In vitro cytotoxicity of amorphous carbon films

Amorphous carbon (a-C), carbon nitride (a-CN) and titanium films were deposited on stainless steel substrates (SS) using a dc magnetron sputtering system attached to a high vacuum chamber. Films were deposited using a base pressure of 1.3x10(-4) Pa. For the carbon films a pure graphite target was eroded in an Argon plasma. For the case of the a-CN films, the Ar flux was substituted by 100% N2 gas. Titanium films were deposited in a different chamber, using a pure Ti target and an argon plasma. In vitro studies were carried out on the coated samples using human osteoblasts cells. Cytotoxicity of carbon films was assessed by cellular adhesion and proliferation, as determined by direct cellular counting using a spectroscopic technique and a well-defined standard curve. Osteoblasts cells were also grown on uncoated steel and prepared Petri dishes for comparison. The percentage of osteoblasts adhesion measured at 24 hrs attained maximum values for the a-C films. Similarly, cellular proliferation evaluated at three, five and seven days showed an outstanding increase of osteoblasts cells for the a-C and Ti coatings in contrast to the uncoated steel. The cell functionality was evaluated by the MTT test after incubation periods of 3, 5 and 7 days. The absorbance values obtained for a-C, a-CN and Ti surfaces resulted significantly higher with respect to the positive control, indicating that the surface did not induce any toxic effect. Preliminary bio-mineralization was evaluated by measuring the elemental composition of the mineral grown on the substrates after periods up to 14 days.     

Layer-by-layer assembly of polyelectrolyte films improving cytocompatibility to neural cells

The using of layer-by-layer assembly polyelectrolyte (PE) films has been suggested as a new versatile technique for surface modification aimed at tissue engineering and cell-based chips. In this study, we investigated the surface morphology of the hyaluronic acid (HA)-based PE films deposited on the amino-functionalized glass slides using atomic force microscopy. These thin films (bilayer number <9) were measured to have nanoscale roughness ranging from 10 to 100 nm. Then the primary hippocampal and cortical neural cells were cultured on the PE films, respectively. After 5 days of culturing, the cytocompatibility to neural cells was evaluated by cellular morphology, neurite outgrowth, and microtubule-associated protein 2 expressions. From the present results, the HA-based PE films were found to be able to support neural cell adhesion and neurite development, especially for the polycation-ending films. It is suggested these HA-based multilayer PE films or similar build-ups could thus be used in the future as a way to modify surfaces for nerve scaffolds and neuron-based chips.     

豚鼠骨髓间充质干细胞与纳米羟基磷灰石体外构建组织工程人工听骨

背景：人工合成的纳米羟基磷灰石具有良好的生物活性和相容性,植入体内后能在短时间内与体内的软硬组织形成紧密结合,因此成为广泛应用的骨组织工程材料。 目的：观察豚鼠骨髓间充质干细胞与纳米羟基磷灰石复合多孔陶瓷材料体外复合培养的结合程度,并分析其构建组织工程人工听骨的可行性。 方法：采用细胞差速贴壁法分离培养豚鼠骨髓间充质干细胞,使用流式细胞仪检测CD29、CD45、CD44进行间充质干细胞表面标记物鉴定,并检测其骨细胞分化能力。将骨髓间充质干细胞与纳米羟基磷灰石复合多孔陶瓷共培养1,3,5,7,10 d,电镜观察骨髓间充质干细胞与此种支架材料的复合情况。 结果与结论：培养3 d后,可见大量骨髓间充质干细胞贴附在材料表层,并且形态稳定,生长旺盛,增殖力强,具有极佳的延伸性;培养5 d后可见材料表面已全部覆盖骨髓间充质干细胞,细胞结合紧密,细胞表面可见大量分泌颗粒,胞体明显增大,边缘完整,呈纤维样伸长;7 d后细胞逐渐从材料表面脱落,仍附在材料表面的细胞出现“星形”改变,“伪足”增多;10 d后细胞呈片状脱落。说明纳米羟基磷灰石材料保持了它良好的生物相容性,利于细胞黏附、增殖,可结合大量的骨髓间充质干细胞,用于构建组织工程人工听骨。     

