In this study, multilayers from polyethylene imine, heparin and chitosan are prepared at three di... more In this study, multilayers from polyethylene imine, heparin and chitosan are prepared at three different pH values of 5, 7 and 9. Water contact angle and quartz microbalance measurements show that resulting multilayers differ in terms of wetting behaviour, layer mass and mechanical properties. The multilayer is then formed within a gradient generation microfluidic (μFL) device. Polyethylene imine or heparin solutions of pH 5 are introduced into one inlet and the same solutions but at pH 9 into another inlet of the μFL device. The pH gradient established during the multilayer formation can be visualized inside the microchamber by pH sensitive fluorophores and confocal laser scanning microscopy. From this setup it is expected that properties of multilayers displayed at distinct pH values can be realised in a gradient manner inside the μFL device. Behaviour of the osteoblast cell line MG-63 seeded and cultured on top of multilayers created inside the μFL device support this hypothesis. It is observed that more cells adhere and spread on multilayers build-up at the basic side of the μFL channel, while those cells on top of multilayers built at pH 5 are fewer and smaller. These results are consistent with the behaviour of MG-63 cells seeded on multilayers formed at discrete pH values. It is particularly interesting to see that cells start to migrate from multilayers built at pH 5 to those built at pH 9 during 6 h of culture. Overall, the presented multilayer formation setup applying pH gradients leads to surfaces that promote migration of cells.
Some Pluronics, particularly F127, are known to stabilize nanospheres and prolong their circulati... more Some Pluronics, particularly F127, are known to stabilize nanospheres and prolong their circulation time in vivo. However, these copolymers of poly(ethylene glycol) (PEG) and poly(propylene glycol) are not biodegradable, and despite the long history, there is no approved commercial product using F127 for parenteral administration until now. Meanwhile, hydroxyethyl starch (HES) is a biodegradable polymer that is currently investigated as a substitute for PEG. In order to produce a fully biodegradable amphiphilic polymer, we esterified different molar masses of HES with lauric acid to get different molar substitutions. These polymers, as well as Pluronic F68 and F127, were used to stabilize poly(lactic-co-glycolic acid) (PLGA) nanospheres prepared by nanoprecipitation. For physicochemical characterization, the particle size, zeta potential, and the thickness of the adsorbed polymer layer were measured. The ability of the polymer coating to prevent the adsorption of human serum albumin (HSA) and fibrinogen (FBG) was evaluated. Finally, the phagocytosis of the stabilized nanospheres by a monocyte macrophage cell line (J774.2) was assessed. Results show that the PLGA nanospheres had an average particle size of 110-140 nm. The thickness of the adsorbed polymer layer increases with the increase in molar mass, and is generally higher for HES laurates than the studied Pluronics. Pluronic F68, F127 as well as the HES laurates with low molar substitution prevented the adsorption of HSA. HES laurates with low molar substitution and F127, but not F68, prevented the adsorption of FBG. The phagocytosis experiments showed that the HES laurates, particularly the one with the highest molar mass, could reduce the uptake of the nanospheres better than F68 and comparable to F127. Finally, these results warrant in vivo experiments to evaluate how the HES laurates can affect the pharmacokinetics and fate of the nanospheres.
Biochimica Et Biophysica Acta-general Subjects, 2007
The maintenance of endothelial cell (EC) monolayer architecture requires stable adhesions not onl... more The maintenance of endothelial cell (EC) monolayer architecture requires stable adhesions not only between neighboring cells but also between cells and the extracellular matrix. While the influence of biomaterials surface wettability on cell–substratum adhesion is rather well studied, its impact on cell–cell cohesion has not been extensively investigated. In the present study a model system consisting of hydrophilic and hydrophobic
Support membranes in bioartificial organs contact blood or plasma on one-side and adhesion depend... more Support membranes in bioartificial organs contact blood or plasma on one-side and adhesion dependent cells on the other side. Since membranes for biomedical applications, such as for haemodialysis, are optimised for blood contact and membranes for biotechnological ...
