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Item does not contain fulltext, Nanoparticulate drug delivery systems are currently explored to overcome critical challenges associated with classical administration forms. In this study, we present a drug delivery system based on a novel class of proteinaceous biodegradable nano/micro capsules, lyophilisomes. Lyophilisomes can be prepared from biomolecules without the need for amphiphilicity. Albumin-based lyophilisomes were prepared by freezing, annealing and lyophilizing, resulting in capsules ranging from 100 to 3000nm. Lyophilisomes were loaded with the anti-tumor drugs doxorubicin and curcumin using different concentrations and time/temperature regimes. Incubation in 0.1mg/ml doxorubicin or 1.0mg/ml curcumin resulted in an entrapment efficiency of 95+/-1% and 4+/-1%, respectively. This corresponds to a drug loading of 0.24mg doxorubicin per milligram albumin and 0.10mg curcumin per milligram albumin. Drug release profiles from doxorubicin and curcumin-loaded lyophilisomes were studied in culture medium and showed slow release for doxorubicin (2.7% after 72h), and rapid release for curcumin (55% after 72h). When applied to cells, non-loaded lyophilisomes did not influence cell viability, even at high concentrations (1mg/ml). Lyophilisomes were internalized by cells. When loaded with doxorubicin and curcumin, lyophilisomes strongly reduced cell proliferation and viability of SKOV-3 and HeLa cells, respectively, to a level similar or better compared to an equal amount of free drugs. In conclusion, albumin lyophilisomes show potential as (nano)carriers of drugs for tumor cell elimination.

Item does not contain fulltext, Glycosaminoglycans (GAGs) are long, anionic polysaccharides involved in many basic aspects of mammalian physiology and pathology. Here we describe a method to extract GAGs from formalin-fixed, paraffin-embedded tissues and found that they are structurally comparable with GAGs extracted from frozen tissues. We employed this method to structurally characterize GAGs in tissues, including laser-dissected layers of skin and pathological specimens. This method enables the use of the archival paraffin-embedded material for detailed (structural) analysis of GAGs.

Contains fulltext : 108918.pdf (Publisher’s version ) (Open Access), Heparan sulfate endosulfatases Sulf1 and Sulf2 hydrolyze 6-O-sulfate in heparan sulfate, thereby regulating cellular signaling. Previous studies have revealed that Sulfs act predominantly on UA2S-GlcNS6S disaccharides and weakly on UA-GlcNS6S disaccharides. However, the specificity of Sulfs and their role in sulfation patterning of heparan sulfate in vivo remained unknown. Here, we performed disaccharide analysis of heparan sulfate in Sulf1 and Sulf2 knock-out mice. Significant increases in DeltaUA2S-GlcNS6S were observed in the brain, small intestine, lung, spleen, testis, and skeletal muscle of adult Sulf1(-/-) mice and in the brain, liver, kidney, spleen, and testis of adult Sulf2(-/-) mice. In addition, increases in DeltaUA-GlcNS6S were seen in the Sulf1(-/-) lung and small intestine. In contrast, the disaccharide compositions of chondroitin sulfate were not primarily altered, indicating specificity of Sulfs for heparan sulfate. For Sulf1, but not for Sulf2, mRNA expression levels in eight organs of wild-type mice were highly correlated with increases in DeltaUA2S-GlcNS6S in the corresponding organs of knock-out mice. Moreover, overall changes in heparan sulfate compositions were greater in Sulf1(-/-) mice than in Sulf2(-/-) mice despite lower levels of Sulf1 mRNA expression, suggesting predominant roles of Sulf1 in heparan sulfate desulfation and distinct regulation of Sulf activities in vivo. Sulf1 and Sulf2 mRNAs were differentially expressed in restricted types of cells in organs, and consequently, the sulfation patterns of heparan sulfate were locally and distinctly altered in Sulf1 and Sulf2 knock-out mice. These findings indicate that Sulf1 and Sulf2 differentially contribute to the generation of organ-specific sulfation patterns of heparan sulfate.

