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Palladium (Pd) is a metal frequently used for dental alloys. In order to elucidate controversial options about Pd concerning its biological performances, our study consists in the evaluation of commercial and experimental PFM and C&B precious and semi-precious dental alloys. This investigation was also designated to the establishment of a cytotoxicity index (CI) such as it was described for hemocompatibility testing. The following materials were tested: 36 commercial alloys (Au-, Pd- and Ag-base), 14 experimental alloys (Pd-base established by an experience plan) and pure metals (Ag, Au, Cu, Ni, Cr, In, Sn, Pt, Ti, Zn). The cells culture experiments were carried out with epithelial L132 cells and NIH 3T3 fibroblasts. In vitro cell viability tests show that Pt, Sn, In, Ti, Au and Pd have no cytotoxic effect; Cr, Cu and Ag are toxic, Ni, Zn, and Co are highly toxic. An identical ranking was found with the inflammatory and proliferation tests. Toxic and highly toxic metals induced slight or strong prosthetic dental restoration morphological alterations after 3-days cultures and mostly cell death after 6-days cultures. These effects are dependent on the leakage of the element into the culture medium as revealed by ICP. The addition of Au gives benefit to Pd-Ag alloys, but does not produce any major effect on Pd-Cu alloys. This qualitative ranking can quantitatively be confirmed by cytocompatibility testing after application of a CI.

We report a case of malignant fibrous histiocytoma of the bone that developed 20 years after a femoral fracture treated by plate-screw fixation. Similar cases reported over the past fifteen years in the literature suggest the possible mechanisms of sarcomatous degeneration. The possible carcinogenic effect of corrosion products is emphasized. Dispersion energy spectrometry of intracellular particles on the periphery and at the center of the tumor demonstrated the presence of chromium, iron and nickel at different concentrations. The association with other elements clearly demonstrates that the corrosion products were metabolized. The presence of metallic components in tumoral cells suggests a possible relationship between metallic implants and malignancy. These observations emphasize the importance of creating a national, or even international, registry of malignant tumors that develop in contact with metallic implants in order to search for a possible cause and effect relationship.

Proliferation and adhesion of mouse (MC3T3-E1) osteoblastic cells and primary human osteoblastic cells were carried out on Ti6Al4V titanium alloy samples with varied surface roughnesses. Mechanically or manually polished surfaces were prepared to produce respectively non-oriented or oriented residual polishing grooves. Sand-blasted surfaces were prepared using 500 microm or 3 mm alumina particles. Surface roughness parameters showed a negative correlation in comparison to proliferation and adhesion parameters. X-ray microprobe chemical surface microanalysis showed complete disturbance of the surface element composition of the Ti6Al4V alloy following sand-blasting treatment. An AlOx-enriched layer was observed on sample surfaces. This may lead to the suspicion that the concomittant effect of surface roughness amplitude and AlOx surface concentration has an effect on osteoblastic cell proliferation and adhesion. These findings show the significance of chemical surface analysis after any surface treatment of titanium-based implants before any biological use.

Background: Chédiak-Higashi syndrome (C-HS) is a rare congenital disease characterized by defective neutrophil function with abnormal lysosomal inclusions, neutropenia, and reduced chemotaxis. The complete syndrome includes oculocutaneous albinism with photophobia, neurologic features, recurrent infections, and enterocolitis., Methods: A 14-year-old male C-HS patient was referred to us because of serious periodontal destruction with acute inflamed gingiva and ulcers. Clinical and biological investigations were performed, leading to the diagnosis of C-HS., Results: Laboratory findings included neutropenia and hypergammaglobulinemia. Peripheral blood smears showed giant granules in neutrophils, eosinophils, and granulocytes. Bone marrow smears showed giant inclusions in leukocyte precursor cells. These granules and inclusions were characteristic of Chédiak-Higashi syndrome. Oral radiographic status showed extensive loss of alveolar bone leading, in most cases, to tooth exfoliation. Bacteria often associated with periodontitis were detected in subgingival plaque samples, including Fusobacterium nucleatum, Campylobacter rectus, Prevotella melaninogenica, Peptostreptococcus anaerobius, and Clostridium sp. Biopsies of periodontal tissues for light and electronic microscopic examinations revealed massive bacterial invasion of the epithelial tissue, epithelial cells, and connective tissue. Ultrastructural observations of periodontal polymorphonuclear leukocytes showed defective granulation, with abnormal granules not discharging their lysosomal content against engulfed bacteria. Viable dividing bacteria were found in the cytoplasm., Conclusions: In this case, early-onset periodontitis seems to be the expression of C-HS granulocyte deficiency. Periodontal treatment of these patients is often unsuccessful. This case report illustrates the importance of the dentist in initiating clinical and biological investigations in such early aggressive periodontitis in young patients.

In order to develop bone substitutes, the design of biomaterials like calcium phosphate ceramic loaded with bone growth factor are of great interest. However, it is necessary to control the amount of growth factor adsorbed onto ceramics and the kinetics of its release. Radiolabeling of insulin-like growth factor-I (IGF-I) with 125-iodine ([(125)I]-IGF-I) and its adsorption onto porous tricalcium phosphate (TCP) cylinders enabled us to establish the time-adsorption and time-release curves using various concentrations of IGF-I. The adsorption curve increased rapidly and then flattened out at 72 h; 90% of the maximum was already reached at 24 h; and 20% of the adsorbed IGF-I was released in water within 4 days. In human serum the release was faster at 82% within 4 days. In vivo evaluation on an animal model was then performed. Rabbits' bilateral femoral cylindrical bone defects were filled with the TCP cylinders, which were either carrying IGF-I or implanted alone as a control in each rabbit. Bone turnover and ceramic resorption were stimulated by IGF-I loaded TCP according to standard radiography, dual-energy X-ray absorptiometry, histology, and histomorphometry., (Copyright 2000 John Wiley & Sons, Inc.)

