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Properties of retinal pigment epithelium (RPE) are relevant for the development of cell culture models concerning an exact reproduction of the ocular cell biology. Here, we want to investigate how different carrier materials and coatings influence proliferation, differentiation and functions of RPE in regard to development of a three-dimensional cell culture model based on primary porcine RPE. Human RPE cell line ARPE-19 and primary porcine RPE were used. Cells were cultivated on plates which were coated with collagen I, collagen IV, laminin or fibronectin, respectively, and cell numbers were assessed after different time periods via trypan blue staining. Also, the ARPE-19 were cultivated on polydimethylsiloxane (PDMS), alginate, gelatin methacrylate (GelMA), poly-N-isopropylacrylamide (PNIPAM) and cells number were assessed. Primary RPE were cultured on PDMS material. Supernatants were collected and analyzed via ELISA for their vascular endothelial growth factor (VEGF) and transforming growth factor β (TGF-β) content. After day 14 cells were lysed and retinal pigment epithelium-specific 65 kDa protein (RPE65) and bestrophin-1 (BEST1) expression was investigated via Western blot. Cellular functions were tested on collagen I, collagen IV, laminin and fibronectin with and without PDMS. Scratch assay was performed to detect wound healing 24 and 48 h after scratch application. Immunolabeling was used to highlight tight junctions in concert with Hoechst staining and phalloidin to label cell nuclei and actin filaments, respectively. Phagocytosis of fluorescently labeled latex beads opsonized with photoreceptor outer segments (POS) was assessed via fluorescence microscopy. Transepithelial electrical resistance was measured for detection of cellular barrier. Gene expression of RDH11 (retinol dehydrogenase 11), BEST1 (bestrophin 1) and TGFB1 (transforming growth factor beta 1) was investigated via real-time PCR. Only PDMS carrier material was appropriate for primary RPE and ARPE-19 cell cultivation. Coating of PDMS with laminin led to increased proliferation. In primary RPE, VEGF secretion was increased if PDMS was coated with laminin or fibronectin compared to uncoated PDMS. No significant changes in phagocytic ability and generation of tight junctions were detected between different coatings, but RPE65 expression was reduced on fibronectin coated PDMS. Laminin coating decreased TGF-β and increased BEST1 protein expression. Also, RPE on collagen IV showed highest TEER on transwell plates. The genes RDH11 and TGFB1 were decreased when coated with collagen IV without PDMS as well as coated PDMS. Laminin and collagen IV coating led to an increased wound healing. Cultivation of RPE and ARPE-1 on PDMS is a possible alternative for cell culture models whereas alginate, GelMA and PNIPAM were not suitable. Coating with laminin increased the proliferation, wound healing and VEGF secretion of the cells. The results suggest that laminin coated PDMS as carrier material is suitable for the development of 3D culture model systems.

Objective We tested the adhesion properties of living gingival fibroblasts on three different implant abutment materials, adhesive resin used to bond bi-partite abutments, and human dentin. Methods Discs of lithium disilicate (LS), zirconium dioxide (Zr), adhesive resin cement (AR), titanium (Ti), and human dentin (HD) were fabricated with three different levels of surface roughness (rough, machined, and polished). Ra and Rz, water contact angle, and cell detachment forces were measured. Cell detachment force was measured for single cells using single-cell force spectroscopy. Data were statistically analyzed using parametric tests (ANOVA, MANOVA, Bonferroni post-hoc tests). Results Surface roughness significantly influenced the water contact angle for all materials (P ≤ 0.05). Overall, HD showed the lowest contact angle, followed by LS, Ti, Zr, and AR (P ≤ 0.05). Comparison of cell detachment forces between materials with rough and machined surfaces revealed no significant differences (P > 0.05), with the exception of Zr compared to HD with rough surfaces (P = 0.006). For polished surfaces, HD showed the highest detachment force (P ≤ 0.0001), followed by Ti, AR, and Zr, which did not significantly differ from each other (P > 0.05) and LS; Ti/AR was significantly different from LS (P ≤ 0.05). Except for HD, where polished surfaces exhibited the highest cell detachment force (P ≤ 0.002), most machined surfaces showed higher cell detachment forces than polished or rough surfaces. Significance Implant abutments should ideally be provided with a machined like surface roughness for best cell adhesion.