The role of osmotic forces and cell swelling in the influenza virus-induced fusion of unsealed or... more The role of osmotic forces and cell swelling in the influenza virus-induced fusion of unsealed or resealed ghosts of human erythrocytes was investigated under isotonic and hypotonic conditions using a recently developed fluorescence assay (Hoekstra, D., De Boer, T., Klappe, K., Wilschut, J. (1984) Biochemistry 23, 5675-5681). The method is based on the relief of fluorescence selfquenching of the fluorescent amphiphile octadecyl rhodamine B chloride (R18) incorporated into the ghost membrane as occurs when labeled membranes fuse with unlabeled membranes. No effect neither of the external osmotic pressure nor of cell swelling on virally mediated ghost fusion was established. Influenza virus fused unsealed ghosts as effectively as resealed ghosts. It is concluded that neither osmotic forces nor osmotic swelling of cells is necessary for virus-induced cell fusion. This is supported by microscopic observations of virus-induced fusion of intact erythrocytes in hypotonic and hypertonic media. A disruption of the spectrin-actin network did not cause an enhanced cell fusion at acidic pH of about 5 or any fusion at pH 7.4.
A universal method for reproducibly directing stem cell differentiation remains a major challenge... more A universal method for reproducibly directing stem cell differentiation remains a major challenge for clinical applications involving cell-based therapies. The standard approach for chondrogenic induction by micromass pellet culture is highly susceptible to interdonor variability. A novel method for the fabrication of condensation-like engineered microtissues (EMTs) that utilizes hydrophilic polysaccharides to induce cell aggregation is reported here. Chondrogenesis of mesenchymal stem cells (MSCs) in EMTs is signifi cantly enhanced compared to micromass pellets made by centrifugation measured by type II collagen gene expression, dimethylmethylene blue assay, and histology. MSCs from aged donors that fail to differentiate in pellet culture are successfully induced to synthesize cartilage-specifi c matrix in EMTs under identical media conditions. Furthermore, the EMT polysaccharides support the loading and release of the chondroinduction factor transforming growth factor β3 (TGF-β3). TGF-β-loaded EMTs (EMT +TGF ) facilitate cartilaginous tissue formation during culture in media not supplemented with the growth factor. The clinical potential of this approach is demonstrated in an explant defect model where EMT +TGF from aged MSCs synthesize de novo tissue containing sulfated glycosaminoglycans and type II collagen in situ.
A cell carrier made from synthetic material supporting selective growth of keratinocytes is a pro... more A cell carrier made from synthetic material supporting selective growth of keratinocytes is a promising approach to avoid the phenomenon of fibroblast overgrowth during in vitro culture of skin substitutes. Therefore, we investigated polymer membranes made of polyacrylonitrile and copolymers of acrylonitrile and N-vinylpyrrolidone (NVP) for their ability to support selectively the growth of keratinocytes. It was found that a copolymer with an NVP-content of 30% (NVP30) supports growth of human keratinocyte cell line (HaCaT) cells and inhibits fibroblast growth under serum-containing conditions. Cell proliferation of HaCaT cells was measured over 14 days. If both cell types were cultured under serum-free conditions for initial adhesion over 6 h on these NVP30 polymers, they adhered to the same extent. Long-term experiments over 7 days were performed as a coculture of both cell types showing that HaCaT cells had a growth advantage that seems to be related to the paracrine activity of contaminating fibroblasts. As a result, confluent layers of HaCaT cells were obtained with small numbers of remaining fibroblasts. The new poly [acrylonitrile-co(NVP) membranes seem to be a promising culture system for the production of epidermal transplants.
Regioselective derivatization was carried out introducing sulfate, phosphate or quaternary ammoni... more Regioselective derivatization was carried out introducing sulfate, phosphate or quaternary ammonia groups in C2-, C3- and C6-position of the anhydroglycose unit of cellulose. The anticoagulant potential of these derivatives was estimated with common clotting assays, such as thrombin time and partial thromboplastin time. It was found that a pronounced anticoagulant activity of cellulose derivatives could be achieved if the degree of substitution (DS) with sulfate was above 1.0. The anticoagulant activity was maximal at a DS of about 1.5 and then decreased again. Further, it was detected that particularly sulfation in C2-position resulted in a pronounced anticoagulant activity of cellulose derivatives. Development and application of assays specific for thrombin and factor Xa indicated that the anticoagulant potential of these cellulose derivatives was mainly due to anti-thrombin activity. The comparison of cellulose sulfates and cellulose derivatized with phosphate and quaternary ammonium groups demonstrated that the negative charge and type of the substituent is an important prerequisite for the anticoagulant activity of cellulose derivatives. Indeed, derivatization with sulfate produced superior activity in comparison with phosphate.
