Vincenzo Conte

Biological performance of a novel biodegradable polyamidoamine hydrogel as guide for peripheral nerve regeneration.

Polyamidoamines (PAAs) are a well-known family of synthetic biocompatible and biodegradable polymers, which can be prepared as soft hydrogels characterized by low interfacial tension and tunable elasticity. For the first time we report here on the in vivo performance of a PAA hydrogel implant as scaffold for tissue engineering. In particular, an amphoteric agmatine-deriving PAA hydrogel shaped as small tubing was obtained by radical polymerization of a soluble functional oligomeric precursor and used as conduit for nerve regeneration in a rat sciatic nerve cut model. The animals were analyzed at 30, 90, and 180 days post-surgery. PAA tubing proved to facilitate nerve regeneration. Good surgical outcomes were achieved with no signs of inflammation or neuroma. Moreover, nerve regeneration was morphologically sound and the quality of functional recovery satisfactory. In conclusion, PAA hydrogel scaffolds may represent a novel and promising material for peripheral nerve regeneration.

Age-related reduction of the satellite cell sheath around spinal ganglion neurons in the rabbit

SummaryThe volumes of the nerve cell bodies and those of the enveloping satellite cell sheaths from spinal ganglia of young adult and aged rabbits were determined by morphometric methods using the electron microscope. The mean volume of the nerve cell bodies was greater in the old rabbits than in young adults; this is probably related to the larger body size of the old animals. The mean volume of the satellite cell sheaths was, however, smaller in the aged rabbits than in the young adults. Consequently the volume ratio between the satellite cell sheaths and the related nerve cell bodies was significantly smaller in the aged animals. Since satellite cells play an important role in the support of the neuron, the reduction in volume of the perineuronal sheath could be associated with a decrease in the trophic activity of satellite cells towards the enveloped neuron with consequences for neuronal activity. Furthermore, in the satellite cell sheaths of old rabbits, the number and extension of gaps that leave the neuronal surface directly exposed to the basal lamina were significantly increased. Since spinal ganglia lack a blood-nervous tissue barrier, only the satellite cell sheath controls the traffic of material to the nerve cell body. Because the neuronal surface unprotected by the satellite cell envelopment is significantly more extensive in the spinal ganglia of old rabbits than in those of young adults, the nerve cells of the former are more exposed to potential damage by harmful substances. A dense undercoating was seen very frequently beneath the portions of the neuronal plasma membrane not covered by satellite cells.

Scanning electron-microscope observations of the perikaryal projections of rabbit spinal ganglion neurons after enzymatic removal of connective tissue and satellite cells

SummaryThe true surface of rabbit spinal ganglion neurons has been made directly accessible to scanning electronmicroscope observation after removal of both the connective tissue and satellite cells that normally cover it. The neuronal surface is characterized by a profusion of slender projections whose shapes have been determined and whose length and width have been quantified. Controls carried out with transmission electron microscopy demonstrate that the procedure employed in this study satisfactorily preserves neuronal structure.

The perikaryal projections of rabbit spinal ganglion neurons. A comparison of thin section reconstructions and scanning microscopy views.

SummaryShape, length and width of the perikaryal projections of spinal ganglion neurons from adult rabbits fixed in situ by perfusion have been evaluated by means of serial section electron microscopy. The results thus obtained have been compared with those obtained by enzymatic removal of ganglionic connective tissue and satellite cells followed by direct observation of the true neuronal surface under the scanning electron microscope. The comparison has shown that the perikaryal projections exhibit a similar shape and similar size with both techniques.

The percentage of nerve cell bodies arranged in clusters decreases with age in the spinal ganglia of adult rabbits

In the spinal ganglia of the rabbit the nerve cell bodies, which in early developmental stages are mutually in contact, come to be completely isolated from each other by a satellite cell sheath and by a connective envelope before birth. The present study demonstrates that in the early postnatal months some nerve cell bodies are still arranged in clusters, and that the percentage of these decreases progressively throughout adult life. This decrease probably arises because in some of the ganglion neurons the process of envelopment of the perikaryon by an individual sheath begins later, or takes place more slowly, than in the majority of cases. Therefore, the relationship between neurons and between neurons and satellite cells may change in certain clusters of nerve cell bodies under normal circumstances during adult life.

