P217 In vitro study on a tissue engineered osteochondral composite

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Gut peculiarities of feed deprived white sturgeons (Acipenser transmontanus, Richardson 1836)

In the White sturgeon fish farms, some individuals have difficulty in getting access to food: such sturgeons are called \u201crunts\u201d, and they result in a slower growth rate than normally feeding fish. In this paper, we have studied the gut pecu- liarities of runt sturgeons. Utilizing in paralleling an analysis of diatom populations in both the fish gut tissues and the rearing tank waters, we hypothesized a causative relation between the occurrence of runt sturgeons and periodic diatom blooms. In fact, we have observed that the diatom species identified in the aquatic environment were also detected in organs (Fragilaria spp and Rhoicosfenia spp for both glandular body, mid-intestine) of the runt sturgeon\u2019s gut, but not in tissues of normally feeding individuals. Owing to their siliceous wall, diatoms can be responsible for areas of epithet- lial detachment in the mucosal surfaces of the alimentary canal and a catharral inflammation in both the gastric pits and intestinal folds which may be the cause of secondary bacterial diseases. We suggest that diatom blooms may contribute to the occurrence of runt sturgeons in the studied Italian fish farm

Immunohistochemical aspects of Ito and Kupffer cells in the liver of domesticated and wild ruminants

The mammalian liver is a morphologically and functionally complex organ, made up of not only of the largely pre- dominant parenchymal cells (hepatocytes) but also non-parenchymal cells. Although there are less non-parenchymal cells than hepatocytes, they nevertheless play an important role in regulating many hepatocyte functions, as well as in the immunology of the liver. We investigated the structural aspects of the liver and the morpho-functional characteris- tics of Ito and Kupffer cells in two domesticated ruminant species (cattle and goat) in comparison with four wild rumi- nant species living in captivity in a zoo in northern Italy. The liver specimens were studied using histological, histo- chemical and immunohistochemical methods. The liver parenchyma was structurally normal. Immunohistochemistry was performed for desmin, glial fibrillary acidic protein (GFAP), vimentin, \u3b1-smooth muscle actin (\u3b1-SMA), collagen I, lysozyme, CD 68 and tumor necrosis factor \u3b1 (TNF-\u3b1). In all the studied ruminants, Ito cells reacted with desmin and vimentin antibodies, Kupffer cells were evidenced only with lysozyme-immunopositivity, and both displayed a charac- teristic distribution in the hepatic lobular/acinar structure. The results obtained, not only contribute to the knowledge of ruminant wild species, but also help to define a normal structure reference for the diagnosis and treatment of liver diseases

Effects of different rearing temperatures on muscle development and stress response in the early larval stages of acipenser baerii

The present study aims at investigating muscle development and stress response in early stages of Siberian sturgeon when subjected to different rearing temperatures, by analysing growth and development of the muscle and by assessing the stress response of yolk-sac larvae. Siberian sturgeon larvae were reared at 16\uc2\ub0C, 19\uc2\ub0C and 22\uc2\ub0C until the yolk-sac was completely absorbed. Sampling timepoints were: hatching, schooling and complete yolk-sac absorption stage. Histometrical, histochemical and immunohistochemical analyses were performed in order to characterize muscle growth (total muscle area, TMA; slow muscle area, SMA; fast muscle area, FMA), development (anti-prolif erating cell nuclear antigen -PCNA or anticaspase) as well as stress conditions by specific stress biomarkers (heat shock protein 70 or 90, HSP70 or HSP90). Larvae subjected to the highest water temperature showed a faster yolk-sac absorption. Histometry revealed that both TMA and FMA were larger in the schooling stage at 19\uc2\ub0C while no differences were observed in the SMA at any of the tested rearing temperatures. PCNA quantification revealed a significantly higher number of proliferating cells in the yolk-sac absorption phase at 22\uc2\ub0C than at 16\uc2\ub0C. HSP90 immunopositivity seems to be particularly evident at 19\uc2\ub0C. HPS70 immunopositivity was never observed in the developing lateral muscle

