Analysis of living cells grown on different titanium surfaces by time-lapse confocal microscopy.

In this study we have combined fluorescence- and reflection-confocal laser scanning microscopy for the simultaneous visualization of living cells and surface topography beneath them. To this purpose we have designed a specific flow chamber and we have tested it with osteoblasts grown on an opaque, thick support, made of smooth or sandblasted titanium. Cells were loaded with Calcein-AM or tetramethylrhodamine methyl ester (TMRM), two probes employed as indicators of cell viability/morphology and mitochondrial membrane potential, respectively. Besides the acquisition of stacks of confocal sections, the system allowed also vertical views and faithful three-dimensional reconstruction of the samples. Confocal microscope implemented with our flow chamber proved to be a promising tool for time-lapse investigation of cell-biomaterial interactions.

Fluorescent in situ hybridisation on tissue sections: a quantitative approach with confocal laser scanning microscopy.

The use of fluorescent detection methods in association with digital microscopy technologies is an innovative approach for tissue localisation of messenger RNA. The success of such methods relies on the tissue preservation, local availability of the probe and on the existence of high resolution tridimensional analysis systems. Cryostatic sections, mild denaturation, short oligonucleotide probes (20mer) and confocal laser scanning microscopy allow the fulfillment of all these conditions avoiding photobleaching and tissue autofluorescence. In this paper, we describe in detail a method for in situ hybridisation set up with digoxigenin-coupled oligonucleotide complementary to beta-actin mRNA as a probe and an anti-hapten fluorescent antibody as second step for detecting specific hybridisation. Fluorescence was analysed by means of a confocal laser scanning microscope (CLSM) that provides images with low out-of-focus blurring also with relatively low numerical aperture (NA) objectives. We propose also an easy method to perform semi-quantitative thresholding analysis which allows to discriminate between background and specific signal.

Low-temperature heat-deproteinated compact bone to heal large bone defects.

The potential of low-temperature (400 degrees C), heat-treated bone matrix in osteorepair has been evaluated in vivo by implantation into defects artificially created in rodent tibia. Histological and ultrastructural analysis of the bone--implant interface has been carried out on samples obtained at 1 to 6 weeks from operation. The obtained data showed that calcined bone is well tolerated and does not cause acute or chronic inflammatory reactions. Osteoid tissue, tightly adhered to the implant, appears within 2 weeks of the operation, while after 6 weeks newly formed bone surrounds and infiltrates the implant. Of greater note, the detection of good adhesion between bone and implant ultrastructurally is demonstrated by the absence of fibrillar connective tissue at the interface. For these reasons, our preliminary observations suggest that low-temperature calcined bone (biological apatite or heat-deproteinated bone) may have a rightful place among the osteointegrators.

Pulsed electromagnetic field stimulation on posterior spinal fusions: a histological study in rats.

This study reports the histological data relative to the effect of pulsed electromagnetic fields (PEMFs) on the evolution of posterior arthrodesis induced in the lumbar vertebrae of 12 adult male Sprague-Dawley rats. After the operation, one group of six rats was stimulated with PEMFs for 18 h per day, by means of a pair of coils fixed to the outside of the cage. A control group of six rats was given no stimulation after surgery. In the groups stimulated with PEMFs an acceleration of the process of bone callus organization was already observed after 4 weeks, and even more so after 8: An early replacement was in fact observed of the newly formed cartilage tissue with primary bone (at 4 weeks) and subsequently with secondary bone (after 8 weeks).

[The development of the vertebral articulations in the fetal period]. / Lo sviluppo delle articolazioni vertebrali nel periodo fetale.

In this paper the thoracic tract of fetal rachis aging between 9 and 19 weeks has been investigated. The study has been carried out with histological and radiological techniques. The histological examination of these samples reveals that the articular processes are completely formed at week 12, in this period it is also evident a primitive joints between the articular processes. Nevertheless the joint is completely formed only at week 19, when the fibrous capsula and the synovial cavity are well formed. These data are confirmed by the sectional radiography.

