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Introduction. Intraductal papillary mucinous neoplasm (IPMN) is the most common cystic precancerous lesion of pancreatic cancer (PDA). IPMNs can progress from low to high-grade dysplasia, to invasive PDAC, but exact data regarding cancer risks are limited. PDAC is the fourth leading cause of cancer death . The overall 5-year survival is 6%, but survival of patients with early stage PDAC is significantly better. AIM We used an approach coupling high resolution cytogenetic analysis (Affymetrix Oncoscan FFPE Array) with a clinically-oriented bioinformatic interpretation of data to understand what are the most relevant pathways altered in precursor lesions. Materials and methods High resolution cytogenetic analysis was performed in 20 formalin fixed paraffin embedded samples of IPMNs by Oncoscan FFPE assay. Genomic data were analyzed for copy number gains and losses, loss of heterozygosity and for a panel of recurrent somatic mutations. Results were validated by qPCR and FISH analysis. Results Twenty samples, including 14 mixed-type IPMNs (70%), 4 branch-duct IPMNs (20%) and 2 main-duct IPMNs (10%), were collected. We identified micro-invasive carcinoma (< 1 mm) and invasive carcinoma in 25% and in 10% of IPMNs respectively. By our high resolution cytogenetic analysis we observed that 8 IPMNs (40%) presented a nearly normal karyotype (< 4 copy number alterations), while 12 IPMNs (60%) showed a complex karyotype (> 10 alterations). In the latter subgroup were detected specific copy number gains of TERC and c-MYC oncogenes. Interestingly we noticed that TERC and c-MYC overexpression, present in 92% and 45% of complex karyotype samples respectively, were only observed in high-grade IPMNs, suggesting a possible role in a progression to malignancy. Oncoscan data were confirmed by Real Time and FISH analysis. Conclusions Those results suggest a role of TERC and c-MYC overexpression as a possible biomarkers in the early identification of patients with complex karyotype IPMNs in order to better stratification of cystic lesions that could require surgery

The estimation of the post-mortem interval (PMI) is a critical step in forensic medicine and remains one of the most challenging variables to determine. In general, the numerous methods currently used in estimating post-mortem interval yield to large post-mortem windows, are influenced by several factors and sometime contradict each other. With the aim to obtain a much more accurate determination of the post-mortem interval we combined morphological ultrastructural and immunoistochemical analyses to reach a more detailed knowledge on tissue organization and degradation after death. Samples of gingival tissues obtained from 20 cadavers at different post-mortem intervals were processed for transmission electron microscopy to evaluate ultrastructural modifications in the epithelium and connective tissue. Gingival cells and the extracellular matrix of gingival tissues have been observed by a transmission electron microscopy (TEM) in combination with the evaluation of potential post-mortem biochemical markers, with the final goal to find a tight correlation between the ultrastructural modifications, the biomarker expression and the time of death. All the samples were also immunostained with anti-hypoxia-induced factor 1-α (HIF1-α) antibody, a transcription factor expressed in response to hypoxia, in order to evaluate the expression of HIF1-α, and to establish a correlation between the protein presence and the time of death. Results showed nuclear chromatin changes and cytoplasmic vacuolization in both epithelial and connective tissues and a different pattern of expression of HIF1alpha protein that correlate to the time of death. In conclusion, our preliminary findings suggest that ultrastructural investigations in combination with immunohistochemistry techniques in gingival specimens may represent a new tool to accurately and systematically estimate post-mortem interval., Italian Journal of Anatomy and Embryology, Vol. 122, No. 1 (Supplement) 2017

Articular cartilage is an avascular tissue without innervations, characterized by low cell density and abundant extracellular matrix (ECM). These characteristics leave articular cartilage with very limited capacity of repair and regeneration. Multipotent stem/stromal cells (MSC) are considered promising for cartilage tissue engineering. Stem cells are resided in a special microenvironment known as the stem-cell niche, characterized by the presence of low oxygen concentration. Previous studies have reported that hypoxic conditions could enhance the chondrogenic differentiation of mesenchymal stem cells in the presence of an inductive medium. Cobalt chloride (CoCl2) imitates hypoxia in vitro by preventing hypoxia-inducible factor-alpha (HIF-a) from being destroyed by oxygen. However, the long-term hypoxic culture of stem cells is difficult and requires special attention to avoid cell death due to cobalt treatment. In this study we investigated if CoCl2 affected MSCs isolated from dental pulp, umbilical cord and adipose tissue in their potential to differentiate toward the chondrogenic phenotype. Cells were treated with concentrations of CoCl2 ranging from 50 to 400 uM. Cell proliferation, mRNA expression of stem-cell marker and chondrogenic associated genes were analyzed by RT-PCR and Real-time PCR. The results showed that the CoCl2 supplementation had no effect on the proliferation of all the three type of cells analyzed, while the up-regulation of chondrogenic markers such as aggrecan, sox9, and type II collagen, was dependent on the cellular source. This study shows that hypoxia induced by CoCl2 treatment can differently influence the behavior of MSCs of different sources in their chondrogenic potential. These findings should be taken into consideration in the treatment of cartilage repair and regeneration based on stem cell therapies., Italian Journal of Anatomy and Embryology, Vol. 122, No. 1 (Supplement) 2017