Expression of vaccine antigens to edema disease in tobacco seeds and evaluation of immunogenicity on mouse model

Plant-derived vaccines present many potential advantages related to the management in intensive livestock. They could be administered without restraint of the animals, with low stress and without labour costs related to multiple injections of traditional vaccines. The aim of this study was the construction and subsequent evaluation in mouse model of transgenic tobacco seeds as edible vaccines for swine Edema disease. We focalized our attention on Verocitotoxic Escherichia coli strains (O138, O139, O141), responsible of Edema disease, that occurs in pigs approximately one week after weaning and is characterized by edema in various sites and by damages to vascular endothelium. The adhesion of bacterial strains is related to different fimbriae and Shiga-like toxins (VT2e), that play an important role in the pathogenesis. Structural parts of F18 fimbriae and B-subunit of VT2e genes were inserted in expression vectors, under control of GLOB promoter to obtain specific seed accumulation of heterologous proteins, and transformed in tobacco by agroinfection. We obtained two stable lines of transformed tobacco expressing the proteins in the seed: one included F18 gene (F18+) and another one included B-subunit of Vt2e gene (VT2e-B+). Tobacco lines were characterized by molecular and immunoenzymatic techniques for the expression of F18 and VT2e-b proteins. The amount of transgenic proteins was estimated at around 10ug/g of seeds. 14 Balb-c mice were divided randomly in two groups Control (CG) and Treatment (TG), with 7 mice each. Treatment diet, prepared as pellet to avoid different feed intakes in animals, contained 10% of tobacco seeds from F18+ and 10% of tobacco seeds from VT2e-b+. CG received a diet containing 20% of not-transgenic tobacco seeds. Treatments were administered on days 0,5,8,14,19,23. TG revealed an increment of fecal IgA at day 26, while CG at the same period decreased. The histometric data of the small intestine showed that TG crypts of the duodenum were significantly deeper than those of the CG. Immunostaining of the intestine showed that administration of transgenic tobacco seeds promotes a significant increase in the IgA-positive plasma cells production of the tonaca propria if compared to control group. In conclusion our findings suggest that tobacco seeds might be a potential source of oral vaccines

Oral administration of tobacco seeds expressing antigenic proteins in mice Balb-C : a model of edible vaccines for oedema disease

The aim of this study was the evaluation of immunogenic activity of tobacco seeds as edbile vaccine against oedema disease in mice

Meniscus maturation in the swine model: role of endostatin in cellular differentiation

The development of an engineered meniscus derives from the need to regenerate a tissue which is largely unable to self-repair with consequent loss of functionality. Hence a deeper knowledge of the native meniscus morphology and biomechanics in its different regions, including molecules involved in regulation of the maturation process, is essential. The meniscus is a complex tissue, displaying great regional variation in extracellular matrix components and in vascularization, as a result of several biomechanical stimuli. Its biochemical composition is modulated to adapt the tissue to the different functions that are required throughout growth, until a \u201cmature\u201d phase is reached in adulthood. The aim of this work is to evaluate the biological role of Endostatin in the regulation of angiogenesis as in the fibro-chondrogenic differentiation of neonatal meniscal cells in the pig. The swine is an attractive model for meniscal repair studies, as its knee joint is closely comparable to the human one in terms of anatomical structure, vascularization, and healing potential. Our preliminary data show that Endostatin contributes to the acquisition of chondrocyte phenotype in an undifferentiated but committed cellular population. Thus, a better understanding of the role of Endostatin in cell metabolism might lead to a deeper knowledge of the events regulating meniscus maturation. These findings may be crucial for the development of an engineered scaffold able to induce meniscal cell differentiation by releasing Endostatin-rich microspheres

Administration of a novel plant extract product via drinking water to post-weaning piglets : effects on performance and gut health

