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To facilitate research and development (R&D) and to expedite the review processes of medical devices, the Ministry of Health, Labor and Welfare (MHLW) and the Ministry of Economy, Trade and Industry (METI) founded a joint committee to establish guidance for newly emerging technology. From 2005 to 2007, two working groups held discussions on ventricular assist devices and total artificial hearts, including out-of-hospital programs, based on previous guidance documents and standards. Based on this discussion, the METI published the R&D Guidelines for innovative artificial hearts in 2007, and in 2008 the MHLW published a Notification by Director regarding the evaluation criteria for emerging technology.

The international standard ISO 8124-3: 1997 "Safety of toys -Part 3: Migration of certain elements" and "Interim Enforcement Policy for Children's Metal Jewelry Containing Lead- 2/3/2005" by the U.S. Consumer Product Safety Commission (CPSC) to control the amount of eluted lead from metal accessories cannot be simply compared, because the acid extraction methods and the limit values are different from each other. Therefore acid extraction tests based on the ISO standard and the CPSC policy were conducted and the amounts of eluted lead from small metal products were compared between both tests. There was less eluted lead in the ISO method than in the CPSC method. Moreover, the amount of eluted lead in the ISO method did not even reach that of the first elution in the CPSC method. It became clear that the acid extraction test of the ISO standard was not as good as that of the CPSC policy, because of the difference in test conditions. In 16 small metal products, seven products were unsuitable for the ISO standard and 14 products were unsuitable for the CPSC policy; however, all these products were originally inapplicable to the ISO standard and the CPSC policy. The calculation grounds of the limit values were also different between the ISO standard and the CPSC policy. The standardization of an acid extraction test that simulates the lead elution to gastric juice is required so as to prevent adverse health effects in children due to their accidental ingestion of small metal products containing lead.

Many studies have been performed to accelerate osteoinduction and osteoconduction into porous ceramic scaffolds by seeding them with cells. In this study, we compared available cell-seeding methods on a porous β-tricalcium phosphate (β-TCP) scaffold and evaluated the effects of cell-seeding on the mechanical properties of the porous β-TCP scaffold. Three types of porous bioceramic scaffolds were used: dry scaffold, scaffold wetted with media, and scaffold cultivated with normal human osteoblasts (NHOs). Cell-seeding into the porous β-TCP scaffolds was performed by conventional, centrifuge, high-density, and vacuum methods. After confirming cell proliferation with MTT assay and cell staining, a compressive test was performed after 2 and 4 weeks of cell culture. The vacuum method based on the high-density cell culture inserted effectively NHOs into the β-TCP scaffolds. The compressive elastic modulus of wetted β-TCP scaffolds decreased significantly (p < 0.05) about 20∼30% after 2 and 4 weeks of incubation in comparison with that of the dry scaffold. However, the compressive strength of the scaffolds cultivated with NHOs for 3 weeks was significantly (p < 0.05) higher than that of scaffolds without NHOs. The vacuum with the high-density of cell-seeding seems to be a suitable method for seeding cells into complex porous ceramic scaffolds. Cell proliferation and uniform distribution in the scaffolds can change the initial mechanical properties of porous ceramic scaffolds.

The use of tributyltin (TBT) and triphenyltin (TPT) in some household products are prohibited by "Act on the Control of Household Products Containing Harmful Substances" in Japan. In this study, methods for determination of TBT and TPT in water soluble paints and adhesives were developed by GC-MS. These compounds in paints and adhesives, which were mainly composed of vinyl acetate, urethane and acryl resins, and chloroprene rubber, were firstly extracted with HCl-acetone, and then extracted with hexane. On the other hand, the adhesive composed of natural rubber was firstly dispersed in water before acidification. The organotins were extracted with hexane from this solution and then these compounds were extracted with acetonitrile from hexane extract. These extracts were purified by a florisil cartridge column after ethyl-derivation with sodium tetraethylborate, and analyzed by GC-MS. The quantifications using deuterated compound of both organotins as surrogate standard were conducted, and good results were obtained. The recoveries were 81 to 118% and the coefficients of variation were 0.83 to 4.3% (TBT and TPT added; 5 microg/g). The method quantification limits were 0.0090 to 0.025 microg/g, which were lower than those of an official method. These methods were applied to monobutyltin (MBT), dibutyltin (DBT), monophenyltin (MPT), and diphenyltin (DPT). DBT and DPT in paints and adhesives were quantified, except for DPT in natural rubber. These methods were applied to commercial products. DBT was detected at low concentrations (t.r.-0.19 microg/g) in some paint samples, while TBT and TPT were not detected in all samples.

We examined the effects of serum-free medium on the gene expression changes in human mesenchymal stem cells (hMSCs) during the in vitro culture using a DNA microarray analysis. In this study, we cultured hMSCs with two kinds of medium; 1) MSCGM (contain 10% fetal bovine serum) or 2) STK2 (serum-free medium developed for mesechymal stem cells multiplication), and compared hMSCs proliferation, cell morphology, and gene expression changes until 50 days culture. Expression analysis was performed with Affymetrix GeneChip Human Genome U133 Plus 2.0 Array. hMSC proliferation was significantly higher in STK2 medium than in MSCGM medium. The cell morphology of hMSC cultured with STK2 was not significantly changed in 50 days culture. The gene expression changes in hMSCs during the in vitro culture were significantly higher in STK2 than in MSCGM. After 50 days culture, 1991 genes were significantly changed the expression levels compared with 3 days in STK2 but not MSCGM. The expressions of genes related to cell cycle, cancer, proliferation, and cell growth were significantly changed by STK2 for 50 days culture. It was also changed by STK2 that the expressions of genes related to the signaling pathways contain various growth factors, such as IGF-1, FGF, TGF-β, EGF, proliferation, and cell cycle. These results suggest that STK2 may be useful to obtain an enough number of hMSC cells for tissue engineered medical devices in short-term, however, it should be recognized that STK2 would alter the expressions of genes related to a variety of signaling pathways in hMSC if the culture period would be extended to obtain a large number of cells.

Ultra-high molecular weight polyethylene (UHMWPE) has been widely used as a bearing surface of joint implants. Since wear of UHMWPE component has been the primal cause of implant failure, crosslinking of UHMWPE is now widely used to improve its wear property. On the other hand, crosslinking degrades its fatigue property and increase of fatigue related failure, such as delamination, is possible. However, the relationship between degraded fatigue property and implant failure is not clear, primarily because the fatigue properties of failed and retrieved UHMWPE components are not known. In this study, we propose a new test method to evaluate the fatigue property of retrieved UHMWPE components or final products using small specimens. First, the effects of test conditions were investigated using virgin UHMWPE. Then, tests were repeated using specimens from UHMWPE components of retrieved knee implants. The fatigue properties of virgin and retrieved UHMWPE were suc- cessfully evaluated by the new test method and a relationship was found among the extent of oxidation, the fatigue property, and occurrence of delamination. This result suggested that evaluation of fatigue property of UHMWPE component can predict the occurrence of fatigue related implant failure during operation in vivo.