Effect of vitamin E addition to poly(D,L)-lactic acid on surface properties and osteoblast behaviour

Vitamin E (Vit.E, alpha-tocoferol) is a natural agent with anti-oxidative and anti-inflammatory properties and it has been suggested that it could act as a stimulating factor for osteoblast proliferation and maturation. We produced poly(D,L)-lactic acid films enriched with Vit.E (1, 5 and 10% w/w) and investigated their surface properties using the FTIR analysis, sessile measure of wettability and serum protein adsorption, and evaluated attachment and spreading of MC-3T3 E1 murine osteoblast cells. FTIR analysis showed the presence of Vit.E on the polymer surface and Vit.E increased the polymer wettability in a concentration-dependent manner. The serum total protein adsorption increased significantly onto the 10% Vit.E P(D,L)-LA and the main protein adsorbed was albumin. The presence of albumin, considered as an anti-adhesive protein, on the surface of Vit.E enriched P(D,L)-LA films (especially 5 and 10% Vit.E) could explain, at least in part, the behaviour of MC-3T3 osteoblast cells seeded onto the polymers. Cell adhesion and spreading were strongly decreased by Vit.E (5 and 10%) in spite of the increased wettability. This reaction could be cell type-specific, independent by the surface wettability and linked to cell-specific characteristics (membrane phospholipid composition, integrins expression). Moreover a direct effect of Vit.E on cell adhesion and spreading cannot be completely excluded.     

Adhesion and growth of human Schwann cells on trimethylene carbonate (co)polymers

Seeding of artificial nerve grafts with Schwann cells is a promising strategy for bridging large nerve defects. The aim of the present study was to evaluate the adhesion and growth of human Schwann cells (HSCs) on 1,3-trimethylene carbonate (TMC) and epsilon-caprolactone copolymers, with the final goal of using these materials in the development of an artificial nerve graft. The adhesion, proliferation, and morphology of HSCs on copolymers containing 10 and 82 mol % of TMC and on the parent homopolymers were investigated. HSCs adhered faster and in greater numbers on the copolymer with 82 mol % of TMC and on the TMC homopolymer compared with the other (co)polymers. On all polymer films, cell adhesion was lower than on gelatin (positive control). Despite differences in cell adhesion, cells displayed exponential growth on all tested surfaces, with similar growth rates. Cell numbers doubled approximately every 3 days on all substrates. When the polymer films were coated with fibronectin, no significant differences in cell adhesion and proliferation were observed between coated polymer surfaces and gelatin. The results indicate that all tested materials support the adhesion and proliferation of HSCs and can in principle be used for the preparation of flexible and slowly degrading nerve guides.     

Evaluation of the antiadhesion potential of UV cross-linked gelatin films in a rat abdominal model

Among five kinds of rat adhesion models tested, the following model was selected. The epigastric vein 2.5 cm from the midline of the abdomen was cut by sharp scissors, and the lateral side of the cut epigastric vein was ligated using a 3-0 silk suture. This model could be easily prepared and gave a rate of adhesion formation of 90%, which was useful for screening antiadhesive materials. For the kinetic study of tissue adhesion in this model, an injured site was covered with a non-degradable poly(vinyl alcohol) (PVA) film. The incidence rate of adhesion was 18%, when the PVA film covered the injured site for 2 days. This suggests that an antiadhesive barrier should cover the injured site for at least 2 days. The antiadhesion efficacy of cross-linked gelatin films were evaluated using this adhesion model. The UV cross-linked gelatin film which was designed to exist for 2 days but to disappear at day 3 in the rat abdominal cavity showed the highest antiadhesion efficacy.     

