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In un suo aspetto più generale la presente invenzione riguarda un reattore per la coltura di cellule. Come è noto, il forte sviluppo avuto negli ultimi anni da quelle che sono genericamente conosciute con il termine di biotecnologie, ha portato alla realizzazione di nuove apparecchiature per attuare le metodologie di coltura di cellule o la formazione di tessuti biologici. In talune applicazioni queste apparecchiature, dette anche comunemente bioreattori, comprendono una camera di coltura dove le cellule vengono mantenute in una soluzione liquida su un substrato di supporto; la camera viene inoltre mantenuta in condizioni di temperatura ed atmosfera controllata, in modo da assicurare la corretta riproduzione delle cellule. Questa breve premessa serve per meglio introdurre l’invenzione, che in un suo aspetto più particolare riguarda un bioreattore per la stimolazione elettromeccanica di cellule staminali. Come è noto, queste cellule hanno la capacità di mutare le proprie caratteristiche per assumere quelle delle cellule di un diverso fenotipo, in seguito ad un trans-differenziamento. Sfruttando questa capacità, è prassi usuale quella di riprodurre “in vitro” un microambiente in grado di avvicinarsi alle caratteristiche del tessuto verso il quale si voglia indirizzare la popolazione cellulare. Così, ad esempio, nel tentativo di ottenere cellule di tessuto muscolare cardiaco, cellule staminali possono essere sottoposte a cicli periodici di espansione e contrazione che riflette quello delle pulsazioni cardiache. A tal fine le cellule vengono supportate su un substrato di materiale elastico biocompatibile in un bagno di coltura; il substrato viene allungato e rilasciato elasticamente con cicli di frequenza prestabilita, così che il tessuto cellulare posato su di esso subisca anch’esso cicli analoghi di deformazione. Il bioreattore 0001395109 soddisfa questi requisiti, trovando applicazione nei laboratori di ricerca pubblici e privati che lavorano nel campo della medicina rigenerativa. In commercio sono presenti bioreattori in grado di stimolare meccanicamente cellule coltivate su di un supporto elastico ma presentano alcune limitazioni significative: 1) necessitano di un incubatore in cui porre il bioreattore; 2) impiegano supporti elastici esclusivamente 2D; 3) sono privi di un sistema di controllo della forza di stiramento applicata. Il bioreattore oggetto dell'invenzione supera tutte queste limitazioni, permettendo di ottimizzare la ricerca nell'ambito dell'ingegneria dei tessuti e della medicina rigenerativa.

Ischemic preconditioning is a cardioprotective phenomenon involving adaptive changes in cells and molecules that occurs in a biphasic pattern: an early phase (1-2 h), and a late phase (12-24 h). Reactive oxygen species (ROS) are strongly involved in triggering ischemic preconditioning, but it is not clear if they play a major role in the early or late phase of preconditioning and which are the molecular mechanisms involved and the cellular effects generated. In this study we investigated the mechanisms behind H2O2 induced cardioprotection in cultured rat cardiomyocytes demonstrating the induction of phase II enzymes and the involvement of Nuclear factor E2-related factor 1 (Nrf1) and Nrf2. Rat neonatal cardiomyocytes were grown until confluence. Preconditioning was simulated with 100 µM H2O2 for 10 min and after different times oxidative stress was generated by 100 µM H2O2 for 30 min. Cell viability was evaluated by MTT and ROS production by 2’,7’-dichlorofluorescein-diacetate. Phase II enzyme expression and Nrf1 and Nrf2 nuclear translocation were determined by immunoblotting. Moreover, Nrf1 and Nrf2 were silenced with Nrf1-siRNA and Nrf2-siRNA, to evaluate the involvement in phase II enzyme induction. H2O2-preconditioning showed a higher ability to counteract oxidative stress in the late than in the early phase of adaptation. H2O2-preconditioning led to a decrease in intracellular ROS production related to the induction of glutathione reductase, catalase, thioredoxin reductase, and NADPH quinone oxidoreductase 1 expression. H2O2-preconditioning induced the nuclear translocation of both Nrf1 and Nrf2. The down-regulation of Nrf1 and Nrf2 by Nrf1-siRNA and Nrf2-siRNA inhibited the enzyme induction. These findings demonstrate that, both Nrf1 nd Nrf2 are involved in the induction of phase II enzymes by preconditioning, thus contributing to the cardioprotection elicited by these important antioxidant/detoxifying enzymes.