Porous beta-tricalcium phosphate (betaTCP) has osteoconductive properties. The adsorption of human recombinant bone morphogenetic protein-2 (rhBMP-2) onto TCP could realize an osteoinductive bone substitute. We evaluated it on an animal model using dual-energy X-ray absorptiometry (DEXA) and solid-state 31P nuclear magnetic resonance (NMR) spectroscopy. BetaTCP cylinders loaded with rhBMP-2 were implanted into rabbits' femoral condyle bone defects, and betaTCP alone as control into the contralateral femur. We studied two different doses of rhBMP-2 (10 and 40 microg) on two groups of four animals. Evaluation consisted in radiography, histology, and histomorphometry, DEXA, and NMR spectroscopy using an original method of quantification. With both doses of rhBMP-2, we observed on radiographs an increase of trabecular bone around implants. Histology showed resorption of the ceramic, trabecular bone with osteoblasts and osteoid substance around the implants, and colonization inside the porous betaTCP by new bone formed. Histomorphometry showed that the osteoid surface (OS/BS) was greatest with the high dose of rhBMP-2. The difference was slight between the low dose of rhBMP-2 and control. DEXA showed a dose-dependent increase of bone mineral density of rhBMP-2-loaded betaTCP vs. control. NMR spectroscopy confirmed that the amount of new bone formed in betaTCP was greater when betaTCP carried rhBMP-2, and increased with the dose of rhBMP-2 used. We showed that betaTCP was a good matrix for rhBMP-2, which gave it osteoinductive properties in an orthotopic site, in a dose-dependent manner. Thus, such composite biomaterial seems to be of great interest in reconstructive bone surgery. Further studies are needed in clinical practice to determine optimal doses.

The aim of this paper is to present the potentialities of ultrasounds to investigate the mechanical properties of a cell/substrate interface. The adhesion process plays a major role in the development of osteoblastic cells on various substrates used in orthopedic applications such as metals, bioceramics, etc. Particularly, cell adherence appears to be a critical factor in the colonization process. High-frequency and low-power ultrasounds seem to be an appropriate tool for a nondestructive evaluation of interface properties. First, we present the results obtained with bulk longitudinal and shear waves under an arbitrary incidence over an aluminum-adhesive interface. This study was performed for an industrial application of bonding. The results clearly show the sensitivity of shear waves for the evaluation of the adhesion quality owing to the shear solicitations at the interface they induce. A model of ultrasound interactions with a boundary subject to varying degrees of adhesion has been developed and compared to the experiments. Second, we investigated osteoblastic cell cultures with a high-frequency acoustic microscope working at 50 MHz. The images obtained in the shear mode reveal a better contrast than those obtained in the longitudinal mode. For the time being, these results are qualitative, and theoretical models have to be developed according to the point of view of biologists.

The cytoxicity and the 50% lethal concentration (LC50) has been assessed in vitro by the colony forming method in extracts of 5 precious or semiprecious alloys and of oxides and chlorides of the metal contained in these alloys: Pd, Ag, Cu, Zn, Sn, In and Ga. All alloy extracts in culture medium induced cell death depending on their composition. In, Ga and Sn compounds showed the most favorable cell viability, Cu and Ag salts induced the lowest survival rate.

The in vitro tests on cell viability reveal a favorable position for steel and hydroxyapatite (HA)? However, the roughness induces a negative effect on cell proliferation. Sandblasting of the stainless steel considerably decreased cell number compared with the polished substrate. HA showed a better percentage of proliferation in spite of the surface effect compared with controls. For in vivo biocompatibility, intramuscular implants revealed localized inflammatory reactions for the HA treated stainless steel but nor for the untreated alloy.

An in vitro study has been carried out in different cell systems to determine the biological response of TiNb30 alloy before and after a surface treatment with hydroxyapatite (HA) and tricalcium phosphate (TCP) by the sol-gel method. TiNb30 pure Ti induce favorable cell viability with respect to pure Ni showing a high cytotoxic effect. After surface treatment with HA or HA-TCP mixtures, no difference in cell proliferation can be observed between amorphous and cristalline forms. However, HA decreases (75 +/- 15%) and HA-TCP mixtures increase (133 +/- 11%) significantly cell proliferation compared with controls.

The electrochemical behavior in artificial saliva of TiNb30 and TiTa30 alloys were compared with that of commercial pure titanium. The anodic potential, the current density, the passivation potential and the galvanic corrosion vs. Au were determined. Both alloys have a similar behavior to that of pure titanium. Crevace corrosion, which is very weak in pure Ti, is completely inhibited by the addition of Nb or Ta.

