S. aureus develops biofilms on titanium within 2 weeks in vitro.

BACKGROUND: The formation of biofilms, characterized by cell aggregation and extracellular polymeric substance (EPS) production, is a common feature of periprosthetic joint infections (PJI). OBJECTIVE: The current study aimed to investigate the development of biofilm features in vitro within less than 3 weeks by Staphylococcus aureus isolated from PJIs. METHODS: Biofilms were grown on sandblasted titanium discs, and fluorescence spectroscopy and microscopy were used to observe biofilm maturation for 21 days. RESULTS: DNA mass decreased initially, then increased from day 5 onwards, and decreased again after day 7. The proportion of living to dead bacteria oscillated until day 7 and increased at day 10 for strain A and day 14 for strain B. EPS mass decreased initially and then continuously increased. Multilayer bacterial organization was observed at day 7. CONCLUSION: Cell aggregation occurred during the first week, followed by EPS production in the second week, and characteristic biofilm features were observed within 1 to 2 weeks.

From Shape to Function: The Next Step in Bioprinting.

In 2013, the "biofabrication window" was introduced to reflect the processing challenge for the fields of biofabrication and bioprinting. At that time, the lack of printable materials that could serve as cell-laden bioinks, as well as the limitations of printing and assembly methods, presented a major constraint. However, recent developments have now resulted in the availability of a plethora of bioinks, new printing approaches, and the technological advancement of established techniques. Nevertheless, it remains largely unknown which materials and technical parameters are essential for the fabrication of intrinsically hierarchical cell-material constructs that truly mimic biologically functional tissue. In order to achieve this, it is urged that the field now shift its focus from materials and technologies toward the biological development of the resulting constructs. Therefore, herein, the recent material and technological advances since the introduction of the biofabrication window are briefly summarized, i.e., approaches how to generate shape, to then focus the discussion on how to acquire the biological function within this context. In particular, a vision of how biological function can evolve from the possibility to determine shape is outlined.

Thiol-Ene Clickable Gelatin: A Platform Bioink for Multiple 3D Biofabrication Technologies.

Bioprinting can be defined as the art of combining materials and cells to fabricate designed, hierarchical 3D hybrid constructs. Suitable materials, so called bioinks, have to comply with challenging rheological processing demands and rapidly form a stable hydrogel postprinting in a cytocompatible manner. Gelatin is often adopted for this purpose, usually modified with (meth-)acryloyl functionalities for postfabrication curing by free radical photopolymerization, resulting in a hydrogel that is cross-linked via nondegradable polymer chains of uncontrolled length. The application of allylated gelatin (GelAGE) as a thiol-ene clickable bioink for distinct biofabrication applications is reported. Curing of this system occurs via dimerization and yields a network with flexible properties that offer a wider biofabrication window than (meth-)acryloyl chemistry, and without additional nondegradable components. An in-depth analysis of GelAGE synthesis is conducted, and standard UV-initiation is further compared with a recently described visible-light-initiator system for GelAGE hydrogel formation. It is demonstrated that GelAGE may serve as a platform bioink for several biofabrication technologies by fabricating constructs with high shape fidelity via lithography-based (digital light processing) 3D printing and extrusion-based 3D bioprinting, the latter supporting long-term viability postprinting of encapsulated chondrocytes.

Additive Manufacturing of a Photo-Cross-Linkable Polymer via Direct Melt Electrospinning Writing for Producing High Strength Structures.

Melt electrospinning writing (MEW) is an emerging additive manufacturing technique that enables the design and fabrication of micrometer-thin fibrous scaffolds made of biocompatible and biodegradable polymers. By using a computer-aided deposition process, a unique control over pore size and interconnectivity of the resulting scaffolds is achieved, features highly interesting for tissue engineering applications. However, MEW has been mainly used to process low melting point thermoplastics such as poly(ε-caprolactone). Since this polymer exhibits creep and a reduction in modulus upon hydration, we manufactured scaffolds of poly(L-lactide-co-ε-caprolactone-co-acryloyl carbonate) (poly(LLA-ε-CL-AC)), a photo-cross-linkable and biodegradable polymer, for the first time. We show that the stiffness of the scaffolds increases significantly (up to ∼10-fold) after cross-linking by UV irradiation at room temperature, compared with un-cross-linked microfiber scaffolds. The preservation of stiffness and high average fiber modulus (370 ± 166 MPa) within the cross-linked hydrated scaffolds upon repetitive loading (10% strain at 1 Hz up to 200,000 cycles) suggests that the prepared scaffolds may be of potential interest for soft connective tissue engineering applications. Moreover, the approach can be readily adapted through manipulation of polymer properties and scaffold geometry to prepare structures with mechanical properties suitable for other tissue engineering applications.