Long term effects of lipopolysaccharide on satellite glial cells in mouse dorsal root ganglia

Abstract Lipopolysaccharide (LPS) has been used extensively to study neuroinflammation, but usually its effects were examined acutely (24 h<). We have shown previously that a single intraperitoneal LPS injection activated satellite glial cells (SGCs) in mouse dorsal root ganglia (DRG) and altered several functional parameters in these cells for at least one week. Here we asked whether the LPS effects would persist for 1 month. We injected mice with a single LPS dose and tested pain behavior, assessed SGCs activation in DRG using glial fibrillary acidic protein (GFAP) immunostaining, and injected a fluorescent dye intracellularly to study intercellular coupling. Electron microscopy was used to quantitate changes in gap junctions. We found that at 30 days post‐LPS the threshold to mechanical stimulation was lower than in controls. GFAP expression, as well as the magnitude of dye coupling among SGCs were greater than in controls. Electron microscopy analysis supported these results, showing a greater number of gap junctions and an abnormal growth of SGC processes. These changes were significant, but less prominent than at 7 days post‐LPS. We conclude that a single LPS injection exerts long‐term behavioral and cellular changes. The results are consistent with the idea that SGC activation contributes to hyperalgesia. HighlightsA single lipopolysaccharides injection activated glia in mouse dorsal root ganglia for 30 days.This was accompanied by increased communications by gap junctions among glia and by hyperalgesia.Glial activation and coupling may contribute to chronic pain.

Epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma: Characterization in a 3D-cell culture model

AIM To analyze the effect of three-dimensional (3D)-arrangement on the expression of epithelial-to-mesenchymal transition markers in pancreatic adenocarcinoma (PDAC) cells. METHODS HPAF-II, HPAC, and PL45 PDAC cells were cultured in either 2D-monolayers or 3D-spheroids. Ultrastructure was analyzed by transmission electron microscopy. The expression of E-cadherin, β-catenin, N-cadherin, collagen type I (COL-I), vimentin, α-smooth muscle actin (αSMA), and podoplanin was assayed by confocal microscopy in cells cultured on 12-mm diameter round coverslips and in 3D-spheroids. Gene expression for E-cadherin, Snail, Slug, Twist, Zeb1, and Zeb2 was quantified by real-time PCR. E-cadherin protein level and its electrophoretic pattern were studied by Western blot in cell lysates obtained from cells grown in 2D-monolayers and 3D-spheroids. RESULTS The E-cadherin/β-catenin complex was expressed in a similar way in plasma membrane cell boundaries in both 2D-monolayers and 3D-spheroids. E-cadherin increased in lysates obtained from 3D-spheroids, while cleavage fragments were more evident in 2D-monolayers. N-cadherin expression was observed in very few PDAC cells grown in 2D-monolayers, but was more evident in 3D-spheroids. Some cells expressing COL-I were observed in 3D-spheroids. Podoplanin, expressed in collectively migrating cells, and αSMA were similarly expressed in both experimental conditions. The concomitant maintenance of the E-cadherin/β-catenin complex at cell boundaries supports the hypothesis of a collective migration for these cells, which is consistent with podoplanin expression. CONCLUSION We show that a 3D-cell culture model could provide deeper insight into understanding the biology of PDAC and allow for the detection of marked differences in the phenotype of PDAC cells grown in 3D-spheroids.

3D-spheroids: What can they tell us about pancreatic ductal adenocarcinoma cell phenotype?