Workers are occupationally exposed to Pd and Pd-complexes during the refining process, in particular in Pt-refineries. The metal is increasingly used as component in fine jewellery, is commonly present in dental alloys, and is also employed in telecommunication systems.In view of the toxicity of Pd when absorbed and anticipating the possible entry of some of this material into man's environment from the use of automotive catalitic converters, the retention, tissue distribution, excretion and placental transfer of Pd were determined by several authors following different administration routes in animal experiments. Most investigations on Pd and the Pt group metals were performed with simple and complex salts (chlorides and water-soluble ammoniacal compounds of Pt, Ir, Os, Pd, Rh and Ru). The highest retention for Pd was obtained following intravenous dosing, and the lowest retention occured after oral administration. Following a single oral dose, almost all Pd is excreted in the faeces, whereas after i.v. injections, similar quantities are excreted in both urine and faeces. Tissues containing the highest concentrations were the kidneys, spleen and liver. Following i.v. dosing of pregnant rats, a small amount is found in the fetuses.Pd and its compounds are considered to have hepatotoxic and nephrotoxic effects, acting by way of the -SH groups on complex biosystems: proteins, enzymes, etc. Oral administration of PdCh2 has obvious effects on the hepatic mono-oxygenase system and causes notable decreases in hepatic content of cytochrome-P450. Pd++ salts have also metabolic effects on a lot of other enzymes by inhibiting the activity of prolyl-hydroxylase, creatine kinase, aldolase, succinic dehydrogenase, carbonic anhydrase and alkaline phosphatase. These studies suggest that Pd may interfere with energy metabolism, acidlbase and electrolyte eqUilibria. In addition, Pd significantly increases the hepatic Se content and has a strong interaction within the Se-Hg competition. Proposed exposure limit for Pd compounds is 0.03 mg/m3.It is highly important to make the difference between soluble Pd-salts and metallic Pd. Although simple and complex Pd-salts have obvious hepatotoxic and nephrotoxic effects, no biological incidences have been demonstrated for metallic Pd. This is important especially for its increasing use in dental alloys. Our own experiences on cell culture systems with powders of pure metallic Pd and Pd-containing dental alloys have never shown any influence on the cell viability and on the induction of inflammatory effects. Its biocompatibility is comparable to that of Au, Pt or Ti.In the last few years, however, Pd has caused a lot of controversial discussion with respect to its sensitizing effects. The present state-of-the-art of this problem is that Pd sensitization may coincide with Ni sensitization.

Metal ion release was assessed in 31 patients with loosening of total hip replacements (THR). Three types of alloys were used for these prostheses: stainless steel (9/31), Ni-Cr-Mo (5/31) and Co-Cr (17/31). The exposure periods were from 2 to 15 years.Intracellular deposits were observed in macrophages, fibroblasts, histiocytes and numerous multinucleated giant cells. X-ray microprobe analyses by energy dispersive spectrometry (EDS) on ultrathin sections revealed the presence of elements contained in the alloys (Ni, Cr, Fe, Mo) and of additional elements, in particular P, CI, Ca and S. Co was only detected in wear particles. Metal distribution in tissues and body fluids was related to the alloys used. Stainless steel THR induced the lowest metal concentrations, Ni-Cr-Mo alloys showed high increases of Ni and Cr. Co-Cr alloys induced very high Co levels: 100- to 400-fold concentrations in body fluids and 600- to 1000-fold concentrations in tissues with respect to normal upper levels. The metal clearing was studied in three patients with Co-Cr-THR. Two years after removal, only Ni reached normal values. The sometimes alarming high concentrations should lead to a systematic follow up and surveillance of patients.

The biological performance of titanium (Ti) particles has been investigated in vitro on murine peritoneal macrophages in a primary culture system. The ultrastructural study revealed an unchanged morphology with respect to controls and the presence of numerous phagocytic vacuoles containing Ti particles as confirmed by X-ray microprobe analyses. The activities of beta-glucuronidase, acid phosphatase, lactate dehydrogenase and glucose-6-phosphate dehydrogenase were determined. All of the enzymes were found to be activated after different exposure times to various Ti concentrations. These activations are qualified as the consequence of cell defence rather than a significant cytotoxic effect. Nevertheless, they indicate a possible inflammatory action of short duration. This investigation provides new arguments for the high biological performance of Ti.

Many materials are used in bone reconstruction. According to their physico-chemical structure, their activity is based on one of the three main mechanisms of bone repair: osteogenesis, osteoconduction, osteo-induction. The materials can be classified in two categories: tissues and substitution biomaterials. Tissues may be living ones (mainly, autograft) or non living ones (mainly, allograft or xenograft). Among the substitution biomaterials, we find synthetic materials (calcium based ceramics, vitreous ceramics and bioglasses, polymers) and composite materials made of a mixture of synthetic and natural materials or a mixture of different synthetic materials or a mixture of different synthetic materials. At last, membranes provide a new technic in bone reconstruction. They come from natural origin (human or animal) or synthetic origin (resorbable or non resorbable).

In the present paper, the authors call in mind the definitions of biocompatibility and the essential qualities required for biomaterials. The materials mostly used in implantology and maxillofacial surgery are numerous alloys, bioceramics, resorbable and non-resorbable polymers, and finally osseous substitutes of human or animal origin. As to synthetic and non-living materials, the risks in patients are generated by the degradation products. These may induce tissular reactions of inflammatory or immune origin owing to toxic effects. Concerning osseous substitutes, rejections are mostly of immune origin, for allografts and in particular for xenografts. Infections may be another major risk and in spite of all precautionary measures viral infections by hepatitis B, HIV and transmissible spongiform encephalopathy are not yet got under total control. It is just in these domains that one can state juristic lacks which national and european organisation of standardisation and homologation have to cover during the next few years.

