D. Deponti

The effects of dietary verbascoside on blood and liver oxidative stress status induced by a high n-6 polyunsaturated fatty acids diet in piglets12

Twenty-four weaned female Hypor piglets (10.9 ± 0.1 kg mean BW) were used to evaluate the antioxidant effect of a natural extract, titrated in verbascoside, on blood and liver oxidative status in relation to a high intake of n-6 PUFA, inducing oxidative stress. Piglets were assigned to 1 of 3 experimental groups; the first group was fed a diet with 9% sunflower oil (T1) and the second received the sunflower oil diet supplemented with 5 mg of verbascoside/kg feed from Verbenaceae extract (Lippia spp.; T2). The third group was fed a control diet (CTR), in which an isoenergetic replacement of oil by starch was done. Blood samples were collected at the beginning and the end of the trial (30 d). At the end of the trial, the animals were slaughtered and the liver specimens were collected. Oxidative stress markers, including total antiradical activity, superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT) activities, were determined in blood samples. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and γ-glutamyl transferase (GGT) plasma levels were also evaluated. Immunohistochemistry and western blot analyses were performed in liver to evaluate heat shock protein (Hsp) 70, Hsp90, and Kupffer and Ito cell activation. Liver activities of SOD, GPX, and CAT were also determined. Total antiradical activity in blood and red blood cells were affected (P < 0.01) by dietary treatments. The n-6 PUFA supplementation at a high dosage for 30 d induced oxidative stress, decreasing total antiradical activity in blood and red blood cells (CTR vs. T1 + T2; P < 0.01) and plasma CAT activity (CTR vs. T1 + T2; P = 0.088) and increasing ALT value (CTR vs. T1 + T2; P < 0.01). Also, in liver, the CAT and GPX activities tended to be lower in pigs fed n-6 PUFA diets than pigs fed a control diet (CTR vs. T1 + T2; = 0.090 and = 0.085, respectively). The liver samples presented a normal architecture and no Ito and Kupffer cell activations were observed. In liver, the SOD activity tended to be lower in the T1 group (P = 0.064) than in the CTR and T2 groups. Moreover, the level of Hsp70 was higher (P < 0.01) in the T1 group than the CTR and T2 groups. These data suggest that the dose of dietary verbascoside partially restores the antioxidant status of the liver without affecting the systemic responses to oxidative stress induced by a high-fat diet.

Age-related modulation of angiogenesis-regulating factors in the swine meniscus

An in‐depth knowledge of the native meniscus morphology and biomechanics in its different areas is essential to develop an engineered tissue. Meniscus is characterized by a great regional variation in extracellular matrix components and in vascularization. Then, the aim of this work was to characterize the expression of factors involved in angiogenesis in different areas during meniscus maturation in pigs. The menisci were removed from the knee joints of neonatal, young and adult pigs, and they were divided into the inner, intermediate and outer areas. Vascular characterization and meniscal maturation were evaluated by immunohistochemistry and Western blot analysis. In particular, expression of the angiogenic factor Vascular Endothelial Growth Factor (VEGF) and the anti‐angiogenic marker Endostatin (ENDO) was analysed, as well as the vascular endothelial cadherin (Ve‐CAD). In addition, expression of Collagen II (COLL II) and SOX9 was examined, as markers of the fibro‐cartilaginous differentiation. Expression of VEGF and Ve‐CAD had a similar pattern in all animals, with a significant increase from the inner to the outer part of the meniscus. Pooling the zones, expression of both proteins was significantly higher in the neonatal meniscus than in young and adult menisci. Conversely, the young meniscus revealed a significantly higher expression of ENDO compared to the neonatal and adult ones. Analysis of tissue maturation markers showed an increase in COLL II and a decrease in SOX9 expression with age. These preliminary data highlight some of the changes that occur in the swine meniscus during growth, in particular the ensemble of regulatory factors involved in angiogenesis.

Tissue engineering for cartilage repair: in vitro development of an osteochondral scaffold. Abstracts of the 3rd TERMIS (Tissue Engineering & Regenerative Medicine International Society) World Congress 2012. September 5-8, 2012. Vienna, Austria