&NA; We aimed at analyzing the effect of the 3D‐arrangement on the expression of some genes and proteins which play a key role in pancreatic adenocarcinoma (PDAC) progression in HPAF‐II, HPAC and PL45 PDAC cells cultured in either 2D‐monolayers or 3D‐spheroids. Cytokeratins 7, 8, 18, 19 were differently expressed in 3D‐spheroids compared to 2D‐monolayers. Syndecan 1 was upregulated in HPAF‐II and PL45 3D‐spheroids, and downregulated in HPAC. Heparanase mRNA levels were almost unchanged in HPAF‐II, and increased in HPAC and PL45 3D‐spheroids. Hyaluronan synthase (HAS) 2 and 3 mRNA increased in all 3D‐spheroids compared to 2D‐monolayers. CD44 and CD44s were expressed to a lower extent in HPAF‐II and HPAC 3D‐spheroids. By contrast, the CD44s/v3 and the CD44s/v6 ratio increased in HPAC and PL45 3D‐spheroids, compared to 2D‐monolayers. The expression of MMP‐7 was strongly upregulated in 3D‐spheroids. STAT3 was similarly expressed 3D‐spheroids or 2D‐monolayers, while pSTAT3 was almost undetectable in 2D‐monolayers and strongly upregulated in 3D‐spheroids. These results suggest that 3D‐spheroids represent a cell culture model that allows the characterization of PDAC cell phenotype, adding new information that contributes to a better understanding of the biology and behavior of PDAC cells. Highlights3D‐spheroids allow the detection of key characteristics of PDAC cell phenotype.3D‐spheroids contribute to a better understanding of PDAC cell biology.the first study describing the relationship between MMP‐7 and STAT3 in PDAC 3D‐spheroids.

Friction and morphology of pleural mesothelia

To verify the hypothesis that by enmeshing lubricants, microvilli reduce the coefficient of kinetic friction (μ) of pleural mesothelium, μ was measured during reciprocating sliding of rabbits visceral against parietal pleura before and after addition of hyaluronan, and related to the morphological features of the microvillar network. Because no relation was found between μ or μ changes after hyaluronan and microvillar characteristics, the latter are not determinants of the frictional forces which oppose sliding of normal mesothelial surfaces under physiological conditions, nor of the effects of hyaluronan. Addition of hyaluronan increased μ slightly but significantly in normal specimens, probably by altering the physiological mix of lubricants, but decreased μ of damaged mesothelia, suggesting protective, anti-abrasion properties. Indeed, while sliding of an injured against a normal pleura heavily damaged the latter and increased μ when Ringer was interposed between the surfaces, both effects were limited or prevented when hyaluronan was interposed between the injured and normal pleura before onset of sliding.

Characterization of pancreatic ductal adenocarcinoma cells in a 3D-cell culture model: focus on epithelial-to-mesenchymal transition

Three-dimensional (3D) cell cultures provides a key to the information encoded in the tissue architecture, therefore mimicking the functions of living tissues [1]. Considered the key role of epithelial-to-mesenchymal transition (EMT) in carcinoma progression [2], we aimed at analyzing the effect of the 3D-arrangement on the expression of some key markers of EMT in pancreatic adenocarcinoma (PDAC) cells cultured in either 2D-monolayers or in 3D-spheroids by morphological and molecular methods. HPAF-II, HPAC, and PL45 cell ultrastructure was analyzed by transmission electron microscopy. The main EMT markers E-cadherin, β-catenin, N-cadherin, collagen type I (COL-I), vimentin, α-smooth muscle actin (αSMA), Snail, Slug, Twist, Zeb1 and Zeb2 were evaluated by confocal microscopy and molecular methods. Moreover, the expression of cytokeratins was characterized in PDAC cells grown in 2D-monolayers and 3D-spheroids to better understand PDAC cell behaviour. We show important differences in the phenotype of PDAC cells grown in 3D-spheroids or in 2D-monolayers, especially providing additional correlative evidence of EMT marker expression in PDAC cells and contributing to a clarification of the role of EMT in PDAC progression. Considered as a whole, our results suggest that a 3D cell culture model could provide deeper insight into the understanding of the biology of PDAC.