Scaffolds for tissue engineering enable the possibility to fabricate and form biomedical implants in vitro, which fulfill special functionality in vivo. In this study, free-standing Nickel–Titanium(NiTi) thin film mesheswere produced by means of magnetron sputter deposition.Meshes contained precisely defined rhombic holes in the size of 440 to 1309 μm2 and a strut width ranging from 5.3 to 9.2 μm. The effective mechanical properties of the microstructured superelastic NiTi thin film were examined by tensile testing. These results will be adapted for the design of the holes in the film. The influence of hole and strut dimensions on the adhesion of sheep autologous cells (CD133+) was studied after 24 h and after seven days of incubation. Optical analysis using fluorescence microscopy and scanning electron microscopy showed that cell adhesion depends on the structural parameters of the mesh. After 7 days in cell culture a large part of the mesh was covered with aligned fibrous material. Cell adhesion is particularly facilitated on meshes with small rhombic holes of 440 μm2 and a strut width of 5.3 μm. Our results demonstrate that free-standing NiTi thin film meshes have a promising potential for applicationsin cardiovascular tissue engineering, particularly for the fabrication of heart valves.

Superparamagnetic iron oxide nanoparticles (SPIONs) are an important tool for labeling cells and tissues in many therapeutic and diagnostic applications, such as magnetic resonance imaging (MRI) and magnetic particle imaging (MPI). However, these methods require large and expensive instrumentation. Here we show that our magnetic susceptibility particle mapping (MSPM) system can achieve the detection of magnetic nanoparticles in an inexpensive and small device. The system is based on magnetoelectric (ME) sensors utilizing the ΔE effect in combination with a permanent magnet that is generating a bias field for the sensor and at the same time is magnetizing the SPIONs in the sample. The permanent magnet is placed above the sensor, and the sample is rotated through the gap in between. The magnetized SPIONs in the sample generate an additional magnetic field that can be detected by the ME sensor. The clear novelty of our approach is the use of a rotating sample, generating a periodic signal, which enables an easy separation of the desired signal from the background signal and the possibility to compensate drift, which is commonly observed in ME sensor measurements. With this improvement and the use of a ME sensor that is sensitive for low frequencies the setup is able to measure significantly smaller amounts of magnetic nanoparticles than previous approaches described in the literature and we are even able to reconstruct 2D nanoparticle distributions. The noise floor, also referred to as limit of detection (LOD), of this measurement system is around 500 pT/(Hz)1/2. The detection threshold of our MSPM system is 20 μg SPIONs in a volume of 200 mm3 and the spatial resolution is in the range of a few mm. The spatial resolution is determined by reconstructing the particle distribution in the sample layer by solving the inverse problem. To demonstrate the feasibility of the method for detecting living cells, we measured the field distribution originating from SPION-labeled fibroblast cells in an alginate-gelatin matrix, thus demonstrating the potential of our method for biomaterial applications.

We have studied the initial phase of cell adhesion as a function of the lateral organization of individual integrin molecules with single-cell force microscopy. Nanostructures, consisting of hexagonally ordered gold dots, were prepared with diblock-copolymer micelle lithography and functionalized with arginine- glycine-aspartate peptides, thus defining integrin position with nanometer resolution. Adhesion strength was characterized with an atomic force microscope and both cell detachment forces and work of detachment showed a reinforcement of adhesion if the distance between integrin molecules was

The mammalian inflammatory response significantly relies on integrin-mediated T-lymphocyte adhesion to endothelial cells. Whereas the outside-in signalling pathway of integrins in response to the proinflammatory cytokine tumor necrosis factor (TNF) has already been studied in detail, little knowledge exists about the inside-out signalling pathway of integrins in lymphocyte activation by TNF. We here show single-cell force spectroscopy (SCFS) data of T-lymphocyte (Jurkat E6-1) adhesion to fibronectin. Fibronectin is present on top of endothelial cell layers and is therefore a crucial player in mediating T-lymphocyte binding to endothelial cells. Our results show that activating integrins with TNF significantly increases the maximum adhesion force and detachment energy in Jurkat cell adhesion to fibronectin-coated surfaces. Analysis of single-molecule ruptures further proves that TNF reinforces integrin binding strength, particularly at sub-second timescales. Hence, our results provide quantitative evidence for the significant impact of TNF-induced inside-out signalling in the T-lymphocyte adhesion machinery.