Application of Linear and Branched Poly(Ethylene Glycol)-Poly(Lactide) Block Copolymers for the Preparation of Films and Solution Electrospun Meshes.

Poly(ethylene glycol)-poly(lactide) (PEG-PLA) block copolymers are processed to solvent cast films and solution electrospun meshes. The effect of polymer composition, architecture, and number of anchoring points for the plasticizer on swelling, degradation, and mechanical properties of these films and meshes is investigated as potential barrier device for the prevention of peritoneal adhesions. As a result, adequate properties are achieved for the massive films with a longer retention of the plasticizer PEG for star-shaped block copolymers than for the linear triblock copolymers and consequently more endurable mechanical properties during degradation. For electrospun meshes fabricated using the same polymers, similar trends are observed, but with an earlier start of fragmentation and lower tensile strengths. To overcome the poor mechanical strengths and an occurring shrinkage during incubation, which may impair the coverage of the wound, further adaptions of the meshes and the fabrication process are necessary.

Nanogels with high active ß-cyclodextrin content as physical coating system with sustained release properties.

We present the application of nanogels with high functional ß-cyclodextrin (ß-CD) content as new and versatile method for the modification and protection of textiles. The complexation potential of covalently embedded ß-CD in nanogels is demonstrated for the common insecticide permethrin in aqueous environment. It is shown that permethrin containing ß-CD nanogels can be applied easily, homogeneously and safely on keratin fibers like wool fabrics and human hairs. The permethrin concentration on fibers is directly controlled by the permethrin content in nanogels. We tested the permanence of permethrin on treated fibers with regard to washing and UV fastness. Our results show that permethrin complexed in nanogels is removed from the textile during washing, but that the complexation of permethrin by ß-CD domains in the nanogels protects the active ingredient from UV degradation. Bioassay tests against the larvae of Tineola bisselliella and Anthrenocerus australis show that the activity of the ingredients does not decrease after complexation in ß-CD gels and it results in protection of the wool fibers against degradation by the insect larvae.

Unidirectional control of anisotropic wetting through surface modification of PDMS microstructures.

It has been shown before that anisotropically microstructured surfaces exhibit anisotropic wetting phenomena. This study presents a possibility to control the anisotropy of wetting by tailoring the surface chemistry. PDMS microchannels were permanently hydrophilized and subsequently functionalized further. Thereby, systematic studies on the effect of the surface modification on the wetting properties of microstructures have been possible. Importantly, we found that the wetting parallel to the groove strongly depended on the chemical modification of the structure although the wetting perpendicular to the groove is almost unaffected. Through immobilization of a monolayer of Si nanoparticles (SiNPs) exclusively on the elevations of the hydrogel-coated microstructured PDMS substrate, the anisotropic wetting could be selectively altered unidirectionally along the pattern direction.

Bone tissue engineering in osteoporosis.

Osteoporosis is a polygenetic, environmentally modifiable disease, which precipitates into fragility fractures of vertebrae, hip and radius and also confers a high risk of fractures in accidents and trauma. Aging and the genetic molecular background of osteoporosis cause delayed healing and impair regeneration. The worldwide burden of disease is huge and steadily increasing while the average life expectancy is also on the rise. The clinical need for bone regeneration applications, systemic or in situ guided bone regeneration and bone tissue engineering, will increase and become a challenge for health care systems. Apart from in situ guided tissue regeneration classical ex vivo tissue engineering of bone has not yet reached the level of routine clinical application although a wealth of scaffolds and growth factors has been developed. Engineering of complex bone constructs in vitro requires scaffolds, growth and differentiation factors, precursor cells for angiogenesis and osteogenesis and suitable bioreactors in various combinations. The development of applications for ex vivo tissue engineering of bone faces technical challenges concerning rapid vascularization for the survival of constructs in vivo. Recent new ideas and developments in the fields of bone biology, materials science and bioreactor technology will enable us to develop standard operating procedures for ex vivo tissue engineering of bone in the near future. Once prototyped such applications will rapidly be tailored for compromised conditions like vitamin D and sex hormone deficiencies, cellular deficits and high production of regeneration inhibitors, as they are prevalent in osteoporosis and in higher age.

Tenside-free preparation of nanogels with high functional ß-cyclodextrin content.

We present the preparation of ultrafine (R(h), 50 -150 nm) nanogels through tenside-free condensation of reactive prepolymers with ß-cyclodextrin (ß-CD) in water. These nanogels possess a maximum content of 60 wt % functional ß-CD that can form inclusion complexes as demonstrated by dye sorption with phenolphthalein. Aside of this extremely high uptake capacity to hydrophobic molecules, the nanogels also show good adhesion to surfaces in homogeneous distribution with size of R(h) of 25 nm under dry conditions.

