Genotoxic and mutagenic potential of camphorquinone in L5178/TK(+/-) mouse lymphoma cells

OBJECTIVES: Camphorquinone (CQ) is the most important photoinitiator used in dental composite resins. Sparse data indicate a mutagenic potential of CQ. Therefore, it was aim of this study to evaluate the cytotoxicity, genotoxicity, and mutagenicity of CQ in L5178Y TK(+/-) mouse lymphoma cells. METHODS: L5178Y/TK(+/-) cells were exposed to different concentrations of non-irradiated CQ (0.25-2.5mM). Cytotoxicity was evaluated by propidium iodide assay, determination of suspension growth rate, relative total growth and the mitotic index. Intracellular levels of reactive oxygen/nitrogen species (ROS/RNS) were quantified by 2',7'-dichlorofluoresceine diacetate (DCFH-DA). Early induction of DNA strand breaks and oxidative DNA base lesions was assessed using the 8-hydroxyguanine DNA-glycosylase 1 (hOGG1)-modified alkaline comet assay, whereas mutagenicity of CQ was determined in the mouse lymphoma TK assay (MLA), according to OECD Guideline No. 490. RESULTS: CQ (0.5-2.5mM) induced concentration- and time-dependent inhibition of cell growth associated with increased ROS/RNS production, amounting to 2342%+/-1108% of controls after 90min at 2.5mM. Additionally, CQ concentration-dependently caused direct DNA-damage, i.e. formation of DNA strand breaks and 8-hydroxy-2'-deoxyguanosine. Whereas the MLA indicated lack of mutagenicity of CQ after a 4h of treatment, CQ concentration-dependently increased total mutant frequency (MF) after 24h (about 2-fold at 2.5mM). But, based on the global evaluation factor concept, increase in MF did not reach biologically relevance. SIGNIFICANCE: CQ induced concentration-dependent, cytotoxic and genotoxic effects in L5178Y/TK(+/-) cells, most likely due to oxidative stress, but without mediating obvious biological relevant mutagenicity

Cytotoxicity of precious and nonprecious alloys - Experimental comparison of In vitro data from two laboratories

The aim of this investigation was to evaluate and compare the reproducibility of cytotoxicity data generated in two different laboratories using the same testing protocols. A series of dental alloys that are widely used in both countries were chosen. These alloys (five precious, two nonprecious) were wet ground up to 1200 grit SiC, sterilized in 70% ethanol, and extracted in sterile culture medium for 7 days. Pure copper was used as a positive control and Teflon® and media only were used as negative controls. Test and control samples were randomized and blinded to each laboratory. Cells, primary human gingival fibroblasts, and immortalized 3T3 fibroblasts, were exposed to the extracts for 24 h. Extract cytotoxicity was evaluated spectrophotometrically with the use of a mitochondrial enzyme activity assay. Data were collected from both laboratories, combined, and subjected to a mixed-model analysis of variance. No statistical difference was obtained for the immortalized 3T3 cells, except for two extracts in which differences between the two labs were significant but were still not cytotoxic. Furthermore, no statistical differences were found for the primary cells. These data strongly suggest that cytotoxicity tests performed in different laboratories with the use of the same test materials may lead to comparable results if sample preparation, cells, test procedures, and data analyses are carefully considered. © 2002 John Wiley & Sons, Inc

Isolation and prolonged expansion of oral mesenchymal stem cells under clinical-grade, GMP-compliant conditions differentially affects “stemness” properties

Abstract Background Development of clinical-grade cell preparations is central to meeting the regulatory requirements for cellular therapies under good manufacturing practice-compliant (cGMP) conditions. Since addition of animal serum in culture media may compromise safe and efficient expansion of mesenchymal stem cells (MSCs) for clinical use, this study aimed to investigate the potential of two serum/xeno-free, cGMP culture systems to maintain long-term “stemness” of oral MSCs (dental pulp stem cells (DPSCs) and alveolar bone marrow MSCs (aBMMSCs)), compared to conventional serum-based expansion. Methods DPSC and aBMMSC cultures (n = 6/cell type) were established from pulp and alveolar osseous biopsies respectively. Three culture systems were used: StemPro_MSC/SFM_XenoFree (Life Technologies); StemMacs_MSC/XF (Miltenyi Biotek); and α-MEM (Life Technologies) with 15% fetal bovine serum. Growth (population doublings (PDs)), immunophenotypic (flow cytometric analysis of MSC markers) and senescence (β-galactosidase (SA-β-gal) activity; telomere length) characteristics were determined during prolonged expansion. Gene expression patterns of osteogenic (ALP, BMP-2), adipogenic (LPL, PPAR-γ) and chondrogenic (ACAN, SOX-9) markers and maintenance of multilineage differentiation potential were determined by real-time PCR. Results Similar isolation efficiency and stable growth dynamics up to passage 10 were observed for DPSCs under all expansion conditions. aBMMSCs showed lower cumulative PDs compared to DPSCs, and when StemMacs was used substantial delays in cell proliferation were noted after passages 6–7. Serum/xeno-free expansion produced cultures with homogeneous spindle-shaped phenotypes, while serum-based expansion preserved differential heterogeneous characteristics of each MSC population. Prolonged expansion of both MSC types but in particular the serum/xeno-free-expanded aBMMSCs was associated with downregulation of CD146, CD105, Stro-1, SSEA-1 and SSEA-4, but not CD90, CD73 and CD49f, in parallel with an increase of SA-gal-positive cells, cell size and granularity and a decrease in telomere length. Expansion under both serum-free systems resulted in “osteogenic pre-disposition”, evidenced by upregulation of osteogenic markers and elimination of chondrogenic and adipogenic markers, while serum-based expansion produced only minor changes. DPSCs retained a diminishing (CCM, StemPro) or increasing (StemMacs) mineralization potential with passaging, while aBMMSCs lost this potential after passages 6–7 under all expansion conditions. Conclusions These findings indicate there is still a vacant role for development of qualified protocols for clinical-grade expansion of oral MSCs; a key milestone achievement for translation of research from the bench to clinics

Additional file 1: of Isolation and prolonged expansion of oral mesenchymal stem cells under clinical-grade, GMP-compliant conditions differentially affects “stemness” properties

is representative flow cytometry diagrams showing alterations in MSC marker expression in DPSCs during long-term expansion with CCM, StemMacs and StemPro (green line, unstained control; red line, marker of interest). Similar trends were observed for all DPSC and aBMMSC donors (PDF 418 kb