Characterization and application of chondroitin sulfate/polyvinyl alcohol nanofibres prepared by electrospinning.

Composite nanofibres were prepared by electrospinning from a solution of chondroitin sulfate and polyvinyl alcohol. The chondroitin sulfate/polyvinyl alcohol (CS/PVA) mass ratios of 7/3 has a uniform and smooth morphology, and the average diameter of the nanofibres was 136nm. Combretastatin A-4 phosphate was loaded on the nanofibres and used as a model for testing drug release from the nanofibres crosslinked with glutaric dialdehyde. The morphology and structure of the nanofibres was determined using scanning electron microscopy. In order to assess their possible application to tissue engineering scaffolds, the toxicity and cytocompatibility of the nanofibres were tested by methylthiazolydiphenyl-tetrazolium bromide assay.

Photocrosslinkable bioadhesive based on dextran and PEG derivatives.

In this study, a hybrid photopolymeric bioadhesive system consisting of urethane methacrylated dextran (Dex-U) and 3, 4-Dihydroxyphenyl-l-alanine (DOPA) modified three-arm poly (ethylene glycol) s (PEG-DOPAs) was designed. The process of photopolymerization was detected by Photo-Differential Scanning Calorimetry (Photo-DSC). The adhesion strength was evaluated by the lap shear tests. The surface tension of the solutions, burst pressures and the cytotoxicity assays were also investigated. The addition of PEG-DOPAs significantly improved the properties of Dex-U especially in the field of adhesion strength and burst pressure. And materials variation could be tailored to match the demands for tissue repair. Compared to the Dex-U systems, the maximum adhesion strength of the copolymeric system increased from 2.7±0.1 MPa to 4.0±0.6 MPa. Owing to its strong adhesion strength, rapid curing rate and good biocompatibility, such photocrosslinkable hydrogelsa could be applied to the areas of bioadhesive.

Biomimetic composite scaffolds based mineralization of hydroxyapatite on electrospun calcium-containing poly(vinyl alcohol) nanofibers.

Nanocomposite materials consisting of polymer matrix and inorganic salts in the form of nanocrystals of hydroxyapatite (HA) are regarded as superior candidates for bone treatment. A biomimetic nanocomposite scaffold with HA formation on the electrospun poly(vinyl alcohol) (PVA) nanofibrous structure by employing a Ca-P alternate soaking method was developed in this work. The calcium-containing PVA nanofibers were prepared by adding calcium nitrate to the starting solution prior to electrospinning, and then mineralized by Ca-P treatment in incubation solution. With this rapid and effective procedure, a continuous biomimetic crystalline HA layer could be formed successfully without the need of a prior chemical modification of the substrate surface under very mild reaction conditions. Moreover, the HA formed with a relatively accelerated growth had a carbonated and poor crystalline structure, resembling biological apatite in the bone mineral. The introduction of calcium ions in nanofibers by electrospinning was a favorable approach to induce the deposition of calcium phosphate and improve the distribution, nucleation, and growth of crystalline HA layer on nanofibrous scaffolds. Bioactivity tests revealed that these mineralized PVA/HA composite scaffolds improved the biocompatibility. The porous polymer/HA composite scaffolds produced in the present study might have potential applications in bone tissue engineering.

The photocrosslinkable tissue adhesive based on copolymeric dextran/HEMA.

We developed a copolymeric bioadhesive system with the potential to be used as a tissue adhesive based on biopolymer dextran. Copolymeric hydrogels comprising a urethane dextran (Dex-U) and 2-hydroxyethyl methacrylate (HEMA) were prepared and crosslinked under the ultraviolet (UV) irradiation. In this study, the photopolymerization process was monitored by real time infrared spectroscopy (RTIR). The adhesion strength was evaluated by lap-shear-test. The surface tension, viscosity of the solutions and the cytotoxicity assays were investigated. Compared to Dex-U system, the addition of HEMA remarkably improved the properties of Dex-H system especially the adhesion strength and the nontoxicity. And materials variation could be tailored to match the need of tissues. The copolymeric tissue adhesives demonstrated promising adhesion strength and nontoxicity. The maximum adhesion strength reached to 4.33±0.47 Mpa which was 86 times higher than that of Tisseel. The obtained products have the potential to serve as tissue adhesive in the future.

