Potential role of culture mediums for successful isolation and neuronal differentiation of amniotic fluid stem cells.

In recent years, the use of stem cells has generated increasing interest in regenerative medicine and cancer therapies. The most potent stem cells derive from the inner cell mass during embryonic development and their use yields serious ethical and methodological problems. Recently, a number of reports suggests that another suitable source of multipotent stem cells may be the amniotic fluid. Amniotic fluid mesenchymal stem cells (AFMSCs) are capable of extensive self-renewal, able to differentiate in specialized cells representative of all three germ layers, do not show ethical restriction, and display minimal risks of teratomas and a very low immunogenity. For all these reasons, amniotic fluid appears as a promising alternative source for stem cell therapy. Their recent discovery implies a lack of knowledge of their specific features as well as the existence of a protocol universally recognized as the most suitable for their isolation, growth and long-term conservation. In this study, we isolated stem cells from six amniotic fluids; these cells were cultured with three different culture mediums (Mesenchymal Stem Cell Medium (MSCGM), PC-1 and RPMI-1640), characterized by cytofluorimetric analysis, and then either frozen or induced to neuronal differentiation. Even if the immunophenotype seemed not to be influenced by culture medium (all six samples cultured in the above-mentioned mediums expressed surface antigens commonly found on stem cells), cells showed different abilities to differentiate into neuron-like cells and to re-start the culture after short/long-term storage. Cells isolated and cultured in MSCGM showed the highest proliferation rate, and formed neuron-like cells when sub-plated with neuronal differentiation medium. Cells from PC-1, on the contrary, displayed an increased ability to re-start culture after short/long term storage. Finally, cells from RPMI-1640, even if expressing stem cells markers, were not able to differentiate in neuron-like cells. Further studies are still needed in order to assess the effective role of culture medium for a successful isolation, growth, differentiation and storage of AFMSCs, but our data underline the importance of finding a universally accepted protocol for the use of these cells.

In vitro study of biofunctional indicators after exposure to asbestos-like fluoro-edenite fibres.

The in vitro biological response to fluoro-edenite (FE) fibres, an asbestos-like amphibole, was evaluated in lung alveolar epithelial A549, mesothelial MeT-5A and monocyte-macrophage J774 cell lines. The mineral has been found in the vicinity of the town of Biancavilla (Catania, Sicily), where an abnormal incidence of mesothelioma has been documented. Cell motility, distribution of polymerized actin, and synthesis of vascular endothelial growth factor (VEGF) and of beta-catenin, critical parameters for tumour development, progression and survival, were investigated in A549 and MeT-5A cells exposed to 50 microg/ml FE fibres for 24 hr and 48 hr. The levels of cyclooxygenase (COX-2) and prostaglandin (PGE2), two molecules involved in cancer pathogenesis by affecting mitogenesis, cell adhesion, immune surveillance and apoptosis, were measured in J774 cells treated with FE fibres under the same experimental conditions. Finally, FE fibres were studied by SEM and EDS analysis to investigate their chemical composition. Exposure of A549 and MeT-5A cells to FE fibres affected differentially phalloidin-stained cytoplasmic F-actin networks, cell motility and VEGF and beta-catenin expression according to the different sensitivity of the two cell lines. In J774 cells it induced a significant increase in COX-2 expression, as assessed by Western blot analysis, and in the concentration of PGE2, measured in culture media by ELISA. SEM-EDS investigations demonstrated two types of FE fibres, edenite and fluoro-edenite, differing in chemical composition and both recognizable as calcic amphiboles. Fibre width ranged from less than 1 microm (prevalently 0.5 microm) to 2-3 microm (edenite) up to several microm (fluoro-edenite); length ranged from about 6 to 80 microm (edenite) up to some hundred microm (fluoro-edenite). Results provide convincing evidence that FE fibres are capable of inducing in vitro functional modifications in a number of parameters with crucial roles in cancer development and progression. Inhaled FE fibres have the potential to induce mesothelioma, even though their ability to penetrate lung alveoli depends on their aerodynamic diameter.

Mitochondrial changes induced by natural and synthetic asbestos fibers: studies on isolated mitochondria.

