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Nanofiber nonwovens are highly promising to serve as biomimetic scaffolds for pioneering cardiac implants such as drug-eluting stent systems or heart valve prosthetics. For successful implant integration, rapid and homogeneous endothelialization is of utmost importance as it forms a hemocompatible surface. This study aims at physicochemical and biological evaluation of various electrospun polymer scaffolds, made of FDA approved medical-grade plastics. Human endothelial cells (EA.hy926) were examined for cell attachment, morphology, viability, as well as actin and PECAM 1 expression. The appraisal of the untreated poly-L-lactide (PLLA L210), poly-ε-caprolactone (PCL) and polyamide-6 (PA-6) nonwovens shows that the hydrophilicity (water contact angle > 80°) and surface free energy (

Magnetic nanoparticles are highly promising for the usage in various biomedical applications including magnetic particle imaging (MPI), cancer hyperthermia treatment or as drug carriers. The present study aims at assessing in vitro biocompatibility of two commercially available magnetic iron oxide nanoparticle formulations: dextran-based magnetic nanoparticle synomag-D and bionized nanoferrite BNF-starch. Biological performance of both nanoparticle formulations were studied in human endothelial cells by analyzing cell viability and nanoparticle internalization in order to judge their suitability as theranostics.

Implants that feature a drug delivery system loaded with antifibrotic active drugs provide a promising approach to address postoperative complications caused by fibrosis. This study is intended to clarify whether Actinomycin D has an impact specifically on components of the extracellular matrix (ECM) and its formation in human primary fibroblasts of the Tenon capsule (hTF). Furthermore, the suitability of this agent in poly(N-vinylpyrrolidone)- poly(methylmethacrylate)(PVP-co-PMMA) as a drug delivery model is evaluated in drug incorporation and release studies. RT-qPCR revealed a significant downregulation of the fibrotic marker genes ACTA2, COL1A1 and FN1 in cells stimulated with TGF- β1 and additionally treated with Actinomycin D. However, these findings could only be confirmed on α- SMA protein level. collagen I and Fibronectin synthesis stayed unaffected. The diffusion based incorporation of Actinomycin D into the polymer model proved to be very effective. The release of the agent was retarded with a slightly prolonged kinetic. These findings make Actinomycin D a promising antifibrotic agent in ophthalmic implant surgery.

Resveratrol is a member of stilbenoids with promising anti-atherosclerotic properties. This hallmark makes it an extremely interesting candidate for local drug delivery to damaged tissue adjacent to the implant in order to reduce implant-related complications. For the regulatory approval drug-eluting medical devices have to be thoroughly tested for safety, efficacy and interactions with the surrounding tissue, including tests for sensitization among others. Studies for sensitization help to estimate the risk for an allergic reaction upon prolonged exposure to a chemical compound. Due to increased social and regulatory demand for replacement of animal experiments by in vitro approaches a number of reliable predictive non-animal tests have been developed. Here, we assessed the skin sensitization potential of resveratrol by the direct peptide reactivity assay (DPRA), one of the first non-animal tests adopted by the OECD.

On the protection of animals used for scientific purposes, the EU Parliament adopted Directive 2010/63/EU. The essential factor is the 3R principle: Replacement, Reduction and Refinement. In 2013, the third amendment to the German Animal Welfare Act was revised and adapted to the European Directive. The majority of animals in science are used in basic research, as well as in translational and applied research. In medical research, animal experimentation is conducted to clarify previously unknown life processes and basic biological relationships, in order to improve diagnostics and treatment of human diseases. Before an animal experiment can be performed, it must be reported to and approved by the responsible authorities. The planned research project must be justified scientifically, and it must be demonstrated that the personnel and spatial/ technical prerequisites are in place to successfully complete the project. If all conditions are met, the approval can be granted, but may be subject to conditions. The guiding principle of essentiality also affects the procedure of the experiments: The number of animals used and the pain, suffering and damage caused to these animals must be limited to what is absolutely necessary. In this context, the 3R principle has to be applied. To obtain reliable results, it is essential that the laboratory animals are in normal physiological conditions and free of pain and fear. Scientific interest and animal welfare are therefore not in opposition, but rather mutually dependent. In our GLP (Good Laboratory Practice) laboratory we test new drug release systems for different biomedical applications in rabbits after careful selection of the animal model. Stress during animal experiments must be avoided as far as possible. Providing pain-killers and ensuring the best possible husbandry and care conditions are crucial for the animal’s wellbeing and absence of pain and anxiety. In the present work we report our different experience in a GLPcertified biomaterial test laboratory.