Differences of crystal structure and dynamics between a soft porous nanocrystal and a bulk crystal.

We have demonstrated downsizing effects of the soft porous crystal, [Zn(isophthalate)(4,4'-bipyridyl)](n) (CID-1), on the adsorption behavior between CID-1 and CID-1 nanocrystal (NCID-1). The difference results from the packing crystal structures and the dynamics of the frameworks.

Rapid preparation of flexible porous coordination polymer nanocrystals with accelerated guest adsorption kinetics.

Porous coordination polymers, in particular flexible porous coordination polymer networks that change their network structure on guest adsorption, have enormous potential in applications involving selective storage, separation and sensing. Despite the expected significant differences in their adsorption properties, porous coordination polymer nanocrystals remain largely unexplored, and there have been no reports about studies on flexible porous coordination polymer nanocrystals, mainly due to a lack of preparation methods. Here, we present a new technique for the rapid preparation of porous coordination polymer nanocrystals that combines non-aqueous inverse microemulsion with ultrasonication. Uniform nanocrystals of {[Zn(ip)(bpy)]}(n) (ip = isophthalate, bpy = 4,4'-bipyridyl; CID-1), a flexible porous coordination polymer, have been prepared by this method and analysed using field-emission scanning electron microscopy, energy-dispersive X-ray analysis, infrared spectroscopy, Raman spectroscopy and X-ray powder diffraction. A model for particle formation and growth is presented and discussed. Adsorption experiments with methanol show that the overall adsorption capacities of nanoparticles and bulk are almost identical, but the shapes of the sorption isotherms differ significantly and the adsorption kinetics increase dramatically.

Patterned melt electrospun substrates for tissue engineering.

Tissue engineering scaffolds can be built with patterning techniques that allow discrete placement of structures. In this study, electrospun fibres are collected in focused spots; the patterning and drawing of a cell adhesive scaffold is shown. Blends of biodegradable poly(ethylene glycol)-block-poly(epsilon-caprolactone) (PEG-b-PCL) and PCL were melt electrospun onto glass collectors, and the optimal electrospinning parameters determined. The quality of the fibre was largely influenced by the flow rate of the melt to the spinneret; however, this can be adjusted with the voltage. A collection distance between 3 cm and 5 cm was optimal, and at 10 cm the fibres became unfocused in their deposition although the diameter remained similar (0.96 +/- 0.19 microm). Aligned lines of electrospun fibres 200-400 microm in width could be applied onto the slide with an x-y stage, continuously and discretely. Lines of electrospun fibres could be applied on top of one another and were very uniform in diameter. Fibroblasts adhered primarily in the fibre region, due to the poor cell adhesion to the PEG substrate. Improvements in depositing hydrophilic electrospun fibres that wet and adhere to in vitro substrates and the use of stage automation for the writing interface could provide scaffold-building devices suitable for tissue engineering applications.

Structure and properties of urea-crosslinked star poly[(ethylene oxide)-ran-(propylene oxide)] hydrogels.

Isocyanate-terminated six armed star shaped macromers with a statistical copolymer backbone consisting of 80% EO and 20% PO have previously demonstrated excellent protein and cell repellence as nano-layered surfaces. In this study, various macromers are mixed with water and provide a spectrum of materials that range from particles to uniform hydrogels. Due to hydrophobic end groups, 3 kDa molecular weight macromers result in micro and nano-particles, while 18 kDa macromers completely dissolve and consequently uniform, transparent, high water content hydrogels are formed. Oriented channels may be induced into these hydrogels through the controlled freezing of water in the preformed hydrogel.

Ultrathin Coatings with Change in Reactivity over Time Enable Functional In Vitro Networks Of Insect Neurons.

It's just not cricket! A novel coating system that enables covalent attachment of biomolecules in a nonfouling environment without use of additional chemical crosslinkers is presented. Concanavalin A is patterned on the coatings to direct cell adhesion and growth of neurons from the cricket Gryllus bimaculatus and generate functional, patterned in vitro insect neuronal networks for the first time.

Nanostructured ordering of fluorescent markers and single proteins on substrates.

Highly ordered hexagonal nanopatterns of gold clusters on glass substrates were used as anchoring points for the specific attachment of fluorescence dyes and proteins labeled with fluorescence dyes. Thiol- or disulfide-containing linker molecules were used for the binding to the gold dots. In order to ensure specific binding on the gold dots only, the surface area in between the dots was protected against unspecific adsorption. For the attachment of polar low-molecular-weight fluorescence dyes, an octadecyltrichlorosilane self-assembled monolayer was prepared on the surface in between the gold dots, whereas a layer prepared from star-shaped poly(ethylene oxide-stat-propylene oxide) prepolymers was used to prevent unspecific adsorption of proteins between the gold dots. Fluorescence microscopy proved the specific binding of the dyes as well as of the proteins. Scanning force microscopy studies show that each gold dot is only capable of binding one protein at a time.

