Hemocompatible albumin-heparin coatings prepared by the layer-by-layer technique. The effect of layer ordering on thrombin inhibition and platelet adhesion

Using the layer-by-layer technique, ELISA polystyrene plates were coated with multilayer assemblies of albumin with various heparins or with multilayer assemblies of albumin. The coatings containing heparin were tested for their ability to potentiate thrombin inhibition by antithrombin and its dependence on the layer arrangement. The order of activities of surface bound heparins matched their order in solution; however their activity was reduced to less than 10% due to binding. The increasing number of layers increased the activity of the coatings suggesting that heparin inside the assemblies is available for the interaction. The albumin-heparin assemblies overcoated with albumin layers preserved about half of heparin activity. Platelets adhered in similar amounts to albumin-heparin and albumin coatings; however, in both cases platelets adhered more to single layer than to multilayer coatings. The adhesion of platelets to single layer coatings was also affected by the crosslinking of the coatings; more platelets adhered to less crosslinked single layer coatings while multilayer coatings remained essentially unaffected by crosslinking. If the coatings were dried and reswollen, a substantial number of platelets adhered to the reconditioned single layer coatings but the two layer coatings were affected much less and the adhesion of platelets to the coatings with three layers was close to normal. A minimum of three albumin-heparin or albumin layers is apparently required to shield the underlying surface and to achieve proper functioning of the coatings.     

Construction of antithrombogenic polyelectrolyte multilayer on thermoplastic polyurethane via layer-by-layer self-assembly technique

The improvement of hydrophilicity and hemocompatibility of thermoplastic polyurethane (TPU) film was developed using surface modification of polyelectrolyte multilayers (PEMs) deposition. The polysaccharide PEMs included chitosan (CS, as a positive-charged agent) and dextran sulfate (DS, as a negative-charged and an antiadhesive agent) that were successfully prepared on the aminolyzed TPU film in a layer-by-layer (LBL) self-assembly manner. X-ray photoelectron spectroscopy (XPS), field-emission scanning electronic microscopy (FE-SEM), and atomic force microscopy (AFM) data will verify the progressive buildup of the PEMs film. The obtained results showed that the contact angle and Zeta-potential reached the steady value after four bilayers of coating, hence proving that the full coverage of coating with PEM layers was achieved. It could be found that the PEMs-deposited TPU films with DS as the outmost layer could resist the platelet adhesion and human plasma fibrinogen (HPF) adsorption, thereby prolonging effectively the blood coagulation times. Besides, the results of growth inhibition index (GI) of L929 fibroblast proliferation suggested that the as-fabricated TPU films were noncytotoxic. Overall results demonstrated that such an easy, valid, shape-independent, and noncytotoxic processing should be potential for the ion of TPU substrate in the application of hemodialysis or cardiovascular devices.     

In vitro hemocompatibility testing of UV-modified hyaluronan hydrogels

Hydrogels (hylans) based on cross-linked hyaluronan (HA) are potentially good biomaterials for vascular tissue engineering applications because they are highly non-antigenic and -immunogenic. To facilitate surface endothelialization, vital to vascular deployment, we irradiated the gel surface with low wavelength UV light. This process micro-textures the smooth gel surface to provide sites for cell anchorage and causes limited scission of native long-chain HA yielding smaller fragments that elicit an enhanced cell response. In the current in vitro study, we assessed the effects of UV irradiation on the short-term (<45 min) interaction between hylan gels and human blood cells (RBCs, platelets) and coagulation proteins at physiologic temperature. Although the lowered hydrophilicity of irradiated (UV) hylans elicited greater vascular cell response relative to unmodified (U) hylans, platelet deposition was unaffected and much lower compared to collagen-coated glass controls. The adhered platelets were rounded or mildly pseudopodic and did not express p-selectin, an activation marker. Both gel types induced identical, and minimal platelet release as measured using an platelet factor 4 ELISA, and identically deferred the intrinsic and extrinsic coagulation pathways. Both gel types induced elevated levels of contact activation of bound, but not plasma-phase factor XII relative to controls. Hemolysis rates were also identical and within accepted standards. We conclude that UV-treatment of hylans, useful to improve surface endothelialization, does not compromise their short-term hemocompatibility, vital to their use as vascular implant materials.     

