Modulazione del differenziamento osteogenico di precursori mesenchimali umani per applicazioni di ingegneria tissutale

Lo scheletro è un tessuto dinamico, capace di adattarsi alle richieste funzionali grazie a fenomeni di rimodellamento ed alla peculiare proprietà rigenerativa. Tali processi avvengono attraverso l’azione coordinata di osteoclasti ed osteoblasti. Queste popolazioni cellulari cooperano allo scopo di mantenere l’ equilibrio indispensabile per garantire l’omeostasi dello scheletro. La perdita di tale equilibrio può portare ad una diminuzione della massa ossea e, ad una maggiore suscettibilità alle fratture, come avviene nel caso dell’osteoporosi. E’ noto che, nella fisiopatologia dell’osso, un ruolo cruciale è svolto da fattori endocrini e paracrini. Dati recenti suggeriscono che il rimodellamento osseo potrebbe essere influenzato dal sistema nervoso. L’ipotesi è supportata dalla presenza, nelle vicinanze dell’osso, di fibre nervose sensoriali responsabili del rilascio di alcuni neuro peptidi, tra i quali ricordiamo la sostanza P. Inoltre in modelli animali è stato dimostrato il diretto coinvolgimento del sistema nervoso nel mantenimento dell’omeostasi ossea, infatti ratti sottoposti a denervazione hanno mostrato una perdita dell’equilibrio esistente tra osteoblasti ed osteoclasti. Per tali ragioni negli ultimi anni si è andata intensificando la ricerca in questo campo cercando di comprendere il ruolo dei neuropeptidi nel processo di differenziamento dei precursori mesenchimali in senso osteogenico. Le cellule stromali mesenchimali adulte sono indifferenziate multipotenti che risiedono in maniera predominante nel midollo osseo, ma che possono anche essere isolate da tessuto adiposo, cordone ombelicale e polpa dentale. In questi distretti le MSC sono in uno stato non proliferativo fino a quando non sono richieste per processi locali di riparo e rigenerazione tessutale. MSC, opportunamente stimolate, possono differenziare in diversi tipi di tessuto connettivo quali, tessuto osseo, cartilagineo ed adiposo. L’attività di ricerca è stata finalizzata all’ottimizzazione di un protocollo di espansione ex vivo ed alla valutazione dell’influenza della sostanza P, neuropeptide presente a livello delle terminazioni sensoriali nelle vicinanze dell’osso, nel processo di commissionamento osteogenico.

Musculoskeletal tissue regeneration by human non-embryonic stem cells

The aim of this thesis was to investigate the regenerative potential of alternative sources of stem cells, derived from human dental pulp (hDPSCs) and amniotic fluid (hAFSCs) and, specifically, to evaluate their capability to be committed towards osteogenic and myogenic lineages, for the eventual applicability of these stem cells to translational strategies in regenerative medicine of bone and skeletal muscle tissues. The in vitro bone production by stem cells may represent a radical breakthrough in the treatment of pathologies and traumas characterized by critical bone mass defects, with no medical or surgical solution. Human DPSCs and AFSCs were seeded and pre-differentiated on different scaffolds to test their capability to subsequently reach the osteogenic differentiation in vivo, in order to recover critical size bone defects. Fibroin scaffold resulted to be the best scaffold promoting mature bone formation and defect correction when combined to both hDPSCs and hAFSCs. This study also described a culture condition that might allow human DPSCs to be used for human cell therapy in compliance with good manufacturing practices (GMPs): the use of human serum (HS) promoted the expansion and the osteogenic differentiation of hDPSCs in vitro and, furthermore, allowed pre-differentiated hDPSCs to regenerate critical size bone defects in vivo. This thesis also showed that hDPSCs and hAFSCs can be differentiated towards the myogenic lineage in vitro, either when co-cultured with murine myoblasts and when differentiated alone after DNA demethylation treatment. Interestingly, when injected into dystrophic muscles of SCID/mdx mice - animal model of Duchenne Muscular Dystrophy (DMD) - hDPSCs and hAFSCs pre-differentiated after demethylating treatment were able to regenerate the skeletal muscle tissue and, particularly, to restore dystrophin expression. These observations suggest that human DPSCs and AFSCs might be eventually applied to translational strategies, in order to enhance the repair of injured skeletal muscles in DMD patients.

Sindrome da insensitività completa agli androgeni e massa ossea. Valutazione basale e longitudinale dopo oltre 12 mesi di terapia estrogenica

