Ilaria Iacopetti

Neoarteries grown in vivo using a tissue-engineered hyaluronan-based scaffold

Vascular tissue engineering has emerged as a promising technology for the design of an ideal, responsive, living conduit with properties similar to that of native tissue. The missing link in tissue‐engineered blood vessels is elastin biosynthesis. Several biomaterials are currently used but few support elastin biosynthesis in a 3‐D array. In previous studies, we demonstrated that a hyaluronan‐based scaffold (HYAFF‐11™) grafted in the infrarenal rat aorta successfully guided the complete regeneration of a well‐functioning smalldiameter (2 mm) neoartery. The aim of the present study was to test the ability of HYAFF‐11 biodegradable grafts to develop into neovessels of larger size (4 mm) in a porcine model, focusing on extracellular matrix (ECM) remodeling and elastin biosynthesis. HYAFF‐11 tubes (diameter 4 mm, length 5 cm) were implanted in an end‐to‐end fashion in the common carotid artery. Grafts were analyzed for patency with a Duplex scan every 15 days. ECM components were evaluated by histological and molecular biological methods. All the animals survived the observation period without complications. Intimal hyperplasia (initiating at the anastomotic site) and graft thrombosis led to 3 cases of partial or complete occlusion, as demonstrated by histological examination. There were no signs of stenoses or aneurysms in the remaining grafts. After 5 months, the biomaterial was almost completely degraded and replaced by a neoartery segment composed of mature smooth muscle cells, collagen, and elastin fibers organized in layers and was completely covered on the luminal surface by endothelial cells (vWF+). Whereas in previous small animal studies, patency rates were not optimal, those obtained in the present study using hyaluronan‐based grafts of larger size confirmed the ability of these constructs to guide the development of a well‐functioning neoartery, with the remarkable additional attribute of facilitating the formation of organized layers of elastin fibers.—Zavan, B., Vindigni, V., Lepidi, S., Iacopetti, I., Avruscio, G., Abatangelo, G., Cortivo, R. Neoarteries grown in vivo using a tissueengineered hyaluronan‐based scaffold. FASEB J. 22, 2853–2861 (2008)

Effects of in vivo applications of peripheral blood-derived mesenchymal stromal cells (PB-MSCs) and platlet-rich plasma (PRP) on experimentally injured deep digital flexor tendons of sheep.

Tendon injuries, degenerative tendinopathies, and overuse tendinitis are common in races horses. Novel therapies aim to restore tendon functionality by means of cell‐based therapy, growth factor delivery, and tissue engineering approaches. This study examined the use of autologous mesenchymal stromal cells derived from peripheral blood (PB‐MSCs), platelet‐rich plasma (PRP) and a combination of both for ameliorating experimental lesions on deep digital flexor tendons (DDFT) of Bergamasca sheep. In particular, testing the combination of blood‐derived MSCs and PRP in an experimental animal model represents one of the few studies exploring a putative synergistic action of these treatments. Effectiveness of treatments was evaluated at 30 and 120 days comparing clinical, ultrasonographic, and histological features together with immunohistochemical expression of collagen types 1 and 3, and cartilage oligomeric matrix protein (COMP). Significant differences were found between treated groups and their corresponding controls (placebo) regarding tendon morphology and extracellular matrix (ECM) composition. However, our results indicate that the combined use of PRP and MSCs did not produce an additive or synergistic regenerative response and highlighted the predominant effect of MSCs on tendon healing, enhanced tissue remodeling and improved structural organization.

Ureteral Stenosis in HDAF Pig‐to‐Primate Renal Xenotransplantation: A Phenomenon Related to Immunological Events?

The aim of this study was to analyze the incidence of ureteral stenosis in a life‐supporting human decay‐accelerating factor (hDAF) transgenic pig‐to‐cynomolgus monkey kidney transplantation model and determine the role of possible immunological events in its pathogenesis.

Influence of temperature, time and different media on mesenchymal stromal cells shipped for clinical application.

