Helen Fink

Bacterial cellulose modified with xyloglucan bearing the adhesion peptide RGD promotes endothelial cell adhesion and metabolism—a promising modification for vascular grafts

Today, biomaterials such as polytetrafluorethylene (ePTFE) are used clinically as prosthetic grafts for vascular surgery of large vessels (>5 mm). In small diameter vessels, however, their performance is poor due to early thrombosis. Bacterial‐derived cellulose (BC) is a new promising material as a replacement for blood vessels. This material is highly biocompatible in vivo but shows poor cell adhesion. In the native blood vessel, the endothelium creates a smooth non‐thrombogenic surface. In order to sustain cell adhesion, BC has to be modified. With a novel xyloglucan (XG) glycoconjugate method, it is possible to introduce the cell adhesion peptide RGD (Arg‐Gly‐Asp) onto bacterial cellulose. The advantage of the XG‐technique is that it is an easy one‐step procedure carried out in water and it does not weaken or alter the fiber structure of the hydrogel. In this study, BC was modified with XG and XGRGD to asses primary human vascular endothelial cell adhesion, proliferation, and metabolism as compared with unmodified BC. This XG‐RGD‐modification significantly increased cell adhesion and the metabolism of seeded primary endothelial cells as compared with unmodified BC whereas the proliferation rate was affected only to some extent. The introduction of an RGD‐peptide to the BC surface further resulted in enhanced cell spreading with more pronounced stress fiber formation and mature phenotype. This makes BC together with the XG‐method a promising material for synthetic grafts in vascular surgery and cardiovascular research. Copyright

Sequence-Specific Crosslinking of Electrospun, Elastin-Like Protein Preserves Bioactivity and Native-Like Mechanics

A nanoscale mimic of the extracellular matrix is electrospun from a highly tunable family of elastin-like proteins. A sequence-specific, two-step crosslinking procedure is developed to preserve the nanofiber morphology, elastin-like mechanics, and specific bioactivity. Rodent marrow stromal cells show sequence-specific adhesion on the matrices, which are imaged using label-free coherent anti-Stokes Raman scattering (CARS) microscopy.

An in vitro study of blood compatibility of vascular grafts made of bacterial cellulose in comparison with conventionally-used graft materials

In this study we analyzed the blood compatibility of bacterial cellulose (BC) as a new biosynthetic material for use as a vascular graft. As reference materials we used expanded polytetrafluoroethylene (ePTFE) and poly(ethylene terephthalate) (PET) vascular grafts. These materials are in clinical use today. Tubes with inner diameters of both 4 (not PET) and 6 mm were tested. Heparin-coated PVC tubes (hepPVC) were used as a negative control. Platelet consumption and thrombin-antithrombin complex (TAT) were used as parameters of coagulation and for complement activation, sC3a and sC5b-9 were used. The investigated parameters were measured after 1-h exposure to freshly drawn human blood supplemented with a low dose of heparin in a Chandler loop system. The results showed that BC exhibits no significant difference in platelet consumption, as compared with PET (6 mm), ePTFE and hepPVC. The PET material consumed more platelets than any of the other materials. The TAT generation for 4 mm tubes was not significantly different between BC and the other materials. For 6 mm tubes, however, differences were observed between hepPVC and PET (p < 0.0001); BC and hepPVC (p = 0.0016); ePTFE and PET (p < 0.0001); BC and ePTFE (p = 0.0029); BC and PET (p = 0.0141). Surprisingly, considering the low platelet consumption, the complement activation parameters (sC3a and sC5b-9) were much higher for BC, as compared with the other materials for both 4 and 6 mm tubes.

Intravital fluorescent microscopic evaluation of bacterial cellulose as scaffold for vascular grafts.

Although commonly used synthetic vascular grafts perform satisfactorily in large caliber blood vessels, they are prone to thrombosis in small diameter vessels. Therefore, small vessels might benefit from tissue engineered vascular grafts. This study evaluated bacterial cellulose (BC) as a potential biomaterial for biosynthetic blood vessels. We implanted the dorsal skinfold chambers in three groups of Syrian golden hamsters with BC (experimental group), polyglycolic acid, or expanded polytetrafluorethylene (control groups). Following implantation, we used intravital fluorescence microscopy, histology, and immunohistochemistry to analyze the biocompatibility, neovascularization, and incorporation of each material over a time period of 2 weeks. Biocompatibility was good in all groups, as indicated by the absence of leukocyte activation upon implantation. All groups displayed angiogenic response in the host tissue, but that response was highest in the polyglycolic acid group. Histology revealed vascularized granulation tissue surrounding all three biomaterials, with many proliferating cells and a lack of apoptotic cell death 2 weeks after implantation. In conclusion, BC offers good biocompatibility and material incorporation compared with commonly used materials in vascular surgery. Thus, BC represents a promising new biomaterial for tissue engineering of vascular grafts.