Asbestos fibers, such as chrysotile and crocidolite, are known to have cytotoxic effects on different cell types. In vivo exposure to asbestos fibers can induce both fibrotic and malignant lung diseases , however, the mechanisms linking exposure to the subsequent development of the diseases are unknown. Numerous investigations suggest the involvement of reactive oxygen species (ROS). ROS are known to damage biological macromolecules including proteins, cell membrane lipids and nucleic acids; alterations of these essential cellular components can alter cell function and can drive the cell to neoplastic transformation or to cell death. Because the mitochondrial respiratory chain is an important source of ROS and RNS (reactive nitogen species) in the cells, we have investigated the effects of aqueous extracts of asbestos (natural and synthetic) fibers on some mitochondrial activities. Our data show that crocidolite fibers release substances in solution that may interfere directly with the mitochondrial cytochrome oxidase complex. Moreover, the calcium ions released from these fibers induce opening of the permeability transition pore of the inner membrane leading to a possible cytotoxic effect due to the release of apoptotic factors normally localized in the mitochondrial intermembrane space. In addition, crocidolite extracts enhance the mitochondrial production of ROS. No significant biochemical effects are exerted by chrysotile, either natural or synthetic, on isolated mitochondria. Nevertheless, all asbestos fibers tested induce morphological alterations visualized by transmission electron microscopy and morphometric analysis.

In vitro evaluation of bio-functional performances of Ghimas titanium implants.

Titanium is the most widely used material for dental implants. The natural formation, in presence of oxygen, of different oxide films (passivation films) is correlated to titanium implant biocompatibility, resistance to corrosion and is responsible for implant bacteriostatic action. Surface roughness is another surface property of Ti-implants that, affecting implant-to-bone contact, improves integration. In the present study data concerning composition, surface roughness and biocompatibility of Ghimas implants and mini-implants undergoing sandblasting with Calcium Magnesium Carbonate (CaMg(CO3)2) are reported. AFM, SEM/EDX, XRD analyses and morpho-functional tests (MTT and ALP) were performed. Cell actin cytoskeletal modification (fluorescence phalloidin staining) was also observed with confocal laser microscopy (CLSM). Data related to surface geometry and chemical properties, associated with evidence of high purity of all the tested materials (XRD and EDX), highlighted the elevated biocompatibility of tested implants and mini-implants. CLSM investigation confirmed osteoblast features of an active cell behavior able to fit cell to chemico-mechanical stimuli present at the bone/implant interface and suggests an effective implant/alveolar bone integration in vivo.

A biochemical-morphological study on microvillus plasma membrane development.

The microvillus plasma membrane of the human placental syncytiotrophoblast at term has been extensively studied, while little is known about the characteristics of its development. The aim of the present work was to compare functional and structural properties of this membrane at early and term gestational age. Ten normal term placentas (40 weeks) and ten placentas at 10 weeks of gestational age were studied. The Na+/K+-ATPase activity is significantly decreased in the syncytiotrophoblast plasma membrane obtained from term placentas as compared to the early ones, with significant variation of maximum velocity (Vmax). The microviscosity, evaluated by the P parameter of DPH and Sn parameters of 5- and 16-NS, is increased in the term placentas compared to the early placentas. This alteration is accompanied by an increased cholesterol to phospholipids ratio in term placentas, while there is a decreased unsaturated to saturated fatty acid ratio. As follows from morphological studies, an increased mean diameter in the E face was observed in the term placenta with respect to the early placenta. The distribution factor DF, which indicates the particle aggregation state, decreased in the E face in the term placenta as compared to the early one. The present biochemical morphological study shows that a deep modification of the membrane is at the basis of the syncytiotrophoblast plasma membrane development.

Decrease of rotenone inhibition is a sensitive parameter of complex I damage in brain non-synaptic mitochondria of aged rats.

We investigated NADH oxidation in non-synaptic and synaptic mitochondria from brain cortex of 4- and 24-month-old rats. The NADH oxidase activity was significantly lower in non-synaptic mitochondria from aged rats; we also found a significant decrease of sensitivity of NADH oxidation to the specific Complex I inhibitor, rotenone. Since the rotenone-binding site encompasses Complex I subunits encoded by mtDNA, these results are in accordance with the mitochondrial theory of aging, whereby somatic mtDNA mutations are at the basis of cellular senescence. Accordingly, a 5 kb deletion was detected only in the cortex of the aged animals.

In vitro study of gingival fibroblasts from normal and inflamed tissue: age-related responsiveness.