Implants of heterologous demineralized bone matrix for induction of posterior spinal fusion in rats.

The authors tested the osteoinductive capacity of powdered heterologous (bovine) demineralized bone matrix in rats. The first part of the study concerned a monolateral posterior spinal implant after decortication of three vertebrae, using as a control area the animal's contralateral side, in which neither bone graft nor any other material were placed. In another group of rats, a comparative evaluation was made of powdered heterologous demineralized bone matrix and fresh autologous bone. In the same animal, autologous bone was implanted to realize a thoracic posterior fusion and demineralized bone matrix, to induce a posterior fusion in the lumbar area. All data obtained suggested a good osteoinductive activity of heterologous powdered demineralized bone matrix. The two posterior spinal fusions done in the same animal with heterologous demineralized bone matrix or autologous bone, respectively, had similar callus development and required the same time for formation.

[Immunoelectronmicroscopic characterization of a subpopulation of Langerhans cells bearing the CD23 antigen]. / Caratterizzazione immunoelettronmicroscopica di una sottopopolazione di cellule di Langerhans portatrice dell'antigene CD23.

The present study demonstrates that a consistent percentage (over 30%) of freshly isolated human Langerhans cells express the CD23 moiety. This was achieved employing a pre-embedding immunoelectronmicroscopy, using the peroxidase reaction product as a marker, assay on suspended trypsinized epidermal cells isolated from normal human skin. The possibility that the CD23 molecule on the surface of Langerhans cells could play a role in the antigen-presentation function of dendritic epidermal cells to T lymphocytes is proposed.

[Effects of pulsing electromagnetic fields (PEMF) on the course of vertebral fusion callus. A histological study]. / Effetti dei campi elettromagnetici pulsanti (CEMP) sull'evoluzione del callo di fusione vertebrale. Studio istologico.

In this paper the findings concerning the effectiveness of PEMF on the evolution of the vertebral fusion callus are reported. The study has been carried on by preparing postero-lateral arthrodesis in the lumbar spinal tract in rats. In this tract the laminae have been decorticated, the articular processes prepared by decortication and removal of the articular cartilage, and the spinal processes removed and employed as osteoinductive material. The rats sacrificed after 4 and 8 weeks, show how the decorticated areas are clearly influenced from PEMF, an early appearance of the bony fusion callus is already evident in the treated group just after 4 weeks. Also the articular areas are influenced from PEMF but less markedly than the decorticated one; in these areas after 8 weeks the fusion callus is prevalently cartilaginous even if areas of calcification are detectable inside. This different behaviour can be explained with the absence of any form of spinal fusion by means of surgical tools.

[The IGSS (immunogold silver stain) method in the study of the immunophenotype of free cells. Electron microscopy study of human venous blood]. / Il metodo IGSS (immunogold-silver staining) nello studio dell'immunofenotipo di cellule libere. Ricerche al microscopio elettronico sul sangue venoso di uomo.

In the present study a pre-embedding immunoelectron microscopical method ideally suited to detect suspended cell surface-associated antigens, is described. In this method 5nm colloidal gold particles have been enlarged by means of silver enhancement yielding a large marker that is easily detectable at the TEM level. The present method is particularly suited when investigation are performed with a low percentage of labeled cells as well as low antigen expression.

Characterization of two morphologically distinct Leu-7+ cell subsets with respect to Leu-15 antigen. Evaluation of Leu-15 determinant distribution on both E rosetting and non-adherent non-E rosetting cell populations.

