Remodeling of the Cardiac Extracellular Matrix Proteome During Chronological and Pathological Aging.

Impaired extracellular matrix (ECM) remodeling is a hallmark of many chronic inflammatory disorders that can lead to cellular dysfunction, aging, and disease progression. The ECM of the aged heart and its effects on cardiac cells during chronological and pathological aging are poorly understood across species. For this purpose, we first used mass spectrometry-based proteomics to quantitatively characterize age-related remodeling of the left ventricle (LV) of mice and humans during chronological and pathological (Hutchinson-Gilford progeria syndrome (HGPS)) aging. Of the approximately 300 ECM and ECM-associated proteins quantified (named as Matrisome), we identified 13 proteins that were increased during aging, including lactadherin (MFGE8), collagen VI α6 (COL6A6), vitronectin (VTN) and immunoglobulin heavy constant mu (IGHM), whereas fibulin-5 (FBLN5) was decreased in most of the data sets analyzed. We show that lactadherin accumulates with age in large cardiac blood vessels and when immobilized, triggers phosphorylation of several phosphosites of GSK3B, MAPK isoforms 1, 3, and 14, and MTOR kinases in aortic endothelial cells (ECs). In addition, immobilized lactadherin increased the expression of pro-inflammatory markers associated with an aging phenotype. These results extend our knowledge of the LV proteome remodeling induced by chronological and pathological aging in different species (mouse and human). The lactadherin-triggered changes in the proteome and phosphoproteome of ECs suggest a straight link between ECM component remodeling and the aging process of ECs, which may provide an additional layer to prevent cardiac aging.

Entropic repulsion of cholesterol-containing layers counteracts bioadhesion.

Control of adhesion is a striking feature of living matter that is of particular interest regarding technological translation1-3. We discovered that entropic repulsion caused by interfacial orientational fluctuations of cholesterol layers restricts protein adsorption and bacterial adhesion. Moreover, we found that intrinsically adhesive wax ester layers become similarly antibioadhesive when containing small quantities (under 10 wt%) of cholesterol. Wetting, adsorption and adhesion experiments, as well as atomistic simulations, showed that repulsive characteristics depend on the specific molecular structure of cholesterol that encodes a finely balanced fluctuating reorientation at the interface of unconstrained supramolecular assemblies: layers of cholesterol analogues differing only in minute molecular variations showed markedly different interfacial mobility and no antiadhesive effects. Also, orientationally fixed cholesterol layers did not resist bioadhesion. Our insights provide a conceptually new physicochemical perspective on biointerfaces and may guide future material design in regulation of adhesion.

Development of a synthesis strategy for sulfamethoxazole derivatives and their coupling with hydrogel microparticles.

Sulfonamides were the first synthetic antibiotics broadly applied in veterinary and human medicine. Their increased use over the last few decades and limited technology to degrade them after entering the sewage system have led to their accumulation in the environment. A new hydrogel microparticle based biosensing application for sulfonamides is developed to overcome existing labour-intensive, and expensive detection methods to analyse and quantify their environmental distribution. This biosensing assay is based on the soft colloidal probe principle and requires microparticle functionalization strategies with target molecules. In this study, we developed a step-wise synthesis approach for sulfamethoxazole (SMX) derivatives in high yield, with SMX being one of the most ubiquitous sulfonamide antibiotics. After de novo synthesis of the SMX derivative, two coupling schemes to poly(ethylene glycol) (PEG) hydrogel microparticles bearing maleimide and thiol groups were investigated. In one approach, we coupled a cysteamine linker to a carboxyl group at the SMX derivative allowing for subsequent binding via the thiol-functionality to the maleimide groups of the microparticles in a mild, high-yielding thiol-ene "click" reaction. In a second approach, an additional 1,11-bis(maleimido)-3,6,9-trioxaundecane linker was coupled to the cysteamine to target the hydrolytically more stable thiol-groups of the microparticles. Successful PEG microparticle functionalization with the SMX derivatives was proven by IR spectroscopy and fluorescence microscopy. SMX-functionalized microparticles will be used in future applications for sulfonamide detection as well as for pull-down assays and screenings for new sulfomethoxazole binding targets.

