Fatigue-Crack Detection in a Multi-Riveted Strap-Joint Aluminium Aircraft Panel Using Amplitude Characteristics of Diffuse Lamb Wave Field.

Structural health monitoring of riveted aircraft panels is a real challenge for maintenance engineers. Here, a diffused Lamb wave field is used for fatigue-crack detection in a multi-riveted strap-joint aircraft panel. The panel is instrumented with a network of low-profile surface-bonded piezoceramic transducers. Various amplitude characteristics of Lamb waves are used to extract information on fatigue damage. A statistical outlier analysis based on these characteristics is also performed to detect damage. The experimental work is supported by simplified modelling of wave scattering from crack tips to explain complex response features. The Local Interaction Simulation Approach (LISA) is used for this modelling task. The results demonstrate the potential and limitations of the method for reliable fatigue-crack detection in complex aircraft components.

Air contrast of the intervillous space enables non-disruptive Micro-CT analysis of paraffin-embedded archival placental tissue.

The morphometric parameters of the villous tree are a strong indicator of deviant placentas. Methods have been established to digitally reconstruct small peripheral branches by tracing with 3D Microscopy at subcellular resolution. Micro-CT can help scale up the scanning of villous trees with resolution in the range of a few micrometers. As placental tissue samples are routinely conserved and archived by fixation and paraffin embedding, the villous structures are inaccessible to Micro-CT imaging due to poor contrast between paraffin and paraffinized tissue. We present a novel procedure for contrast enhancement by selectively replacing wax by air in the intervillous space.

The volume of villi with γ-sm-actin positive perivascular cells correlates with placental weight and thickness.

INTRODUCTION: The classification of histologically stained villous cross sections in villous types (terminal, intermediate and stem villi) by stromal peculiarities is known to be observer predicated. Therefore, quantitative histology of villous trees has not become a routine endpoint of studies on the role of the placenta in prenatal programming, as opposed to the gross placental parameters weight and thickness. The classification of villous cross sections in central (stem) and peripheral (terminal) parts based on the presence or absence, respectively, of immunohistochemical detection of myofibroblasts in perivascular position is less observer dependent. We hypothesized that it will, possibly, identify microscopic correlates of placental weight and thickness within the villous tree. METHODS: 50 placentas from clinically normal pregnancies were processed for the present study. Thin villous cross sections, obtained in a systematic random manner, were stained immunohistochemically to detect γ-smooth muscle (sm) actin and to classify them subsequently as part of central or peripheral villous tree. The volume fractions of histological structures visible in villous cross sections (stroma, lumen, endothelium and syncytium) were estimated by design-based stereology. RESULTS: The present study reveals a significant correlation of placental weight and thickness with the volume estimate of stroma that have myofibroblasts in perivascular position. DISCUSSION: The positive linear correlation between the volume of central parts of villous trees and the placental weight and thickness is new. Surprisingly, the volume of more peripheral parts of villous trees, which is the main site of materno-fetal exchange does not correlate with placental weight and thickness.

Syncytial nuclei accumulate at the villous surface in IUGR while proliferation is unchanged.

INTRODUCTION: Placental syncytiotrophoblast is responsible for feto-maternal nutrient exchange during pregnancy. It is assumed that in IUGR, placental dysfunction is crucially bound to compromised stability and function of syncytiotrophoblast, the latter being related to altered proliferation of villous trophoblast. Cell cycle data obtained on conventional thin sections has produced inconsistent results. In the present study we investigated cell cycle markers found in the villous trophoblast using a novel 3D histological quantification method. METHODS AND FINDINGS: We analyzed 40 placentas from IUGR pregnancies and 42 placentas from clinically normal pregnancies by immunohistochemical detection of the cell cycle marker PCNA. Nuclei immuno-positive for PCNA were quantified using 3D microscopy, and the results were compared to corresponding results obtained on conventional thin histological sections. These data did not show any evidence of altered trophoblast proliferation in IUGR, while the density of post-proliferative (i.e. PCNA-negative) trophoblast nuclei was statistically significantly increased in IUGR. The latter could be revealed by 3D topological microscopy, but not by conventional histology of thin sections. DISCUSSION: The data of the present study indicate a previously unknown type of regulation of syncytial stability and function, independent of proliferation. We hypothesize that in IUGR, post-proliferative trophoblast nuclei accumulate at the villous surface of peripheral villous branches. This could possibly reflect the presence of an unknown mechanism controlling syncytial function and stability by modulation of syncytial passage time rather than by modulation of proliferative supply.