In the present study the fine structures of Leu-7+-Leu-15+ and Leu-7+-Leu-15- cell subpopulations were characterized by using an immunogold-immunoperoxidase double labelling in electron microscopy. The densities of Leu-15 antigenic sites on both E rosetting (Er) and non-adherent/non-E rosetting (non-A/non-Er) Leu-15 positive cell surfaces were also evaluated by using an immunogold analysis in electron microscopy. A majority of Leu-7+ cells co-expressed the Leu-15 antigen and showed an ultrastructural pattern specific for mature natural killer (NK) cells, i.e. abundant cytoplasm with many organelles, numerous electron dense granules, and irregular outline. On the other hand, a minority of Leu-7+ cells did not express the Leu-15 antigen and showed a clearly different ultrastructural feature in comparison to Leu-7+-Leu-15+ cells. Thus, the presence of the Leu-15 antigen on Leu-7+ cell surface corresponds to ultrastructural features specific to differentiated NK cells and may represent an expression of Leu-7+ cell differentiation. An alternative hypothesis may be that Leu-7+-Leu-15+ and Leu-7+-Leu-15- cells represent distinct cell lineages within non-A/non-Er Leu-7+ cells. Finally, the results of the present study provide proof that Leu-15 antigen is more frequently represented on non-A/non-Er Leu-15+ cells than on Er Leu-15+ cells.

Leu-7-positive cells with monocyte phenotype show different ultrastructural features in comparison to Leu-7-positive cells with T-cell phenotype.

The ultrastructural features of the Leu-7-positive - Leu-M3-positive cell subpopulation and the Leu-7-positive - Leu-4-positive cell subpopulation were characterized and compared using immunogold-immunoperoxidase double labelling with immunoelectron microscopy. The majority of Leu-7-positive cells coexpressed a monocyte phenotype and showed an ultrastructural pattern specific for functional natural killer (NK) cells, i.e. a low nuclear/cytoplasmic (N/C) ratio, an irregular outline, many cytoplasmic organelles and electron-dense granules. In contrast, only a minority of Leu-7-positive cells coexpressed a T phenotype, and these were characterized by a high N/C ratio, an even surface and the absence of electron-dense granules. Thus, Leu-7-positive - Leu-4-positive cells may by an immature form of NK cells, and Leu-7-positive - Leu-4-positive and Leu-7-positive - Leu-M3-positive cell subpopulations may represent different stages of Leu-7-positive cell differentiation.

Immunoperoxidase-immunogold double labelling in immunoelectronmicroscopy of large granular lymphocytes.

A method for better characterization of mononuclear cell subpopulations using detection of 2 surface antigens simultaneously in electron microscopy was developed with immunoperoxidase-immunogold double labelling. Monoclonal antibodies of IgG and IgM classes were used in the first step, and colloidal gold-labelled anti-mouse IgG antibody and peroxidase-labelled anti-mouse IgM antibody (mu chain-specific) in the second step. Immunoelectronmicroscopy with such double labelling improves ultrastructural analysis of mononuclear cell subpopulations.

The fine structure of HNK-1 (Leu7) positive cells. A study using an immunoperoxidase technique.

The ultrastructural characteristics of HNK-1 (Leu7) positive cells, visualized with a peroxidase labelled anti-mouse IgM serum, were analysed. Our investigation demonstrates: 1) the majority of Leu7 positive cells has a low nuclear/cytoplasmic ratio (N/C), an irregular outline, a well developed smooth endoplasmic reticulum, parallel tubular arrays (PTA) and electron dense granules; 2) the minority of Leu7 positive cells has a high N/C, regular profiles and lacks electron dense granules. The presence of two distinct ultrastructural patterns within Leu7 positive cells may represent: 1) the expression of subsequent stages of cell differentiation; 2) two distinct Leu7 positive cell subpopulations.

[Progesterone-induced changes in the endometrium. Ultrastructural studies of castrated, adrenalectomized and hypophysectomized impuberal rats]. / Sulle modificazioni indotte dal progesterone nell'endometrio. Ricerche ultrastrutturali sul ratto impubere castrato, surrenalectomizzato ed ipofisectomizzato.