Phenotype Switching of Breast Cancer Cells upon Matrix Interface Crossing.

Tumor cell growth, invasion, and metastasis are dependent on the tumor microenvironment. Many studies emphasize a correlation between the material characteristics of the tumor extracellular matrix (ECM) and the invasive properties of tumor cells and even a trigger of tumor aggressiveness. Herein, we report that the previously observed trigger of migration characteristics of MDA-MB-231 breast cancer cells during transmigration across interfaces of two differently porous matrices is strongly correlated with a persistent change in cell invasiveness and aggressiveness. Using an in vitro 3D model of fibrillar collagen-I matrices, we found an increase in migration directionality, strongly elongated morphology, higher proliferation, and an increase in aggressive markers in the genetic profile after cells crossed the interface from dense to open porous matrix microstructure. Moreover, our results indicate strong nuclear deformation and increased DNA damage during transmigration of the matrix interface as a possible trigger of the more aggressive phenotype. These findings suggest that distinct tissue interfaces or altered ECM conditions with differences in microstructure may instruct or even reprogram tumor cells toward more aggressive phenotypes in vivo. The biomedical relevance of our results is corroborated by additional findings that the transmigrated cells exhibit an increased resistance against a common breast cancer therapeutic.

Biomaterial approaches for engineering and analyzing structure and metabolic states of microbial consortia within biofilms.

Microbial consortia within biofilms are frequently found in structured organization in nature and are thought to bear great potential for productive biotechnological applications, such as the degradation of complex substrates, biosensing, or the production of chemical compounds. However, in-depth understanding of their organizational principles, as well as comprehensive design criteria of structured microbial consortia for industrial applications are still limited. It is hypothesized that biomaterial engineering of such consortia within scaffolds can advance the field by providing defined in vitro mimics of naturally occurring and industrially applicable biofilms. Such systems will allow for adjustment of important microenvironmental parameters and in-depth analysis with high temporal and spatial resolution. In this review, we provide the background of biomaterial engineering of structured biofilm consortia, show approaches for their design, and demonstrate tools to analyze their metabolic state.

Correlation between Fibrin Fibrillation Kinetics and the Resulting Fibrin Network Microstructure.

Fibrin, the prominent extracellular matrix in early wound tissue, is discussed to influence immune cells and healing. The nature of fibrinogen/fibrin to form fibrillary networks is frequently exploited to engineer microenvironments for cellular analysis. This study focuses on revealing the correlation of fibril formation kinetic and the resulting network microstructure of engineered 3D fibrin networks. Different concentrations of fibrinogen (1-3 mg mL-1 ), thrombin (0.01-0.15 U mL-1 ), sodium chloride (40-120 mm), and calcium chloride (1-10 mm) are applied to assess the impact on the fibril growth kinetics by turbidity analysis and on the resulting fibril and pore diameter by laser scanning microscopy. The results highlight a direct influence of the sodium chloride concentration on fibrillation kinetics and reveal a strong correlation between fibrillation kinetics and network microstructure. With the assumption of a first-order growth kinetic, an increase of the growth constant k (0.015-0.04 min-1 ) is found to correlate to a decrease in fibril diameter (1-0.65 µm) and pore diameter (11-5 µm). The new findings enable an easy prediction of 3D fibrin network microstructure by the fibril formation kinetic and contribute to an improved engineering of defined scaffolds for tissue engineering and cell culture applications.

Microfluidics-assisted synthesis and functionalization of monodisperse colloidal hydrogel particles for optomechanical biosensors.