Journal of Materials Science: Materials in Medicine, 2008
Materials for blood contacting applications have to meet high requirements in terms to prevent th... more Materials for blood contacting applications have to meet high requirements in terms to prevent thrombotic complications after the medical treatment. Surface induced thrombosis, e.g. after application of cardiovascular devices, is linked clearly to the activation of coagulation system and platelet adhesion and activation. The flat sheet poly(ether imide) membrane (PEI) was modified by binding of iminodiacetic acid (IDA) for different periods of time to obtain surfaces with carboxylic (-COOH) groups, namely PEI-1 (modified for 1 min) and PEI-2 (modified for 30 min). The successful binding of the ligands was monitored by thionin acetate assay. The physico-chemical characteristics of the materials were analyzed by SEM, AFM, water contact angle and Zeta-potential measurements. Hemocompatibility of the polymer materials was studied by analyzing the activation of coagulation system (plasma kallikrein-like activity) and platelet adhesion/activation by using immunofluorescence technique. The blood response to PEI membranes was compared to that of a commercial poly(ethylene terephthalate) (PET) membrane. Our results showed that the increase of the negative charges on the modified PEI membrane surfaces (number of COOH groups) caused a higher contact activation of the coagulation system and a higher rate of platelet adhesion and activation. However, overall the hemocompatibility of all PEI membranes was higher than that of PET.
Particles with specific ligands for the adsorption of plasma proteins can be used in therapeutic ... more Particles with specific ligands for the adsorption of plasma proteins can be used in therapeutic or preparative apheresis. The development of these particles may benefit from an improved knowledge of the relationship between protein adsorption and the structure of ligands. Nanoparticles were functionalized with aliphatic diamines of increasing chain length; with the amino acids lysine, tryptophan, histidine, and their corresponding amines; and with tryptophan and histidine spaced with diamines of different length. Suitable protocols were developed for the washing of particles and the subsequent desorption of proteins adsorbed from human plasma. The adsorption pattern, as well as the quantification of the overall adsorption of proteins on these modified particles, was investigated with gel electrophoresis. This was followed by immunoblotting which yielded specific assessments of bound human serum albumin and fibrinogen. The comparison of protein adsorption with surface charge density and measured hydrophobicities yielded no simple correlations although in general more hydrophobic ligands bound higher quantities of protein. The detection of human serum albumin yielded similar results because it was observed for overall protein adsorption while the adsorption of fibrinogen expressed a different pattern. In this case, particular nanoparticles functionalized with aliphatic diamines bound significantly higher amounts of fibrinogen than all other ligands.
Little is known about the influence of substratum properties and composition on the ability of ce... more Little is known about the influence of substratum properties and composition on the ability of cells to translocate α5β1 integrins and to form fibrillar adhesion. We have examined the impact of self-assembled monolayers (SAMs) bearing different functional end groups (amines (NH2) or carboxylic acids (COOH)) on the presence of fibrillar adhesions in human fibroblasts attached to fibronectin-coated SAMs. Most of
The ability of human fibroblasts to remove and reorganize fibronectin (FN) bound on material surf... more The ability of human fibroblasts to remove and reorganize fibronectin (FN) bound on material surfaces was studied as a novel feature of material surface biocompatibility. Other traditional parameters of biocompatibility analyzed included cell spreading, clustering of fibronectin ...
Proteins and glycosaminoglycans (GAGs) are main constituents of the extracellular matrix (ECM). T... more Proteins and glycosaminoglycans (GAGs) are main constituents of the extracellular matrix (ECM). They act in synergism and are equally critical for the development, growth, function or survival of an organism. In this work, we developed surfaces that display these two classes of biomacromolecules, namely GAGs and proteins, in spatially controlled fashion. The generated surfaces can be used as a minimalistic but straightforward model aiding the elucidation of cell-ECM interactions. GAGs (hyaluronic acid and heparin) were covalently bound to amino func-tionalized surfaces and albumin or fibronectin were patterned by micro-contact printing on top of them. We demonstrate that adipose-derived stem cells (ASCs) can adhere either on protein or GAG pattern as a function of the patterned molecules. ASCs found on the GAG pattern had different morphology and expressed different surface markers than the cells adherent on the protein pattern. ASCs morphology and spreading were also dependent on t...