Cell-based therapies, such as the use of mesenchymal stromal cells (MSCs), are becoming popular in veterinary medicine. When MSCs are not cryopreserved, they are shipped in suspension, but no previous studies have analyzed MSC viability during delivery. Here, the impact of several experimental shipping conditions on the number of equine blood-derived (ePB-MSC) and canine adipose-derived (cA-MSC) MSCs were evaluated. Among the different parameters tested, only time and temperature influenced MSC number during the experimental shipping conditions. Cells were monitored over different time intervals for gene expression of typical MSC markers and to evaluate acquired resistance to apoptosis and beta-galactosidase activity. Overall, these results indicate that ePB-MSC and cA-MSC should be delivered in phosphate buffered saline at room temperature and within 9-12 h.

Methotrexate for immunosuppression in life-supporting pig-to-cynomolgus monkey renal xenotransplantation.

Abstract: Methotrexate (MTX) has been used successfully as an immunosuppressant in rodent xenotransplantation models, but the data generated so far with MTX in pig‐to‐baboon cardiac transplantation studies have been disappointing. The potential of this agent was consequently explored in a life‐supporting pig‐to‐primate renal model using the cynomolgus monkey as the recipient species. Introductory in vitro and in vivo pharmacokinetic and pharmacodynamic studies with MTX were conducted in three cynomolgus monkeys. Subsequently,10 cynomolgus monkey recipients of a life‐supporting kidney from human decay‐accelerating factor transgenic pigs were administered MTX intravenously according to three different regimens. All the animals also received cyclosporine A and steroids. In addition, mycophenolate sodium (MPS) was administered post‐operatively in two of the three groups of transplanted animals. At clinically relevant concentrations, MTX is able in vitro to inhibit the mixed lymphocyte reactions (MLR) in cynomolgus monkeys. After intravenous administration, moreover, exposure of cynomolgus monkeys to MTX appeared to be higher than had been previously reported in baboons. Graft function was observed in the transplanted animals, which survived from 0 to 41 days. All but two animals revealed acute humoral rejection in the explanted graft and developed diarrhea. Diarrhea was the cause of euthanasia in five cases. It was unrelated to the administration of MPS and associated with severe histopathological signs of enteritis. This study demonstrates that the pharmacokinetic and pharmacodynamic profiles of MTX vary substantially between non‐human primate species. In vitro, MTX has immunosuppressive properties in the cynomolgus monkey at clinically relevant concentrations. In vivo, MTX has a very narrow therapeutic window in cynomolgus monkeys, however, as it does in baboons. We conclude that MTX is scarcely effective as an immunosuppressant, be it for induction or maintenance, in pig‐to‐cynomolgus monkey renal xenotransplantation.

Description of a double centrifugation tube method for concentrating canine platelets

BackgroundTo evaluate the efficiency of platelet-rich plasma preparations by means of a double centrifugation tube method to obtain platelet-rich canine plasma at a concentration at least 4 times higher than the baseline value and a concentration of white blood cells not exceeding twice the reference range. A complete blood count was carried out for each sample and each concentrate. Whole blood samples were collected from 12 clinically healthy dogs (consenting blood donors). Blood was processed by a double centrifugation tube method to obtain platelet concentrates, which were then analyzed by a flow cytometry haematology system for haemogram. Platelet concentration and white blood cell count were determined in all samples.ResultsPlatelet concentration at least 4 times higher than the baseline value and a white blood cell count not exceeding twice the reference range were obtained respectively in 10 cases out of 12 (83.3%) and 11 cases out of 12 (91.6%).ConclusionsThis double centrifugation tube method is a relatively simple and inexpensive method for obtaining platelet-rich canine plasma, potentially available for therapeutic use to improve the healing process.