The soft colloidal probe (SCP) assay is a highly versatile sensing principle employing micrometer-sized hydrogel particles as optomechanical transducer elements. We report the synthesis, optimization, and conjugation of SCPs with defined narrow size distribution and specifically tailored mechanical properties and functionalities for integration into a microinterferometric optomechanical biosensor platform. Droplet-based microfluidics was used to crosslink polyethylene glycol (PEG) macromonomers by photocrosslinking and thiol-Michael addition. The effect of several synthesis parameters, i.e. PEG and radical initiator solid content, molecular weight and architecture of macromonomers, as well as UV exposure time and energy, were examined. SCPs were characterized with regard to the conversion of contained functional groups, morphology and mechanical properties by bright-field, confocal laser scanning and reflection interference contrast microscopy, as well as force spectroscopy. Functional groups were introduced during SCP synthesis and by several post-synthesis procedures, based on photoradical grafting and thiol-Michael addition. Preparation of SCPs by thiol-Michael addition and subsequent coupling of maleimide derivatives to unreacted thiols proved to be the superior strategy, while other approaches were associated with changes in the properties of the SCP. The newly developed SCPs were tested for their sensing capabilities employing the biotin-streptavidin-system. Biotin detection in the range of 10-7 to 10-10 M verified the concept of the synthesis strategy and the advantage of using monodisperse SCPs for easier and faster sensing applications of the SCP assay.

Stiffness Variation of 3D Collagen Networks by Surface Functionalization of Network Fibrils with Sulfonated Polymers.

Fibrillar collagen is the most prominent protein in the mammalian extracellular matrix. Therefore, it is also widely used for cell culture research and clinical therapy as a biomimetic 3D scaffold. Charged biopolymers, such as sulfated glycosaminoglycans, occur in vivo in close contact with collagen fibrils, affecting many functional properties such as mechanics and binding of growth factors. For in vitro application, the functions of sulfated biopolymer decorations of fibrillar collagen materials are hardly understood. Herein, we report new results on the stiffness dependence of 3D collagen I networks by surface functionalization of the network fibrils with synthetic sulfonated polymers, namely, poly(styrene sulfonate) (PSS) and poly(vinyl sulfonate) (PVS). A non-monotonic stiffness dependence on the amount of adsorbed polymer was found for both polymers. The stiffness dependence correlated to a transition from mono- to multilayer adsorption of sulfonated polymers on the fibrils, which was most prominent for PVS. PVS mono- and multilayers caused a network stiffness change by a factor of 0.3 and 2, respectively. A charge-dependent weakening of intrafibrillar salt bridges by the adsorbed sulfonated polymers leading to fibrillar softening is discussed as the mechanism for the stiffness decrease in the monolayer regime. In contrast, multilayer adsorption can be assumed to induce interfibrillar bridging and an increase in network stiffness. Our in vitro results have a strong implication on in vivo characteristics of fibrillar collagen I, as sulfated glycosaminoglycans frequently attach to collagen fibrils in various tissues, calling for an up to now overlooked impact on matrix and tendon mechanics.

Fibrillar biopolymer-based scaffolds to study macrophage-fibroblast crosstalk in wound repair.

Controlled wound healing requires a temporal and spatial coordination of cellular activities within the surrounding extracellular matrix (ECM). Disruption of cell-cell and cell-matrix communication results in defective repair, like chronic or fibrotic wounds. Activities of macrophages and fibroblasts crucially contribute to the fate of closing wounds. To investigate the influence of the ECM as an active part controlling cellular behavior, coculture models based on fibrillar 3D biopolymers such as collagen have already been successfully used. With well-defined biochemical and biophysical properties such 3D scaffolds enable in vitro studies on cellular processes including infiltration and differentiation in an in vivo like microenvironment. Further, paracrine and autocrine signaling as well as modulation of soluble mediator transport inside the ECM can be modeled using fibrillar 3D scaffolds. Herein, we review the usage of these scaffolds in in vitro coculture models allowing in-depth studies on the crosstalk between macrophages and fibroblasts during different stages of cutaneous wound healing. A more accurate mimicry of the various processes of cellular crosstalk at the different stages of wound healing will contribute to a better understanding of the impact of biochemical and biophysical environmental parameters and help to develop further strategies against diseases such as fibrosis.

Functional label-free assessment of fibroblast differentiation in 3D collagen-I-matrices using particle image velocimetry.

