Mihaela R. Cimpan

Induction of cell death by TiO2 nanoparticles : Studies on a human monoblastoid cell line

The cellular responses to degradation products from titanium (Ti) implants are important indicators for the biocompatibility of these widely used implantable medical devices. The potential toxicity of nanoparticulate matter released from implants has been scarcely studied. The aim of this study was to investigate the potential of TiO2 nanoparticles to induce modifications characteristic for death by apoptosis and/or necrosis in U937 human monoblastoid cells. Suspensions of TiO2 nanoparticles with a diameter <100nm were prepared in RPMI cell culture medium at concentrations that covered a range (0.005-4mg/ml) corresponding to concentrations found in blood, plasma, or in tissues surrounding Ti implants. The cells were exposed to the nanoparticulate suspensions for 24 and 48h and the responses were evaluated by flow cytometry and transmission electron microscopy. TiO2 nanoparticles induced both apoptotic and necrotic modifications in U937 cells.

Role of physicochemical characteristics in the uptake of TiO2 nanoparticles by fibroblasts.

The relation between the physico-chemical properties of nanoparticles (NPs) and the degree of cellular uptake is incompletely elucidated. In this study, we investigated the influence on the cellular uptake of a wide range of fully characterized TiO2 NPs. L929 fibroblasts were exposed for 24 h to clinically relevant concentrations of nano-TiO2 and the degree of their association was assessed by ultrahigh resolution imaging microscopy (URI), scanning (SEM) and transmission (TEM) electron microscopy, as well as inductivity coupled plasma-mass spectroscopy (ICP-MS). The role of actin polymerization, a central feature of active internalization, was also studied and the results indicated that the internalization of TiO2 NPs involves a combination of actin-dependent uptake of large agglomerates as well as non actin-dependent uptake of small agglomerates. SEM and TEM revealed that the agglomerates of all NPs types were attached to the cellular membrane as well as internalized and confined inside cytoplasmic vesicles. URI and ICP-MS demonstrated that the particle association with cells was dose-dependent. The highest association was observed for spherical particles having mixed anatase-rutile crystallographic phase and the lowest for spindle-shaped rutile particles. ICP-MS revealed that the association was size-dependent in the order 5>10>40 nm for anatase spherical nanoparticles.

High throughput toxicity screening and intracellular detection of nanomaterials

With the growing numbers of nanomaterials (NMs), there is a great demand for rapid and reliable ways of testing NM safety—preferably using in vitro approaches, to avoid the ethical dilemmas associated with animal research. Data are needed for developing intelligent testing strategies for risk assessment of NMs, based on grouping and read‐across approaches. The adoption of high throughput screening (HTS) and high content analysis (HCA) for NM toxicity testing allows the testing of numerous materials at different concentrations and on different types of cells, reduces the effect of inter‐experimental variation, and makes substantial savings in time and cost. HTS/HCA approaches facilitate the classification of key biological indicators of NM‐cell interactions. Validation of in vitro HTS tests is required, taking account of relevance to in vivo results. HTS/HCA approaches are needed to assess dose‐ and time‐dependent toxicity, allowing prediction of in vivo adverse effects. Several HTS/HCA methods are being validated and applied for NM testing in the FP7 project NANoREG, including Label‐free cellular screening of NM uptake, HCA, High throughput flow cytometry, Impedance‐based monitoring, Multiplex analysis of secreted products, and genotoxicity methods—namely High throughput comet assay, High throughput in vitro micronucleus assay, and γH2AX assay. There are several technical challenges with HTS/HCA for NM testing, as toxicity screening needs to be coupled with characterization of NMs in exposure medium prior to the test; possible interference of NMs with HTS/HCA techniques is another concern. Advantages and challenges of HTS/HCA approaches in NM safety are discussed. WIREs Nanomed Nanobiotechnol 2017, 9:e1413. doi: 10.1002/wnan.1413 For further resources related to this article, please visit the WIREs website.

