In vitro characterization of the three-dimensional strain pattern in human vertebrae affected by metastases

La colonna vertebrale è la principale sede di metastasi, le quali possono alterare la normale distribuzione dei tessuti ossei e ridurre la capacità della vertebra di sostenere carichi. L’instabilità spinale causata dalle metastasi, tuttavia, è di difficile determinazione. La caratterizzazione meccanica delle vertebre metastatiche permetterebbe di identificare e, di conseguenza trattare, quelle ad alto rischio di frattura. In questo studio, ho valutato il comportamento meccanico a rottura di vertebre umane affette da metastasi misurando in vitro il campo di deformazione. Undici provini, costituiti da due vertebre centrali, una metastatica e una sana, sono stati preparati e scansionati applicando carichi graduali di compressione in una micro-tomografia computerizzata (μCT). Le deformazioni principali sono state misurate attraverso un algoritmo globale di Digital Volume Correlation (DVC) e successivamente sono state analizzate. Lo studio ha rivelato che le vertebre con metastasi litiche raggiungono deformazioni maggiori delle vertebre sane. Invece, le metastasi miste non assicurano un comportamento univoco in quanto combinano gli effetti antagonisti delle lesioni litiche e blastiche. Dunque la valutazione è stata estesa a possibili correlazioni tra il campo di deformazione e la microstruttura della vertebra. L'analisi ha identificato le regioni in cui parte la frattura (a più alta deformazione), senza identificare, in termini microstrutturali, una zona preferenziale di rottura a priori. Infatti, alcune zone con un pattern trabecolare denso, presunte più rigide, hanno mostrato deformazioni maggiori di quelle dei tessuti sani, sottolineando l’importanza della valutazione della qualità del tessuto osseo. Questi risultati, generalizzati su un campione più ampio, potrebbero essere utilizzati per implementare nuovi criteri negli attuali sistemi di valutazione dell'instabilità spinale.

The three-dimensional single-bin-size bin packing problem: combining metaheuristic and machine learning approaches

The Three-Dimensional Single-Bin-Size Bin Packing Problem is one of the most studied problem in the Cutting & Packing category. From a strictly mathematical point of view, it consists of packing a finite set of strongly heterogeneous “small” boxes, called items, into a finite set of identical “large” rectangles, called bins, minimizing the unused volume and requiring that the items are packed without overlapping. The great interest is mainly due to the number of real-world applications in which it arises, such as pallet and container loading, cutting objects out of a piece of material and packaging design. Depending on these real-world applications, more objective functions and more practical constraints could be needed. After a brief discussion about the real-world applications of the problem and a exhaustive literature review, the design of a two-stage algorithm to solve the aforementioned problem is presented. The algorithm must be able to provide the spatial coordinates of the placed boxes vertices and also the optimal boxes input sequence, while guaranteeing geometric, stability, fragility constraints and a reduced computational time. Due to NP-hard complexity of this type of combinatorial problems, a fusion of metaheuristic and machine learning techniques is adopted. In particular, a hybrid genetic algorithm coupled with a feedforward neural network is used. In the first stage, a rich dataset is created starting from a set of real input instances provided by an industrial company and the feedforward neural network is trained on it. After its training, given a new input instance, the hybrid genetic algorithm is able to run using the neural network output as input parameter vector, providing as output the optimal solution. The effectiveness of the proposed works is confirmed via several experimental tests.

Resistance of multicellular aggregates to pharmorubicin observed in human hepatocarcinoma cells

The objective of the present study was to investigate the multicellular resistance of human hepatocarcinoma cells BEL-7402 to pharmorubicin. Cells (1 x 10(4)) and 200 microcarrier Cytodex-3 beads were seeded onto a 24-well plate and cultured in RPMI 1640 medium. After the formation of multicellular aggregates, morphology and cell viability were analyzed by scanning electron microscopy, transmission electron microscopy and flow cytometry, respectively. The IC50 was determined by flow cytometry and MTT assay after the cells cultured in aggregates and monolayers were treated with pharmorubicin. The culture products exhibited structural characteristics somewhat similar to those of trabecular hepatocarcinoma in vivo. Among the microcarriers, cells were organized into several layers. Intercellular spaces were 0.5-2.0 µm wide and filled with many microvilli. The percent of viable cells was 87%. The cells cultured as multicellular aggregates were resistant to pharmorubicin with IC50 4.5-fold and 7.7-fold that of monolayer culture as determined by flow cytometry and MTT assay, respectively. This three-dimensional culture model may be used to investigate the mechanisms of multicellular drug resistance of hepatocarcinoma and to screen new anticancer drugs.

