Plasma-polymerized surfaces for culture of human keratinocytes and transfer of cells to an in vitro wound-bed model.

The aim of this study was to develop plasma-polymerized surfaces suitable for the attachment and culture of human keratinocytes and that would allow their subsequent transfer to a wound-bed model. Keratinocyte attachment has been assessed on a carrier polymer, either untreated or treated with a hydrocarbon plasma polymer, collagen I, or carboxylic-acid-containing plasma copolymers. Cell attachment was poor on the "bare" carrier polymer and hydrocarbon plasma polymer (PP) surfaces. Cell attachment was good and comparable on collagen I-coated carrier polymer and carrier polymer plasma coated with carboxylic acid functionalities. After 24 h of cell culture, surfaces were inverted so that cells were adjacent to a de-epidermalized dermis (DED) for 4 days. After 4 days in contact with DED, the surfaces were removed and the level of residual cells and cells transferred to DED were assessed using a cell viability assay. Cell transfer from the collagen I-coated surface was on the order of 90%. Transfer from the carrier polymer surface and the hydrocarbon-coated surface was poor while cells cultured on acid-containing surfaces showed high levels of transfer. Cell transfer was greatest from those surfaces containing the highest level of acid functionality (ca. 21%). Cell transfer was not significantly affected by the choice of carrier polymer material although some sample-to-sample variation was seen. To determine that plasma-polymerized surfaces could be used clinically, selected samples were sterilized with ethylene oxide. Subsequent analysis and cell culture indicated that the surface chemistry and cell-transfer capability of these plasma-polymerized surfaces were unaffected by the sterilization procedure. Plasma-polymerized carboxylic-acid-containing surfaces show great promise in the field of wound healing, encouraging keratinocyte attachment and permitting keratinocyte transfer to a wound bed.

XPS analysis of the surface of leucite-reinforced feldspathic ceramics.

OBJECTIVE: The bond of a silane-coupling agent to a ceramic surface is expected to be influenced by the composition and chemical state of the ceramic surface. The purpose of this study was to determine the variation in the composition and the chemical states of the surfaces of a range of leucite-reinforced feldspathic (LRF) ceramics using X-ray photoelectron spectroscopy (XPS). METHODS: Five LRF ceramic discs (IPS Empress,Optec HSP,VMK 68, Mirage, and a modified Mirage) were produced and polished to a 1 microm finish. A further nine discs of the modified Mirage were produced. The discs were stored for 48h in a vacuum oven at 110 degrees C to remove absorbed water. The surfaces of these discs were analysed by XPS. Survey scans at 30 degrees take-off angle were taken and surface composition (in at%) was calculated from the narrow scans for Si 2p, O 1s, Al 2p, Mg 2s, K 2p, Na 1s, Ca 2p and N 1s. RESULTS: Atomic concentration of elements (after exclusion of C) for the five LRF ceramics were in the range: O, 45.0-51.6%; Si, 26.7-35.6%; Al, 6.3-9.7%; Mg, 4.9-8.8%; K, 0.5-2.2%; N, 0.9-2.9% and less than 1% of Na and Ca. The shapes of the O 1s and Si 2p narrow scan core lines of the five LRF ceramics were virtually identical. SIGNIFICANCE: All five LRF ceramics were found to have a silica-rich surface layer due to a reduction in K and Na relative to the bulk composition. Both the composition and chemical states of the surfaces for the five LRF ceramics were very similar.

Comparison of proliferation and growth of human keratinocytes on plasma copolymers of acrylic acid/1,7-octadiene and self-assembled monolayers.