The effect of heat- and auto-polymerized denture base polymers on clonogenicity, apoptosis, and necrosis in fibroblasts: denture base polymers induce apoptosis and necrosis.

Eluates from poly(methyl methacrylate)-based denture base polymers have recently been found to enhance death by apoptosis and necrosis in U-937 human monoblastoid cells. The present study investigated the potential of such polymers to induce apoptosis and/or necrosis and to alter clonogenicity in L929 murine fibroblasts. A fibroblast cell line was chosen because the impairment of fibroblasts subjacent to denture bases may result in a weaker or more permeable mucosa. Two aspects were addressed: the effect of direct contact with the denture base polymers and the effect of eluates extracted from the polymers. For this purpose L929 fibroblasts were seeded on disks manufactured from three heat-polymerized and four autopolymerized denture base polymers or in different concentrations of their eluates. The effects were evaluated by light, fluorescent, confocal and electron microscopy, counting of colonies, and flow cytometry. Disks and eluates of all polymers enhanced cell death by apoptosis and necrosis in L929 cells and decreased their clonogenic potential in a dose-dependent manner. Apoptosis was the main form of cell death. In general, the deleterious effects were stronger when cells were plated directly on the polymer disks than in the eluates. The autopolymerized polymers, except one, yielded higher percentages of apoptosis and necrosis than the heat-polymerized polymers. The results of the study indicated that poly(methyl methacrylate)-based denture base polymers trigger death-signals in L929 fibroblasts and open doors for possible modulation of the cell/biomaterial interaction.Eluates from poly(methyl methacrylate)-based denture base polymers have recently been found to enhance death by apoptosis and necrosis in U-937 human monoblastoid cells. The present study investigated the potential of such polymers to induce apoptosis and/or necrosis and to alter clonogenicity in L929 murine fibroblasts. A fibroblast cell line was chosen because the impairment of fibroblasts subjacent to denture bases may result in a weaker or more permeable mucosa. Two aspects were addressed: the effect of direct contact with the denture base polymers and the effect of eluates extracted from the polymers. For this purpose L929 fibroblasts were seeded on disks manufactured from three heat-polymerized and four autopolymerized denture base polymers or in different concentrations of their eluates. The effects were evaluated by light, fluorescent, confocal and electron microscopy, counting of colonies, and flow cytometry. Disks and eluates of all polymers enhanced cell death by apoptosis and necrosis in L929 cells and decreased their clonogenic potential in a dose-dependent manner. Apoptosis was the main form of cell death. In general, the deleterious effects were stronger when cells were plated directly on the polymer disks than in the eluates. The autopolymerized polymers, except one, yielded higher percentages of apoptosis and necrosis than the heat-polymerized polymers. The results of the study indicated that poly(methyl methacrylate)-based denture base polymers trigger death-signals in L929 fibroblasts and open doors for possible modulation of the cell/biomaterial interaction.

The effect of blood protein adsorption on cellular uptake of anatase TiO2 nanoparticles

Protein adsorption onto nanoparticles (NPs) in biological fluids has emerged as an important factor when testing biological responses to NPs, as this may influence both uptake and subsequent toxicity. The aim of the present study was to quantify the adsorption of proteins onto TiO2 NPs and to test the influence on cellular uptake. The surface composition of the particles was characterized by thermal analysis and by X-ray photoelectron spectroscopy. The adsorption of three blood proteins, ie, human serum albumin (HSA), γ-globulins (Glbs), and fibrinogen (Fib), onto three types of anatase NPs of different sizes was quantified for each protein. The concentration of the adsorbed protein was measured by ultraviolet-visible spectrophotometry using the Bradford method. The degree of cellular uptake was quantified by inductivity coupled plasma mass spectroscopy, and visualized by an ultra-high resolution imaging system. The proteins were adsorbed onto all of the anatase NPs. The quantity adsorbed increased with time and was higher for the smaller particles. Fib and Glbs showed the highest affinity to TiO2 NPs, while the lowest was seen for HSA. The adsorption of proteins affected the surface charge and the hydrodynamic diameter of the NPs in cell culture medium. The degree of particle uptake was highest in protein-free medium and in the presence HSA, followed by culture medium supplemented with Glbs, and lowest in the presence of Fib. The results indicate that the uptake of anatase NPs by fibroblasts is influenced by the identity of the adsorbed protein.

