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Human mesenchymal stem cells (hMSCs) have the capacity to proliferate and differentiate into multiple cells etc. Polysaccharides can modulate the cell proliferation of human endothelial cell. Here, we investigated the role of different kinds of new polysaccharides to regulate the gap junctional intercellular communication (GJIC) and cell proliferation of cultured normal human dermal fibroblasts (NHDF) cells and hMSCs. The NHDF cells and hMSCs were cultured for 4 days with new polysaccharides. The cultures were then analyzed to verify the extent of GJIC by the scrape- loading dye transfer (SLDT) method, using Lucifer yellow. Alamar blue staining was performed to determine the proliferation of the cultured cells. In NHDF cells, the GJIC was significantly inhibited in cells treated with different kinds of new polysaccharides. On the contrary, in hMSCs, the GJIC was slightly inhibited in all cultured treated cells. But proliferation was enhanced in both cells with different polysaccharides, the extents of cell proliferation was stronger in hMSCs than in NHDF cells. These findings reveal that new polysaccharides seem to play an important role in hMSCs, thus provide a novel tool on tissue engineering.

It is important to investigate a cause of formaldehyde contamination exceeding a regulation limit value in a textile product. If formaldehyde was released from a textile product itself by treatment or processing with formaldehyde, an administrative guidance is given to a manufacture. On the other hand, when the formaldehyde migrated from other textile products or a furniture stand during displaying, an improvement instruction is performed to the store. Iwama et al. [Ann. Rep. Nagoya City Public Res. Inst., 42, 11-16 (1996)] developed a method for distinguishing fabric processing and migration by additional hydrolytic extraction using hydrochloric acid solution. This study was to confirm the reliability and stability of the method for knowing formaldehyde processing on textiles. Five laboratories evaluated three samples: unprocessed textile, processed textile and unprocessed but formaldehyde-migrated textile. For a processed textile sample, amounts of formaldehyde increased by additional extractions with acidic solution, so all laboratories judged that the sample had been treated with formaldehyde. In the cases of the other two samples, such increases were not observed in the extracts using acidic solution. All laboratories reported that these samples were not processed using formaldehyde but had absorbed a different level of formaldehyde by migration. In a series of experiments, the judgement about the existence of formaldehyde processing or migration is comparatively consistent among all laboratories. This validation study concluded that the distinguishing method adopting additional extractions with acidic solution is useful to find formaldehyde processing of textile, and to deal with processing and migration separately as a cause of formaldehyde contamination.

The transplantation of bioartificial pancreas has the potential to restore endogenous insulin secretion in type I diabetes. The bioartificial pancreas is constructed in vitro from cells and a support matrix. Hyaluronic acid (HA) is an extremely ubiquitous polysaccharide of extracellular matrix in the body and plays various biological roles. It has been suggested that high molecular weight (HMW) HA increases in the function of gap-junctional intercellular communications (GJIC) and the expression of connexin-43 (Cx43). To determine whether the function of pancreatic β -cells is affected by gap junctions after HMW HA-treatment, we exposed HIT-T15, a clonal pancreatic β -cell line, in various concentrations of HA for 24 h, and then detected the insulin secretion and content, using an insulin assay kit by ELISA technique. The cellular functions of GJIC were assayed by dye-transfer method using the dye solution of Lucifer yellow. HA-treatment resulted in the enhancement of GJIC function, the increase of insulin release and insulin content. The results obtained in this study suggest that HA-coating increases the insulin secretion of HIT-T15 cells by the enhancement of Cx43-mediated GJIC. The results give useful information on design biocompatibility of HA when is used as a biomaterial for bioartificial pancreas.

Recombinant P2X2 receptor was observed by atomic force microscope in the aqueous phase. The P2X2 receptor was expressed in an insect cell line, and recombinant proteins were prepared under native conditions. The membrane fractions were extracted, and histidine-tagged receptor protein was purified from the fractions by column chromatography. When the purified protein fraction was diluted with water and served for atomic force microscopy, dispersed particles of about 3 nm in height were observed. In the presence of 1 mM ATP, the assembly-like images of the particles were obtained. More densely assembled images of the particles were achieved when the protein was dissolved in a Tris buffer containing 1 mM ATP. Under this condition, imaging of the surface of the particles exhibited a circular structure with a diameter of about 10 nm having a pore-like structure. These results suggest that atomic force microscopy provides structural information about P2X2 receptor in aqueous phase.

In order to study the interaction of fullerenes with biological molecules, a novel photoaffinity labeling agent derived from C60 was designed and synthesized. As photosensitive functional groups, azide group, and aziridine group are utilized. A convenient synthetic route via fulleropyrrolidine 2 was employed to obtain compounds labeling agents 5 and 9 .

