An X-ray micro-fluorescence study to investigate the distribution of Al, Si, P and Ca ions in the surrounding soft tissue after implantation of a calcium phosphate-mullite ceramic composite in a rabbit animal model

Synthetic calcium phosphates, despite their bioactivity, are brittle. Calcium phosphate-mullite composites have been suggested as potential dental and bone replacement materials which exhibit increased toughness. Aluminium, present in mullite, has however been linked to bone demineralisation and neurotoxicity: it is therefore important to characterise the materials fully in order to understand their in vivo behaviour. The present work reports the compositional mapping of the interfacial region of a calcium phosphate-20 wt% mullite biocomposite/soft tissue interface, obtained from the samples implanted into the long bones of healthy rabbits according to standard protocols (ISO-10993) for up to 12 weeks. X-ray micro-fluorescence was used to map simultaneously the distribution of Al, P, Si and Ca across the ceramic-soft tissue interface. A well defined and sharp interface region was present between the ceramic and the surrounding soft tissue for each time period examined. The concentration of Al in the surrounding tissue was found to fall by two orders of magnitude, to the background level, within similar to 35 mu m of the implanted ceramic.

Microspheres as a vehicle for biomolecule delivery to neural stem cells

Neural stem cells (NSC) are a valuable model system for understanding the intrinsic and extrinsic controls for self-renewal and differentiation choice. They also offer a platform for drug screening and neurotoxicity studies, and hold promise for cell replacement therapies for the treatment of neurodegenerative diseases. Fully exploiting the potential of this experimental tool often requires the manipulation of intrinsic cues of interest using transfection methods, to which NSC are relatively resistant. In this paper, we show that mouse and human NSC readily take up polystyrene-based microspheres which can be loaded with a range of chemical or biological cargoes. This uptake can take place in the undifferentiated stage without affecting NSC proliferation and their capacity to give rise to neurons and glia. We demonstrate that ß-galactosidase-loaded microspheres could be efficiently introduced into NSC with no apparent toxic effect, thus providing proof-of-concept for the use of microspheres as an alternative biomolecule delivery system.

The toxicity of hexanedione isomers in neural and astrocytic cell lines

The metabolite 2,5-hexanedione (HD) is the cause of neurotoxicity linked with chronic n-hexane exposure. Acute exposure to high levels of 2,5-HD, have also shown toxic effects in neuronal cells and non-neuronal cells. Isomers of 2,5-HD, 2,3- and 3,4-HD, added to foodstuffs, are reported to be non-toxic. The acute cytotoxic effects of 2,5-, 2,3- and 3,4-HD were evaluated in neural (NT2.N, SK-N-SH), astrocytic (CCF-STTG1) and non-neural (NT2.D1) cell lines. All the cell lines were highly resistant to 2,5-HD (34-426 mM) at 4-h exposure, although sensitivity was greatest with NT2.D1, then SK-N-SH, NT2.N and finally the CCF-STTG1 line. At 24-h exposure, cell vulnerability increased 5-10-fold. The NT2.D1 cells were again the most sensitive, followed by NT2.N, SK-N-SH and then the CCF-STTG1 cells. 2,3- and 3,4-HD (8-84 mM), were significantly more toxic towards all four cell lines compared with 2,5-HD, after 4-h exposure. After 24-h exposure there was a 12-fold increase in inhibition of MTT turnover in the SK-N-SH cells and a 4-fold increase in the CCF-STTG1 cells, compared with 2,5-HD exposure. 2,3- and 3,4-HD, were significantly less toxic to the NT2.N cells than the SK-N-SH cells after 24-h exposure to the compounds, demonstrating a differing toxin vulnerability between these neural and neuroblastoma cell lines. This study indicates that these non-neuronal and neuronal cells are acutely resistant to 2,5-HD cytotoxicity, whilst the previously unreported sensitivity of all four cell lines to the 2,3- and 3,4- isomers of HD to has been shown to be significantly greater than that of 2,5-HD. © 2006 Elsevier B.V. All rights reserved.

Phytoestrogens modulate breast cancer resistance protein expression and function at the blood-cerebrospinal fluid barrier

PURPOSE: Breast cancer resistance protein (BCRP/ABCG2) is a drug efflux transporter expressed at the blood cerebrospinal fluid barrier (BCSFB), and influences distribution of drugs into the central nervous systems (CNS). Current inhibitors have failed clinically due to neurotoxicity. Novel approaches are needed to identify new modulators to enhance CNS delivery. This study examines 18 compounds (mainly phytoestrogens) as modulators of the expression/function of BCRP in an in vitro rat choroid plexus BCSFB model. METHODS: Modulators were initially subject to cytotoxicity (MTT) assessment to determine optimal non-toxic concentrations. Reverse-transcriptase PCR and confocal microscopy were used to identify the presence of BCRP in Z310 cells. Thereafter modulation of the intracellular accumulation of the fluorescent BCRP probe substrate Hoechst 33342 (H33342), changes in protein expression of BCRP (western blotting) and the functional activity of BCRP (membrane insert model) were assessed under modulator exposure. RESULTS: A 24 hour cytotoxicity assay (0.001 µM-1000 µM) demonstrated the majority of modulators possessed a cellular viability IC50 > 148 µM. Intracellular accumulation of H33342 was significantly increased in the presence of the known BCRP inhibitor Ko143 and, following a 24 hour pre-incubation, all modulators demonstrated statistically significant increases in H33342 accumulation (P < 0.001), when compared to control and Ko143. After a 24 hour pre-incubation with modulators alone, a 0.16-2.5-fold change in BCRP expression was observed for test compounds. The functional consequences of this were confirmed in a permeable insert model of the BCSFB which demonstrated that 17-β-estradiol, naringin and silymarin (down-regulators) and baicalin (up-regulator) can modulate BCRP-mediated transport function at the BCSFB. CONCLUSION: We have successfully confirmed the gene and protein expression of BCRP in Z310 cells and demonstrated the potential for phytoestrogen modulators to influence the functionality of BCRP at the BCSFB and thereby potentially allowing manipulation of CNS drug disposition.

Phytochemical mediated-modulation of the expression and transporter function of breast cancer resistance protein at the blood-brain barrier:an in-vitro study

Clinical translation of BCRP inhibitors have failed due to neurotoxicity and novel approaches are required to identify suitable modulators of BCRP to enhance CNS drug delivery. In this study we examine 18 compounds, primarily phytochemicals, as potential novel modulators of AhR-mediated regulation of BCRP expression and function in immortalised and primary porcine brain microvascular endothelial cells as a mechanism to enhance CNS drug delivery. The majority of modulators possessed a cellular viability IC50 > 100 µM in both cell systems. BCRP activity, when exposed to modulators for 1 hour, was diminished for most modulators through significant increases in H33342 accumulation at < 10 µM with 2,6,4-trimethoflavone increasing H33342 intracellular accumulation by 3.7–6.6 fold over 1–100 µM. Western blotting and qPCR identified two inducers of BCRP (quercetin and naringin) and two down-regulators (17-β-estradiol and curcumin) with associated changes in BCRP efflux transport function further confirmed in both cell lines. siRNA downregulation of AhR resulted in a 1.75 ± 0.08 fold change in BCRP expression, confirming the role of AhR in the regulation of BCRP. These findings establish the regulatory role AhR of in controlling BCRP expression at the BBB and confirm quercetin, naringin, 17-β-estradiol, and curcumin as novel inducers and down-regulators of BCRP gene, protein expression and functional transporter activity and hence potential novel target sites and candidates for enhancing CNS drug delivery.