Differentiation and extracellular matrix interactions of chondrocytes in response to signaling molecules and biomaterials for cartilage repair = Differenzierung und Interaktionen von Chondrozyten mit der extrazellulären Matrix als Reaktion auf Signalmoleküle und Biomaterialien für die Wiederinstandsetzung von Knorpel

Chondrocytes live isolated in the voluminous extracellular matrix of cartilage, which they secrete and is neither vascularized nor innervated. Nutrient and waste exchanges occur through diffusion leading to low oxygen tension around the cells. Consequently even normal cartilage under normal physiological conditions suffers from a poor reparative potential that predisposes to degenerative conditions, such as osteoarthritis of the joints, with significant clinical effects.rnOne of the key challenges in medicine is the structural and functional replacement of lost or damaged tissues. Current therapeutical approaches are to transplant cells, implant bioartificial tissues, and chemically induce regeneration at the site of the injury. None of them reproduces well the biological and biomechanical properties of hyaline cartilage.rnThis thesis investigates the re-differentiation of chondrocytes and the repair of cartilage mediated by signaling molecules, biomaterials, and factors provided in mixed cellular cultures (co-culture systems). As signaling molecules we have applied prostaglandin E2 (PGE2) and bone morphogenetic protein 1 (BMP-1) and we have transfected chondrocytes with BMP-1 expressing vectors. Our biomaterials have been hydrogels of type-I collagen and gelatin-based scaffolds designed to mimic the architecture and biochemistry of native cartilage and provide a suitable three-dimensional environment for the cells. We have brought chondrocytes to interact with osteosarcoma Cal 72 cells or with murine preosteoblastic KS483 cells, either in a cell-to-cell or in a paracrine manner.rnExogenous stimulation with PGE2 or BMP-1 did not improve the differentiation or the proliferation of human articular chondrocytes. BMP-1 induced chondrocytic de-differentiation in a dose-dependent manner. Prostaglandin stimulation from gelatin-based scaffolds (three-dimensional culture) showed a certain degree of chondrocyte re-differentiaton. Murine preosteoblastic KS483 cells had no beneficial effect on human articular chondrocytes jointly cultivated with them in hydrogels of type I collagen. Although the hydrogels provided the chondrocytes with a proper matrix in which the cells adopted their native morphology; additionally, the expression of chondrocytic proteoglycan increased in the co-cultures after two weeks. The co-culture of chondrocytes with osteoblast-like cells (in transwell systems) resulted in suppression of the regular de-differentiation program that passaged chondrocytes undergo when cultured in monolayers. Under these conditions, the extracellular matrix of the chondrocytes, rich in type-II collagen and aggrecan, was not transformed into the extracellular matrix characteristic of de-differentiated human articular chondrocytes, which is rich in type-I collagen and versican.rnThis thesis suggests novel strategies of tissue engineering for clinical attempts to improve cartilage repair. Since implants are prepared in vitro (ex-vivo) by expanding human articular chondrocytes (autologous or allogeneic), we conclude that it will be convenient to provide a proper three-dimensional support to the chondrocytes in culture, to supplement the culture medium with PGE2, and to stimulate chondrocytes with osteoblastic factors by cultivating them with osteoblasts.rn

Mandibular reconstruction using a combination graft of rhBMP-2 with bone marrow cells expanded in vitro