Poly(N-isopropylacrylamide) copolymer films as vehicles for the sustained delivery of proteins to vascular endothelial cells

The aim of this study was to establish the capacity of thermoresponsive poly(N-isopropylacrylamide) copolymer films to deliver bioactive concentrations of vascular endothelial growth factor (VEGF165) to human aortic endothelial cells (HAEC) over an extended time period. Films were prepared using a 50:50 (w/w) mixture of non-crosslinkable and crosslinkable copolymers of the following monomer compositions (w/w): 85:15, N-isopropylacrylamide (NiPAAm):N-tert-butylacrylamide (NtBAAm); and 85:13:2 NiPAAm:NtBAAm:acrylamidobenzophenone (ABzPh, crosslinking agent), respectively. After crosslinking by UV irradiation, the ability of films to incorporate a fluorescently labeled carrier protein (FITC-labeled BSA, 1 mg loaded per film), at 4 degrees C, was first established. Incorporation into the matrix was confirmed by the observation that increasing film thickness from 5 to 10 microm increased release from collapsed films at 37 degrees C (1.76 +/- 0.15 and 10.98 +/- 3.38 microg/mL, respectively, at 24 h postloading) and that this difference was maintained at 5 days postloading (1.81 +/- 0.25 and 13.8 +/- 2.3 microg/mL, respectively). Incorporation was also confirmed by visualization using confocal microscopy. When 10-microm films were loaded with a BSA solution (1 mg/mL) containing VEGF165 (3 microg/mL), sustained release of VEGF165 was observed (10.75 +/- 3.11 ng at 24 h; a total of 31.32 +/- 8.50 ng over 7 days). Furthermore, eluted VEGF165 increased HAEC proliferation by 18.2% over control. The absence of cytotoxic species in medium released from the copolymer films was confirmed by the lack of effect of medium (incubated with copolymer films for 3 days) on HAEC viability. In conclusion this study has shown that NiPAAm:NtBAAm copolymers can be loaded with a therapeutic protein and can deliver bioactive concentrations to human vascular endothelial cells over an extended time period.     

A novel star PEG-derived surface coating for specific cell adhesion

In this study a novel approach for the coating and functionalization of substrates for cell culture and tissue engineering is presented. Glass, silicon, and titanium panes were coated with an ultrathin film (30 +/- 5 nm) of reactive star-shaped poly(ethylene glycol) prepolymers (Star PEG). Homogeneity of the films was checked by optical microscopy and scanning force microscopy. These coatings prevent unspecific protein adsorption as monitored by fluorescence microscopy and ellipsometry. In order to allow specific cell adhesion the films were modified with linear RGD peptides (gRGDsc) in different concentrations. After sterilization, fibroblast, SaOS, and human mesenchymal stem cells (hMSC) were seeded on these substrates. Cell adhesion, spreading, and survival was observed for up to 30 days on linear RGD peptide (gRGDsc)-modified coatings, whereas no cell adhesion could be detected on unmodified Star PEG layers. By variation of the RGD concentration within the film the amount of cells that became adhesive could be controlled. When differentiation conditions are used for cultivation of hMSCs the cells show the expression of osteogenic marker genes after 14 days which is comparable to cultivation on cell culture plastic. Thus, the Star PEG/RGD film did not negatively influence the differentiation process. The high flexibility of the system considering the incorporation of biologically active compounds opens a broad field of future experiments.     

Nanocomposite Ti/hydrocarbon plasma polymer films from reactive magnetron sputtering as growth support for osteoblast-like and endothelial cells

Nanocomposite Ti/hydrocarbon plasma polymer (Ti/ppCH) films were deposited by DC magnetron sputtering of titanium target in n-hexane, argon, or a mixture of these two gases. The resultant films were heterogeneous, with inorganic regions of nanometer scale distributed within a plasma polymer matrix. The titanium content was controlled by adjusting the argon/n-hexane ratio in the working gas. In the pure n-hexane atmosphere, the Ti concentration was found to be below 1 at %, whereas in pure argon it reached 20 at %, as measured by Rutherford backscattering spectroscopy and elastic recoil detection analysis (RBS/ERDA). A high level of titanium oxidation is detected with TiO(2), substoichiometric titania, and titanium carbide, composing an inorganic phase of the composite films. In addition, high hydrogen content is detected in films rich with titanium. Ti-deficient and Ti-rich films proved equally good substrates for adhesion and growth of cultured human osteoblast-like MG 63 cells. In these cells, the population densities on days 1, 3, and 7 after seeding, spreading area on day 1, formation of talin-containing focal adhesion plaques as well as concentrations of talin and osteocalcin (per mg of protein) were comparable to the values obtained in cells on the reference cell culture materials, represented by microscopic glass coverslips or a polystyrene dish. An interesting finding was made when the Ti/ppCH films were seeded with calf pulmonary artery endothelial cells of the line CPAE. The cell population densities, the spreading area and also the concentration of von Willebrand factor, a marker of endothelial cell maturation, were significantly higher on Ti-rich than on Ti-deficient films. On Ti-rich films, these parameters were also higher or similar in comparison with the reference cell culture materials. Thus, both types of films could be used for coating bone implants, of which the Ti-rich film remains effective in enhancing the endothelialization of blood contacting artificial materials.     

