A comparative study of different in vitro lung cell culture systems to assess the most beneficial tool for screening the potential adverse effects of carbon nanotubes

To determine the potential inhalatory risk posed by carbon nanotubes (CNTs), a tier-based approach beginning with an in vitro assessment must be adopted. The purpose of this study therefore was to compare 4 commonly used in vitro systems of the human lung (human blood monocyte-derived macrophages [MDM] and monocyte-derived dendritic cells [MDDC], 16HBE14o- epithelial cells, and a sophisticated triple cell co-culture model [TCC-C]) via assessment of the biological impact of different CNTs (single-walled CNTs [SWCNTs] and multiwalled CNTs [MWCNTs]) over 24h. No significant cytotoxicity was observed with any of the cell types tested, although a significant (p < .05), dose-dependent increase in tumor necrosis factor (TNF)-α following SWCNT and MWCNT exposure at concentrations up to 0.02mg/ml to MDM, MDDC, and the TCC-C was found. The concentration of TNF-α released by the MDM and MDDC was significantly higher (p < .05) than the TCC-C. Significant increases (p < .05) in interleukin (IL)-8 were also found for both 16HBE14o- epithelial cells and the TCC-C after SWCNTs and MWCNTs exposure up to 0.02mg/ml. The TCC-C, however, elicited a significantly (p < .05) higher IL-8 release than the epithelial cells. The oxidative potential of both SWCNTs and MWCNTs (0.005-0.02mg/ml) measured by reduced glutathione (GSH) content showed a significant difference (p < .05) between each monoculture and the TCC-C. It was concluded that because only the co-culture system could assess each endpoint adequately, that, in comparison with monoculture systems, multicellular systems that take into consideration important cell type-to-cell type interactions could be used as predictive in vitro screening tools for determining the potential deleterious effects associated with CNTs.

The shape of a bone scraper: an in vitro pilot study using porcine bone chips.

Bone scrapers are commonly used to harvest autologous bone in oral and implant surgery. The angle of the cutting blade is a variable that distinguishes bone scrapers. In the present study, the impact of the angle of the cutting blade on the in vitro characteristics of harvested bone was determined. Bone scrapers with blade angles of 15°, 25°, 35°, 45°, and 55° were used to harvest porcine cortical mandibular bone. The number and characteristics of the cells that grew out from the bone chips were examined. The data showed that, independent of the angle of the cutting blade, viable cells were barely detectable in fresh bone grafts. However, cells with a fibroblastic morphology appeared within 1 week in the culture dishes. After 21 days, the number of cells did not differ significantly between the five preparations. Moreover, cells responded to incubation with bone morphogenetic protein 7 (BMP-7) with an increased alkaline phosphatase activity, irrespective of the preparation. The data suggest that bone scrapers with different cutting angles produce bone chips with comparable in vitro characteristics.

Human bone chips release of sclerostin and FGF-23 into the culture medium: an in vitro pilot study

Abstract OBJECTIVE: Signaling molecules derived from osteocytes have been proposed as a mechanism by which autografts contribute to bone regeneration. However, there have been no studies that determined the role of osteocytes in bone grafts. MATERIAL AND METHOD: Herein, it was examined whether bone chips and demineralized bone matrix release sclerostin and FGF-23, both of which are highly expressed by osteocytes. RESULTS: Bone grafts from seven donors were placed in culture medium. Immunoassay showed that bone chips released sclerostin (median 1.0 ng/ml) and FGF-23 (median 9.8 relative units/ml) within the first day, with declining levels overtime. Demineralized bone matrix also released detectable amounts of sclerostin into culture medium, while FGF-23 remained close to the detection limit. In vitro expanded isolated bone cells failed to release detectable amounts of sclerostin and FGF-23. CONCLUSION: These results suggest that autografts but also demineralized bone matrix can release signaling molecules that are characteristically produced by osteocytes. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. KEYWORDS: FGF-23; autologous bone; bone grafts; demineralized bone matrix; osteocytes; sclerostin

Differential influence of maternal and fetal pregnancy factors on the in-vitro induction of human regulatory T cells: a preliminary study.