Research has been made on the progesterone-induced effects upon endometrium on impuberal rats (either intact, or castrated, or castrated and adrenalectomized, or hypophysectomized) after an acute or a protracted treatment, which were made every third hour all through the day. It resulted that the hormone basically shows two distinct activities. The first one causes a modest awakening of the anabolical functions both in the lining and in the glandular epithelia, which results in the building up of small amounts of lipid and glycogen. The second one involves the lamina propria as well, and brings about morphological effects which can be superposed on those induced by estrogens. The former seems an autonomous activity and can mainly be found after long-lasting treatments. The latter, on the contrary is influenced by the administration time and can be detected only during a.m. treatments whereas it cannot be detected during p.m. and night treatments. Besides it is influenced by the hypophysal activity and in particular by gonadotropins. The possible mechanism of these influences are discussed.

Identification of macrophages and of a T-lymphocytes subpopulation in normal human lymphnode by histochemical demonstration of alpha-naphthyl-acetate-esterasic activity.

The cell population of normal human lymphnode was studied by a histochemical technique showing alpha-naphthyl-acetate-esterasic activity. Two distinct cell populations were evidenced: a) macrophagic cells, bearing a diffused ANAE cytoplasmic positivity, most of which were in the subcapsular area, whereas a minority of them was found within the lymphatic nodules and the paracortical area; b) lymphocytic cells, bearing on ANAE positive cytoplasmic spot, most of which were in the paracortical area, whereas a minority of them was found within the lymphatic nodules. A discussion of the results we got, as compared with those which are reported by literature, confirms what is already well-known about the cell colonization of the various areas in the lymphnode. Besides it gives a chance to identify lymphocytes bearing an ANAE positive cytoplasmic spot with T lymphocytes belonging to the "helper-inducer" subclass.

Cell subpopulations in the paracortical area of the normal human lymphnode defined by monoclonal antibodies.

The cell subpopulations of normal human lymphnode were studied by immunohistochemical and immunoelectronmicroscopy techniques carried out by using monoclonal antibodies. OKT3 positive cells were demonstrated overwhelming in comparison to both OKT4 positive cells and OKT8 positive cells; furthermore la positive cells, dendritic in shape, were observed. Our results, compared to literature data, confirm that paracortex of limphnode is a T-dependent area. Moreover a possible role of the la positive dendritic cells in the immunological education of T helper-inducer lymphocytes was hypotised. Finally, our immunoelectronmicroscopic findings prove that OKT4 positive cells and OKT8 positive cells show different ultrastructural patterns.

Fine structure of granular cell tumor of Abrikosov.

The case report illustrates the microscopical and ultrastructural findings obtained in a case of Abrikossoff's tumor. On the basis of the light and ultrastructural data a neural histogenesis is suggested.

Ultrastructural analysis of the lining layer of the rat pulmonary alveolus.

Using electron microscopy we have examined the lining layer of the rat pulmonary alveolus. This layer appears as a morphological entity 1-3 days after birth: it is composed at first of a filamentous Ruthenium Red-negative material derived from lamellar bodies, and subsequently (4 days after birth) of a homogeneous Ruthenium Red-positive material. This latter material, which corresponds to the epithelia lining of the alveolus typical of adult rats, is presumably derived from a mixture of the filamentous material produced by the lamellar bodies, and a material produced by the alveolar cells 4 days after birth which contains acidic groups which bind Ruthenium Red.

[The effects of testosterone on the adenohypophysis of female rat (author's transl)]. / Aspetti morfo-funzionali dell'adenoipofisi di ratto femmina trattato con testosterone.

The research regards the action of rapidly and slowly absorbed testosterone in the adenohypophysis of female impuberal rats. The results are that the rapidly adsorbed testosterone if administered in small quantities stimulates the activity of synthesis in the FSH cells, if administered in large quantities stimulates the activity of synthesis both in the FSH cells and in the LH cells. The slowly absorbed testosterone stimulates again the activity of synthesis only of the FSH cells. Both rapidly absorbed and slowly absorbed testosterone inhibit the release activity in both the FSH cells and the LH cells.