Bacterial Adhesion,Cell Adhesion and Biocompatibility of Nafion Films

We investigated bioadhesion (bacterial and cell adhesion) and biocompatibility of poly(tetrafluoroethylene-co-perfluoro-3,6-dioxa-4-methyl-7-octenesulfonic acid) (Nafion^?) and compared the results with those obtained with poly(vinylidene fluoride-co-hexafluoropropylene) (PVFHFP). When incubated with bacteria for 4 h to 7 days, Nafion film exhibited scarce bacterial adhesion at 6 h, after which the adhesion gradually increasing to relatively low levels. In contrast, significant bacterial adhesion to PVFHFP film was observed at 4 h, and much higher adhesion levels were shown thereafter. Although HEp-2 human cells adhered normally to both films, reaching confluence in 7–8 days, the cells adhered to Nafion appeared more lively and stable than those to PVFHFP. Subcutaneous implantation in mice revealed that Nafion elicited a mild acute inflammatory reaction without chronic inflammation or tissue necrosis, indicating excellent biocompatibility in mice. PVFHFP, however, provoked a moderate and prolonged acute inflammatory response. These differences in the biological characteristics of Nafion and PVFHFP films may be attributable to the differences in the chemical and physical natures of these polymer films. Nafion film provided a sufficiently solid support, expressing a high surface charge density and good water-wettability. In summary, Nafion is suitable for use in biomedical applications that require biocompatibility with a reduced possibility of post-operative infections.     

Synthesis of poly(ester-carbonate) with a pendant acetylcholine analog for promoting neurite growth

The modification of biodegradable polyesters with bioactive molecules has become an important strategy for controlling neuron adhesion and neurite outgrowth in nerve regeneration. In this study we report a biodegradable poly(ester-carbonate) with a pendant acetylcholine analog, which a neurotransmitter for the enhancement of neuron adhesion and outgrowth. The acetylcholine-functionalized poly(ester-carbonate) (Ach-P(LA-ClTMC)) was prepared by copolymerizing l-lactide (LA) and 5-methyl-5-chloroethoxycarbonyl trimethylene carbonate (ClTMC), followed by quaternization with trimethylamine. The acetylcholine analog content could be modulated by changing the molar feeding fraction of ClTMC. The incorporation of the acetylcholine analog improved the hydrophilicity of the films, but the acetylcholine analog content did not significantly influence the surface morphology of the acetylcholine-functionalized films. The results of PC12 cell culture showed that the acetylcholine analog promoted cell viability and neurite outgrowth in a concentration-dependent manner. The longest length of neurite and the percentage of cells bearing neurites were obtained on the Ach-P(LA-ClTMC)-10 film. All the results indicate that the integration of the acetylcholine analog at an appropriate fraction could be an effective strategy for optimizing the existing biodegradable polyesters for nerve regeneration applications.     