Item does not contain fulltext, Skin substitutes are of great benefit in the treatment of patients with full thickness wounds, but there is a need for improvement with respect to wound closure with minimal contraction, early vascularisation, and elastin formation. In this study we designed and developed an acellular double-layered skin construct, using matrix molecules and growth factors to target specific biological processes. The epidermal layer was prepared using type I collagen, heparin and fibroblast growth factor 7 (FGF7), while the porous dermal layer was prepared using type I collagen, solubilised elastin, dermatan sulfate, heparin, fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF). The construct was biochemically and morphologically characterised and evaluated in vivo using a rat full thickness wound model. The results were compared with the commercial skin substitute IntegraDRT and untreated wounds. The double-layered construct was prepared according to the design specifications. The epidermal layer was about 40 mum thick, containing 9% heparin and 0.2 mug FGF7 mg per layer, localised at the periphery. The dermal layer was 2.5 mm thick, had rounded pores and contained 10% dermatan sulfate+heparin, and 0.7 mug FGF2+VEGF mg per layer. The double-layered skin construct was implanted in a skin defect and on day 7, 14, 28 and 112 the (remaining) wound area was photographed, excised and (immuno) histologically evaluated. The double-layered skin construct showed more cell influx, significantly less contraction and increased blood vessel formation at early time points in comparison with IntegraDRT and/or the untreated wound. On day 14 the double-layered skin construct also had the fewest myofibroblasts present. On day 112 the double-layered skin construct contained more elastic fibres than IntegraDRT and the untreated wound. Structures resembling hair follicles and sebaceous glands were found in the double-layered skin construct and the untreated wound, but hardly a

01 november 2010, Contains fulltext : 89668.pdf (publisher's version ) (Closed access), OBJECTIVE: Despite the efficacy of collagen in femoral artery pseudoaneurysm treatment, as reported in one patient study, its use has not yet gained wide acceptance in clinical practice. In this particular study, the collagen was not described in detail. To further investigate the potential of collagen preparations, we prepared and characterized highly purified injectable fibrillar type I collagen and evaluated its use for femoral artery pseudoaneurysm (PSA) treatment in vivo using a pig model. METHODS: Purified fibrillar type I collagen was characterized using electron microscopy. The effect of three different sterilization procedures, ie, hydrogen peroxide gas plasma (H2O2), ethylene oxide gas (EtO), and gamma irradiation, was studied on both SDS-PAGE and platelet aggregation. Different collagen injectables were prepared (3%, 4%, and 5%) and tested using an injection force test applying a 21-gauge needle. To evaluate the network characteristics of the injectable collagen, the collagen was suspended in phosphate buffered saline (PBS) at 37 degrees C and studied both macroscopically and electron microscopically. To determine whether the collagen induced hemostasis in vivo, a pig PSA model was used applying a 4% EtO sterilized collagen injectable, and evaluation by angiography and routine histology. RESULTS: Electron microscopy of the purified type I collagen revealed intact fibrils with a distinct striated pattern and a length<300 mum. Both SDS-PAGE and platelet aggregation analysis of the sterilized collagen indicated no major differences between EtO and H2O2 sterilization, although gamma-irradiated collagen showed degradation products. Both 3% and 4% (w/v) collagen suspensions were acceptable with respect to the force used (<50 N). The 4% suspension was selected as the preferred injectable collagen, which formed a dense network under physiologic conditions. Testing the collagen in vivo (n=5), the angiograms revealed that the PSA partly or completely coagulated. Hi

01 februari 2010, Contains fulltext : 87807.pdf (publisher's version ) (Closed access), Endocytosis, subsequent protein sorting into multivesicular bodies (MVBs), and eventual degradation in lysosomes compose an important mechanism for controlling protein expression on the plasma membrane. The lysosomal trafficking regulator interacting protein-5 (LIP5) is part of the complex protein machinery involved in MVB biosynthesis. LIP5 interacts with other players of the ESCRT machinery as well as with two known cargo proteins, AQP2 and EGFR, whose degradation is affected upon reduction of LIP5 expression. To investigate the expression and localization pattern of LIP5, we studied LIP5 protein expression in a mouse tissue panel and subjected various rodent and human tissues to immunohistochemistry. Immunoblotting revealed that, except for jejunum, LIP5 is expressed as a 42 kDa protein in all mouse tissues tested. Alternatively-spliced gene products could not be detected. Immunohistochemical studies revealed that in tissues positive for LIP5, LIP5 is detected in virtually all epithelial cells of the examined rodent and human tissues. The observed LIP5 expression in epithelial tissues suggests that LIP5 is of particular importance in the MVB sorting and degradation of proteins expressed in polarized cells.