Pharmacokinetics of gallium nitrate after oral administration in adult horses – pilot study

Gallium (Ga), a metal in group IIIA of the periodic table, has shown a remarkable activity against bone resorption and could therefore possibly prove useful in the treatment of certain diseases in sport horses, for example navicular disease. The aim of this study was to gain more information concerning the kinetics of Ga after oral administration of gallium nitrate (GaN) in adult horses. Six horses received a single dose of 10 mg/kg of GaN mixed with the food ration. Absorption was slow (T(max) = 10 ± 3 h, T(½abs) = 2 ± 0.8 h), and a C(max) of 26 ± 11 μg/L was achieved. Excretion followed a one-phase elimination model, with a long half-life (T(½el) = 52 ± 14 h). By means of a mathematical model, we estimated that the plasmatic levels should reach 93 μg/L (1.33 μm) at steady state, following the repeated daily administration of 10 mg/kg of GaN. A three times lower concentration has been demonstrated as effective in inhibiting the osteolytic activity of osteoclasts in vitro. The results of this study suggest that the administration of oral GaN at a rate of 10 mg/kg per day may be considered for future clinical studies.

Morphological description of limbal epithelium: searching for stem cells crypts in the dog, cat, pig, cow, sheep and horse

The cornea provides protection and transparency to the eye, allowing an optimal sharpness view. In some pathological conditions the cornea is able to regenerate thanks to the presence of a stem cells reservoir present at the level of the transition area between cornea and sclera (limbus). Corneal cell therapies in Veterinary Medicine are really limited due to the lacking of knowledge about the anatomy of the limbal area, the putative presence of stem cells and their identification in domestic species. The aim of this study was to provide an overview of the main distinctive structural features of the sclero-corneal junction and conjunctival-corneal junction areas in some species of veterinary importance, using optic microscope observations of histological sections. The resulting data were compared with cornea from humans adapting protocols already used to identify stem cells by means of a specific cellular marker. We tested the expression of ΔNp63α isoform in the cornea basal cells, trying to correlate the distribution profile with areas of highly proliferative turnover. The results obtained from this study represent a first step towards the identification of a corneal stem cells reservoir in different animals.

Cytology of the healthy canine and feline ocular surface: comparison between cytobrush and impression technique.

BACKGROUND Impression cytology (IC) is a noninvasive technique in which filters are used to sample superficial layers of ocular epithelium. OBJECTIVES The aim of this study was to compare cytology specimens obtained by IC and cytobrush from healthy canine and feline eyes. METHODS Dogs and cats were prospectively sampled using polytetrafluorethylene filters on the right eye, and cytobrush on the left eye. Wright-Giemsa-stained specimens were evaluated by 2 observers. Cellularity, preservation, and morphology of cells and presence of goblet and inflammatory cells were scored with a 4-grade scale. Inter-observer agreement and effects of topical anesthesia were analyzed. RESULTS In 20 canine IC samples, 10 showed good cellularity (score 2-3) and 13 good preservation. Superficial epithelial cells (SEC) were present in 13/20 of IC, while basal-intermediate cells (BIC) were seen in 14/20. In 6/20 and 7/20, goblet and inflammatory cells were noted, respectively. In 20 cats, 15 of IC showed good cellularity and 14 good preservation, and SEC were present in 16/20 of IC and BIC in 17/20. In 13/20 and 3/20 cats, goblet cells and inflammatory cells were noted, respectively. Canine cytobrush specimens appeared well preserved (9/20) and had good cellularity (8/20). In feline cytobrush specimens, good preservation and cellularity were observed in 16/20 and 14/20, respectively. In both species, all cell types were present without a clear separation. There was moderate to fair agreement about cellular morphology in IC between observers. Specimens obtained with and without anesthesia were comparable. CONCLUSION Impression cytology allowed collection of samples with maintained cytoarchitecture, while cytoplasmatic and nuclear details were often difficult to evaluate.

Pocket technique or pocket technique combined with modified orbital rim anchorage for the replacement of a prolapsed gland of the third eyelid in dogs: 353 dogs

The aim of this retrospective study was to evaluate the results obtained in 353 dogs (420 eyes) using two different surgical techniques for correction of a prolapsed gland of the third eyelid: the Morgans pocket technique and a technique combining Morgans approach with a slightly modified periosteal anchoring technique of Stanley and Kaswan. The pocket technique was used in 234 eyes and the combined technique in 186 eyes. Successful repositioning was obtained in 95% of all cases, with recurrence occurring in 5%. The recurrence rate in large breed dogs such as the English Bulldog and Boxer was lower with the combined technique than with the pocket technique.