Manipulation of the adhesive behaviour of skeletal muscle cells on soft and stiff polyelectrolyte multilayers

Polyelectrolyte multilayer coatings have emerged as substrates to control a variety of cell behaviour, including adhesion, proliferation and differentiation. In particular, it is possible to modulate film stiffness by physical or chemical cross-linking. In this study, we evaluate the adhesive behaviour of skeletal muscle cells (C2C12 myoblasts) during the initial steps of spreading on layer-by-layer films of controlled stiffness made of poly(l-lysine) and hyaluronan as model biomaterial surfaces for muscle tissue engineering. We show that integrin clustering, integrin actin cytoskeleton connection and focal adhesion formation for cell spreading can be decoupled by controlling film stiffness. This made it possible to switch the cells morphologically between round and spreading shapes depending on the stiffness of the microenvironment. Although hyaluronan is one of the main components of cross-linked multilayer films, the HA receptor CD44 did not appear to mediate early adhesion as suggested by the use of blocking antibodies. In contrast, integrins were found to play a pivotal role in early adhesion: their activation significantly enhanced C2C12 myoblast spreading on soft films, where they were otherwise round. Integrin clustering was also induced by the softer films and enhanced on the stiffest films. Conversely, the use of soluble inhibitors or blocking antibodies directed against integrins induced a round phenotype on stiff films, where cells were well spread out in control conditions. We show that specific integrins were involved in the adhesion process as blocking β₃, but not β₁, integrins inhibited cell adhesion. These soft, stiff films can thus be used to tune the adhesion of C2C12 myoblasts, an early key event in myogenesis, via integrin clustering and subsequent signalling. They may be further used to decorticate the signalling pathways associated with β₃ integrins.     

Electrochemical preparation and characterization of poly(2,5-thiophenediyl)

A highly conductive poly(2,5-thiophenydily) film was obtained when thiophene was electrochemically polymerized at 5°C in nitrobenzene with Bu₄NBF₄ or Bu₄NClO₄ as a supporting electrolyte. A platinum plate or indium oxide glass was used as an electrode. The current efficiency was 62%. The IR, UV, and ESR spectra were compared with those of a poly(2,5-thiophenediyl) obtained from 2,5-dibromothiophene by polycondensation. The electrochemically prepared poly(2,5-thiophenediyl) was identified by its IR spectrum. In its UV spectrum, two bands are observed at 440 nm and 740 nm. The band at 740 nm disappears in an undoped film of poly(2,5-thiophenediyl), which resembles that of the polymer obtained by polycondensation. The ESR spectra were also measured for undoped and partly undoped films. The undoped film shows an asymmetrical signal, Whereas the signal of the polymer resulting from polycondensation is symmetrical. In the air the conductivity decreases by a factor of 10^?2 of the initial value in about 70 days, but an undoped film is quite stable. The undoped film can be re-doped with iodine or sulfur trioxide and attains higher conductivity than the pellets of polymers obtained by polycondensation. This may be attributed to the difference in morphology.     

Impact of nanopore morphology on cell viability on mesoporous polymer and carbon surfaces

Topography at the nanoscale can lead to dramatic changes in the adhesion of cells to surfaces and their subsequent viability. For biological applications, including tissue engineering and cell-based sensing, the large internal surface area of ordered mesoporous carbons provides an opportunity for enhanced sensitivity and performance, but the mesostructure also affects the topography of the material. In this work, we probe the viability and adhesion of osteoblasts on ordered mesoporous materials with different morphologies and matrix chemistries. FDU-15 (hexagonal) and FDU-16 (cubic) films were processed at either 350 °C (polymeric) or 800 °C (carbon) to provide these different materials. For the films processed at 350 °C, the cell adhesion was markedly improved on the mesoporous films in comparison to a dense film analog, consistent with many reports in the literature that nanostructuring of surfaces improves the viability and adhesion of osteoblasts. Conversely, osteoblast adhesion was reduced on the carbonized surfaces processed at 800 °C when ordered mesopores were introduced, particularly for the cubic mesostructure (FDU-16). We attribute the decrease in cell adhesion to the propensity of the ordered mesoporous carbon films to sorb organics from aqueous solution, which could lead to removal of adhesion-promoting compounds at the film surface. These results suggest that cell viability on mesoporous polymer and carbon films can be controlled through simple changes in the pyrolysis temperature.     

Comparison of Chlamydia subgroup A detection from clinical specimens after 40 and 64 hours of incubation in 5-iodo-2-deoxyuridine-treated McCoy''s cells.