Fibroblasts are a diverse population of connective tissue cells that are a key component in physiological wound healing. Myofibroblasts are differentiated fibroblasts occurring in various physiological and pathological conditions, like in the healing of wounds or in the tumour microenvironment. They exhibit important functions compared to fibroblasts in terms of proliferation, protein secretion, and contractility. The gold standard to distinguish myofibroblasts is alpha-smooth muscle actin (αSMA) expression and its incorporation in stress fibres, which is only revealed by gene expression analysis and immunostaining. Here, we introduce an approach to functionally determine the myofibroblast status of live fibroblasts directly in in vitro cell culture by analysing their ability to contract the extracellular matrix around them without the need for labelling. It is based on particle image velocimetry algorithms applied to dynamic deformations of the extracellular matrix network structure imaged by phase contrast microscopy. Advanced image analysis allows us to distinguish between various differentiation stages of fibroblasts including the dynamic change over several days. We further apply machine learning classification to automatically evaluate different cell culture conditions. With this new method, we provide a versatile tool to functionally evaluate the dynamic process of fibroblast differentiation. It can be applied for in vitro screening studies in biomimetic 3D cell cultures with options to extend it to other cell systems with contractile phenotypes.

Biomimetic estrogen sensor based on soft colloidal probes.

An increasing number of reports substantiate the link between emerging estrogenic pollutants and a variety of adverse effects including developmental disorders, infertility, cancer and neurological disorders, threatening public health as well as environment. The detection of the diverse classes of estrogenic and antiestrogenic substances is still challenging due to analytics which needs to cover the whole range of compounds acting on estrogen receptors and the complex estrogen pathways. In this proof-of-concept study, we report a novel biomimetic detection scheme based on the specific recognition of estrogenic ligands by estrogen sulfotransferase 1E1 (SULT1E1), which acts as one of the key enzymes in estrogen homeostasis. SULT1E1 was site-specifically immobilized on transparent glass slides via a hexahistidine-tag in a multi-step procedure. Soft colloidal probes (SCPs) covalently functionalized with ligands of SULT1E1, namely estrone and estradiol 17-(ß-D-glucuronide), served as adhesion probes. The various functionalization steps were analyzed and optimized using epifluorescence, confocal laser scanning as well as reflection interference contrast microscopy (RICM). A competitive SCP binding assay probing the elastic SCP deformation driven by the specific interaction between SCPs and the SULT1E1 decorated glass slides was employed in conjunction with an optical readout by RICM and automated image analysis to detect estrogenic compounds by their inhibition of SCP adhesion. This sensing concept has demonstrated exceptional specificity for estrogenic steroid compounds compared to structurally related substance classes and provides promising options for multiplexed assays and incorporation of other proteins of the endocrine system to fully capture the whole ensemble of hormonally active substances.

Impact of binding mode of low-sulfated hyaluronan to 3D collagen matrices on its osteoinductive effect for human bone marrow stromal cells.

Synthetically sulfated hyaluronan derivatives were shown to facilitate osteogenic differentiation of human bone marrow stromal cells (hBMSC) by application in solution or incorporated in thin collagen-based coatings. In the presented study, using a biomimetic three-dimensional (3D) cell culture model based on fibrillary collagen I (3D Col matrix), we asked on the impact of binding mode of low sulfated hyaluronan (sHA) in terms of adsorptive and covalent binding on osteogenic differentiation of hBMSC. Both binding modes of sHA induced osteogenic differentiation. Although for adsorptive binding of sHA a strong intracellular uptake of sHA was observed, implicating an intracellular mode of action, covalent binding of sHA to the 3D matrix induced also intense osteoinductive effects pointing towards an extracellular mode of action of sHA in osteogenic differentiation. In summary, the results emphasize the relevance of fibrillary 3D Col matrices as a model to study hBMSC differentiation in vitro in a physiological-like environment and that sHA can display dose-dependent osteoinductive effects in dependence on presentation mode in cell culture scaffolds.

Fibronectin-functionalization of 3D collagen networks supports immune tolerance and inflammation suppression in human monocyte-derived macrophages.