The aim of the current study was to assess the in vitro effects of nickel hydroxy carbonate (NiHC) at noncytotoxic concentrations on the production of cytokines such as tumour necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) in alveolar macrophages (AMs). The effect of NiHC was evaluated in both unstimulated AMs and cells activated by lipopolysaccharide (LPS). Cytotoxicity was related to lactate dehydrogenase release and ATP cell content. The results confirm that NiHC at concentrations of 0.125, 1.25 and 3.125 micrograms NiHC 10(-6) cells was not cytotoxic. The NiHC exposure of unstimulated AMs significantly increased the release of TNF-alpha at all concentrations and that of IL-6 at 1.25 micrograms NiHC 10(-6) cells. LPS addition significantly increased the secretion of both cytokines. However, NiHC did not cause a significant increase in the release of TNF-alpha and IL-6 in LPS-stimulated cells. In conclusion, the ability of NiHC to activate AMs and to release increased amounts of pro-inflammatory mediators may be responsible, at least partly, for inflammation and pneumotoxicity associated with nickel exposure.

The aim of this investigation was to assess the cytotoxic effect of nickel hydroxy carbonate (NiHC) on guinea pig alveolar macrophages (AMs) by studying ultrastructural modifications and by determining beta-glucuronidase (BG) and lactate dehydrogenase (LDH) activities, as well as cellular ATP content. The ultrastructural studies revealed phagocytosis of NiHC particles and a general vacuolisation of the cells, especially at high concentrations. X-ray microprobe analyses of these particles demonstrated the presence of Ni, P and Ca which suggests the formation of Ni-P-Ca complexes. In exposed cells, a biphasic change in intracellular ATP concentrations was observed which could indicate 'activation' of AMs at low concentrations and inhibition of energy generation at higher concentrations. As for enzymatic activities, a dose-dependent increase in LDH release was observed except at low doses which increased ATP. There was a good correlation between ATP decrease and LDH release, consistent with a dose-dependent cytotoxic effect of NiHC. However beta-glucuronidase activity remained unchanged at all NiHC concentrations. It has been concluded that NiHC undergoes an intracellular, biological transformation to form Ni-P-Ca. Further investigations are needed to determine the precise nature and importance of these complexes.

Our previous investigations have shown evidence of an interaction between alpha Ni3S2 and membranous and cellular lipids of lung cells with a significant increase in the linoleic, linolenic and arachidonic acid pool. The present work is designed to follow the metabolic fate of arachidonic acid in alpha Ni3S2-exposed human embryonic pulmonary epithelial cells (L132) in culture (50 microM alpha Ni3S2 for 3 days). The metabolites of arachidonic acid were assessed by HPLC determination coupled with UV or electrochemical detection. We determined malondialdehyde (MDA), hydroxyeicosatetraenoic acid (HETE), leukotrienes (LT) and reduced glutathione (GSH). In exposed cells we observed a significant increase of MDA, which is a breakdown product of lipid peroxidation. In addition, we noted significant increases of 5-HETE and 15-HETE in L132 cells resulting from the enzymatic reduction of 5-HPETE and 15-HPETE respectively. There was also a simultaneous decrease of GSH--confirmed by a strong decrease of GSH in exposed cells with respect to controls. 5-HPETE is furthermore converted to epoxides such as leukotriene A4 and we also quantified in exposed cells a significant increase of its subsequent catabolites LTB4, LTC4 and LTE4. These investigations show clearly that exposure of L132 cells to alpha Ni3S2 enhances lipid peroxidation based upon direct measurements of MDA and other metabolites of arachidonic acid. This lipid peroxidation is an autocatalytic free-radical process and could be responsible for DNA damage.

In environmental health, inhalation is the most prominent route of ethylene oxide (EO) exposure. In the current study, human embryonic pulmonary epithelial cells (L132 cell line) were exposed to EO following a prior adaptation to atmospheric conditions. A comparative study between two EO exposure conditions (0.07 and 0.18 g/m3 gas injections) was carried out after a 1-h incubation. Whereas control cells were exposed to a pure air stream. The EO cytotoxicity was established by electron microscopy and LDH and ATP determinations after 1, 3, 6 and 24 h following the exposure. The ultrastructural examination revealed a remarkable vacuolisation of the exposed cells leading to cell death. In spite of this modification, the number of mitochondria and the content of endoplasmic reticulum increased in the L132 cells. For both exposure concentrations LDH was released into the extracellular milieu. In the presence of the high EO concentration, LDH activity increased with respect to the post-exposure time involving alteration of the membrane and permeability. For low EO exposure, ATP synthesis was significantly increased after 1 h of post-exposure (P < 0.01) and decreased to normal levels after 6 h. For the high EO concentration, however, ATP continually increased with respect to the post-exposure time. This indicates cellular stimulation and suggests the activation of a defense mechanism. This study shows a direct EO cytotoxicity on L132 cells cultured in atmospheric conditions.

The metabolic effects of different concentrations of nickel hydroxycarbonate (NiHC) on guinea pig alveolar macrophages (GPAMs) were investigated. Exposure to high concentrations of NiHC (6.25 and 12.5 micrograms 10(-6) cells) led to cell vacuolization. Morphological changes were associated with a dramatic reduction in the steady-state level of cellular adenosine triphosphate (ATP), i.e. ATP levels were reduced by 35% (P less than 0.001) and 53% (P less than 0.01), respectively. Low concentrations of NiHC (0.0625 and 0.125 microgram 10(-6) cells) did not induce morphological changes but increased cellular ATP content by 19% (P less than 0.01) and 12% (P less than 0.05), respectively. Effects of NiHC (0.125 and 6.25 micrograms 10(-6) cells) on cell oxidative metabolism were studied. The chemiluminescence was significantly increased (P less than 0.001) by the lower but not the higher concentration. A slight inhibition of total superoxide dismutase (P less than 0.05) and a decrease of catalase activity were demonstrated (P less than 0.05) for the high dose, while the low dose decreased the levels of reduced and total glutathione. In conclusion, the effects of NiHC on alveolar macrophages are characterized by an overproduction of free radicals for low concentrations and the depletion of cellular reserve energy, particularly ATP, for high concentrations.