Stability of surface coatings against environmental stress, such as pH, high ionic strength, mech... more Stability of surface coatings against environmental stress, such as pH, high ionic strength, mechanical forces, and so forth, is crucial for biomedical application of implants. Here, a novel extracellular-matrix-like polyelectrolyte multilayer (PEM) system composed of collagen I (Col I) and oxidized glycosaminoglycans (oGAGs) was stabilized by intrinsic cross-linking due to formation of imine bonds between aldehydes of oxidized chondroitin sulfate (oCS) or hyaluronan (oHA) and amino groups of Col I. It was also found that Col I contributed significantly more to overall mass in CS-Col I than in HA-Col I multilayer systems and fibrillized particularly in the presence of native and oxidized CS. Adhesion and proliferation studies with murine C3H10T1/2 embryonic fibroblasts demonstrated that covalent cross-linking of oGAG with Col I had no adverse effects on cell behavior. By contrast, it was found that cell size and polarization was more pronounced on oGAG-based multilayer systems, whic...
The endothelization of cardiovascular implants is desirable to improve their blood compatibility.... more The endothelization of cardiovascular implants is desirable to improve their blood compatibility. The capacity of the endothelial cells to attach, migrate, proliferate and function on the implant surface depends on the presence of matrix proteins such as ®bronectin (FN) and ®brinogen (FNG). In this study, we show that the deposition of ®brinogen into extracellular matrix-like structures by human umbilical vein endothelial cells (HUVEC) is dependent on FN matrix formation. We found further that the process of organization of both adsorbed and soluble FN and FNG is dependent on the wettability of materials since it was observed only on a hydrophilic and not on a hydrophobic model surface. b 3 integrin was involved in the process of cell attachment to adsorbed FNG, while the mechanism of FNG ®brillogenesis required the activity of the b 1 integrin. Studies of EC morphology showed the predominant peripheral organization of actin ®laments and the formation of distinct leading and trailing cell edges suggesting a motile phenotype of cells when they are seeded on FNG. In summary, we concluded that adsorbed ®brinogen may enhance the motility of HUVEC and that soluble FNG requires FN matrix assembly to be organized in ®brilar structures.
Journal of Biomaterials Science-polymer Edition, 2002
The hemocompatibility of four different wettable polymer membranes, namely Cuprophan (CE), polyet... more The hemocompatibility of four different wettable polymer membranes, namely Cuprophan (CE), polyether-polycarbonate (PC-PE), polysulfone (PSU), and polyetherimide (PEI), was investigated with respect to brinogen (Fng) adsorption and platelet adhesion/ activation. In order to estimate the polar and dispersion components of the surface free energy, contact angles using water/ vapor and water/ n-hexadecane systems were measured. Adsorption of brinogen was studied using uorescence-labeled protein. The adsorption isotherms showed that the amount and the af nity of adsorbed Fng increased with decreasing surface wettability of the membranes, which correlates with the dispersion and polar components of the surface free energy. The conformational changes of adsorbed Fng were detected by measuring the difference between monoclonal antibody binding to the conformation-sensitive epitope in the D-domain and the binding of polyclonal anti-Fng antibody. The anticipated conformational / orientational changes were greater for PEI and PSU membranes (the least wettable membranes) and negligible for the more wettable PC-PE and CE membranes. In addition, a possible relationship with the degree of platelet activation was found, showing negligible platelet adhesion on PC-PE and CE, but high platelet adhesion on PEI and PSU. Furthermore, platelets were spread to a large extent on PEI, while the formation of aggregates was observed on PSU. This may correspond to the anticipated differences in the conformational state of Fng on both membranes.
NATO Science for Peace and Security Series A: Chemistry and Biology, 2010
Abstract The initial cellular events that take place at the biomaterials interface mimic to a cer... more Abstract The initial cellular events that take place at the biomaterials interface mimic to a certain extent the natural interaction of cells with the extracellular matrix (ECM). The cells adhering to the adsorbed soluble matrix proteins, such as fibronectin (FN) and fibrinogen (FNG) tend ...
NATO Science for Peace and Security Series A: Chemistry and Biology, 2010
... 1990). For example it was shown that hydrophobic methyl-terminated alkanethiol SAMs on gold i... more ... 1990). For example it was shown that hydrophobic methyl-terminated alkanethiol SAMs on gold induce mini-mal cell ... 13.3 Structure of self assembled monolayers composed of coupling unit, assembling structure and terminal group ... 13 Surface Modification and Cell Adhesion ...
