Modification of human pericardium by chemical crosslinking.

Autologous and allogenic human pericardia used as biomaterials for cardiovascular surgery are traditionally crosslinked with glutaraldehyde. In this work, we have evaluated the resistivity to collagenase digestion and the cytotoxicity of human pericardium crosslinked with various concentrations of glutaraldehyde in comparison with pericardium crosslinked by genipin, nordihydroguaiaretic acid, tannic acid, and in comparison with unmodified pericardium. Crosslinking retained the wavy-like morphology of native pericardium visualized by second harmonic generation microscopy. The collagenase digestion products were analyzed using SDS-PAGE, capillary electrophoresis, and a hydroxyproline assay. Glutaraldehyde and genipin crosslinking protected the native pericardium efficiently against digestion with collagenase III. Only low protection was provided by the other crosslinking agents. The cytotoxicity of crosslinked pericardium was evaluated using xCELLigence by monitoring the viability of porcine valve interstitial cells cultured in eluates from crosslinked pericardium. The highest cell index, reflecting both the number and the shape of the monitored cells was observed in eluates from genipin. Crosslinking pericardium grafts with genipin therefore seems to be a promising alternative procedure to the traditional crosslinking with glutaraldehyde, because it provides similarly high protection against degradation with collagenase, without cytotoxic effects.

Fibrin nanostructures for biomedical applications.

Fibrin is a versatile biopolymer that has been extensively used in tissue engineering. In this paper fibrin nanostructures prepared using a technique based on the catalytic effect of fibrin-bound thrombin are presented. This technique enables surface-attached thin fibrin networks to form with precisely regulated morphology without the development of fibrin gel in bulk solution. Moreover, the influence of changing the polymerization time, along with the antithrombin III and heparin concentrations on the morphology of fibrin nanostructures was explored. The binding of bioactive molecules (fibronectin, laminin, collagen, VEGF, bFGF, and heparin) to fibrin nanostructures was confirmed. These nanostructures can be used for the surface modification of artificial biomaterials designed for different biomedical applications (e.g. artificial vessels, stents, heart valves, bone and cartilage constructs, skin grafts, etc.) in order to promote the therapeutic outcome.

Attachment of human endothelial cells to polyester vascular grafts: pre-coating with adhesive protein assemblies and resistance to short-term shear stress.

Cardiovascular prosthetic bypass grafts do not endothelialize spontaneously in humans, and so they pose a thrombotic risk. Seeding with cells improves their performance, particularly in small-caliber applications. Knitted tubular polyethylene-terephthalate (PET) vascular prostheses (6 mm) with commercial type I collagen (PET/Co) were modified in the lumen by the adsorption of laminin (LM), by coating with a fibrin network (Fb) or a combination of Fb and fibronectin (Fb/FN). Primary human saphenous vein endothelial cells were seeded (1.50 × 10(5)/cm2), cultured for 72 h and exposed to laminar shear stress 15 dyn/cm(2) for 40 and 120 min. The control static grafts were excluded from shearing. The cell adherence after 4 h on PET/Co, PET/Co +LM, PET/Co +Fb and PET/Co +Fb/FN was 22%, 30%, 19% and 27% of seeding, respectively. Compared to the static grafts, the cell density on PET/Co and PET/Co +LM dropped to 61% and 50%, respectively, after 120 min of flow. The cells on PET/Co +Fb and PET/Co +Fb/FN did not show any detachment during 2 h of shear stress. Pre-coating the clinically-used PET/Co vascular prosthesis with LM or Fb/FN adhesive protein assemblies promotes the adherence of endothelium. Cell retention under flow is improved particularly on fibrin-containing (Fb and Fb/FN) surfaces.

A periadventitial sirolimus-releasing mesh decreased intimal hyperplasia in a rabbit model.

Autologous vein grafts used as aortocoronary bypasses are often prone to intimal hyperplasia, which results in stenosis and occlusion of the vein. The aim of this study was to prevent intimal hyperplasia using a newly developed perivascular system with sustained release of sirolimus. This system of controlled drug release consists of a polyester mesh coated with a copolymer of L-lactic acid and epsilon-caprolactone that releases sirolimus. The mesh is intended for wrapping around the vein graft during surgery. The mesh releasing sirolimus was implanted in periadventitial position onto arteria carotis communis of rabbits, and neointimal hyperplasia was then assessed. We found that implanted sirolimus-releasing meshes reduced intima thickness by 47+/-10 % compared to a vein graft after 3 weeks. The pure polyester mesh decreased vein intima thickness by 35+/-9 %. Thus, our periadventitial system for controlled release of sirolimus prevented the development of intimal hyperplasia in autologous vein grafts in vivo in rabbits. A perivascularly applied mesh releasing sirolimus is a promising device for preventing stenosis of autologous vein grafts.