The genotoxic activity of alpha-Ni3S2 was assessed on human lymphocytes from nickel-hypersensitized (SSL) and nickel-unsensitized (USL) subjects. Three genotoxicity tests were performed: the sister chromatid exchange (SCE) test, the metaphase analysis test and the micronucleus test. (i) The SCE test (3-100 micrograms/ml) showed a weak but statistically significant increase in the number of SCE in both lymphocyte types with respect to controls, USL presenting a slightly higher SCE incidence but only at one concentration. (ii) The metaphase analysis test demonstrated a high dose-dependent clastogenic activity of alpha-Ni3S2 in both lymphocyte types. The frequency of chromosomal anomalies was significantly higher in USL than in SSL for all concentrations applied. (iii) The micronucleus test confirmed the dose-dependent clastogenic activity of alpha-Ni3S2 and the differences already observed between USL and SSL, i.e. the number of cells with micronuclei was statistically higher in USL. Finally, the incorporation study with alpha-63Ni3S2 showed a higher uptake of its solubilized fraction by USL. This allows an explanation of the different genotoxic action of nickel on the two cell types. In this study we demonstrated that hypersensitivity has an influence on the incorporation of alpha-Ni3S2 and subsequently on the different induction of chromosomal aberrations in human lymphocytes.

Biomaterial research has been mostly concerned with biocompatibility, i.e. tissue response, of materials for dental or orthopaedic implantation. In this study, the effects of titanium powder on seven bacterial species commonly found in dental plaque or gingival sulcus, were determined by agar incorporation and by liquid medium culture. In neither culture system could any inhibitory or stimulatory activity be detected.

The uptake and biological transformation of Ni3S2 was studied on guinea pig alveolar macrophages (GPAM) in primary culture. Two different pathways are observed: (i) phagocytosis of alpha Ni3S2 crystals and subsequent degradation to minute particles, which are recovered bound to the membranes of phagocytic vacuoles and to lysosomal membranes. These degradation products contain sulphur in very reduced quantities, as revealed by energy-dispersive spectrometry (EDS). (ii) Extracellular degradation to regular round particles (0.1-0.2 microns) and irregular minute particles (10-30 nm). Round particles may enter the cell by pinocytosis and are characterized by the loss of sulphur. Minute particles are bound preferentially to cell membranes, to cytoplasmic organelles, such as endoplasmic reticulum, mitochondria and peroxysomes, to liposomes and to the euchromatinic part of nuclei. EDS analyses in these particles revealed the substitution of sulphur by phosphorus. This observation suggests the formation of an Ni-P complex with the phosphate groups of membranous and liposomal phospholipids and of the euchromatinic part of DNA or RNA. Steady-state fluorescence polarization analysis were carried out on GPAM and, for comparative purposes, on human embryonic pulmonary epithelial cells (L132 cell line). In both cell types they revealed a significant increase of membrane fluidity, induced either by desaturation of aliphatic chains or directly by the cleavage of fatty acid chains.

Methylmethacrylate (MMA) is a volatile liquid widely used in the manufacture of acrylic polymers. Apart from its general toxicity (cardiac, respiratory, cutaneous, etc.), MMA is a potential mutagenic agent. Data on the mutagenicity of MMA is available almost exclusively with in vitro systems, thus the demonstration of genetic effects produced by monomeric MMA vapours in exposed workers is valuable additional information. An in vivo sister chromatid exchange (SCE) test was performed using lymphocytes from 31 workers occupationally exposed to MMA and a control group of 31 men whose mean age and smoking habits were similar. Our results indicate that the number of SCE in exposed workers (7.85 +/- 2.66) was not higher than the control group (7.49 +/- 2.33). However, the rate of SCE was significantly higher in the group exposed to MMA at peak concentrations ranging from 114 to 400 p.p.m. A few cells with a large number of SCE, called 'high frequency cells' (HFC), are responsible for this increase.

The uptake, the biological transformation and the interaction with cellular constituents of alpha Ni3S2 and beta NiS have been studied in vitro and in vivo on rat lymphocytes. beta NiS crystals are phagocytized in vitro and no structural degradation is observed within the first 3 days of exposure. Energy dispersive spectrometry (EDS) reveals a slight dissolution characterized by the loss of sulfur. alpha Ni3S2 is degraded in the extracellular space to minute particles (50-100 nm) covering the cell membrane. Smaller intracellular particles (10-30 nm) are found selectively bound to mitochondria, endoplasmic reticulum, Golgi vesicles, nuclear membranes, and the euchromatinic part of nuclei. EDS analyses reveal that the particles bound to cell membranes and euchromatin no longer contain sulfur but phosphorus and nickel as inorganic compounds. This observation suggests the formation of a Ni/P complex with the phosphate groups either of membranous phospholipids or of nuclear RNA or DNA. A similar uptake and transformation process of alpha Ni3S2 is observed on lymphocytes after in vivo incubation. This leads us to consider lymphocytes as target cells, as compared with other cell types where the alpha Ni3S2 uptake occurs only partially. The present findings show a difference of uptake and biological transformation between alpha Ni3S2 and beta NiS. The identical results obtained after in vitro and in vivo bioassays enhance the in vitro experiments, at least for this cell type.

