Colony assay using V79 cells, the agar diffusion assay with L929 cells, and the 7-day rabbit muscle implantation test were employed to evaluate the cytotoxicity and tissue toxicity of natural rubber latex (NRL) materials. The in vivo implantation test showed that, among 13 histological parameters, thickness of inflammatory layer was the most useful index to evaluate tissue responses quantitatively. A comparison of the in vivo and in vitro parameters revealed the following correlations between the thickness of the inflammatory layer and cytotoxicity indices: Colony assay of the extracts, IC50: r = 0.80; Agar diffusion assay, Zone index: r = 0.73; Lysis index: r = 0.61. From these results, it appears that the colony assay provides a more reliable prediction of the tissue response than the agar diffusion assay.
Polyurethane films that contained various amounts of zinc diethyldithiocarbamate (ZDEC) and zinc dibutyldithiocarbamate (ZDBC) were prepared as standard reference materials (SRM). Using three cell lines of V79, L929, and Balb/3T3 cells, the cytotoxicity of the dithiocarbamates and the SRM films were compared by agar diffusion assay, filter diffusion assay, neutral red assay, cell growth assay, and colony assay. Among these in vitro cytotoxicity tests, colony assay was found to be the most sensitive method for detecting the cytotoxicity. The cytotoxic potentials of extracts from SRM films correlated well with the concentrations of ZDEC or ZDBC involved in SRM. When various rubber materials including SRM and surgical rubber latex materials were tested, cytotoxic potentials of these extracts were also correlated with the inflammatory tissue capsule thickness in short-term implantation tests. On the basis of these results, the SRM is judged to be useful for validating test sensitivity, and comparing the correlation between in vitro and in vivo responses.
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.
The biocompatibility of nine different materials, including positive and negative references, 4 polyurethane based and 3 latex based materials was investigated by (1) cytotoxic assay using V79 chinese hamster cells, (2) the thickness of inflammatory layer at 3 and 7 days after intramuscular implantation test, and (3) the course of the blood flow in tissue reaction around subcutaneously implanted materials using Laser Doppler Flowmetry (LDF) over 14 days following implantation. In addition, for some materials, different modes of sterilization were compared. Although the three methods explore different reactive systems, the material ranking obtained was highly similar for the three methods, suggesting a relative accuracy between them. For one latex however, an absence of cytotoxic effect in culture and a highly intense response by LDF investigation of the same order of magnitude as for the positive reference implant suggest that bioincompatibility may result from the material itself and cannot be exclusively investigated by the leaching of toxic components.
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