For the development of intelligent implant systems hydrogels (HG) from crosslinked ionic liquids feature a high potential to be utilised as a drug depot. Biocompatibility of the HGs is one key prerequisite for biomedical applications. HGs were polymerised from a variety of different ionic monomers based on methacrylate, methacrylamide, styrene or vinyl imidazolium derivatives in aqueous solution. N,N'-methylenebisacrylamide was used as crosslinker. CellQuanti-Blue™ Cell Viability Assay Kit was implemented to proof viability of L929 mouse fibroblasts. The predominant part of the HG eluates generated only a marginal reduction of less than 15% cell viability at 100% eluate concentration. This underlines the excellent suitability of these HGs for biomedical applications and revealed some promising candidates for the development of drug depots for implants.

For a long-term efficient drug-eluting stent for vascular applications, the development of drug-loaded coating, combining the effective inhibition of smooth muscle cell proliferation while promoting the re-endothelialization, is a promising concept. However the mostly required simultaneous incorporation of drugs can influence decisively the stability, efficacy and release of the respective drug. Therefore, the mutual influence of a dual local drug delivery coatings based on poly(L-lactide-co-ε-caprolactone) (PLLA-co-CL) containing vascular endothelial growth factor (VEGF165) coupled to the surface and an embedded drug, such as fluorescein diacetate (FDAc) instead of Paclitaxel (PTX) on the in vitro drug release was investigated. Surprisingly, for the investigated coating the immobilized VEGF loading was enhanced and the release profile was accelerated by FDAc incorporation. Even a manifold increase for the in vitro released amounts of VEGF was detected. In contrast, the immobilization of VEGF seems to have a negligible influence on the in vitro FDAc release profiles.

Phenotype by genotype prediction based on ecophysiological models, which account for allelic gene, QTL, or marker effects, have many possible applications in plant breeding programs. The goal of the present study was to predict heading date of individual lines of a Hordeum vulgare x H. vulgare ssp. spontaneum BC2DH-population using a phenology model parameterized with marker effects derived from ridge regression best linear unbiased prediction. The genetic linkage map included SSR markers and flowering-time genes. Effects of photoperiod and temperature on heading date were measured under controlled conditions on a subset of the population comprising the recurrent parent and 36 BC2DH candidate introgression lines covering the H. spontaneum genome. Marker effects, which were subsequently used for model parameterization, were estimated. Model evaluation was carried out on already published field trial data comprising the 36 BC2DH lines and 266 independent BC2DH lines from the same cross. Applying the model on the lines used for model parameterization explained 33–51% of heading-date variation in three of the four evaluation environments but only 20% of the variation in the fourth environment. Heading dates of the 266 independent lines were predicted with less accuracy. Between 20 and 25% of phenotypic variation was explained by the model in three environments and only 8% of heading date variation in the fourth environment. The root mean squared error (RMSE) was slightly higher for independent lines than for the lines used for model parameterization. Dissecting RMSE into its components revealed that RMSE was largely influenced by a systematic bias in most environments and by the missing ability of the model to describe the observed variation within the set of genotypes in all environments. Comparing the combined genome-wide prediction (GWP) and phenology model with a conventional GWP model gave similar prediction accuracies if the training set had the same size.

Biomedical devices in the blood flow disturb the fine-tuned balance of pro- and anti-coagulant factors in blood and vessel wall. Numerous technologies have been suggested to reduce coagulant and inflammatory responses of the body towards the device material, ranging from camouflage effects to permanent activity and further to a responsive interaction with the host systems. However, not all types of modification are suitable for all types of medical products. This review has a focus on application-oriented considerations of hemocompatible surface fittings. Thus, passive versus bioactive modifications are discussed along with the control of protein adsorption, stability of the immobilization, and the type of bioactive substance, biological or synthetic. Further considerations are related to the target system, whether enzymes or cells should be addressed in arterial or venous system, or whether the blood vessel wall is addressed. Recent developments like feedback controlled or self-renewing systems for drug release or addressing cellular regulation pathways of blood platelets and endothelial cells are paradigms for a generation of blood contacting devices, which are hemocompatible by cooperation with the host system. STATEMENT OF SIGNIFICANCE: This paper is part 4 of a series of 4 reviews discussing the problem of biomaterial associated thrombogenicity. The objective was to highlight features of broad agreement and provide commentary on those aspects of the problem that were subject to dispute. We hope that future investigators will update these reviews as new scholarship resolves the uncertainties of today.