Adequate cellular in-growth into biomaterials is one of the fundamental requirements in regenerative medicine. Type-I-collagen is the most commonly used material for soft tissue engineering, because it is nonimmunogenic and a highly porous network for cellular support. However, adequate cell in-growth and cell seeding has been suboptimal. Different densities of collagen scaffolds (0.3% to 0.8% (w/v)) with/without polymer knitting (poly-caprolactone (PCL)) were prepared. The structure of collagen scaffolds was characterized using scanning electronic microscopy (SEM) and HE staining. The mechanical strength of hybrid scaffolds was determined using tensile strength analysis. Cellular penetration and interconnectivity were evaluated using fluorescent bead distribution and human bladder smooth muscle cells and urothelium seeding. SEM and HE analysis showed the honeycomb structure and the hybrid scaffolds were adequately connected. The hybrid scaffolds were much stronger than collagen alone. The distribution of the beads and cells were highly dependent on the collagen density: at lower densities the beads and cells were more evenly distributed and penetrated deeper into the scaffold. The lower density collagen scaffolds showed remarkably deeper cellular penetration and by combining it with PCL knitting the tensile strength was enhanced. This study indicated that a 0.4% hybrid scaffold strengthened with knitting achieved the best cellular distribution.Human adult heart harbors a population of resident progenitor cells that can be isolated by Sca-1 antibody and expanded in culture. These cells can differentiate into cardiomyocytes and vascular cells in vitro and contribute to cardiac regeneration in vivo. However, when directly injected as single cell suspension, the survival rate and retention is really poor, less than 1% of injected cells being detectable in the hosttissue within few weeks. The present study aimed at investigating the possibility to produce scaffoldless, thick cardiac progenitor cell-derived cardiac patches by thermo-responsive technology. Human cardiac progenitors obtained from the auricles of patients were cultured as scaffoldless engineered tissues fabricated using temperature-responsive surfaces obtained by poly-N-isopropylacrylamide (PNIPAAm) surface immobilization. In the engineered tissue, progenitor cells established proper three-dimensional intercellular relationships and produced abundant extracellular matrix, while preserving their phenotype and plasticity. Cell phenotype and viability within the 3D construct were followed for 1 week, showing that no significant differentiation or apoptotic events occurred within the construct. After engineered tissues were leant on visceral pericardium, a number of cells migrated into the myocardium and in the vascular walls, where they integrated in the respective textures. The study demonstrates the suitability of such approach to deliver stem cells.Spinal cord injury and repair is one of the important focus areas in tissue regeneration. Mechanical trauma caused due to factors such as contusion, compression or involuntary stretching induce post-traumatic secondary tissue damage in many Spinal Cord Injury (SCI) patients. Therefore, there is a need for scaffolds that provide a conducive threedimensionsal (3D) environment for injured cells to attach and grow. In this study we propose to synthesize 3D polymeric scaffolds in order to study the mechanical and adhesive properties & the nature of the interactions between hyaluronan-based (HY) biomaterials and cells and tissues both in vitroandin vivo. Here we have synthesized 3D HY-based hydrogels with robust mechanical and adhesive properties and demonstrate the use of this material for neuronal-related applications such as the treatment of SCI. Cell culture and survivability studies were done with NSC-34 cells. Live/Dead assay performed on the cells revealed significant differences in the staining of live cells and showed increased viability and proliferation. The number of live cells in the HY-based hydrogels with 0.1% collagen showed higher cell numbers compared with the other hydrogels. In this study we show that Injectable HYbased hydrogels with high elasticity, comparable to the mechanical properties of nervous tissue have been used in this study to study their biocompatibility and neuroprotective properties and they show better affinity for neuronal cells.Calcium phosphates (CaP) obtained by biomineralisation in Simulated Boby Fluid have been used for decades to assess the mineralisation capability of biomaterials. Recently, they have been envisioned as potential agents to promote bone formation. In this study, we have fabricated and coated with calcium phosphate melt electrospun scaffolds whereby macropores permit adequate cell migration and nutrient transfer. We have systematically investigated the effect of coating and osteoinduction onto the response of ovine osteoblasts and we observed that the coating up-regulated alkaline phosphatase activity regardless of the in vitro culture conditions. Micro Computed Tomography revealed that only scaffolds cultured in an osteoinductive cocktail were capable of depositing mineralised matrix, and that CaP coated scaffolds were more efficient at promoting mineralisation. Theses scaffolds were subcutaneously implanted in athymic rats and this demonstrated that the osteoinduction was a pre-requisite for bone formation in this ectopic model. It showed that although the bone formation was not significantly different after 8 weeks, the CaP coated scaffolds were superior at inducing bone formation as evidenced by higher levels of mineralisation at earlier time points. This work demonstrated that CaP coating is not sufficient to induce bone formation; however the combination of osteoinduction and CaP coating resulted in earlier bone formation in an ectopic model.Introduction: Bladder regeneration using minced bladder mucosa is an alternative to costly and time-consuming conventional in vitro culturing of urothelial cells. In this method, the uroepithelium ...

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.