Claudia Matschegewski

Cell architecture-cell function dependencies on titanium arrays with regular geometry

Knowledge about biocomplexity of cell behavior in dependence on topographical characteristics is of clinical relevance for the development of implant designs in tissue engineering. The aim of this study was to find out cell architecture-cell function dependencies of human MG-63 osteoblasts on titanium (Ti) arrays with regular geometry. We compared cubic pillar structures (SU-8, dimension 3 x 3 x 5 and 5 x 5 x 5 mum) with planar samples. Electrochemical surface characterization revealed a low amount of surface energy (including polar component) for the pillar-structured surfaces, which correlated with a reduced initial cell adhesion and spreading. Confocal microscopy of cells actin cytoskeleton revealed no stress fiber organization instead, the actin was concentrated in a surface geometry-dependent manner as local spots around the pillar edges. This altered cell architecture resulted in an impaired MG-63 cell function - the extracellular matrix proteins collagen-I and bone sialo protein (BSP-2) were synthesized at a significantly lower level on SU-8 pillar structures; this was accompanied by reduced beta3-integrin expression. To find out physicochemical factors pertaining to geometrically microstructured surfaces and their influence on adjoining biosystems is important for the development of biorelevant implant surfaces.

Automatic Actin Filament Quantification of Osteoblasts and Their Morphometric Analysis on Microtextured Silicon-Titanium Arrays

Microtexturing of implant surfaces is of major relevance in the endeavor to improve biorelevant implant designs. In order to elucidate the role of biomaterial’s topography on cell physiology, obtaining quantitative correlations between cellular behavior and distinct microarchitectural properties is in great demand. Until now, the microscopically observed reorganization of the cytoskeleton on structured biomaterials has been difficult to convert into data. We used geometrically microtextured silicon-titanium arrays as a model system. Samples were prepared by deep reactive-ion etching of silicon wafers, resulting in rectangular grooves (width and height: 2 µm) and cubic pillars (pillar dimensions: 2 × 2 × 5 and 5 × 5 × 5 µm); finally sputter-coated with 100 nm titanium. We focused on the morphometric analysis of MG-63 osteoblasts, including a quantification of the actin cytoskeleton. By means of our novel software FilaQuant, especially developed for automatic actin filament recognition, we were first able to quantify the alterations of the actin network dependent on the microtexture of a material surface. The cells’ actin fibers were significantly reduced in length on the pillared surfaces versus the grooved array (4–5 fold) and completely reorganized on the micropillars, but without altering the orientation of cells. Our morpho-functional approach opens new possibilities for the data correlation of cell-material interactions.

Genetic variation of temperature-regulated curd induction in cauliflower: elucidation of floral transition by genome-wide association mapping and gene expression analysis.

Cauliflower (Brassica oleracea var. botrytis) is a vernalization-responsive crop. High ambient temperatures delay harvest time. The elucidation of the genetic regulation of floral transition is highly interesting for a precise harvest scheduling and to ensure stable market supply. This study aims at genetic dissection of temperature-dependent curd induction in cauliflower by genome-wide association studies and gene expression analysis. To assess temperature-dependent curd induction, two greenhouse trials under distinct temperature regimes were conducted on a diversity panel consisting of 111 cauliflower commercial parent lines, genotyped with 14,385 SNPs. Broad phenotypic variation and high heritability (0.93) were observed for temperature-related curd induction within the cauliflower population. GWA mapping identified a total of 18 QTL localized on chromosomes O1, O2, O3, O4, O6, O8, and O9 for curding time under two distinct temperature regimes. Among those, several QTL are localized within regions of promising candidate flowering genes. Inferring population structure and genetic relatedness among the diversity set assigned three main genetic clusters. Linkage disequilibrium (LD) patterns estimated global LD extent of r2 = 0.06 and a maximum physical distance of 400 kb for genetic linkage. Transcriptional profiling of flowering genes FLOWERING LOCUS C (BoFLC) and VERNALIZATION 2 (BoVRN2) was performed, showing increased expression levels of BoVRN2 in genotypes with faster curding. However, functional relevance of BoVRN2 and BoFLC2 could not consistently be supported, which probably suggests to act facultative and/or might evidence for BoVRN2/BoFLC-independent mechanisms in temperature-regulated floral transition in cauliflower. Genetic insights in temperature-regulated curd induction can underpin genetically informed phenology models and benefit molecular breeding strategies toward the development of thermo-tolerant cultivars.

Quantification of Actin Filament Organization by Estimating Graph Structures in Confocal Microscopic Images

With the progress in implant technology, the understanding of the interaction between implants and living cells and tissue raised in relevance. To get insight into the biocomplexity of the underlying cellular mechanisms in the cell-biomaterial dialogue, an automatic quantification of cell parameters is required as a first step. An ad hoc designed texture analysis procedure is implemented to assess the development of actin filaments in human osteoblasts grown on titanium surfaces from confocal microscopic images.

