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IntroductionGrowth Differentiation Factor 15 (GDF15) is a mitochondrial-stress-responsive molecule whose expression strongly increases with aging and age-related diseases. However, its role in neurodegenerative diseases, including Alzheimer’s disease (AD), is still debated.MethodsWe have characterized the expression of GDF15 in brain samples from AD patients and non-demented subjects (controls) of different ages.ResultsAlthough no difference in CSF levels of GDF15 was found between AD patients and controls, GDF15 was expressed in different brain areas and seems to be predominantly localized in neurons. The ratio between its mature and precursor form was higher in the frontal cortex of AD patients compared to age-matched controls (p p GDF15 and IL-6 mRNA levels was observed. Finally, when GDF15 was silenced in vitro in dermal fibroblasts, a decrease in OXPHOS complexes transcript levels and an increase in IL-6 levels were observed.DiscussionAlthough GDF15 seems not to be a reliable CSF marker for AD, it is highly expressed in aging and AD brains, likely as a part of stress response aimed at counteracting mitochondrial dysfunction and neuroinflammation.

The most recent discoveries in the biochemical field are highlighting the increasingly important role of lipid droplets (LDs) in several regulatory mechanisms in living cells. LDs are dynamic organelles and therefore their complete characteriza- tion in terms of number, size, spatial positioning and relative distribution in the cell volume can shed light on the roles played by LDs. Until now, fluorescence microscopy and transmission electron microscopy are assessed as the gold standard methods for identifying LDs due to their high sensitivity and specificity. However, such methods generally only provide 2D assays and partial measurements. Furthermore, both can be destructive and with low productivity, thus limit- ing analysis of large cell numbers in a sample. Here we demonstrate for the first time the capability of 3D visualization and the full LD characterization in high-throughput with a tomographic phase-contrast flow-cytometer, by using ovarian cancer cells and monocyte cell lines as models. A strategy for retrieving significant parameters on spatial correlations and LD 3D positioning inside each cell volume is reported. The information gathered by this new method could allow more in depth understanding and lead to new discoveries on how LDs are correlated to cellular functions.

Neisseria gonorrhoeae (GC) is the agent of one of the most common bacterial sexually transmitted infections worldwide. The possible development of 'untreatable' infections points out the need for antibiotic-sparing methods to reduce the number of gonococcal infections. In this context, fatty acids are interesting candidates as next-generation antibacterial agents. The aim of this study was to investigate the bactericidal effects of selected fatty acids on GC viability, as well as to observe their biological effects by means of transmission electron microscopy. The cytotoxicity of these compounds on human cervical cells (HeLa), chosen as a model of genital mucosa, was assessed as well. Lauric, myristic, and palmitic acid displayed high killing activity against GC in concentrations ranging between 100 μM and 25 μM, whereas the antimicrobial effect of oleic and butyric acids was present in concentrations between 1 mM and 0.25 mM. Modifications induced by fatty acids on the GC cell included the disorganization of the cytoplasmic structure, the distortion of pili/fimbriae, and the separation of the inner and outer membrane layers. For concentrations active against GC, fatty acids were not toxic for cervical cells. Our data can help in promoting innovative antibiotic-free compounds for the treatment of GC infections.

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Background and aim of the study: Nursing students are at high risk of injury while on clinical placement. The incidence of injury is three times higher in comparison to other healthcare workers, with a prevalence ranging from 25% up to 33,2%. Lack of knowledge and of experience in delivering nursing care, anxiety and unfamiliarity with the clinical environment all represent risk factors for injury. This study describes the number and type of injuries occurring in an organizational setting where nursing students are trained in simulation laboratories before being exposed to a real clinical environment. Methods: An eight year retrospective observational study (2009 to 2017) was conducted on a non-probabilistic population of 1,403 nursing students from a university in the north of Italy. Incident reports were anonymised and entered in a database. Results: Overall the percentage of students who reported an injury (6,2%) was much lower than the one reported in the literature. Conclusions: The nursing programme curriculum of the above mentioned university consists of frontal lectures, group works, simulation based sessions and supervised clinical practice designed to equip students with all the necessary knowledge and clinical skills to prevent health care injuries. Despite the encouraging results of this study, further actions in order to protect first year students, who are more subject to risk of injuries, are recommended.