In Japan, the amount of formaldehyde in textile products was regulated by the low for the control of household products containing harmful substances. Formaldehyde was determined by measuring the optical density of acetylacetone derivative of formaldehyde extracted from textiles. The household products low stated that the increase in the optical density of color development of the extract from the textile products for babies or infants within 24 months after birth should not be more than 0.05. Collaborative study decided the amount of formaldehyde equivalent to the increase in absorbance described above, and the amount was 16 ppm. There are some reports that formaldehyde causes an allergic reaction even at a very low concentration, so continuous regulation for formaldehyde in the textiles was desirable using this level of amount. We developed HPLC method for the determination of formaldehyde in textile products. Formaldehyde was determined by the direct injection of acetylacetone derivative of samples into the system equipped with ODS column and UV-VIS detector (detection wavelength 413 nm) using the mixture of acetonitrile and water as mobile phase. The linearity was obtained between a peak area or height and the concentrations of formaldehyde solution in the range of 0.0625-2 micrograms/ml. The regulation level was sufficiently detected by the present HPLC method. We recommended that the HPLC test was adopted as a reexamination method for the products may violate the regulation as well as a dimedone test.

In this study, we examined the interaction of the osteoblast which forms bone and sulfated hyaluronan (SHya). For the purpose of the creation of a new functional polysaccharide, we introduced a sulfate group in hyaluronan (Hya) of high molecular weight, and SHya of high molecular weight could be obtained for the first time. When rat calvarial osteoblast (rOB) cells were cultured with a high concentration of SHya, they formed aggregated spheroids after 4h and the spheroids grew to about 200microm after 24h. We examined the expression of cell adhesion molecules in order to clarify the mechanism of aggregate formation. The N-cadherin (N-cad) and Connexin43 (Cx43) expression level of rOB cells cultured with SHya remarkably increased after 2h. A difference in the expression of Integrin beta1 (Intbeta1) could not be observed between the SHya addition and control group. The alkaline phosphatase (ALPase) activity of rOB cells cultured with SHya after 8h was significantly enhanced in comparison with control. Therefore, the sulfate group of SHya seems to enhance expression of cell adhesion protein such as N-cad and Cx43, resulting in aggregate formation and further remarkable induction of the ALPase activity of rOB cells.

The relative standard deviation (R.S.D.) of measurements is estimated in the kinetic-colorimetric assay of bacterial endotoxins without recourse to the usual repeated experiments. The measurements are the slopes of kinetic curves and two major factors are considered to cause the uncertainty of the measurements: (1) the pipetting of the sample and color development reagent; and (2) noise in the detection unit. The measurement R.S.D. is formulated as a function of endotoxin concentration. Two parameters (S.D. of the pipetted volumes and S.D. of the detector noise) are also required in the uncertainty equation, but no arbitrary coefficients are included. Since the S.D. values for pipettes and detector noise can be determined independently of the endotoxin assays, the measurement R.S.D. can be estimated by the above equation without repeating the assays. However, the calibration curve is necessary. The theoretical estimation is shown to be in good agreement with the experimental R.S.D. (n = 12) over a wide concentration range.

To ensure the effects of multipurpose solutions (MPS) for hydrogel contact lenses on the cornea, the inhibitory activity of three types of MPS on corneal cells has been evaluated with the use of scrape loading and dye transfer assay (SLDT assay) and Western blotting on rabbit corneal keratocytes (RC4). In SLDT assay, MPS-A and poloxamine showed dose-dependent inhibitory activity, suggesting the inhibitory action of MPS-A and poloxamine to gap junctional intercellular communication (GJIC) in the tested cells. Moreover, after treatment with MPS-A, the GJIC was initially inhibited within 4 h, and thereafter gradually turned to an approximately 60% level of the initial value. When MPS-A was removed from the incubation media after exposure of the cells, the recovery of GJIC was time dependent and returned to approximately initial levels at 8 h. Complete recovery was established after approximately 24 h. These findings suggested that the inhibitory action of MPS-A on corneal keratocytes was reversible. This inhibition was accompanied by a decrease in the quantity of connexin-43, which is a major protein constituting the gap junctional channel of these cells, and its change in the phosphorylation state. Taken together, it was suggested that MPS-A interacts with connexin-43, inducing an inhibitory action on GJIC.

Connexin 32 (Cx32) is the main gap junction protein in hepatocytes and plays an important role in the regulation of liver gap junctional communication (GJIC). In this study, the human Cx32 gene was transfected in a hepatoma cell line (HepG2) that is aberrant expression of Cx32 and deficient in GJIC. Cx32-transfected HepG2 showed the increased GJIC comparing with HepG2 and the vector-transfected HepG2. Furthermore, the liver functions of ammonia removal activity of HepG2 were remarkably enhanced with Cx32 gene transfection. It may be expected to improve the cellular functions of the hepatoma cell line by Cx32 gene transfection and serve to develop an efficacious bioartificial liver.

We studied the influence of multi-purpose solutions (MPS) on gap-junctional intercellular communication (GJIC) of cornea cells and its cell toxicity such as cell metabolism and proliferation. This study evaluates long-term safety of three types of multi-purpose solutions (MPS) in the cornea cells. Three types of MPS were investigated using scrape-loading dye transfer (SLDT) method to measure GJIC. Cell toxicity was evaluated for cell viability according to colony method and USP elution method (MEM Elution Test). In the SLDT method, one type solution was significantly showed an inhibitory effect on the GJIC in cornea cells but not the other type solutions. Comparative cytotoxicity potentials of three types of MPS also indicated similar tendency in the result of SLDT method. These results suggest that the evaluation methods are considered to be effective to confirm the safety of MPS.