Plaque-associated lipids in Alzheimer’s diseased brain tissue visualized by nonlinear microscopy

By simultaneous coherent anti-Stokes Raman scattering (CARS) and 2-photon fluorescence microscopy of Thioflavin-S stained Alzheimer´s diseased human brain tissues, we show evidence of lipid deposits co-localizing with fibrillar β-amyloid (Aβ) plaques. Two lipid morphologies can be observed; lamellar structures and coalescing macro-aggregates of sub-micron sizes to ~25 μm. No significant lipid deposits were observed in non-fibrillar, diffuse plaques identified by Aβ immuno-staining. CARS microscopy of unlabeled samples confirms the lamellar and macro-aggregate lipid morphologies. The composition of the plaques was analyzed by CARS microspectroscopy and Raman microscopy; vibrational signatures of lipids with long acyl chains co-localize with the β-sheet vibrations. The lipid fluidity was evaluated from the CARS spectra, illustrating that the lipid composition/organization varies throughout the plaques. Altogether this indicates close amyloid-lipid interplay in fibrillar Aβ plaques, rendering them more dynamic compositions than previously believed and, hence, potential sources of toxic oligomers.

Imaging of Lipids in Microalgae with Coherent Anti-Stokes Raman Scattering Microscopy

Lipids are accumulated as giant droplets alongside coalescing emerging droplets under excessive lipid storage, in contrast to the multiple micron-sized droplets formed at normal conditions. Microalgae have great prospects as a sustainable resource of lipids for refinement into nutraceuticals and biodiesel, which increases the need for detailed insights into their intracellular lipid synthesis/storage mechanisms. As an alternative strategy to solvent- and label-based lipid quantification techniques, we introduce time-gated coherent anti-Stokes Raman scattering (CARS) microscopy for monitoring lipid contents in living algae, despite strong autofluorescence from the chloroplasts, at approximately picogram and subcellular levels by probing inherent molecular vibrations. Intracellular lipid droplet synthesis was followed in Phaeodactylum tricornutum algae grown under (1) light/nutrient-replete (control [Ctrl]), (2) light-limited (LL), and (3) nitrogen-starved (NS) conditions. Good correlation (r2 = 0.924) was found between lipid volume data yielded by CARS microscopy and total fatty acid content obtained from gas chromatography-mass spectrometry analysis. In Ctrl and LL cells, micron-sized lipid droplets were found to increase in number throughout the growth phases, particularly in the stationary phase. During more excessive lipid accumulation, as observed in NS cells, promising commercial harvest as biofuels and nutritional lipids, several micron-sized droplets were present already initially during cultivation, which then fused into a single giant droplet toward stationary phase alongside with new droplets emerging. CARS microspectroscopy further indicated lower lipid fluidity in NS cells than in Ctrl and LL cells, potentially due to higher fatty acid saturation. This agreed with the fatty acid profiles gathered by gas chromatography-mass spectrometry. CARS microscopy could thus provide quantitative and semiqualitative data at the single-cell level along with important insights into lipid-accumulating mechanisms, here revealing two different modes for normal and excessive lipid accumulation.

Self-Assembled Arrays of Dendrimer–Gold-Nanoparticle Hybrids for Functional Cell Studies†