Background and aim of the study. COVID-19 is characterized by super spread events occurring in communities, e.g., hospitals. To limit virus diffusion among healthcare workers the use of personal protective equipment and screening tests are highly advised; also, isolation of virus positive professionals while monitoring their health condition is recommended. This study aims to assess, in a cohort of COVID-19 positive quarantined healthcare workers, the perceived source of infection and exposure risk as well as the clinical evolution of the disease through a surveillance interview. Methods. A retrospective observational study accounting 896 observations on 93 healthcare professionals tested positive for COVID-19. Data were collected from the Nursing and Technical Directorate of Romagna, Ravenna, Local Health Company, Italy. Results. 99.5% of the positive workers accepted phone interviews with management staff. 2.6% of workers were positive with increasing records in the specialist medical area. Nurses and social health professionals were mostly affected. Patient exposure at a distance 2 hours was the first cause of positivity. In COVID-19 and territorial emergency departments, the first cause was the contact with colleagues. At the time of the infection, most of the staff wore a surgical mask. Cough, asthenia, fever, anosmia, dysgeusia, and rhinitis were common symptoms. Asymptomatic percentage was about 10%. The self-perceived physical condition was high (>7) and improved during the observation period. Conclusions. The diffusion rate of COVID-19 among healthcare workers is relatively low, probably due to the use of personal protective equipment. The distancing, also among colleagues, is a fundamental measure to reduce the possibility of infection. Symptoms are mild and can be controlled by surveillance measures. Constant contact with the organization is an essential strategy for promoting recovering of workers and reducing the spread of the virus within the healthcare organization. (www.actabiomedica.it)

Late calcification of vascular synthetic grafts is a recently underscored but not sufficiently investigated issue involving vascular substitutes. Most of the research in this field has been focused on material biocompatibility and development of strategies that allow an effective prosthetic incorporation into the recipient vascular tissues through neointima formation. In this short review we report the most generally accepted mechanisms leading to graft calcification with consequent detrimental effect on graft outcome. Besides to the role exerted by circulating or induced resident osteogenic progenitors, and by inflammatory cells, i.e. macrophages and multinuclear giant cells, endothelial cells are gaining attention as mediators of vascular diseases. Here we highlight the role of Endothelial-Mesenchymal Transition (End-MT), a process whereby endothelial cells acquire mesenchymal properties, in vascular calcification. End-MT has been observed in vascular graft, suggesting its involvement during remodelling response and calcification. We therefore tested this hypothesis by culturing an endothelial cell line (HUVEC) on poly-lactic-L-acid (PLLA) scaffolds under inflammatory milieu and investigating End-MT (SLUG, MMP-9, VIMENTIN) and osteogenic (BMP-2, ALP, RUNX-2) markers. Interestingly, HUVEC underwent an up-regulation of mesenchymal and osteogenic genes when in contact with PLLA, even in absence of inflammatory stimulation. As expected, the inflammatory cytokine combination (TNF-α + IL-1β) enhanced the End-MT and the osteogenic switch in HUVEC grown on PLLA. These preliminary data support a tight association between inflammation, calcification and polymer graft, also implicating a remodelling cell response to the biomaterial. Research in this direction needs to be improved, in the view of a future prevention strategy of vascular graft calcification and failure.