To evaluate the role of particle size in cytotoxicity tests of nanomaterials (NMs), we exposed Chinese hamster cells to polystyrene (PS) spheres with defined diameters ranging from 0.1 to 9.2 microm. We found that the 4.45-microm PS particles were most cytotoxic while sizes 0.1 and 0.2 microm showed no cytotoxicity up to 1000 microg/ml. In the chromosome aberration test, the 4.45-microm PS particles induced polyploidy in a mass concentration-dependent manner in 24- and 48-h treatments. The 5.26-microm PS particles induced polyploidy only at 1000 microg/ml for 48 h. Next, we performed the cytotoxicity test with as-grown single walled carbon nanohorns (NHas). These were suspended in DMSO and then transferred into the culture medium followed by sonication. Six suspensions differently sonicated showed the same apparent toxicity, although the total particle size distributions differed. However, the sizes of NHas particles predicted to be most toxic from the experiments with PS particles, i.e. 1.01-4.47 microm constituted 40-60% of all particles in all six suspensions. The results suggest that the cytotoxicity of NMs in suspension depends on specific sizes of aggregates and therefore suspensions should be checked with regard to particle size distributions in assays of toxic effects. The uptake of particles into cells was confirmed by confocal microscopy.

The purpose of tumorigenicity testing, as applied not only to cell substrates used for viral vaccine manufacture but also stem cells used for cell-based therapy, is to discriminate between cells that have the capacity to form tumors and cells that do not. Therefore, tumorigenicity testing is essential in assessing the safety of these biological materials. Recently developed NOD/Shi-scid IL2Rg(null) (NOG) mice have been shown to be superior to NOD/Shi-scid (SCID) mice for xenotransplantation of both normal and cancerous cells. To select a suitable mouse strain as a xenogenic host for tumorigenicity testing, we compared the susceptibility of NOG (T, B, and NK cell-defective), SCID (T and B cell-defective), and the traditionally used nude (T cell-defective) mice to tumor formation from xenotransplanted HeLa S3 cells. When 10(4) HeLa S3 cells were subcutaneously inoculated into the flanks of these mice, the tumor incidence on day 22 was 10/10 (100%) in NOG, 2/10 (20%) in SCID, and 0/10 (0%) in nude mice. The subcutaneous tumors formed reproducibly and semiquantitatively in a dose-dependent manner. Unexpectedly, half of the NOG mice (5/10) that had been inoculated with a mere 10(1) HeLa S3 cells formed progressively growing subcutaneous tumors on day 78. We confirmed that the engrafted tumors originated from inoculated HeLa S3 cells by immunohistochemical staining with anti-HLA antibodies. These data suggest that NOG mice may be the best choice as a suitable strain for testing tumorigenicity.

We investigated mRNA expression of c-myc and chromosome aberrations at the c-myc locus in the same passage number of human mesenchymal stem cells (hMSCs). To understand the sensitivity of mRNA expression and the induction of chromosome aberrations, we first tested them in hMSC and cancer cell lines (HeLa S3, HOS, and OUMS-27). The c-myc mRNA expressions in HeLa S3 and OUMS-27 were significantly higher than those in hMSC, but then those in HOS were not. On the other hand, c-myc aberrant cells detected by fluorescence in situ hybridization in HeLa S3, HOS, and OUMS-27 were significantly higher than that in hMSC. Both analyses were performed in hMSCs derived from five donors for the culture period of 50 days. In hMSCs from one donor, the frequency of c-myc aberrant cells significantly increased at 20 and 50 days respectively, and each mRNA expressions had a tendency to increase, but there is no significant change among 3, 20 and 50 days. In hMSCs from the others, both endpoints did not change for 50 days. For safe use of somatic stem cells in the regenerative medicine, the investigation of characteristic change of them during the in vitro culture is important. In the present study, we showed the mRNA expressions and chromosome aberrations of hMSCs in in vitro culture as the first step for establishing of safety evaluation of tissue engineered medical devices using normal hMSCs.

Stannous 2-ethylhexanoate [Sn(Oct)2] is used as a catalyst for production of poly-L-lactic acid and copolymers that are implanted in cranial surgery, but reports on its effects on the central nervous system are few. We examined the effects of Sn(Oct)2 on cell viability in vitro and on neurotransmission and behavior in the rat. Treatment of normal human astrocytes with 10 mg/mL Sn(Oct)2 reduced mitochondrial activity to 16% of the control. Injection of Sn(Oct)2 at 6.28 mg/kg BW (2 mg/kg BW Sn) into right lateral ventricle of the rat brain tended to increase the ambulation distance after 30 days when compared with the control group. The turnover of dopamine neurotransmission was increased in the cerebral cortex. These results suggest that Sn(Oct)2 is cytotoxic to astrocytes in vitro. Injection of Sn(Oct)2 into the brain had no or very weak immediate neurotoxicity, but long-term exposure to Sn(Oct)2 increased dopamine neurotransmission turnover. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2008

We demonstrated the effect of synthesized sulfated hyaluronan (SHya), which is composed of a sulfated group and hyaluronan, and basic fibroblast growth factor 2 (FGF-2) on normal human astrocytes (NHA) activity and its morphological transformation in vitro study. Astrocyte is a kind of glial cell and stellated astrocyte (activating astrocyte) supports axons network, neurons survival and synaptic plasticity. Treatment of SHya hardly affected NHA proliferation. However combination treatment of SHya and FGF-2 increased NHA proliferation. Treatment of SHya promoted transformation of normal astrocyte into a stella morphology (stellation) and combination treatment of SHya and FGF-2 promoted stellation than that of SHya only. Treatment of SHya increased glial fibrillary acidic protein (GFAP), nestin mRNA and GFAP protein expression in the stellated NHA. The cell-cell adhesion of NHA increased by treatment of SHya. Treatment of SHya increased heparin-binding trophic factors FGF-2, midkine, and some other trophic factors mRNA level in the NHA. These results suggested that the treatment of SHya promoted NHA activity due to enhancing neurotrophins production and the morphological transformation of NHA and the effect of SHya on astrocytes partly involved FGF-2 activity. These findings indicate that SHya may be involved in the astrocyte activity and support neurons survivals.

Fullerenes are condensed ring aromatic compounds with extended π systems and unique cage structures. Fullerenes are used for medical devices such as carbon nanotubes because they are very flexible and suitable for drug delivery systems. Recently, fullerene derivatives and tube-shaped materials have been used for neuroregeneration studies, and we expect that fullerenes and carbon nanotubes have potential uses as materials in novel medical devices targeting the brain. However, little information on the effects of fullerenes on brain function is available; thus, we examined the effects of [60]fullerene (C60) on the central nervous system in this study. In a V79 cell colony Asia, the IC50 of C60 was 1620 μg/ml. In an in vivo study, 0.25 mg/kg B.W. of C60 was injected into the lateral brain ventricle or abdominal cavity of rats. The intracerebral injection of C60 increased the locomotor behavior of the rats on days 1 and 30 after the injection. The intraperitoneal injection of C60 did not change the locomotor behavior of rats acutely, but it was decreased on day 30. The intracerebral injection of C60 affected monoamine concentrations in the rat brain. In particular, serotonin turnover rates were increased in the hypothalamus, cerebral cortex, striatum, and hippocampus, and dopamine turnover rates were increased in the hypothalamus, cerebral cortex, and striatum. The intraperitoneal injection of C60 decreased only the dopamine turnover rate in the hippocampus. These results suggest that intracerebral injection of C60 had different effects on the central nervous system than intraperitoneal injection. In conclusion, it was suggested that fullerene did not cross the blood-brain barrier. The intracerebral injection of C60 affected neurotransmission in the brain widely, and the monoamine dysbolism might be related to changes in locomotor activity.