In this study, multilayers from polyethylene imine, heparin and chitosan are prepared at three di... more In this study, multilayers from polyethylene imine, heparin and chitosan are prepared at three different pH values of 5, 7 and 9. Water contact angle and quartz microbalance measurements show that resulting multilayers differ in terms of wetting behaviour, layer mass and mechanical properties. The multilayer is then formed within a gradient generation microfluidic (μFL) device. Polyethylene imine or heparin solutions of pH 5 are introduced into one inlet and the same solutions but at pH 9 into another inlet of the μFL device. The pH gradient established during the multilayer formation can be visualized inside the microchamber by pH sensitive fluorophores and confocal laser scanning microscopy. From this setup it is expected that properties of multilayers displayed at distinct pH values can be realised in a gradient manner inside the μFL device. Behaviour of the osteoblast cell line MG-63 seeded and cultured on top of multilayers created inside the μFL device support this hypothesis. It is observed that more cells adhere and spread on multilayers build-up at the basic side of the μFL channel, while those cells on top of multilayers built at pH 5 are fewer and smaller. These results are consistent with the behaviour of MG-63 cells seeded on multilayers formed at discrete pH values. It is particularly interesting to see that cells start to migrate from multilayers built at pH 5 to those built at pH 9 during 6 h of culture. Overall, the presented multilayer formation setup applying pH gradients leads to surfaces that promote migration of cells.
Some Pluronics, particularly F127, are known to stabilize nanospheres and prolong their circulati... more Some Pluronics, particularly F127, are known to stabilize nanospheres and prolong their circulation time in vivo. However, these copolymers of poly(ethylene glycol) (PEG) and poly(propylene glycol) are not biodegradable, and despite the long history, there is no approved commercial product using F127 for parenteral administration until now. Meanwhile, hydroxyethyl starch (HES) is a biodegradable polymer that is currently investigated as a substitute for PEG. In order to produce a fully biodegradable amphiphilic polymer, we esterified different molar masses of HES with lauric acid to get different molar substitutions. These polymers, as well as Pluronic F68 and F127, were used to stabilize poly(lactic-co-glycolic acid) (PLGA) nanospheres prepared by nanoprecipitation. For physicochemical characterization, the particle size, zeta potential, and the thickness of the adsorbed polymer layer were measured. The ability of the polymer coating to prevent the adsorption of human serum albumin (HSA) and fibrinogen (FBG) was evaluated. Finally, the phagocytosis of the stabilized nanospheres by a monocyte macrophage cell line (J774.2) was assessed. Results show that the PLGA nanospheres had an average particle size of 110-140 nm. The thickness of the adsorbed polymer layer increases with the increase in molar mass, and is generally higher for HES laurates than the studied Pluronics. Pluronic F68, F127 as well as the HES laurates with low molar substitution prevented the adsorption of HSA. HES laurates with low molar substitution and F127, but not F68, prevented the adsorption of FBG. The phagocytosis experiments showed that the HES laurates, particularly the one with the highest molar mass, could reduce the uptake of the nanospheres better than F68 and comparable to F127. Finally, these results warrant in vivo experiments to evaluate how the HES laurates can affect the pharmacokinetics and fate of the nanospheres.
Biochimica Et Biophysica Acta-general Subjects, 2007
The maintenance of endothelial cell (EC) monolayer architecture requires stable adhesions not onl... more The maintenance of endothelial cell (EC) monolayer architecture requires stable adhesions not only between neighboring cells but also between cells and the extracellular matrix. While the influence of biomaterials surface wettability on cell–substratum adhesion is rather well studied, its impact on cell–cell cohesion has not been extensively investigated. In the present study a model system consisting of hydrophilic and hydrophobic
Support membranes in bioartificial organs contact blood or plasma on one-side and adhesion depend... more Support membranes in bioartificial organs contact blood or plasma on one-side and adhesion dependent cells on the other side. Since membranes for biomedical applications, such as for haemodialysis, are optimised for blood contact and membranes for biotechnological ...