Repeat oral dose toxicity studies of melamine in rats and monkeys.

Melamine is an important and widely used organic industrial chemical. Recently, clinical findings of renal failure and kidney stones in infants have been associated with ingestion of melamine-contaminated infant formula. To understand the toxicity and clinical outcome of melamine exposure, repeated oral dose studies in rats and monkeys were performed to characterize the subchronic toxicity of melamine. Assessment of toxicity was based on mortality, clinical signs, body weights, ophthalmic findings, clinical pathology, gross pathology, organ weights, and microscopic observations. The first rat study was intended to be a 14-day oral study followed by an 8-day recovery period. The dose levels were 140, 700, and 1,400 mg/kg/day (lowered to 1,000 mg/kg/day subsequently due to mortality). Oral administration of melamine at 700 mg/kg/day for 14 consecutive days in rats produced compound-related clinical signs (red urine), decreased body weights, and changes in clinical pathology (increased serum urea nitrogen and creatinine) and anatomical pathology (renal tubular cell debris, crystal deposition, and hyperactive regeneration of renal tubular epithelium). The kidney was identified as the target organ. Oral administration at 1,400 mg/kg/day (subsequently lowered to 1,000 mg/kg/day) resulted in animal death and moribundity. There were no treatment-related findings in the 140 mg/kg/day group. There were no compound-related findings in the high-dose recovery animals. The second rat study was a 5-day oral toxicity study with genomic biomarkers assayed in the kidney tissues. At the top dose of 1,050 mg/kg/day, similar clinical and anatomical pathology findings as described above were observed. The genes measured, Kim-1, Clu, Spp1, A2m, Lcn2, Tcfrsf12a, Gpnmb, and CD44, were significantly up-regulated (fivefold to 550-fold), while Tff3 was significantly down-regulated (fivefold). These results indicated that genomic markers could sensitively diagnose melamine-induced kidney injury. A 3-month oral study with 4-week recovery in monkeys was also conducted. In this monkey study, the animals were treated with melamine at doses of 60, 200, or 700 mg/kg/day. The administration of 700 mg/kg/day melamine by nasal-gastric gavage to monkeys resulted in test article-related clinical signs including turbid and whitish urine, urine crystals, red blood cell changes, increased serum alanine aminotransferase and kidney and/or liver weights, and microscopic findings including nephrotoxicity, pericarditis, and increased hematopoiesis. Nephrotoxicity was also noted at 200 mg/kg/day. It was concluded that the kidney is the primary target organ and the NOAEL was estimated to be 140 mg/kg/day in rats following a 14-day oral administration and 60 mg/kg/day in the monkey study.

Electric field induced phase separation on electrospinning polyelectrolyte based core-shell nanofibers.

In the present study, we report a facile method to fabricate polyelectrolyte based core-shell nanofibers with the assistance of the high gradient electric potential between the tip of capillary and the collector. The core-shell structure and the composition of each layer have been supported by TEM and XPS studies. The effect of the electric field on the phase separation was considered to be the major factor. An electric field experiment was introduced to elucidate the influence of electric field on the phase separation process. It was assumed the polyelectrolyte based core-shell nanofibers membrane could be applied in wound care and tissue engineering.

Gene expression profiling of androgen receptor antagonists in the rat fetal testis reveals few common gene targets.

The androgen receptor (AR) is expressed in the fetal testis; however, the role of AR in fetal testicular development is poorly understood. Disrupted AR activity and subsequent gene expression alterations may disturb developmental programming of the fetal testis and result in testicular abnormalities later in life. The present study was performed to examine global gene expression patterns in rat fetal testis following in utero exposure to various AR antagonists. Pregnant Sprague-Dawley rats were treated with flutamide (50 mg/kg/day), linuron (50 mg/kg/day), vinclozolin (200 mg/kg/day), p,p'-DDE (100 mg/kg/day) or corn oil vehicle by gavage daily from gestation day (GD) 12-19. Testes were isolated on GD 19, and AR immunostaining, histology, and global changes in gene expression were determined. There were no alterations in the pattern or expression level of AR and no apparent histological changes in the fetal testes in any treatment group. Microarray analysis using Dunnett's test with multiple testing correction revealed no significant gene expression alterations following exposure to flutamide, linuron, vinclozolin, and p,p'-DDE. A less stringent analysis yielded some chemical specific effects on gene expression, and these effects were further evaluated by real-time RT-PCR. Vinclozolin treatment reduced the expression of several genes involved in cholesterol biosynthesis, though the testosterone levels were unchanged in the fetal testes in any treatment group. In flutamide, linuron, and p,p'-DDE treatment groups, the expression of hemoglobin Y, beta-like embryonic chain (Hbb-y) was reduced. Myomesin 2 (Myom2) expression was increased following linuron treatment. Given the lack of a common set of genes and the absence of overt histopathology, we conclude that the fetal testis is not a major target for AR activity at this stage of development although some cell-type specific gene expression changes cannot be ruled out.