The implantation of a biomaterial often induces host inflammatory responses. Some adverse effects by the biomaterials, such as poly-L-lactic acid (PLLA) and polyurethanes (PUs) were reported in animal experiments. PLLA produced tumorigenicity in rats after long-term implantation. The purpose of this study was to determine the in vitro effect of PLAO3 (high-molecular weights of PLLA) and PU8 (PTMO/MDI/BD) on the function of the normal human dermal fibroblasts (NHDF) and the in vivo effect of PLAO3 on the function of the cells originated from the subcutaneous tissue in the two female mouse strains, BALB/cJ and SJL/J. The results with Scrape-loading and dye transfer (SLDT) assay, Western Blot and RT-PCR analysis clearly demonstrated that gap-junctional intercellular communication (GJIC) and the expression of Cx43 were significantly suppressed in PLAO3-implanted group of BALB/cJ mice in compared to the control mice. While, no significant difference was found in GJIC and the expression of mRNA level but a little bit difference was observed in the Cx43 protein expression between the SJL/J implanted and the control mice. We considered that the PLAO3 suppressed irreversibly gap junctional protein connexin43 at the earlier stage after implantation and the suppression of connexin43 gene-expression might play a vital role in the inhibition of GJIC and thus promotes the tumorigenesis.

This study investigated the usefulness of modified polyurethane (MPU) coating micropore membrane bags for diminishing the immunological responses following organ or tissue transplantation in allogeneic setting. Spleen from Brown Norway (BN) rats (donor) were placed into the peritoneal cavity of Lewis rat (recipient) either directly or inside of MPU coated bags. Lewis rat with Sham’s operation served as control. After 12 and 24 weeks, cytokines of IL -4, IL -13, TNF -α and IFN -γ, and flow cytometric evaluations for CD4+ and CD8+ cells of the recipient blood were carried out. TNF -α levels proved polyurethane coating effective in reducing inflammatory reaction at 12 weeks. Twelve week IFN -γ and, CD4+ and CD8 cells indicated that graft-versus-host-reaction (GVHR) took place but polyurethane coated bag did not prevent or reduced this reaction. Thus, this study shows that MPU coating might be functional in preventing inflammatory reaction but is not useful for preventing GVHR.

Biocompatibility of the biomaterials for the differentiation of the human articular chondrocytes were estimated by reverse transcription-polymerase chain reaction (RT-PCR). We used five biodegradable polymers for culturing with human articular chondrocytes. In addition to these five materials, we also estimated aquous type of fullerene, namely C60 dimalonic acid (C60DMA). Cultures were carried out using micromass culture method for 4 weeks. Collagen type II, aggrecan and connexin43 gene levels were estimated using RT-PCR methods. Among the biomaterials, Poly glycolic acid (PGA) showed the highest expression level of the collagen type II gene. On the contraty, C60DMA showed the lowest expression level among six kinds of test substances. In the case of the aggrecan gene, PGA also showed the highest levels, and C60DMA showed the lowest ones. However, the expression patterns of the connexin 43 gene were different from previous two genes. Using the multi regression analysis was carried out between differentiation and these three gene expression levels. There was a high correlation between cellular differentiation and three gene expression levels.

Since gold sodium thiosulfate (GST) has been included in a standard patch test series for diagnosis of allergic contact dermatitis from gold, the incidence of patients showing positive reactions to gold is increasing. However, there were little reports on induction of gold sensitization in animals. In this study, we have examined the sensitization potential of GST using mice and guinea pigs. In the guinea pig maximization test, 2 or 6 out of 10 animals showed positive skin responses, mainly edema, by challenge with 2% or 5% GST in 50% ethanol solution, respectively. In the mouse ear swelling test, positive ear swelling (20% greater increase in ear thickness) after challenge with GST was shown in 2 out of 6 mice those previously treated with GST. Topical exposure of mice to GST in 70% dimethylsulfoxide solution induced small increases in the lymph node weight and the lymph node cell (LNC) number in the murine local lymph node assay (LLNA). A greater degree of LNC responses were observed in the sensitive mouse lymph node assay (SLNA) compared with the LLNA, but the stimulation index of total lymph node response by GST was not so high. From these results, GST was identified as a contact allergen, but the sensitization potential was not so strong. In the mouse IgE test, treatment of mice with GST resulted in a statistically significant increase in the serum IgE antibody concentration that associated with immediate-type hypersensitivity reaction. It may suggest that the sensitization responses from gold would appear not only at the contact site but also systematically.