We prepared polyurethane (PU) containing 0.4% or 4% 4,4'-methylenedianiline (MDA) as model materials to investigate the effectiveness of sample preparation by organic-solvent extraction for the in vitro chromosome aberration (CA) test. MDA itself (0.4 mg/mL) was positive only in the presence of an exogenous metabolizing system (S9 mix). The culture medium extract of PU containing 4% MDA (PU/4% MDA) was negative with and without S9 mix. Methanol and acetone extracts, on the other hand, induced structural CAs without S9 mix, which we did not expect because MDA requires S9 mix for activity. On chemical analysis, however, we found that the ratio of MDA extracted by the organic solvents to that extracted by the culture medium of PU/4% MDA was about 15:1. Interestingly, oligomers consisting of poly(tetramethyleneglycol) derivatives (OTMG) were also extracted by the organic solvents. The data suggest that the induction of structural CAs in the absence of S9 mix may have been partly due to synergism of MDA and OTMG. CA tests of MDA and PTMG-1000 in combination confirmed that to be the case. Thus, organic-solvent extraction may be more effective than medium extraction in evaluating the biological safety of biomaterials. Detailed chemical analysis of extracts was performed.

Accurate measurement of the mechanical properties of artificial or cultivated cartilage is a major factor for determining successive regeneration of defective soft tissues. In this study, we developed a novel method that enabled the bulk modulus (k-modulus) to be measured nondestructively using the relationship between volume and pressure of living soft tissues. In order to validate this method we estimated the bulk modulus of soft silicone rubbers using our new method and a conventional method. The results showed a 5 ~ 10% difference between the results obtained with the two methods. Our method was used subsequently to measure the mechanical properties of cultivated cartilage samples (collagen gel type), that had been incubated for four weeks in the presence or absence of human articular chondrocytes (HACs). Our experiments showed that cultivated cartilage tissues grown in the presence of HACs had a higher bulk modulus (120 ± 20 kPa) than samples grown without HACs (90 ± 15 kPa). The results indicated that our novel method offered an effective method for measurement of volume changes in minute living soft tissues, with the measurements having a high degree of accuracy and precision. Furthermore, this method has significant advantages over conventional approaches as it can be used to rapidly and accurately evaluate the strength of soft tissues during cultivation without causing damage to the specimen.

Biodegradable polymers are often used as scaffolds for tissue engineering and these polymers are in class IV under the revised Pharmaceutical Affairs Law. From the point of view of safety and efficacy, recent problems in the development of tissue-engineered products using biodegradable polymers are summarized in this report.

To promote the osteogenic properties of osteoblasts, we synthesized a hydroxyapatite (HAp) with beta-tricalcium phosphate (beta-TCP) biphasic calcium phosphate containing Nb ions (NbTCP/HAp). NbTCP/HAp was prepared by annealing precipitates obtained by coprecipitation of an aqueous solution of Ca(NO(3))(2) and a mixture of (NH(4))(2)HPO(4) and aqueous Nb solution. The precipitates can be regarded as a calcium-deficient HAp, the PO(4) sites of which are partly occupied by Nb ions. NbTCP/HAp was successfully synthesized by thermal decomposition of the precipitates. NbTCP/HAp enhanced the calcification of normal human osteoblasts (NHOst), and the amount of calcified tissue increased in proportion to the Nb ion concentration in the NbTCP/HAp. The alkaline phosphatase (ALP) activity of NHOst was also enhanced by NbTCP/HAp. Because Nb ions significantly enhance the ALP activity of NHOst, calcification by NbTCP/HAp is considered to be due to enhancement of ALP activity induced by Nb ions dissolved from NbTCP/HAp. These results indicate that NbTCP/HAp can be an effective bone repair material.

We investigated the expression levels of several genes related to cell proliferation in human mesenchymal stem cells (hMSCs) during in vitro culture for use in clinical applications. In this study, we focused on the relationship between hMSC proliferation and transforming growth factor beta (TGFbeta) signaling during in vitro culture. The proliferation rate of hMSCs gradually decreased and marked changes in hMSC morphology were not observed in 3 months of in vitro culture. The mRNA expressions of TGFbeta1, TGFbeta2, and TGFbeta receptor type I (TGFbetaRI) in hMSCs increased with the length of cell culture. There had been no change in the TGFbeta3, TGFbetaRII, and TGFbetaRIII mRNA expressions by the 12th passage from the primary culture (at about 3 months). The mRNA expression of Smad3 increased, but those of c-myc and nucleostemin decreased with the length of hMSC in vitro culture. In addition, the expression profiles of the genes that regulate cellular proliferation in hMSCs were significantly different from those of cancer cells. In conclusion, hMSCs derived from bone marrow seldom underwent spontaneous transformation during 1-2 months of in vitro culture for use in clinical applications. In hMSCs as well as in epithelial cells, growth might be controlled by the TGFbeta family signaling.

The cytotoxicity of five calcium phosphate ceramics, hydroxyapatite (HAp), flouroapatite (FAp), α-tricalcium phosphate (α-TCP), β-tricalcium phosphate (β-TCP) and tetracalcium phosphate (TTCP), was investigated. Based on the guidelines of biological test for medical devices in Japan, a cytotoxicity test of these calcium phosphates was carried out using Chinese hamster V79 lung fibroblasts. The cytotoxic study revealed that FAp and α-TCP showed high cytotoxicities. From various analyses, it was considered that the cytotoxicity of the FAp was due to fluorine ions extracted in a culture medium and the cytotoxicity of α-TCP resulted from a decrease in pH of the medium by the phosphoric acid, which produced by hydrolysis of( the α-TCP.

The aseptic loosening of artificial joints with associated periprosthetic bone resorption may be partly due to the suppression of osteoblast function to form new bone by wear debris derived from the joint. To assess the effect of wear debris on osteoblasts, we cultured normal human osteoblasts (NHOst) in contact with several kinds of microspheres as models of wear debris. The NHOst in contact with polystyrene, polyethylene, and alumina microspheres showed a lower differentiation level than NHOst alone as estimated from the amounts of deposited calcium. On the other hand, hydroxyapatite particles enhanced the differentiation of NHOst. In addition, sintered hydroxyapatite enhanced expression of osteocalcin mRNA and gap junctional communication of NHOst. This study suggests that polystyrene, polyethylene, and alumina microspheres have the potential to disorder not only the differentiation but also the homeostasis of NHOst in contact with them. However, hydroxyapatite enhanced the differentiation as well as the homeostasis of NHOst, even in microsphere form, suggesting its good biocompatibility as biomaterials for bone tissues.