Background and aim of the study: Nursing has not yet received full social recognition and in general the public opinion does not have a completely positive perception of it, which regards it as an auxiliary profession to the medical profession. This study aims to investigate the image of the nurse among year 4 and 5 high school students. Methods: Two groups of students were interviewed, one at their institution without any previous orientation (n = 102) and one at the University of Bologna during orientation day (n = 388). A validated questionnaire (Nursing Attitude Questionnaire) of 30 items was delivered. Two additional questions were added to explore the possibility of advising relatives and acquaintances to become a nurse and therefore to choose the nursing degree course. Results: All areas of the NAQ scored significantly higher in the group where students attended the orientation day, especially the ones related to the professional role, stereotypes and professional value items. Female students ≥ 21 had a more positive image of the nurse. Both groups recognized that nurses are important figures for patients and that they are intelligent people, who must have a degree in order to carry out their work duties. Differences between the groups were recorded. Students in group 1 did not accept nursing autonomy while students in group 2 understood differences between nursing and medical careers, attributing to them the same value. A positive correlation between the positive image of the nursing profession and the interest in becoming nurses or advising others to undertake the nursing course was found. In conclusion, orientation is effective in helping high school students in the choice of their university courses. Regarding nursing, strategies to improve the image of the nurse and enrollment should be combined during orientation day with current up to date communication and learning tools, such as social media and simulation laboratories. Furthermore, it appears increasingly important to extend orientation and tutoring activities to junior high school students, families and teachers.

The human arterial wall contains progenitors and mesenchymal stem cells (MSCs) acting as a postnatal reservoir of stem cells during lifetime. They are nestled in distinct arterial zones close to blood support, that is, the intima and the media-adventitia vasa vasorum plexus, representing vascular stem cell niches. In previous studies, MSCs were successfully isolated from fresh and cadaveric human large- and middle-sized arteries; these cells have a mesenchymal phenotype, self-renewal ability, and tri-lineage plasticity with high endothelial and smooth muscle cell differentiation potential. Here we present an overview of human MSCs derived from the vascular wall (hVW-MSCs) of different anatomical sites focusing on their phenotypic expression, multilineage potency, and stemness properties based on the localization in the arterial tree. We describe the isolation protocols as well as immunophenotyping, functional, and ultrastructure methods used to investigate these cell properties. hVW-MSCs from distinct portions of the vascular tree exhibit distinct phenotypic expression, multilineage potency, and stemness properties. This observation may contribute to explain the regional differences seen in vascular disease; moreover the different attitudes that hVW-MSCs exhibit in vascular differentiation should be taken in consideration whenever cell therapy, regenerative medicine, and tissue engineering strategies are attempted to replace tissues and organs.

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L’obiettivo principale di quest’opera è quello di presentare in modo completo e approfondito il quadro attuale relativo alla ricerca sulle cellule staminali e le loro possibili applicazioni terapeutiche. Il testo in particolare è strutturato seguendo un percorso che parte dalle basi molecolari e le proprietà biologiche delle cellule staminali pluripotenti, embrionali o riprogrammate, e mette a confronto i diversi gradi della staminalità, descrivendo le popolazioni staminali “adulte” residenti nei vari tessuti ed organi del corpo umano. Partendo dalla ricerca di base vengono presentati esempi di medicina rigenerativa che traslano le scoperte sperimentali rendendole possibili nelle applicazioni cliniche di terapia cellulare. Le cellule staminali infine possono rappresentare un modello di studio per comprendere non solo i meccanismi fisiologici che controllano il loro destino ma anche quelli patologici coinvolti nella biologia aberrante delle cellule staminali tumorali. Ogni capitolo è stato affidato ad illustri ricercatori del campo, cosicché ogni parte risulta rigorosamente approfondita, aggiornata e arricchita dal contributo personale di ciascun autore .

Il capitolo prende in esame le caratteristiche genetiche, molecolari, morfologiche e immunofenotipiche delle cellule progenitrici endoteliali, linfatiche e delle cellule mesenchimali stromali circolanti e residenti nella parete vascolare.

Feulgen image analysis densitometry protocol with automatic measurement program wizard. (PDF 55 kb)

International audience; Data on the in situ structural-functional organization of ribosomal genes in the mammalian cell nucleolus are reviewed here. Major findings on chromatin structure in situ come from investigations carried out using the Feulgen-like osmium ammine reaction as a highly specific electron-opaque DNA tracer. Intranucleolar chromatin shows three different levels of organization: compact clumps, fibers ranging from 11 to 30 nm, and loose agglomerates of extended DNA filaments. Both clumps and fibers of chromatin exhibit a nucleosomal organization that is lacking in the loose agglomerates of extended DNA filaments. In fact, these filaments constantly show a thickness of 2-3 nm, the same as a DNA double-helix molecule. The loose agglomerates of DNA filaments are located in the fibrillar centers, the interphase counterpart of metaphase NORs, therefore being constituted by ribosomal DNA. The extended, non-nucleosomal configuration of this rDNA has been shown to be independent of transcriptional activity and characterizes ribosome genes that are either transcribed or transcriptionally silent. Data reviewed are consistent with a model of control for ribosome gene activity that is not mediated by changes in chromatin structure. The presence of rDNA in mammalian cells always structurally ready for transcription might facilitate a more rapid adjustment of the ribosome production in response to the metabolic needs of the cell.