BACKGROUND: The aim of this study was to evaluate the efficacy of a combination graft, using recombinant human bone morphogenetic protein-2 (rhBMP-2) and culture-expanded cells derived from bone marrow, for bone regeneration in a nonhuman primate mandible. METHODS: Five Japanese monkeys were used. Three milliliters of bone marrow was obtained from the tibia and plated into culture flasks. Adherent cells were cultured until near confluence; then, the proliferated cells were transferred to a three-dimensional culture system using collagen beads as the cell carrier. The medium was supplemented with ascorbic acid, beta-glycerophosphate, and dexamethasone to promote osteoblastic differentiation. After further proliferation on beads, the cells were mixed with a collagen sponge that was impregnated with rhBMP-2 and grafted into surgically created segmental bone defects of the mandibles. Three animals received this treatment, and either culture-expanded cells alone or collagen beads without cells were implanted into the remaining two monkeys as controls. The animals were killed 24 weeks after surgery, and the results were assessed by radiographic and histologic evaluation. RESULTS: The combination graft of culture-expanded bone marrow cells with rhBMP-2 in a collagen sponge regenerated the mandibular bone completely. By contrast, the graft of culture-expanded cells alone resulted in only a small amount of bone formation, and the implantation of collagen beads alone led to no bone formation. CONCLUSION: The combination graft of rhBMP-2 and culture-expanded cells, which requires only a small amount of bone marrow, is a reliable method for the reconstruction of segmental bone defects of the mandible.

E-cadherin induces mesenchymal-to-epithelial transition in human ovarian surface epithelium

Ovarian carcinomas are thought to arise in the ovarian surface epithelium (OSE). Although this tissue forms a simple epithelial covering on the ovarian surface, OSE cells exhibit some mesenchymal characteristics and contain little or no E-cadherin. However, E-cadherin is present in metaplastic OSE cells that resemble the more complex epithelia of the oviduct, endometrium and endocervix, and in primary epithelial ovarian carcinomas. To determine whether E-cadherin was a cause or consequence of OSE metaplasia, we expressed this cell-adhesion molecule in simian virus 40-immortalized OSE cells. In these cells the exogenous E-cadherin, all three catenins, and F-actin localized at sites of cell–cell contact, indicating the formation of functional adherens junctions. Unlike the parent OSE cell line, which had undergone a typical mesenchymal transformation in culture, E-cadherin-expressing cells contained cytokeratins and the tight-junction protein occludin. They also formed cobblestone monolayers in two-dimensional culture and simple epithelia in three-dimensional culture that produced CA125 and shed it into the culture medium. CA125 is a normal epithelial-differentiation product of the oviduct, endometrium, and endocervix, but not of normal OSE. It is also a tumor antigen that is produced by ovarian neoplasms and by metaplastic OSE. Thus, E-cadherin restored some normal characteristics of OSE, such as keratin, and it also induced epithelial-differentiation markers associated with weakly preneoplastic, metaplastic OSE and OSE-derived primary carcinomas. The results suggest an unexpected role for E-cadherin in ovarian neoplastic progression.

Nuclear Factor-κB (NF-κB) Regulates Proliferation and Branching in Mouse Mammary Epithelium

The nuclear factor-κB (NF-κB) family of transcription factors has been shown to regulate proliferation in several cell types. Although recent studies have demonstrated aberrant expression or activity of NF-κB in human breast cancer cell lines and tumors, little is known regarding the precise role of NF-κB in normal proliferation and development of the mammary epithelium. We investigated the function of NF-κB during murine early postnatal mammary gland development by observing the consequences of increased NF-κB activity in mouse mammary epithelium lacking the gene encoding IκBα, a major inhibitor of NF-κB. Mammary tissue containing epithelium from inhibitor κBα (IκBα)-deficient female donors was transplanted into the gland-free mammary stroma of wild-type mice, resulting in an increase in lateral ductal branching and pervasive intraductal hyperplasia. A two- to threefold increase in epithelial cell number was observed in IκBα-deficient epithelium compared with controls. Epithelial cell proliferation was strikingly increased in IκBα-deficient epithelium, and no alteration in apoptosis was detected. The extracellular matrix adjacent to IκBα-deficient epithelium was reduced. Consistent with in vivo data, a fourfold increase in epithelial branching was also observed in purified IκBα-deficient primary epithelial cells in three-dimensional culture. These data demonstrate that NF-κB positively regulates mammary epithelial proliferation, branching, and functions in maintenance of normal epithelial architecture during early postnatal development.