Polymeric nanocapsules ultra stable in complex biological media.

Non-specific protein adsorption from complex biological media, especially from blood plasma, is an urgent challenge for the application of nanoparticles as delivery systems, diagnostics, and other biomedical application. Nanocapsules (NC) prepared from FDA-approved degradable poly(ɛ-caprolactone) shell and Mygliol 812(®) oil in the core were coated with mono-methoxy terminated oligo(ethylene glycol) methacrylate (poly(MeOEGMA)) polymer brush layers with a well-controlled thickness at the nanometer scale up to 350 nm using surface initiated atom transfer radical polymerization in water or phosphate buffered saline. Incubation of uncoated NC with human serum albumin solution, fetal bovine serum, or human blood plasma resulted in fast aggregation observed by dynamic light scattering as an increase in diameter of particles present in the solutions. Conversely, these biological fluids affected only marginally the size distribution of the NC coated with a 60 nm thick poly(MeOEGMA) layer. The high suspension stability of the coated NC in complex biological fluids was related to the suppressed deposition of proteins from these fluids observed by surface plasmon resonance (SPR) on analogous poly(MeOEGMA) layer prepared on flat surfaces of SPR chips.

Interaction of blood plasma with antifouling surfaces.

Nonspecific adsorption of proteins is a crucial problem in the detection of analytes in complex biological media by affinity sensors operating with label-free detection. We modified the gold surface of surface plasmon resonance (SPR) sensors with three types of promising antifouling coatings: self-assembled monolayers (SAM)s of alkanethiolates terminated with diethylene glycol and carboxylic groups, poly(ethylene glycol) (PEG) grafted onto the SAMs, and zwitterionic polymer brushes of poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), and poly(phosphorylcholine methacrylate). Using SPR, we compared the efficacy of the coatings to reduce nonspecific adsorption from human blood plasma and from single-protein solutions of human serum albumin, immunoglobulin G, fibrinogen, and lysozyme. There was no direct relationship between values of water contact angles and plasma deposition on the coated surfaces. A rather high plasma deposition on SAMs was decreased by grafting PEG chains. Fouling on PEG was observed only from plasma fractions containing proteins with molecular mass higher than 350 000 Da. The adsorption kinetics from plasma collected from different healthy donors differed. Poly(carboxybetaine methacrylate) completely prevented the deposition from plasma, but the other more hydrophilic zwitterionic polymers prevented single-protein adsorption but did not prevent plasma deposition. The results suggest that neither wettability nor adsorption of the main plasma proteins was the main indicator of deposition from blood plasma.

pH dependence and protein selectivity of poly(ethyleneimine)/poly(acrylic acid) multilayers studied by in situ ATR-FTIR spectroscopy.

The selective interaction between polyelectrolyte multilayers (PEM) consecutively adsorbed from poly(ethyleneimine) (PEI) and poly(acrylic acid) (PAC) and a binary mixture containing concanavalin A (COA) and lysozyme (LYZ) based on electrostatic interaction is reported. The composition and structure of the PEM and the uptake of proteins were analyzed by in situ attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy, and the morphology and thickness were characterized by atomic force microscopy (AFM) and ellipsometry. The PEM dissociation degree and charge state and the protein adsorption were shown to be highly dependent on the outermost layer type and the pH in solution. High protein uptake was obtained under electrostatically attractive conditions. This was used to bind selectively one protein from a binary mixture of LYZ/COA. In detail it could be demonstrated that six-layered PEM-6 at pH = 7.3 showed a preferential sorption of positively charged LYZ, while at PEM-5 and pH = 7.3 negatively charged COA could be selectively bound. No protein sorption from the binary mixture was observed at pH = 4.0 for both PEM, when COA, LYZ, and the outermost PEI layer of PEM-5 were positively charged or the outermost PAC layer of PEM-6 was neutral. Furthermore, from factor analysis of the spectral data the higher selectivity was found for PEM-5 compared to PEM-6. Increasing the ionic strength revealed a drastic decrease in the selectivity of both PEM. Evidence was found that the proteins were predominantly bound at the surface and to a minor extent in the bulk phase of PEM. These results suggest possible working regimes and application fields of PEI/PAC multilayer assemblies related to the preparative separation of binary and multicomponent protein mixtures (biofluids, food) as well as to the design of selective protein-resistant surfaces.

