Osteoblast response to titanium surfaces functionalized with extracellular matrix peptide biomimetics.

OBJECTIVE: Functionalizing surfaces with specific peptides may aid osteointegration of orthopedic implants by favoring attachment of osteoprogenitor cells and promoting osteoblastic differentiation. This study addressed the hypothesis that implant surfaces functionalized with peptides targeting multiple ligands will enhance osteoblast attachment and/or differentiation. To test this hypothesis, we used titanium (Ti) surfaces coated with poly-l-lysine-grafted polyethylene glycol (PLL-g-PEG) and functionalized with two peptides found in extracellular matrix proteins, arginine-glycine-aspartic acid (RGD) and lysine-arginine-serine-arginine (KRSR), which have been shown to increase osteoblast attachment. KSSR, which does not promote osteoblast attachment, was used as a control. MATERIALS AND METHODS: Sandblasted acid-etched titanium surfaces were coated with PLL-g-PEG functionalized with varying combinations of RGD and KRSR, as well as KSSR. Effects of these surfaces on osteoblasts were assessed by measuring cell number, alkaline phosphatase-specific activity, and levels of osteocalcin, transforming growth factor beta-1 (TGF-ß1), and PGE(2). RESULTS: RGD increased cell number, but decreased markers for osteoblast differentiation. KRSR alone had no effect on cell number, but decreased levels of TGF-ß1 and PGE(2). KRSR and RGD/KRSR coatings inhibited osteoblast differentiation vs. PLL-g-PEG. KSSR decreased cell number and increased osteoblast differentiation, indicated by increased levels of osteocalcin and PGE(2). CONCLUSIONS: The RGD and KRSR functionalized surfaces supported attachment but did not enhance osteoblast differentiation, whereas KSSR increased differentiation. RGD decreased this effect, suggesting that multifunctional peptide surfaces can be designed that improve peri-implant healing by optimizing attachment and proliferation as well as differentiation of osteoblasts, but peptide combination, dose and presentation are critical variables.

MICROPATTERNING OF GOLD SUBSTRATES BASED ON POLY(PROPYLENE SULFIDE-BL-ETHYLENE GLYCOL), (PPS-PEG) BACKGROUND PASSIVATION AND THE MOLECULAR-ASSEMBLY PATTERNING BY LIFT-OFF (MAPL) TECHNIQUE.

Poly(propylene sulfide-bl-ethylene glycol (PPS-PEG) is an amphiphilic block copolymer that spontaneously adsorbs onto gold from solution. This results in the formation of a stable polymeric layer that renders the surface protein resistant when an appropriate architecture is chosen. The established molecular assembly patterning by lift-off (MAPL) technique can convert a prestructured resist film into a pattern of biointeractive chemistry and a noninteractive background. Employing the MAPL technique, we produced a micron-scale PPS-PEG pattern on a gold substrate, and then characterized the patterned structure with Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) and Atomic Force Microscopy (AFM). Subsequent exposure of the PPS-PEG/gold pattern to protein adsorption (full human serum) was monitored in situ; SPR-imaging (i-SPR) shows a selective adsorption of proteins on gold, but not on PPS-PEG areas. Analysis shows a reduction of serum adsorption up to 93% on the PPS-PEG areas as compared to gold, in good agreement with previous analysis of homogenously adsorbed PPS-PEG on gold. MAPL patterning of PPS-PEG block copolymers is straightforward, versatile and reproducible, and may be incorporated into biosensor-based surface analysis methods.

Reduced medical infection related bacterial strains adhesion on bioactive RGD modified titanium surfaces: a first step toward cell selective surfaces.

