Static 3D Osteoblast Cell Culture on 3D Printed Titanium Scaffolds.

In vitro cell cultures are a very useful tool for the validation of biomaterial cytocompatibility, especially for bone tissue engineering scaffolds and bone implants. In this chapter, a protocol for a static three-dimensional osteoblast cell culture on titanium scaffolds and subsequent analysis of osteogenic capacity is presented. The protocol is explained for additively manufactured titanium scaffolds, but it can be extrapolated to other scaffolds with similar size and structure, while differing in composition or manufactured technology. Additionally, the protocol can be used for culture of other adherent cell types beyond osteoblast cells such as mesenchymal stem cells.

Assessment of Streptococcus mutans biofilm formation on calcium phosphate ceramics: The role of crystalline composition and microstructure.

Streptococcus mutans is one of the bacteria that initiates the colonization of the pellicle at the tooth surface. It forms a plaque, together with other bacteria, which gradually dissolves the pellicle and leaves the tooth surface unprotected against the acidic oral environment. Calcium phosphate ceramics are excellent synthetic materials for the study of biofilm formation in dentistry because they are comparable to teeth in chemical composition and structure. Calcium phosphates can be processed to achieve a variety of crystalline compounds with biologically relevant ionic substitutions and structures that allow study of the effect of the surface chemistry and the topography independently. In this article, we describe the preparation and characterization of three types of calcium phosphate-based materials as a suitable surface for the formation of the S. mutans biofilm: beta-tricalcium phosphate (ß-TCP); sintered hydroxyapatite (SHA); and calcium-deficient hydroxyapatite (CDHA). The densest biofilms were formed on the surfaces of SHA and CDHA, with no significant differences due to the stoichiometry or microstructure. In contrast, ß-TCP showed a lower susceptibility to S. mutans biofilm formation, suggesting that the crystalline structure is the controlling parameter. Subsequently, SHA was selected to develop a dental biofilm model that allowed study of S. mutans biofilm susceptibility to chlorhexidine and ethanol.

Lyophilized Polyvinylpyrrolidone Hydrogel for Culture of Human Oral Mucosa Stem Cells.

This work shows the synthesis of a polyvinylpyrrolidone (PVP) hydrogel by heat-activated polymerization and explores the production of hydrogels with an open porous network by lyophilisation to allow the three-dimensional culture of human oral mucosa stem cells (hOMSCs). The swollen hydrogel showed a storage modulus similar to oral mucosa and elastic solid rheological behaviour without sol transition. A comprehensive characterization of porosity by scanning electron microscopy, mercury intrusion porosimetry and nano-computed tomography (with spatial resolution below 1 µm) showed that lyophilisation resulted in the heterogeneous incorporation of closed oval-like pores in the hydrogel with broad size distribution (5 to 180 µm, d50 = 65 µm). Human oral mucosa biopsies were used to isolate hOMSCs, expressing typical markers of mesenchymal stem cells in more than 95% of the cell population. Direct contact cytotoxicity assay demonstrated that PVP hydrogel have no negative effect on cell metabolic activity, allowing the culture of hOMSCs with normal fusiform morphology. Pore connectivity should be improved in future to allow cell growth in the bulk of the PVP hydrogel.

Ecotoxicological sediment evaluations in marine aquaculture areas of Chile.

Given its geographic characteristics, the southern Chilean fjord area is subjected to growing environmental pressure from the development of diverse forms of aquaculture (i.e., fish, algae, shellfish). The sediments accumulate substances as a natural sink, and ecotoxicology assays offer a reliable and robust proxy for sediment quality analyses. This study's objective was to establish a mid-range toxicity base line for the sediments in the region by applying a battery of non-specific ecotoxicological assays. Sediment samples (28) were collected in the channels and fjords studied during the CIMAR-Fiordos 11 cruise (July 2005). The sediments were evaluated using different species endemic to the eastern Pacific as targets: Ampelisca araucana, Tisbe longicornis, Arbacia spatuligera, and Dunaliella tertiolecta. The conditions for each assay were reported previously. Of the four species used as ecotoxicological tools, only D. tertiolecta differed significantly from the control group (negative) in terms of its growth. This difference could be attributed to nutrient enrichment. In general, we concluded that, although local changes occurred in the sediments, the mesoscale magnitude of the ecotoxicological alterations was small. Nonetheless, a surveillance program should be implemented that would allow us to follow-up and analyze the changes that are taking place in the systems on broader scales of time and space.