Functionalized surfaces of polylactide modified by Langmuir-Blodgett films of amphiphilic block copolymers.

To modify the surface of poly(L-lactide) (PLA) supports, we have investigated the feasibility to deposit on the PLA surface Langmuir-Blodgett films of amphiphilic block copolymers based on poly(L-lactide). AB and ABA block copolymers were prepared with PLA as the A block and either poly(ethylene oxide), alpha-methoxy-omega-hydroxy poly(ethylene oxide), alpha-carboxy-omega-hydroxy poly(ethylene oxide) or poly(L-aspartic acid) as the B blocks. Films with phase-separated hydrophilic and hydrophobic blocks in a bilayer "brush" structure were prepared by compression of the copolymer Langmuir films on the water/air interface. The interfacial behavior of the monolayers and the effect of the copolymer composition on the phase separation was followed by measurements of the surface-pressure/area isotherms using a Langmuir trough and by contact angle measurement of deposited Langmuir-Blodgett (LB) films. The phase separation of the hydrophilic and PLA blocks is more effective in diblock AB copolymers compared with triblock ABA copolymers. The presence of ionic groups in the hydrophilic chains facilitates penetration of hydrophilic segments into the water subphase. Dynamic contact angle measurements were used to study the stability of the LB-films transferred on the PLA support and the changes in the surface properties upon incubation of surfaces in water.

Optical biosensors for real-time measurement of analytes in blood plasma.

The preparation of assemblies consisting of multiple molecular layers of bovine serum albumin (BSA), monoclonal antibodies against horseradish peroxidase (anti-HRP), and monoclonal antibodies against methotrexate (anti-MTT), as well as interaction of the assemblies with human blood plasma were observed using a grating coupler and Young interferometer (YI). The assemblies could be arranged according to decreasing amounts of nonspecific deposits bound irreversibly to them from blood plasma as follows-an adsorbed antibody monolayer saturated with adsorbed BSA, antibody multilayers linked with polycations, antibodies covalently immobilized on a BSA layer densely crosslinked with glutaraldehyde (GA), slightly crosslinked BSA double layer, slightly crosslinked antibody double layers. The occurrence of human serum albumin (HSA), human fibrinogen (Fg), IgG, and IgM in the plasma deposits was studied by binding the respective antibodies. IgG, IgM, and Fg were detected in plasma deposits on the immobilized assemblies while the composition of a plasma deposit on the unmodified sensor surface reflected roughly the plasma composition containing mainly adsorbed HSA and Fg. A crosslinked anti-HRP double layer was immobilized on a waveguiding branch of YI and a similar anti-MTT double layer was immobilized on the other branch. The sensor response to blood plasma was fairly decreased owing to a compensation of the respective optical changes in the two branches, in which a similar non-specific adsorption took place. The addition of HRP or MTT to plasma induced specific responses of the corresponding branches.

A miniature fiber optic surface plasmon resonance sensor for fast detection of Staphylococcal enterotoxin B.

A fiber optic surface plasmon resonance (SPR) biosensor for detection of Staphylococcal enterotoxin B (SEB) is reported. The sensor is based on spectral interrogation of surface plasmons in a miniature sensing element based on a side-polished single-mode optical fiber with a thin metal overlayer. For specific detection of SEB, the SPR sensor is functionalized with a covalently crosslinked double-layer of antibodies against SEB. The SPR biosensor is demonstrated to be able to detect ng/ml concentrations of SEB in less than 10 min.

Albumin and heparin multilayer coatings for blood-contacting medical devices.