The present study evaluated the effects of a novel plant extract (PE) product (GrazixTM) on the performance and gut health of weaned piglets challenged with Escherichia coli. The PE was a standardised mixture of green tea leaves (Camellia sinensis) and pomegranate fruit (Punica granatum) obtained by using the LiveXtractTM process. A total of 144 piglets were weaned at 24 days and allocated to 8 for a 35-day experiment with a 2 7 2 7 2 factorial design comparing different treatments (water without product (CT) or 8 \u3bcl/kg per day PE in drinking water (PE)), feeding regimens (ad libitum (AD) or restricted (RE)) and oral E. coli challenges on day 9 (sham ( 12 ) or infected ( +)). There were six pens per group with three piglets per pen. On day 35, 24 of the RE feeding piglets were slaughtered. It was found that PE supplementation increased the average daily gain (ADG) from day 28 to day 35 ( P =0.03) and increased the gain to feed ratio (G : F) from day 7 to day 14 ( P = 0.02). RE feeding led to lower feed intake in piglets during the 1st week ( P<0.01), 2nd week ( P = 0.06), 3rd week ( P = 0.05), and throughout the course of the overall study period ( P = 0.05). E. coli challenge decreased the ADG and G : F ratio from day 7 to day 14 ( P = 0.08 and <0.01, respectively) and increased the faecal score (higher values indicate more severe diarrhoea) on days 14, 21, 28 and 35 ( P<0.01). PE supplementation decreased the faecal score in the challenged piglets during the 1st week post-challenge ( P<0.01). E. coli challenge increased the faecal E. coli level on day 14 ( P = 0.03) and increased the Enterobacteriaceae level on day 35 ( P<0.01). Reduced faecal E. coli was observed on days 14 and 35 ( P = 0.05 and 0.02, respectively), and reduced Enterobacteriaceae ( P<0.01) was found on day 35 in the PE animals. RE feeding increased the faecal Lactobacillus, Enterobacteriaceae and E. coli levels on day 35 ( P = 0.02, <0.01 and <0.01, respectively). These results suggest that PE supplementation may improve the gut health status of post-weaning piglets and counteract some of the negative effects that occur when piglets are challenged with E. coli

A tissue engineered osteochondral composite for cartilage repair: an in vivo study

This work aimed to validate the efficacy of a tissue engineered osteochondral composite for the treatment of cartilage lesion produced in adult pigs. The osteochondral composite was manufactured by combining an osteo-compatible cylinder and a neocartilagineous tissue obtained by seeding swine articular chondrocytes into a collagen scaffold. Articular cartilage was harvested from the trochlea of six adult pigs and was enzymatically digested to isolate the chondrocytes [Deponti D.et al. 2005]. The cells were then expanded in monolayer culture in chondrogenic medium and seeded onto a collagen scaffold. The collagen scaffold was preintegrated in vitro, macroscopically and microscopically, to a an osteo-compatible cylinder. The seeded osteochondral scaffolds were left in standard culture condition for 3 weeks with the addition of growth factors. At the end of culture time the osteochondral scaffolds were surgically implanted in osteochondral lesion performed in the trochlea of the same pigs from which the cartilage was initially harvested. As control, some osteochondral lesions were treated with acellular scaffolds and others were left untreated. After 3 months, the repair tissue of the three experimental groups was macroscopically analyzed and processed for histological and biochemical analysis. The hystologic ICRS II scale showed a statistically significant difference between the three experimental groups only in the parameters regarding the cell morphology and the surface/superficial assessment: the lesion treated with the unseeded osteochondral scaffolds showed higher values in chondrocytes morphology and in the superficial layer recovery, with respect to the lesions treated with the seeded scaffolds or left untreated. The biochemical analysis showed a higher DNA content in the lesion repaired with cellular scaffold and a higher GAGs/DNA ratio in the lesions with a spontaneous repair. The result of this study demonstrate that an osteochondral scaffold was able to repair an osteochondral lesion in an in vivo model of adult pigs, showing a good integration with the surrounding tissue. The quality of the repair was higher when the scaffold was not seeded with chondrocytes, but filled with cells migrated from subchondral bone. This tissue engineered osteochondral composite could represent a valuable model for further in vivo studies on the repair of chondral/osteochondral lesion