Gap junctional intercellular communication (GJIC) is inhibited by 4,4'-di(ethoxycarboamide) diphenylmethane (MDU) and polytetramethylene oxide 1000 (PTMO1000), which are model chemicals of hard and soft segments of polyetherurethane (PEU), respectively. In our previous study, we suggested that the inhibition of GJIC induced by MDU and PTMO1000 may lead to accelerate promotion step by both segments after the initiation step by hard segment, MDU. To examine this hypothesis, we established connexin 43 overexpressed clones from Balb/c 3T3 A31-1-1 clones (A31-1-1 cells) by transfection. Here we show that these clones acquired much higher GJIC ability than parental A31-1-1 cells and kept them even if MDU or PTMO1000 was added to the culture. We also found that Mutation of Cx43 at Tyr-265 resulted in reduced inhibition of GJIC induced by MDU and PTMO1000. These findings suggest that inhibition of GJIC by PEU may be caused by Tyr-265 phosphorylation of Cx43 molecule.

The effects of different molecular weights of hyaluronic acid (HA), a major component of extracellular matrix, on gap junctional intercellular communication (GJIC) in normal human dermal fibroblasts (NHDF cells) were investigated. NHDF cells were cultured for 4 days with different molecular weights of HA and then the extent of GJIC was assessed by the scrape-loading dye transfer method, using Lucifer yellow. The area of dye transfer was greater in the dishes coated with HA than in those to which HA was added. Thus, NHDF cells cultured on surfaces coated with high molecular weight (HMW) HA (MW, 800 kDa) showed greatly enhanced GJIC. Furthermore, another aim of this study was to evaluate the effects of different molecular weights of HA on the production of FGF-2 and KGF, because both are important cytokines produced by NHDF cells. When FGF-2 and KGF cultured levels of cell extracts and media were determined by ELISA, both levels were significantly enhanced when cells were grown on plates coated with HMW HA. This finding indicated that the function of gap junction channels in NHDF cells grown on plates coated with HMW HA may promote the biosynthesis of growth factors such as FGF-2 and KGF.

Normal human dermal fibroblast (NHDF) cells were used to detect differences in gap-junctional intercellular communication (GJIC) by hyaluronic acid (HA), a linear polymer built from repeating disaccharide units that consist of N-acetyl-D-glucosamine (GlcNa) and D-glucuronic acid (GlcA) linked by a β1-4 glycosidic bond. The NHDF cells were cultured with different molecular weights (MW) of HA for 4 days. The rates of cell attachment in dishes coated with high-molecular-weight (HMW; 310 kDa or 800 kDa) HA at 2 mg/dish were significantly reduced at an early time point compared with low-molecular-weight (LMW; 4.8 kDa or 48 kDa) HA with the same coating amounts. HA-coated surfaces were observed by atomic force microscopy (AFM) under air and showed that HA molecules ran parallel in the dish coated with LMW HA and had an aggregated island structure in the dish coated with HMW HA surfaces. The cell functions of GJIC were assayed by a scrape-loading dye transfer (SLDT) method using a dye solution of Lucifer yellow. Promotion of the dye transfer was clearly obtained in the cell monolayer grown on the surface coated with HMW HA. These results suggest that HMW HA promotes the function of GJIC in NHDF cells. In contrast, when HMW HA was added to the monolayer of NHDF cells, the functions of GJIC clearly were lowered in comparison with the cells grown in the control dish or with those grown on the surface of HMW HA. Therefore it is concluded that the MW size of HA and its application method are important factors for generating biocompatible tissue-engineered products because of the manner in which the GJIC participates in cell differentiation and cell growth rate. © 2002 Wiley Periodicals, Inc. J Biomed Mater Res 60: 541–547, 2002

The biocompatibility of poly-L-lactic acid (PLLA) and copolymer of poly(DL-lactic-coglycolic acid) 50:50 (PLGA), and the effects of basic fibroblast growth factor (bFGF) and ferrous sulfate (FeSO4) on human articular chondrocytes (HAC) proliferation and differentiation were investigated in a 4 weeks micromass culture system. Aliquots of 20 µl (per well) containing 4×105 cells were spotted onto 24-well tissue culture plates and supplemented with the media containing dimethyl sulphoxide (as a vehicle), PLLA, PLGA, bFGF, or FeSO4, respectively. Cells cultured with medium only was used as a control. Alamar blue and alcian blue staining were done to determine the chondrocyte proliferation and differentiation, respectively. All cultures showed sufficient cell proliferation close to control level. The cultures exposed to PLLA, PLGA, bFGF and FeSO4 increased HAC differentiations up to the levels of 1.2-, 1.6-, 1.3- and 2-fold of the control, respectively. These results suggest that both the PLLA and PLGA are suitable as scaffold for tissue-engineered cartilage, and bFGF and FeSO4 also possessed the stimulatory activities on HAC chondrogenesis.

Representative biodegradable-polymers are assayed to clarify the property when used as the materials for the nerve regeneration using the midbrain cell differentiation systems. As for polyglycolic acid [PGA], both the proliferation and differentiation of the midbrain cells were dose-dependently inhibited by PGA3000(Mw=3000). Poly(L-lactic acid)[PLLA]5000 (Mw=5000)showed slightly inhibitory effects on the proliferation and differentiation. In the case of the poly (L-lactic acid-co- e -caprolactone)25 10000[P(LA-CL)25 10000] (Mw=10000), the differentiation was inhibited at 7.5,μg/ml to the levels of about 50% of the controls. But, P(LA-CA)50 18000 (Mw=18000) showed weakly inhibitory action on the differentiation of midbrain cells in comparison with P(LA-CA)2510000. The inhibitory activity of these polymers on neuronal cell differentiation were in the following order: P(LA-CA)2510000 > PGA3000> P(LA-CA)50 18000= PLLA5000. The catalyst of SnCl2 showed the strongest inhibitory action on the midbrain cell differentiation among test chemicals.