Birth weight correlates with size but not shape of the normal human placenta.

INTRODUCTION: Studies on developmental programming rely on various measures of size and form of the human placenta. Size and form are not independent of each other and covariation patterns were not determined systematically. METHODS: Twenty-two morphologic parameters were determined on 418 placentas from uncomplicated singleton pregnancies. We determined (i) placenta weight and birth weight, (ii) form parameters such as diameters, thickness, roundness, and eccentricity of cord insertion, and (iii) shape variability by geometric morphometry. Geometric morphometry analyzes shape variability independent of size. We define the technical terms form and shape according to the language of geometric morphometry. RESULTS: Placenta weight correlated with birth weight. The form parameters correlated variably with placenta weight and shape. Shape variability did not correlate with birth weight and placenta weight. DISCUSSION AND CONCLUSIONS: The correlation of placenta weight with birth weight stays a cornerstone of prenatal programming. Shape analysis shows that form parameters are hybrids of size and shape. Shape variability can be interpreted as an outcome of adaptation of a placenta to maternal factors and the associated uterine habitat. Correlation analysis of the whole data array provides a rigorous statistical frame to interpret published data and plan new studies.

Placental syncytiotrophoblast maintains a specific type of glycocalyx at the fetomaternal border: the glycocalyx at the fetomaternal interface in healthy women and patients with HELLP syndrome.

Recent studies showed that considerable amounts of glycosaminoglycans are released into maternal blood during normal pregnancy and in hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome. Maternal endothelia and the syncytiotrophoblast layer have been discussed as a possible origin of these glycocalyx components. Our study aimed to visualize the glycocalyx on the syncytiotrophoblast by electron microscopy, to analyze its structure and composition by immunohistochemistry, and to determine potential differences between healthy women and women with HELLP syndrome. For electron microscopy, a cotyledon was fixed by perfusion of the intervillous space with a 2% lanthanum-nitrate glutaraldehyde solution followed by immersion fixation in the same fixative. For immunohistochemistry, sections of 16 placentas (HELLP patients/healthy women, n = 8 each) were stained with monoclonal antibodies against the main glycocalyx constituents syndecan 1, hyaluronic acid, and heparan sulfate. Semiquantitative evaluation of staining intensity focused on the apical surface of the syncytiotrophoblast and fetal intravillous endothelia as possible localizations of a placental glycocalyx. Electron microscopy revealed a glycocalyx of approximately 250 nm, covering the syncytiotrophoblast layer. This was found to contain large amounts of syndecan 1, but neither hyaluronic acid nor heparan sulfate as major components. Intravillous fetal endothelium did not express any of the investigated glycosaminoglycans. Healthy women and patients with HELLP showed no differences concerning glycocalyx composition and thickness of the syncytiotrophoblast. The composition of the "placental" glycocalyx differs from the adult and fetal vascular glycocalyx. Obviously, the human placental syncytiotrophoblast maintains a special kind of glycocalyx at the fetomaternal interface.

High-mobility group protein HMGA2-derived fragments stimulate the proliferation of chondrocytes and adipose tissue-derived stem cells.