The aim of this study was to characterize some phenotypic expressions of fibroblasts from the human oral mucosa. Gingival and lower forearm fibroblasts from young (20-30 years) and elderly (> 60 years) subjects were analyzed. Gingival fibroblasts were taken from donors with (P) and without (NP) periodontal disease, while skin biopsies were taken from healthy subjects. Cell proliferation was assessed by evaluating the cell multiplication coefficient (C.M.C.). The proliferation potential of gingival fibroblasts from elderly individuals with and without periodontopathy did not differ from that of young subjects in the same condition but differed significantly in the skin samples. Enzyme neutral endopeptidase (EC 3.4.24.11) (NEP) activity, studied as a possible marker of cell ageing, showed an age-related increase in human skin fibroblasts but not consistently in gingival fibroblasts from individuals with or without periodontal disease. Cell area and substrate adhesion were evaluated by morphometric analysis. There were no significant differences between elderly P and NP subjects, while significant differences were observed between young and elderly P subjects. In conclusion, proliferative capacity and NEP activity in gingival fibroblasts did not appear to be age-related, probably because their microenvironment is continually moistened by saliva, which continues to contain growth factors, notably EGF, even into senescence. Tissue reaction and repair are important clinical and therapeutic implications.

Biochemistry, histology and clinical uses of chitins and chitosans in wound healing.

Biodegradability, biocompatibility and capacity to promote the synthesis of hyaluronan are main characteristics of chitin-derived wound healing materials, whose biological significance in the human body depends largely on the actions that certain hydrolases exert on them. The resulting chitooligomers stimulate various cells, while the released monomers are phosphorylated and incorporated into hyaluronan, keratan sulphate and chondroitin sulphate, components of the intracellular matrix and connective tissue. The healing process favoured by these materials is examined in terms of macrophage activation, cytokine production by macrophages and fibroblasts, antiinflammatory action, angiogenesis stimulation, granulation and scar formation. Current biomedical applications are illustrated by the treatment of leg ulcers, the use of skin substitutes, and the regeneration of bone, nerve and meniscus tissues.

Morphological study of the capsular organization around tissue expanders coated with N-carboxybutyl chitosan.

Expanders coated with N-carboxybutyl chitosan were inserted into surgical wounds in the dorsal skin of rabbits and the formation of capsular tissue was studied by scanning electron microscopy and transmission electron microscopy. N-carboxybutyl chitosan, in the course of the capsular organization, favours and potentiates the correct proliferation and organization of the tissue, rather than sustaining reactive processes leading to scar formation. N-carboxybutyl chitosan stimulates physiologically the tissue repair process and favours angiogenesis, whilst depressing fibrogenesis to a certain extent. Applications are envisaged in the treatment of wounds and in plastic surgery.

Osteoconductive properties of methylpyrrolidinone chitosan in an animal model.

Bone defects were surgically produced in the tibiae of rabbits and medicated with freeze-dried methylpyrrolidinone chitosan. Histological observations 60 d after surgery showed a considerable presence of neoformed bone tissue, as opposed to controls, originating from the pre-existing bone as well as from the periosteum. The cationic nature and the chelating ability of the methylpyrrolidinone chitosan apparently favoured mineralization. Endosteal-periosteal and bone marrow osteoblast-like precursors, stimulated by growth factors entrapped in the coagulum-polysaccharide mixture, gave rise to intramembranous bone formation. The ultrastructural examination evidenced that bone osteoid was followed by mineralization of the tissue.

Reconstruction of parodontal tissue with chitosan.

Chitosan ascorbate, obtained by mixing chitosan with ascorbic acid and sodium ascorbate, was produced in a gel form suitable for the treatment of periodontitis according to current dental surgery. While chitosan ascorbate underwent degradation in vitro, especially in the presence of atmospheric oxygen and at pH 6.0, the protection from oxygen offered by the surgical cements and the physiological pH value permitted chitosan ascorbate to play an important biological role in vivo, where it kept a honeycomb structure, as indicated by SEM on biopsies taken on 10 patients. The proliferation and organization of the cells were thus favoured with a subsequent enhanced capability of reconstructing a histoarchitectural tissue. Chitosan was progressively reabsorbed by the host, with very satisfactory clinical recoveries of the 52 defects treated, for which tooth mobility and pocket depths were significantly reduced.

An electron microscopic study of clinical and laboratory-derived strains of teicoplanin-resistant Staphylococcus haemolyticus.

