How stereochemistry of lipid components can affect lipid organization and the route of liposome internalization into cells.

Though liposome-based drugs are in clinical use, the mechanism of cell internalization of liposomes is yet an object of controversy. The present experimental investigation, carried out on human glioblastoma cells, indicated different internalization routes for two diastereomeric liposomes. Molecular dynamics simulations of the lipid bilayers of the two formulations indicated that the different stereochemistry of a lipid component controls some parameters such as area per lipid molecule and fluidity of lipid membranes, surface potential and water organization at the lipid/water interface, all of which affect the interaction with biomolecules and cell components.

CD34 human hematopoietic progenitor cell line, MUTZ-3, differentiates into functional osteoclasts.

OBJECTIVE: CD14(+) monocyte cell lines can differentiate into an osteoclast (OC)-like lineage. However, the identification of human cell lines with stem cell characteristics, capable of differentiating into OCs, would provide a tool for the study of the molecular mechanisms regulating their commitment, differentiation, and function. Since the human acute myeloid leukemia cell line MUTZ-3 contains both CD34(+) stem cell and CD14(+) cell populations, we investigated the capacity of the stem/progenitor CD34(+) population to differentiate into functional OCs. MATERIALS AND METHODS: Sorted MUTZ-3-CD34(+) and MUTZ-3-CD14(+) cells were cultured in presence of M-CSF, RANK-L, and TNF-alpha to generate OCs. Differentiation was evaluated by TRAP staining and RT-PCR, which assessed the expression of c-fms, RANK, MMP-9, CATK, TRAP, and CTR in -CD34(+)OC and -CD14(+)OC cells. Resorption pit formation was also evaluated. CD34, CD14, M-CSF-R, RANK, and CTR expression was assessed by FACS analysis. RESULTS: MUTZ-3-CD34(+) differentiated into OCs, displaying the full range of differentiation markers; MMP-9, CATK, TRAP, and RANK mRNA were detected from day 3 of culture, whereas CTR from day 12. Stimulated MUTZ-3-CD34(+) generated functional osteoclasts that formed extensive resorption lacunae on both mineralized surface and bone slices. Surprisingly, in both sorted populations we identified a population M-CSF-R(+)/RANK(+) that at the same time co-expressed CD14 and CD34. CONCLUSIONS: These findings demonstrate that MUTZ-3 cells constitute an invaluable model to study the expression pattern in different developmental stages of commitment and differentiation. Importantly, the data indicate that the CD14(+)CD34(+)M-CSF-R(+)RANK(+) population represents an intermediate stage of differentiation from CD34 precursors and monocytes to osteoclast.

Biomimetic Implant Surface Functionalization with Liquid L-PRF Products: In Vitro Study.

OBJECTIVE: Platelet-rich fibrin (PRF) clots and membranes are autologous blood concentrates widely used in oral surgical procedures; less is known, however, about the liquid formulations of such products. The aim of this in vitro study is to assess the behavior of different implant surfaces when in contact with two liquid leucocyte- and platelet-rich fibrin (L-PRF) products. METHODS: Six commercial pure titanium discs, of 9.5 mm diameter and 1.5 mm thickness, were used. Three of these samples had a micro/nano-rough surface; three were machined. Three different protocols were tested. Protocols involved the immersion of the samples in (1) a platelets, lymphocytes, and fibrinogen liquid concentrate (PLyF) for 10 minutes, (2) an exudate obtained from L-PRF clots rich in fibronectin and vitronectin for 5 minutes, and (3) the fibronectin/vitronectin exudate for 2 minutes followed by immersion in the PLyF concentrate for further 8 minutes. After these treatments, the samples were fixed and observed using a scanning electron microscope (SEM). RESULTS: Under microscopic observation, (1) the samples treated with the PLyF concentrate revealed a dense fibrin network in direct contact with the implant surface and a significant number of formed elements of blood; (2) in the samples treated with the fibronectin/vitronectin exudates, only a small number of white and red blood cells were detectable; and (3) in samples exposed to the combined treatment, there was an apparent increase in the thickness of the fibrin layer. When compared to the machined surface, the micro/nano-rough samples showed an overall increased retention of fibrin, leading to a thicker coating. CONCLUSIONS: Liquid L-PRF products promote the formation of a dense fibrin clot on micro/nano-rough implant surfaces in vitro. The adjunctive treatment of surfaces with the fibronectin/vitronectin exudate could provide support to contact of the fibrin with the surface, though it is not essential for the clot formation. Further studies are necessary to better elucidate the properties and benefits of liquid L-PRF products.

Ultrastructural modifications of cell membranes induced by "electroporation" on melanoma xenografts.