Gap junctional intercellular communication (GJIC) is a function that plays an important role in maintaining cell and tissue homeostasis and in regulating cell growth, development, and differentiation. Change in this function of V79 fibroblasts cultured on polyethylene films modified with albumin or collagen was estimated using fluorescence redistribution after photobleaching (FRAP) analysis. The GJIC function of V79 cells on nontreated polyethylene was strongly inhibited in comparison with those on a glass coverslip. When the cells were culture on collagen-immobilized polyethylene film, this function was recovered to about 70% of the cells cultured on the coverslip. However, albumin immobilization did not recover the function as much as collagen immobilization. Western blotting analysis and immunostaining of connexin 43, which is a major protein constituting gap junctional channel of these cells, revealed its abnormal expression and distribution in the cells on nontreated polyethylene, whereas its almost normal distribution was observed in the cells on collagen-immobilized polyethylene. This abnormal expression and distribution of connexin 43 induced by the surface of polyethylene may be ascribed to a strong inhibition of GJIC of V79 fibroblasts.

Recent study has shown that biodegradable polymers are attractive candidates for chondrocyte fixation and further transplantation in cartilage tissue engineering. Poly (glycolic acid) (PGA), a polymer of glycolic acid, is widely used in orthopedic applications as a biodegradable polymer. Organotin, lead, antimony, and zinc are catalysts commonly used in synthesizing PGA. Here, we investigated the biocompatibility of PGA, synthesized with and without inorganic tin as a catalyst in chondrogenesis of human articular chondrocytes in a micromass culture system. Significant enhancement of chondrocyte proliferation and expression of the collagen type II protein gene were observed in cultures treated with PGA synthesized with a tin catalyst. However, aggrecan gene expression was very similar to the control culture. Amount of collagen type II protein was also increased in the same group of cultured chondrocytes. In contrast, PGA without a catalyst caused overall inhibition of chondrogenesis. Despite several positive findings, extensive investigations are essential for the feasibility of this PGA(Sn) in future clinical practice.

[60]Fullerene (C60) was solubilized with poly(vinylpyrrolidone) (PVP) in water, and the aqueous solution was applied to a mouse midbrain cell differentiation system. On incubation of C60 with various concentrations of PVP, cell differentiation and proliferation were potently inhibited, although weaker than the vehicle controls. C60 was clearly distributed into the yolk sac and embryos by intraperitoneal administration to pregnant mice at 50 mg/kg and had a harmful effect on both conceptuses by microscopical evaluation. This in vivo and in vitro action on embryogenesis is a novel and seriously harmful activity of C60.

A new sterilization system using vapour-phase hydrogen peroxide (VPHP) was recently developed. The cytotoxicity of various medical materials sterilized by the VPHP sterilization system was investigated. After VPHP sterilization, polystyrene, Polyurethane (PU8), blend material of silicone and Polyurethane (Sil/PU6), poly(methyl methacrylate) (PMMA), fluorosilicone acrylate and poly(2-hydroxyethyl methacrylate) (HEMA) showed strong cytotoxicity, whereas polyethylene and polypropylene did not. Although the cytotoxic potential of most materials is reduced by extension of the aeration time, HEMA and PMMA still retained strong cytotoxicity after 12 h aeration. Addition of catalase to the cell culture eliminated the cytotoxicity of sterilized polystyrene and PU8. Hydrogen peroxide (H 2 O 2 ) residues remaining in the sterilized materials were determined. Large amounts of H 2 O 2 (5.1–186 μg) were detected in HEMA, PU8, Sil/PU6 and PMMA. In contrast, silicone and polyethylene contained low levels of H 2 O 2 . The amounts of residual H 2 O 2 in the materials decreased with increasing aeration time, but the elimination rate of H 2 O 2 differed among the test materials. The cytotoxic potential of the VPHP-sterilized materials correlated with the amounts of residual H 2 O 2 present. These results indicated that the cytotoxicity of VPHP-sterilized materials was caused by the residual H 2 O 2 . To generalize the developed VPHP sterilization system for various medical devices, it is important to validate the aeration of materials for removal of cytotoxic residuals.