Expression and functional significance of VE-cadherin in aggressive human melanoma cells: Role in vasculogenic mimicry

We recently have introduced the term vasculogenic mimicry to describe the unique ability of aggressive melanoma tumor cells to form tubular structures and patterned networks in three-dimensional culture, which “mimics” embryonic vasculogenic networks formed by differentiating endothelial cells. In the current study, we address the biological significance of several endothelial-associated molecules (revealed by microarray analysis) with respect to expression and function in highly aggressive and poorly aggressive human cutaneous melanoma cell lines (established from the same patient). In a comparative analysis, CD31 was not expressed by any of the melanoma cell lines, whereas TIE-1 (tyrosine kinase with Ig and epidermal growth factor homology domains-1) was strongly expressed in the highly aggressive tumor cells with a low level of expression in one of the poorly aggressive cell lines. Vascular endothelial (VE)-cadherin was exclusively expressed by highly aggressive melanoma cells and was undetectable in the poorly aggressive tumor cells, suggesting the possibility of a vasculogenic switch. Down-regulation of VE-cadherin expression in the aggressive melanoma cells abrogated their ability to form vasculogenic networks and directly tested the hypothesis that VE-cadherin is critical in melanoma vasculogenic mimicry. These results highlight the plasticity of aggressive melanoma cells and call into question their possible genetic reversion to an embryonic phenotype. This finding could pose a significant clinical challenge in targeting tumor cells that may masquerade as circulating endothelial cells or other embryonic-like stem cells.

Adjustment of Mineral Elements in the Culture Medium for the Micropropagation of Three Vriesea Bromeliads from the Brazilian Atlantic Forest: The Importance of Calcium

Many different species of Bromeliaceae are endangered and their conservation requires specific knowledge of their growth habits and propagation. In vitro culture of bromeliads is an important method for efficient clonal propagation and ill vitro seed g,germination can be used to maintain genetic variability. The present work aims to evaluate the in vitro growth and nutrient concentration in leaves of the epiphyte bromeliads Vriesea friburguensis Mez, Vriesea hieroglyphica (Carriere) E. Morren, and Vriesea unilateralis Mez, which exhibit slow rates of growth in vivo and in vitro. Initially, we compared the endogenous mineral composition of bromeliad plantlets grown in half-strength Murashige and Skoog (MS) medium and the mineral composition considered adequate in the literature. This approach suggested that calcium (Ca) is a critical nutrient and this was considered for new media formulation. Three new culture media were defined in which the main changes to half-strength MS medium were an increase in Ca, magnesium, sulfur, copper, and chloride and a decrease in iron, maintaining the nitrate: ammonium rate at approximate to 2:1. The main difference among the three new media formulated was Ca concentration, which varied from 1.5 mm in half-strength MS to 3.0, 6.0, and 12 mm in M2, M3, and M4 media, respectively. Consistently, all three species exhibited significantly higher fresh and dry weight on M4, the newly defined medium with the highest level of Ca (12 mm). Leaf nitrogen, potassium, zinc, magnesium and boron concentrations increased as Ca concentration in the medium increased from 1.5 to 12 mm.

Development of Chitosan and Poly (Vinyl Alcohol) Blended scaffolds for cell culture using supercritical fluids technology

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Mestrado Integrado em Engenharia Química e Bioquímica

Design and validation of a biomechanical bioreactor for cartilage tissue culture

Specific tissues, such as cartilage undergo mechanical solicitation under their normal performance in human body. In this sense, it seems necessary that proper tissue engineering strategies of these tissues should incorporate mechanical solicitations during cell culture, in order to properly evaluate the influence of the mechanical stimulus. This work reports on a user-friendly bioreactor suitable for applying controlled mechanical stimulation - amplitude and frequency - to three dimensional scaffolds. Its design and main components are described, as well as its operation characteristics. The modular design allows easy cleaning and operating under laminar hood. Different protocols for the sterilization of the hermetic enclosure are tested and ensure lack of observable contaminations, complying with the requirements to be used for cell culture. The cell viability study was performed with KUM5 cells.

Effects of the naturally occurring food mycotoxin ochratoxin A on brain cells in culture.