Human keratinocytes were cultured on plasma copolymers (PCPs), self-assembled monolayers (SAMs), and tissue culture poly(styrene) (TCPS). Plasma copolymerization was used to deposit films with controlled concentrations of carboxylic acid functional groups (<5%). Human keratinocytes were cultured onto these PCP surfaces, TCPS, and collagen I. A hydrocarbon plasma polymer surface was used as the negative control. Keratinocyte attachment was measured at 24 h and cell proliferation and growth at 3 and 7 days using optical microscopy and DNA concentrations. The PCP surfaces were compared with two SAM systems comprising pure acid and pure hydrocarbon functionalities, and pure gold was used as a control surface. PCP surfaces containing carboxylic acid functionalities promoted keratinocyte attachment. The level of attachment on these surfaces was comparable to that seen on collagen I, a preferred substratum for the culturing of keratinocytes. After several days in culture the cells were well attached and proliferative, forming confluent sheets of keratinocytes. This result was confirmed by DNA assays that suggested the acid PCP surfaces were performing as well as collagen I. Keratinocytes attached well to gold and acid-terminated SAMs but attached poorly to methyl-terminated SAMs. The acid functionality also promoted proliferation and growth of keratinocytes after several days in culture. DNA assays revealed that keratinocyte growth on the acid surface was higher than on collagen I.

Surface chemistry of a high-copper dental amalgam.

In amalgam, mercury is intended to take the form of stable intermetallic compounds. Any mercury leakage must therefore come from free mercury not involved in such compounds. Thus, a knowledge of the exact surface chemistry of dental amalgam is necessary if this phenomenon is to be understood. From XPS and EDS analyses, a model of the surface chemistry of amalgam is proposed which fully characterizes all the phases that are present. The data show the surface to have a composition different from that of the bulk, being comprised of a hydrocarbon deposit and adsorbed water covering the intermetallic phase gamma2 (Sn(6-8)Hg), tin (iv) oxide, and mercury in a free state. After amalgamation, the amount of mercury at the surface decreases with time and eventually attains a constant concentration, where it is all involved in the gamma2 phase, leaving no free mercury. A model is proposed for the surface of amalgam and the changes in this model with time.

Plasma copolymer surfaces of acrylic acid/1,7 octadiene: surface characterisation and the attachment of ROS 17/2.8 osteoblast-like cells.

The purpose of this study was: (a) to examine the effect of plasma-gas composition on plasma polymer oxygen/carbon (O/C) ratio, functional group composition and stability in water, and then (b) to examine cell attachment to surfaces containing different concentrations of O/C and functional groups. Oxygen-functionalised surfaces were deposited by means of the plasma copolymerisation of acrylic acid/1,7-octadiene. The use of a diluent hydrocarbon allowed the deposition of surfaces with a range of O/C concentrations. Plasma copolymer surfaces were characterised by X-ray photoelectron spectroscopy (XPS). Changes in functional group composition with % acrylic acid monomer and the non-dispersive and dispersive parts of the surface energy of these plasma copolymers were measured. The solubility of the plasma copolymers was assessed by means of XPS. The degree of attachment of ROS 17/2.8 osteoblast-like cells to plasma copolymer surfaces deemed to be 'stable' in aqueous medium was measured. Tissue culture polystyrene (TCPS) was included as a control. Attachment was found to be greatest to the plasma copolymer surface with an O/C of 0.11. This surface had a carboxylic acid concentration of ca. 3%. Attachment did not correlate with increased surface wettability (i.e. the non-dispersive component of the surface energy).

Synthetic implant surfaces. 1. The formation and characterization of sol-gel titania films.

Sol-gel has been used to prepare thin titania films. We have investigated the effects of dip rate, sintering temperature and time on the chemical composition of the films, their physical structure and thickness, and adherence to a silica substrate. Our aim has been to produce films that mimic as closely as possible the natural oxide layer that is found on titanium. These films are to be used as substrates in an in vitro model of osseointegration.

Cytotoxicity of ethylene oxide/propylene oxide copolymers in cultured mammalian cells.