In vitro hemocompatibility of self-assembled monolayers displaying various functional groups

Self-assembled monolayers (SAMs) of alkanethiols with various terminating groups (-OH, -CH3, -COOH) and binary mixtures of these alkanethiols were studied with respect to their hemocompatibility in vitro by means of freshly taken human whole blood. The set of smooth monomolecular films with graded surface characteristics was applied to scrutinize hypotheses on the impact of surface chemical-physical properties on distinct blood activation cascades, i.e. to analyze -OH surface groups vs. complement activation, acidic surface sites vs. contact activation/coagulation and surface hydrophobicity vs. thrombogenicity. Blood and model surfaces were analyzed after incubation for the related hemocompatibility parameters. Our results show that the adhesion of leukocytes is abolished on a -CH3 surface and greatly enhanced on surfaces with -OH groups. The opposite was detected for the adhesion of platelets. A strong correlation between the activation of the complement system and the adhesion of leukocytes with the content of -OH groups could be observed. The contact activation for hydrophilic surfaces was found to scale with the amount of acidic surface sites. However, the coagulation and platelet activation did not simply correlate with any surface property and were therefore concluded to be determined by a superposition of contact activation and platelet adhesion.     

In vitro hemocompatibility studies of drug-loaded poly-(L-lactic acid) fibers

Our objective was to evaluate the hemocompatibility of biodegradable stent fibers, employing a closed-loop circulation system filled with human blood. We also investigated the effects of the anti-inflammatory and anti-proliferative drugs curcumin and paclitaxel, incorporated into stent fibers. Fresh whole blood was circulated in four parallel closed-loop systems: the empty tube circuit (control) and tubes containing either a PLLA fiber coil (PLLA), a curcumin-loaded PLLA coil (C-PLLA) or a paclitaxel-loaded PLLA coil (P-PLLA). The influence of PLLA fiber, alone or loaded with drug incorporated during melt-extrusion, on leukocyte and platelet adhesion and activation was determined by flow cytometry. The effects of blood flow and fiber properties on cell deposition were assessed by scanning electron microscopy (SEM). The flow cytometry results clearly demonstrated that PLLA triggers blood cell activation at the site of deployment, as shown by increases in CD11b, CD62P and leukocyte-platelet aggregates, compared to controls. Curcumin and paclitaxel treatments both significantly reduced leukocyte and platelet activation and adhesion to PLLA fibers, as shown by flow cytometry and SEM. Activated leukocytes and platelets revealed significantly lower CD11b and CD62P receptor binding for C-PLLA compared with PLLA alone, and slightly lower for P-PLLA. Reductions in platelet-leukocyte aggregates were observed as well. In addition, there was less leukocyte and platelet adhesion to C-PLLA, compared with PLLA fiber controls, as shown by SEM. A continuous linear thrombus, composed of platelets, leukocytes, red blood cells and fibrin was occasionally detected along the line of tangency between the coil and the tube wall. Flow separation and eddying, proximal and distal to the line of tangency of coil and tube, is thought to contribute to this deposit. Curcumin was more effective than paclitaxel in reducing leukocyte and platelet activation and adhesion to PLLA stent fibers in this setting. However there was evidence of paclitaxel degeneration during melt extrusion that may have inhibited its effectiveness. Incorporation of the anti-inflammatory and anti-proliferative drug curcumin into bioresorbable stent fibers is proposed to prevent thrombosis and in-stent restenosis.     

In vitro study on hemocompatibility and cytocompatibility of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)