The extracellular matrix (ECM) is dynamically reorganized during wound healing. Concomitantly, recruited monocytes differentiate into macrophages. However, the role of the wound's ECM during this transition remain to be fully understood. Fibronectin is a multifunctional glycoprotein present in early wound ECM with a potential immunomodulatory role during monocyte-to-macrophage differentiation. Hence, to investigate the impact of fibronectin during this differentiation step, 3D fibrillar collagen type I networks with or without fibronectin-functionalization were engineered with defined topology (fibril and pore diameter: 0.8 µm; 7 µm) and amount of adsorbed fibronectin (0.15 µg per µg collagen). Primary, human monocytes were then differentiated into macrophages inside these networks. The immunological imprinting of the resulting macrophages was monitored by means of the expression of FABP4, CLEC4E, SLC2A6, and SOD2 which discriminate naïve and tolerized macrophages, as well pro-inflammatory (M1) and anti-inflammatory (M2) macrophage polarization. The analyses indicate that fibronectin-functionalization of collagen I networks induces macrophage tolerance rather than M1 or M2 macrophage phenotypes. This finding was confirmed by release profiles of pro- and anti-inflammatory cytokines such as IL6, IL8, CXCL10, and IL10. Nevertheless, upon LPS challenge, immune suppression by fibronectin was overridden since these macrophages could then deploy an efficient immune response. Our results therefore provide new perspectives in biomaterial science of wound healing scaffolds and the design of instructive materials for human monocyte-derived cells.

Construction of a 3D brain extracellular matrix model to study the interaction between microglia and T cells in co-culture.

Neurodegenerative disorders are characterised by the activation of brain-resident microglia cells and by the infiltration of peripheral T cells. However, their interplay in disease has not been clarified yet. It is difficult to investigate complex cellular dynamics in living animals, and simple two-dimensional (2D) cell culture models do not resemble the soft 3D structure of brain tissue. Therefore, we developed a biomimetic 3D in vitro culture system for co-cultivation of microglia and T cells. As the activation and/or migration of immune cells in the brain might be affected by components of the extracellular matrix, defined 3D fibrillar collagen I-based matrices were constructed and modified with hyaluronan and/or chondroitin sulphate, resembling aspects of brain extracellular matrix. Murine microglia and spleen-derived T cells were cultured alone or in co-culture on the constructed matrices. Microglia exhibited in vivo-like morphology and T cells showed enhanced survival when co-cultured with microglia or to a minor degree in the presence of glia-conditioned medium. The open and porous fibrillar structure of the matrix allowed for cell invasion and direct cell-cell interaction, with stronger invasion of T cells. Both cell types showed no dependence on the matrix modifications. Microglia could be activated on the matrices by lipopolysaccharide resulting in interleukin-6 and tumour necrosis factor-α secretion. The findings herein indicate that biomimetic 3D matrices allow for co-cultivation and activation of primary microglia and T cells and provide useful tools to study their interaction in vitro.

Matrix Remodeling and Hyaluronan Production by Myofibroblasts and Cancer-Associated Fibroblasts in 3D Collagen Matrices.

The tumor microenvironment is a key modulator in cancer progression and has become a novel target in cancer therapy. An increase in hyaluronan (HA) accumulation and metabolism can be found in advancing tumor progression and are often associated with aggressive malignancy, drug resistance and poor prognosis. Wound-healing related myofibroblasts or activated cancer-associated fibroblasts (CAF) are assumed to be the major sources of HA. Both cell types are capable to synthesize new matrix components as well as reorganize the extracellular matrix. However, to which extent myofibroblasts and CAF perform these actions are still unclear. In this work, we investigated the matrix remodeling and HA production potential in normal human dermal fibroblasts (NHFB) and CAF in the absence and presence of transforming growth factor beta -1 (TGF-ß1), with TGF-ß1 being a major factor of regulating fibroblast differentiation. Three-dimensional (3D) collagen matrix was utilized to mimic the extracellular matrix of the tumor microenvironment. We found that CAF appeared to response insensitively towards TGF-ß1 in terms of cell proliferation and matrix remodeling when compared to NHFB. In regards of HA production, we found that both cell types were capable to produce matrix bound HA, rather than a soluble counterpart, in response to TGF-ß1. However, activated CAF demonstrated higher HA production when compared to myofibroblasts. The average molecular weight of produced HA was found in the range of 480 kDa for both cells. By analyzing gene expression of HA metabolizing enzymes, namely hyaluronan synthase (HAS1-3) and hyaluronidase (HYAL1-3) isoforms, we found expression of specific isoforms in dependence of TGF-ß1 present in both cells. In addition, HAS2 and HYAL1 are highly expressed in CAF, which might contribute to a higher production and degradation of HA in CAF matrix. Overall, our results suggested a distinct behavior of NHFB and CAF in 3D collagen matrices in the presence of TGF-ß1 in terms of matrix remodeling and HA production pointing to a specific impact on tumor modulation.