Il recente ed esplosivo sviluppo delle conoscenze nel campo della terapia cellulare, dell’ingegneria dei tessuti e della medicina rigenerativa ci ha indotto a coagulare le diverse competenze acquisite in vari ambiti di ricerca per contribuire alla stesura di un testo sulle caratteristiche delle cellule staminali e il loro potenziale applicativo nella medicina rigenerativa. In questo volume, data la vastità dell’argomento, abbiamo focalizzato l’attenzione su alcune delle diverse tipologie e sorgenti di cellule staminali e le loro applicazioni. Il testo è rivolto in particolare agli studenti dei corsi magistrali di Biotecnologie e Scienze Biomediche; dovrà essere una guida per lo studente che seguirà un corso di “cellule staminali” svolto da ricercatori coinvolti sperimentalmente nella comprensione dei meccanismi che regolano il rinnovamenti dei tessuti. Ogni capitolo è corredato da letture consigliate solitamente review e lavori scientifici pubblicati su riviste internazionali per avere una visione più ampia e completa delle dinamiche e delle prospettive che si vanno delineando in questo ambito.

BACKGROUND: Human adipose-derived stem cells (hASCs) may represent an easy-to-harvest tool for cell therapy of peripheral artery disease. In most clinical trials, stem cells undergo prolonged ex vivo expansion, with significant senescence, and decline in multipotency, leading to clinical results below expectations. METHODS: We have developed a non-enzymatic method yielding a microfractured fat tissue (Lipogems), harboring intact stromal-vascular niche and pericyte/mesenchymal stem cells. Human Lipogems, Lipogems-derived hASCs preconditioned with or without a vasculogenic mixture, including hyaluronan, butyric and retinoic acids (H+B+R), were transplanted into the gracilis muscle of 16 rats subjected to chronic hind limb ischemia. After two weeks, tissue rescue was assessed by a perivascular flow probe and immunohistochemistry. Coculture of HUVECs with Lipogems or Lipogems-derived hASCs was performed. Vasculogenic gene transcription and secreted cytokines were assessed. RESULTS: Xenogeneic transplantation of human Lipogems elicited significantly higher muscle tissue repair and lower inflammatory infiltration than expanded Lipogems-derived hASCs, even preconditioned with H+B+R. Lipogems action involved a significantly higher arteriogenic response than that elicited by untreated or preconditioned hASCs. Although transplanted Lipogems bordered within the external side of collating primary fibers, it led to remarkably better preservation of muscular fiber size than untreated or preconditioned hASCs. Lipogems-derived hASCs responded to vasculogenic agents with increased VEGF, KDR, and HGF transcription, associated with early and long-lasting secretion of the encoded cytokines. In coculture with HUVECs, Lipogems enhanced endothelial cell proliferation remarkably more than untreated or preconditioned hASCs. CONCLUSIONS: Lipogems emerges as a transplantable immunomodulatory fat tissue product with remarkable arteriogenic and paracrine properties for the rescue of ischemic limb.

Il volume raccoglie il contenuto delle lezioni di Medicina di Laboratorio svolte dagli autori nei Corsi di Laurea Triennali della Scuola di Medicina e Chirurgia

Il capitolo prende in esame le caratteristiche genetiche, molecolari, morfologiche e immunofenotipiche delle cellule progenitrici endoteliali e delle cellule mesenchimali stromali circolanti e residenti nella parete vascolare.