To promote the activity of normal human osteoblasts (NHOst), the novel HAp ceramics containing Nb ions (NbHAp) were synthesized by wet chemical process, which reacting aqueous solution containing a mixture of Ca(NO3)2, (NH4)2HPO4, and the Nb aqueous solution. X-ray diffraction patterns indicated that NbHAp had a monolithic apatitic structure, although crystallite decreased as Nb content increased. From inductively coupled plasma analysis, maximum amount of Nb ions in the sample was almost 8.2atom% of P ions. The NbHAps were presented as aggregates and composed of fine crystal of

A source of morphologically and functionally available human cartilagenous tissue for implantation is required in the field of tissue engineering. To achieve this goal, we evaluated the effects of hyaluronic acid (HA-810 and 1680 kDa), and chondroitin sulfate (CS-A 16 and C-34 kDa) on human articular chondrocytes (HC) in micromass and rotation culture conditions. Cell proliferation was increased by CS-A 16 kDa under micromass and rotation cultures, while cell differentiation was increased under rotation but not micromass conditions. Proliferation and differentiation due to CS-C 34 kDa were very similar to the control under both culture conditions. With HA, cell proliferation was increased depending on the molecular weight under micromass and rotation conditions. In contrast, chondrocyte differentiation was enhanced under rotation conditions, but decreased under micromass conditions depending on the molecular weight of HA. In both culture conditions, aggrecan gene was continuously expressed. However, the collagen type II gene was more weakly expressed in rotation than the micromass culture conditions. Thus, the chemical structures of polysaccharides, and the culture condition, rotation or micromass, caused differences in chondrogenesis.

The aim of this study was to evaluate the potential role of polyglycolic acid (PGA), poly(glycolic acid-epsilon-caprolactone) (PGCL), poly(L-lactic acid-glycolic acid) (PLGA), poly(L-lactic acid-epsilon-caprolactone, 75:25 (w/w)) [P(LA-CL)25], poly-epsilon-caprolactone (tetrabutoxy titanium) [PCL(Ti)], and fullerene C-60 dimalonic acid (DMA) in cartilage transplants. After 4 weeks of culture of human articular cartilage, the levels of cell proliferation and differentiation and the expression of cartilage-specific matrix genes were estimated. The relationship between cell differentiation and gap junction protein connexin 43 (Cx43) was also evaluated. All materials except PCL(Ti) retained cell proliferation activities similar to the controls. Cell differentiation levels from the highest to the lowest were in the following order: PGA >> PLGA > PGCL > Control = DMSO > P(LA-CL)25 = PCL(Ti) >> fullerene C-60 DMA. Expression of the collagen type II gene was selectively upregulated for PGA, PGCL, and PLGA and slightly increased for P(LA-CL)25 polymers but was downregulated for fullerene C-60 DMA. Aggrecan gene expression was strongest with PGA and was consistently expressed with other matrices, especially with PGCL and PLGA. However, the expression patterns of the connexin 43 gene were different from the former two genes. Multiple regression analysis revealed a high correlation between cartilage proteoglycans production and expression levels of these three genes.

We attempted to determine the processing conditions for decreasing the migration of phthalate esters, particularly di-2-ethylhexyl phthalate (DEHP), from polyvinyl chloride (PVC) products using a drug solvent after dilution based on the package insert. PVC sheets and PVC tubing were subjected to optical irradiation (ultraviolet (UV), visible light irradiation) and heat treatment to determine whether they are deteriorated by these treatments. UV irradiation to one side of the PVC sheet decreased the levels of DEHP migration from the sheets by almost 50%, although the amount of DEHP content in PVC sheet was observed no significant change. On the other hand, the levels of DEHP migrating from the inner surface of PVC tubing UV-irradiated from the outer surface were not decreased compared with the control. Therefore, the surface structure was examined by conducting Fourier transform infrared spectroscopy (FT-IR), electron spectroscopy for chemical analysis (ESCA) and static angle of contact measurement. In FT-IR analysis, we found that the UV-irradiated PVC sheets were exhibited broadened absorption bands with time. In ESCA analysis, the chlorine content was decreased and the oxygen content was increased with time in UV-irradiated PVC sheets. Moreover, the other treated PVC sheets shows no significant change compared with the non-UV-irradiated PVC sheet. Therefore, the surface structure of the UV-irradiated PVC sheet was changed. As a result, the migration of DEHP from PVC products can be decreased with simple treatment, such as UV-irradiation. This could be a useful method to develop novel PVC products.

Polyelectrolyte complexes (PEC) were prepared from chitosan as the polycation and several synthesized functional anion polysaccharides, and their effects on cell attachment, morphology, proliferation and differentiation were estimated using normal human osteoblasts (NHOst). After a 1-week incubation, PEC made from polysaccharides having carboxyl groups as polyanions showed low viability of NHOst on it although the NHOst on it showed an enhancement in their differentiation level. On the other hand, NHOst on PEC made from sulfated or phosphated polysaccharides showed similar attachment and morphology to those on the collagen-coated dish. When the number of NHOst was estimated after 1 week, the number on the PEC was ranged from 70% to 130% of those on the collagen-coated dish, indicating few effects of these PEC on cell proliferation. In addition, NHOst on PEC films made from sulfated polysaccharides differentiated to a level very similar to that observed on the collagen-coated dish, indicating that these PEC films maintain the normal potential of NHOst to both proliferate and differentiate. Measurement of gap junctional intercellular communication of NHOst on PEC revealed that PEC did not inhibit communication, suggesting that PEC films have few effects on cell homeostasis. Thus, PEC made from the sulfated polysaccharide may be a useful material as a new scaffold for bone regeneration.

Technology developed to allow recovery of cells without enzyme treatment, involving a dish grafted with a thermoreactive polymer gel of poly-N-isopropylacrylamide (PIPAAm), was found to significantly enhance gap junctional intercellular communication (GJIC) in normal human dermal fibroblasts (NHDF cells). NHDF cells were cultured for 4 days on PIPAAm-grafted dishes irradiated with various doses of electron beams, and GJIC was assayed by the scrape-loading dye transfer method. The area of dye transfer was greater in the PIPAAm-grafted dishes than in the control culture dishes, indicating that the PIPAAm-grafted dishes enhanced the GJIC of NHDF cells. Connexin-43 (Cx43) expression was analyzed because Cx43 is considered to be a main component of the gap junctional channel. PIPAAm-grafted dishes irradiated with 100, 250, or 500 kGy of electron beams showed significantly enhanced expression of Cx43-NP, Cx43-P1, and especially Cx43-P2. Enhanced expression of Cx43-P2, a functional transmembrane protein, may be related to the promotion of GJIC. These results suggest that the PIPAAm-grafted dish not only enables the enzyme-free recovery of a cell monolayer for use in the construction of a three-dimensional artificial tissue, but also significantly contributes to the enhancement of GJIC, which may partly promote tissue strength on the surface of the PIPAAm-grafted dish.

The recycling of disused railway sleepers treated with wood preservatives such as creosote as exterior wood for use in gardens has recently become popular in Japan. Creosote contains high quantities of polycyclic aromatic hydrocarbons (PAHs), and can lead to skin irritation and disease. In this work we have determined the amount of PAHs and water-extractable phenols in creosote and creosote-treated wood products such as railway sleepers and stakes for agricultural use that are either made or are procurable in Japan. PAHs were extracted with dichloromethane and analyzed by gas chromatography-mass spectrometry. Among carcinogenic PAHs, benz(a)anthracene was detected in the highest concentration, varying between 228 and 6328 microg/g in creosotes. Benzo(b)fluoranthene, benzo(k)fluoranthene and benzo(a)pyrene (BaP) were found in the range of 67-3541 microg/g. Almost all creosotes contained more than 50 microg/g of BaP, which is the upper limit level that is permitted in the European Union (EU). Creosote-impregnated wood products, such as brand-new or secondhand railway sleepers and foundations, contained large amounts of BaP (58-749 microg/g) and benz(a)anthracene (250-1282 microg/g). Concentrations of between 692 and 2489 microg/g of phenols were determined in the water extracts from creosotes, but the level was considerably less than the EU control value (3% by mass), and there was no correlation between the amount of water-extractable phenols and the amount of PAHs detected in each sample. The situation that consumers are free to use the creosotes containing a high concentration of carcinogens such as BaP may cause unacceptable damage to the health of persons handling these creosote products.