Picomolar glyphosate sensitivity of an optical particle-based sensor utilizing biomimetic interaction principles.

The continually growing use of glyphosate and its critically discussed health and biodiversity risks ask for fast, low cost, on-site sensing technologies for food and water. To address this problem, we designed a highly sensitive sensor built on the remarkably specific recognition of glyphosate by its physiological target enzyme 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPs). This principle is implemented in an interferometric sensor by using the recently established soft colloidal probe (SCP) technique. EPSPs was site-specifically immobilized on a transparent surface utilizing the self-assembling properties of circadian clock gene 2 hydrophobin chimera and homogeneity of the layer was evidenced by atomic force microscopy. Exposure of the enzyme decorated biochip to glyphosate containing samples causes formation of enzyme-analyte complexes and a competitive loss of available binding sites for glyphosate-functionalized poly(ethylene glycol) SCPs. Functionalization of the SCPs with different types of linker molecules and glyphosate was assessed employing confocal laser scanning microscopy as well as confocal Raman microspectroscopy. Overall, reflection interference contrast microscopy analysis of SCP-biochip interactions revealed a strong influence of linker length and glyphosate coupling position on the sensitivity of the sensor. In employing a combination of pentaglycine linker and tethering glyphosate via its secondary amino group, concentrations in aqueous solutions down to 100 pM could be measured by the differential adhesion between SCP and biochip surface, supported by automated image analysis algorithms. This sensing concept could even prove its exceptional pM sensitivity in combination with a superior discrimination against structurally related compounds.

3D Scaffold-Based Macrophage Fibroblast Coculture Model Reveals IL-10 Dependence of Wound Resolution Phase.

Persistent inflammation and impaired repair in dermal wound healing are frequently associated with cell-cell and cell-matrix miscommunication. A direct coculture model of primary human myofibroblasts (MyoFB) and M-CSF-differentiated macrophages (M-Mɸ) in fibrillar three-dimensional Collagen I (Coll I) matrices is developed to study intercellular interactions. The coculture experiments reveal the number of M-Mɸ regulated MyoFB dedifferentiation in a dose-dependent manner. The amount of MyoFB decreases in dependence of the number of cocultured M-Mɸ, even in the presence of MyoFB-inducing transforming growth factor ß1 (TGF-ß1 ). Gene expression analysis of matrix proteins (collagen I, collagen III, ED-A-fibronectin) confirms the results of an altered MyoFB phenotype. Additionally, M-Mɸ is shown to be the main source of secreted cytokine interleukin-10 (IL-10), which is suggested to affect MyoFB dedifferentiation. These findings indicate a paracrine impact of IL-10 secretion by M-Mɸ on the MyoFB differentiation status counteracting the TGF-ß1 -driven MyoFB activation. Hence, the in vitro coculture model simulates physiological situations during wound resolution and underlines the importance of paracrine IL-10 signals by M-Mɸ. In sum, the 3D Coll I-based matrices with a MyoFB-M-Mɸ coculture form a highly relevant biomimetic model of late stages of wound healing.

Biomimetic tissue models reveal the role of hyaluronan in melanoma proliferation and invasion.