Introduction: The application of stem cells in various medical fields is a well-known challenge for many researchers; hence the need to find more efficient cell sources for experimental manipulation as well as regenerative medicine and transplantation procedures. Tissues harvested from donors could represent an innovative and unlimited reservoir of different type of stem cells. Since tissues are routinely derived from cadaveric donors they can be used for the isolation of mesenchymal stromal/stem cell population (MSCs). In this study, we described the successful isolation, in vitro expansion and characterization of cadaver mesenchymal stromal/stem cells (hC-MSCs) derived by long-time frozen human post mortem arterial segments. Materials and Methods: hC-MSCs were obtained from explanted variously-sized arteries derived from post mortem donors after 4 days post mortem and following liquid nitrogen storage in tissue-banking facilities for more than 5 years. Immunophenotype, self-renewal and differentiation potential were determined using flow cytometry, in vitro assays, histological and immunological staining, molecular and ultrastructural approaches. Results: We successful isolated a cells population that survived to a prolonged ischemic insult and long-time cryopreservation. hC-MSCs appear with spindle-shape morphology, plastic adherent growth forming multiple confluence colonies and high proliferation rate. Phenotypically, they displayed numerous mesenchymal surface antigens (CD44, CD73, CD90, CD105, HLA), stemness (Stro-1, Oct-4, Notch-1), pericyte (CD146, PDGFR-β, NG2) and neuronal (Nestin) markers and lacked the expression of hematopoietic and vascular markers. In addition, these cells exhibited colony-forming ability using extreme cell seeding diluition; to form free floating embryoid-like bodies when grown in suspension; high immunosuppressive properties and multipotency for their ability to differentiate in multiple mesegenic lineages, i.e., adipogenic, osteo/chondrogenic, leiomyogenic and angiogenic. Discussion: Based on these results in addition to the easy accessibility, non-controversial, safety and abundant stem cell number, the procurement of plentiful hC-MSCs from cadaveric vascular tissues could be a scientific revolution in the field of cell therapy for many patients with severe diseases.

Intro Mönckeberg sclerosis is an uncommon form of arterial calcification occurring in the media of large to middle-sized vessels that has been erroneously associated with atherosclerosis. It has been described in generalized metabolic disorders, including hypervitaminosis D, end/stage renal disease and diabetes; however, it has been reported exceptionally even in normal young patients with no overt metabolic disease. Aim: To establish the occurrence of Mönckeberg sclerosis in a consecutive series of 111 human femoral arteries from multiorgan donors and determine whether there is a correlation between media calcification and the most common risk factors for cardiovascular disease. M&M One-hundred-eleven femoral arteries from multiorgan donors (age: 14 to 59 yrs, mean 36,5 yrs; 70 male) were histologically examined to determine the quality of the graft before cryopreservation. Selected samples were processed for electron microscopy. Immunohistochemistry was performed using a polyclonal antibody (Osx, 1:100, Abcam, Cambridge, UK) which recognizes Osterix, a transcription factor of osteoblast differentiation. The antigen-antibody reaction was revealed using a polymer system (Novolink, New Castle, UK). The percentage of Osx-positive cells was calculated on the total number of cells on 10 HPF each sample. Statistical analyses were performed using GraphPad Prism. Results Twenty-eight arteries had calcifications as the only pathological feature. Two patterns: one involving the inner elastic lamina (95%), the other the media (5%). More frequent in women (OR=1,70); young people (20-29 years; OR=1,35) affected as well; no correlation with diabetes, tabagism, hypercholesterolemia; strong correlation with hypertension (OR=1,68). Ultrastructurally calcifying matrix vesicles close to inner elastic lamina. Osx expressed in smooth muscle and mural cells of adventitia. In calcified arteries, the percentage of Osx+ cells was higher when hypertension was present (P