The time course of formation of inclusion bodies produced by Chlamydia in 5-iodo-2-deoxyuridine (IUdR)-treated McCoy's cells was studied with the use of a known isolate of Chlamydia trachomatis D/UW-184/Ur and 47 frozen clinical urethral specimens previously shown to be either positive or negative for chlamydial inclusions after 3 days of incubation. Subsequent examination of 369 clinical specimens from the genitourinary tract over a 6-month period revealed 47 (13%) Chlamydia-positive cultures, all of which demonstrated inclusion bodies by iodine staining at 40 and 64 h postinoculation. Another 146 similar detected by iodine staining from 22 (15%). This study indicates that, although Chlamydia subgroup A inclusions are larger at 64 h, they can be readily detected from clinical specimens in IUdR-treated McCoy's cells at 40 h postinfection.     

Flexible polymeric ultrathin film for mesenchymal stem cell differentiation

Ultrathin films (also called nanofilms) are two-dimensional (2-D) polymeric structures with potential application in biology, biotechnology, cosmetics and tissue engineering. Since they can be handled in liquid form with micropipettes or tweezers they have been proposed as flexible systems for cell adhesion and proliferation. In particular, with the aim of designing a novel patch for bone or tendon repair and healing, in this work the biocompatibility, adhesion and proliferation activity of Saos-2, MRC-5 and human and rat mesenchymal stem cells on poly(lactic acid) nanofilms were evaluated. The nanofilms did not impair the growth and differentiation of osteoblasts and chondrocytes. Moreover, nanofilm adhesion to rabbit joints was evident under ex vivo conditions.     

Biocompatibility and stability of disulfide-crosslinked hyaluronan films

Hyaluronan (HA) can be chemically modified to engineer robust materials with pre-selected mechanical properties and resorption rates that can be dictated by the intended clinical use. Disulfide-crosslinked HA films were prepared by air oxidation of thiol-modified HA, followed by treatment with 0.3% hydrogen peroxide. The degradation of the disulfide-crosslinked films in vitro was very slow (<10% in 7 days) in buffer alone and shorter (t1/2=3-5 days) in the presence of hyaluronidase (HAse). The cytocompatibility of the disulfide-crosslinked HA films was determined using two separate conditions: (i) in vitro culture of mouse fibroblasts in indirect contract with the films, and (ii) in vitro culture of fibroblasts directly on films coated with poly d-lysine. Excellent cytocompatibility was observed in murine fibroblasts that were cultured in indirect contact with thiolated HA films. Although cells were unable to attach and spread on thiolated HA films, pre-coating the thiolated HA films with poly D-lysine resulted in attachment and spreading equivalent to that observed on polystyrene. Rates of resorption in vivo were obtained by subcutaneous implantation of disulfide-crosslinked HA films into the backs of Wistar rats. Biocompatibility in vivo was determined in both subcutaneous flank and peritoneal cavity implantation of the films in Wistar rats. The disulfide-crosslinked HA films were less than 30% resorbed after 42 days in vivo, and histochemical and cytochemical analysis indicated that the films were well-tolerated with mild inflammatory response at both sites of implantation.     

多孔钽棒表面犬骨髓间充质干细胞的附着与增殖

背景：具有高孔隙率及与骨小梁高度相似弹性模量的多孔钽棒,不仅可为股骨头提供有效机械支撑,还可在坏死区域增强再血管化,降低应力遮挡,为骨长入坏死区提供保证。 目的：为评价多孔钽棒的细胞相容性,观察犬骨髓间充质干细胞在多孔钽棒表面的附着与增殖。 方法：采用密度梯度离心法分离培养犬骨髓间充质干细胞,鉴定后取第3代细胞,以1.5×109 L-1的细胞浓度种植在多孔钽棒表面,联合培养5,10,15 d,分别采用倒置显微镜和扫描电镜观察细胞的附着与增殖情况。 结果与结论：①倒置显微镜：1-5 d 培养期内骨髓间充质干细胞开始增殖,材料近周细胞量少,远周细胞量较多;6-10 d 细胞明显增多并逐渐向材料移行,部分细胞贴附于材料边缘;14 d以后细胞连接成片,填充材料周边及凹陷,材料边缘部分区域还可见细胞层叠成团,出现细胞钙化灶。②扫描电镜：联合培养5 d,钽棒表面无细胞附着;联合培养10 d,细胞形态多样,分散在钽棒表面,细胞间无连接,有多个突起;联合培养15 d,细胞呈多角形,数量增多,连接成片,延展良好,可见多孔钽棒上的细胞分泌大量胶原纤维,细胞被大量细胞外基质包围,细胞形态多为梭形及多角形。结果表明犬骨髓间充质干细胞在钽棒表面有较好的黏附与增殖能力。     