The role of osmotic forces and cell swelling in the influenza virus-induced fusion of unsealed or... more The role of osmotic forces and cell swelling in the influenza virus-induced fusion of unsealed or resealed ghosts of human erythrocytes was investigated under isotonic and hypotonic conditions using a recently developed fluorescence assay (Hoekstra, D., De Boer, T., Klappe, K., Wilschut, J. (1984) Biochemistry 23, 5675-5681). The method is based on the relief of fluorescence selfquenching of the fluorescent amphiphile octadecyl rhodamine B chloride (R18) incorporated into the ghost membrane as occurs when labeled membranes fuse with unlabeled membranes. No effect neither of the external osmotic pressure nor of cell swelling on virally mediated ghost fusion was established. Influenza virus fused unsealed ghosts as effectively as resealed ghosts. It is concluded that neither osmotic forces nor osmotic swelling of cells is necessary for virus-induced cell fusion. This is supported by microscopic observations of virus-induced fusion of intact erythrocytes in hypotonic and hypertonic media. A disruption of the spectrin-actin network did not cause an enhanced cell fusion at acidic pH of about 5 or any fusion at pH 7.4.
A universal method for reproducibly directing stem cell differentiation remains a major challenge... more A universal method for reproducibly directing stem cell differentiation remains a major challenge for clinical applications involving cell-based therapies. The standard approach for chondrogenic induction by micromass pellet culture is highly susceptible to interdonor variability. A novel method for the fabrication of condensation-like engineered microtissues (EMTs) that utilizes hydrophilic polysaccharides to induce cell aggregation is reported here. Chondrogenesis of mesenchymal stem cells (MSCs) in EMTs is signifi cantly enhanced compared to micromass pellets made by centrifugation measured by type II collagen gene expression, dimethylmethylene blue assay, and histology. MSCs from aged donors that fail to differentiate in pellet culture are successfully induced to synthesize cartilage-specifi c matrix in EMTs under identical media conditions. Furthermore, the EMT polysaccharides support the loading and release of the chondroinduction factor transforming growth factor β3 (TGF-β3). TGF-β-loaded EMTs (EMT +TGF ) facilitate cartilaginous tissue formation during culture in media not supplemented with the growth factor. The clinical potential of this approach is demonstrated in an explant defect model where EMT +TGF from aged MSCs synthesize de novo tissue containing sulfated glycosaminoglycans and type II collagen in situ.
A cell carrier made from synthetic material supporting selective growth of keratinocytes is a pro... more A cell carrier made from synthetic material supporting selective growth of keratinocytes is a promising approach to avoid the phenomenon of fibroblast overgrowth during in vitro culture of skin substitutes. Therefore, we investigated polymer membranes made of polyacrylonitrile and copolymers of acrylonitrile and N-vinylpyrrolidone (NVP) for their ability to support selectively the growth of keratinocytes. It was found that a copolymer with an NVP-content of 30% (NVP30) supports growth of human keratinocyte cell line (HaCaT) cells and inhibits fibroblast growth under serum-containing conditions. Cell proliferation of HaCaT cells was measured over 14 days. If both cell types were cultured under serum-free conditions for initial adhesion over 6 h on these NVP30 polymers, they adhered to the same extent. Long-term experiments over 7 days were performed as a coculture of both cell types showing that HaCaT cells had a growth advantage that seems to be related to the paracrine activity of contaminating fibroblasts. As a result, confluent layers of HaCaT cells were obtained with small numbers of remaining fibroblasts. The new poly [acrylonitrile-co(NVP) membranes seem to be a promising culture system for the production of epidermal transplants.
Regioselective derivatization was carried out introducing sulfate, phosphate or quaternary ammoni... more Regioselective derivatization was carried out introducing sulfate, phosphate or quaternary ammonia groups in C2-, C3- and C6-position of the anhydroglycose unit of cellulose. The anticoagulant potential of these derivatives was estimated with common clotting assays, such as thrombin time and partial thromboplastin time. It was found that a pronounced anticoagulant activity of cellulose derivatives could be achieved if the degree of substitution (DS) with sulfate was above 1.0. The anticoagulant activity was maximal at a DS of about 1.5 and then decreased again. Further, it was detected that particularly sulfation in C2-position resulted in a pronounced anticoagulant activity of cellulose derivatives. Development and application of assays specific for thrombin and factor Xa indicated that the anticoagulant potential of these cellulose derivatives was mainly due to anti-thrombin activity. The comparison of cellulose sulfates and cellulose derivatized with phosphate and quaternary ammonium groups demonstrated that the negative charge and type of the substituent is an important prerequisite for the anticoagulant activity of cellulose derivatives. Indeed, derivatization with sulfate produced superior activity in comparison with phosphate.