Four mutagenicity tests of alpha-Ni3S2 were performed: the Ames test on five Salmonella typhimurium strains, HPRT test on V79 cells, in vitro chromosomal aberrations on human lymphocytes and the in vivo micronucleus test in mice. The in vitro tests were carried out without metabolic activation. (i) The Ames test (5-1500 micrograms/plate) demonstrated no mutagenic activity, thus confirming previous observations by other authors. (ii) The HPRT test was carried out under standard conditions (3 h exposure) with alpha-Ni3S2 concentrations from 30 to 1000 micrograms/ml. No significant difference was observed with the control cells. Ultrastructural examination revealed alpha-Ni3S2 binding to the cell membrane. Very few particles were found in the cytoplasm but not in the nucleus. (iii) In vitro metaphase analysis in human lymphocytes were performed after exposure to total alpha-Ni3S2 suspension and to the soluble fraction in culture medium with or without fetal calf serum (FCS) (3-100 micrograms/ml, 24 h exposure). Nickel concentrations in the soluble fraction were determined by electrothermal atomic absorption spectrometry. A clastogenic effect of alpha-Ni3S2 became evident under all experimental conditions with a significant additional increase of chromosomal aberrations in 20% FCS complemented medium. No difference was observed between total suspension and soluble fraction. (iv) The micronucleus test confirmed the clastogenic effect of alpha-Ni3S2 in vivo after administration of 250 mg/kg (i.p.). This test revealed a clear increase of micronuclei frequency in polychromatic erythrocytes 24, 48 and 72 h after the treatment. In addition, we observed a statistically significant decrease in the number (%) of polychromatic erythrocytes after 24 and 48 h exposure. The soluble, i.e. cell-entering, fraction seems to play a great part in the clastogenic effect, as has also been shown with other test methods by other authors.

The cytotoxicity, biological transformation and interaction with plasma membranes of alpha Ni3S2 and beta NiS were studied on human embryonic pulmonary epithelial cells (L132 cell line) in culture. By establishing growth curves and survival curves, it was found that at equal molarity beta NiS has a higher inhibitory effect on cell growth than alpha Ni3S2 but a lower cytotoxic effect: the CL50 being 60 and 40 mumols/l respectively. As to their uptake, beta NiS crystals are preferentially phagocytized in their original form and then probably dissolved in the vacuoles, whereas alpha Ni3S2 is transformed in the extracellular space and in the phagocytic vacuoles into minute particles (10 nm) that are recovered bound to the cell membrane, phagocytic vacuoles and lysosomal membranes respectively. Energy dispersive spectrometry revealed that the particles bound to cell membranes no longer contain sulfur but only phosphorus and nickel as inorganic compounds. This observation suggests the formation of a Ni/P complex with the phosphate groups of membranous phospholipids and/or phosphotransferring proteins. The steady-state fluorescence polarization analysis, however, revealed a significant increase of membrane fluidity either induced by desaturation of aliphatic chains or directly by the cleavage of the fatty acid chains. These results clearly show a difference between beta NiS and alpha Ni3S2 concerning their cytotoxic effects, uptake, biological transformation and interaction with cell membranes.

Corrosion on orthopaedic implants has been studied. Twelve intramedullary nails and twelve osteosynthesis plates were implanted up to eight and thirteen years, respectively. Analysis of biological fluids from all patients was carried out and revealed high concentrations of nickel and chromium that correlated to the implantation time. All implants were corroded by both crevice and intergranular corrosion. Intergranular corrosion is related to mechanical and heating treatments. Crevice-like corrosion is probably enhanced by sulphur present in amino-acids. Electronic probe analysis shows the reaction study between both sulphur and nickel and sulphur and chromium. These results are compared to the metal distribution in body fluids. After a certain incubation time the corrosion accelerates as is characteristic for crevice-like corrosion processes. Studies of the distribution rate of two Ni-Fe-Cr dental alloys in a cell culture system give similar results: metal-ion release increases with the exposure time.

Pig heart myosins isolated from the free wall of the right ventricle and the free wall of the left ventricle were compared with respect to structural and enzymatic properties. The following parameters were studied (1) activation of myosin ATase by Ca2+ and K+j(2) molecular weight of the heavy and light chains of myosins as determined by electrophoretic migration in polyacrylamide sodium dodecyl sulfate (SDS) gels; (3) ability of the heavy chains to form aggregates at low ionic strength as revealed by electron microscopy; (4) sensitivity to the action of chymotrypsin. Differences were observed between left and right ventricular myosins (L-myosin and R-myosin) for all these parameters except for the molecular weight of heavy and light chains. The existence of large amounts of short synthetic filaments for R-myosin compared with L-myosin as revealed by the length repartition of the filaments, and the production of smaller quantities of HMM-S by chymotryptic digestion for R-myosin, strongly suggest the presence of different cardiac myosin heavy chain species.