Cardiovascular diseases (CVDs) are the major cause of death in developed countries. Oxidative stress plays a major role in the pathophysiology of cardiac disorders. Several studies have highlighted the cardinal role played by the overproduction of reactive oxygen species in the pathogenesis of ischemic myocardial damage and consequent cardiac dysfunction. Sulforaphane (SF) is a molecule within the isothiocyanate (ITC) group of organosulfur compound commonly found in cruciferous vegetables. It is reported to be capable of stimulating cellular antioxidant defenses and inducing phase 2 detoxifying enzymes, which can protect cells against oxidative damage. Although SF is known for its anticancer benefits, its role in cardioprotection is emerging. The aim of this study was to investigate the effects of SF in preventing cell damage induced by oxidative stress. Primary rat cardiomyocytes were exposed to different concentrations of SF for 24 h and subsequently treated with H2O2 to induce oxidative stress. Cell viability, and the expression of oxidative stress markers were studied. A transmission electron microscopy (TEM) analysis was carried out to evaluate the effect of SF on cell morphology. Results showed an higher cell viability and a lower level of oxidative stress in cells pre-treated with SF before peroxide exposure in respect to H2O2 treated cardiomyocytes. TEM analysis showed a well preserved morphology in cells pre-treated with SF before H2O2. These findings demonstrate that sulforaphane prevents H2O2 - induced oxidative stress and cell death in rat cardiomyocytes, suggesting a potential protective role of SF in CVDs, Italian Journal of Anatomy and Embryology, Vol. 121, No. 1 (Supplement) 2016

Mesenchymal stem cells (MSCs) have the capability to differentiate into wide range of specialized cells of mesodermal origin such as osteocytes, chondrocytes, adipocytes, cardiomyocytes, muscle fibers. Due to these properties, MSCs are considered as a new emerging treatment option and therapeutic agent in regenerative medicine. Promising results have been obtained after application of MSCs for treating tendon and joint disease in the equine model, making it favorable for therapeutic application. While the horse is considered a highly suitable model for orthopedic diseases, knowledge is lacking regarding the level of analogy of equine MSCs and their human counterparts. Therefore, the aim of this study was to assess the properties of human and equine Wharton’s jelly derived MSCs in a direct comparison. Obtained MSCs, were characterized for their staminal markers, proliferation and adhesion potential, ultrastructural morphology and their ability in differentiate towards osteogenic, chondrogenic and adipogenic lineages. Results showed a similar pattern in the expression of staminal markers, while a light difference was observed in the proliferation and adhesion potential. Ultramorphological analysis showed nuclear and citoplasmatic features comparable in human and equine MSCs. Finally, both MSCs were able to differentiate towards osteogenic, chondrogenic and adipogenic lineages. In conclusion, although revealing some potentially relevant differences, the study demonstrates a high level of analogy between human and equine MSCs, providing a basis for translational research in the equine model., Italian Journal of Anatomy and Embryology, Vol. 121, No. 1 (Supplement) 2016

Hydrogels have attracted considerable attention in biomedical engineering applications due to their many favorable biomimetic properties. Hydrogels based on proteins or carbohydrates can also function as effective extracellular matrices to direct cellular behavior. Recently, polysaccharide based hydrogels have become particularly interesting as matrices for the repair and regeneration of a wide variety of tissues and organs. The incorporation of inorganic minerals as hydroxyapatite nanoparticles can modulate the performance of the hydrogel with potential applications for bone tissue engineering. The aim of this study was to verify the biological potential of a new carboxymethyl cellulose—hydroxyapatite hybrid hydrogel in bone tissue regeneration. Mesenchymal stem cells were seeded on hydrogel scaffold, in presence and absence of hydroxyapatite, for 7, 14 and 21 days. Cell viability assay and morphological analysis were carried out to evaluate biocompatibility and cell adhesion of the materials. Real Time PCR for genes involved in tissue regeneration was carried out to assay the influence of the scaffold in cell differentiation. Results showed a high cell viability and biocompatibility of the tested material, confirmed by morphological analysis. The evaluation of osteoblast markers demonstrated the osteogenic induction of the 3D material enriched with hydroxyapatite in the production of mineralized extracellular matrix compared to the carboxymethyl cellulose based material. In conclusion, our data show that carboxymethyl cellulose—hydroxyapatite hybrid hydrogel may have great potential in bone tissue engineering applications., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