Journal of Materials Science: Materials in Medicine, 2008
Materials for blood contacting applications have to meet high requirements in terms to prevent th... more Materials for blood contacting applications have to meet high requirements in terms to prevent thrombotic complications after the medical treatment. Surface induced thrombosis, e.g. after application of cardiovascular devices, is linked clearly to the activation of coagulation system and platelet adhesion and activation. The flat sheet poly(ether imide) membrane (PEI) was modified by binding of iminodiacetic acid (IDA) for different periods of time to obtain surfaces with carboxylic (-COOH) groups, namely PEI-1 (modified for 1 min) and PEI-2 (modified for 30 min). The successful binding of the ligands was monitored by thionin acetate assay. The physico-chemical characteristics of the materials were analyzed by SEM, AFM, water contact angle and Zeta-potential measurements. Hemocompatibility of the polymer materials was studied by analyzing the activation of coagulation system (plasma kallikrein-like activity) and platelet adhesion/activation by using immunofluorescence technique. The blood response to PEI membranes was compared to that of a commercial poly(ethylene terephthalate) (PET) membrane. Our results showed that the increase of the negative charges on the modified PEI membrane surfaces (number of COOH groups) caused a higher contact activation of the coagulation system and a higher rate of platelet adhesion and activation. However, overall the hemocompatibility of all PEI membranes was higher than that of PET.
Particles with specific ligands for the adsorption of plasma proteins can be used in therapeutic ... more Particles with specific ligands for the adsorption of plasma proteins can be used in therapeutic or preparative apheresis. The development of these particles may benefit from an improved knowledge of the relationship between protein adsorption and the structure of ligands. Nanoparticles were functionalized with aliphatic diamines of increasing chain length; with the amino acids lysine, tryptophan, histidine, and their corresponding amines; and with tryptophan and histidine spaced with diamines of different length. Suitable protocols were developed for the washing of particles and the subsequent desorption of proteins adsorbed from human plasma. The adsorption pattern, as well as the quantification of the overall adsorption of proteins on these modified particles, was investigated with gel electrophoresis. This was followed by immunoblotting which yielded specific assessments of bound human serum albumin and fibrinogen. The comparison of protein adsorption with surface charge density and measured hydrophobicities yielded no simple correlations although in general more hydrophobic ligands bound higher quantities of protein. The detection of human serum albumin yielded similar results because it was observed for overall protein adsorption while the adsorption of fibrinogen expressed a different pattern. In this case, particular nanoparticles functionalized with aliphatic diamines bound significantly higher amounts of fibrinogen than all other ligands.
Little is known about the influence of substratum properties and composition on the ability of ce... more Little is known about the influence of substratum properties and composition on the ability of cells to translocate α5β1 integrins and to form fibrillar adhesion. We have examined the impact of self-assembled monolayers (SAMs) bearing different functional end groups (amines (NH2) or carboxylic acids (COOH)) on the presence of fibrillar adhesions in human fibroblasts attached to fibronectin-coated SAMs. Most of
The ability of human fibroblasts to remove and reorganize fibronectin (FN) bound on material surf... more The ability of human fibroblasts to remove and reorganize fibronectin (FN) bound on material surfaces was studied as a novel feature of material surface biocompatibility. Other traditional parameters of biocompatibility analyzed included cell spreading, clustering of fibronectin ...
Proteins and glycosaminoglycans (GAGs) are main constituents of the extracellular matrix (ECM). T... more Proteins and glycosaminoglycans (GAGs) are main constituents of the extracellular matrix (ECM). They act in synergism and are equally critical for the development, growth, function or survival of an organism. In this work, we developed surfaces that display these two classes of biomacromolecules, namely GAGs and proteins, in spatially controlled fashion. The generated surfaces can be used as a minimalistic but straightforward model aiding the elucidation of cell-ECM interactions. GAGs (hyaluronic acid and heparin) were covalently bound to amino func-tionalized surfaces and albumin or fibronectin were patterned by micro-contact printing on top of them. We demonstrate that adipose-derived stem cells (ASCs) can adhere either on protein or GAG pattern as a function of the patterned molecules. ASCs found on the GAG pattern had different morphology and expressed different surface markers than the cells adherent on the protein pattern. ASCs morphology and spreading were also dependent on t...