The fine structure of the epicyte of D. gigantea was investigated. The motility of the gregarine and the contractile elements are described. Four essential types of movements can be observed in this gregarine: (1) rolling up and pendular movements, (2) locomotion by gliding forward, (3) cytoplasmic streaming (Fig. 1), (4) peristaltic contractions (Fig. 2) which seem to be accompanied by the contraction of annular myonemes (Fig. 2). The epicyte is formed by the folding of the parasitic cell wall which is made from three membranes (Figs. 3 and 4). At the top of each fold one can see apical struts between the outer and middle membrane and apical filaments under the inner membrane (Fig. 3). In addition, the epicytic folds are covered by a cell coat which is made from tubular structures (Fig. 5). At the base of the epicytic folds can be observed the basal lamina (Fig. 3) composed of very fine fibrillar material with an average thickness of 2.5 nm (Fig. 6). These fibrils are oriented in the longitudinal axis of the gregarine. Beneath the epicytic fold in the ectoplasm are found the annular myonemes with a width of up to 0.5 micrometers (Fig. 7). They are composed of many fine fibrils with an average thickness of 5 nm. In young trophozoites, the myonemes also contain microtubuli (Fig. 8). Between the epicytic folds, the cell wall is interrupted by three different types of vesicles: the vesicles with an electrondense content (Fig. 9), the three-membranous vesicles (Fig. 10), and the hose-shaped vesicles (Fig. 11). Glycerol-extraction of the parasites was performed in order to define the contractile structures. After extraction the annular myonemes are difficult to recognize (Fig. 13). When ATP is added, the gregarine does not contract but the myonemes reappear after 3 to 4 min (Fig. 14). Differences can also be observed in the myoneme structure using electron microscopy: After extraction, the myonemes are composed of a very limp fibrillar network (Fig. 15) which becomes very dense after the action of ATP (Fig. 16). Glycerol extraction does not disturb either the apical struts and apical filaments or the fibrils of the basal lamina (Figs. 15--17). In addition, cytoplasmic fibrillar structures appear after glycerol extraction (Figs. 15 and 16). The experimental and electron microscope results indicate that the motility of the gregarine depends upon four different systems: (1) the ectoplasmic annular myonemes, (2) the apical structures in the undulating epicytic folds, (3) the cytoplasmic fibrils, and (4) the basal lamina.

The aim of this investigation is the ultrastructural analysis of biopsies of human gingiva pigmented by the metallic deposits from dental amalgam. The size, the nature and the distribution of the particles are studied by light and electron microscopy and by energy dispersive spectrometry (EDS). Fragments and particles of different size and composition are observed. No traces of mercury are detected in any examined samples. In contrast, very fine grains (0.01 to 0.05 microns in diameter) of a silver compound are observed to be preferentially bound to intracellular membranes and to extracellular structural components. Tissue modifications in the gingival epithelium as well as in the connective tissue can accompany the pigmentation phenomenon. The different metabolic pathways of the metals diffusing from the amalgams are discussed.

Tumoral myosins were isolated from rat and rabbit rhabdomyosarcomas and compared with normal adult and fetal skeletal myosins. The synthetic filaments, the light-chain composition and the Ca2+ ATP-ase activity were studied. In the presence of Mg2+, normal myosins precipitated as bipolar filaments (0.5 micrometer), fetal and tumoral myosins, however, precipitated as long fusiform filaments (1 to 10 micron). SDS-PAGE revealed that tumoral myosins contain the same light-chains as fetal myosin (25000 and 18000 daltons, L25-L18). The third light-chain of the normal muscle myosin (16000 daltons, L16) was absent. In addition, Urea-PAGE revealed the absence of the phosphorylated form of the L18 in fetal and tumoral myosins. Ca2+ ATPase activity measurements performed in function of the Ca2+ concentration showed similarities between fetal and adult muscle myosins. The Ca2+-ATPase activity of tumoral myosins, however, was very low and slightly activated by increasing the Ca2+ concentration (0.01 to 10 mM). The investigation has shown that fetal and tumoral myosins are identical concerning the ultrastructure of their synthetic filaments and their light-chain composition. This was not so in regard to the Ca2+ ATPase activity. This is probably the result of the expression of a new myosin- or of one of its polypeptides-, which has a different Ca2+-ATPase activity.

This study examined the effects of N-methyl-N'-nitro-N-nitrosoguanidine (M.N.N.G.) on the oral mucosa of Wistar rats. The rats were born from dams treated by i.p. injections of M.N.N.G. during gestation. Each pregnant rat of a first group received a total dose of 39.60 mg/kg of M.N.N.G. administered throughout the gestation period. Each pregnant rat of a second group received a total dose of 82.50 mg/kg of M.N.N.G. by i.p. injections during the first ten days of gestation. In the progeny of dams from both groups, the morphology of the gingiva was modified. The lingual gingiva from the lower jaw was most impaired. The epithelium always displayed hyperplasia: acanthosis, papillomatosis and hyperkeratosis. These results indicate a pathogenic effect of M.N.N.G. on lingual, mandibular gingiva. This effect was slight but permanent and irreversible. No carcinogenic effect was noticed on the oral mucosa. Similarities between the lesions described in this work and human epithelial dysfunction makes this experimental model interesting.

A fibrogranular material associated with the inner surface of the plasma membrane of adult Rat liver cells in long-term culture is described and studied. The electrophoretic pattern of the membrane indicates that this structure might contain actin. The filamentous material had not been observed in electron microscope preparations of cell surface membranes isolated from adult Rat liver.

Two patients with clear-cell acanthoma with multiple lesions are reported; histologic and histochemical findings are similar to previous descriptions. The ultrastructural study confirms the overload of glycogen in keratinocytes, associated with an increase of mitochondria and nuclear deformations. The abundance of Langerhans' cells is emphasized. Extrusion of glycogen by keratinocytes and its phagocytosis by Langerhans' cells is suggested.