Cytotoxicity was measured in vitro for 8 ethylene oxide/propylene oxide copolymers (EO/PO copolymers) using lactate dehydrogenase release from cultured mammalian cells as the endpoint. Three cell types were used in these assays: Chinese hamster ovary cell line (AS52), rat lung epithelial cell line (LEC), and freshly isolated rat alveolar macrophages (RAM). A range of cytotoxicity was seen with toxic effects observed from 20 to > 20,000 micrograms/ml. The same relative order of toxicities were observed for all 3 cell lines although RAM cells appeared to be somewhat more sensitive. The in vitro cytotoxicity, as measured by LDH release and microscopic observations of the cells, correlated poorly with the in vivo inhalation toxicity. The most lethal compounds following acute inhalation (UCON 50-HB-5100 and UCON 50-HB-2000) were among the least toxic in the in vitro cytotoxicity screen. Conversely the 2 compounds which were the most toxic in vitro (Pluronic 17 R1 and Pluronic L64) did not produce any unusual degree of toxicity in inhalation studies. The results of these experiments indicate that these in vitro mammalian cell assays will not be useful, at least for these classes of chemistry, in prediction of in vivo inhalation toxicity.

The surface analysis of implant materials. 1. The surface composition of a titanium dental implant material.

The surfaces of titanium (Ti) plates, as models for Ti implants, have been characterized by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF SIMS). Plates were prepared with rough and smooth surface topographies--the rough being similar to that of an implant. The XPS data has been used to construct a model of the plates' surface chemical structure, from the gas-solid interface through to pure Ti metal. At the surface of as-received plates, which underwent the same preparative procedure as an implant, considerable surface contamination was detected. In particular, high levels of carbon (C) contaminants were detected; the nature of this C was elucidated by fitting the C 1s core line and from the secondary ion mass spectra. The oxygen (O) 1s core line could not be fitted using a minimum of 2 gaussian peaks, demonstrating the multiplicity of O environments. The detection of other elements in the XPS analysis further demonstrated that, in nominally pure Ti plates, the surface chemical composition deviates considerably from that of the bulk. The data obtained from the plates were confirmed by examination of a Ti abutment. The handling of Ti plates with stainless steel tweezers was investigated. No obvious change in surface chemistry was detected. All the above results bring into serious questions the validity of rigorous protocols demanded, in some techniques, in the handling and use of Ti implants.

Structure-activity relationship of a series of sensory irritants.

The relative potencies for a series of sensory irritants, with structures based on toluene, were determined by measuring the airborne concentrations that caused a 50% decrease in respiratory rate in Swiss-Webster mice. This concentration is referred to as the RD50. Toluene, a relatively nonirritating compound, and compounds with chlorine, two chlorines, bromine, and iodine atoms substituted on the alpha carbon of toluene were tested. The RD50s for these compound types were determined to be 4900, 27, 27, 5.2, and 4.3 ppm, respectively. In addition, compounds with chlorine substituted at the ortho, meta, and para positions on the toluene ring were also tested. The RD50s were determined to be 4.9, 13, and 14 ppm, respectively. The structure-activity relationships of the compounds studied are explained by a model (Abraham et al., 1990; Nielsen and Alarie, 1982) that relates the interaction of sensory irritants with a receptor protein in a lipid bilayer. The trends in the RD50s, and thus sensory irritation, for the compounds studies are related to the development of a partial positive charge on the toluene alpha carbon by the positioning of a ring chlorine and the bond dissociation energies of the alpha carbon-halogen bond for the iodo, bromo, and chloro isomers of benzyl halide.

A two-generation reproduction study in rats receiving diets containing hexamethylenediamine.

Rats received diets containing average daily doses of 0, 50, 150, and 500 mg/kg/day of hexamethylenediamine over two generations. Although no treatment-related mortality was observed in any of the groups, the weight gain of adults and pups was slightly reduced in the high dose group. While the litter size was also slightly reduced at birth in the high dose group, there was no adverse effect on survival during lactation in any of the treated groups. Thus, the dietary administration of up to 150 mg/kg/day of hexamethylenediamine over two generations did not adversely affect reproduction or fertility in rats.

13-week inhalation toxicity study and fertility assessment in rats exposed to atmospheres containing adiponitrile.