This study deals with the development of a simple method for predicting the elution levels of di-2-ethylhexyl phthalate (DEHP) from medical devices made of polyvinyl chloride (PVC) by using the physicochemical properties of pharmaceutical injections as a marker. GC-MS analysis showed that the release of DEHP from medical grade PVC product was concentration-dependently increased by extraction with two kinds of lipophilic injections (Sandimmun and Prograf) and three kinds of surfactants (HCO-60, Tween 80, and SDS). The solubility of lipophilic pigments such as Sudan III, methyl yellow, and 1,4-diamino-anthraquinone against these solutions were also increased in a concentration-dependent manner, in which methyl yellow showed the highest response regarding the increase of optical density (O.D.). Further, electrical conductivity and static contact angle to the PVC sheet of the solutions were also increased or decreased in the same manner. As a result of the comparative study, significant correlation was found between DEHP release levels and these three physicochemical properties, particularly methyl yellow solubility, of the solutions tested. To evaluate the relationship in detail, DEHP release levels from PVC tubing and methyl yellow solubility of 53 injections used in gynecologic and obstetric fields were determined. None of the hydrophilic medicines showed any significant release of DEHP, and all showed low solubility of methyl yellow. On the other hand, the lipophilic medicines releasing a large amount of DEHP showed high solubility of methyl yellow (greater than O.D. 0.8). These results indicate that a significant proportional relationship exists between DEHP release potency and methyl yellow solubility of pharmaceutical solutions, and the risk of DEHP exposure to the patients administered pharmaceuticals through transfusion set could be easily predicted by the solubility test without complicated elution tests of DEHP using GC-MS or LC-MS.

The effects of the γ-irradiatted PLLA on the osteoblasts and apatite formation were investigated in vitro. The PLLA sheet was γ-ray irradiated at the dose of 10, 25 or 50 kGy. The mouse osteoblast-like MC3T3-E1 cells and normal human osteoblast NHOst cells were micromass cultured on the PLLA sheet for 2 weeks, and then the proliferation and differentiation of the cells were determined. The proliferations of MC3T3-E1 and NHOst cells hardly changed with increasing irradiation dose. However, the differentiations of MC3T3-E1 and NHOst cells increased with irradiation dose. On the other hand, the surface of the PLLA sheet after soaking in the medium without the cells was characterized by SEM, EDX, FT-IR and XPS. The hydroxyapatite was formed on the surface of the PLLA sheet after soaking, and the amount of hydroxyapatite increased with irradiation dose. In summary, the γ-irradiated PLLA increased the differentiation of osteoblasts and also increased apatite-forming ability even without the osteoblasts. The osteoblast differentiation was enhanced well in the apatite formation on the surface of PLLA after the γ-irradiation.

The aseptic loosening of artificial joints with associated periprosthetic bone resorption may be partly due to the suppression of osteoblast function to form new bone by wear debris from the joint. To assess the effect of wear debris on osteoblasts, effects of model wear debris on gap junctional intercellular communication (GJIC) of normal human osteoblasts were estimated. The GJIC activity of the osteoblasts after a 1-day incubation with the microspheres was similar to that of normal osteoblasts. However, hydroxy apatite particles, which have been reported to enhance the differentiation of osteoblasts in contact with them, enhanced the GJIC function of the osteoblasts. From RT-PCR studies, not only connexin 43 but also connexin 45 is suggested to play a role in the GJIC of the osteoblasts in an early stage of coculture with the microspheres, although it is still unclear how these connexins work and are regulated in the GJIC and differentiation. However, this study suggests that there is a relationship between the early levels of GJIC and the differentiation of the cells. Therefore, estimating the effect of biomaterials, even in the microsphere form, on the GJIC of model cells, with which the biomaterials may be in contact in vivo, can provide important information about their biocompatibility.

We tested extracts of custom-made natural rubber samples for cytotoxicity using V79 cells and for chromosome aberration (CA) induction using CHL cells in compliance with the Japanese guidelines for basic biological tests of medical materials and devices. The samples were formulated with a high level of 2-mercaptobenzothiazole (MBT) (A); a low level of MBT (B); or zinc dibutyldithiocarbamate (ZDBC) (C). In the CA test, MBT induced mainly polyploidy, including endoreduplication, and ZDBC induced structural CAs. In the cytotoxicity test, culture medium extracts of A, B, and C suppressed colony formation to 50% of the control value at 53.1%, 94.3%, and >100%, respectively. Culture medium extracts of sample A induced polyploidy and structural CAs in the absence of an exogenous metabolic activation system (S9 mix), but at lower concentrations in its presence, indicating the existence of other leachable promutagens. The extracts of sample B induced structural CAs at the highest concentration and only with S9 mix. Sample C was negative. The facts suggest that sample A may be a candidate for a positive control for genotoxicity tests. The high frequency of polyploidy induced by sample A was not predicted by MBT, suggesting the usefulness of the test for safety evaluation of medical devices. Numerical CAs induced by MBT and sample A are discussed.

Titanium–zirconium (Ti–Zr) binary alloy has better corrosion resistance and mechanical properties than commercially pure Ti. The present study was designed to determine the biocompatibility of Ti–Zr alloy by an implantation test in animal bodies in comparison with pure Ti, Zr, and chromium (Cr) implants as positive controls. Sample specimens were placed in a subcutaneous position in rats for 8 months. No significant decreases in body weight, the weight of any organ, or the weight of any organ relative to body weight were found in the implant groups compared to a no-implant control group. On hematological examination, small differences in several parameters were found in some groups, but these changes were not attributable to the materials implanted. Mitogen-induced blastogenesis was observed in similar degrees among all implant groups. These results suggest that the implantation of test samples did not cause systemic toxicity or a decrease in immune activity. The fibrous capsule membranes around the Ti and Ti–Zr alloy implants were thinner than those around Cr implants. The numbers of macrophages, inflammatory cells, and other cells involved in immune responses in and around the fibrous capsules of the Cr- and Ti-implant groups were higher than those of the Ti–Zr alloy- and Zr-implant groups. The Ti–Zr alloy had the lowest total score of tissue responses among the materials tested. None of the animals from the Ti-, Zr-, and Ti–Zr alloy-implant groups exhibited a skin reaction following exposure to Ti or Zr salt solutions. These results indicate the Ti–Zr alloy has better biocompatibility than Ti for use as an artificial surgical implant.