Samples of polyhydroxybutyrate (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) containing 4–20% (mol/mol) 3-hydroxyhexanoate (3HHx) were characterized as potential components of blood-contact biomaterials. In an erythrocyte contact hemolysis assay, all tested PHBHHx films had substantially reduced reactivity, typically displaying about 2-fold less hemolytic activity compared with that of PHBV. Both 12% and 20% containing PHBHHx also bound less platelets than other films. After a 120-min exposure to platelet-rich plasma (PRP), few platelets adhered to the 12% and 20% containing PHBHHx films, while numerous platelets were seen on PHBV. Surface properties investigation suggested along with increasing 3HHx content, PHBHHx co-polymer films became smoother and smoother, which may contribute to lower platelet adhesion of PHBHHx containing high HHx content in a short-term contact to platelet-rich plasma. In a long-term contact to PRP, the difference in crystallization of PHBVand PHBHHx can be a critical parameter for platelet adhesion. Human umbilical vein endothelial cells (HUVECs) grew well on PHBHHx containing high content of 3HHx, indicating that both had good biocompatibility with HUVECs. While gelatin-coated or lipase-treated polyesters improved HUVECs proliferation compared with that on uncoated films, platelet adhesion was also decreased on gelatin-coated polyester. The hemocompatibility and biocompatibility of PHBHHx film were markedly improved. Thus, PHBHHx, particularly the surface-modified PHBHHx film, is promising for blood-contact materials.     

In vivo performance of two different hydroxyapatite coatings on titanium prepared by discharging in electrolytes

This study reports a discharging method for bone-like carbonated HA (cHA)-coating (Ca/P 1.71) and stoichiometric HA (sHA)-coating (Ca/P 1.67) with micrometer order thicknesses on titanium plates, using modified body fluid and acidic calcium phosphate solutions, respectively. In vivo histological performance of the HA coatings prepared by discharging in electrolytes was evaluated. Bone-contact indexes of HA coatings were measured microscopically. Additionally, bone-coating interface was analyzed by scanning electron microscopy and the use of an electron probe microanalyzer. Results demonstrated that there was no significant difference in contact index between HA coatings. However, the cHA coating was practically replaced by immature bone, and the titanium metal substrate was directly connected to the bone structure whereas the sHA coating layer remained and was partially detached from the titanium metal substrate. Since detached coating particles are pathogens, and can cause peri-implantitis, the cHA coating was more favorable than the sHA coating even if contact index was equivalent to that of the sHA coating. It is thought that coating thickness and chemical composition of coatings are important for biological stability of implants. In conclusion, since bone-like thin cHA coating showed high osteoconductivity and bone replacement, bone-like HA is superior to sHA coating for use in dental implants.     

In vitro and in vivo hemocompatibility evaluation of graphite coated polyester vascular grafts

Attempts have been made in this study to prepare a homogeneous and stable coating of graphite on polyester vascular grafts (GPVG) using an electrophoresis method to evaluate thromboresistant and blood compatibility of GPVG in comparison to non-coated PVG and InterGard (collagen sealed PVG) as control. Lactate dehydrogenase (LDH) activity measurement was carried out on all PVG types to evaluate platelet adhesion. To examine tissue reaction GPVG and non-coated sheets of knitted polyester fabric were implanted simultaneously in the dorsal flank of rats subcutaneously. The GPVG, non-coated and control were implanted in descending aorta as end-to-end or end-to-side implantation substitution in 25 sheep for 4-60 weeks. Results showed that the graphite coating on polyester vascular grafts reduced the number of adherent platelets and prevent platelet activation and spreading on the surface in comparison with non-coated and control. Pathological investigation showed inflammatory reactions were totally resolved after 12 weeks and there was no difference in the tissue reaction between graphite coated, non-coated and control patches. All grafts remained patent and there was no significant difference in patency rate between these three types of PVG. We found that GPVG has no need for pre-clotting and it showed lower platelet aggregation, thinner capsule formation and lower calcification after 15 months. However, suturing of GPVG was more difficult in comparison with the other types.     

In vitro behavior of osteoblast-like cells on fluoridated hydroxyapatite coatings

In this work, fluoridated hydroxyapatite (Ca10(PO4)6Fx(OH)(2-x) or FHA) coatings are prepared by sol-gel method for study of the influence of F content on the behavior of osteoblast-like cells. The results show that the cells well attach and proliferate on the FHA coatings studied (Ca10(PO4)6F(0.67-2.00)(OH)(0-1.33)). With increasing F content in the FHA coatings, percentage of cell in S period increases, indicating F in the coating favors the proliferation process of the cells. On the other hand, the proliferation rate increases inversely with zeta potential of the coating surface. As tested from the MTT of the cells cultured in the leaching out solution, increase of F content in the FHA coatings results in a slight decrease in cell proliferation rate, which is most probably due to reduction in release of Ca2+ ions. As a compromise among cell attachment, cell proliferation, apatite deposition and ability to resist dissolution, it is suggested that FHA coatings (Ca10(PO4)6Fx(OH)(2-x)) with x in the range of 0.67-1.48, from the results of this study, may be most suitable for real case implantation.     