Staphylococcal resistance to glycopeptides (which involves more teicoplanin than vancomycin) is uncommon and largely confined to Staphylococcus haemolyticus, an emerging nosocomial pathogen with a tendency to develop antibiotic resistance. In this study, six S. haemolyticus strains, including two isogenic pairs of teicoplanin-susceptible/-resistant strains and two resistant clinical isolates, were used in a morphologic and morphometric electron microscope investigation. Cells from both clinical and laboratory-derived teicoplanin-resistant strains exhibited abnormally roughened, irregular outlines when observed by transmission electron microscopy. However, no significant differences in cell wall thickness resulted from morphometric analysis when the susceptible/resistant cells of the two isogenic pairs were compared. By scanning electron microscopy, an abnormally roughened, blistered surface was associated with teicoplanin-resistant cocci. A certain variability was noted between strains, not clearly related to the resistance level. In freeze-fracture investigations, a higher number per square micrometer of intramembrane particles, more significant in the E than in the P membrane fracture face, was observed in the laboratory-derived resistant clones as compared to susceptible parent strains. Further studies are needed to understand the cause-effect relation between these ultrastructural alterations and staphylococcal resistance to teicoplanin (but not to vancomycin).

Characterization of large unilamellar vesicles as models for studies of lipid peroxidation initiated by azocompounds.

The aim of this work was to characterize large unilamellar vesicles (LUVETs) prepared by a hand-driven extrusion device in order to use them for studies of lipid peroxidation and antioxidant activity. Vesicle structure and size were examined by electron microscopy. Lipid and antioxidant content was determined before and after the extrusion procedure. Then LUVETs were subjected to autoxidation initiated by both the lipid-soluble 2,2'-azobis(2,4-dimethylvaleronitrile) and the water-soluble 2,2'-azobis(2-amidinopropane hydrochloride) azocompounds. The results demonstrated that: i) LUVETs prepared with lipid concentrations ranging between 25 and 150 mM were essentially unilamellar and reasonably homogeneous, with an average diameter of 90 nm; ii) the phospholipid, cholesterol and antioxidant amounts retained by filters were about 10-15%; iii) LUVETs were suitable for autoxidation studies initiated by the water-soluble azocompound both in the absence and presence of antioxidants. The lipid-soluble azocompound could be used only at low concentrations and its vesicle content had to be determined since part of the initiator was not incorporated into the lipid bilayer. These data suggest that LUVETs seem to be recommended for studies of lipid peroxidation and antioxidant activity.

Osteoblast behaviour in the presence of bisphosphonates: ultrastructural and biochemical in vitro studies.

OBJECTIVE: A positive balance in bone remodelling is an important goal of bone metabolism both in the presence of the osteoporotic processes characteristic of ageing and, especially, of prosthetic implants. The aim of the present work was to obtain new information about the initial steps of osteoblastic growth in an in vitro osteoblastic model in the presence of two bisphosphonates. METHODS: Experiments were performed with Alendronate and Neridronate, two molecules used in the therapy of osteoporosis. Since differentiating features into osteoblastic cells are known to parallel the presence in the cytoplasm of alkaline phosphatase and osteocalcin, we also carried out immunohistochemical typing. RESULTS: Good differentiation and osteoblastic activity were generally observed in the cells in contact with these compounds, except for 10(-4) Neridronate, where biochemical data clearly indicated its toxic effect on the cells. CONCLUSION: The detection of osteoblastic markers associated with an ultrastructural picture of correct organellar morphology in our cultures further supports the hypothesis of a metabolically positive action of these molecules on osteoblasts.

Tyrosine phosphorylation in type-1 diabetes by immunogold detection: an in vitro human aortic endothelial cell (HAEC) study in the presence of diabetic low density lipoproteins (LDL).

An immunomorphometric study of tyrosine phosphorylation was performed by the immunogold technique on cultured human aortic endothelial cells (HAEC) with a view to demonstrating their impaired signal transduction status, induced in vitro by incubation with low-density lipoproteins from the plasma of Type-1 diabetic patients. The results seem to sustain the hypothesis that extranuclear bioenergetic derangement induced by low-density lipoproteins from Type-1 diabetic patients may be associated with an up-regulation of the nuclear energetic machinery aimed at maintaining intracellular metabolic equilibrium. Our data demonstrate that phosphorylated tyrosine is a useful marker to monitor this metabolic condition.

Lymphocyte dysmetabolism: an immunocytochemical comparative approach in IDDM and control subjects.