Ideally, implants should inhibit nonspecific protein adsorption, bacterial adhesion, and at the same time, depending on the final application be selective toward cellular adhesion and spreading for all or only selected cell types. Poly(L-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG) polymers have been shown to adsorb from aqueous solution onto negatively charged metal oxide surfaces, reducing protein adsorption as well as fibroblast, osteoblast and epithelial cell adhesion significantly. PLL-g-PEG can be functionalized with bioligands such as RGD (Arg-Gly-Asp), which then restores host cell adhesion, but the surface remains resistant to nonspecific protein adsorption. Previously, it was also shown that both nonfunctionalized PLL-g-PEG and RGD-peptide functionalized PLL-g-PEG reduced the adhesion of Staphylococcus aureus to titanium (Ti) surfaces. The present study looked at the effect of other implant associated infection relevant bacteria, Staphylococcus epidermidis, Streptococcus mutans and Pseudomonas aeruginosa towards the same surface chemistries. The different surfaces were exposed to the bacteria for 1-24 h, and bacteria surface density was evaluated using scanning electron microscopy (SEM) and fluorescence light microscopy (FM). The adhesion of all bacteria strains tested was reduced on Ti surfaces coated with PLL-g-PEG compared to uncoated Ti surfaces even in the presence of RGD. The percentage reduction in bacterial adhesion over the 24-h culture time investigated was 88%-98%, depending on the bacteria type. Therefore, coating surfaces with PLL-g-PEG/PEG-RGD allows cells such as fibroblasts and osteoblasts to attach but not bacteria, resulting in a selective biointeractive pattern that may be useful on medical implants.

Patterned cell adhesion by self-assembled structures for use with a CMOS cell-based biosensor.

A strategy for patterned cell adhesion based on chemical surface modification is presented. To confine cell adhesion to specific locations, an engineered surface for high-contrast protein adsorption and, hence, cell attachment has been developed. Surface functionalization is based on selective molecular-assembly patterning (SMAP). An amine-terminated self-assembled monolayer is used to define areas of cell adhesion. A protein-repellent grafted copolymer, poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG), is used to render the surrounding silicon dioxide resistant to protein adsorption. X-ray photoelectron spectroscopy, scanning ellipsometry and fluorescence microscopy techniques were used to monitor the individual steps of the patterning process. Successful guided growth using these layers is demonstrated with primary neonatal rat cardiomyocytes, up to 4 days in vitro, and with the HL-1 cardiomyocyte cell line, up to 7 days in vitro. The advantage of the presented method is that high-resolution engineered surfaces can be realized using a simple, cost-effective, dip-and-rinse process. The technique has been developed for application on a CMOS cell-based biosensor, which comprises an array of microelectrodes to extracellularly record electrical activity from cardiomyocytes.

Functionalization of titanium oxide surfaces by means of poly(alkyl-phosphonates).

The use of a multiple attachment sites strategy is considered in order to improve the stability of monomolecular adlayers. The hypothesis was tested in the case of PEG-ylated compounds carrying phosphonate groups, known for their affinity toward titanium oxide surfaces. As a result, a new class of co- and terpolymers were synthesized by free-radical polymerization of three different monomers: dialkyl(methacryloyloxyalkyl)phosphonates, PEG methyl ether methacrylate, and/or butyl methacrylate monomers. Adlayers were formed following a simple dip-and-rinse protocol using diluted aqueous polymer solutions and were characterized by evaluating their thicknesses with variable angle spectroscopic ellipsometry (VASE) and their elemental compositions with X-ray photoelectron spectroscopy (XPS). The same techniques were used to determine changes of the adlayer as a function of exposure to electrolytes at different pH values and to monitor nonspecific protein adsorption upon serum exposures. The results indicated that the poly(alkyl-phosphonate)-based adlayers combine multiple site attachment of phosphonic groups and presentation of PEG side chains to the aqueous environment, resulting in both improved stability over a wide pH range in comparison to the tested reference surfaces and excellent resistance to protein adsorption when exposed to full human serum.

Staphylococcus aureus adhesion to titanium oxide surfaces coated with non-functionalized and peptide-functionalized poly(L-lysine)-grafted-poly(ethylene glycol) copolymers.

