| PartⅠ. Comparative study of a novel contraceptive composite versus HCu280 IUD on cytotoxicity and genotoxicityThis study was aimed to investigate the underlying cytotoxicity and genotoxicity of a novel copper-containing composite for contraception and provide sound evidence for clinical application. Moreover, given that copper wire with regard to its antifertility effect has been introduced to copper-containing IUD for decades, a comparative study was conducted between the novel composite and copper wires from HCu280 IUD which is commercial available under the brand-name Xinxin(Shenyang Liren Medical Science&Technology Co. Ltd, Shenyang, China).ExperimentⅠ. In vitro cytotoxicity test: MTT AssayObjective: To investigate and compare in vitro cytotoxicity of a novel copper-containing composite for contraception and copper wires from HCu280 IUD and select concentrations suitable for genotoxicity assays.Method(s): The mitochondrial activity testing method(MTT test) were performed to evaluate the levels of cell viability after treating TM4 cells with different specimens for 24h. Copper concentration in the extracts was measured by flame atomic absorption spectrophotometry. Following this, a range of concentrations [100%(8.54μg/ml), 50%(4.27μg/ml), 25%(2.135μg/ml)] of novel composite were prepared by a serial dilution with complete growth medium. In terms of copper concentration, an initial extract(106.54μg/ml) of copper wires and subsequent doubling dilutions[50%(53.27μg/ml), 25%(26.64μg/ml), 12.5%(13.32μg/ml), 6.25%(6.66μg/ml), 3.125%(3.33μg/ml), 1.5625%(1.67μg/ml)] were made to facilitate an accurate comparative analysis. By measuring the optical density(OD) at 492nm using microplate reader, cell viability was represented by relative growth rate (RGR) of OD(exposed) to OD(control receiving media alone) expressed as a percentage. Consequently, the cytotoxicity was graded by RGR(%) into 1 of 6 categories.Result(s): Relative growth rate[RGR(%)] of novel copper-containing composite ranged from 87.3% to 92.1%, indicating that in our experiments there were no significant effect towards the cell viability, at all concentrations studied. Whereas copper wire extracts, of the seven concentrations tested, displayed a concentration-response pattern and was considered to be excessively cytotoxic at the top concentration which gave almost zero percent cell survival. Dilutions of the copper extracts were clearly devoid of cytotoxic activity at concentrations ranging from 1.5625%(1.67μg/ml) to 6.25%(6.66μg/ml) with their corresponding CTG Grade 1.Conclusion:Extracts derived from novel copper-containing composite showed no sign of cytotoxicity, which yielded a safety profile consistent with that required in guidelines ISO10993-5:2009 and GB/T16886.5-2003. Test concentrations for copper wire samples were narrowed to 1.5625%, 3.125%, and 6.25% to investigate genetic alterations in the absence of cell death, in the following comet assay. eExperimentⅡ. In vitro genotoxicity test: Alkaline single cell gel electrophoresis assay (comet assay)Objective: To investigate and compare in vitro genotoxicity of a novel copper-containing composite for contraception and copper wires from HCu280 IUD.Method(s): In view of results from MTT assay, concentrations of [100%(8.54μg /ml),50%(4.27μg/ml),25%(2.135μg /ml)] for novel copper-containing composite and three retained concentrations[6.25%(6.66μg/ml), 3.125%(3.33μg/ml),1.5625% (1.67μg/ml)] of copper wire extracts were selected to investigate their ability to damage DNA with comet assay. Before performing the comet assay, viability of TM4 cells was estimated using trypan blue exclusion. For quantitative analysis, Olive Tail Moment(OTM) was chosen to characterize DNA damage in individual cells.Result(s): Light microscopic examination of the cells after staining with trypan blue demonstrated stable viability over all concentrations, with percent viability consistently over 90% in all samples. The OTM of positive control-treated cells was 13.78±2.21, i.e. approximately 16-fold more than the OTM of negative control cells. Without affecting the cell viability, extractables of composite were found to induce significant DNA damage(P<0.05) at concentration as low as 25%, and a concentration-effect profile was also observed. The OTM for an initial concentration of composite was 10-fold compared to that of negative control. Similarly, the copper wire extracts with positive call induced elevated DNA migration in the absence of excessive cytotoxicity, at all concentrations with the exception of the lowest retained concentration(1.5625%) which demonstrated a slight though not significant increase(P>0.05) in OTM as