Electroporation (EP) has been widely employed in the past years as a safe and effective technique to drive drugs and DNA plasmids into target cells both for experimental and therapeutic purposes. Despite the large bulk of literature on this topic, often describing successful outcomes, there is a lack of knowledge about the intimate mechanism(s) controlling this phenomenon. In this paper, we describe a number of ultrastructural alterations in the cellular membranes following the exposure of orthotopic melanomas and red blood cells to trains of biphasic pulses. Specifically, melanoma xenografts grown in nude mice were subject to trains of eight biphasic pulses using an electric field of 1250 or 2450 V/cm, excised after 5 min and processed for electron microscopy. The freeze-fracturing analysis of both cell types evidenced defects in the dynamic assembly of lipids and proteins, which generate "areas with rough structure" and intensive clustering of intramembrane proteins. Such modifications could be the hallmarks of lipid and protein alterations, of protein cohesion reduction, and of changes in lipid orientation inside cell membranes, as postulated in several mathematical models applied to electroporation, and warrant further investigations.

Biocompatibility assessment of liquid artificial vitreous replacements: relevance of in vitro studies.

The biocompatibility of liquid artificial vitreous replacements is generally assessed by performing tests in animal models before their clinical use, whereas in vitro experimentation is seldom carried out due to their physico-chemical characteristics. Since their introduction in vitreoretinal surgery, however, the use of some certified vitreous replacements has been discouraged after clinical trials, because of the occurrence of serious side effects. This observation suggests that the tests currently performed for biocompatibility assessment cannot fully guarantee their safety when they are used in humans. Here we review the available literature on in vitro biocompatibility testing of liquid artificial vitreous replacements and survey our own experience on the subject, obtained by using primary retinal cell cultures, seeded on micro-porous inserts. We suggest that in vitro biocompatibility assessment, conducted before experiments in animal models, could improve the required safety evaluation and decrease the risk of undesired side effects, as well as providing a beneficial reduction of animal experimentation.

Physical Profile and Impact of a Calcium-Incorporated Implant Surface on Preosteoblastic Cell Morphologic and Differentiation Parameters: A Comparative Analysis.

PURPOSE: To assess and compare topographic features and preosteoblastic cell responses of a new hydrothermally treated, calcium-incorporated surface against other commercially available implant surfaces. MATERIALS AND METHODS: Four different surfaces were the subject of comparison in this study: machined (MC), resorbable blast media (RBM), sandblasted/large-grit/acid-etched (SLA), and calcium-incorporated SLA (Ca-SLA). Surface morphology and roughness were first characterized by scanning electron microscope (SEM) and white light interferometer, respectively. Preosteoblastic MC3T3-E1 cells were then cultured on the titanium surfaces. Cell morphology was observed at 24 hours, 48 hours, 7 days, and 15 days by SEM; differentiation was assessed at 7, 11, and 15 days by assaying alkaline phosphatase (ALP) activity and osteocalcin (OCN) levels. RESULTS: Surface characterization revealed nanotopographic features on Ca-SLA. At topographic analysis, SLA and Ca-SLA showed similar roughness values. Significant differences in cell differentiation parameters were found only at 15 days between the SLA surfaces (both Ca-incorporated and nonincorporated) and MC. CONCLUSION: Collectively, this study demonstrated that hydrothermal treatment determines the formation of nanotopography without altering the SLA microtopography. Moreover, Ca-SLA and SLA induce MC3T3-E1 cell differentiation at comparable levels.

Induction of apoptosis in rat retinal cell cultures by partially fluorinated alkanes.

PURPOSE: To test whether the partially fluorinated alkanes (PFAs) perfluorobutylbutane (O(44)), perfluorohexylethan (O(62)), and the oligomer OL(62HV), recently proposed as artificial vitreous replacements (AVRs), have pro-apoptotic effect in rat retinal cultures. DESIGN: Laboratory investigation. METHODS: Rat retinal cell cultures were seeded onto microporous inserts to study AVR-cell interaction without impairing cell survival. Cells were treated for 24 hours with O(62), O(44), and OL(62HV). Apoptosis was analyzed by transferase-mediated dUTP-biotin nick end-labeling assay and Hoechst stain. RESULTS: O(44) and O(62) did not affect structural organization and cell survival in retinal cell cultures; however, OL(62HV) induced increased apoptosis compared with control cultures. CONCLUSIONS: OL(62HV), a high-viscosity PFA, induces severe retinal damage in human eyes, although it successfully passed animal experimentation. Our in vitro study showed a remarkable pro-apoptotic effect of OL(62HV) and suggests that in vitro tests can contribute to AVR biocompatibility assessment.

Primary retinal cultures as a tool for modeling diabetic retinopathy: an overview.

Experimental models of diabetic retinopathy (DR) have had a crucial role in the comprehension of the pathophysiology of the disease and the identification of new therapeutic strategies. Most of these studies have been conducted in vivo, in animal models. However, a significant contribution has also been provided by studies on retinal cultures, especially regarding the effects of the potentially toxic components of the diabetic milieu on retinal cell homeostasis, the characterization of the mechanisms on the basis of retinal damage, and the identification of potentially protective molecules. In this review, we highlight the contribution given by primary retinal cultures to the study of DR, focusing on early neuroglial impairment. We also speculate on possible themes into which studies based on retinal cell cultures could provide deeper insight.

