Cellular transcriptional response to zirconia-based implant materials.

OBJECTIVE: To adequately address clinically important issues such as osseointegration and soft tissue integration, we screened for the direct biological cell response by culturing human osteoblasts and gingival fibroblasts on novel zirconia-based dental implant biomaterials and subjecting them to transcriptional analysis. METHODS: Biomaterials used for osteoblasts involved micro-roughened surfaces made of a new type of ceria-stabilized zirconia composite with two different topographies, zirconium dioxide, and yttria-stabilized zirconia (control). For fibroblasts smooth ceria- and yttria-stabilized zirconia surface were used. The expression of 90 issue-relevant genes was determined on mRNA transcription level by real-time PCR Array technology after growth periods of 1 and 7 days. RESULTS: Generally, modulation of gene transcription exhibited a dual dependence, first by time and second by the biomaterial, whereas biomaterial-triggered changes were predominantly caused by the biomaterials' chemistry rather than surface topography. Per se, modulated genes assigned to regenerative tissue processes such as fracture healing and wound healing and in detail included colony stimulating factors (CSF2 and CSF3), growth factors, which regulate bone matrix properties (e.g. BMP3 and TGFB1), osteogenic BMPs (BMP2/4/6/7) and transcription factors (RUNX2 and SP7), matrix collagens and osteocalcin, laminins as well as integrin ß1 and MMP-2. SIGNIFICANCE: With respect to the biomaterials under study, the screening showed that a new zirconia-based composite stabilized with ceria may be promising to provide clinically desired periodontal tissue integration. Moreover, by detecting biomarkers modulated in a time- and/or biomaterial-dependent manner, we identified candidate genes for the targeted analysis of cell-implant bioresponse during biomaterial research and development.

Kinetics of calcium oxalate crystal formation in urine.

It is routinely observed that persons with increased urinary stone risk factors do not necessarily form uroliths. Furthermore, stone formers can present with urinalyses that do not reflect the clinical picture. We explain this discrepancy by differences in crystallization kinetics. In 1162 urines, crystallization of Ca-oxalate was induced according to the BONN-Risk-Index (BRI) method. The urine's relative light transmissivity (RLT) was recorded from 100 % at start of titration to 95 % due to nuclei formation and crystal growth. From the RLT changes, a measure of the thermodynamic inhibition threshold of crystal formation (BRI) and of crystal growth kinetics is derived ("turbidity slope" after crystallization onset). On average, subjects presenting with a low inhibition threshold, i.e., high BRI, also present significantly higher crystal growth rates compared with subjects in lower BRI classes. Only subjects in the highest BRI class show a lower growth rate than expected, probably due to a depletion of supersaturation by massive initial nucleation. With increasing thermodynamic risk of crystal formation (i.e., increasing BRI) due to an imbalance between inhibitors and promoters of crystal formation, an increase in the imbalance between inhibitors and promoters of crystal growth (i.e., increasing growth rate) is observed. Both lead to an increased urolith formation risk. Healthy subjects with increased BRI are an exception to this trend: their urine is thermodynamically prone to form stones, but they show a kinetic inhibition preventing nuclei from significant growth.

Hydrophobic forces as a key factor in crystalline biofilm formation on ureteral stents.

BACKGROUND: Current discussions about biofilm formation focus on the solid/liquid interface between a medical device and body fluids. Yet it has been shown that gas bubbles (GB) can stably form on ureteral stents in artificial urine and that their fate depends on the stent's surface properties. The liquid/gas interface constitutes an adhesion site for precipitating salts as well as hydrophobic organic molecules. MATERIALS AND METHODS: The surface wettability of polyurethane stents is varied by coating with amorphous hydrogenated carbon (a-C:H). GB and crystalline biofilm formation on the stents are investigated in a novel encrustation device which avoids gravitation- or sample-position-related influences on the results. RESULTS: Bigger and more stable GB form on hydrophobic stents than on hydrophilic, coated stents. Appearance and amount of crystalline deposits differ significantly between the surfaces. With decreasing wettability the number of hollow crystalline spheres and the mass of precipitate increase. CONCLUSIONS: On hydrophobic surfaces, stable GB increase precipitation of salts and become incorporated in the growing encrustation layer in vitro. In contrast, GB quickly lift off from hydrophilic surfaces taking part of the precipitate with them. This self-cleaning mechanism slows down the encrustation process. A similar effect may explain the prolonged complication-free indwelling time of amorphous-carbon coated stents in vivo.