Biomedical devices in the blood flow disturb the fine-tuned balance of pro- and anti-coagulant factors in blood and vessel wall. Numerous technologies have been suggested to reduce coagulant and inflammatory responses of the body towards the device material, ranging from camouflage effects to permanent activity and further to a responsive interaction with the host systems. However, not all types of modification are suitable for all types of medical products.This review has a focus on application-oriented considerations of hemocompatible surface fittings. Thus, passive versus bioactive modifications are discussed along with the control of protein adsorption, stability of the immobilization, and the type of bioactive substance,biological or synthetic. Further considerations are related to the target system, whether enzymes or cells should be addressed in arterial or venous system, or whether the blood vessel wall is addressed. Recent developments like feedback controlled or self-renewing systems for drug release or addressing cellular regulation pathways of blood platelets and endothelial cells are paradigms for a generation of blood contacting devices, which are hemocompatible by cooperation with the host system.

The usage of electrospun polymer scaffolds is a promising approach for artificial heart valve design. This study aims at the evaluation of biological performance of nanofibrous polymer scaffolds poly(L-lactide) PLLA L210, PLLA L214 and polyamide-6 fabricated by electrospinning via analyzing viability, adhesion and morphology of human umbilical vein endothelial cells (EA.hy926). Nanofibrous surface topography was shown to influence cell phenotype and cell viability according to the observation of diminished cell spreading accompanied with reduced cell viability on nonwovens. Among those, highest biocompatibility was assessed for PLLA L214, although being generally low when compared to the planar control surface. Electrospinning was demonstrated as an innovative technique for the fabrication of advanced biomaterials aiming at guided cellular behavior as well as the design of novel implant platforms. A better understanding of cell–biomaterial interactions is desired to further improve implant development.

Improved cold tolerance during the juvenile phase is a major breeding goal to develop new sorghum cultivars suitable as an alternative energy crop in temperate regions. The objectives of this study were to identify marker-trait associations for cold tolerance in a sorghum diversity panel fingerprinted with 2620 single nucleotide polymorphism (SNP) markers and to detect quantitative trait loci (QTL) in two F2:3 populations. Traits of interest were dry matter growth rate (DMGR), leaf appearance rate (LAR), chlorophyll content (SPAD) and chlorophyll fluorescence (Fv′/Fm′ and ФPSII) in relation to temperature. The association panel comprised 194 genotypes, while the F2:3 populations consisted of 80 and 92 genotypes. All populations were tested under controlled conditions in a minimum of four temperature regimes ranging from 9.4°C to 20.8°C. QTL were identified for means across environments and regression parameters describing temperature effects. Several marker-trait associations for mean (m) DMGR, base temperature (Tb) of SPAD and ФPSII and temperature effect on LAR were validated by QTL detected in population 1 or 2. Promising QTL regions were found on chromosomes SBI-01, SBI-02, SBI-03, SBI-04, SBI-06 and SBI-09, among them genomic regions where candidate genes involved in the C-repeat binding pathway or encoding cold-shock proteins are located.

An ongoing challenge in drug delivery systems for a variety of medical applications, including cardiovascular diseases, is the delivery of multiple drugs to address numerous phases of a treatment or healing process. Therefore, an extended dual drug delivery system (DDDS) based on our previously reported cardiac DDDS was generated. Here we use the polymer poly(L-lactide) (PLLA) as drug carrier with the cytostatic drug Paclitaxel (PTX) and the endothelial cell proliferation enhancing growth factor, human vascular endothelial growth factor (VEGF), to overcome typical in-stent restenosis complications. We succeeded in using one solution to generate two separate DDDS via spray coating (film) and electrospinning (nonwoven) with the same content of PTX and the same post processing for VEGF immobilisation. Both processes are suitable as coating techniques for implants. The contact angle analysis revealed differences between films and nonwovens. Whereas, the morphological analysis demonstrated nearly no changes occurred after immobilisation of both drugs. Glass transition temperatures (Tg ) and degree of crystallinity (χ) show only minor changes. The amount of immobilised VEGF on nonwovens was over 300% higher compared to the films. Also, the nonwovens revealed a much faster and over three times higher PTX release over 70 d compared to the films. The almost equal physical properties of nonwovens and films allow the comparison of both DDDS independently of their fabrication process. Both films and nonwovens have significantly increased in vitro cell viability for human umbilical vein endothelial cells (EA.hy926) with dual loaded PTX and VEGF compared to PTX-only loaded samples.

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.

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.