Interactions of hyaluronan (HA) and tumor and stromal cells are highly discussed as one of the major contributors in tumor progression and metastasis. The balance of HA in the tissue is highly regulated by two key enzyme classes; hyaluronan synthases (HAS) and hyaluronidases (HYAL). Current reports hint that the HA amount in the tissue is correlated with poor prognosis in melanoma, the most life-threatening skin tumor. In this work, we generated in vivo mouse models with low and high expression of Has2 and used the models for studying melanoma proliferation of the B78D14 melanoma cell line. We found that a strong reduction of HA amount in the skin was correlated to decreased tissue stiffness and a reduction in tumor weight. Since tumor cells have a direct contact to the HA in the tumor and at the stroma interface, we reconstituted different biomimetic in vitro models using fibroblasts derived from a mouse model to recapitulate melanoma cell behavior at the tumor boundary, namely, (i) decellularized fibroblast matrix (FbECM), (ii) fibroblast embedded into 3D collagen matrices (FbColl), and (iii) well-defined HA-functionalized 3D collagen matrices (HAColl). We found no considerable effect of high and low amounts of fibroblast-derived HA in the matrices on melanoma proliferation and invasion. However, HYAL1-treated FbECM and FbColl, as well as HAColl functionalized with low molecular weight HA (34 kDa) promoted proliferation and invasion of melanoma cells in a concentration dependent manner. Our results emphasize the molecular weight specific effects of HA in regulation of melanoma behavior and provide an alternative explanation for the in vivo observation of HA dependent tumor growth.

Remodeling of Three-Dimensional Collagen I Matrices by Human Bone Marrow Stromal Cells during Osteogenic Differentiation In Vitro.

Cell fate is triggered by the characteristics of the surrounding extracellular matrix (ECM) including its composition and topological and mechanical properties. Human bone marrow stromal cells (hBMSC) are known to reside in a niche environment where they are maintained in a quiescent, multipotent state, also controlled by the ECM characteristics. In this in vitro study, three-dimensional (3D) fibrillary collagen I (Col)-based matrices with defined topological and mechanical characteristics were used (pore size of 3-4 µm, fibril diameter of ∼0.7 µm, ∼90 Pa (non-cross-linked), and ∼160 Pa (cross-linked)), mimicking conditions of the environment in the bone marrow. The performance of non-cross-linked and cross-linked scaffolds during osteogenic differentiation of hBMSC in terms of matrix stiffness and proteolytic degradability was investigated. Cell adhesion, morphology, and invasion as well as matrix remodeling were investigated on cross-linked and non-cross-linked Col matrices over 22 days. About 25% of the cells invaded the matrices and showed a spread morphology independent of cross-linking. Cellular proteolytic matrix degradation in terms of a decreased matrix layer thickness was only found for non-cross-linked matrices at constant pore size and fibril diameter. Osteogenic differentiation of hBMSC was examined by alkaline phosphatase staining and enzyme activity (early marker) and calcium phosphate deposition (late marker) and was similarly supported in both scaffolds. Furthermore, both matrices were strongly stiffened by about 10-fold because of high mineralization under osteogenic conditions. In summary, these results emphasize that fibrillary 3D Col matrices are a suitable model to study primary hBMSC behavior in terms of ECM remodeling during osteogenesis at defined in vitro conditions.

Surface Functionalization by Hydrophobin-EPSPS Fusion Protein Allows for the Fast and Simple Detection of Glyphosate.

Glyphosate, the most widely used pesticide worldwide, is under debate due to its potentially cancerogenic effects and harmful influence on biodiversity and environment. Therefore, the detection of glyphosate in water, food or environmental probes is of high interest. Currently detection of glyphosate usually requires specialized, costly instruments, is labor intensive and time consuming. Here we present a fast and simple method to detect glyphosate in the nanomolar range based on the surface immobilization of glyphosate's target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) via fusion to the hydrophobin Ccg2 and determination of enzyme activity with a malachite green assay, which is a common photometric technique to measure inorganic phosphate (Pi). The assay demonstrates a new approach for a fast and simple detection of pesticides.