For the detection of tumor-promoting activities of phenolic antioxidants, the inhibitory activities on the intercellular gap-junctional communication were investigated using the V79 metabolic cooperation (MC) assay. Among eight antioxidants, 4,4'-butylidene-bis(3-methyl-6-tert-butyl-phenol), 2,2'-methylene-bis(4-methyl-6-tert-butylphenol) (MBMBP), and styrenated phenol (SP) showed stronger inhibitory activities than lithocholic acid, which is known to be a tumor promotor. However, 4,4'-thio-bis(3-methyl-6-tert-butylphenol), Irganox 1010, and 1330 did not inhibit at any concentrations. When the single-electron oxidation potentials were compared among antioxidants, the electrochemical ease estimated with the first oxidation potential was correlated with the cytotoxic potentials (r = 0.88), but not with the inhibitory activities in an MC assay. The tumor-promoting activity of MBMBP was also investigated using an in vitro, two-stage Balb/c 3T3 transformation assay. MBMBP did not show initiating activity, but significant promoting activity at concentrations of both 1 and 2.5 micrograms/ml were noted. These concentrations were close to the lowest effective inhibitory concentration (1.3 micrograms/ml) of MBMBP in an MC assay. In conclusion, there is a possibility that the phenolic antioxidants that show inhibitory activities in an MC assay contribute to the enhancement of tumor incidence induced by biomaterials.

Standard reference materials (SRM) made of polyurethane films containing various amounts of cytotoxic compounds such as zinc diethyldithiocarbamate (ZDEC) and zinc dibutyldithiocarbamate (ZDBC) are proposed. The cytotoxicity indices of five dithiocarbamates and the SRM films .obtained by colony assay were compared with those by published standards, i.e., neutral red assay (NF S 90-702), agar diffusion assay (USP XXII), and millipore filter diffusion assay (DIN-V 13 930). Among them, colony assay using 3 cell lines of Balb 3T3, L929 and V79 was found to be most sensitive. The extraction method and direct contact method were proposed as two test methods of colony assay. In the former, culture media after incubation with test materials were used as test solution, and in the latter, cells were directly cultured on test materials. The extraction method gave a linear relationship between the cytotoxic potentials of the SRM and its concentration of ZDEC or ZDBC. The cytotoxic potentials of the SRM correlated well with the thickness of inflammatory layer in rabbit muscle implantation tests of the SRM. The direct contact method was found to be useful to detect weak cytotoxicity because of its high sensitivity. Based on these data, we propose the colony assay and the two kinds of new SRMs as Japanese standard test method for evaluating the cytotoxicity of biomaterials.

Contact sensitivity of and cross-sensitivity between 2-(2′-hydroxy-5′-methylphenyl)benzotriazole (Tinuvin ® P, TP) and 2-(2′-hydroxy-3′- tert -butyl-5′-methylphenyl)-5-chlorobenzotriazole (Tinuvin ® 326, T326) were investigated in BALB/c mice. Mice received an intradermal injection and subsequent topical application of the test chemical. Following final application, auricular lymph node cell (LNC) proliferation was measured. In another experiment, the appearance of contact sensitivity was assessed by increases in ear thickness following challenge of the test chemical. Application of TP to mice caused a significant increase in LNC proliferation. Challenge of these mice with TP resulted in an increase in ear thickness. However, TP-sensitized mice did not show an increase in ear thickness by challenge with T326. Under the same conditions, application of T326 failed to induce either LNC proliferation or ear swelling response. The induction of LNC proliferative response was closely related to the ear swelling response by challenge. Following intradermal injection of TP, topical application of T326 to mice did not induce LNC proliferation. A significant ear swelling response was not induced by first challenge with TP or T326 but induced by rechallenge with TP. Topical exposure of mice to TP following injection of T326 failed to induce LNC proliferation or any ear responses by challenge with T326 and TP. These data suggest that TP is a sensitizer, while T326 is not a sensitizer, and these chemicals do not cross-sensitize.

The contact sensitizing potential of 2-(2′-hydroxy-5′-methylphenyl)benzotriazole (Tinuvin® P) was investigated using the marine local lymph node assay (LLNA) and the mouse ear swelling test with some modification in the induction procedure (modified MEST), Topical exposure of the ears to Tinuvin® P in acetone: olive oil (AOO) for 3 consecutive days induced no increase in auricular lymph node weight and lymph node cell proliferation. In the LLNA, Tinuvin® P was negative, and its sensitizing potential was determined as not strong. In the modified MEST, mice were sensitized with a combination of an intradermal injection of 0.2% Tinuvin® P in Freund's complete adjuvant into the shaved abdomen and topical application of 1% Tinuvin® P in AOO on both ears for 3 consecutive days. The ear thickness in mice previously treated with Tinuvin® P increased significantly on challenge with Tinuvin® P. A cross-reaction test between Tinuvin® P and various substituted benzotriazole ultraviolet light (UV) absorbers was performed. Challenge with these benzotriazole compounds on the ears of the mice previously sensitized to Tinuvin® P showed no increase in ear thickness. These data indicated that Tinuvin® P had a contact sensitizing ability, while Tinuvin® P-sensitized mice did not cross-react with the other benzotriazole UV absorbers.

Modified Eagle's minimum essential medium supplemented with 5% fetal calf serum was highly sensitive to cytotoxicity and formed large control colonies in the V79 colony assay. A highly sensitive cytotoxicity test was developed using 96-well microtiter plates. Test chemicals or extracts of polyurethane materials containing the same chemicals were added 24 h after inoculation of cell suspensions. The cells were fixed and stained with crystal violet after additional culture for 6 days (V79 cells) or 10 days (Balb/3T3 cells). In terms of sensitivity and rapid quantitative measurement, this modified colony microassay, using a low cell density in 96-well microplates, was superior to various cytotxicity tests such as colony, growth inhibition, cytolethality, and agar diffusion assays. © 1994 John Wiley & Sons, Inc.