01 december 2010, Contains fulltext : 88948.pdf (publisher's version ) (Closed access), There is a consistent need for a suitable natural biomaterial to function as an arterial prosthesis in achieving arterial regeneration. Natural grafts are generally obtained by decellularization of native blood vessels, but batch to batch variations may occur and the nature/content of remaining contaminants is generally unknown. In this study we fabricated a molecularly defined natural arterial graft from scratch resembling the native three layered architecture from the fibrillar extracellular matrix components collagen and elastin. Using casting, moulding, freezing and lyophilization techniques, a triple layered construct was prepared consisting of an inner layer of elastin fibres, a middle (porous) film layer of collagen fibrils and an outer scaffold layer of collagen fibrils. The construct was carbodiimide cross-linked and heparinized. Characterization included biochemical/biophysical analyses, scanning electron microscopy, micro-computed tomography, (immuno)histology and haemocompatibility. Burst pressures were up to 400mm Hg and largely conferred by the intermediate porous collagen film layer. The highly purified type I collagen fibrils and elastin fibres used did not evoke platelet aggregation in vitro. Suturability of the graft in end to side anastomosis was successful and considered adequate for in vivo application.

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01 november 2010, Contains fulltext : 88354.pdf (publisher's version ) (Closed access), The in vivo performance of tissue-engineered constructs is often based on generally accepted read-out parameters, like (immuno)histology. In this study, high-density gene expression microarrays and gene ontology (GO) analysis were used as a read-out tool to identify the biological processes occurring after implantation of an acellular collagen-based skin construct using a rat full-thickness wound model. A freely-available program (DAVID) was used to identify up/downregulated biological processes (GO-terms) and results were compared to wound healing/regeneration without a construct. The entire process from RNA isolation to biological interpretation is explained step-by-step. Conventional (immuno)histology was used to validate the biological processes identified and indicate that microarray analysis may provide a valuable, fast and unbiased tool to evaluate the in vivo performance of tissue-engineered constructs. However, challenges remain e.g. with regards to the development of specific GO-terms and annotation of the (rat) genome.

01 juli 2010, Contains fulltext : 88102.pdf (publisher's version ) (Closed access), PURPOSE: To prepare a porcine model for femoral artery pseudoaneurysm via a one-step surgical procedure without the need for microsurgery. MATERIALS AND METHODS: This pseudoaneurysm model involves the preparation of an arteriovenous shunt between the femoral artery and femoral vein in which approximately 2 cm of the vein is segmented by proximal and distal closure with the use of ligatures. The femoral pseudoaneurysm models were evaluated by angiography, Doppler auscultation, and histologic examination. RESULTS: In seven of eight pigs, angiography and Doppler auscultation showed that the pseudoaneurysm models were open and that there was communication between the pseudoaneurysm model and the femoral artery. The mean length (+/-SD) of the pseudoaneurysm model was 1.9 cm +/- 0.3 (n= 7), with a neck region of 4 mm. Histologic analysis confirmed that pseudoaneurysm models were open and no thrombi were observed. CONCLUSIONS: The principal advantages of this model are the location of the pseudoaneurysm model, the short period of clamping, and the controllable size. The pig pseudoaneurysm model is straightforward and reproducible, and may serve as a useful tool in the evaluation of interventional strategies for treatment of pseudoaneurysms.

Contains fulltext : 79581.pdf (publisher's version ) (Open Access), The aim of this work was to introduce high-resolution computed tomography (micro-CT) for scaffolds made from soft natural biomaterials, and to compare these data with the conventional techniques scanning electron microscopy and light microscopy. Collagen-based scaffolds were used as examples. Unlike mineralized tissues, collagen scaffolds do not provide enough X-ray attenuation for micro-CT imaging. Therefore, various metal-based contrast agents were applied and evaluated using two structurally distinct scaffolds, one with round pores and one with unidirectional lamellae. The optimal contrast techniques for obtaining high-resolution three-dimensional images were either a combination of osmium tetroxide and uranyl acetate, or a combination of uranyl acetate and lead citrate. The data obtained by micro-CT analysis were in line with data obtained by light and electron microscopy. However, small structures (less than a few mum) could not be visualized due to limitation of the spot size of the micro-CT apparatus. In conclusion, reliable three-dimensional images of scaffolds prepared from soft natural biomaterials can be obtained using appropriate contrast protocols. This extends the use of micro-CT analysis to soft materials, such as protein-based biomaterials.