Covert signal disruption: anti-ecdysteroidal activity of bisphenol A involves cross talk between signaling pathways.

Bisphenol A is a key industrial chemical used in the manufacture of polycarbonate plastics and other products. Several recent reports ascribe toxicological properties to this compound that have been attributed to the disruption of endocrine-related processes. In the present study, the toxicity of bisphenol A was definitively characterized in the water flea (Daphnia magna) in an effort to discern whether this compound may elicit endocrine toxicity in an invertebrate species and to establish the mechanism by which this toxicity is elicited. The ability of bisphenol A to interfere with two ecdysteroid-dependent physiological processes--molting and embryonic development--was evaluated. Bisphenol A elicited antiecdysteroidal activity as indicated by its prolongation of the intermolt period and interference with embryonic development. This apparent antiecdysteroidal activity was not due to reduced availability of endogenous ecdysteroid nor due to ecdysteroid-receptor antagonism. The ability of bisphenol A to elicit antiecdysteroidal activity by functioning as a juvenoid hormone was next evaluated. Bisphenol A, alone, did not elicit juvenoid activity. However, bisphenol A did enhance the activity of the crustacean juvenoid hormone methyl farnesoate. A definitive assessment of the effects of bisphenol A on the reproductive capacity of daphnids revealed a concentration-response relationship that extended at least one order of magnitude below exposure levels that were overtly toxic to the maternal organisms. These results demonstrate that bisphenol A is chronically toxic to daphnids, probably through its ability to interfere with ecdysteroid/juvenoid regulated processes. However, effects are elicited at levels that are not likely to pose environmental concern.

Cross communication between signaling pathways: juvenoid hormones modulate ecdysteroid activity in a crustacean.

Methyl farnesoate is a juvenoid hormone that regulates a variety of processes in crustaceans including male sex determination among daphnids (Branchiopoda, Cladocera). The synthetic juvenoids pyriproxyfen and fenoxycarb mimic the action of methyl farnesoate in daphnids. In the present study we tested the hypothesis that juvenoids also can regulate ecdysteroid activity in a crustacean (Daphnia magna). Methyl farnesoate, pyriproxyfen, and fenoxycarb all disrupted ecdysteroid-regulated aspects of embryo development in daphnids. Exposure of ecdysteroid-responsive cells to 20-hydroxyecdysone reduced cell proliferation and increased mRNA levels of the ecdysone receptor and its partner protein ultraspiracle. Co-treatment of cells with the juvenoid pyriproxyfen attenuated all of these ecdysteroid mediated responses. While juvenoids functioned as anti-ecdysteroids in both intact embryos and in cultured cells, 20-hydroxyecdysone showed no evidence of acting as an anti-juvenoid. The combined effects of pyroproxyfen with the ecdysteroid synthesis inhibitor fenarimol and the ecdysteroid receptor antagonist testosterone were evaluated in an effort to discern whether the action of the juvenoids were additive with those of know anti-ecdysteroids. The anti-ecdysteroid effects of pyriproxyfen were non-additive with those of either anti-ecdysteroid. Rather, joint effects conformed to a model of synergy. These results demonstrated that juvenoids elicit anti-ecdysteroidal activity in a crustacean through a unique mechanism of action. A model involving receptor partner deprivation is proposed that explains the synergistic interactions observed.

Synergistic interaction of endocrine-disrupting chemicals: model development using an ecdysone receptor antagonist and a hormone synthesis inhibitor.