Implanted biomaterials are coated immediately with host plasma constituents, including extracellular matrix (ECM); this reaction may be undesirable in some cases. Poly(L-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG) has been shown to spontaneously adsorb from aqueous solution onto metal oxide surfaces, effectively reducing the degree of non-specific adsorption of blood and ECM proteins, and decreasing the adhesion of fibroblastic and osteoblastic cells to the coated surfaces. Cell adhesion through specific peptide-integrin receptors could be restored on surfaces coated with PLL-g-PEG functionalized with peptides of the RGD (Arg-Asp-Gly) type. To date, no study has examined the effect of surface modifications by PLL-g-PEG-based polymers on bacterial adhesion. The ability of Staphylococcus aureus to adhere to the ECM and plasma proteins deposited on biomaterials is a significant factor in the pathogenesis of medical-device-related infections. This study describes methods for visualizing and quantifying the adhesion of S. aureus to smooth and rough (chemically etched) titanium surfaces without and with monomolecular coatings of PLL-g-PEG, PLL-g-PEG/PEG-RGD and PLL-g-PEG/PEG-RDG. The different surfaces were exposed to S. aureus cultures for 1-24h and bacteria surface density was evaluated using scanning electron microscopy and fluorescence microscopy. Coating titanium surfaces with any of the three types of copolymers significantly decreased the adhesion of S. aureus to the surfaces by 89-93% for PLL-g-PEG, and 69% for PLL-g-PEG/PEG-RGD. Therefore, surfaces coated with PLL-g-PEG/PEG-RGD have the ability to attach cells such as fibroblasts and osteoblasts while showing reduced S. aureus adhesion, resulting in a selective biointeraction pattern that may be useful for applications in the area of osteosynthesis, orthopaedic and dental implantology.

RGD-containing peptide GCRGYGRGDSPG reduces enhancement of osteoblast differentiation by poly(L-lysine)-graft-poly(ethylene glycol)-coated titanium surfaces.

Osteoblasts exhibit a more differentiated morphology on surfaces with rough microtopographies. Surface effects are often mediated through integrins that bind the RGD motif in cell attachment proteins. Here, we tested the hypothesis that modulating access to RGD binding sites can modify the response of osteoblasts to surface microtopography. MG63 immature osteoblast-like cells were cultured on smooth (Ti sputter-coated Si wafers) and rough (grit blasted/acid etched) Ti surfaces that were modified with adsorbed monomolecular layers of a comb-like graft copolymer, poly-(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG), to limit nonspecific protein adsorption. PLL-g-PEG coatings were functionalized with varying amounts of an integrin-receptor-binding RGD peptide GCRGYGRGDSPG (PLL-g-PEG/PEG-RGD) or a nonbinding RDG control sequence GCRGYGRDGSPG (PLL-g-PEG/PEG-RDG). Response to PLL-g-PEG alone was compared with response to surfaces on which 2-18% of the polymer sidechains were functionalized with the RGD peptide or the RDG peptide. To examine RGD dose-response, peptide surface concentration was varied between 0 and 6.4 pmol/cm(2). In addition, cells were cultured on uncoated Ti or Ti coated with PLL-g-PEG or PLL-g-PEG/PEG-RGD at an RGD surface concentration of 0.7 pmol/cm(2), and free RGDS was added to the media to block integrin binding. Analyses were performed 24 h after cultures had achieved confluence on the tissue culture plastic surface. Cell number was reduced on smooth Ti compared to plastic or glass and further decreased on surfaces coated with PLL-g-PEG or PLL-g-PEG/PEG-RDG, but was restored to control levels when PLL-g-PEG/PEG-RGD was present. Alkaline phosphatase specific activity and osteocalcin levels were increased on PLL-g-PEG alone or PLL-g-PEG/PEG-RDG, but PLL-g-PEG/PEG-RGD reduced the parameters to control levels. On rough Ti surfaces, cell number was reduced to a greater extent than on smooth Ti. PLL-g-PEG coatings reduced alkaline phosphatase and increased osteocalcin in a manner that was synergistic with surface roughness. The RDG peptide did not alter the PLL-g-PEG effect but the RGD peptide restored these markers to their control levels. PLL-g-PEG coatings also increased TGF-beta1 and PGE(2) in conditioned media of cells cultured on smooth or rough Ti; there was a 20x increase on rough Ti coated with PLL-g-PEG. PLL-g-PEG effects were inhibited dose dependently by addition of the RGD peptide to the surface. Free RGDS did not decrease the effect elicited by PLL-g-PEG surfaces. These unexpected results suggest that PLL-g-PEG may have osteogenic properties, perhaps correlated with effects that alter cell attachment and spreading, and promote a more differentiated morphology.