In previous research, it was shown that recombinant HMGA2 protein enhances the proliferation of porcine chondrocytes grown in vitro, opening up promising applications of this embryonic architectural transcription factor for tissue engineering, such as in cartilage repair. In this paper, we describe the development and analyses of two synthetic fragments comprising the functional AT-hook motifs of the HMGA2 protein, as well as the nuclear transport domain. They can be synthesised up to large scales, while eliminating some of the problems of recombinant protein production, including unwanted modification or contamination by the expression hosts, or of gene therapy approaches such as uncontrolled viral integration and transgene expression even after therapy. Application of one of these peptides onto porcine hyaline cartilage chondrocytes, grown in in vitro monolayer cell culture, showed a growth-promoting effect similar to that of the wild type HMGA2 protein. Furthermore, it also promoted cell growth of adult adipose tissue derived stem cells. Due to its proliferation inducing function and vast availability, this peptide is thus suitable for further application and investigation in various fields such as tissue engineering and stem cell research.

Trophoblastic invasion in vitro and in vivo: similarities and differences.

BACKGROUND: The basic mechanisms of trophoblast invasion are not completely understood. This may be due to the lack of suitable in vitro models which enable experimental modulation of this complex process. In the present study, a three-dimensional co-culture model is used for comparing two factors considered to be implicated in the regulation of trophoblast invasion, the expression of HLA-G and apoptosis, in vitro and in vivo. METHODS: Tissue fragments from human first trimester decidua parietalis were put in close contact with spheroids of AC-1M59 trophoblast/choriocarcinoma hybrid cells as a model of the invasive trophoblast. Cryostat sections from these co-cultures were immunohistochemically stained and compared with first trimester placentation sites in vivo. RESULTS: Only the invasive trophoblast-derived cells showed an intensive staining for HLA-G, whereas the cells on the periphery of the confrontation culture exhibited only a weak staining. A similar staining pattern was found in vivo. Both in vitro and in vivo CD45(+) apoptotic leukocytes were frequently detected in close proximity to the invasive trophoblastic cells. CONCLUSIONS: In this co-culture system, key factors considered to be implicated in trophoblast invasion in vivo can also be demonstrated in vitro. Therefore, it may help in finding strategies for the management of diseases associated with deficient trophoblast invasion.

Genome multiplication of extravillous trophoblast cells in human placenta in the course of differentiation and invasion into endometrium and myometrium. II. Mechanisms of polyploidization.

Peculiarities of the structure of interphase nuclei, mitotic activity, and Ki-67 protein intranuclear immunolocalization were studied to elucidate mechanisms of genome multiplication in proliferative and differentiating invasive extravillous trophoblast cells in the human placenta. The presence of numerous chromocenters was shown to be a characteristic feature of proliferative cell nuclei of both villous and extravillous trophoblast. At the beginning of extravillous trophoblast cell differentiation, i.e. in the proximal part of cell columns, some amount of cells with large nuclei containing enlarged chromocenters were found. DNA content was measured simultaneously with counting the number of chromocenters in similarly looking nuclei of squash preparations of placental villi. The increase in the ploidy level up to 4c-8c, accompanied by a slight increase in the number of chromocenters being not proportional to the ploidy level and not exceeding the diploid number of chromosomes of the human genome, was demonstrated. This suggests that genome multiplication of extravillous trophoblast cells may be accomplished by endoreduplication. In addition, pictures of endomitosis were seen at early steps of differentiation of EVT cells. The lack of polyploid mitotic figures or any obvious polyploidizing or restitutional mitoses suggests that these are not of considerable importance in genome multiplication of human EVT cells. However, the prevalence of metaphases at the boundary of the distal part of cell columns suggests that restitutional mitoses may be involved, even partly, in human trophoblast cell polyploidization. At later steps of differentiation, i.e. in the distal part of cell columns, the nuclear structure obviously changes, with a uniform "network" chromatin arrangement prevailing, whereas numerous chromocenters and features of endomitosis are no longer seen. The pattern of Ki-67 protein immunolocalization is also changing along the invasive pathway. In the proliferating stem cells and trophoblast cells of the proximal part of cell columns, Ki-67 was localized in the karyoplasm, chromocenters and numerous small nucleoli, whereas in the distal part of cell columns this protein was detected predominantly in 1-2 large nucleoli. The comparative analysis of the literature data on Ki-67 localization at different stages of cell cycle provided another evidence that EVT cells in the course of invasion may switch to the endoreduplication cycle. In agreement with the relevant report on rodent placentation, our present data suggest that acquirement of an invasive phenotype of EVT cells is accompanied by switching from mitotic division to endoreduplication cycle.