The kinetic studies of 63Ni[II]-incorporation in whole tissue show that after 6 days lung has the highest affinity to nickel of all studied organs. The same observation was made when 63Ni[II]-incorporation was carried out by 7 daily injections. In both experiments, kidneys take the second place in the relative distribution of nickel. For the studies of Ni-binding proteins in lung and liver cytosols, three different types of 63Ni[II]-incorporation were performed: (i) in vitro incorporation, (ii) kinetic study of in vivo incorporation where the animals were sacrificed after 30 min, 1, 2 and 4 h after a single i.p. injection of 63NiCl2, (iii) in vitro incorporation by 7 daily i.p. injections of 63NiCl2 with sacrifice of the animals 24 h after the last injection. Several Ni-binding proteins could be observed without regard to the type of incorporation performed. In liver cytosol most of these proteins can be revealed in vitro as well as in vivo. The in vivo labelled proteins are not the same at the beginning of the incorporation (30 min) and after an elapsed period (1 to 2 h). In lung cytosol in vitro and in vivo incorporation gave different results: although an intense labelling occurs after in vitro incubation, only few proteins are labelled after in vivo incorporation, two of which are only revealed after continuous exposure to 63Ni[II]. Most of the labelled proteins of lung and liver cytosols can be recovered after different fractionation experiments. These investigations confirm that nickel is preferentially bound to lung and demonstrate that nickel-incorporation is different in lung and in liver cytosols. It is shown here that several cellular proteins are implicated in the nickel-transport. The evolution of this phenomenon suggests the existence of a nickel-metabolism in the cell.

Nickel-sulfid-induced rhabdomyosarcomas were studied by both light and electron microscopy. The successive stages differentiating tumor cells were described, and two differentiation types of rhabdomyoblasts could be observed 1) with the characteristic pattern of fetal differentiation--i.e., myofilament apparation before Z-line formation--and 2) with synthesis of these elements in the reverse order. An organized T-system is not evident. The sarcoplasmic reticulum is irregular and its cisternae often contain a granular substance or microcrystals. The only well-developed element of tumoral myofibrils is the Z-line; the other zones of sarcomeres are seldom clearly defined. Several unusual granular structures were observed. No virus particles were found. The formation of cilia appears only in interphase rhabdomyoblasts and has to be considered as aberrant and temporary formations from centrioles. They generally possess a "9 + 0" microtubular pattern, but often could be observed as rudimentary forms with a "7 + 2" microtubular arrangement. This case is another example demonstrating the relationship of cilia formation with cell division, especially after suppression of mitotic control. The histology and the electron microscopy results are discussed in relation to the differentiation pattern and the ultrastructural features of embryonic, regenerating and pathological muscle differentiating in vitro.

Histological and ultrastructural studies have been undertaken on a perineal rhabdomyosarcoma from a newborn child. The spontaneous tumour has the typical feature of mesenchymoma. The recurrent tumour, however, displays some rhabdopoietic characteristics. The myosin of the recurrent tumour has been extracted and compared with human fetal myosin. These two myosins are identical in their synthetic filaments and their light-chain composition. Nevertheless, whereas the ATPase activity of fetal myosin can be stimulated considerably by increasing the ca2+ concentration, that of tumoral myosin remains very low. These results show that there are isoenzymes of myosins and there must be differences in the myosin heavy chains, particularly in the active sites. These findings are identical with those seen in experimental rhabdomyosarcoma.

Nickel sub-sulphide-induced leiomyosarcomas in rabbit white skeletal muscle were studied by both light and electron microscopy. Two types of tumor cells, small spindle cells and elongated smooth muscle cells, are revealed by light microscopy. Nevertheless, their ultrastructure displays the same general feature. The most differentiated cells have abundant cytoplasmic filaments 7 nm in diameter, kept together in bundles by dense bodies. There also exist many 10 nm filaments and a large number of microtubules. The nuclei have prominent nucleoli with an extensive nucleolonema which form an irregular tridimensional network. Distinct fibrillar nuclear bodies were observed. Sometimes there exist desmosomes or gap junctions. The Golgi apparatus produces coated vesicles with secretory function. In the tumors were generally found the Ni3S2 implantations surrounded by a capsule, the major component of which were collagen fibers, degenerated nuclei and rod-like structures with a transverse periodicity of 15.5 nm. From these observations, several characteristics should be pointed out: 1) Many tumor cells contain large nucleoli and distinct intranuclear inclusions of undetermined nature. 2) The coated vesicles represent a secretory activity of the tumor cells; the coat material is probably used during the formation of cell membranes. Another possible function of coated vesicles could be the sequestering of calcium ions. 3) The rod-like structures in the Ni3S2-including capsule are not of Z-line material. 4) The tumoral stem myoblast in heart and skeletal muscle arise from mesenchymal cells.

After a short description of the fundamental basis of allergy, particularly in relation to contact allergy, the authors review the 41 published cases of allergy to dental amalgam consisting of 30 female and 11 male patients. 20 of these patients recovered on removal of the dental amalgam. The most frequent symptoms were of the remote cutaneous type (38/41 cases) while local symptoms, particularly gingivitis and stomatitis, occurred in 17 cases. These probably underestimate the true prevalence of the condition for several reported cases have not been published. The authors then go on to describe the sensitizing property of amalgam in relation to the occupational environment and the length of time that the amalgam has been in the mouth. Mercury is the most common sensitizing agent, but other metals, particularly cooper, zinc or silver could also be implicated. The authors attempted to explain the physio-pathological mechanism of sensitization via the cutaneous as well as the oral, digestive and respiratory tracts by describing the chelating properties of metals with cellular or tissue constituents. In the last chapter, the prevention and clinical treatment of sensitization to dental amalgams is described.