Peptide functionalized poly(L-lysine)-g-poly(ethylene glycol) on titanium: resistance to protein adsorption in full heparinized human blood plasma.

The graft copolymer poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) and its RGD- and RDG-functionalized derivatives (PLL-g-PEG/PEG-peptide) were assembled from aqueous solutions on titanium (oxide) surfaces. The polymers were characterized by NMR in order to determine quantitatively the grafting ratio, g (Lys monomer units/PEG side chains), and the fraction of the PEG side chains carrying the terminal peptide group. The titanium surfaces modified with the polymeric monomolecular adlayers were exposed to full heparinized blood plasma. The adsorbed masses were measured by in situ ellipsometry. The different PLL-g-PEG-coated surfaces showed, within the detection limit of the ellipsometric technique, no statistically significant protein adsorption during exposure to plasma for 30 min at 22 degrees C or 37 degrees C, whereas clean, uncoated titanium surfaces adsorbed approximately 350 ng/cm2 of plasma proteins. The high degree of resistance of the PEGylated surface to non-specific adsorption makes peptide-modified PLL-g-PEG a useful candidate for the surface modification of biomedical devices such as implants that are capable of eliciting specific interactions with integrin-type cell receptors even in the presence of full blood plasma. The results refer to short-term blood plasma exposure that cannot be extrapolated a priori to long-term clinical performance.

[In vitro experimentation of a new model of radial flow bioreactor containing isolated hepatocytes]. / Sperimentazione in vitro di un nuovo modello di bioreattore a flusso radiale contenente epatociti isolati.

Hepatocyte based artificial liver support systems are under investigation to support acute liver failure patients. The main purpose of such systems is to serve as a bridge to liver transplantation, or to promote spontaneous liver recovery. Limitation in mass-transfer capacity makes hollow-fiber bioreactors unsuited for long-term functioning of hybrid devices. We developed a novel radial-flow bioreactor in which the fluid perfuses the module from the center to the periphery, after having diffused through a space occupied by a three-dimensional structure filled with the hepatocytes. Five grams of freshly isolated porcine hepatocytes were seeded into uncoated, woven-non woven, hydrophilic polyester fabric, overlaid by two polyethersulfone membranes. Liver cells were perfused with 37 degrees C-warm, oxygenated, serum-free tissue culture medium, in which NH4Cl and Lidocaine were added at the final concentration of 1 mM and 60 micrograms/ml, respectively. Ammonium chloride removal, urea synthesis, monoethylglycinexylide (MEGX), pO2, pCO2, and pH were measured throughout the 14 day duration of the study. In a separate set of experiments, a scaled-up version of the radial flow bioreactor containing 150 grams of cells was perfused for 7 h with recirculating human plasma and MEGX production was monitored. During the 2 weeks of the study, an increasing production of urea was paralleled by constant ammonium removal. MEGX concentration after Lidocaine addition increased throughout the 14 days of perfusion with tissue culture medium, as well as after 7 hour perfusion with human plasma. Under transmission and scanning electron microscopy cells appeared attached to the polyester and one to each other, displaying ultrastructural features typical of functioning hepatocytes. Our study showed that liver cells were metabolically active when perfused into the radial-flow bioreactor. This configuration allowed close contact between media, or plasma, and cells at a physiological flow rate, by equalizing the concentration of the perfusing components, including O2, throughout the module. Our results suggest a potential use of this system for temporary extracorporeal liver support in acute hepatic failure patients.

[Transplantation of the left lung in a 2-month-old piglet. The Study Group for Lung Transplantation]. / Il trapianto di polmone sinistro nel suinetto di due mesi. Gruppo di Studio per il Trapianto di polmone.

Experimental surgery in animals remains an irreplaceable model for the clinical application of a new technique. We performed a single lung allotransplantation in young pigs. The pigs were organized into groups: 1) 6 to assess the surgical anatomy; 2) 10 to receive left transplantation; 3) 3 to study the pathophysiology of the transplanted lung. The preliminary results demonstrated that the surgical model is useful.