Polyurethane films that contained various amounts of zinc diethyldithiocarbamate (ZDEC) and zinc dibutyldithiocarbamate (ZDBC) were prepared as standard reference materials (SRM). Using three cell lines of V79, L929, and Balb/3T3 cells, the cytotoxicity of the dithiocarbamates and the SRM films were compared by agar diffusion assay, filter diffusion assay, neutral red assay, cell growth assay, and colony assay. Among these in vitro cytotoxicity tests, colony assay was found to be the most sensitive method for detecting the cytotoxicity. The cytotoxic potentials of extracts from SRM films correlated well with the concentrations of ZDEC or ZDBC involved in SRM. When various rubber materials including SRM and surgical rubber latex materials were tested, cytotoxic potentials of these extracts were also correlated with the inflammatory tissue capsule thickness in short-term implantation tests. On the basis of these results, the SRM is judged to be useful for validating test sensitivity, and comparing the correlation between in vitro and in vivo responses. © 1993 John Wiley & Sons, Inc.

To clarify the relationship between eye irritancy and cytotoxic potential induced by irritant materials, we made lenses coated with standard reference materials (SRMs) prepared from various amounts of zinc diethyldithiocarbamate (ZDEC) and polyurethane (PU). Zinc diethyldithiocarbamate was classified as a mild irritant by the Draize eye irritation test. When ZDEC-SRM coated contact lenses were applied to rabbit eyes, Draize scores increased in proportion to both the ZDEC and PU concentrations used for coating. Furthermore, correlation with the cytotoxic potential (gamma = -0.93) was better than with lactate dehydrogenase (LDH) activities of tears from rabbit eyes wearing these coated lenses (gamma = 0.78). In conclusion, in vivo eye irritancy induced by wearing lenses could be estimated quantitatively with the cytotoxic potentials using a colony assay. Furthermore, we could compare different sensitivities caused by the same set of SRMs among three different sites of tissue. As a result, the order of sensitivity was eye > muscle >> skin.

Methyl methacrylate polymer (PolyMMA) is widely used as the composite resin for the dental plate. During the fabrication process of PolyMMA for the polymerization reaction, benzoylperoxide (BPO) and N,N-dimethyl p-toluidine (DMPT) are added as the initiator and the stimulator, respectively. Because these compounds exhibit toxicity as well as a residue potential, their use raises concerns regarding human safety. The degree of dissolution into serum was determined to evaluate risk to the user. Analysis was by HPLC combined with solid-phase extraction using a C-18 column. The eluted compounds were found to be in the order of 10 to 100 ppm.

The sensitizing abilities of 4 rubber additives, tetramethylthiuram disulfide (TMTD). 2-mercaptobenzothiazole (MBT), N-isopropyl-N'-phenyl-p-phenylenediamine (IPPD) and zinc diethyldithiocarbamate (ZDEC), were evaluated using the murine local lymph node assay. Exposure to IPPD induced a significant increase of lymph node cell proliferation in the draining lymph nodes even at a low concentration. Exposure to TMTD and MBT induced moderate proliferative responses, while ZDEC induced a weak proliferation even at the higher concentrations. The sensitizing potency of each chemical was described in terms of the concentration that increased lymph node cell proliferation by a factor of 2 over that in the vehicle-treated control group. The concentrations of IPPD, TMTD, MBT and ZDEC were 0.14%, 0.35%, 4.2% and > 10%, respectively.

A predictive test using mice for the identification of contact sensitizing chemicals was developed. Contact sensitizing activity is measured as a function of draining lymph node activation following application of test chemical. Experimental conditions for assessment of induced lymph node cell (LNC) responses have been optimized. BALB/c mice were initially treated with intradermal injections of test chemical in Freund's complete adjuvant emulsion. Five days after intradermal injection, mice were exposed topically to chemical in vehicle on the ears daily for 3 consecutive days. Next day following the final exposure, changes in lymph node weight, total cell number in the draining lymph nodes and LNC proliferation for 24 h culture were assessed. The performance of the method was evaluated with ten sensitizing chemicals and a non-sensitizing irritant, sodium lauryl sulfate (SLS). The LNC proliferation induced by combination of intradermal injection and topical application of sensitizing chemicals was more clearly increased than that following only topical application. With the single exception of sulfanilic acid, the method developed was able to detect the sensitizing capacity of chemicals that failed to induce sensitization in the local lymph node assay. Under the conditions used, SLS did not induce measurable lymph node responses. These results suggest that the mouse lymph node assay can provide a sensitive screening test for weak to moderate sensitizers. In addition, the assay offers the advantages of objective and quantitative endpoints, and is suitable for the evaluation of colored or irritant chemicals.