Three types of covalently crosslinked assemblies consisting of multiple (1) molecular layers of human serum albumin (HSA); (2) alternating layers of HSA and unfractionated heparin; and (3) alternating layers of HSA and partly depolymerized heparin fixed with one end to HSA were prepared on various surfaces. Adsorption of fibrinogen, IgG, and antithrombin (ATIII) from human citrated plasma on coated surfaces was evaluated by ELISA. Fibrinogen adsorption on coated ELISA plates was lower than that on bare polystyrene. There was no IgG adsorption on the HSA coating alone, but considerably high IgG adsorption was detected on the heparin-containing surface. The adsorption of ATIII increased with increasing heparin on the surface. The effect of multilayer coatings on platelets was tested by incubation of modified vascular prostheses with citrated blood. The most favorable interaction with platelets was observed on the HSA assembly. The interaction of platelets with the surface bearing unfractionated heparin was higher than that of the surface covered with partly depolymerized heparin. The long-term durability of the HSA-heparin coating was proven by a 21-day implantation of coated polyurethane plates in goat heart.

Capillary zone electrophoresis with electroosmotic flow controlled by external radial electric field.

A new way of regulation of electroosmotic flow (EOF) in capillary zone electrophoresis (CZE) by external electric field has been developed. A set of three high-voltage power supplies is used to form a radial electric field across the capillary wall. One power supply is applied in the usual way as a driving force of CZE and EOF to the ends of the inner capillary compartment dipped into the electrode vessels and filled with background electrolyte. Two power supplies are connected to the ends of the outer low-conductivity coating of the capillary which is formed by the dispersion of copolymer of aniline and p-phenylenediamine in polystyrene matrix. The difference between electric potentials on the outer capillary surface and inside the capillary determines the voltage of radial electric field across the capillary wall and affects the electrokinetic potential at the solid-liquid interface inside the capillary. The effect of magnitude and polarity of external radial electric field on the flow rate of EOF, on the migration times of charged analytes and on the separation efficiency and resolution of CZE separations of synthetic oligopeptides, diglycine, triglycine and octapeptide fragments of human insulin was evaluated. Through the EOF control by external electric field the dynamic effective length of the capillary was obtained and the speed of analysis and resolution of CZE separations of peptide analytes could be optimized.

The detection of human beta 2-microglobulin by grating coupler immunosensor with three dimensional antibody networks.

Immunosensors for the detection of human beta 2-microglobulin (B2M) were prepared by immobilisation of covalently crosslinked assemblies containing various numbers of molecular layers of monoclonal antibody against B2M (anti-B2M) on the surface of a Ta2O5 grating coupler sensor. The immobilisation procedure consisted of repeated successive adsorption of anti-B2M and dextran sulfate (DS) followed by glutaraldehyde (GA) crosslinking of anti-B2M and washing out DS. The flexibility of the resulting anti-B2M networks was evaluated from the sensor response to the reversible expansion and contraction of the networks induced by changing pH of the ambient solution. A decreased GA concentration and the use of a higher-molecular-mass DS increased the network flexibility. The sensor sensitivity to B2M increased with increasing flexibility of the antibody networks and with increasing number of anti-B2M molecular layers, indicating that B2M can penetrate inside the antibody network.

Immobilisation of multilayer bioreceptor assemblies on solid substrates.

Multilayer assemblies were prepared by alternating adsorption of monolayers of monoclonal antibody against horse radish peroxidase (anti-HRP) and dextran sulfate (DS) on solid supports at acid pH. After crosslinking with glutaraldehyde, DS was washed out of the film with buffered physiological saline, while the antibody remained immobilised on the support. Assembly was monitored in situ on germanium supports by infrared multi-internal reflection spectroscopy. The binding capacity of the immobilised antibodies for HRP was measured by ELISA and by optical waveguide light mode spectroscopy. The activity of an immobilised anti-HRP bilayer was approximately twice that of a monolayer prepared by simple physiosorption. An addition of further anti-HRP layers could increase the activity only up to 2.5 of the monolayer activity independently of a number of layers in the assembly. The non-specific adsorption of proteins from human blood plasma was three times lower on the immobilised anti-HRP multilayer film than on the surface covered only with a physiosorbed anti-HRP monolayer.

Modulation of complement activation on hemodialysis membranes by immobilized heparin.