[Vecuronium bromide in pediatric anesthesia]. / Bromuro di vecuronio in anestesia pediatrica.

Ninety patients were included in a study to assess the clinical characteristics of vecuronium bromide used in children. The myorelaxant was administered to all patients using different routes. The use of vecuronium at a dose approximately equal to 1ED95 was characterised by a duration of action sufficient to allow its use in short operations; on the other hand, it also produced a long induction-intubation interval and not optimal conditions in which to perform intubation. Conditions for intubation improved during induction via inhalation and there was a reduced induction-intubation interval compared to intravenous induction using the same dose of vecuronium. A further reduction in intubation time was obtained by increasing the dose from 50 to 150 micrograms/kg-1 together with an increased clinical duration of action. The "priming principle" technique also allowed intubation time to be shortened without variations in the duration of action provided a full dose of vecuronium, 100 micrograms/kg-1, was used. However, this was also associated with a notable incidence of adverse reactions. Of the various combinations examined, the most advantageous association of pre-dose and interval between doses was the association of a pre-dose of 10 micrograms/kg-1 and an interval of 4 min between doses. Lower doses countered the advantages of priming, whereas higher doses resulted in an increased number of adverse reactions without producing notable changes in the intubation time.

[Microbiologic flora in two different periods in intensive care units]. / Indagine microbiologica in due diversi periodi di degenza in rianimazione.

Bacterial examinations performed in two periods (Nov. 1985-Feb. 1986) and Nov. 1988-Feb. 1989) on materials taken from patients admitted into two similar Intensive Care Units (ICU) of the same hospital were evaluated. Excluding oropharyngeal swab cultures, which were performed only in the second period, Gram positive bacteria were 45.3% in 1985-1986 and 62.3% in 1988-1989 and Gram negative fell from 41.9% to 29%. The percentage of fungi remained unchanged. Microorganisms did not grow in 33.3% of tracheal aspirate specimens in 1985-1986 and in 54.3% in 1988-1989, probably depending on the larger number of patients with decompensated chronic respiratory failure treated in the second period. Usually the microorganisms found in the trachea was also in the oropharynx; Gram negative were only 5.1% of the microorganism collected in the oropharinx. The authors emphasize the increase of Gram positive bacteria in ICUs and suggest that the low rate of pneumonia observed in this study depends mostly on the good neurologic state of their patients.

[Pollution levels of the anesthetic agent fluorurate (gas chromatography study)]. / Tassi di inquinamento da agente anestetico fluorurato (Indagine gas-cromatografica).

Using a simple, highly sensitive technique, the rate of pollution due to enflurane in an operating theatre with two beds and two respirators but without an air exchange system has been measured. After much debate and after seven serious cases of viral hepatitis among medical staff, the facility was closed because it was judged to be unusable and because the surgical department using it was moved elsewhere.

Disorders in peripheral arterial system in asymptomatic elderly: plethysmographic semiology at rest, during postural, effort and pharmacological tests.

The vascular system of 30 old asymptomatic patients (average age 62.5 years) was studied by reflection plethysmography. The plethysmogram (PTG) was recorded from the forefinger of the left hand at rest, during two postural tests (+45 degrees arm-up and -45 degrees arm-down), after physical work, and during the infusion of nitroglycerin and the beta-agonist metaproterenol. 50% of the subjects presented a normal PTG under basal conditions. However, pathological PTGs were recorded after the various tests: 23.3% with the arm-up test, 6.6% with the arm-down test, 52% with the effort test, 31% with the nitroglycerin test and 73.9% with the metaproterenol test. Clearly, the effort and metaproterenol tests are pathological in the majority of cases, both tests inducing physiologically vasodilation and increase in the peripheral pulse. The arm-down test, which usually induces vasoconstriction, is almost always normal. Since the percentage of pathological responses to the nitroglycerin test is significantly smaller than that to the effort and metaproterenol tests, it is concluded that the vascular changes induced by aging are primarily functional, at least in asymptomatic subjects. Aging more negatively influences the vasodilating than the vasoconstricting ability.