Stability of surface coatings against environmental stress, such as pH, high ionic strength, mech... more Stability of surface coatings against environmental stress, such as pH, high ionic strength, mechanical forces, and so forth, is crucial for biomedical application of implants. Here, a novel extracellular-matrix-like polyelectrolyte multilayer (PEM) system composed of collagen I (Col I) and oxidized glycosaminoglycans (oGAGs) was stabilized by intrinsic cross-linking due to formation of imine bonds between aldehydes of oxidized chondroitin sulfate (oCS) or hyaluronan (oHA) and amino groups of Col I. It was also found that Col I contributed significantly more to overall mass in CS-Col I than in HA-Col I multilayer systems and fibrillized particularly in the presence of native and oxidized CS. Adhesion and proliferation studies with murine C3H10T1/2 embryonic fibroblasts demonstrated that covalent cross-linking of oGAG with Col I had no adverse effects on cell behavior. By contrast, it was found that cell size and polarization was more pronounced on oGAG-based multilayer systems, whic...
The endothelization of cardiovascular implants is desirable to improve their blood compatibility.... more The endothelization of cardiovascular implants is desirable to improve their blood compatibility. The capacity of the endothelial cells to attach, migrate, proliferate and function on the implant surface depends on the presence of matrix proteins such as ®bronectin (FN) and ®brinogen (FNG). In this study, we show that the deposition of ®brinogen into extracellular matrix-like structures by human umbilical vein endothelial cells (HUVEC) is dependent on FN matrix formation. We found further that the process of organization of both adsorbed and soluble FN and FNG is dependent on the wettability of materials since it was observed only on a hydrophilic and not on a hydrophobic model surface. b 3 integrin was involved in the process of cell attachment to adsorbed FNG, while the mechanism of FNG ®brillogenesis required the activity of the b 1 integrin. Studies of EC morphology showed the predominant peripheral organization of actin ®laments and the formation of distinct leading and trailing cell edges suggesting a motile phenotype of cells when they are seeded on FNG. In summary, we concluded that adsorbed ®brinogen may enhance the motility of HUVEC and that soluble FNG requires FN matrix assembly to be organized in ®brilar structures.
Journal of Biomaterials Science-polymer Edition, 2002
The hemocompatibility of four different wettable polymer membranes, namely Cuprophan (CE), polyet... more The hemocompatibility of four different wettable polymer membranes, namely Cuprophan (CE), polyether-polycarbonate (PC-PE), polysulfone (PSU), and polyetherimide (PEI), was investigated with respect to brinogen (Fng) adsorption and platelet adhesion/ activation. In order to estimate the polar and dispersion components of the surface free energy, contact angles using water/ vapor and water/ n-hexadecane systems were measured. Adsorption of brinogen was studied using uorescence-labeled protein. The adsorption isotherms showed that the amount and the af nity of adsorbed Fng increased with decreasing surface wettability of the membranes, which correlates with the dispersion and polar components of the surface free energy. The conformational changes of adsorbed Fng were detected by measuring the difference between monoclonal antibody binding to the conformation-sensitive epitope in the D-domain and the binding of polyclonal anti-Fng antibody. The anticipated conformational / orientational changes were greater for PEI and PSU membranes (the least wettable membranes) and negligible for the more wettable PC-PE and CE membranes. In addition, a possible relationship with the degree of platelet activation was found, showing negligible platelet adhesion on PC-PE and CE, but high platelet adhesion on PEI and PSU. Furthermore, platelets were spread to a large extent on PEI, while the formation of aggregates was observed on PSU. This may correspond to the anticipated differences in the conformational state of Fng on both membranes.
NATO Science for Peace and Security Series A: Chemistry and Biology, 2010
Abstract The initial cellular events that take place at the biomaterials interface mimic to a cer... more Abstract The initial cellular events that take place at the biomaterials interface mimic to a certain extent the natural interaction of cells with the extracellular matrix (ECM). The cells adhering to the adsorbed soluble matrix proteins, such as fibronectin (FN) and fibrinogen (FNG) tend ...
NATO Science for Peace and Security Series A: Chemistry and Biology, 2010
... 1990). For example it was shown that hydrophobic methyl-terminated alkanethiol SAMs on gold i... more ... 1990). For example it was shown that hydrophobic methyl-terminated alkanethiol SAMs on gold induce mini-mal cell ... 13.3 Structure of self assembled monolayers composed of coupling unit, assembling structure and terminal group ... 13 Surface Modification and Cell Adhesion ...
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Papers by Thomas Groth