Microbial adhesion on novel yttria-stabilized tetragonal zirconia (Y-TZP) implant surfaces with nitrogen-doped hydrogenated amorphous carbon (a-C:H:N) coatings.

OBJECTIVES: Biomaterial surfaces are at high risk for initial microbial colonization, persistence, and concomitant infection. The rationale of this study was to assess the initial adhesion on novel implant surfaces of Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans upon incubation. MATERIALS AND METHODS: The tested samples were 3 mol% yttria-stabilized tetragonal zirconia polycrystal (3Y-TZP) samples with nitrogen-doped hydrogenated amorphous carbon (a-C:H:N) coating (A) and 3Y-TZP samples coated with ceria-stabilized zirconia-based (Ce-TZP) composite and a-C:H:N (B). Uncoated 3Y-TZP samples (C) and bovine enamel slabs (BES) served as controls. Once the surface was characterized, the adherent microorganisms were quantified by estimating the colony-forming units (CFUs). Microbial vitality was assessed by live/dead staining, and microbial-biomaterial surface topography was visualized by scanning electron microscopy (SEM). RESULTS: Overall, A and B presented the lowest CFU values for all microorganisms, while C sheltered significantly less E. faecalis, P. aeruginosa, and C. albicans than BES. Compared to the controls, B demonstrated the lowest vitality values for E. coli (54.12 %) and C. albicans (67.99 %). Interestingly, A (29.24 %) exhibited higher eradication rates for S. aureus than B (13.95 %). CONCLUSIONS: Within the limitations of this study, a-C:H:N-coated 3Y-TZP surfaces tended to harbor less initially adherent microorganisms and selectively interfered with their vitality. CLINICAL RELEVANCE: This could enable further investigation of the new multi-functional zirconia surfaces to confirm their favorable antimicrobial properties in vivo.

Ureteral stents should be soaked for several minutes before placement.

PURPOSE: Placement of ureteral stents (DJ-stents) may lead to complications. Inappropriate friction properties of the implant are, inter alia, made responsible for primary injuries, injury-related inflammation and a cascade of consecutive side effects. Hydrophilicity is considered to be related to low friction. The question arises, whether the various products on the market show their respective maximum hydrophilicity directly after unwrapping or a pre-use moistening, as already routinely done with the guide wire, is necessary. METHODS: The surface wettability of commercial and experimental DJ-stents was determined by water contact angle (WCA) measurements using the sessile drop method. One reference surface and 11 different stent surface types were tested. In order to determine the influence of moistening on the stents' surface wettability, WCAs were measured twice, with dry, and soaked (30 min, 0.9%-NaCl) specimens. Each sample of a surface type was tested at three different positions to avoid effects of surface heterogeneities. Up to six samples of the same surface type were examined. RESULTS: Mean WCAs on fresh and soaked stent surfaces ranged from 75°-103° and 71°-99°. In every case the WCAs on soaked surfaces were lower. On average the WCAs decrease by 7%, the individual decreases differ considerably, from 2 to 16%. For 7/12 of the examined surface types, the decrease in contact angle is statistically significant with p ≤ 0.01. CONCLUSIONS: DJ-stents freshly unwrapped show less hydrophilic properties compared to DJ-stents soaked in saline. To obtain maximum hydrophilicity at stent placement, DJ-stents should be soaked. The results may advocate a similar approach for other urological equipment.

Induced urinary crystal formation as an analytical strategy for the prediction and monitoring of urolithiasis and other metabolism-related disorders.

Crystal formation reflects the entire composition of the surrounding solution. In case of urolithiasis, induced crystal formation in native urine has led to the development of the Bonn-Risk-Index (BRI), a valuable tool to quantify an individual's risk of calcium oxalate urolithiasis. If the progression of a disease is associated with characteristic changes in the activities of urinary components, this leads to an altered urinary crystallisation capacity. Therefore, the results of induced urinary crystal formation can be used to detect and monitor any disease linked to the altered urinary composition. Since crystal formation inherently takes into account the entire urinary composition, the influence of the disease on individual urinary parameters does not have to be known in order to monitor the consequent pathologic alterations. In this paper, we review the background of urinary crystal formation analysis and describe its established application in urolithiasis monitoring as well as potential further fields of clinical application.