RUNX-1 and CD44 as markers of resident stem cell derivation in undifferentiated intimal sarcoma of pulmonary artery Aims: To report a rare case of undifferentiated intimal sarcoma (UIS) of the pulmonary artery in a 44-year-old woman, and to study it by electron microscopy and a novel immunohistochemical (IHC) panel that recognizes markers of endothelial and haematopoietic stemness, in order to extend current knowledge about the histogenesis of this rare neoplasm. Methods and results: Immunohistochemical reactions for CD31, CD34, a-smooth muscle actin (a-SMA), caldesmon, calponin, actin, desmin, epithelial membrane antigen, WT1, CD117, Ki67, nestin, RUNX-1, platelet-derived growth factor, NG2, CD44, CD90 and CD105 were performed manually or automatically. Neoplastic cells were negative for CD31 and CD34, but positive for calponin, nestin, WT1, CD44, and RUNX-1. Electron microscopy was performed after osmium tetroxide fixation and staining with lead citrate and uranyl acetate. Ultrastructurally, tumour cells had slightly irregular nuclei, cisternae of rough endoplasmic reticulum, and punctate intercellular junctions. Conclusions: We report a case of pulmonary artery UIS expressing previously unreported markers, i.e. RUNX-1, nestin, WT1, and CD44, that are commonly seen in different stages of the vascular differentiation hierarchy. These findings, together with the negativity for mature endothelial and smooth muscle markers, raise the question of whether this neoplasm may derive from a vessel wall-resident stem cell, such as the haemangioblast or an embryonic-like stem cell.

In the last few years, we and other few groups have built a solid scientific background aimed at demonstrating the existence of resident stem cells in adult large and medium-sized human arteries. These cells are engaged in the regulation of normal arterial wall homeostasis but under stressful circumstances they are also supposed to contribute to arterial wall disease. In our experience, electron microscopy (EM) has been of great help in solving several critical issues that have arisen in the execution of research; further EM was invaluable in making critical decisions for choosing the best method for subsequent study of proteins and transcripts. In this study, we report some of the situations where we feel that EM plays a major role in vascular stem cell research. Characterization of the primary cell isolate. There are numerous methods used for the isolation of cells from the arterial wall. The more selective make use of magnetic beads coated with specific antibodies directed against surface molecules selectively expressed by the cell population of interest. In the case of CD34 positive populations EM has allowed us to verify how the cells isolated by this method are homogenous. For other investigation purposes, methods of isolation making use of enzymes are more appropriate; in this case, it is often necessary grow cells in suspension or in adhesion to plastic. Before establishing of culture and during the early passages (1-3), EM shows that cell isolates are composed of several cytotypes which are then lost in the course of the next passages. The selection of, for example, mesenchymal stem cells (MSC) that is achieved by cell adhesion to plastic, is never absolute and this raises doubts about the true differentiation potential of these cells. Characterization of resident vascular wall-mesenchymal cells (vw-MSC). The MSC are considered necessary to keep the architecture of the stem cell niche and to maintain in good health more embryonic-like stem cells and progenitors. MSC are currently identified by using a panel of monoclonal antibodies against surface molecules, i.e., CD44, CD90, CD105, CD166. EM allows to reveal the subcellular morphology of the MSC that is mesenchymal in nature and to assume, for comparison with the normal cell architecture of the arterial wall, the putative anatomical location of the niche stem. Our studies have thus allowed us to focus attention on the adventitia and in particular on the vasa vasorum compartment. Identification of the stem cell niche. The vasa vasorum constitute a branched vascular system that has the function of oxygenate and nourish the arterial wall. The vasa vasorum can be distinguished on the basis of their size in first order vasa vasorum, with a diameter greater than 100 micrometers, and second order vasa vasorum, with a diameter of less than 100 micrometers. These classes of vasa vasorum have a similar cell composition with endothelial cells and pericytes. By means of immunohistochemistry and monoclonal antibodies against nestin, an intermediate filament originally seen in brain stem cells, and its promoter WT1, we have observed that some vasa vasorum cells intensely co-express these two molecules; these vasa vasorum are collected in hot spots placed within a particular organoid structure that aggregates nerva vasorum, arterioles, venules and less than 50 micrometers-sized vasa vasorum. Using EM, we observed prominent perivascular collections of spindle cells with extremely thin and long cytoplasmic projections which establish functional contacts between structures belonging to different morphological and functional compartments. These cells have the potential to correspond to the MSC that we isolate from the arterial wall. This organoid adventitia area may represent the putative vascular stem cell niche. Establishing the differentiating potential of MSC. To exploit the differentiating potential and plasticity of MSC classical histochemical methods, i.e. oil red O, von Kossa and Alcian-PAS staining, are frequently employed. In our opinion EM is the gold standard to validate adipogenic and osteo-chondrogenic differentiation protocols. Features of early subcellular mesengenic differentiation can be derived from the past knowledge that pathologists have accumulated studying poorly differentiated sarcomas.