Limited in-depth invasion of Fusobacterium nucleatum into in vitro reconstructed human gingiva.

OBJECTIVE Fusobacterium nucleatum is an opportunistic pathogen with a key role in subgingival plaque formation and it is found in increased numbers in periodontally affected sites. This study aimed to investigate the potential of F. nucleatum to penetrate and induce alterations in an in vitro reconstructed human gingival mucosa model. METHODS Three-dimensional (3D) organotypic models of human gingiva were engineered using primary gingival keratinocytes and fibroblasts. The reconstructed tissues were challenged with four different strains of fluorescently labelled F. nucleatum in suspension placed on top of epithelial layers. Confocal laser scanning was used to assess the presence of fusobacteria through the organotypic model. Apoptosis (cleaved caspase-3) and cell proliferation (Ki-67) were evaluated by the use of immunohistochemistry in 3D-tissue models. Quantitative real-time PCR was performed to investigate the mRNA expression for MMP-13 and E-cadherin in both 3D-tissues and monolayers. RESULTS F. nucleatum invaded the superficial epithelial layers of gingival 3D-tissue models. Challenged tissues showed accentuated shedding of superficial layers and increased number of cleaved caspase-3 and Ki-67 positive cells than controls, although not statistically significant. Levels of E-cadherin and MMP-13 mRNA were not significantly perturbed in multilayer culture. A variable and disproportionate response of MMP-13 mRNA level resulted in challenged primary keratinocytes in monolayers, compared to multilayer culture. CONCLUSION These results indicate that F. nucleatum is able to invade superficially a differentiated, stratified gingival epithelium in vitro and triggers the efficient elimination of bacterial infection through epithelial shredding without causing a permanent damage of the tissue.

In vitro reconstruction of human junctional and sulcular epithelium

BACKGROUND The aim of this study was to develop and characterize standardized in vitro three-dimensional organotypic models of human junctional epithelium (JE) and sulcular epithelium (SE). METHODS Organotypic models were constructed by growing human normal gingival keratinocytes on top of collagen matrices populated with gingival fibroblasts (GF) or periodontal ligament fibroblasts (PLF). Tissues obtained were harvested at different time points and assessed for epithelial morphology, proliferation (Ki67), expression of JE-specific markers (ODAM and FDC-SP), cytokeratins (CK), transglutaminase, filaggrin, and basement membrane proteins (collagen IV and laminin1). RESULTS The epithelial component in 3- and 5-day organotypics showed limited differentiation and expressed Ki-67, ODAM, FDC-SP, CK 8, 13, 16, 19, and transglutaminase in a similar fashion to control JE samples. PLF supported better than GF expression of CK19 and suprabasal proliferation, although statistically significant only at day 5. Basement membrane proteins started to be deposited only from day 5. The rate of proliferating cells as well as the percentage of CK19-expressing cells decreased significantly in 7- and 9-day cultures. Day 7 organotypics presented higher number of epithelial cell layers, proliferating cells in suprabasal layers, and CK expression pattern similar to SE. CONCLUSION Both time in culture and fibroblast type had impact on epithelial phenotype. Five-day cultures with PLF are suggested as JE models, 7-day cultures with PLF or GF as SE models, while 9-day cultures with GF as gingival epithelium (GE) models. Such standard, reproducible models represent useful tools to study periodontal bacteria–host interactions in vitro.

