Comparison of tetrazolium colorimetric and 51Cr release assays for cytotoxicity determination of dental biomaterials.

OBJECTIVES: The purpose of this study was to compare a methylthiazole tetrazolium (MTT) dye colorimetric method with the standard 51Cr assay as methods of assessing cytotoxicity of dental materials. METHODS: Two MTT-based colorimetric formats, test tube and 96-well microplate methods, were compared to the 51Cr release assay. A series of eight dental materials were evaluated. Cytotoxicity profiles were determined for each test material. A TC50 value (Toxic Concentration required to kill 50% of the cells) was determined for each biomaterial, and these results were used to make statistical comparisons between the methods. RESULTS: The three methods were statistically correlated (p<0.005) by comparison of the eight samples tested. That is, the same rank in toxicity was given by the two tetrazolium sample formats and the 51Cr method. SIGNIFICANCE: The MTT assay was found to have several advantages in comparison to the current standard 51Cr release assay. Optimized in the 96-well format, complete dose response curves and greater sample comparisons can be made rapidly, making the MTT method more economical in time and cost. Furthermore, the MTT method is based on intracellular biochemical changes, measuring cell viability rather than cell morbidity, and has lower detectable limits than the 51Cr release method. There is also less detector chemical binding interference than encountered in the 51Cr release method.

The effect of spiroorthocarbonate volume modifier co-monomers on the in vitro toxicology of trial non-shrinking dental epoxy co-polymers.

A major improvement in dental restoratives is possible through the development of biomaterials that do not shrink upon polymerization, hence, avoid leakage and subsequent breakdown. Polymers containing spiroorthocarbonates (SOCs) show promise in this respect, but their toxicology in copolymerized materials has not been explored. In this study, the in vitro toxicology of these materials in homopolymer form and in two trial non-shrinking epoxy co-polymers was evaluated for cytotoxicity and mutagenicity. Cytotoxicity was determined by the MTT test to measure the lethality effect on mouse L929 cells. Mutagenicity was evaluated using the Ames-Salmonella Test. For comparison, commercial composite and adhesive materials as well as several other materials of current interest in dentistry were also evaluated. Epoxy resin samples containing 5% of either T/T SOC or Dp SOC reduced the cytotoxicity (TC50) from approximately 400 to 800 micrograms/200 microliters. The epoxy-spiro copolymers had more favorable TC50 values than the commercial product Super-Bond. They showed TC50 values on the order of 35% greater than Super-Bond and 45% less than Scotchbond 2, the latter two being materials currently used in the clinic. These two comparatives demonstrated dose response curves with lower doses at maximum cell kill values than the spiro materials. The epoxy formulations all showed weak mutagenesis, but this is attributed to the epoxy formulation and not the SOCs. Although considerable toxicology is yet be conducted, these in vitro results suggest that biocompatible copolymer formulations for spiroorthocarbonates are a developmental reality.

Effect of esterase on methacrylates and methacrylate polymers in an enzyme simulator for biodurability and biocompatibility testing.

Current in vitro biocompatibility methods do not evaluate the degradation of biomaterials after contact with enzymes that might be present in the oral or systemic environment. In this study, two methods of in vitro enzyme degradation and a method for the separation of the degradative products by high performance thin-layer chromatography (HPTLC) are reported. In the first method two dental adhesives, Scotchbond and Scotchbond II, and two dental composites, Heliomolar and P-50, were evaluated. These materials were incubated with four different enzymatic preparations for periods of up to 72 h. The enzymes were lipase, esterase, and liver enzyme extracts from both mouse and rat. Chloroform soluble products extracted from the aqueous phase were examined by HPTLC for decomposition products resulting from enzyme activity. The second method was similar, but analyzed the aqueous fraction directly without chloroform extraction. In this method five dental restorative materials, P-50, P-30, Scotchbond II, Silux, and Silux Plus, were incubated with a nonspecific porcine liver esterase. In addition to the polymerized biomaterials, monomers containing methacrylic acid units were also hydrolyzed with esterase and analyzed by ion chromatography to establish the sensitivity of the enzyme simulator. Each biomaterial presented thin-layer zones not present before enzymatic action. These experiments provide support that aqueous enzymatic action may facilitate the hydrolytic weakening of polymeric biomaterials.

Human aflatoxin B1 metabolism: an investigation of the importance of aflatoxin Q1 as a metabolite of hepatic post-mitochondrial fraction.

The metabolism of aflatoxin B1 by the post-mitochondrial fraction of human autopsy or biopsy liver and monkey necropsy liver was investigated. Aflatoxin Q1, a hydroxy metabolite of B1, was produced by 18 of 22 human liver samples and by 5 of 7 species of non-human primates investigated. Human specimens from both sexes, ages 20-80 years, with a variety of hepatic histological and historical influences produced this metabolite. Aflatoxin Q1 was of variable quantitative importance. Yields seldom exceeded 10% of initial B1. However, based upon its persistent formation in vitro, these studies identify the need to clarify the role of Q1 in aflatoxin epidemiology.