Initial Bacterial Adhesion on Different Yttria-Stabilized Tetragonal Zirconia Implant Surfaces in Vitro.

Bacterial adhesion to implant biomaterials constitutes a virulence factor leading to biofilm formation, infection and treatment failure. The aim of this study was to examine the initial bacterial adhesion on different implant materials in vitro. Four implant biomaterials were incubated with Enterococcus faecalis, Staphylococcus aureus and Candida albicans for 2 h: 3 mol % yttria-stabilized tetragonal zirconia polycrystal surface (B1a), B1a with zirconium oxide (ZrO2) coating (B2a), B1a with zirconia-based composite coating (B1b) and B1a with zirconia-based composite and ZrO2 coatings (B2b). Bovine enamel slabs (BES) served as control. The adherent microorganisms were quantified and visualized using scanning electron microscopy (SEM); DAPI and live/dead staining. The lowest bacterial count of E. faecalis was detected on BES and the highest on B1a. The fewest vital C. albicans strains (42.22%) were detected on B2a surfaces, while most E. faecalis and S. aureus strains (approximately 80%) were vital overall. Compared to BES; coated and uncoated zirconia substrata exhibited no anti-adhesive properties. Further improvement of the material surface characteristics is essential.

Dopamine-melanin film deposition depends on the used oxidant and buffer solution.

The deposition of "polydopamine" films, from an aqueous solution containing dopamine or other catecholamines, constitutes a new and versatile way to functionalize solid-liquid interfaces. Indeed such films can be deposited on almost all kinds of materials. Their deposition kinetics does not depend markedly on the surface chemistry of the substrate, and the films can reach thickness of a few tens of nanometers in a single reaction step. Up to now, even if a lot is known about the oxidation mechanism of dopamine in solution, only little information is available to describe the deposition mechanism on surfaces either by oxidation in solution or by electrodeposition. The deposition kinetics of melanin was only investigated from dopamine solutions using oxygen or ammonium persulfate as an oxidant and from a tris(hydroxymethyl) aminomethane (Tris) containing buffer solutions at pH 8.5. Many other oxidants could be used, and the buffer agent containing a primary amine group may influence the deposition process. Herein we show that the deposition kinetics of melanin from dopamine containing buffers at pH 8.5 can be markedly modified using Cu(2+) instead of O2 as an oxidant: the deposition kinetics remains linear up to thicknesses of more than 70 nm, whereas the film growth stops at 45 ± 5 nm in the presence of 02. In addition, the films prepared from Cu(2+) containing solutions display an absorption spectrum with defined peaks at 320 and 370 nm, which are absent in the spectra of films prepared in oxygenated solutions. The replacement of Tris buffer by phosphate buffer also has a marked effect on the melanin deposition kinetics.

Melanin-containing films: growth from dopamine solutions versus layer-by-layer deposition.

Films formed by oxidation of dopamine are of interest for functionalisation of solid-liquid interfaces owing to their versatility. However, the ability to modulate the properties of such films, for example, permeability to ionic species and the absorption coefficient, is urgently needed. Indeed, melanin films produced by oxidation of dopamine absorb strongly over the whole UV/Vis part of the electromagnetic spectrum and are impermeable to anions even for a film thickness as low as a few nanometers. Herein we combine oxidation of dopamine to produce a solution containing dopamine-melanin particles and their alternating deposition with poly(diallyldimethylammonium chloride) to produce films which have nearly the same morphology as pure dopamine-melanin films but are less compact, more transparent and more permeable to ferrocyanide anions.

Protein adsorption on dopamine-melanin films: role of electrostatic interactions inferred from zeta-potential measurements versus chemisorption.