PROBLEM Given the important role of regulatory T cells (Treg) for successful pregnancy, the ability of soluble maternal and fetal pregnancy factors to induce human Treg was investigated. METHOD OF STUDY Peripheral blood mononuclear cells (PBMCs) or isolated CD4+CD25‒ cells were cultured in the presence of pooled second or third trimester pregnancy sera, steroid hormones or supernatants from placental explants, and the numbers and function of induced CD4+CD25+FOXP3+ Treg were analysed. RESULTS Third trimester pregnancy sera and supernatants of early placental explants, but not sex steroid hormones, induced an increase of Tregs from PBMCs. Early placental supernatant containing high levels of tumour necrosis factor-α, interferon-γ, interleukins -1, -6 and -17, soluble human leucocyte antigen-G, and transforming growth factor-β1, increased the proportion of Treg most effectively and was able to induce interleukin-10-secreting-Treg from CD4+CD25‒cells. CONCLUSIONS Compared with circulating maternal factors, placental- and fetal-derived factors appear to exert a more powerful effect on numerical changes of Treg, thereby supporting fetomaternal tolerance during human pregnancy.

LRH-1 May Rescue SF-1 Deficiency for Steroidogenesis: An in vitro and in vivo Study

Steroidogenic factor 1 (NR5A1/SF-1) mutations usually manifest in 46,XY individuals with variable degrees of disordered sex development and in 46,XX women with ovarian insufficiency. So far, there is no genotype-phenotype correlation. The broad spectrum of phenotype with NR5A1 mutations may be due to a second hit in a gene with similar function to NR5A1/SF-1. Liver receptor homologue-1 (LRH-1/NR5A2) might be a good candidate. We performed in vitro studies for the interplay between SF-1, LRH-1 and DAX-1, expression profiles in human steroidogenic tissues, and NR5A2 genetic studies in a cohort (11 patients, 8 relatives, 11 families) harboring heterozygote NR5A1/SF-1 mutations. LRH-1 isoforms transactivate the CYP17A1 and HSD3B2 promoters similarly to SF-1 and compensate for SF-1 deficiency. DAX-1 inhibits SF-1- and LRH-1-mediated transactivation. LRH-1 is found expressed in human adult and fetal adrenals and testes. However, no NR5A2/LRH-1 mutations were detected in 14 individuals with heterozygote NR5A1/SF-1 mutations. These findings demonstrate that in vitro LRH-1 can act like SF-1 and compensate for its deficiency. Expression of LRH-1 in fetal testis suggests a role in male gonadal development. However, as we found no NR5A2/LRH-1 mutations, the 'second genetic hit' in SF-1 patients explaining the broad phenotypic variability remains elusive.

Modeling immune functions of the mouse blood-cerebrospinal fluid barrier in vitro: primary rather than immortalized mouse choroid plexus epithelial cells are suited to study immune cell migration across this brain barrier.

BACKGROUND The blood-cerebrospinal fluid barrier (BCSFB) established by the choroid plexus (CP) epithelium has been recognized as a potential entry site of immune cells into the central nervous system during immunosurveillance and neuroinflammation. The location of the choroid plexus impedes in vivo analysis of immune cell trafficking across the BCSFB. Thus, research on cellular and molecular mechanisms of immune cell migration across the BCSFB is largely limited to in vitro models. In addition to forming contact-inhibited epithelial monolayers that express adhesion molecules, the optimal in vitro model must establish a tight permeability barrier as this influences immune cell diapedesis. METHODS We compared cell line models of the mouse BCSFB derived from the Immortomouse(®) and the ECPC4 line to primary mouse choroid plexus epithelial cell (pmCPEC) cultures for their ability to establish differentiated and tight in vitro models of the BCSFB. RESULTS We found that inducible cell line models established from the Immortomouse(®) or the ECPC4 tumor cell line did not express characteristic epithelial proteins such as cytokeratin and E-cadherin and failed to reproducibly establish contact-inhibited epithelial monolayers that formed a tight permeability barrier. In contrast, cultures of highly-purified pmCPECs expressed cytokeratin and displayed mature BCSFB characteristic junctional complexes as visualized by the junctional localization of E-cadherin, β-catenin and claudins-1, -2, -3 and -11. pmCPECs formed a tight barrier with low permeability and high electrical resistance. When grown in inverted filter cultures, pmCPECs were suitable to study T cell migration from the basolateral to the apical side of the BCSFB, thus correctly modelling in vivo migration of immune cells from the blood to the CSF. CONCLUSIONS Our study excludes inducible and tumor cell line mouse models as suitable to study immune functions of the BCSFB in vitro. Rather, we introduce here an in vitro inverted filter model of the primary mouse BCSFB suited to study the cellular and molecular mechanisms mediating immune cell migration across the BCSFB during immunosurveillance and neuroinflammation.