The amount of benzo[a]pyrene (BaP), benz[a]anthracene (BaA), and dibenz[a,h]anthracene (DBA) has been restricted to a concentration of 10 g/g each in creosotes, and 3 g/g each in creosote-treated woods, respectively, because of the possibility of the risk of skin cancer in consumers, and creosotes can otherwise contain high concentrations of each chemical. We already reported the content of 16 polycyclic aromatic hydrocarbons (PAHs) and phenols in creosotes and creosote-treated wood as determined by gas chromatography-mass spectrometry (GC-MS) and absorptiometry [Chemosphere, 60, 1279–1287 (2005)]. However, the limit of determination of each PAH per sample was > 40 g/g according to that method, the sensitivity of which was insufficient for determining the allowable levels of these 3 compounds. Moreover, a substantial amount of time was needed for GC-MS analysis. In the present study, we improved upon our previous analytical method in order to increase the sensitivity of the test and to reduce the duration of GC-MS analysis. Creosote was extracted from treated wood samples by dichloromethane-soak incubation, and was placed on a Sep-Pak silica cartridge and eluted with dichloromethane. The eluates were evaporated and dissolved in dichloromethane. The sample solution spiked with the internal standard solution was injected into the GC-MS system. The limit of determination of each chemical in the test solution was approximately 0.2 g/ml, which corresponded to 1–2 g/g in each sample. The duration of GC-MS analysis was approximately 17 min. A collaborative study was also carried out in order to evaluate the reproducibility of the method for determining low levels of BaP and related compounds in creosotes. The present method was applied for the analysis of certain commercially available creosotes and creosote-treated wood samples in Japan. It was confirmed that some creosotes and railway sleepers contained these compounds in high concentrations, thus exceeding the allowed control value.

This study deals with in vitro investigation of the release of di(2-ethylhexyl)phthalate (DEHP) during hemodialysis and pump-oxygenation therapy using medical grade PVC tubing. High resolution GC-MS analysis showed that the release of DEHP was time-dependently increased by circulation of bovine blood into a major system for the hemodialysis that is used in Japan, and the amount of DEHP released into the blood had reached 7.3 mg by 4 h of circulation. No significant difference was observed in the release patterns of DEHP under the conditions with and without fluid removal treatment during hemodialysis, indicating that the treatment seems not to be effective for eliminating DEHP from the blood through the hemodialysis membrane. Mono(2-ethylhexyl)phthalate (MEHP) analysis revealed that a small amount of DEHP (3-4%) was converted to MEHP by hydrolysis during the circulation of blood. A considerable amount of DEHP was also released from the PVC circuit mimicking the pump-oxygenation system, and 7.5-12.1 mg of DEHP had migrated into bovine blood from the circuit by 6 h. It was noticed, however, that the release was obviously suppressed by covalently coating the inner surface of the PVC tubing with heparin, though this effect was not observed with ionic bond type-heparin coating. Covalent bond type-heparin coating of PVC tubing seems to offer the advantage of decreasing the amount of DEHP exposure to patients during treatment using a PVC circuit.

Poly-L-lactic acid (PLLA) is a widely used promising material for surgical implants such as tissue-engineered scaffolds. In this study, we aimed to determine the in vivo effect of PLLA plates on the cellular function of subcutaneous tissue in the two mouse strains, BALB/cJ and SJL/J, higher and lower tumorigenic strains, respectively. Gap-junctional intercellular communication (GJIC) and the expression of connexin 43 (Cx43) protein were significantly suppressed, whereas the secretion of transforming growth factor-beta 1 (TGF-beta 1) level was significantly increased in PLLA-implanted BALB/cJ mice compared with BALB/cJ controls. However, no significant difference in TGF-beta 1 secretion was observed between the SJL/J-implanted and SJL/J control mice. We found for the first time that a significant difference was observed between the two strains; thus, the PLLA increased the secretion of TGF-beta 1 and suppressed the mRNA expression of Cx43 at the earlier stage after implantation into the higher-tumorigenic strain, BALB/cJ mice. This novel mechanism might have a vital role in the inhibition of GJIC and promote the tumorigenesis in BALB/cJ mice.

Background: Extensive analysis of allergenic proteins is generally time-consuming and labor-intensive. Accordingly, a rapid and easy procedure for allergen identification is required. As sequence information on proteins and genes is accumulated in databases, it is becoming easier to identify a candidate protein using proteomic strategies, i.e. two-dimensional gel electrophoresis, site-specific fragmentation, mass spectrometry and then database search. In this study, we evaluated the usefulness of a proteomic strategy for identifying putative allergens through its application to latex proteins. Methods: Latex proteins were separated with two-dimensional gel electrophoresis, and putative allergens were visualized by IgE immunoblotting using pooled serum from latex-sensitive patients. The IgE-interactive proteins were cut out from the negatively stained two-dimensional gel and subjected to in-gel digestion by trypsin. Then the resulting peptides were analyzed with mass spectrometry. Based on the mass spectrometric data we obtained, the allergen candidates were assigned by a database search. Results: Five previously reported allergens and five new allergen candidates were identified with the proteomic approach without isolating the individual proteins. Less than 1 mg of crude latex protein was sufficient for the entire protocol. Because plural proteins can be processed in parallel, analysis of about 50 IgE-interactive proteins was accomplished within 1 week. Conclusions: Analysis of putative allergens with proteomic strategies (allergenomics) is a promising avenue for rapid and exhaustive research. The high resolving power of two-dimensional gel electrophoresis is superior to conventional gel electrophoresis. Moreover, the notable sensitivity and speed of mass spectrometry have pronounced advantages over the N-terminal sequencing that has generally been used for protein identification.

Connexin 32 (Cx32) is the main gap junction protein in hepatocytes and plays an important role in the regulation of signal transfer and growth control in the liver by constructing gap junction channels and gap junctional intercellular communication (GJIC). In this study, the human Cx32 gene was transfected into a hepatoma cell line (HepG2) that showed aberrant expression of Cx32 and was deficient in GJIC. Cx32-transfected HepG2 not only expressed a higher level of Cx32 mRNA, but also showed increased GJIC compared with HepG2 and vector-transfected HepG2. Furthermore, the liver functions of ammonia removal and albumin secretion of HepG2 were markedly 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.

Contamination by endotoxin of nine kinds of wound dressings made of natural biomaterials (calcium alginate, collagen, chitin, and poly-L-leucine) was examined with the use of water extracts. By applying the Limulus amoebocyte lysate (LAL) test, high concentrations of endotoxin were detected in extracts from three kinds of products made of calcium alginate. These extracts evoked fever in rabbits and induced the release of a proinflammatory (pyrogenic) cytokine, interleukin-6 (IL-6), from human monocytic cells (MM6-CA8). The effects disappeared when the extracts were treated with endotoxin-removing gel column chromatography or with an endotoxin antagonist, B464, confirming that the contaminating pyrogen was endotoxin. A noteworthy finding was that one of the endotoxin-containing extracts showed very weak IL-6-inducibility in human monocytic cells in contrast to its high pyrogenicity to rabbits. The discrepancy could be explained based on differences between humans and rabbits in sensitivity to the endotoxin, because the extract showed higher proinflammatory-cytokine (TNF-alpha)-inducibility in rabbit whole-blood cells (WBCs) than human WBCs. The results suggest that the LAL test is a useful method of detecting endotoxin contamination in wound dressings and the MM6-CA8 assay is a good supplement to the LAL test for evaluating pyrogenicity in humans accurately.

We have previously reported that transforming growth factor beta (TGF-beta) triggers two independent senescence programs, 1) replicative senescence dependent upon telomere shortening and 2) premature senescence independent of telomere shortening, in the cell line of A549 human lung adenocarcinoma. In this study, we examined the possibility that cancer cell tumor phenotypes could be suppressed by forced senescence. We used A549 cells treated with TGF-beta for a long time (over 50 days), where senescence was induced in a telomere-shortening-dependent or an independent way. Fully senescent A549 cells were elongated, acquired contact inhibition capabilities when reaching confluence, and secreted the senescence-associated cytokine IL-6. Furthermore, senescent A549 cells had no tumorigenicity in nude mice. These results indicate that the forced induction of senescence in cancer cells may be a novel and potentially powerful method for advancing anti-cancer therapy.