In vitro cytocompatibility studies of Diamond Like Carbon coatings on titanium

Diamond like carbon (DLC) films were deposited on to titanium (Ti) substrates by Plasma Enhanced Chemical Vapour Deposition (PECVD) process. The quality of the films were checked by Raman spectra and nano-hardness tests. The cytocompatibility of titanium and DLC coated titanium were studied using continuous cell lines of mouse fibroblast cells ( L-929), Human Osteoblast cells (HOS) and primary human umbilical cord vein endothelial cells (HUVEC). The cellular responses to the materials were assessed both quantitatively and qualitatively. The adhesion and spreading of cells on materials were compared using Ti as a control. Present study indicates an improved cytocompatibility of DLC coated Ti in comparison to bare Ti.     

In vitro cross-allergenicity of major aeroallergenic pollens by the radioallergosorbent technique.

Cross-reactivity between major classes of aeroallergenic pollens has been estimated by comparing the inhibitory effects of selected allergens upon the reaginic activity of other native and non-native varieties, as measured by the radioallergosorbent technique (RAST). Common allergenic determinants were demonstrated in indigenous and nonindigenous ragween species. Although patterns of inhibitory capacity were less uniform among grass pollens, endemic species tended to have more common allergenic properties than nonendemic species. Tree pollens exhibited the greatest degree of heterogeneity, confirming the previous view that these pollens tend to be less cross-reactive. Concomitant skin test threshold studies utilizing the same groups of pollens were in general agreement with in vitro results. Because, threshold skin testing was affected by a number of uncontrollable variables, however, the current modification of RAST in estimating cross-allergenicity was more accurate and reliable than data based upon cutaneous tests. It was concluded that reagin neutralization by the RAST method is the best currently available method of assessing cross-allergenic properties of pollens.     

Processing and in vitro behavior of hydroxyapatite coatings prepared by electrostatic spray assisted vapor deposition method

Hydroxyapatite (HA) bioactive coatings are often used to improve bone attachment and reduce corrosion of metal prosthesis implants. This paper reports the preparation of HA coatings onto titanium substrates using a novel electrostatic spray assisted vapor deposition (ESAVD)-based method. The deposited coatings are characterized using a combination of Fourier transform infrared spectrometer, X-ray diffraction, Raman spectroscopy, scanning electron microscopy, and scratch test. The results confirm that well-crystallized HA coatings can be produced directly via the single-step ESAVD process, without further heat-treatment. The in vitro behavior of the as-deposited HA coating in simulated body fluid (SBF) is also presented. After 14-day immersion in SBF, the adhesion of the HA coating to the substrate increases significantly.     

改良自乳化溶剂扩散法制备MePEG-PLA纳米粒对成骨细胞的毒性

背景：两亲性嵌段聚合物由于其较强的载药能力强、纳米级大小、血液中长循环等优点在载药系统中得到广泛的应用。 目的：评估改良自乳化溶剂扩散法制备的甲氧基封端的聚乙二醇-聚乳酸 (MePEG-PLA)纳米粒对人骨肉瘤细胞MG63的毒性。 方法：通过改良自乳化溶剂扩散法制备MePEG-PLA纳米粒,MTS法测定纳米粒培养1,2,3 d后对MG63的毒性。激光粒度分析仪测定纳米颗粒的粒径大小、粒径分布及Zeta电位;透射电镜表征纳米胶束外观形态;酶标仪检测培养1,2,3 d细胞吸光度值。 结果与结论：MePEG-PLA纳米粒的平均粒径为25.7 nm,分布均匀,呈球形,Zeta电位为-8.06 mV,MePEG-PLA毒性为 0级。提示改良自乳化溶剂扩散法制备纳米粒简单易行,制备的纳米粒无毒,具有良好的应用前景。     

A layer-by-layer approach to natural polymer-derived bioactive coatings on magnesium alloys