We recently showed the possibility to build dopamine-melanin films of controlled thickness by successive immersions of a substrate in alkaline solutions of dopamine [F. Bernsmann, A. Ponche, C. Ringwald, J. Hemmerlé, J. Raya, B. Bechinger, J.-C. Voegel, P. Schaaf, V. Ball, J. Phys. Chem. C 113 (2009) 8234-8242]. In this work the structure and properties of such films are further explored. The zeta-potential of dopamine-melanin films is measured as a function of the total immersion time to build the film. It appears that the film bears a constant zeta-potential of (-39+/-3) mV after 12 immersion steps. These data are used to calculate the surface density of charged groups of the dopamine-melanin films at pH 8.5 that are mostly catechol or quinone imine chemical groups. Furthermore the zeta-potential is used to explain the adsorption of three model proteins (lysozyme, myoglobin, alpha-lactalbumin), which is monitored by quartz crystal microbalance. We come to the conclusion that protein adsorption on dopamine-melanin is not only determined by possible covalent binding between amino groups of the proteins and catechol groups of dopamine-melanin but that electrostatic interactions contribute to protein binding. Part of the adsorbed proteins can be desorbed by sodium dodecylsulfate solutions at the critical micellar concentration. The fraction of weakly bound proteins decreases with their isoelectric point. Additionally the number of available sites for covalent binding of amino groups on melanin grains is quantified.

Polyelectrolyte multilayer films built from poly(L-lysine) and a two-component anionic polysaccharide blend.

The buildup of polyelectrolyte multilayer films made from poly(L-lysine) (PLL) as a polycation and from a blend of two anionic polysaccharides, namely, beta-1,3 glycan sulfate (GlyS) and alginate (Alg), was investigated as a function of the mass fraction, x, of GlyS in the blend, at a constant total weight concentration in polyanions. We find that the film thickness, after the deposition of a given number of layer pairs, reaches a minimum for x values lower than 0.1 (the position of this minimum could not be more precisely localized) and that the film thickness at intermediate values of x is the same as that of films built at the same concentration of GlyS in the absence of Alg (pure GlyS solution). Infrared spectroscopy in the attenuated total reflection mode shows that the weight fraction of GlyS in the multilayer films is much higher than its weight fraction, x, in the blend used to build the film. This preferential incorporation of GlyS over Alg is related to preferential interactions of GlyS as compared to Alg with PLL in solution, as measured by means of isothermal titration calorimetry. We also demonstrate that GlyS is able to displace Alg almost quantitatively from (PLL/Alg)n films but that in contrast Alg is not able to exchange GlyS from (PLL/GlyS)n films. These results, which combine adsorption from blended polyanion solutions, exchange of one polyanion already present in the film by the other in solution, and thermodynamic measurements, suggest that sulfated polymers are able to interact with polycations preferentially over polymers carrying carboxylated charged groups. These results give a first structural basis to the mechanism of preferential incorporation of a given polyanion with respect to another.

Protein films adsorbed on experimental dental materials: ToF-SIMS with multivariate data analysis.

The proteins lysozyme, amylase, and bovine serum albumin (BSA) were adsorbed on two experimental dental materials, made of fluoroapatite particles embedded in polymer matrices, and on silicon wafers. The protein films were prepared as single-component layers, as binary mixtures, and as double layers. These systems were investigated by time-of-flight secondary ion mass spectrometry (ToF-SIMS) and the multivariate data analysis technique of discriminant principal-component analysis (DPCA). During adsorption of a single protein film on to the solid surfaces, the three proteins could be clearly distinguished by the scores of their mass spectra after selection of amino acid-related peaks and DPCA. Furthermore, very similar results were obtained on the two different fluoroapatite substrates. For samples coated with binary layers of two proteins adsorbed simultaneously, it was found for both substrate types that BSA shows the strongest ability to adsorb followed by lysozyme, while amylase has the smallest ability. By contrast, the consecutive adsorption of two protein layers showed a strong influence of substrate type on the adsorption ability of the proteins.

Use of polymerisation to produce free-standing membranes from exponentially growing multilayer films.

In this communication, we demonstrate that dopamine is able to undergo a polymerisation process in (PLL-HA)n polyelectrolyte multilayer films, and that this polymerisation is of the same nature as in solution at pH 8.5. This polymerisation changes the chemical composition and decreases the mobility of the PLL chains in the film, and ultimately allows the easy detachment of the film as free-standing membranes with 0.1 M HCl solutions.