Contains fulltext : 79597.pdf (publisher's version ) (Closed access), Micromechanical properties of single elastic fibers and fibrillin-microfibrils, isolated from equine ligamentum nuchae using chemical and enzymatic methods, were determined with atomic force microscopy (AFM). Young's moduli of single elastic fibers immersed in water, devoid of or containing fibrillin-microfibrils, were determined using bending tests. Bending freely suspended elastic fibers on a micro-channeled substrate by a tip-less AFM cantilever generated a force versus displacement curve from which Young's moduli were calculated. For single elastic fibers, Young's moduli in the range of 0.3-1.5 MPa were determined, values not significantly affected by the presence of fibrillin-microfibrils. To further understand the role of fibrillin-microfibrils in vertebrate elastic fibers, layers of fibrillin-microfibrils were subjected to nano-indentation tests. From the slope of the force versus indentation curves, Young's moduli ranging between 0.56 and 0.74 MPa were calculated. The results suggest that fibrillin-microfibrils are not essential for the mechanical properties of single vertebrate elastic fibers.

Contains fulltext : 80686.pdf (publisher's version ) (Closed access), We recently demonstrated aberrant staining of fibrillin-1 in lung tissue specimens with emphysematous lesions. In this study, we have extended this observation by an elaborate analysis of the elastic fibre. Using domain-specific antibodies to fibrillin-1, and to other elastin fibre-associated molecules, lung tissue derived from patients without obvious clinical emphysema, but harbouring various degrees of microscopical emphysematous lesions, was analysed. In addition, the fibrillin-regulated growth factor TGF-beta was studied. Electron microscopy and biochemical analysis of desmosine (a marker for elastin) were also performed. Results were compared with lung tissue derived from patients with clinical emphysema. Domain-specific antibodies recognizing the C-terminal, N-terminal, and middle part of fibrillin-1 showed aberrant staining patterns associated with increasing degrees of microscopical emphysema. Staining for elastin, emilin-1, and fibulin-2 was, however, not aberrant. TGF-beta staining was markedly increased. On the electron microscopic, but not light microscopical, level, initial elastic fibre degradation was noticed in specimens with microscopical emphysema. Lung specimens from patients with clinical emphysema also displayed fragmented fibrillin-1 staining and, in addition, displayed extensive degradation of the elastic fibre. The results suggest that fibrillin-1 anomalies and TGF-beta overexpression are associated with initial events occurring during the emphysematous process. Based on these and other data, a mechanism for emphysematogenesis is proposed.

Contains fulltext : 70670.pdf (publisher's version ) (Open Access), Elastin is the prime protein in elastic tissues that contributes to elasticity of, for example, lung, aorta, and skin. Upon injury, elastic fibers are not readily replaced, which hampers tissue regeneration. Incorporation of solubilized elastin (hydrolyzed insoluble elastin fibers or elastin peptides) in biomaterials may improve regeneration, because solubilized elastin is able to promote proliferation as well as elastin synthesis. Porous biomaterials composed of highly purified collagen without and without elastin fibers or solubilized elastin were prepared by freezing and lyophilization. Solubilized elastin formed spherical structures that were incorporated in the collagenous part of the scaffolds and that persisted after chemical crosslinking of the scaffolds. Crosslinked scaffolds were subcutaneously implanted in young Sprague Dawley rats. Collagen-solubilized elastin and collagen scaffolds showed no calcification in this sensitive calcification model, in contrast to scaffolds containing elastin fibers. Collagen-solubilized elastin scaffolds also induced angiogenesis, as revealed by type IV collagen staining, and promoted elastic fiber synthesis, as shown with antibodies against rat elastin and fibrillin-1. It is concluded that scaffolds produced from collagen and solubilized elastin present a non-calcifying biomaterial with a capacity for soft-tissue regeneration, especially in relation to elastic fiber synthesis.

Contains fulltext : 71200.pdf (publisher's version ) (Open Access), Heparan sulfate in the glomerular basement membrane has been considered crucial for charge-selective filtration. In many proteinuric diseases, increased glomerular expression of heparanase is associated with decreased heparan sulfate. Here, we used mice overexpressing heparanase and evaluated the expression of different heparan sulfate domains in the kidney and other tissues measured with anti-heparan sulfate antibodies. Glycosaminoglycan-associated anionic sites were visualized by the cationic dye cupromeronic blue. Transgenic mice showed a differential loss of heparan sulfate domains in several tissues. An unmodified and a sulfated heparan sulfate domain resisted heparanase action in vivo and in vitro. Glycosaminoglycan-associated anionic sites were reduced about fivefold in the glomerular basement membrane of transgenic mice, whereas glomerular ultrastructure and renal function remained normal. Heparanase-resistant heparan sulfate domains may represent remnant chains or chains not susceptible to cleavage. Importantly, the strong reduction of glycosaminoglycan-associated anionic sites in the glomerular basement membrane without development of a clear renal phenotype questions the primary role of heparan sulfate in charge-selective filtration. We cannot, however, exclude that overexpression of heparanase and heparan sulfate loss in the basement membrane in glomerular diseases contributes to proteinuria.