Design, characterization and testing of Ti-based multicomponent coatings for load-bearing medical applications

A comparative investigation of multicomponent thin films based on the systems Ti-Ca-C-O-(N), Ti-Zr-C-O-(N), Ti-Si-Zr-O-(N) and Ti-Nb-C-(N) is presented. TiC(0.5) + 10%CaO, TiC0.5 + 20%CaO, TiC0.5 + 10%ZrO2, TiC0.5 + 20%ZrO2, Ti5Si3 + 10%ZrO2, TiC0.5 + 10%Nb2C and TiC0.5 + 30%Nb2C composite targets were manufactured by means of self-propagating high-temperature synthesis, followed by DC magnetron sputtering in an atmosphere of argon or in a gaseous mixture of argon and nitrogen. The films were characterized in terms of their structure, chemical composition, surface topography, hardness, elastic modulus, elastic recovery, surface charge, friction coefficient, and wear rate. The biocompatibility of the films was evaluated by both in vitro and in vivo experiments. In vitro studies involved the investigation of the proliferation of Rat-1 fibroblasts and IAR-2 epithelial cells on the tested films, morphometric analysis and actin cytoskeleton staining of the cells cultivated on the films. In vivo studies were fulfilled by subcutaneous implantation of Teflon plates coated with the tested films in mice and analysis of the population of cells on the surfaces. The films deposited under optimal conditions showed high hardness in the range of 30-37 GPa, significant reduced Young's modulus, low friction coefficient down to 0.1-0.2 and low wear rate in comparison with conventional magnetron-sputtered TiC and TiN films. The surface of all films was negatively charged with an outstanding shift between the Ar and Ar + N2 Zeta potential curves that reaches 5 mV at the highest pH values. We did not detect statistically significant differences in the attachment, spreading and cell shape of cultured IAR-2 and Rat-1 cells on the Ti-Ca-C-O-(N), Ti-Zr-C-O-(N) (TiC0.5 + 10%ZrO2 target), Ti-Si-Zr-O-(N) films and the uncoated substrata. The adhesion and proliferation of cultured cells in vitro was perfect at all investigated films. Assessment of the population of cells covering on the Teflon plates coated with the Ti-Ca-C-O-(N) and Ti-Zr-C-O-(N) films after 16 weeks of subcutaneous implantation revealed the high biocompatibility level of tested films and absence of inflammatory reactions in mice. Contrary, the epitheliocytes and fibroblasts cultivated on the Ti-Zr-C-O-(N) (TiC0.5 + 20%ZrO2 target) and Ti-Nb-C-(N) films had disturbing actin cytoskeleton.     

Development of a high capacity microassay for measurement of neutrophil adhesion

A high capacity semiautomated assay for neutrophil adhesion was developed utilizing the 96 well microtiter plate format. Optimal adhesion occurred with about 150 microliters/well of neutrophils at 5 X 10(6) cells/ml in tissue culture plates that had been precoated with 5% serum. Optimal incubation times were 10 min for f-Met-Leu-Phe-treated cells and 20 min for A23187 or phorbol myristate acetate stimulation. Optimal washing occurred after three washes with a Cetus Pro/pette pump. Adhesion could be effectively blocked by the inhibitors of cellular protein kinase C, an enzyme known to be necessary for the occurrence of neutrophil adhesion.