Endocrine toxicants can interfere with hormone signaling through various mechanisms. Some of these mechanisms are interrelated in a manner that might result in synergistic interactions. Here we tested the hypothesis that combined exposure to chemicals that inhibit hormone synthesis and that function as hormone receptor antagonists would result in greater-than-additive toxicity. This hypothesis was tested by assessing the effects of the ecdysteroid-synthesis inhibitor fenarimol and the ecdysteroid receptor antagonist testosterone on ecdysteroid-regulated development in the crustacean Daphnia magna. Both compounds were individually characterized for effects on the development of isolated embryos. Fenarimol caused late developmental abnormalities, consistent with its effect on offspring-derived ecdysone in the maturing embryo. Testosterone interfered with both early and late development of embryos, consistent with its ability to inhibit ecdysone provided by maternal transfer (responsible for early developmental events) or de novo ecdysone synthesis (responsible for late developmental events). We predicted that, by decreasing endogenous levels of hormone, fenarimol would enhance the likelihood of testosterone binding to and inhibiting the ecdysone receptor. Indeed, fenarimol enhanced the toxicity of testosterone, while testosterone had no effect on the toxicity of fenarimol. Algorithms were developed to predict the toxicity of combinations of these two compounds based on independent joint action (IJA) alone as well as IJA with fenarimol-on-testosterone synergy (IJA+SYN). The IJA+SYN model was highly predictive of the experimentally determined combined effects of the two compounds. These results demonstrate that some endocrine toxicants can synergize, and this synergy can be accurately predicted.

Developmental toxicity of testosterone in the crustacean Daphnia magna involves anti-ecdysteroidal activity.

Testosterone has been shown to cause developmental arrest of embryonic daphnids (Daphnia magna). The present study was undertaken to determine whether this toxicity might be due to anti-ecdysteroidal activity associated with testosterone. The effect of testosterone on molt frequency of early instar daphnids was first evaluated to determine whether testosterone interfered with this ecdysteroid-regulated process. Molt frequency was delayed by exposure to testosterone and this effect was mitigated by co-exposure to the ecdysteroid 20-hydroxyecdysone. Testosterone exposure concentrations that interfered with molting also elicited developmental abnormalities among neonatal organisms produced by maternal organisms that were continuously exposed to testosterone or among embryos that were removed from unexposed mothers and exposed directly to the hormone. Embryos were significantly protected against the developmental toxicity of testosterone by co-exposure to 20-hydroxyecdysone. Taken together, these results demonstrated that the embryo toxicity of testosterone to daphnids is due largely to its ability to interfere with ecdysteroid control of development. Experiments next were conducted to determine whether testosterone interfered with ecdysteroidal activity by acting as an ecdysone receptor antagonist or by reducing endogenous ecdysone levels. Testosterone significantly antagonized the action of 20-hydroxyecdysone in an ecdysone-responsive cell line. Testosterone had no discernable effect on endogenous ecdysone levels in daphnids. These results demonstrated that (1). ecdysteroids regulate critical processes in daphnid embryo development, (2). testosterone elicits embryo toxicity to daphnids by interfering with ecdysteroid activity, and (3). ecdysteroid receptor antagonism could be one mechanism by which testosterone elicits these effects.

Environmental antiecdysteroids alter embryo development in the crustacean Daphnia magna.

The role of ecdysteroids in crustacean embryo development and the susceptibility of the developing embryo to the antiecdysteroidal properties of an environmental chemical were evaluated. The agricultural fungicide fenarimol was shown to exhibit antiecdysteroidal activity to the crustacean Daphnia magna by lowering endogenous ecdysone levels and delaying molting in a concentration-dependent fashion that was mitigated by co-exposure to exogenous 20-hydroxyecdysone. Exposure of either gravid maternal organisms or isolated embryos to fenarimol resulted in embryo abnormalities ranging from early partial developmental arrest to incomplete development of antennae and shell spines. Developmental abnormalities were associated with suppressed ecdysone levels in the embryos and the abnormalities could be prevented by co-exposure to 20-hydroxyecdysone. Developmental abnormalities caused by the antiecdysteroid were associated with reduced fecundity of the parental organisms. These results demonstrate that ecdysteroids are critical to normal crustacean embryo development and environmental antiecdysteroids can disrupt normal embryo development and compromise the production of viable offspring. Antiecdysteroidal activity may provide a means by which environmental chemicals impact crustacean species while not affecting vertebrates.