Contains fulltext : 51552.pdf (publisher's version ) (Open Access), Heparan sulfate (HS) within the glomerular basement membrane (GBM) is thought to play a major role in the charge-selective properties of the glomerular capillary wall. Recent data, however, raise questions regarding the direct role of HS in glomerular filtration. For example, in situ studies suggest that HS may prevent plasma macromolecules from clogging the GBM, keeping it in an "open" state. We evaluated this potential role of HS in vivo by studying the passage of protein through the glomerular capillary wall in the presence and absence of HS. Intravenous administration of neuraminidase removed neuraminic acid--but not HS--from the GBM, and this led to albuminuria. Concomitant removal of HS with heparinase III, confirmed by ultrastructural imaging, prevented the development of albuminuria in response to neuraminidase treatment. Taken together, these results suggest that HS keeps the GBM in an open state, facilitating passage of proteins through the glomerular capillary wall.

Contains fulltext : 34565.pdf (publisher's version ) (Closed access)

Contains fulltext : 53032.pdf (publisher's version ) (Open Access), Heparan sulfates (HS) are long, unbranched, negatively charged polysaccharides that are bound to core proteins. HS in the glomerular basement membrane (GBM) is reported to be important for charge-selective permeability. Aberrant GBM HS expression has been observed in several glomerular diseases, such as diabetic nephropathy and membranous glomerulopathy, and a decrease in HS generally is associated with proteinuria. This study, with the use of a controlled in vivo approach, evaluated whether degradation of HS in rat GBM resulted in acute proteinuria. Rats received two intravenous injections of either heparinase III to digest HS or neuraminidase to remove neuraminic acids (positive control). Urine samples were taken at various time points, and at the end of the experiment, kidneys were removed and analyzed. Injection with heparinase III resulted in a complete loss of glomerular HS as demonstrated by immunofluorescence staining using anti-HS antibodies and by electron microscopy using cupromeronic blue in a critical electrolyte concentration mode. In the urine, a strong increase in HS was found within 2 h after the first injection. Staining for agrin, the major HS proteoglycan core protein in the GBM, was unaltered. No urinary albumin or other proteins were detected at any time point, and no changes in glomerular morphology were noticed. Injection of rats with neuraminidase, however, resulted in a major increase of urinary albumin and was associated with an increase in urinary free neuraminic acid. An increased glomerular staining with Peanut agglutinin lectin, indicative of removal of neuraminic acid, was noted. In conclusion, removal of HS from the GBM does not result in acute albuminuria, whereas removal of neuraminic acid does.

Contains fulltext : 51608schalkwijk.pdf (publisher's version ) (Closed access), An important issue in tissue engineering is the vascularisation of the implanted construct, which often takes several weeks. In vivo, the growth factors VEGF and FGF2 show a combined effect on both angiogenesis and maturation of blood vessels. Therefore, we hypothesise that the addition of these growth factors to an acellular construct increases blood vessel formation and maturation. To systematically evaluate the contribution of each scaffold component with respect to tissue response and in particular to blood vessel formation, five porous scaffolds were prepared and characterised, viz.: collagen, collagen with heparin, and collagen with heparin plus one or two growth factors (rrFGF2 and rrVEGF). Scaffolds were subcutaneously implanted in 3 months old Wistar rats. Of all scaffolds tested, the one with a combination of growth factors displayed the highest density of blood vessels (type IV collagen) and most mature blood vessels (smooth muscle actin). In addition, no hypoxic cells were found in this scaffold at day 7 and 21 (hypoxia inducible factor 1-alpha). These results indicate that the addition of both FGF2 and VEGF to an acellular construct enhances an early mature vasculature. This opens prospects for (acellular) tissue-engineered constructs in conditions as ischaemic heart disease or diabetic ulcers.

Contains fulltext : 35041.pdf (publisher's version ) (Open Access)

Contains fulltext : 50909schalkwijk.pdf (publisher's version ) (Closed access)

Contains fulltext : 49565.pdf (publisher's version ) (Open Access)