A PPARs cross-talk concertedly commits C6 glioma cells to oligodendrocytes and induces enzymes involved in myelin synthesis.

Peroxisome proliferator activated receptors (PPARs, alpha, beta/delta, gamma) control lipid homeostasis and differentiation in various tissues and tumor cells. PPARbeta and PPARgamma increase oligodendrocyte maturation in glial mixed populations and spinal cord oligodendrocytes, respectively, and PPARbeta is known to modulate the activity of other PPARs. To assess a possible interaction between PPARs in glial cell differentiation we used the undifferentiated C6 glioma cell line as model. These cells express all three PPARs, but only PPARgamma shows transcriptional activity in agonist-based reporter gene assay. Agonist-activated PPARgamma up-regulates oligodendrocyte markers, down-regulates an astrocyte marker, and increases alkyl-dihydroxyacetone phosphate synthase, enzyme involved in the synthesis of myelin-rich plasmalogens. Similar effects are induced in PPARgamma overexpressing cells, which in addition show PPARbeta up-regulation. PPARbeta or PPARalpha agonists show no effect. Nevertheless, PPARbeta overexpression up-regulates PPARgamma and commits C6 cells to oligodendrocytes; effect that is abrogated by a PPARgamma antagonist or PPARgamma interference RNA. Moreover, PPARbeta overexpression also induces PPARalpha and its target genes, including acyl-CoA oxidase, enzyme involved in very long chain fatty acid recycling, and in the synthesis of myelin components such as docosahexaenoic acid. These results indicate for the first time, that PPARs concertedly cooperate in C6 glioma cell differentiation to oligodendrocytes. Further, they suggest that active PPARbeta might be essential for increasing oligodendrocyte distinctive markers and enzymes required for myelin synthesis in C6 glioma cells through up-regulation of PPARgamma and PPARalpha.

On Biophysical Properties and Sensitivity to Gap Junction Blockers of Connexin 39 Hemichannels Expressed in HeLa Cells.

Although connexins (Cxs) are broadly expressed by cells of mammalian organisms, Cx39 has a very restricted pattern of expression and the biophysical properties of Cx39-based channels [hemichannels (HCs) and gap junction channels (GJCs)] remain largely unknown. Here, we used HeLa cells transfected with Cx39 (HeLa-Cx39 cells) in which intercellular electrical coupling was not detected, indicating the absence of GJCs. However, functional HCs were found on the surface of cells exposed to conditions known to increase the open probability of other Cx HCs (e.g., extracellular divalent cationic-free solution (DCFS), extracellular alkaline pH, mechanical stimulus and depolarization to positive membrane potentials). Cx39 HCs were blocked by some traditional Cx HC blockers, but not by others or a pannexin1 channel blocker. HeLa-Cx39 cells showed similar resting membrane potentials (RMPs) to those of parental cells, and exposure to DCFS reduced RMPs in Cx39 transfectants, but not in parental cells. Under these conditions, unitary events of ~75 pS were frequent in HeLa-Cx39 cells and absent in parental cells. Real-time cellular uptake experiments of dyes with different physicochemical features, as well as the application of a machine-learning approach revealed that Cx39 HCs are preferentially permeable to molecules characterized by six categories of descriptors, namely: (1) electronegativity, (2) ionization potential, (3) polarizability, (4) size and geometry, (5) topological flexibility and (6) valence. However, Cx39 HCs opened by mechanical stimulation or alkaline pH were impermeable to Ca2+. Molecular modeling of Cx39-based channels suggest that a constriction present at the intracellular portion of the para helix region co-localizes with an electronegative patch, imposing an energetic and steric barrier, which in the case of GJCs may hinder channel function. Results reported here demonstrate that Cx39 form HCs and add to our understanding of the functional roles of Cx39 HCs under physiological and pathological conditions in cells that express them.