Colony assay using V79 cells, the agar diffusion assay with L929 cells, and the 7-day rabbit muscle implantation test were employed to evaluate the cytotoxicity and tissue toxicity of natural rubber latex (NRL) materials. The in vivo implantation test showed that, among 13 histological parameters, thickness of inflammatory layer was the most useful index to evaluate tissue responses quantitatively. A comparison of the in vivo and in vitro parameters revealed the following correlations between the thickness of the inflammatory layer and cytotoxicity indices: Colony assay of the extracts, IC50: r = 0.80; Agar diffusion assay, Zone index: r = 0.73; Lysis index: r = 0.61. From these results, it appears that the colony assay provides a more reliable prediction of the tissue response than the agar diffusion assay.

The biocompatibility of nine different materials, including positive and negative references, 4 polyurethane based and 3 latex based materials was investigated by (1) cytotoxic assay using V79 chinese hamster cells, (2) the thickness of inflammatory layer at 3 and 7 days after intramuscular implantation test, and (3) the course of the blood flow in tissue reaction around subcutaneously implanted materials using Laser Doppler Flowmetry (LDF) over 14 days following implantation. In addition, for some materials, different modes of sterilization were compared. Although the three methods explore different reactive systems, the material ranking obtained was highly similar for the three methods, suggesting a relative accuracy between them. For one latex however, an absence of cytotoxic effect in culture and a highly intense response by LDF investigation of the same order of magnitude as for the positive reference implant suggest that bioincompatibility may result from the material itself and cannot be exclusively investigated by the leaching of toxic components.

The effects of ethylenethiourea (ETU) were investigated using rat (Wistar-imamichi) embryos cultured from days 11 to 13 of gestation or cultured rat embryonic cells extracted on day 11. Malformations in cultured embryos at the concentration of 30 micrograms/ml of ETU were found in the head and tail, which were severely affected, as well as the limb and face. All embryos exposed to 150 and 300 micrograms/ml of ETU had malformed heads, tails, limbs, and facial configurations. Protein contents of the cultured embryos were decreased dose-dependently at the concentrations ranging from 30 to 300 micrograms/ml. In the histological studies of the cultured embryos with ETU, thinner neuroepithelium in head was observed. In the embryonic cells extracted on day 11 of gestation, ETU dose-dependently inhibited the differentiation of midbrain (MB) cells into neurons and that of limb bud (LB) cells into chondrocytes at the concentrations ranging from 30 to 600 micrograms/ml of ETU. The concentrations of ETU that inhibited the production of differentiated foci by 50% (IC50) were 170 micrograms/ml in LB cells of day 11, and greater than 600 micrograms/ml in LB cells on day 12 of development. Therefore, differentiation of MB cells was more sensitive to ETU than the differentiation of LB cells. These results indicated that there was a reasonable correlation of ETU induced changes in cultured whole embryos and embryonic cells.

New herbicidal compounds (11 pyrimidine-diones, 3 benzoates and 1 sulfonamide) were found to be embryolethal but not teratogenic in rats. The range of the embryolethal dose varied from 0.2 to greater than 200 mg/kg. This broad range enabled us to validate whether proposed in vitro teratogen tests can detect the embryolethality of these herbicides. The IC50 values (inhibition concentration 50%) for both differentiation and proliferation of midbrain and limb bud cells of rat embryos were determined and found to be above 50 micrograms/ml in all cases, confirming that the herbicides were not teratogenic. No correlation, however, was observed between the embryolethality in vivo and the activities in these cells. In order to test whether the potential to cause embryolethality could be predicted and detected as a general cytotoxic effect, the inhibition of colony forming ability in V79 cells was determined. The results indicated that cytotoxicity in V79 cells may be useful for preliminary testing of the embryolethal effect of herbicides.

Rationale As sequence information on genes and proteins is accumulated in databases, it becomes easy to identify a large number of proteins in a short time with proteomics approaches. Application of such techniques to allergenic proteins is referred to allergenomics. In this study, we tried to find unidentified latex allergens with an allergenomics method. Methods Proteins extracted from non-ammoniated rubber latex was two-dimensionally separated according to their isoelectric points (pI) and molecular weights then transferred onto a membrane. IgE-recognizing proteins were visualized by immunoblotting using a pooled serum from latex-allergic people. Spots of the allergenic proteins were cut out form the gel and the allergens were site-specifically in-gel digested by trypsin. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) spectra of the resultant fragments and MS/MS spectra of some prominent peptide-ions were recorded on a TOF-TOF instrument. From these mass-spectrometric data, the allergens were assigned by database searches. Results A few new latex allergens as well as several previously reported allergens such as patatin-like proteins (Hev b 7), enolase (Hev b 9), and havamine were identified with this approach. One of the novel latex allergens (32 kD pI 5.2) was sequentially homologous to plant defense-related reductases like isoflavone reductase and phenylcoumaran benzylic ether reductase. Conclusions We first identified a defense-related reductase as a latex allergen. Several allergens belonging to the plant defense-related reductase family (Bet v 6-related allergens) have already been found from fruits, vegetables, and pollen. Considering the sequence similarities among them, we can predict the cross-reactivity of the defense-related reductase found from natural rubber.