[Chorioallantoic membrane of fertilized avian eggs as a substrate for assessment of cancerous invasiveness]. / Die Chorioallantoismembran befruchteter Vogeleier als Substrat zur Testung der Invasivität von Karzinomen.

PURPOSE: Invasiveness is a characteristic feature of malignant tumors considerably determining the prognosis of affected patients. For assessment, apart from in vitro procedures with limited validity, tests on animal models have been established which certainly should be replaced by alternative methods whenever possible. The chorioallantoic membrane (CAM) of fertilized avian eggs represents an epithelial-lined membrane composed of all three blastodermic germ layers. In an "in ovo" assay cancer cells can be applied to this membrane after sinking (CAM assay). Tumor growth and invasiveness should be monitored in succession. MATERIAL AND METHODS: Hybrid chorionic carcinoma trophoblast cells were expanded in cell culture and spread over the CAM of hen's eggs after sinking followed by further incubation at 37 degrees C. The growth and development of the tumors were assessed macroscopically and finally (immuno-)histologically. Additionally, cytokeratin 19 was determined by enzyme-linked immunosorbent assay following homogenization of the tumor cells. RESULTS. Macroscopically, development of solid tumors was evident. Histological and immunohistochemical analysis revealed initial intraepithelial followed by cone-shaped infiltration of the CAM by the tumor cells. Tumor growth could be correlated with quantitative cytokeratin 19 measurements. CONCLUSIONS: Histomorphological appearance of the tumors was comparable with those results achieved in an immunodeficient mouse model. In addition, the CAM assay can be used for qualitative assessment of invasiveness of malignant tumors and yields quantitative results regarding growth kinetics. In contrast to conventional animal models, there is no need for official approval. Finally, this method is economical and facilitates processing many cases within a short time.

A two-colour fluorescence assay for the measurement of syncytial fusion between trophoblast-derived cell lines.

Syncytial fusion is a key event in implantation and placentation. Its regulation is only poorly understood. We present a cell-cell fusion assay based on staining of cells in two portions with a green and a red fluorescent cytoplasmic dye that become intracellularly mixed only after syncytial fusion. We quantified cell-cell fusion by fluorescence microscopy in choriocarcinoma cell lines BeWo, JAR and JEG3 and in some non-trophoblastic cell lines and found clear differences in fusion behaviour. Only BeWo cells fused with each other, while the other cell lines tested did not. BeWo cells also fused with all other cell lines tested. The efficiency of cell-cell fusion of BeWo cells was stimulated by forskolin. We tried to correlate messenger levels of syncytin and its receptor RDR with the fusion index of choriocarcinoma cells. BeWo and JAR cells contained readily detectable and forskolin-inducible levels of syncytin mRNA, whereas this messenger was barely detectable in JEG3 cells. RDR transcript levels were similar in all cell lines tested and were unaffected by forskolin treatment. The data suggests that the expression of syncytin and RDR messengers alone does not guarantee successful fusion. The fusion assay presented in this paper is a useful tool to study syncytial fusion in an accurate and quantitative way.

A positive immunoselection method to isolate villous cytotrophoblast cells from first trimester and term placenta to high purity.