A round-robin study was organized in the international standardization working group, ISO/TC194/WG8, with the purpose to compare the efficacy of different sample preparation procedures for determining the sensitization potency of polymeric medical devices. Three custom-made rubber samples were supplied to the participating laboratories and tested according to predetermined extraction and test protocols: A, formulated with a high level of mercaptobenzothiazole (MBT); B, formulated with a low level of MBT; and C, formulated with zinc dibutyldithiocarbamate. Extraction protocols included the so-called ISO method that applies physiological saline and/or vegetable oil extraction media without concentration (designated here “simple extraction”) and the so-called Japanese method that applies organic solvent extract (in this study chloroform–acetone mixture) after concentration to the test systems (designated here “exhaustive extraction”). Tests included human patch test, guinea pig maximization test (GPMT), adju...

Gap junctional intercellular communication 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 when contacting fibroblasts with various polymer microspheres was estimated using the fluorescence recovery after photobleaching (FRAP) assay system. When the cells were in contact on test dishes, the inhibition level increased as the diameters of polystyrene microspheres decreased, except for a microsphere with 0.5 µm diameter. The function was inclined to be recovered with the increase of the incubation time, while it was not recovered when the cells were cultured with pre-coated polystyrene microspheres. As well as inhibitory activities of the function, cytotoxicity potentials of tested microspheres depended on their diameter and their composition. These findings suggest that the size and the physico-chemical character of polymer microspheres, and how cells recognize them plays important roles in causing influences of the microspheres on both gap junctional intercellular communication and their cytotoxicity. Therefore, estimating the inhibitory effect of biomaterials on the gap junctional intercellular communication will provide valuable information about the biocompatibility of materials even in the form of particles.

We have previously demonstrated that the addition of muramyl dipeptide (MDP), interleukin-2 (IL-2) and IL-4 effectively raises antibody production from L-Leucyl-L-leucine methyl ester (LLME)-treated human peripheral blood lymphocytes (PBLs) against specific soluble antigen when immunized in vitro. However, PBLs from individual donors were separate optimal conditions regarding concentrations for IL-2 and IL-4, which in turn required us to optimize each individual PBLs to effectively produce antigen specific human antibody by in vitro immunization. These individual differences in the requirement for IL-2 and IL-4 reflects the differences in individual immune responses against a specific soluble antigen, which can be elicited by in vitro immunization. In the present study, we investigated these individual differences in the requirement for IL-2 and IL-4 to induce antibody productionin vitro in the PBLs of 12 volunteers (9 healthy donors and 3 allergenic patients). IL-2 requirements for antibody production varied dependent upon each donor, while higher amounts of IL-4 inhibited IgM and IgG production in all of the healthy donors. However, some of the characteristic features for PBLs donated from allergenic included lowered IgM production compared to PBLs derived from healthy donors, and very high IgE production in the absence of cytokines and allergen. These results demonstrate that the sensitivity of PBLs against antigen sensitization differs between healthy donors and atopic patients, which suggests that the frequency of antigen sensitization might be reflected in differing activation states and/or differing subpopulations of lymphocytes in vivo.

The aim of the study is to see whether corrosion of metallic implants in a living body is related to metal sensitivity reactions. As model materials, we prepared iron (Fe)-chromium (Cr) binary alloys containing various amounts of Cr, having different corrosion resistance. As a comparison, 316L type stainless steel (SUS316L) was used. These specimens were subcutaneously implanted into rats, and the tissue reactions around the implants and the sensitivity to Cr were evaluated by histological examination, patch testing and lymphocyte proliferation tests. After 4 months implantation, there was rust on the surface of all 10%Cr–Fe alloys, three 15%Cr–Fe alloys and one 20%Cr–Fe alloys out of 4 specimens per each group. There were no significant toxicological sign in any of the animals. Tissue responses, such as extent of hemorrhage and frequency of inflammatory cells in or around the fibrous capsule, were scored and the sum total score was determined. With regard to total score of tissue response, no significant difference was found among the groups. Concerning the thickness of fibrous capsule, 10% and 20%Cr–Fe alloy group was somewhat higher than other Cr–Fe alloys and SUS316L, but there was not significant difference. In the histological examination of tissue responses, the sign of metal sensitivity could not be obtained. Although there was no significant difference in the lymphocyte proliferation activity by addition of potassium dichromate (K 2Cr2O7)-solution among the implanted groups, two animals exhibited positive skin reactions by topical challenge with K 2Cr2O7 in 10%Cr–Fe alloy-implanted group. In 15% and 20%Cr–Fe alloy groups, each one showed a skin response. The skin reaction observed was mainly edema that is characteristic of metal allergy. This suggests that some animals were sensitized to Cr by the implantation of easily corroded Cr–Fe alloy, and the corrosion products of metallic devices in body fluid is likely responsible for development of metal hypersensitivity.

Biomaterials such as polyetherurethans (PEUs) are the scaffolding, which is indispensable for the development of the bio-artificial organs. However, PEUs can induce tumors in subcutaneous implantation sites in rat. We have shown that the different inhibitory potential of gap junctional intercellular communication (GJIC) on the surface of the biomaterials, including PEUs, is a key step in determining the tumorigenic potential. Here we show that suppression of a gap junctional protein connexin 43 (Cx43) plays an important role in in vivo tumorigenesis induced by PEUs for the first time and that Cx43 transfection may be an effective strategy for preventing tumorigenesis induced by biomaterials. Rat tumor cell line U41 is derived from tumors in the subcutaneous implantation of PEU films. The GJIC and the expression of Cx43 were suppressed in U41. The restoration of normal phenotype, such as reduction of growth rate, recovery of contact inhibition and loss of colony formation ability in soft agar, was achieved by Cx43 transfection. These results strongly suggest that suppression of Cx43 expression plays an important role in the development of rat malignant fibrous histiocytoma (MFHC) caused by PEUs and that Cx43 transfection is effective for prevention of tumorigenesis induced by PEUs.

Binary alloys of chromium-iron (Cr–Fe) are no longer used in dental and orthopaedic applications, and were used only for comparative purposes in the present study. Four kinds of Cr–Fe binary alloys, namely, 10 mass%, 15 mass%, 20 mass% and 30 mass%Cr in Fe, and 100%Cr and SUS316L were prepared. These metals were subcutaneously implanted into rats for four months. There was no significant difference in body weights and organ weights among control (sham-operation) and implant-groups, and no significant accumulation of Cr and Fe in the blood, liver or kidney of the alloy implant groups. To clarify the usefulness of urinary monitoring, the Cr and Fe concentrations in rat urine after implantation were analyzed. Alloy implant groups, including the SUS316L group, intended to excrete higher urinary Cr amounts than the control group during the 3 weeks after implantation. Urinary Cr and Fe amounts of the 15%Cr-alloy group showed especially high values in comparison not only with the control level but also the other alloy groups. The visual corrosion extents of the retrieved implants showed the following order: 10%Cr > 15%Cr > 20%Cr > 30%Cr > 100%Cr, SUS316L, and did not always correlate with the levels of the urinary amounts of Cr and Fe. From these findings, monitoring of urinary metal amounts is considered to be useful to monitor the in vivo elution property non-invasively.