Cartilage is a highly organized tissue with complex biomechanical properties, but since it has a poor intrinsic capacity of self-healing, injuries at this site usually lead to several problems, often ending in disabling symptoms. Although, different approach- es have been proposed, even now cartilage repair represents a great challenge for orthopaedic surgeons (1, 2). One of the promising approach is given from tissue engineering, employing the combination of biomaterials and cell therapy to develop new therapeutic strategies. In this paper, we describe the behaviour of human adipose derived mesenchymal stem cells encapsulated into Ca/Co alginate beads as potential chondrogenic inducing biomaterial tacking advance on the synergy between alginate matrix and Co+2 ions without employing other expensive growth factors such as TGFbs or BMPs. Cells were cultured up to 3 weeks into alginate beads at different Ca/Co ratio, Calcein/Ethidium assay was performed to evaluate cell viability, light, and transmis- sion electron microscopy were carried out to check the cells behaviour. The expression of chondrogenic markers such as sox9, collagen type II, and versican was investigated by Real Time PCR. The expression of hif1mRNA was investigated to check the capability of Co+2 ions to induce a chemical hypoxia. Results showed an high cell viability at high Ca/Co ratio value of alginate beads. Real Time PCR data reveal a different cells behaviour on chondrogenic marker expression. In conclusion, the synergic effect of alginate and Co+2 ions can represent a valid strategy for chondrogenic differentiation of stem cells., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

For the enhanced understanding of fundamental cancer biology and improving molecular diagnostics and therapeutics, the role of the microenvironment during the initiation and progression of carcinogenesis is thought to be of critical importance (1, 2). The aim of this study was to establish an in vitro model based on a co-culture of healthy human fibroblasts (HFs) and human osteosarcoma cells (MG-63s) to simulate the microenvironment including tumor and healthy cells. The HFs and MG-63s were in vitro co-cultured for a period of time ranging from 24h to 7 days. Cell morphology and organization were studied by phase contrast microscopy while the expression of Human Cartilage Glycoprotein 39 (YKL-40), Vascular Endothelial Growth Factor (VEGF), Matrix Metalloprotease 1 (MMP1) and IL1 alpha was investigated by Real Time PCR and Western Blotting. To better correlate the role of YKL-40, VEGF, MMP1 and IL1 alpha, siRNA knockdown of YKL-40 in MG-63 cells was performed, and changes in protein expression in the co-cultures were analyzed. Results showed a characteristic disposition of tumor and healthy co-cultured cells in columns which are not visible in tumor and healthy cells grown singularly. The expression of YKL-40, VEGF and MMP1 significantly changed in co-cultured cells compared to HFs and MG-63s separately cultured. siRNA knockdown of YKL-40 was responsible of a down-regulation of VEGF expression on HFs, and an increase of IL1 alpha expression in MG-63s. These results suggest that the tumor microenvironment has an influence on the protein expression of the healthy surrounding tissues and the process of tumorigenicity., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

Recently, tissue engineering has merged with stem cell technology to develop new sources of transplantable material for injury or disease treatment. Eminently interesting, are bone and joint injuries/disorders because of the low self-regenerating capacity of the matrix secreting cells, particularly chondrocytes (1). Gelatin based scaffolds are considered to be a highly suitable 3D material for tissue regeneration, due to high biocompatibility and adaptation to native tissues. In the present study, the chondrogenic and osteogenic potential of a porous gelatin based scaffold (2), alone or in combination with hydroxyapatite crystals, was investigated in human mesenchymal stem cells. Cells were culture up to 4 weeks on the scaffold and on monolayer. MTT assay was performed to evaluate cell viability, light and transmission electron microscopy were carried out to demonstrate cell colonization inside the porous architecture of the biomaterial and scaffold adhesion. The expression of chondrogenic markers such as SOX9, collagen type II, aggregan and versican and osteogenic markers such as Collagen type I, Runx -2, osteopontin and bone matrix protein, were investigated by Real Time PCR. Results showed an high cell viability, adhesion and colonization of the scaffold. Real Time PCR data demonstrated an up-regulation of all the chondrogenic and osteogenic markers analyzed. In conclusion, gelatin porous scaffold provides an improved environment for chondrogenic and osteogenic differentiation of stem cells compared to cell monolayer culture system., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014