We developed a method for isolating highly pure villous cytotrophoblast cells from first trimester and term placenta that excludes extravillous trophoblast and syncytiotrophoblast fragments. The method is based on positive immunoselection using an antibody (mAb C76/18) reacting with hepatocyte growth factor activator inhibitor 1, HAI-1, a membrane antigen on villous cytotrophoblast. As a comparison, we also immunopurified cells using an antibody against CD105, present on syncytiotrophoblast and some extravillous trophoblast cells. The isolates were characterized by flow cytometry. HAI-1-positive cells from first trimester and term placentae were highly pure (>98 per cent cytokeratin 7-positive) mononuclear trophoblast cells. These isolations were contaminated with only very small percentages of vimentin and CD45-positive cells. HAI-1-positive trophoblast cells lacked CD105 and also HLA class I, a marker for extravillous trophoblast. In culture HAI-1-positive cells adhered, displayed an epithelial morphology, and survived for more than three days. In contrast, CD105-positive cell fractions from first trimester placenta were a heterogeneous mixture of mononuclear and multinuclear elements consisting of syncytiotrophoblast fragments, extravillous trophoblast cells, as well as around 5 per cent non-trophoblastic contaminants. In conclusion, the positive immunoselection method using antibody C76/18 yielded highly pure villous cytotrophoblast cells devoid of elements derived from syncytiotrophoblast or extravillous trophoblast.

Mechanisms of syncytial fusion: a review.

Syncytial fusion of trophoblast is a key process in placental morphogenesis and physiology. Disturbed syncytial fusion may lead to a number of pregnancy-associated pathologies. The mechanisms regulating syncytial fusion are only partly understood. This review tries to summarize the available knowledge on trophoblast fusion, originating from different scientific disciplines. Among the themes addressed in this paper are: morphogenesis and functions of syncytiotrophoblast; early apoptotic events and changes in plasmalemmal phospholipid orientation; proteins involved in membrane fusion: ADAMs and retrovirally-derived proteins and short-lived proteolipid intermediates in membrane fusion. Deeper understanding of syncytiotrophoblast fusion in future studies is only to be anticipated from collaborative studies focusing in parallel on physicochemical events in the participating plasmalemmas, early apoptotic/differentiation events preceding the fusion and role of the fusogenic membrane proteins.

Genome multiplication of extravillous trophoblast cells in human placenta in the course of differentiation and invasion into endometrium and myometrium. I. Dynamics of polyploidization.

Polyploidization of the extravillous trophoblast (EVT) cells at different stages of differentiation and invasion into the uterine wall in human placenta has been studied. An increase in the ploidy level of EVT cells in the course of their differentiation within cell columns (CC) was shown. Stem cells were mainly diploid (86.2%); incidence of polyploid nuclei of highly proliferative cells of the proximal part of CC increased progressively. In the distal part of CC, where EVT cells did not divide mitotically, polyploid cells prevailed, with 58.0 and 3.5% nuclei being 4c and 8c, respectively. The highest percentage of polyploid cells was found in the population of EVT cells attached directly to the surface of the decidualized endometrium: percentage of tetraploid cells turned out to be 74.7% and the share of octaploid nuclei rose up to 4.9%; however, there appeared a few (0.3%) 16c cells. The majority of EVT cells invading the decidualized endometrium were polyploid, the share of octaploid and hexadecaploid cells rose up to 9.7 and 1.4%, respectively. On the other hand, the percentage of diploid cells also increased up to 29.2% as compared to EVT cells attached to decidua (20.0%). The same tendency proved to be even stronger in myometrium: the share of diploid EVT cells increased up to 46.0%, a prominent amount of tetraploid (45.1%) and highly polyploid (8c and 16c) cells retained in the EVT cell population (7.4 and 1.1%, respectively). Immunohistochemical staining of Ki-67 protein (MIB1), which labels cells held in the cell cycle, showed a high incidence of MIB1-positive stem cells (93.7%) and the EVT cells of the proximal part of CC (85.5%) characterized by high mitotic activity. A lower MIB1-positivity (43.2%) was found in the distal part of CC, whereas invasive EVT cells showed no MIB1-labeling. The presence of MIB1-positive nuclei in the distal part of CCs in the absence of mitoses, taken together with data on polyploidization of these cells, indicates their switch to the endoreduplication cycle. As a whole, the data obtained evidence that differentiation of EVT cells of the invasive pathway is accompanied by polyploidization. However, in a population of trophoblast cells capable of most profound invasion (up to myometrium), the proportion of diploid cells rose. These results suggest that the human cytotrophoblast invasion into the uterine wall requires an optimum, not the highest, ploidy level, whereas highly polyploid cells may form a subpopulation at the border between the maternal and fetal parts of placenta.