Normal human osteoblast NHOst cells were cultured on the dental Au–Ag–Pd casting alloy using the micromass culture, and the proliferation and differentiation of NHOst cells were determined. 13Au–58Ag–21Pd which met JIS T 6106 and 10Au–62Ag–13Pd which did not meet JIS T 6106 were used in this experiment. 10Au–62Ag–13Pd contained Cu more than 13Au–58Ag–21Pd. The proliferation and differentiation of NHOst cells cultured on 10Au–62Ag–13Pd were significantly decreased more than those on 13Au–58Ag–21Pd, respectively. It was suggested that the content of Cu caused the difference in the proliferation and differentiation between NHOst cells cultured on 13Au–58Ag–21Pd and that on 10Au–62Ag–13Pd. Moreover, the cytotoxicity of the Au–Ag–Pd alloy was evaluated by the MEM elution assay and the colony assay using fibroblast L929 and V79 cells, in order to compare with the cytological effects of the alloy on NHOst cells. 13Au–58Ag–21Pd showed no cytotoxicity to L929 and V79 cells in the MEM elution assay and the colony assay. However, 10Au–62Ag–13Pd showed extremely weak cytotoxicity to L929 cells and weak cytotoxicity to V79 cells in the colony assay, though 10Au–62Ag–13Pd showed no cytotoxicity to L929 cells in the MEM elution assay. NHOst cells expressed the toxicity to the proliferation and differentiation on 10Au–62Ag–13Pd clearly. The in vitro toxicity test based on the proliferation and differentiation of NHOst cells was useful to evaluate the toxicity of medical materials.

Biodegradable polymers are attractive candidates for chondrocyte embedding and transplantation in cartilage tissue engineering. In an attempt to determine the effects of a variety of biodegradable materials on cartilage proliferation and extracellular matrix production, poly-L-lactic acid (PLLA) with a molecular weight of 5,000, polyglycolic acid (PGA) with a molecular weight of 3,000, and copolymer of poly(L-lactic acid-glycolic acid) 50:50 (PLGA) with a molecular weight of 5,000, were dissolved in DMSO and added into the medium for 4 weeks in in vitro high-density micromass culture of multiplied human articular chondrocytes (HAC). PLLA with a molecular weight of 270,000 (PLAO3) was used as thin film. Cell proliferation and differentiation in these biomaterials were compared with tissue culture polystyrene (TCPS) as a control. Alamar blue and alcian blue staining were carried out to determine the chondrocyte proliferation and differentiation, respectively. Samples exposed to these biomaterials promoted cell proliferation in the range of 86-105% of the control proliferation, and a slight but significant increase in cell proliferation was noted only in the culture exposed to PLGA. The sample exposed to PGA elicited a significant 3.7-fold higher (p0.01) cell differentiation than controls and was significantly higher than that of the samples exposed to PLLA, PLAO3, and PLGA. After 4 weeks of culture, the cell differentiation from most to least was in the following order PGAPLAO3PLGA = PLLACont. = DMSO. Chondrocyte differentiation of the samples exposed to various biomaterials were significantly higher compared with controls. Thus, serially passage chondrocytes are competent for cell growth and quantifiable matrix production, and biodegradable polymers, especially PGA, hold promise as suitable substrates for scaffolding materials for human cartilage tissue engineering.

Chondrogenic differentiation of mesenchymal cells is generally thought to be initiated by the inductive action of specific growth factors and depends on intimate cell-cell interactions. The aim of our investigation was to characterize the influences of basic fibroblast growth factor (bFGF) and ferroussulfate (FeSO4) on proliferation and differentiation of human articular chondrocytes (HAC). This is the first report of the effects of FeSO4 on chondrogenesis of HAC. Multiplied chondrocytes of hip and shoulder joints were cultured in chondrocyte growth medium supplemented with bFGF, FeSO4, or both bFGF + FeSO4 for4weeks. A 20 μl aliquot of a cell suspension containing2 × 107 cells ml−1 was delivered onto each well of 24-well tissue culture plates. Cells cultured with the growth medium only was used as a control. Alamar blue and alcian blue staining were done to determine the chondrocyte proliferation and differentiation, respectively, after 4 weeks. The samples exposed to bFGF, FeSO4, and combination of both indicated sufficient cell proliferation similar to the control level. Differentiations of the HAC exposed to bFGF, FeSO4,and bFGF + FeSO4 were 1.2-, 2.0-, and 2.2-fold of the control, respectively. Therefore, chondrocyte differentiation was significantly enhanced by the addition of FeSO4 andbFGF + FeSO4. The combined effects of bFGF and FeSO4 were additive, rather than synergistic. These results suggest that treatment with ferrous sulfate alone or in combination with basic fibroblast growth factor etc, is a powerful tool to promote the differentiation of HAC for the clinical application.

Gap junctional intercellular communication 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 when contacting fibroblasts with various polymer microspheres was estimated using the metabolic cooperation assay system. When the cells were in contact with the microspheres after their adhesion onto a substrate, the function did not alter. However, when they were in contact with precoated microspheres on test dishes, the function was inhibited as the quantity of microspheres increased. Moreover, the inhibition level increased as the diameters of polyethylene and polystyrene microspheres decreased. However, no inhibition was observed if precoated microspheres were composed from poly(L-lactic acid). These findings suggest that the size and the material of microspheres, and how cells recognize the microspheres, are factors affecting cell function of gap junctional intercellular communication. Therefore, estimating this function may provide valuable information about the biocompatibility of many kinds of materials even in the form of particles.

Serum-free mouse embryo (SFME) cells were established by D. Barnes et al., and are known to be a neural stem cell line, which differentiate into astrocytes upon treatment with TGF-β. Therefore, SFME cells is thought to be a model well suited to analyze the differentiation mechanism of neural stem cells. Until now, we have investigated the regulation mechanisms of telomerase activity and telomere length in human cancer and normal cells. Telomerase is the enzyme responsible for the synthesis and maintenance of telomere repeats located at chromosomal ends and is normally expressed in embryonic and germline cells, but not in most normal cells. Here, using SFME cells, we attempted to analyze the regulation mechanism of telomerase activity in neural stem cells and to detect a change upon differentiation into astrocytes. When SFME cells were cultured in the presence of TGF-β, cells showed anelongated morphology and decreased its growth to 50% of control culture. Cells also expressed the glial fibrillary acidic protein (GFAP), a marker for astrocytes,indicating that TGF-β induced differentiation in SFME cells from neural stem cells into astrocytes. At the same time,TGF-β also inhibited telomerase activity and repressed the expression of the mouse telomerase reverse transcriptase(mTERT), demonstrating that SFME cells was vested with a finite replicative life span upon treatment with TGF-β. To understand the mechanisms regulating mTERT levels during differentiation into astrocytes, we have estimated the expression level of c-myc, which is known to be a key molecule in activating the TERT promoter. As a result, TGF-β-treated SFME cells were shown to repress the expression of c-myc. Furthermore, promoter analysis, using the 5′-region of the mTERT gene, which possess two E-box elements bound to c-Myc/Max, demonstrated that mTERT promoter activity greatly decreased in TGF-β-treated SFME cells as compared to non-treated SFME cells. These suggest that c-myc might play a critical role in the expression of mTERT, and that down-regulation of c-myc dependent upon the astrocytic differentiation in SFME cells might cause the repression of mTERT in TGF-β-treated SFME cells.