Liver transplantation is the only definitive treatment for transthyretin amyloidosis, with an excellent 5-year survival in endemic countries where the Met30 mutation is predominant. We report our experience of liver transplantation for transthyretin amyloidosis. We reviewed the clinical records of 17 transplanted patients (11 males, 6 females; age at liver transplant: 45.7±11.7 years). We had a wide spectrum of non-Met30 mutations (52.9%), with a predominance of Gln89 (23.5%). Five-year survival after transplantation was 43.8%; at multivariate analysis, both non-Met30 mutations (HR 17.3, 95% CI 1.03-291.7) and modified BMI (HR 0.50, 95% CI 0.29-0.87) showed significant and independent prognostic roles (P=0.048 and P=0.015, respectively). Five out of the 9 non-Met30 carriers received combined heart transplantation because of severe cardiomyopathy; they showed a trend towards a better prognosis vs. the 4 patients who did not receive combined heart transplantation (although not statistically significant; P=0.095). At follow-up, no significant improvement of transthyretin amyloidosis manifestations was observed. The results of liver transplantation for transthyretin amyloidosis in our population are poorer than those reported in the literature probably because of the high prevalence of non-Met30 mutations.

The dramatic impact that vascular diseases have on human life quality and expectancy nowadays is the reason why both medical and scientific communities put great effort in discovering new and effective ways to fight vascular pathologies. Among the many different treatments, endovascular surgery is a minimally-invasive technique that makes use of X-ray fluoroscopy to obtain real-time images of the patient during interventions. In this context radiopaque biomaterials, i.e. materials able to absorb X-ray radiation, play a fundamental role as they are employed both to enhance visibility of devices during interventions and to protect medical staff and patients from X-ray radiations. Organic-inorganic hybrids are materials that combine characteristics of organic polymers with those of inorganic metal oxides. These materials can be synthesized via the sol-gel process and can be easily applied as thin coatings on different kinds of substrates. Good radiopacity of organic-inorganic hybrids has been recently reported suggesting that these materials might find applications in medical fields where X-ray absorption and visibility is required. The present PhD thesis aimed at developing and characterizing new radiopaque organic-inorganic hybrid materials that can find application in the vascular surgery field as coatings for the improvement of medical devices traceability as well as for the production of X-ray shielding objects and garments. Novel organic-inorganic hybrids based on different polyesters (poly-lactic acid and poly-ε-caprolactone) and polycarbonate (poly-trimethylene carbonate) as the polymeric phase and on titanium oxide as the inorganic phase were synthesized. Study of the phase interactions in these materials allowed to demonstrate that Class II hybrids (where covalent bonds exists between the two phases) can be obtained starting from any kind of polyester or polycarbonate, without the need of polymer pre-functionalization, thanks to the occurrence of transesterification reactions operated by inorganic molecules on ester and carbonate moieties. Polyester based hybrids were successfully coated via dip coating on different kinds of textiles. Coated textiles showed improved radiopacity with respect to the plain fabric while remaining soft to the touch. The hybrid was able to coat single fibers of the yarn rather than coating the yarn as a whole. Openings between yarns were maintained and therefore fabric breathability was preserved. Such coatings are promising for the production of light-weight garments for X-ray protection of medical staff during interventional fluoroscopy, which will help preventing pathologies that stem from chronic X-ray exposure. A means to increase the protection capacity of hybrid-coated fabrics was also investigated and implemented in this thesis. By synthesizing the hybrid in the presence of a suspension of radiopaque tantalum nanoparticles, PDMS-titania hybrid materials with tunable radiopacity were developed and were successfully applied as coatings. A solution for enhancing medical device radiopacity was also successfully investigated. High metal radiopacity was associated with good mechanical and protective properties of organic-inorganic hybrids in the form of a double-layer coating. Tantalum was employed as the constituent of the first layer deposited on sample substrates by means of a sputtering technique. The second layer was composed of a hybrid whose constituents are well-known biocompatible organic and inorganic components, such as the two polymers PCL and PDMS, and titanium oxide, respectively. The metallic layer conferred to the substrate good X-ray visibility. A correlation between radiopacity and coating thickness derived during this study allows to tailor radiopacity simply by controlling the metal layer sputtering deposition time. The applied metal deposition technique also permits easy shaping of the radiopaque layer, allowing production of radiopaque markers for medical devices that can be unambiguously identified by surgeons during implantation and in subsequent radiological investigations. Synthesized PCL-titania and PDMS-titania hybrids strongly adhered to substrates and show good biocompatibility as highlighted by cytotoxicity tests. The PDMS-titania hybrid coating was also characterized by high flexibility that allows it to stand large substrate deformations without detaching nor cracking, thus being suitable for application on flexible medical devices.