Ultrathin functional star PEG coatings for DNA microarrays.

In this study, star PEG coatings on glass substrates have been used as support material for oligonucleotide microarrays. These coatings are prepared from solutions of six armed star shaped prepolymers that carry reactive isocyanate endgroups. As described earlier, such films prevent the adsorption of proteins and the adhesion of cells but can easily be functionalized for specific biological recognition. Here we used the high functionality of these coatings for the covalent immobilization of amino terminated 20mer oligonucleotides, both by microcontact printing and spotting techniques. The permanent immobilization of fluorescently labeled DNA as well as hybridization of 20mer oligonucleotides have been monitored by fluorescence microscopy. The hybridization efficiency as determined by fluorescence intensity varied from 30% to 80% depending on the way of layer preparation. The direct spotting without additional activation and blocking steps of the surface demonstrates the potential of star PEG coatings as ultrathin surface modification for microarrays.

A novel star PEG-derived surface coating for specific cell adhesion.

In this study a novel approach for the coating and functionalization of substrates for cell culture and tissue engineering is presented. Glass, silicon, and titanium panes were coated with an ultrathin film (30 +/- 5 nm) of reactive star-shaped poly(ethylene glycol) prepolymers (Star PEG). Homogeneity of the films was checked by optical microscopy and scanning force microscopy. These coatings prevent unspecific protein adsorption as monitored by fluorescence microscopy and ellipsometry. In order to allow specific cell adhesion the films were modified with linear RGD peptides (gRGDsc) in different concentrations. After sterilization, fibroblast, SaOS, and human mesenchymal stem cells (hMSC) were seeded on these substrates. Cell adhesion, spreading, and survival was observed for up to 30 days on linear RGD peptide (gRGDsc)-modified coatings, whereas no cell adhesion could be detected on unmodified Star PEG layers. By variation of the RGD concentration within the film the amount of cells that became adhesive could be controlled. When differentiation conditions are used for cultivation of hMSCs the cells show the expression of osteogenic marker genes after 14 days which is comparable to cultivation on cell culture plastic. Thus, the Star PEG/RGD film did not negatively influence the differentiation process. The high flexibility of the system considering the incorporation of biologically active compounds opens a broad field of future experiments.

Ultrathin coatings from isocyanate terminated star PEG prepolymers: patterning of proteins on the layers.

This study presents the easy and fast patterning of low molecular weight molecules that act as binding partners for proteins on Star PEG coatings. These coatings are prepared from isocyanate terminated star shaped prepolymers and form a highly cross-linked network on the substrate in which the stars are connected via urea groups and free amino groups are present. Streptavidin has been patterned on these layers by microcontact printing (muCP) of an amino reactive biotin derivative and consecutive binding of streptavidin to the biotin. Patterns of Ni(2+)-nitriltriacetic acid (NTA) receptors have been prepared by printing amino functional NTA molecules in freshly prepared Star PEG layers that still contain amino reactive isocyanate groups. Complexation of the NTA groups with Ni(II) ions enabled the binding of His-tag enhanced green fluorescent protein (EGFP) in the desired pattern on the substrates. Since the unmodified Star PEG layers prevent unspecific protein adsorption, His-EGFP could selectively be bound to the sample by immersion into crude, nonpurified His-tag EGFP containing cell lysate.

Ultrathin coatings from isocyanate-terminated star PEG prepolymers: layer formation and characterization.

In this study we present the preparation of thin and ultrathin coatings from six-arm star-shaped isocyanate-terminated prepolymers on amino-functionalized silicon wafers. The backbone of the stars is a statistical copolymer of ethylene oxide and propylene oxide in the ratio 80:20 (Star PEG). Film preparation by spin coating from aqueous THF resulted in a variety of film morphologies that are determined by the water content of the solvent. Water is indispensable for activation of the isocyanate-terminated stars in solution and for proper cross-linking of the coatings on the substrate. This cross-linking results in a dense network of PEG chains on the substrate linked via urea groups with a mesh size of the network that corresponds to the arm length of the stars. Layer thickness variations between 3 and 500 nm revealed a strong dependence of the contact angle with water on the layer thickness which is explained by the chemical composition of the coatings. Due to the high functionality of the star-shaped prepolymers, free amino groups remain in the films that were detected by fluorescence microscopy after reaction with 4-chloro-7-nitrobenzofurazan (NBF). To test the system for the ability to prevent unspecific interaction with proteins, adsorption of fluorescence-labeled avidin was examined with fluorescence microscopy. For layer thicknesses between 3 and 50 nm, no protein adsorption could be detected.