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 ß-amyloid (Aß) 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 Aß 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 ß-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 Aß 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.

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 (r(2) = 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.

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.

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.

Real-time measurements of coagulation on bacterial cellulose and conventional vascular graft materials.

The search for a functional, small diameter (<5mm) vascular graft has been ongoing for over 30 years, but yet there is no consistently reliable synthetic graft. The primary mechanisms of graft failure are intimal hyperplasia, poor blood flow and surface thrombogenicity. Bacterial cellulose (BC) became therefore a proposed new biosynthetic vascular graft material. Since conventional methods are not suited for coagulation measurements on BC, we have adapted the automated calibrated thrombin generation method for measurements of biomaterial-induced coagulation of BC as compared with clinically used graft materials i.e., expanded poly(tetrafluoroethylene) (ePTFE) and poly(ethyleneterephtalat) (PET). We have also visualized the coagulation propagation at the material surfaces. Thrombin generation experiments revealed dramatic differences between the materials tested. Both ePTFE and BC were found to generate longer lag times and ttpeak values than PET. Most importantly, BC was found to generate the lowest "peak", indicating a slower coagulation process at the surface. These results are also supported by the measurements of factor XIIa generation and analysis of surface coagulation times, which were detected in the following increasing order (mean + or - SD): PET (27 + or - 8 min)

Modification of nanocellulose with a xyloglucan-RGD conjugate enhances adhesion and proliferation of endothelial cells: implications for tissue engineering.

This paper describes a novel method for introducing the RGD cell adhesion peptide to enhance cell adhesion onto bacterial cellulose (BC). BC and cotton linters as reference were modified with xyloglucan (XG) and xyloglugan bearing a GRGDS pentapeptide. The adsorptions followed Langmuir adsorption behavior, where both XGs probably decorate the cellulose surfaces as a monolayer. The adsorption maximum of the XGs reached around 180 mg/g on BC and only about three times as much on cotton linters. The adsorption was verified with colorimetric methods. The specific surface area of BC measured with XG and XG-GRGDS was about 200 m (2)/g and was almost three times less for cotton linters, 60 m (2)/g. The difference in the amounts of XGs adsorbed might be explained by the swollen network of bacterial cellulose and a more exposed and accessible bulk as compared to cotton linters. The nanocellulose material was modified homogeneously throughout the material, as seen by the z-scan in confocal microscopy. Moreover, the modification in the water phase, in comparison with organic solvents, was clearly advantageous for preserving the morphology, as observed with SEM. The modification slightly increased the wettability, which might explain the decrease in or undetectable adsorption of adhesive protein shown by QCM-D. Initial cell studies showed that adhesion of human endothelial cells is enhanced when the BC hydrogel is modified with XG-GRGDS. QCM-D studies further revealed that the cell enhancement is due to the presence of the RGD epitope on XG and not to a nonspecific adsorption of fibronectin from cell culture medium. Optimization and proliferation studies of human endothelial cells onto bacterial cellulose modified with XG-GRGDS are currently being carried out at the Vascular Engineering Center, Sahlgrenska University Hospital, Gothenburg.

Influence of cultivation conditions on mechanical and morphological properties of bacterial cellulose tubes.

Bacterial cellulose (BC) was deposited in tubular form by fermenting Acetobacter xylinum on top of silicone tubes as an oxygenated support and by blowing different concentrations of oxygen, that is, 21% (air), 35%, 50%, and 100%. Mechanical properties such as burst pressure and tensile properties were evaluated for all tubes. The burst pressure of the tubes increased with an increase in oxygen ratio and reached a top value of 880 mmHg at 100% oxygen. The Young's modulus was approximately 5 MPa for all tubes, irrespective of the oxygen ratio. The elongation to break decreased from 30% to 10-20% when the oxygen ratio was increased. The morphology of the tubes was characterized by Scanning Electron Microscopy (SEM). All tubes had an even inner side and a more porous outer side. The cross section indicated that the tubes are composed of layers and that the amount of layers and the yield of cellulose increased with an increase in oxygen ratio. We propose that an internal vessel wall with high density is required for the tube to sustain a certain pressure. An increase in wall thickness by an increase in oxygen ratio might explain the increasing burst pressure with increasing oxygen ratio. The fermentation method used renders it possible to produce branched tubes, tubes with unlimited length and inner diameters. Endothelial cells (ECs) were grown onto the lumen of the tubes. The cells formed a confluent layer after 7 days. The tubes potential as a vascular graft is currently under investigation in a large animal model at the Centre of Vascular Engineering, Sahlgrenska University