Adiponitrile is an aliphatic dinitrile that is used as an intermediate in the synthesis of hexamethylenediamine. In order to asses potential health effects associated with industrial exposure, rats were exposed 6 h/d, 5 d/wk for 4 or 13 wk to atmospheres containing a range of adiponitrile concentrations and observed for signs of toxicity. A fertility assessment was also included as a component of the 13-wk study. Mortality and reduced weight gain were observed within 1 wk only in rats exposed to 493 mg/m3. Evidence of slight anemia was present in rats exposed to 99 mg/m3 and above. There was no histopathological evidence of organ toxicity in about 30 tissues from both sexes exposed up to 99 mg/m3, the highest concentration tested, for 13 wk. In addition, fertility, as monitored by reproductive performance and litter parameters, was normal in both males and females similarly exposed.

XPS and SSIMS analysis of the surface chemical structure of poly(caprolactone) and poly(beta-hydroxybutyrate-beta-hydroxyvalerate) copolymers.

The surface chemical structures of poly(beta-hydroxybutyrate), poly(caprolactone) and poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) have been analysed using static secondary ion mass spectrometry and X-ray photoelectron spectroscopy. The X-ray photoelectron spectroscopy data confirm the purity of the polyester surfaces and there is close agreement between the stoichiometric and experimentally determined ratios of the peaks and different carbon environments within the C1s envelopes. The static secondary ion mass spectrometry analysis reveals general fragmentation pathways which permit the ready distinction between the different polyesters examined. The differentiation of the different monomer repeat units in the copolymer together with the detection of some ions representative of the random copolymer sequence are also possible in the static secondary ion mass spectrometry analysis.

A 6-month multispecies inhalation study with maleic anhydride.

This study was initiated to assess the safety of atmospheres containing maleic anhydride. Accordingly, rats (15/sex/group), hamsters (15/sex/group), and monkeys (3/sex/group) were treated 6 hr a day 5 days a week for 6 months. Atmospheres were generated by subliming maleic anhydride and were monitored using Tenax collection columns and gas chromatography to detect total maleic; i.e., maleic anhydride plus maleic acid. The mean analytical concentrations were 0, 1.1, 3.3, and 9.8 mg/m3 of total maleic. Dose-related signs of nasal and ocular irritation were observed at each test level in all three species; signs included discharge, sneezing, gasping, and coughing. No significant treatment-related mortality was observed in any species. While reduced weight gains were observed only in mid- and high-dose rats, their terminal body weights were greater than 90% of control values. No treatment-related effects were observed in hematology, clinical chemistry, urinalysis, and pulmonary function tests. Although microscopic evaluation of tissue revealed evidence of nasal irritation in all species, there was no evidence of systemic toxicity which was directly attributed to maleic anhydride. While the results of this study support the current ACGIH TLV and OSHA PEL of 1 mg/m3 regarding systemic toxicity, continuous exposure at this level during the day may produce some signs of irritation.

Metabolic and peroxisome proliferation studies with di(2-ethylhexyl)phthalate in rats and monkeys.

Male Fischer 344 rats and cynomolgus monkeys were treated with various doses of di(2-ethylhexyl)phthalate (DEHP) for at least 21 days. There was metabolic, biochemical, and morphological evidence for peroxisomal proliferation in rats that consumed diets containing 1000 ppm DEHP and above. These diets were estimated to provide average daily doses of about 100 mg/kg of DEHP. In contrast, peroxisomal proliferation was not observed in monkeys that received up to 500 mg/kg/day of DEHP by gavage. The results of this study suggest that rats do not provide a good model for predicting the results of DEHP exposure on peroxisomal proliferation in higher primates.

Further evaluation of an in vivo teratology screen.