Engineered surfaces with nanoscale features of gold on silicon or glass have recently been used to improve the understanding of adhesion-mediated environmental sensing of cells. Often such surfaces present a cell-binding ligand, such as arginine–glycine–aspartic acid (RGD) peptide motifs, at controlled intramolecular distances on an inert background surface such as polyethylene glycol (PEG). The adhesion mechanism of macromolecular ligands in which direct interaction with cells is nonspecific is not known and the cell response is dictated by the type and the concentration of proteins adsorbed from solution. Dendrimers may increase the availability and multivalency of cell-interacting ligands as a consequence of their branched shape and inherently high concentration of end groups. It is therefore interesting to examine the eventual effect of the macromolecular architecture on the cell viability by the controlled reduction of ligands on a surface. Herein, we demonstrate the fabrication of selfassembledmacromolecular hybrid arrays in which the relative position of two anionic macromolecules of different architectures—a carboxy-functionalized dendrimer and a linear polymer—is straightforwardly controlled on a PEG surface. We also show how the interaction of primary human endothelial cells with these surfaces is modulated by the molecular spacing and how protein binding to the macromolecular arrays can be evaluated by using a standard surface plasmon resonance (SPR) technique. Self-assembled, short-range-ordered Au nanoparticle (NP) arrays were used as a versatile template to arrange polymeric entities at the nanometer scale (Figure 1a). This

Effect of Shear Stress on the Expression of Coagulation and Fibrinolytic Factors in Both Smooth Muscle and Endothelial Cells in a Co-Culture Model

Blood vessels are subjected to forces due to the flow. Endothelial cells (EC) are recipients, cross-talk with smooth muscle cells (SMC), and regulate physiology. It was hypothesized that both EC and SMC respond to shear stress, which alters the expression of factors in coagulation and fibrinolysis. Methods: A co-culture of human saphenous vein EC (HSVEC) and human saphenous vein SMC (HSVSMC) was exposed to shear, following which the cells were separated. Gene expression of tissue factor, thrombomodulin (TM), plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) were analyzed with real-time RT-PCR. Protein expression was studied with ELISA. In HSVEC, the expression of PAI-1 (×2.1), tPA (×1.8), uPA (×1.6), tissue factor (×2.5) and TM (×1.9) was upregulated after 4 h of shear compared to controls. After 24 h of shear, expression was still upregulated in tPA (×2.3) and TM (×1.6). In HSVSMC, change in expression of PAI-1 (×2.1) was present after 4 h and in uPA (×2.1), and TM (×0.4) after 24 h. Both HSVEC and HSVSMC responded to shear, which led to altered expression of coagulation and fibrinolytic factors. This indicates that SMC, and interactions between EC and SMC, are more important in the regulation of vascular wall hemostasis than earlier studies have reported.

CARS microscopy of Alzheimer's diseased brain tissue

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder currently without cure, characterized by the presence of extracellular plaques surrounded by dystrophic neurites. In an effort to understand the underlying mechanisms, biochemical analysis (protein immunoblot) of plaque extracts reveals that they consist of amyloid-beta (Aβ) peptides assembled as oligomers, protofibrils and aggregates. Their spatial distribution has been confirmed by Thioflavin-S or immuno-staining with fluorescence microscopy. However, it is increasingly understood that the protein aggregation is only one of several mechanism that causes neuronal dysfunction and death. This raises the need for a more complete biochemical analysis. In this study, we have complemented 2-photon fluorescence microscopy of Thioflavin-S and Aβ immuno-stained human AD plaques with CARS microscopy. We show that the chemical build-up of AD plaques is more complex and that Aβ staining does not provide the complete picture of the spatial distribution or the molecular composition of AD plaques. CARS images provide important complementary information to that obtained by fluorescence microscopy, motivating a broader introduction of CARS microscopy in the AD research field.

Neuronal cell growth on polymeric scaffolds studied by CARS microscopy

For studies of neuronal cell integration and neurite outgrowth in polymeric scaffold materials as a future alternative for the treatment of damages in the neuronal system, we have developed a protocol employing CARS microscopy for imaging of neuronal networks. The benefits of CARS microscopy come here to their best use; (i) the overall three-dimensional (3D) arrangement of multiple cells and their neurites can be visualized without the need for chemical preparations or physical sectioning, potentially affecting the architecture of the soft, fragile scaffolds and (ii) details on the interaction between single cells and scaffold fibrils can be investigated by close-up images at sub-micron resolution. The establishment of biologically more relevant 3D neuronal networks in a soft hydrogel composed of native Extra Cellular Matrix (ECM) components was compared with conventional two-dimensional networks grown on a stiff substrate. Images of cells in the hydrogel scaffold reveal significantly different networking characteristics compared to the 2D networks, raising the question whether the functionality of neurons grown as layers in conventional cultivation dishes represents that of neurons in the central and peripheral nervous systems.