The potential of ochratoxin A (OTA) to damage brain cells was studied by using a three-dimensional cell culture system as model for the developing brain. Aggregating cell cultures of foetal rat telencephalon were tested either during an early developmental period, or during a phase of advanced maturation, over a wide range of OTA concentrations (0.4 nM to 50 microM). By monitoring changes in activities of cell type-specific enzymes (ChAt and GAD, for cholinergic and GABAergic neurones, respectively, GS for astrocytes and CNP for oligodendrocytes), the concentration-dependent toxicity and neurodevelopmental effects of OTA were determined. OTA proved to be highly toxic, since a 10-day treatment at 50 nM caused a general cytotoxicity in both mature and immature cultures. At 10 nM of OTA, cell type-specific effects were observed: in immature cultures, a loss in neuronal and oligodendroglial enzyme activities, and an increase in the activity of the astroglial marker glutamine synthetase were found, Furthermore, at 2 and 10 nM of OTA, a clustering of microglial cells was observed. In mature cultures, OTA was somewhat less potent, but caused a similar pattern of toxic effects. A 24 h-treatment with OTA resulted in a concentration-dependent decrease in protein synthesis, with IC50 values of 25 nM and 33 nM for immature and mature cultures respectively. Acute (24 h) treatment at high OTA concentrations (10 to 50 microM) caused a significant increase in reactive oxygen species formation, as measured by the intracellular oxidation of 2',7'-dichlorofluorescin. These results suggest that OTA has the potential to be a potent toxicant to brain cells, and that its effects at nanomolar concentrations are primarily due to the inhibition of protein synthesis, whereas ROS seem not to be involved in the toxicity mediated by a chronic exposure to OTA at such low concentrations.

Concise reviews: in vitro-produced pancreas organogenesis models in three dimensions: self-organization from few stem cells or progenitors.

Three-dimensional models of organ biogenesis have recently flourished. They promote a balance between stem/progenitor cell expansion and differentiation without the constraints of flat tissue culture vessels, allowing for autonomous self-organization of cells. Such models allow the formation of miniature organs in a dish and are emerging for the pancreas, starting from embryonic progenitors and adult cells. This review focuses on the currently available systems and how these allow new types of questions to be addressed. We discuss the expected advancements including their potential to study human pancreas development and function as well as to develop diabetes models and therapeutic cells.

Ammonium alters creatine transport and synthesis in a 3D culture of developing brain cells, resulting in secondary cerebral creatine deficiency.

Hyperammonemic disorders in pediatric patients lead to poorly understood irreversible effects on the developing brain that may be life-threatening. We showed previously that some of these NH4+-induced irreversible effects might be due to impairment of axonal growth that can be protected under ammonium exposure by creatine co-treatment. The aim of the present work was thus to analyse how the genes of arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT), allowing creatine synthesis, as well as of the creatine transporter SLC6A8, allowing creatine uptake into cells, are regulated in rat brain cells under NH4+ exposure. Reaggregated brain cell three-dimensional cultures exposed to NH4Cl were used as an experimental model of hyperammonemia in the developing central nervous system (CNS). We show here that NH4+ exposure differentially alters AGAT, GAMT and SLC6A8 regulation, in terms of both gene expression and protein activity, in a cell type-specific manner. In particular, we demonstrate that NH4+ exposure decreases both creatine and its synthesis intermediate, guanidinoacetate, in brain cells, probably through the inhibition of AGAT enzymatic activity. Our work also suggests that oligodendrocytes are major actors in the brain in terms of creatine synthesis, trafficking and uptake, which might be affected by hyperammonemia. Finally, we show that NH4+ exposure induces SLC6A8 in astrocytes. This suggests that hyperammonemia increases blood-brain barrier permeability for creatine. This is normally limited due to the absence of SLC6A8 from the astrocyte feet lining microcapillary endothelial cells, and thus creatine supplementation may protect the developing CNS of hyperammonemic patients.

”Potato seeking rice”: language, culture, and identity in gay personal ads in Hong Kong

This paper examines the relationship between language, culture, and identity in a corpus of gay personal ads collected from two publications in Hong Kong in the three years before the 1997 transition of sovereignty. Gay personal ads are seen äs an "island of discourse," whose marginal nature is reflected in the use of language and in turn reflects issues of marginalization in the larger social context. Using Fairclough's (1992, 1993) three- dimensional model for critical discourse analysis, an attempt is made to uncover the relationship between text structure and issues ofpower/ideology in the society that produces the texts. On the level of text, it was found that structural components, particularly the degree of grammatical elaboration, differ according to the stated race or cultural background of the authors and their targets. On the level of discourse practice, authors were found to appropriate a variety of "voices"from the larger culture arena, the use of which amplifies or limits the participation of particular classes of individuals. Finally, on the level of social practice, the ads were found to reflect and re-create both the racial stereotypes and heterosexist ideology found in the dominant culture.