Human trophoblast contains an intracellular protein reactive with an antibody against CD133--a novel marker for trophoblast.

CD133 is a protein expressed on the cell membrane of a subfraction of haematopoietic stem and progenitor cells, as well as on some epithelial cells. Previously available antibodies against CD133 recognized only the glycosylated protein, localized to membrane protrusions or microvilli. Due to this, immature intracellular stages of the CD133 protein could not be visualized using these antibodies. We describe reactivity of a commercially available antibody against CD133, called AC133-2, with an intracellular protein in trophoblast. Both villous and extravillous cytotrophoblast, as well as syncytiotrophoblast were stained by AC133-2 in cryostat sections of first trimester and term placenta. Villous stroma was not stained. AC133-2 reactivity was seen in methanol-fixed primary trophoblast cells and trophoblast-derived cell lines, and was coexpressed with cytokeratin-7. CD133 messenger RNA was present in trophoblast and trophoblast-derived cell lines, but also in cells not displaying any reactivity with CD133 antibodies. AC133-2 recognized a 55-60 kDa protein on Western blots of cell extracts including trophoblast. The exact nature of this protein is not yet understood. However, AC133-2 is applicable as a positive marker for the characterization of all subtypes of trophoblast and for trophoblast cell lines.

Macrophage-induced apoptosis limits endovascular trophoblast invasion in the uterine wall of preeclamptic women.

Impaired invasion of uteroplacental arteries by extravillous trophoblast cells is a key pathogenic mechanism of preeclampsia. We previously demonstrated that reduced trophoblast invasion into uteroplacental spiral arteries was associated with an excess of macrophages in and around these arteries. To explore the significance of these observations, we correlated the extent of extravillous trophoblast apoptosis in placental bed biopsy specimens with macrophage distribution and studied the effect of macrophages upon trophoblast apoptosis in vitro. Extravillous trophoblast hybrid cells were cocultured with activated macrophages exposed to exogenous tumor necrosis factor alpha (TNFalpha), anti-tumor necrosis factor receptor I (TNF-RI), and tryptophan depletion, and the rates of trophoblast apoptosis were measured. Extravillous trophoblast hybrid cells showed increased rates of apoptosis following exposure to exogenous TNFalpha, with tryptophan depletion, and when cocultured with activated macrophages. The proapoptotic effects of macrophages in vitro were completely inhibited only by simultaneous addition of tryptophan and anti-TNF-RI. Our data indicate that macrophages, residing in excess in the placental bed of preeclamptic women, are able to limit extravillous trophoblast invasion of spiral arterial segments through apoptosis mediated by the combination of TNFalpha secretion and tryptophan depletion. The mechanisms by which macrophages are activated and recruited to the placental bed are presently unknown but are likely central to the pathogenesis of preeclampsia.

Characterization of trophoblast cell isolations by a modified flow cytometry assay.

The purity of isolated trophoblast cells is an important checkpoint in in vitro studies on the human placenta. Maintaining viability of primary cells for a prolonged period, or achieving cell proliferation in primary cell cultures, is often a matter of concern. In this paper we present a method for characterizing the purity of isolated trophoblast cells based on the expression of cytoskeletal proteins, as well as for assessing their cell cycle status, by flow cytometry. We show that after a simple permeabilization and fixation step in 70 per cent methanol, staining for cytokeratin 7 and vimentin could discriminate between trophoblast cells and contaminating populations. The method was applicable to trophoblast cells both from villous and extravillous origin. By staining the proliferation-related antigen Ki-67 and DNA, information was gained about the cell cycle status and viability of freshly isolated and cultured villous trophoblast cells. This method may help to quickly and quantitatively characterize preparations of isolated trophoblast, as well as to search for culture conditions favouring long-term survival and proliferation.