L’osso è un tessuto target per estrogeni ed androgeni ma l’azione singola e la sinergia tra i due non sono compresi interamente. Le donne affette da Sindrome da Insensititvità Completa agli Androgeni (CAIS) hanno un cariotipo 46XY ma presentano una completa inattività del recettore degli androgeni. Nello studio abbiamo valutato la densità minerale ossea (BMD) in un gruppo di donne adulte CAIS sottoposte a gonadectomia al momento della prima visita e dopo almeno 12 mesi di terapia estrogenica. Il principale obiettivo è stato di valutare se, nelle donne CAIS, una ottimale estrogenizzazione fosse sufficiente a mantenere/ripristinare una adeguata BMD. 24 donne CAIS sono state sottoposte a DXA lombare e femorale all'arruolamento nello studio (t1), dopo terapia estrogenica di 12mesi(t2) e oltre (t>2). Sono state valutate: BMD(g/cm2) e Zscore lombare e femorale (a t1,t2 e t>2) E’ stato considerato se fossero rilevanti l’essere (gruppo1) o meno (gruppo 2) in terapia ormonale al t1 e l’età della gonadectomia. Risultati: Al t1 BMD e Zscore lombari e femorale erano significativamente ridotti rispetto alla popolazione controllo nel campione totale (lombare 0,900+0,12; -1,976+0,07, femorale 0,831 + 0,14; -1,385+0,98), nel gruppo 1 (lombare 0,918+0,116;-1,924+0,79, femorale 0,824+0,13;-1,40+1,00) e nel gruppo 2 (lombare 0.845+0,11 -2,13+1,15, femorale 0,857+0,17;-1,348+1,05) Al t2 e t>2 la BMD lombare è risultata significativamente aumentata (p=0,05 e p=0,02). Zscore lombare, BMD e Zscore femorale non hanno dimostrato variazioni significative. L’aver effettuato la gonadectomia in età post puberale è associato a Zscore lombare e femorale più elevati al t1. Nelle donne CAIS la terapia estrogenica è indispensabile per prevenire un'ulteriore perdita di BMD ma, da sola, non sembra in grado di ripristinare normali valori di BMD.I risultati del nostro studio supportano la tesi che gli androgeni, mediante l’azione recettoriale, abbiano un' azione diretta nel raggiungere e mantenere la BMD.

Effect of pulsed electromagnetic fields (pemfs) on muscle activity, tissue oxygenation and vo2 during exercise.

PEMF are a medical and non-invasive therapy successfully used for clinical treatments of bone disease, due to the piezoelectric effect that improve bone mass and density, by the stimulation of osteoblastogenesis, with modulation of calcium storages and mineral metabolism. PEMF enhance tissue oxygenation, microcirculation and angiogenesis, in rats and cells erythrocytes, in cells-free assay. Such responses could be caused by a modulation of nitric oxide signal and interaction between PEMF and Ca2+/NO/cGMP/PKG signal. PEMF improve blood flow velocity of smallest vein without changing their diameter. PEMF therapy helpful in patients with diabetes, due to increased microcirculation trough enhance capillary blood velocity and diameter. We investigated the influence of stimulation on muscular activity, tissue oxygenation and pulmonary VO2, during exercise, on different intensity, as heavy or moderate, different subjects, as a athlete or sedentary, and different sport activity, as a cycling or weightlifting. In athletes, we observed a tendency for a greater change and a faster kinetic of HHb concentration. PEMF increased the velocity and the quantity of muscle O2 available, leading to accelerate the HHb kinetics. Stimulation induced a bulk muscle O2 availability and a greater muscle O2 extraction, leading to a reduced time delay of the HHb slow component. Stimulation increased the amplitude of muscle activity under different conditions, likely caused by the effect of PEMF on contraction mechanism of muscular fibers, by the change of membrane permeability and Ca2+ channel conduction. In athletes, we observed an increase of overall activity during warm-up. In sedentary people, stimulation increased the magnitude of muscle activity during moderate constant-load exercise and warm-up. In athletes and weightlifters, stimulation caused an increase of blood lactate concentration during exercise, confirming a possible influence of stimulation on muscle activity and on glycolytic metabolism of type-II muscular fibers.

Characterization of the Staphylococcus aureus bone sialoprotein-binding protein SdrE and the serine protease EpiP

In an attempt to develop a Staphylococcus aureus vaccine, we have applied reverse vaccinology approach, mainly based on in silico screening and proteomics. By using this approach SdrE, a protein belonging to serine-aspartate repeat protein family was identified as potential vaccine antigen against S. aureus. We have investigated the biochemical properties as well as the vaccine potential of SdrE and its highly conserved CnaBE3 domain. We found the protein SdrE to be resistant to trypsin. Further analysis of the resistant fragment revealed that it comprises a CnaBE3 domain, which also showed partial trypsin resistant behavior. Furthermore, intact mass spectrometry of rCnaBE3 suggested the possible presence of isopeptide bond or some other post-translational modification in the protein.However, this observation needs further investigation. Differential Scanning Fluorimetry study reveals that calcium play role in protein folding and provides stability to SdrE. At the end we have demonstrated that SdrE is immunogenic against clinical strain of S. aureus in murine abscess model. In the second part, I characterized a protein, annotated as epidermin leader peptide processing serine protease (EpiP), as a novel S. aureus vaccine candidate. The crystal structure of the rEpiP was solved at 2.05 Å resolution by x-ray crystallography . The structure showed that rEpiP was cleaved somewhere between residues 95 and 100 and cleavage occurs through an autocatalytic intra-molecular mechanism. In addition, the protein expressed by S. aureus cells also appeared to undergo a similar processing event. To determine if the protein acts as a serine protease, we mutated the catalytic serine 393 residue to alanine, generating rEpiP-S393A and solved its crystal structure at a resolution of 1.95 Å. rEpiP-S393A was impaired in its protease activity, as expected. Protective efficacy of rEpiP and the non-cleaving mutant protein was comparable, implying that the two forms are interchangeable for vaccination purposes.