The in vivo teratology screening procedure described previously [Chernoff N, Kavlock RJ: J Toxical Environ Health 10:541-550, 1982] was further evaluated using a total of 46 chemicals in 50 different treatment regimens. Pregnant CD-1 mice were generally treated by oral gavage on days 8-12 of gestation at a dose level predicted from a preliminary range finding study to induce a slight degree of maternal toxicity. The effects on early postnatal growth and viability were compared to results generated from standard mouse teratology bioassays as reported in the literature (there were nine regimens for which no valid comparisons could be made). The procedure correctly categorized 25 of the 30 treatment regimens which were considered developmentally toxic in the mouse, as well as nine of 11 which were considered to be nondevelopmentally toxic in the mouse. Thus, based upon the criteria used in the present study, the assay correctly classified 83% of the chemicals tested as to their effect in a standard mouse bioassay. The five nonconcurring negative findings were likely due to a combination of pharmacokinetic differences between the studies, as well as to the cessation of dosing on day 12, while critical events of organogenesis are still occurring. The assay achieves the requirements for a teratology screening system, but improved predictability would result from the addition of a lower dose level and extension of the dosing period to include later stages of organogenesis.

Teratology and multigeneration reproduction studies with maleic anhydride in rats.

These studies were initiated to evaluate the effects of maleic anhydride on development and reproduction in CD rats. In the teratology study, pregnant rats (19-23/group) received 0, 30, 90, or 140 mg/kg/day maleic anhydride in corn oil orally from Days 6-15 of gestation and fetuses were examined for gross soft tissue and skeletal defects. A reduced weight gain or weight loss was observed in all maleic anhydride-treated groups between Days 6 and 9; however, mean weights of all groups were within 5% of control on Days 15 and 20. No treatment-related effects on fetal development were observed. In the multigeneration study, rats (10 males and 20 females/group) received 0, 20, 55, or 150 mg/kg/day maleic anhydride in corn oil orally and were mated to produce two generations, each with two litters. Groups of the same size from the second litter were used for subsequent generations and were given the same dose of maleic anhydride as were their parents. The high-dose group was terminated during the second generation due to treatment-related mortality in adults. Renal cortical necrosis occurred in high-dose Fo males and females. Increased kidney weights were observed in low- and mid-dose adult F1 females. No treatment-related effects on reproduction were observed with maleic anhydride at doses up to 55 mg/kg/day over two generations.

Feature extraction using problem localization.

Feature extraction is considered as a mean-quare estimation of the Bayes risk vector. The problem is simplified by partitioning the distribution space into local subregions and performing a linear estimation in each subregion. A modified clustering algorithm is used to fimd the partitioning which minimizes the mean-square error.

Developmental toxicity of guthion in rats and mice.

The purpose of this study was to assess the effects of Guthion, a pesticide with anticholinesterase activity, on development in rats and mice. A preliminary toxicity study with Guthion indicated that a 35-day LD50 dose for virgin rats and a 10-day LD50 dose for virgin mice was between 4 and 8 mg/kg/day for both species. On the basis of these data, doses of 0, 1.25, 2.5, and 5.0 mg/kg/day were selected for the developmental study, which consisted of two phases. During the first phase, pregnant rats and mice were treated for 10 days starting on gestational day 6. The high dose affected maternal welfare only in rats. Guthion did not significantly increase in a dose-related manner any of the specific anomalies observed in either rats or mice. During the second phase, pregnant rats were treated from gestational day 6 to postpartum day 21. Dams in the high dose group were more sensitive to Guthion later in gestation with the result that deaths and signs of anticholinesterase toxicity increased during this time. Guthion also adversely affected maternal welfare in this group. As a result of Guthion toxicity, only one litter survived until weaning. The inability to dissociate toxicity in adult and developing animals suggests that Guthion has little primary effect on the development of rats or mice.

Effect of some nitrotoluenes on the biotransformation of xenobiotics in rats.

Male rats were treated with phenobarbital, a dinitrotoluene (2,4-DNT or 2,6-DNT), or 2,4,6-trinitrotoluene (2,4,6-TNT); and the biotransformation of model xenobiotics was determined. Phenobarbital produced a stimulation, and 2,6-DNT produced a biphasic effect in biotransformation.