compared with negative control.Conclusion : Both materials provoked statistically significantly DNA strand breakage in TM4 cells with a concentration-dependent manner. Concerning copper content, we assumed that copper ions displayed in partial aspects a significant connection with genotoxic activity.ExperimentⅢ. In vitro genotoxicity test: Bacterial Reverse Mutation Test (Ames test)Objective: To further investigate and compare in vitro genotoxicity of a novel copper-containing composite for contraception and copper wires from HCu280 IUD.Method(s): The copper concentrations of novel composite and copper wires after immersion in simulated body fluid were 6.84, 197.0μg/ml, respectively. Then concentrations of 100%(6.84μg/ml), 50%(3.42μg/ml), 25%(1.71μg/ml), and 12.5%(0.855μg/ml) were prepared from novel composite by a serial dilution with extraction solvent. Taking into account results obtained in the foregoing assays, copper ions, a critical extractable constituent prepared from both materials, appeared to be causally associated with cytotoxic and genotoxic response. Thereby an initial concentration of copper wire extract was then diluted, yielding the final concentrations of 6.84, 3.42, 1.71, and 0.855μg/ml respectively, to allow for further comparative analysis. A standard plate incorporation procedure was utilized for Ames test with two tester strains TA 98 and TA 100, in the presence and absence of S9. A test sample was considered to be mutagenic if MR≥2 with an observed concentration-dependent relationship.Result(s): All positive-control agents were consistently and unequivocally positive, indicating the viability of the experiments. The number of revertant colonies exceeded the threshold of twice the number of the solvent control following exposure to novel composite extracts in concentrations from 25% to 100% in the TA98 strain, with and without metabolic activation by S9-rat liver homogenate, and a concentration-dependent manner in revertant colony number could be observed, indicating that novel composite may act as a frameshift mutagen. Following treatment in the TA100 strain, a slight and statistically significant increase in the incidence of revertant colonies was observed at the two highest concentrations of 50% and 100% in the presence and absence of S9-mix. The lowest concentration(12.5%) possessed no mutagenic potential, except that a weaker mutagenic activity occurred in the TA98 strain in the absence of S9. Copper wire samples showed similar trend, and as a matter of fact, comparison of mutagenicity ratio (MR) between novel composite and copper wires with respect to similar concentrations of copper ions, indicated no significant difference at any concentration in the presence or absence of metabolic activation(P>0.05). Furthermore, with S9-mix less pronounced mutagenicity occurred for most of the samples, suggesting a contribution of mixed-function monooxygenases leading to detoxification.Conclusion: Most samples from both novel copper-containing composite and copper wires exhibited mutagenic activity in tester strains TA98 and TA100 with and without metabolic activation and corresponding concentrations from both materials achieved intriguingly similar results, further strengthening a causal relationship between copper ions and genotoxic hazards. Objective: To investigate the underlying cytotoxicity and genotoxicity of nano-silica complex/polymer composites for Intra-vas Device (IVD).Method(s): TM4 cells were treated with extracts derived from this novel composite at concentrations of 100%, 50%, 25%. In vitro cytotoxicity was determined firstly with MTT assay. Subsequently, comet assay was performed with these three concentrations. Thereafter, the bacterial reverse mutation test (Ames test) was performed to investigate the ability of extracts to induce gene mutations with two tester strains TA 98 and TA 100, in the presence and absence of S9.Result(s): Extracts derived from nano-silica complex/polymer composite yielded no significant differences whatever the concentration investigated in cell viability as compared with vehicle treated cells. In the comet assay, of three concentrations studied, no sign of DNA damage was observed when comparing OTM with negative control. Furthermore, the specimens tested show no mutagenic activity with and without S9 in two tester strains.Conclusion: Taken together, nano-silica complex/polymer composites exhibited excellent biocompatibility, without eliciting any undesirable effects in a series of in vitro cytotoxicity and genotoxicity tests. The novel material is promising in Intra-vas Device (IVD) application. |
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