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.

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.

Analisi morfologica e molecolare di cellule da colture primarie umane esposte a resine biocompatibili

The cytotoxicity of dental composites has been attributed to the release of residual monomers from polymerized adhesive systems due to degradation processes or the incomplete polymerization of materials. 2-Hydroxyethyl methacrylate (HEMA) is one of the major components released from dental adhesives. Cytotoxic effects due to high concentrations of HEMA have already been investigated, but the influence of minor toxic concentrations for long-term exposition on specific proteins such as type I collagen and tenascin has not been studied in depth. The objective of this project was to study the effect of minor toxic concentrations of HEMA on human gingival fibroblasts (HGFs) and human pulp fibroblasts (HPFs), investigating modification in cell morphology, cell viability, and the influence on type I collagen and tenascin proteins. Different concentrations of the resin monomer and different times of exposition were tested on both cell lines. The cell vitality was determined by MTT assay, and high-resolution scanning electron microscopy analysis was performed to evaluate differences in cell morphology before and after treatment. To evaluate the variability in the expression and synthesis of procollagen α1 type I and tenascin proteins on HGFs and HPFs treated with HEMA at different concentrations immunofluorescence, RT-PCR and western blot analysis, were carried out. The treatments on HGFs with 3mmol/L HEMA, showed a strong reduction of procollagen α1 type I protein at 72h and 96h, demonstrating that HEMA interferes both with the synthesis of the procollagen α1 type I protein and its mRNA expression. The results obtained on HPFs treated with different concentrations of HEMA ranging from 0,5mmol/L to 3mmol/L and for different exposition times showed a strong reduction in cell viability in specimens treated for 96h and 168h, while immunofluorescence and western blotting analysis demonstrated a reduction of procollagen α1 type I and an overexpression of tenascin protein. In conclusion, our results showed that the concentrations of HEMA we tested, effect the normal cell production and activity, such as the synthesis of some dental extracellular matrix proteins.