Stem cells are one of the most fascinating areas of biology today, and since the discover of an adult population, i.e., adult Stem Cells (aSCs), they have generated much interest especially for their application potential as a source for cell based regenerative medicine and tissue engineering. aSCs have been found in different tissues including bone marrow, skin, intestine, central nervous system, where they reside in a special microenviroment termed “niche” which regulate the homeostasis and repair of adult tissues. The arterial wall of the blood vessels is much more plastic than ever before believed. Several animal studies have demonstrated the presence of cells with stem cell characteristics within the adult vessels. Recently, it has been also hypothesized the presence of a “vasculogenic zone” in human adult arteries in which a complete hierarchy of resident stem cells and progenitors could be niched during lifetime. Accordingly, it can be speculated that in that location resident mesenchymal stem cells (MSCs) with the ability to differentiate in smooth muscle cells, surrounding pericytes and fibroblasts are present. The present research was aimed at identifying in situ and isolating MSCs from thoracic aortas of young and healthy heart-beating multiorgan donors. Immunohistochemistry performed on fresh and frozen human thoracic aortas demonstrated the presence of the vasculogenic zone between the media and the adventitial layers in which a well preserved plexus of CD34 positive cells was found. These cells expressed intensely HLA-I antigens both before and after cryopreservation and after 4 days of organ cultures remained viable. Following these preliminary results, we succeeded to isolate mesenchymal cells from multi-organ thoracic aortas using a mechanical and enzymatic combined procedure. Cells had phenotypic characteristics of MSC i.e., CD44+, CD90+, CD105+, CD166+, CD34low, CD45- and revealed a transcript expression of stem cell markers, e.g., OCT4, c-kit, BCRP-1, IL6 and BMI-1. As previously documented using bone marrow derived MSCs, resident vascular wall MSCs were able to differentiate in vitro into endothelial cells in the presence of low-serum supplemented with VEGF-A (50 ng/ml) for 7 days. Under the condition described above, cultured cells showed an increased expression of KDR and eNOS, down-regulation of the CD133 transcript, vWF expression as documented by flow cytometry, immunofluorescence, qPCR and TEM. Moreover, matrigel assay revealed that VEGF induced cells were able to form capillary-like structures within 6 hours of seeding. In summary, these findings indicate that thoracic aortas from heart-beating, multi-organ donors are highly suitable for obtaining MSCs with the ability to differentiate in vitro into endothelial cells. Even though their differentiating potential remains to be fully established, it is believed that their angiogenic ability could be a useful property for allogenic use. These cells can be expanded rapidly, providing numbers which are adequate for therapeutic neovascularization; furthermore they can be cryostored in appropriate cell banking facilities for later use.

The arterial wall contains MSCs with mesengenic and angiogenic abilities. These multipotent precursors have been isolated from variously-sized human adult segments, belying the notion that vessel wall is a relatively quiescent tissue. Recently, our group identified in normal human arteries a vasculogenic niche and subsequently isolated and characterized resident MSCs (VW-MSCs) with angiogenic ability and multilineage potential. To prove that VW-MSCs are involved in normal and pathological vascular remodeling, we used a long-term organ culture system; this method was of critical importance to follow spontaneous 3-D vascular remodeling without any influence of blood cells. Next we tried to identify and localize in situ the VW-MSCs and to understand their role in the vascular remodeling in failed arterial homografts. Subsequently, we isolated this cell population and tested in vitro their multilineage differentiation potential through immunohistochemical, immunofluorescence, RT-PCR and ultrastructural analysis. From 25-30cm2 of each vascular wall homograft sample, we isolated a cell population with MSCs properties; these cells expressed MSC lineage molecules (CD90, CD44, CD105, CD29, CD73), stemness (Notch-1, Oct-4, Sca-1, Stro-1) and pericyte markers (NG2) whilst were negative for hematopoietic and endothelial markers (CD34, CD133, CD45, KDR, CD146, CD31 and vWF). MSCs derived from failed homografts (H-MSCs) exhibited adipogenic, osteogenic and chondrogenic potential but scarce propensity to angiogenic and leiomyogenic differentiation. The present study demonstrates that failed homografts contain MSCs with morphological, phenotypic and functional MSCs properties; H-MSCs are long-lived in culture, highly proliferating and endowed with prompt ability to differentiate into adipocytes, osteocytes and chondrocytes; compared with VW-MSCs from normal arteries, H-MSCs show a failure in angiogenic and leiomyogenic differentiation. A switch in MSCs plasticity could be the basis of pathological remodeling and contribute to aneurysmal failure of arterial homografts. The study of VW-MSCs in a pathological setting indicate that additional mechanisms are involved in vascular diseases; their knowledge will be useful for opening new therapeutic options in cardiovascular diseases.