Pannexin 1 channels in skeletal muscles.

Normal myotubes and adult innervated skeletal myofibers express the glycoprotein pannexin1 (Panx1). Six of them form a "gap junction hemichannel-like" structure that connects the cytoplasm with the extracellular space; here they will be called Panx1 channels. These are poorly selective channels permeable to ions, small metabolic substrate, and signaling molecules. So far little is known about the role of Panx1 channels in muscles but skeletal muscles of Panx1(-/-) mice do not show an evident phenotype. Innervated adult fast and slow skeletal myofibers show Panx1 reactivity in close proximity to dihydropyridine receptors in the sarcolemma of T-tubules. These Panx1 channels are activated by electrical stimulation and extracellular ATP. Panx1 channels play a relevant role in potentiation of muscle contraction because they allow release of ATP and uptake of glucose, two molecules required for this response. In support of this notion, the absence of Panx1 abrogates the potentiation of muscle contraction elicited by repetitive electrical stimulation, which is reversed by exogenously applied ATP. Phosphorylation of Panx1 Thr and Ser residues might be involved in Panx1 channel activation since it is enhanced during potentiation of muscle contraction. Under denervation, Panx1 levels are upregulated and this partially explains the reduction in electrochemical gradient, however its absence does not prevent denervation-induced atrophy but prevents the higher oxidative state. Panx1 also forms functional channels at the cell surface of myotubes and their functional state has been associated with intracellular Ca(2+) signals and regulation of myotube plasticity evoked by electrical stimulation. We proposed that Panx1 channels participate as ATP channels and help to keep a normal oxidative state in skeletal muscles.

Modulation of the Akt pathway reveals a novel link with PERK/eIF2α, which is relevant during hypoxia.

The unfolded protein response (UPR) and the Akt signaling pathway share several regulatory functions and have the capacity to determine cell outcome under specific conditions. However, both pathways have largely been studied independently. Here, we asked whether the Akt pathway regulates the UPR. To this end, we used a series of chemical compounds that modulate PI3K/Akt pathway and monitored the activity of the three UPR branches: PERK, IRE1 and ATF6. The antiproliferative and antiviral drug Akt-IV strongly and persistently activated all three branches of the UPR. We present evidence that activation of PERK/eIF2α requires Akt and that PERK is a direct Akt target. Chemical activation of this novel Akt/PERK pathway by Akt-IV leads to cell death, which was largely dependent on the presence of PERK and IRE1. Finally, we show that hypoxia-induced activation of eIF2α requires Akt, providing a physiologically relevant condition for the interaction between Akt and the PERK branch of the UPR. These data suggest the UPR and the Akt pathway signal to one another as a means of controlling cell fate.

Assessment of EDTA in chromium (III-VI) toxicity on marine intertidal crab (Petrolisthes laevigatus).

Chromium poses a potential threat to coastal ecosystems. We used standard toxicity bioassays (semi-static, chronic) to evaluate EDTA as a chelating agent for reducing trivalent and hexavalent chromium toxicity on Petrolisthes laevigatus. Crab survival decreased linearly with increased chromium concentrations and dropped significantly beginning at 40 mg/L Cr (VI) and 80 mg/L Cr (III). No significant differences were observed with Cr (III) + EDTA as compared with untreated controls. Cr (VI) toxicity was greater than that of Cr (III), with low individual survival rates. The protective effect of EDTA in the medium increased crab survival by 41%-48%.