Objectives: Stem cells are potential agents for the treatment of myocardial infarction and Bone Marrow Cells (BMC) are the most extensively investigated. Engraftment and delivery strategies are still controversial. We present an in vivo experimental model with a swine animal. Technical aspects as, infarction induction and cell transplantation with a biopolymer scaffold, anesthesia management, diagnosis and preliminary results are described Materials and methods: Infarction is created by direct ligature of the interventricularis artery. Simultaneously, BMC are harvested. BMC are left grown during two weeks and are marked with bromodeoxyirudine. Then are sown in a polymeric scaffold surface of hyaluronic acid (HYAFF-11) and left for 15 days. Thirty days after the infarction, a second operation is performed. Through right thoracotomy, the heart is exposed and an intra-myocardial pocket created at the scar level. The scaffold in then implanted inside, and the myocardial flap fixed above. To evaluate ventricular function and wall kinesis, a Q-contrast echocardiography with CEUS method is performed. The first one is performed the day before the infarction, the second one, 30 days later and the last one, 4 weeks after the biopolymer implantation. The animal is then suppressed and histological analysis performed. Until now, infarction has been performed in 17 swines. The scaffold was implanted in 6 cases, with BMC in 4 cases, and without in other 2 cases. Results: Histology demonstrated, in those cases of biopolymer implantation plus BMC, an increased angiogenesis. Echocardiography with Q-contrast shown an increased perfusion in the infarcted area. Conclusions: This experimental model represents an optimal and reproducible “in vivo” strategy of stem cell transplantation or delivery. Our preliminary results confirm the validity of the animal model, in terms of infarction generation and BMC engraftment. Bigger population as well as further investigation is still needed to understand the ongoing neo-angiogenesis and the myocardial regeneration.

The fabrication of biodegradable 3-D scaffolds enriched with multipotent stem cells seems to be a promising strategy for the repair of irreversibly injured tissues. The fine mechanisms of the interaction of rat mesenchymal stem cells (rMSCs) with a hyaluronan-based scaffold, i.e. HYAFF(R)11, were investigated to evaluate the potential clinical application of this kind of engineered construct. rMSCs were seeded (2 x 10(6) cells cm(-2)) on the scaffold, cultured up to 21 days and analysed using appropriate techniques. Light (LM), scanning (SEM) and transmission (TEM) electron microscopy of untreated scaffold samples showed that scaffolds have a highly porous structure and are composed of 15-microm-thick microfibres having a rough surface. As detected by trypan blue stain, cell adhesion was high at day 1. rMSCs were viable up to 14 days as shown by CFDA assay and proliferated steadily on the scaffold as revealed by MTT assay. LM showed rMSCs in the innermost portions of the scaffold at day 3. SEM revealed a subconfluent cell monolayer covering 40 +/- 10% of the scaffold surface at day 21. TEM of early culture showed rMSCs wrapping individual fibres with regularly spaced focal contacts, whereas confocal microscopy showed polarized expression of CD44 hyaluronan receptor; TEM of 14-day cultures evidenced fibronexus formation. Immunohistochemistry of 21-day cultures showed that fibronectin was the main matrix protein secreted in the extracellular space; decorin and versican were seen in the cell cytoplasm only and type IV collagen was minimally expressed. The expression of CD90, a marker of mesenchymal stemness, was found unaffected at the end of cell culture. Our results show that HYAFF(R)11 scaffolds support the adhesion, migration and proliferation of rMSCs, as well as the synthesis and delivery of extracellular matrix components under static culture conditions without any chemical induction. The high retention rate and viability of the seeded cells as well as their fine modality of interaction with the substrate suggest that such scaffolds could be potentially useful when wide tissue defects are to be repaired as in the case of cartilage repair, wound healing and large vessel replacement.