The development of polyelectrolyte multilayered coatings on magnesium alloy substrates that can be used for controlled delivery of growth factors and required biomolecules from the surface of these degradable implants could have a significant impact in the field of bone tissue regeneration. The current work reports on the fabrication of multilayered coatings of alginate and poly-l-lysine on alkaline- and fluoride-pretreated AZ31 substrates using a layer-by-layer (LbL) technique under physiological conditions. Furthermore, these coatings were surface functionalized by chemical cross-linking and fibronectin immobilization, and the resultant changes in surface properties have been shown to influence the cellular activity of these multilayered films. The physicochemical characteristics of these coated substrates have been investigated using attenuated total reflectance Fourier transform infrared spectroscopy, atomic force microscopy, scanning electron microscopy and energy-dispersive X-ray spectroscopy. Cytocompatibility studies using MC3T3-E1 osteoblasts show that the fluoride-pretreated, cross-linked and fibronectin-immobilized LbL-coated substrates are more bioactive and less cytotoxic than the hydroxide-pretreated, cross-linked and fibronectin-immobilized LbL-coated samples. The in vitro degradation results show that the multilayered coatings of these natural polysaccharide- and synthetic polyamino acid-based polyelectrolytes do not alter the degradation kinetics of the substrates; however, the pretreatment conditions have a significant impact on the overall coating degradation behavior. These preliminary results collectively show the potential use of LbL coatings on magnesium-based degradable scaffolds to improve their surface bioactivity.     

In vitro comparative evaluation under static conditions of the hemocompatibility of four types of tubing for cardiopulmonary bypass.

A comparative in vitro assessment of 4 types of tubing representative of the materials currently used in cardiopulmonary bypass (CPB) procedures was conducted under static conditions using liquid extracts of the materials or direct contact with fresh human blood or serum. The parameters monitored were biomarkers of coagulation and fibrinolytic cascades, the complement system and cell activation. Silicone and PVC tubing were shown to be non-cytotoxic and non-hemolytic. Heparin-coated PVC tubing did present a certain degree of cytotoxicity especially when in direct contact. Thrombosis was found to be significantly lower with the same heparin-coated material. To a lesser extent, platinum-cured silicone also showed a reduced thrombotic tendency. None of the materials activated platelets or the complement system. With platinum-cured silicone tubing, constant and lower leukocyte adhesion was evidenced at the different experimental time points. This could reflect reduced cell activation.     

层层组装技术制备牙科硅藻土基纳米复相陶瓷粉体

目的 探讨层层组装技术对硅藻土基牙科陶瓷粉体的改性效果及其与纳米氧化锆的吸附能力的影响,减小硅藻土及纳米氧化锆粉体的团聚现象,制备出分散均匀的纳米复相陶瓷粉体.方法 采用层层组装将2种聚电解质分别组装到硅藻土颗粒表面,再将硅藻土与纳米氧化锆通过异种电荷进行吸附.比较改性前后硅藻土Zeta电位、粒径及粒径分布的变化、红外...     

In vitro assessment of bioactive coatings for neural implant applications

Recent efforts in our laboratory have focused on developing methods for immobilizing bioactive peptides to low cell-adhesive dextran monolayer coatings and promoting biospecific cell adhesion for biomaterial implant applications. In the current study, this dextran-based bioactive coating technology was developed for silicon, polyimide, and gold, the base materials utilized to fabricate our prototype neural implants. Chemical composition of all modified surfaces was verified by X-ray photoelectron spectroscopy (XPS). We observed that surface-immobilized dextran supported very little cell adhesion in vitro (24-h incubation with serum-supplemented medium) on all base materials. Inactive nonadhesion-promoting Gly-Arg-Ala-Asp-Ser-Pro peptides immobilized on dextran-coated materials promoted adhesion and spreading at low levels not significantly different from dextran-coated substrates. Arg-Gly-Asp (RGD) peptide-grafted surfaces were observed to promote substantial fibroblast and glial cell adhesion with minimal PC12 (neuronal cell) adhesion. In contrast, dextran-coated materials with surface-grafted laminin-based, neurite-promoting Ile-Lys-Val-Ala-Val (IKVAV) peptide promoted substantial neuron cell adhesion and minimal fibroblast and glial cell adhesion. It was concluded that neuron-selective substrates are feasible using dextran-based surface chemistry strategies. The chemical surface modifications could be utilized to establish a stable neural tissue-implant interface for long-term performance of neural prosthetic devices.     