The reverse transcription inhibitor abacavir shows anticancer activity in prostate cancer cell lines.

BACKGROUND: Transposable Elements (TEs) comprise nearly 45% of the entire genome and are part of sophisticated regulatory network systems that control developmental processes in normal and pathological conditions. The retroviral/retrotransposon gene machinery consists mainly of Long Interspersed Nuclear Elements (LINEs-1) and Human Endogenous Retroviruses (HERVs) that code for their own endogenous reverse transcriptase (RT). Interestingly, RT is typically expressed at high levels in cancer cells. Recent studies report that RT inhibition by non-nucleoside reverse transcriptase inhibitors (NNRTIs) induces growth arrest and cell differentiation in vitro and antagonizes growth of human tumors in animal model. In the present study we analyze the anticancer activity of Abacavir (ABC), a nucleoside reverse transcription inhibitor (NRTI), on PC3 and LNCaP prostate cancer cell lines. PRINCIPAL FINDINGS: ABC significantly reduces cell growth, migration and invasion processes, considerably slows S phase progression, induces senescence and cell death in prostate cancer cells. Consistent with these observations, microarray analysis on PC3 cells shows that ABC induces specific and dose-dependent changes in gene expression, involving multiple cellular pathways. Notably, by quantitative Real-Time PCR we found that LINE-1 ORF1 and ORF2 mRNA levels were significantly up-regulated by ABC treatment. CONCLUSIONS: Our results demonstrate the potential of ABC as anticancer agent able to induce antiproliferative activity and trigger senescence in prostate cancer cells. Noteworthy, we show that ABC elicits up-regulation of LINE-1 expression, suggesting the involvement of these elements in the observed cellular modifications.

The plant alkaloid voacamine induces apoptosis-independent autophagic cell death on both sensitive and multidrug resistant human osteosarcoma cells.

In our previous studies, the bisindolic alkaloid voacamine (VOA), isolated from the plant Peschiera fuchsiaefolia, proved to exert a chemosensitizing effect on cultured multidrug resistant (MDR) osteosarcoma cells exposed to doxorubicin (DOX). In particular, VOA was capable of inhibiting P-glycoprotein action in a competitive way, thus explaining the enhancement of the cytotoxic effect induced by DOX on MDR cells. Afterwards, preliminary observations suggested that such an enhancement did not involve the apoptotic process but was due instead to the induction of autophagic cell death. The results of the present investigation demonstrate that the plant alkaloid VOA is an autophagy inducer able to exert apoptosis-independent cytotoxic effect on both wild-type and MDR tumor cells. In fact, under treatment condition causing about 50 percent of cell death, no evidence of apoptosis could be revealed by microscopical observations, Annexin V-FITC labeling and analysis of PARP cleavage, whereas the same cells underwent apoptosis when treated with apoptosis inducers, such as doxorubicin and staurosporine. Conversely, VOA-induced autophagy was clearly evidentiated by electron microscopy observations, monodansylcadaverine staining, LC3 expression, and conversion. These results were confirmed by the analysis of the modulating effects of the pretreatment with autophagy inhibitors prior to VOA administration. In addition, transfection of osteosarcoma cells with siRNA against ATG genes reduced VOA cytotoxicity. In conclusion, considering the very debated dual role of autophagy in cancer cells (protective or lethal, pro- or anti- apoptotic) our findings seem to demonstrate, at least in vitro, that a natural product able to induce autophagy can be effective against drug resistant tumors, either used alone or in association with conventional chemotherapeutics.

Biocompatibility assessment of silicone oil and perfluorocarbon liquids used in retinal reattachment surgery in rat retinal cultures.

The effects of silicone oil and perfluorocarbon liquids used in retinal reattachment surgery were studied in vitro using rat retinal cultures seeded on microporous inserts. These inserts allow the cell layer to be in contact with the material to be tested on the apical side and with the nutrient medium on the basal side. The materials tested were silicone oil, the perfluorocarbons perfluorophenanthrene and perfluoroctane, and hydroxypropylmethylcellulose. Perfluorophenanthrene, the heaviest of the compounds, induced a very precocious detachment of the cell layer. All the other tested biomaterials were compatible with cell survival and did not alter the structural organization of the retinal cultures, as revealed by scanning electron microscopy. By immunocytochemical techniques we evaluated the cell composition and the differentiation state of each of the cultures. In both control and treated samples, neuronal cells were well preserved. The expression of microtubule-associated protein 2, a marker of differentiated neuronal cytoskeleton, was not affected. Amacrine neurons, immunolabeled for gamma-aminobutyric acid, still were detectable after treatment. Synapses, marked by immunoreactivity for synapthophysin, were equally preserved. Vimentin-positive glial cells did not show modifications. The apoptotic rate, as determined by the terminal transferase-mediated dUTP-biotin nick end-labeling assay, was similar in treated and control samples. The results confirm that the use of biomaterials with a specific gravity close to intraocular fluids is compatible with retinal cell survival and differentiation in vitro.