Contact-dependent transfer of TiO2 nanoparticles between mammalian cells

Abstract Cellular organelles have been shown to shuttle between cells in co-culture. We hereby show that titanium dioxide (TiO2) nanoparticles (NPs) can be transferred in such a manner, between cells in direct contact, along with endosomes and lysosomes. A co-culture system was employed for this purpose and the NP transfer was observed in mammalian cells including normal rat kidney (NRK) and HeLa cells. We found that the small GTPase Arf6 facilitates the intercellular transfer of smaller NPs and agglomerates. Spherical, anatase nano-TiO2 with sizes of 5 (Ti5) and 40 nm (Ti40) were used in this study. Humans are increasingly exposed to TiO2 NPs from external sources such as constituents of foods, cosmetics, and pharmaceuticals, or from internal sources represented by Ti-based implants, which release NPs upon abrasion. Exposure to 5 mg/l of Ti5 and Ti40 for 24 h did not affect cellular viability but modified their ability to communicate with surrounding cells. Altogether, our results have important implications for the design of nanomedicines, drug delivery and toxicity.

In vitro cytotoxicity assessment of nanodiamond particles and their osteogenic potential: CYTOTOXICITY ASSESSMENT OF NANODIAMOND PARTICLES

Scaffolds functionalized with nanodiamond particles (nDP) hold great promise with regard to bone tissue formation in animal models. Degradation of the scaffolds over time may leave nDP within the tissues, raising concerns about possible long-term unwanted effects. Human SaOS-2 osteoblast-like cells and U937 monoblastoid cells were exposed to five different concentrations (0.002-2 mg/L) of nDP (size range: 2.36-4.42 nm) for 24 h. Cell viability was assessed by impedance-based methods. The differential expression of stress and toxicity-related genes was evaluated by polymerase chain reaction (PCR) super-array, while the expression of selected inflammatory and cell death markers was determined by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Furthermore, the expression of osteogenic genes by SaOS-2 cells, alkaline phosphatase activity and the extracellular calcium nodule deposition in response to nDP were determined in vitro. Cells responded differently to higher nDP concentrations (≥0.02 mg/L), that is, no loss of viability for SaOS-2 cells and significantly reduced viability for U937 cells. Gene expression showed significant upregulation of several cell death and inflammatory markers, among other toxicity reporter genes, indicating inflammatory and cytotoxic responses in U937 cells. Nanodiamond particles improved the osteogenicity of osteoblast-like cells with no evident cytotoxicity. However, concentration-dependent cytotoxic and inflammatory responses were seen in the U937 cells, negatively affecting osteogenicity in co-cultures.

Nano-TiO2 penetration of oral mucosa: in vitro analysis using 3D organotypic human buccal mucosa models.

Background Oral cavity is a doorway for a variety of products containing titanium dioxide (TiO2) nanoparticles (NPs) (nano‐TiO2) such as food additives, oral healthcare products and dental materials. Their potential to penetrate and affect normal human oral mucosa is not yet determined. Objectives To evaluate the ability of nano‐TiO2 to penetrate the in vitro reconstructed normal human buccal mucosa (RNHBM). Methods RNHBM was generated from primary normal human oral keratinocytes and fibroblasts isolated from buccal oral mucosa of healthy patients (n = 6). The reconstructed tissues were exposed after 10 days to clinically relevant concentrations of spherical or spindle rutile nano‐TiO2 in suspension for short (20 min) and longer time (24 h). Ultrahigh‐resolution imaging (URI) microscopy (CytoViva™, Auburn, AL, USA) was used to assess the depth of penetration into reconstructed tissues. Results Ultrahigh‐resolution imaging microscopy demonstrated the presence of nano‐TiO2 mostly in the epithelium of RNHBM at both 20 min and 24‐h exposure, and this was shape and doze dependent at 24 h of exposure. The depth of penetration diminished in time at higher concentrations. The exposed epithelium showed increased desquamation but preserved thickness. Conclusion Nano‐TiO2 is able to penetrate RNHBM and to activate its barrier function in a doze‐ and time‐dependent manner.