In vitro transfection of plasmid DNA by cationized gelatin prepared from different amine compounds

The objective of this paper is to compare the in vitro transfection efficiency of a luciferase plasmid DNA using cationized gelatin prepared from different amine compounds. The compounds used here were ethylenediamine, putrescine, spermidine and spermine, chemically introduced to the carboxyl group of gelatin for the cationization. Complexation of the cationized gelatin with the plasmid DNA was performed by simply mixing the two materials at various N+/P- mixing ratios (the molar number ratio of amino groups of gelatin to the phosphate groups of DNA) in aqueous solution. Gel retardation studies revealed that the formation of cationized-gelatin-plasmid DNA complexes depended on the N+/P- mixing ratio. The stronger interaction of plasmid DNA with the cationized gelatin of spermine compared to the other cationized gelatins was observed by an ethidium bromide intercalation assay and Scatchard binding analysis. When the transfection efficiency of plasmid DNA complexed with the various cationized gelatins at different N+/P- mixing ratios was evaluated for mouse L929 fibroblasts, the highest transfection efficiency was observed for the complex prepared from the cationized gelatin of spermine at a N+/P- mixing ratio of 2. The present study indicates that there is an optimal N+/P- mixing ratio and a type of amine compound or cationization extent of cationized gelatin to enhance the transfection efficiency of plasmid DNA.     

In vitro behavior of silicate glass coatings on Ti6A14V

The in vitro response in simulated body fluid (SBF) of silicate glass coatings on Ti6A14V was evaluated. Glasses belonging to the SiO2-CaO-MgO-Na2O-K2O-P2O5 system were used to prepare 50-70 m thick coatings on Ti6Al4V, employing a simple enameling technique. Glasses with silica content higher than 55 wt% can be used to prepare coatings that do not crack or delaminate and exhibit good adhesion to the alloy. It has been found that coatings with silica content lower than 60 wt% are more susceptible to corrosion and precipitate carbonated hydroxyapatite on their surface during in vitro tests. However, these coatings have a higher thermal expansion than the metal and are under tension. After 2 months in SBF cracks grow in the coating that reach the glass/metal interface and initiate delamination. Glasses with silica content higher than 60 wt% are more resistant to corrosion and have lower thermal expansion. These coatings do not crack but they do not precipitate apatite even after 2 months in SBF.     

In vitro behavior of HVOF sprayed calcium phosphate splats and coatings

Hydroxyapatite (HA) coatings and splats deposited by high velocity oxy-fuel (HVOF) spray technique was investigated in vitro. HA coatings prepared from two different HA powder size range (30+/-5 and 50 +/-5 microm) were immersed in a simulated body fluid with various incubation periods of maximum 6 weeks. The dissolution/precipitation behavior was studied and the degradation of HA coatings caused by in vitro ageing was demonstrated by measuring the changes in flexural modulus through a 3-point bend test. It was found that the dissolution and precipitation behavior of the coatings was significantly dependent upon the incipient coating phase composition and the precipitation of bone-like hydroxyapatite on the coating's surface was found to be directly related to the dissolution process. Higher dissolution rates of tricalcium phosphate, tetracalcium phosphate and amorphous calcium phosphate relative to HA, resulted in accelerated precipitation. Furthermore, analysis of coatings' surface morphology demonstrated that advanced precipitation invariably occurred at regions where dissolution took place. Results showed that the changes in flexural modulus of investigated HA coatings accompanying different incubation duration was not systematic but was found to be dependent upon changes of coating structure and other factors brought about by in vitro ageing. In vitro investigation of individual HA splats collected from different HA particle sizes revealed, after 3 days ageing, that the rate ratio of precipitation to dissolution was directly determined by the local phase composition, and this phenomenon could be effectively used to explain the behavior of thermally sprayed HA coatings in vitro. It implied that the precipitation was strongly dependent on the first molecule attachment. To achieve rapid precipitation in vitro, partial molten state of HA particles during HVOF coating deposition was recommended.