Due to oxidation and adsorption of chloride and hydroxyl anions, the surface of titanium (Ti) implants is negatively charged. A possible mechanism of the attractive interaction between the negatively charged Ti surface and the negatively charged osteoblasts is described theoretically. It is shown that adhesion of positively charged proteins with internal charge distribution may give rise to attractive interaction between the Ti surface and the osteoblast membrane. A dynamic model of the osteoblast attachment is presented in order to study the impact of geometrically structured Ti surfaces on the osteoblasts attachment. It is indicated that membrane-bound protein complexes (PCs) may increase the membrane protrusion growth between the osteoblast and the grooves on titanium (Ti) surface and thereby facilitate the adhesion of osteoblasts to the Ti surface. On the other hand, strong local adhesion due to electrostatic forces may locally trap the osteoblast membrane and hinder the further spreading of osteointegration boundary. We suggest that the synergy between these two processes is responsible for successful osteointegration along the titanium surface implant.

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 × 3 × 5 and 5 × 5 × 5 μm) 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 cell’s 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 β3-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.

Mathematical modeling of the cell-material contact demands a thorough characterization of both the material surface and the cellular reaction. In earlier investigations we used stochastic material surfaces for this purpose. Also, other groups working on the same or similar subject used such stochastically structured material surfaces. In continuation of this work we now use fine-structured material surfaces with well-defined regular geometry because a stepwise and systematic variation of the structural parameters of geometrically defined surfaces makes it easier to find the characteristic parameters for modeling the cell-material interface. To begin with, we used grooves with rectangular profiles and cubic pillars. The grooves were etched in silicon by dry etching and then were sputter coated with 100 nm titanium. Arrays of cubic photo resist pillars with vertical side walls in different dimensions were obtained by a photolithographic process and were also sputter coated with 100 nm titanium. The samples were characterized by SEM and electrochemical methods. Human osteoblastic cells MG-63 (ATCC, LGC promochem, Manassas, USA) were cultured in Dulbecco's modified eagle medium with 10% fetal calf serum (FCS) at 37 °C and 5% CO 2 . MG-63 cells grown on microstructures within 24 h were visualized by SEM. We observed that cells on the grooved surfaces grew along the grooves mainly adhering to the horizontal edges of the material surface, whereas on the pillar structures the cells lie on the top of the pillars and are well spread. But the direction of spreading on the pillars is also determined by the horizontal and vertical edges of the surface structure.

QTL regions on chromosomes C06 and C09 are involved in temperature dependent time to curd induction in cauliflower. Temperature is the main environmental factor influencing curding time of cauliflower (Brassica oleracea var. botrytis). Temperatures above 20–22 °C inhibit development towards curding even in many summer cultivars. To identify quantitative trait loci (QTL) controlling curding time and its related traits in a wide range of different temperature regimes from 12 to 27 °C, a doubled haploid (DH) mapping population segregating for curding time was developed and days to curd initiation (DCI), leaf appearance rate (LAR), and final leaf number (FLN) were measured. The population was genotyped with 176 single nucleotide polymorphism (SNP) markers. Composite interval mapping (CIM) revealed repeatedly detected QTL for DCI on C06 and C09. The estimated additive effect increased at high temperatures. Significant QTL × environment interactions (Q × E) for FLN and DCI on C06 and C09 suggest that these hotspot regions have major influences on temperature mediated curd induction. 25 % of the DH lines did not induce curds at temperatures higher than 22 °C. Applying a binary model revealed a QTL with LOD >15 on C06. Nearly all lines carrying the allele of the reliable early maturing parental line (PL) on that locus induced curds at high temperatures while only half of the DH lines carrying the allele of the unreliable PL reached the generative phase during the experiment. Large variation in LAR was observed. QTL for LAR were detected repeatedly in several environments on C01, C04 and C06. Negative correlations between LAR and DCI and QTL co-localizations on C04 and C06 suggest that LAR has also effects on development towards curd induction.

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.

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.

Seven tumour suppressor genes (Chk1, Chk2, Apaf1, Rb1, p53, p16(INK4a) and p14(ARF)) and two oncogenes (N-ras and BRAF) were screened in nine human malignant melanoma (HMM) cell lines for point mutations or small deletions/insertions by DGGE, TGGE and SCCP analysis. For the first time in human mesothelioma, Chk1 gene mutations were detected in two of the nine investigated HMM cell lines. P53 gene mutations were found in three cell lines and p16(INK4a) mutations in 5. Mutation of the Chk1 gene implies a novel disruption mechanism of the p53 pathway in HMM, without affecting p53 itself. According to our knowledge, this is the first mutation screening of Chk1, Chk2, Apaf1 and Rb1 in human malignant mesothelioma.

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.

Background Intra-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. Results To 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. Conclusion Letting 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.