We have investigated by immuno-electron microscopy the presence of phosphotyrosine in cells as a whole and in different cell districts (nucleus, cytoplasm, plasma membrane, and mitochondria) in peripheral blood lymphocytes of IDDM (insulin-dependent diabetes mellitus) patients and age-matched controls. Immuno-gold particle density was highest in mitochondria and decreased in cytoplasm, nucleus and plasma membrane. The time dependence of phosphotyrosine labelling after cell isolation was very strong in all subcellular populations, with a fall in immunogold staining after 30 min. Staining levels at zero time were similar in controls and IDDM patients; the loss of phosphotyrosine labelling was much stronger in controls, except in the plasma membrane. Plasma membrane NADH oxidoreductase activity, studied using cytosolic NADH as substrate and assayed with DCIP as acceptor, was significantly increased in IDDM patients, suggesting a response to a deficient mitochondrial energetic activity. The fact that NADH oxidoreductase is a growth factor related to tyrosine phosphorylation pathways raises intriguing questions on the cellular derangement occurring in peripheral lymphocytes in IDDM, although the relationships among the immunocytochemical and biochemical changes is still obscure.

rhVEGF and experimental rat skin flaps: systemic or local administration and morphological characteristics.

Skin flap survival is a significant problem in skin surgery; in particular, inadequate arterial or venous blood supply results in necrosis of the distalmost portion. The aim of this study was to evaluate the ability of Vascular Endothelial Growth Factor (VEGF) of modifying the morphological features of skin flaps. Bilateral epigastric skin flaps were raised in 16 Wistar male rats. The epigastric artery and vein of the left flaps were clamped and then injected with rhVEGF (8 rats) or saline (8 rats). The right flaps were not clamped and received rhVEGF or saline systemically. The rats were euthanized on the seventh day and flap skin samples collected. Tissue fragments were subject to immunohistochemical (rhVEGF, VEGFr, VIII factor, CD34 antibodies), ultrastructural and morphostructural investigations. The results showed that rhVEGF improved the condition of flaps and that systemic administration was effective in promoting the development of an adequate vascular network.

Umbilical veins in dichorionic twins. A morpho-functional assessment.

Investigations on singleton and twin pregnancies show different functional behaviour on maternal-fetal relationship. In some ways twin pregnancies may be considered at risk and they may develop associated pathologies such as hypertension. The aim of this work was to evaluate the morpho-functional behaviours of umbilical cord veins in twin and singleton gestations to better understand the role of these extra-embryonic tissues in the regulation of pregnancies. The umbilical cords were studied from singleton pregnancies and from dichorionic twin pregnancies. Biochemical and morphological investigations were carried out. A significant decrease in the anisotropy values was observed in endothelial cells from twins compared with singletons. Our ultrastructural data show immaturity features at the vein vessel wall level in twins. Furthermore, immunohistochemical investigations showed a lower degree of expressivity concerning adhesion molecules such as ICAM-1 and ELAM. Morphogenetic extracellular glycoproteins like fibronectin and tenascin seem over-expressed in twin pregnancies. Our morpho-functional data well testify the lower maturation degree of umbilical cord veins in twins with respect to singletons.

Cultured human trophoblast cells reproduce the initial events of placental biology.

OBJECTIVE: The objective of this study was to determine whether cultured trophoblast cells shared the same morphological and biological properties observed in trophoblast, in vivo. STUDY DESIGN: Trophoblast cells from human term placenta were cultured, morphologically, biochemically and immunochemically monitored for as long as 30 days. RESULTS: Single cells progressively aggregated and fused into a syncytio, the Ca2+ and the Ca(2+)-ATPase activity drooped, and the 72 kDa collagenase (MMP-2) was consistently expressed. CONCLUSIONS: Term placenta trophoblast cultures can be viewed and used as a model system mimicking morphological and biochemical events of placenta biology and differentiation.

[Regeneration of peripheral nerves after interposition of acellular muscle into larger defects]. / Regeneration peripherer Nerven nach Interposition grösserer Defekte durch azellulären Muskel.

Peripheral nerve grafting is an established procedure in reconstructive surgery. Nerve grafts, however, are only available to a limited extent and patients are faced with neurologic deficits at the donor areas. Pretreated skeletal muscle has been proposed as an alternative grafting material. In nine adult Sprague-Dawley rats, a 2 cm gap of the sciatic nerve is grafted with a M. gracilis segment which has been pretreated through repeated freezing and thawing. Regeneration is evaluated after six weeks postoperatively. The results are compared to nine conventional nerve grafts. Regeneration was evident in all grafts. Histologically, the muscle grafts revealed a high proportion of connective tissue, a good vascularisation but an inferior degree of myelinisation. Morphometrically, the muscle grafts proved to be inferior according to axon counts and myelinisation. Muscle grafts provide a substrate comparable to peripheral nerves regarding the tubular architecture based on laminin. There are however no viable Schwann cells within these substitutes, which makes them inferior compared to conventional nerve grafts for peripheral nerve repair. These results are discussed with respect to further experiments concerning allogeneic nerve grafting and peripheral nerve preservation.