The pathogenetic mechanisms underlying gastrointestinal dysmotility in diabetic patients remain poorly understood, although enteric neuropathy, damage to interstitial cells of Cajal (ICC) and smooth muscle cell injury are believed to play a role.The aim of this study was to investigate the morphological and functional changes underlying intestinal dysmotility in RIP-I ⁄ hIFNb transgenic mice treated with multiple very low doses of streptozotocin (20 mg⁄ kg, i.p., 5 days). Compared with vehicle-treated mice, streptozotocin-treated animals developed type 1 diabetes mellitus, with sustained hyperglycaemia for 3.5 months, polyphagia, polydipsia and increased faecal output without changes in faecal water content (metabolic cages). Diabetic mice had a longer intestine, longer ileal villi and wider colonic crypts (conventional microscopy) and displayed faster gastric emptying and intestinal transit. Contractility studies showed selective impaired neurotransmission in the ileum and mid-colon of diabetic mice. Compared with controls, the ileal and colonic myenteric plexus of diabetic mice revealed ultrastructural features of neuronal degeneration and HuD immunohistochemistry on whole-mount preparations showed 15% reduction in neuronal numbers. However, no immunohistochemical changes in apoptosis-related markers were noted. Lower absolute numbers of neuronal nitric oxide synthase- and choline acetyltransferase-immunopositive neurons and enhanced vasoactive intestinal polypeptide and substance P immunopositivity were observed. Ultrastructural and immunohistochemical analyses did not reveal changes in the enteric glial or ICC networks. In conclusion, this model of diabetic enteropathy shows enhanced intestinal transit associated with intestinal remodelling, including neuroplastic changes, and overt myenteric neuropathy. Such abnormalities are likely to reflect neuroadaptive and neuropathological changes occurring in this diabetic model.

AIMS: Pre-treating placenta-derived human mesenchymal stem cells (FMhMSCs) with a hyaluronan mixed ester of butyric and retinoic acid (HBR) potentiates their reparative capacity in rodent hearts. Our aim was to test FMhMSCs in a large-animal model by employing a novel combination of in vivo and ex vivo analyses. METHODS AND RESULTS: Matched regional quantifications of myocardial function and viability were performed by magnetic resonance imaging (MRI) and positron emission tomography (PET) 4 weeks after myocardial infarction combined with intramyocardial injection of FMhMSCs (n = 7), or HBR-pre-treated FMhMSCs (HBR-FMhMSCs, n = 6), or saline solution (PBS, n = 7). Sham-operated pigs (n = 4) were used as control animals. Despite no differences in the ejection fraction and haemodynamics, regional MRI revealed, in pigs treated with HBR-FMhMSCs compared with the other infarcted groups, a 40% smaller infarct scar size and a significant improvement of the end-systolic wall thickening and circumferential shortening of the infarct border zone. Consistently, PET showed that myocardial perfusion and glucose uptake were, respectively, 35 and 23% higher in the border zone of pigs treated with HBR-FMhMSCs compared with the other infarcted groups. Histology supported in vivo imaging; the delivery of HBR-FMhMSCs significantly enhanced capillary density and decreased fibrous tissue by approximately 68%. Moreover, proteomic analysis of the border zone in the HBR-FMhMSCs group and the FMhMSCs group indicated, respectively, 45 and 30% phenotypic homology with healthy tissue, while this homology was only 26% in the border zone of the PBS group. CONCLUSION: Our results support a more pronounced reparative potential of HBR-pre-treated FMhMSCs in a clinically relevant animal model of infarction and highlight the necessity of using combined diagnostic imaging to avoid underestimations of stem cell therapeutic effects in the heart.