Membrane related dynamics and the formation of actin in cells growing on micro-topographies: a spatial computational model

BackgroundIntra-cellular processes of cells at the interface to an implant surface are influenced significantly by their extra-cellular surrounding. Specifically, when growing osteoblasts on titanium surfaces with regular micro-ranged geometry, filaments are shorter, less aligned and they concentrate at the top of the geometric structures. Changes to the cytoskeleton network, i. e., its localization, alignment, orientation, and lengths of the filaments, as well as the overall concentration and distribution of key-actors are induced. For example, integrin is distributed homogeneously, whereas integrin in activated state and vinculin, both components of focal adhesions, have been found clustered on the micro-ranged geometries. Also, the concentration of Rho, an intracellular signaling protein related to focal adhesion regulation, was significantly lower.ResultsTo explore whether regulations associated with the focal adhesion complex can be responsible for the changed actin filament patterns, a spatial computational model has been developed using ML-Space, a rule-based model description language, and its associated Brownian-motion-based simulator. The focus has been on the deactivation of cofilin in the vicinity of the focal adhesion complex. The results underline the importance of sensing mechanisms to support a clustering of actin filament nucleations on the micro-ranged geometries, and of intracellular diffusion processes, which lead to spatially heterogeneous distributions of active (dephosphorylated) cofilin, which in turn influences the organization of the actin network. We find, for example, that the spatial heterogeneity of key molecular actors can explain the difference in filament lengths in cells on different micro-geometries partly, but to explain the full extent, further model assumptions need to be added and experimentally validated. In particular, our findings and hypothesis referring to the role, distribution, and amount of active cofilin have still to be verified in wet-lab experiments.ConclusionLetting cells grow on surface structures is a possibility to shed new light on the intricate mechanisms that relate membrane and actin related dynamics in the cell. Our results demonstrate the need for declarative expressive spatial modeling approaches that allow probing different hypotheses, and the central role of the focal adhesion complex not only for nucleating actin filaments, but also for regulating possible severing agents locally.

Regulation of T-Type Calcium Channels in Osteoblasts on Micro-Structured Surface Topography

Micro- and nanotopography as well as the surface chemistry of biomaterials affect cell adhesion, proliferation and cell differentiation. Furthermore, the organization and localization of intracellular adhesion components such as the actin cytoskeleton are also altered dependent on the material surface topography. However, the detailed influence of the material micro-structure on cellular mechanisms on the molecular level is still unknown. This study is intended to elucidate such effects using regular pillar structures to characterize the modulation of cell responses like the regulation of voltage-sensitive calcium channels as well as signaling molecules in human osteoblasts. To analyze cell behavior on defined biomaterial surfaces, human osteoblastic MG 63 cells were cultured on geometrically micro textured titanium coated silicon wafers, as opposed to planar titanium references. Samples were fabricated by a photolithographic process using the negative tone resist SU 8 and sputter-coated with 100 nm titanium. Immunofluorescence staining and flow cytometry are used to detect the expression levels and the function of T type calcium channels. Knowledge about the biocomplexity of cell behavior dependent on topographical characteristics is of clinical relevance for the development of implant designs in tissue engineering.

Pyramid array substrates for biomedical studies

Cellular reactions to structured surfaces are the subject of intense studies in biomedical research, e.g., for implant technology. Substrates with well-defined structures in relevant materials such as titanium (Ti) are required for these investigations. The pyramidal arrays presented here offer very sharp edges and tips, which appeared to be the main adhesion spots in our earlier cell-surface-interaction studies. Using an etch process based on anisotropic etching of silicon in alkaline solution, the shape of the pyramids is strictly determined by the crystal structure of silicon, while the height as well as the pitch of the pyramids can be precisely controlled and, therefore, be varied in a systematic fashion. Being made from silicon nitride with a thin cover layer of titanium, they offer mechanical stiffness, inertness to all chemicals used in the cell experiments, and a high degree of biocompatibility.

Quantitative Analysis of the Cellular Actin Cytoskeleton on Geometrically Designed Surface Topography

In tissue engineering, topographical modification of implants has been used as a powerful tool for the development of biorelevant implant designs. However, there is still a lack of knowledge about the fundamental principles of the cell–material interaction and quantitative correlations between cell biological parameters and physicochemical surface characteristics. The focus of our studies on cell architecture–cell function dependencies on regular micro-scaled surface structures was to investigate and further quantify the cell phenotype obtained from images of confocal microscopy and scanning electron microscopy. We used periodically structured titanium surfaces with regular cubic pillar geometry (dimension 3x3x5 µm and 5x5x5 µm) in comparison to planar samples. Confocal microscopy revealed a considerable rearrangement of the actin cytoskeleton on the top of the pillars with a reduced filament length. The quantification of different actin filament networks of cells grown on structured surfaces was carried out with a novel software for automatic filament recognition, covering the majority of filaments and their branching in noisy data. The quantitative analysis of cell phenotype changes on surfaces with regular geometry opens new possibilities for the data correlation cell vs. material.