To determine the effects of surface-associated heparin on the capacity of hemodialysis membranes to activate complement, cellulose acetate (CA) membranes that were untreated and CA membranes that had been coated with heparin (HCA) were incubated with C3-depleted serum repleted with radio-labeled C3. Next, the proteins in the supernatant and those eluted from the membranes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. C3 activation was quantified by determining the radioactivity of the C3a-containing band in the gel. Total C3a generation (fluid phase C3a plus membrane-associated C3a) was three times greater in the presence of HCA compared with CA. Most (88%) of the C3a generated in the presence of HCA, however, was adsorbed onto the membrane surface. Consequently, there was more C3a in the CA supernatant than in the HCA supernatant. To determine the mechanism by which heparin enhanced alternative pathway activity, binding studies with radiolabeled factor B and factor H were performed. HCA bound 3.4 times more factor B and 20 times more factor H than did CA. The binding of these proteins, however, was not dependent on complement activation. Studies designed to test the functional activity of isolated factor H and factor B that had been adsorbed to the membrane showed that factor H was active on both CA and HCA, whereas factor B was active only on HCA. These data demonstrate that heparin immobilized onto CA hemodialysis membrane enhances C3 activation but produces low levels of C3a in the fluid phase because of high surface adsorption of the anaphylatoxin. Heparin appears to augment alternative pathway activity by favoring the interactions of factor B with other constituents of the amplification C3 convertase of the alternative pathway of complement.

Irritation effects of residual products derived from p(HEMA) gels. II. Compounds extracted from hydrogels.

Samples of poly(2-hydroxyethyl methacrylate) p(HEMA) hydrogels were prepared using three different polymerization initiators. The gels were washed in water under standard conditions. The extracts were then examined for intradermal irritation in rats using a radioactive indicator (113mIn). The irritation effects were dependent on the concentrations of the irritating substances and also on the gel type. Solid discs made of the gels, washed to varying degrees of purity, were also implanted into rats. Tissue irritation, as well as some other biological responses, were followed in situ using the radioindicator and common histological techniques. The irritation effects were very mild (even with the unextracted gel material). A possible explanation for the events taking place at the site of implantation is presented.

Irritation effects of residual products derived from poly(2-hydroxyethyl methacrylate) gels. I. Testing of some model compounds.

2-Hydroxyethyl methacrylate (HEMA) monomer and sodium benzoate, diluted with saline in the range 0-20%, were tested for intradermal irritation in rats. Radioactive indicator (113mIn) was used to quantify this biological response. At low concentrations (up to 1%) only a little irritation was recorded, while at higher levels (5% or more) a significant adverse reaction developed. The degree of irritation was dose dependent. In the concentration range 0-10%, the response was exponential. Model decomposition products derived from three different polymerization initiators were also tested. How the results obtained with the model irritants relate to real polymerization systems is discussed.

Polyethylene/hydrophilic polymer blends for biomedical applications.

Polyethylene blends with poly(2-hydroxyethyl methacrylate) [poly(HEMA)] or poly(2,3-dihydroxypropyl methacrylate) [poly(DHPMA)] were prepared by swelling polyethylene with HEMA or 2,3-epoxypropyl methacrylate (EPMA) and by polymerization of the respective monomers. Poly(EPMA) in blends was hydrolysed to poly(DHPMA) with acetic acid. The blends had similar surface and bulk compositions. Swelling with water and surface wettability were proportional to the content of the hydrophilic component; at the same content the polyethylene/poly(DHPMA) blends appeared more hydrophilic than those of polyethylene/poly(HEMA). Thrombus formation in contact with blood examined ex vivo and in vivo was considerably slower on the blends than on unmodified polyethylene. The tests indicated optima in composition; the best biological response was achieved with the blends containing about 14% poly(HEMA) or 16% poly(DHPMA).

The removal of residuals and oligomers from poly(2-hydroxyethylmethacrylate).

Poly(2-hydroxyethyl methacrylate) gels obtained by the cross-linking polymerization using four different free-radical initiators were washed with water. Chromatographically, the eluate appeared to be a mixture of low-molecular-weight compounds and of a small amount of the high-molecular-weight component. The UV and IR absorption spectra of compounds present in the eluate were compared with those of model compounds that were assumed to exist in the gel as impurities after the polymerization (monomers and oligomers of hydroxyethyl methacrylate, decomposition products of initiators). Time dependences of the removal of impurities from the gels by washing were measured. Most of the impurities were washed out within a few hours. In addition to the assumed impurities, the eluate was found to contain an unidentified compound that was still washed out after several months. Intracutaneous applications of this compound did not produce local irritation of the tested tissue.