Electrospun (ES) materials are considered highly promising scaffolds for tissue engineering given their particular fibrous morphology that resembles the fibrous component of tissue extracellular matrix. ES scaffolds made of commercial bioresorbable polyesters can be dramatically affected by commonly employed sterilization methods (e.g. autoclaving, ethylene oxide, -rays), thus they are usually disinfected with ethanol (EtOH) before being used in contact with a biological environment. However, even if less extensively, also EtOH disinfection might affect ES scaffold morphology and, consequently, the response of cells cultured onto them. This work aims at investigating the effect of EtOH sterilization on poly(L)lactic acid (PLLA) ES scaffold and in particular on its phase morphology (amorphous to crystalline ratio). The morphological changes of PLLA non-woven mats treated at different temperatures by using EtOH solutions with different concentration, were compared. Moreover, the behaviour of H9c2 cardiac cells seeded onto PLLA scaffolds treated with EtOH was investigated.

La parete vascolare è sede di una completa gerarchia di cellule staminali residenti (CSr) capaci di differenziare in molteplici citotipi mesodermici e non; le CSr sono localizzate in una zona al confine tra tonaca media e tonaca avventizia detta “zona vasculogenica”. In questo studio è stato utilizzato un sistema di coltura 3D di arteria femorale umana per stabilire il ruolo di progenitori vascolari residenti in parete nel rimodellamento spontaneo non influenzato dai normali rapporti con la componente ematica circolante e senza l’impiego di fattori di crescita. Il segmento di arteria femorale umana di donatore multiorgano, fornitoci dalla Banca Regionale dei Tessuti Cardiovascolari del Policlinico S.Orsola - Malpighi di Bologna, dopo decontaminazione, è stato processato per la microscopia ottica (LM) ed elettronica (TEM); la rimanente parte, suddiviso in più porzioni, è stato mantenuto in coltura, in terreno basale di crescita, fino a 70 giorni. A intervalli di 14 giorni, i campioni sono stati processati per le analisi istologiche, immunoistochimiche e ultrastrutturali. L’analisi istologica mostra una rapida perdita dell’endotelio e un decremento significativo della cellularità di parete fino al 35° giorno di coltura. Dal 42° giorno in poi, si osserva la presenza di due monostrati cellulari istologicamente identici ma fenotipicamente distinti a rivestire gli strati più esterni della parete arteriosa; le cellule intimali esprimono molecole endoteliali (CD34, CD31); diversamente, il versante avventiziale assume un fenotipo mesenchimale (vimentin, CD105, CD90, CD44). Queste differenze fenotipiche sono state confermate anche mediante analisi ultrastrutturale. In conclusione questo studio ha evidenziato come la coltura d’organo sia una buona alternativa per studiare l’omeostasi tissutale vascolare; essa ha permesso di seguire i fenomeni di rimodellamento spontaneo suggerendo l’importanza della matrice extracellulare nel guidare in maniera appropriata i processi differenziativi delle CSr in assenza di specifici stimoli chimici.

Nowadays, efforts are dedicated to develop new biodegradable polymers for soft tissues engineering, where scaffolds displaying high elasticity are required in order to resist to long-term cyclic strains. These mechanical performances can be achieved by few synthetic biodegradabile polymers (e.g. Degrapol®, poly(glycerol sebacate) and poly(lactide-co-trimethylene carbonate)) whereas the most extensively used polyester (e.g. polycaprolactone, polylactide and poly(lactide-co-glycolide)) are too stiff to be suitable for engineering soft tissues. Therefore, nowadays, efforts are dedicated to develop new biodegradable elastomers. Reactive blending is a synthetic method that potentially enables to synthesize an infinite number of both block and random copolyesters by exploiting the transesterification reactions occurring at high temperatures. In this work new copolyesters of poly(butylene succinate), synthesized by reactive blending, are used to fabricate electrospun (ES) scaffolds displaying low elastic modulus. Biocompatibility of the new ES scaffolds is evaluated through embrionic rat cardiac cell culture.

In the chapter are reported and discussed the preventive techniques in order to avoid restenosis after endovascular treatment of the superficial femoral artery