Introduzione L’efficacia dei chemio/radioterapici ha aumentato notevolmente l’aspettativa di vita delle pazienti oncologiche, tuttavia, questi trattamenti possono compromettere la funzionalità ovarica. La crioconservazione di tessuto ovarico, con il successivo reimpianto, ha lo scopo di preservare la fertilità delle pazienti a rischio di fallimento ovarico precoce. Scopo dello studio Definire la migliore procedura di crioconservazione e reimpianto in grado di ottenere la neovascolarizzazione del tessuto reimpiantato nel minor tempo possibile al fine di diminuire la perdita follicolare causata dall’ischemia durante la procedura. Materiali e metodi Per ciascuna paziente (3) le biopsie ovariche, sono state prelevate laparoscopicamente e crioconservate secondo il protocollo di congelamento lento/scongelamento rapido. Campioni di corticale ovarica sono stati processati per l’analisi istologica, ultrastrutturale, immuistochimica e confocale per valutare la preservazione morfologiaca del tessuto. Le fettine di corticale ovarica sono state scongelate e reimpiantate ortotopicamente (2), nelle ovaia e in due tasche peritoneali, o eterotopicamente (1), in due tasche create nel sottocute sovrapubico. Risultati Le analisi di microscopia hanno mostrato il mantenimento di una discreta morfologia dello stroma, e dei vasi criopreservati e un lieve ma non significativo danneggiamento dei follicoli scongelati. Tutte le pazienti hanno mostrato la ripresa della funzionalità endocrina rispettivamente dopo 2/4 mesi dal reimpianto. Il color-doppler, inoltre ha rivelato un significativo aumento della vascolarizzazione ovarica rispetto alla quasi totale assenza di vascolarizzazione prima del reimpianto, quando le pazienti mostravano una conclamata menopausa. Conclusioni Lo studio ha confermato la ripresa della vascolarizzazione dell’ovaio in seguito a reimpianto avascolare di fettine di corticale, senza l’impiego di fattori esogeni o meccanici aggiuntivi, in tempi concordanti con i dati della letteratura. I risultati sono incoraggianti e l’avanzare degli studi e della ricerca potranno contribuire allo sviluppo di nuove metodologie di reimpianto che possano avere un successo clinico ed una sicurezza superiori a quelle finora ottenute. / Recent advances in the diagnosis and the introduction of new protocols of chemo/radiotherapy have significantly increased the survival rate of oncological patients. However, these treatments are gonadotoxic and can severely affect the reproductive potential of patients. Ovarian tissue cryopreservation represents a valid strategy to preserve reproductive function and steroidogenic activity in patients with a high risk of POF. The aim of this procedure is to reimplant cryopreserved ovarian cortical strips allowing the recovering of vascularization and ovarian function. Biopsies samples were laparoscopically removed from both ovaries (3 oncological patients) and frozen according to the slow freezing rapid thawing protocol. Samples of ovarian cortex were processed for histological, ultrastructural, confocal and immuistochimica analysis, to evaluate the morfologiacal tissue preservation. The slices of ovarian cortex were thawed and replanted orthotopically (2), in the ovary and in two peritoneal pockets, or eterotopically (1), in two pockets created in the suprapubic subcutaneous tissue. The microscopy analysis have shown maintenance of a discrete morphology of the cryopreserved stroma, and vessels and a slight but not significant damage to the thawed follicles. All patients showed the resumption of endocrine function respectively after 2/4 months after planting. The color-doppler, also revealed a significant increase in ovarian blood flow compared to the almost total absence of vascularization before replanting, when the patients showed n menopause. The study has confirmed the recovery of the vascularization of the ovary after avascular replanting of cortical slices slices, without the use of exogenous factors , in times consistent with the literature data. The results are encouraging and advancing studies and research will contribute to the development of new methods of reimplant that may have better clinical success and security than those obtained.

La ricerca tratta il tema delle modificazioni del corpo (piercing, tatuaggio, scarificazione, chirurgia estetica) nelle sue implicazioni con la questione dell'identità di genere. Attraverso uno studio sul campo condotto con metodo etnografico e interviste in profondità, vengono indagate in chiave etnometodologica le procedure attraverso cui i soggetti elaborano ed esprimono il proprio self in relazione alle rappresentazioni sociali condivise relative alla mascolinità e alla femminilità. / This research is about body modification techniques (piercing, tattooing, scarification, aesthetic surgery) in relation to gender identity. By means of a fieldwork carried out with ethnographies and in depth interviews, procedures of elaboration and expression of individual self are investigated, via an etnomethodological approach, and in a relation to social representations of femininity and masculinity.