensional (3D) scaffold-like structures using the photosensitive organic-inorganic hybrid polymer ORMOCER. The structures were studied with respect to potential applications as scaffold for tissue engineering. Cell counting and comet assay, respectively, demonstrated that doubling time and DNA strand breaks of CHO cells, GFSHR-17 granulosa cells, GM-7373 endothelial cells, and SH-SY5Y neuroblastoma cells were not affected by ORMOCER. ORMOCER related alteration of formation of tissue specific cell-to-cell adhesions like gap junctions was ruled out by double whole-cell patch-clamp technique. Additionally, growth of cells on the vertical surfaces of 3D structures composed of ORMOCER is shown.

Water-soluble photopolymerizable chitosan hydrogels for biofabrication via two-photon polymerization.

Fabrication of three-dimensional (3D) hydrogel microenvironments with predefined geometry and porosity can facilitate important requirements in tissue engineering and regenerative medicine. Chitosan (CH) is well known as a biocompatible hydrogel with prospective biological properties for biomedical aims. So far, microstructuring of this soft material presents a great limitation for its application as functional supporting material for guided tissue formation. Enabling photopolymerization, chemically modified CH can be applied for the biofabrication of reproducible 3D scaffolds using rapid prototyping techniques like two-photon polymerization (2PP) or others. The application of this technique allows precise serial fabrication of computer-designed microstructure geometries by scanning a femtosecond laser beam within a photosensitive material. This work explores a new synthesis of water-soluble photosensitive chitosan and the fabrication of well-defined microstructures from the generated materials. To modulate the mechanical and biochemical properties of the material, CH was combined and cross-linked with synthetic poly(ethylene glycol) diacrylate. For a biological adaption to the in vivo situation, CH was covalently crosslinked with a photosensitive modified vascular endothelial growth factor (VEGF). Performed in vitro studies reveal that modified CH is biocompatible. VEGF enhances CH bioactivity. Furthermore, a 3D CH scaffold can be successfully seeded with cells. Therefore, the established CH holds great promise for future applications in tissue engineering.

Two-photon polymerization of 3-D zirconium oxide hybrid scaffolds for long-term stem cell growth.

Two-photon polymerization is a technique that involves simultaneous absorption of two photons from a femtosecond laser for selective polymerization of a photosensitive material. In this study, two-photon polymerization was used for layer-by-layer fabrication of 3-D scaffolds composed of an inorganic-organic zirconium oxide hybrid material. Four types of scaffold microarchitectures were created, which exhibit layers of parallel line features at various orientations as well as pores between the line features. Long-term cell culture studies involving human bone marrow stromal cells were conducted using these 3-D scaffolds. Cellular adhesion and proliferation were demonstrated on all of the scaffold types; tissuelike structure was shown to span the pores. This study indicates that two-photon polymerization may be used to create microstructured scaffolds out of an inorganic-organic zirconium oxide hybrid material for use in 3-D tissue culture systems.

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.

Two-photon polymerization of polyethylene glycol diacrylate scaffolds with riboflavin and triethanolamine used as a water-soluble photoinitiator.

AIM: In this study, the suitability of a mixture containing riboflavin (vitamin B2) and triethanolamine (TEOHA) as a novel biocompatible photoinitiator for two-photon polymerization (2PP) processing was investigated. MATERIALS & METHODS: Polyethylene glycol diacrylate was crosslinked using Irgacure(®) 369, Irgacure 2959 or a riboflavin-TEOHA mixture; biocompatibility of the photopolymer extract solutions was subsequently assessed via endothelial cell proliferation assay, endothelial cell viability assay and single-cell gel electrophoresis (comet) assay. Use of a riboflavin-TEOHA mixture as a photoinitiator for 2PP processing of a tissue engineering scaffold and subsequent seeding of this scaffold with GM-7373 bovine aortic endothelial cells was also demonstrated. RESULTS: The riboflavin-TEOHA mixture was found to produce much more biocompatible scaffolds than those produced with Irgacure 369 or Irgacure 2959. CONCLUSION: The results suggest that riboflavin is a promising component of photoinitiators for 2PP fabrication of tissue engineering scaffolds and other medically relevant structures (e.g., biomicroelectromechanical systems).

Dynamics of cell attachment: adhesion time and force.

Due to its biological significance, cell adhesion to biomaterial surfaces or scaffolds is the key step in biomedical applications. Here, we describe two sensitive and facile methods that quantify the kinetic and mechanic properties of the entire cell attachment process characterized by two parameters: Adhesion Time T(Ad) and Adhesion Force F(Ad). We demonstrate that both methods can be applied to any adherent cell type (e.g., stem or cancer cells), tissue-engineered substrate, and culture condition in a fast, effective, and reproducible manner. Additional investigations about the role of the extracellular matrix and the formation of focal contacts help in acquiring further interpretations of these parameters from biological and mechanical points of view.

Multiphoton microscopy of transdermal quantum dot delivery using two photon polymerization-fabricated polymer microneedles.

Due to their ability to serve as fluorophores and drug delivery vehicles, quantum dots are a powerful tool for theranostics-based clinical applications. In this study, microneedle devices for transdermal drug delivery were fabricated by means of two-photon polymerization of an acrylate-based polymer. We examined proliferation of cells on this polymer using neonatal human epidermal keratinocytes and human dermal fibroblasts. The microneedle device was used to inject quantum dots into porcine skin; imaging of the quantum dots was performed using multiphoton microscopy.

Laser fabrication of large-scale nanoparticle arrays for sensing applications.

A novel method for high-speed fabrication of large scale periodic arrays of nanoparticles (diameters 40-200 nm) is developed. This method is based on a combination of nanosphere lithography and laser-induced transfer. Fabricated spherical nanoparticles are partially embedded into a polymer substrate. They are arranged into a hexagonal array and can be used for sensing applications. An optical sensor with the sensitivity of 365 nm/RIU and the figure of merit of 21.5 in the visible spectral range is demonstrated.

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.

Two-photon polymerization of microneedles for transdermal drug delivery.

IMPORTANCE OF THE FIELD: Microneedles are small-scale devices that are finding use for transdermal delivery of protein-based pharmacologic agents and nucleic acid-based pharmacologic agents; however, microneedles prepared using conventional microelectronics-based technologies have several shortcomings, which have limited translation of these devices into widespread clinical use. AREAS COVERED IN THIS REVIEW: Two-photon polymerization is a laser-based rapid prototyping technique that has been used recently for direct fabrication of hollow microneedles with a wide variety of geometries. In addition, an indirect rapid prototyping method that involves two-photon polymerization and polydimethyl siloxane micromolding has been used for fabrication of solid microneedles with exceptional mechanical properties. WHAT THE READER WILL GAIN: In this review, the use of two-photon polymerization for fabricating in-plane and out-of-plane hollow microneedle arrays is described. The use of two-photon polymerization-micromolding for fabrication of solid microneedles is also reviewed. In addition, fabrication of microneedles with antimicrobial properties is discussed; antimicrobial microneedles may reduce the risk of infection associated with the formation of channels through the stratum corneum. TAKE HOME MESSAGE: It is anticipated that the use of two-photon polymerization as well as two-photon polymerization-micromolding for fabrication of microneedles and other microstructured drug delivery devices will increase over the coming years.

Fabrication of polymer microneedles using a two-photon polymerization and micromolding process.

BACKGROUND: Microneedle-mediated drug delivery is a promising method for transdermal delivery of insulin, incretin mimetics, and other protein-based pharmacologic agents for treatment of diabetes mellitus. One factor that has limited clinical application of conventional microneedle technology is the poor fracture behavior of microneedles that are created using conventional materials and methods. In this study polymer microneedles for transdermal delivery were created using a two-photon polymerization (2PP) microfabrication and subsequent polydimethylsiloxane (PDMS) micromolding process. METHODS: Solid microneedle arrays, fabricated by means of 2PP, were used to create negative molds from PDMS. Using these molds microneedle arrays were subsequently prepared by molding eShell 200, a photo-reactive acrylate-based polymer that exhibits water and perspiration resistance. RESULTS: The eShell 200 microneedle array demonstrated suitable compressive strength for use in transdermal drug delivery applications. Human epidermal keratinocyte viability on the eShell 200 polymer surfaces was similar to that on polystyrene control surfaces. In vitro studies demonstrated that eShell 200 microneedle arrays fabricated using the 2PP microfabrication and PDMS micromolding process technique successfully penetrated human stratum corneum and epidermis. CONCLUSIONS: Our results suggest that a 2PP microfabrication and subsequent PDMS micromolding process may be used to create microneedle structures with appropriate structural, mechanical, and biological properties for transdermal drug delivery of insulin and other protein-based pharmacologic agents for treatment of diabetes mellitus.

Rapid prototyping of scaphoid and lunate bones.

In this study, a novel rapid prototyping technology was used to fabricate scaphoid and lunate bone prostheses, two carpal bones that are prone to avascular necrosis. Carpal prostheses were fabricated with an Envisiontec Perfactory SXGA stereolithography system using Envisiontec eShell 200 photocurable polymer. Fabrication was guided using 3-D models, which were generated using Mimics software (Materialise NV, Leuven, Belgium) from patient computer tomography data. The prostheses were fabricated in a layer-by-layer manner; approximately 50-microm thick layers were observed in the prostheses. Hardness and Young's modulus values of polymerized eShell 200 material were 93.8 +/- 7.25 MPa and 3050 +/- 90 MPa, respectively. The minimum compressive force required for fracture was 1360 N for the scaphoid prosthesis and 1248 N for the lunate prosthesis. Polymerized Envisiontec eShell material exhibited high human neonatal epidermal keratinocyte cell viability rate in an MTT assay. The results of this study indicate that small bone prostheses fabricated by stereolithography using eShell 200 polymer may have suitable geometry, mechanical properties, and cytocompatibility properties for in vivo use.

Three-dimensional biodegradable structures fabricated by two-photon polymerization.

Two-photon polymerization has been employed to fabricate three-dimensional structures using the biodegradable triblock copolymer poly(epsilon-caprolactone-co-trimethylenecarbonate)-b-poly(ethylene glycol)-b-poly(epsilon-caprolactone-co-trimethylenecarbonate) with 4,4'-bis(diethylamino)benzophenone as the photoinitiator. The fabricated structures were of good quality and had four micron resolution. Initial cytotoxicity tests show that the material does not affect cell proliferation. These studies demonstrate the potential of two-photon polymerization as a technology for the fabrication of biodegradable scaffolds for tissue engineering.

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.