Growth and Differentiation of Circulating Stem Cells After Extensive Ex Vivo Expansion

Background:: Stem cell therapy is gaining momentum as an effective treatment strategy for degenerative diseases. Adult stem cells isolated from various sources (i.e., cord blood, bone marrow, adipose tissue) are being considered as a realistic option due to their well-documented therapeutic potentials. Our previous studies standardized a method to isolate circulating multipotent cells (CMCs) that are able to sustain long term in vitro culture and differentiate towards mesodermal lineages. Methods:: In this work, long-term cultures of CMCs were stimulated to study in vitro neuronal and myogenic differentiation. After induction, cells were analysed at different time points. Morphological studies were performed by scanning electron microscopy and specific neuronal and myogenic marker expression were evaluated using RT-PCR, flow cytometry and western blot. For myogenic plasticity study, CMCs were transplanted into in vivo model of chemically-induced muscle damage. Results:: After neurogenic induction, CMCs showed characteristic dendrite-like morphology and expressed specific neuronal markers both at mRNA and protein level. The calcium flux activity of CMCs under stimulation with potassium chloride and the secretion of noradrenalin confirmed their ability to acquire a functional phenotype. In parallel, the myogenic potential of CMCs was confirmed by their ability to form syncytium-like structures in vitro and express myogenic markers both at early and late phases of differentiation. Interestingly, in a rat model of bupivacaine-induced muscle damage, CMCs integrated within the host tissue taking part in tissue repair. Conclusion:: Overall, collected data demonstrated long-term cultured CMCs retain proliferative and differentiative potentials suggesting to be a good candidate for cell therapy

Electrospun polyphosphazene nanofibers for in vitro osteoblast culture

Since 20 years both natural and syntehtic materials had been studied and applied to bone tissue engineering. In this context, poly(organophosphazene)s, high molecular weight polymers with a backbone of alternating phosphorus and nitrogen atoms and two organic side groups bonded to each phosphorus atom, can represent an attractive alternative to the materials used currently. The polymer degrade in acqueous medium to nontoxic products, including ammonia, phosphate, aminoacids, and the corresponding alchol. Polyphosphazenes polymers were indeed studied for the controlled release of many drugs bath few works concerned tissue engineering applications. In this work a poly[(ethyl phenylalanato)1.4(ethyl glycinato)0.6phosphazene] (PPhe-GlyP) has been prepared and used to assemble scaffolds for bone tissue engineering purposes. Either solvent casting or electrospinning methods were employed. We have also evaluated the effects on osteoblast attachment and proliferation of PPhe-GlyP blends with two widely used polymers, poly(lactic acid) (PLA) and poly(caprolactone) (PLC). PPhe-GlyP disks, obtained by solvent casting method, presented a smooth surface with several holes whose diameter ranged from 0.5 to 2 \uf06dm. The in vitro degradation carried out in phosphate buffer, pH 7.4, at 37\ub0C, displayed a nearly constant degradation kinetics, losing approximately 20% of the disk mass in 100 days. To verify the in vivo biocompatibility, PPhe-GlyP disks were inserted into subcutaneous pocket of BALB/c mice. A thin fibrous capsule around the polymeric disk was still present until 60 days, and no cells were visible inside the implants The in vivo results confirmed the biocompatibility of the PPhe-GlyP polymer, but the solvent casting method was not suitable to obtain PPhe-GlyP scaffolds able to allow host cell ingrowth.On the contrary, PPhe-GlyP scaffolds obtained by electrospinning method showed good porosity and fiber dimensions (600\ub1300 nm) resembling those of the natural extracellular matrix. Osteoblasts collected from bone marrow of femurs of Sprague-Dowley rats were seeded on electrospun scaffolds composed of PLA, PCL alone, or as blends with PPhe-GlyP [PLA/PPhe-GlyP 75:25 (w/w) and PCL/PPhe-GlyP 75/25 (w/w)]. Although PPhe-GlyP supports osteoblast adhesion and growth to a lesser extent than that observed for electrospun PLA, a synergic effect on cell proliferation was noted when osteoblasts were cultured on PLA/PPhe-GlyP 75/25. Since polyphosphazenes can exert a buffering effect on acidic degradation products of PLA, electrospun PPhe-GlyP/PLA blend may represent an interesting material to use for bone tissue engineering. Finally, it must be noted that the poor mechanical properties of nanofibrous scaffolds make these materials useful only to repair defects whereby limited mechanical loading occurs, such as some cranial and maxillofacial defects

Assessment of in vitro temporal corrosion and cytotoxicity of AZ91D alloy

Magnesium alloys represent a valuable option for the production of bioresorbable implantable medical devices aimed to improve the therapeutic approach and minimize the potential risks related to biostable materials. In this regard, the degradation process needs to be carefully evaluated in order to assess the effectiveness of the regenerative support and the eventual toxic effects induced by the released corrosion products. Aluminium is one of the most common alloying element that raised several safety concerns, contributing to shift the investigation toward Al-free alloys. To delve into this issue, a long-term investigation (up to 28 days) was performed using AZ91D alloy, due to its relevant Al content. Immersion tests in phosphate buffered saline (PBS) solution was performed following the ASTM standards and the corrosion behaviour was evaluated at fixed time points by means of electrochemical techniques. Cytotoxic effects were assessed by culturing human neuroblastoma cells with conditioned medium derived from immersion tests at different dilution degree. An increase in the resistance corrosion with the time was observed. In all the investigated cases the presence of Al in the conditioned media did not induce significant toxic effects directly correlated to its content. A decrease of cell viability was only observed in the case of 50 % dilution of PBS conditioned for the longest immersion period (i.e., 28 days)

PERIPHERAL-NERVE REPAIR USING A POLY(ORGANO)PHOSPHAZENE TUBULAR PROSTHESIS

Nerve regeneration experiments were carried out using tubular nerve guides of poly[(ethylalanato)(1.4)(imidazolyl)(0.6)phosphazene] (PEIP). By means of in vivo tests, this polymer was found to be biodegradable and transformed into harmless products. The tubular nerve guides were prepared by deposition of the dissolved polymer on a glass capillary tube, followed by evaporation of the solvent (methylene dichloride). After transectioning, rat sciatic nerve stumps were immediately sutured into the ends of 10-mm-long polymer tubes. On removal bf the prosthesis, after implantation for 45 d, a tissue cable was found bridging the nerve stumps in all cases. Histological analysis revealed that the tissue cable was essentially composed of a regenerated nerve fibre bundle. A parallel series of experiments was undertaken to compare the use of silicone tubes that are not biodegradable and are most frequently used for studies of nerve regeneration with tubulization techniques, The advantages of biodegradable PEIP tubular nerve guides used for peripheral nerve repair are discussed.16534735

Studio sulla potenziale tossicità della lega AZ91D per applicazioni biomedicali

I dispositivi medicali bioriassorbibili possono introdurre un potenziale miglioramento nel trattamento di diverse patologie, fornendo un’azione strutturale-funzionale per un tempo idoneo al completamento del processo terapeutico ed evitando di esporre il paziente a rischi di medio e lungo termine legati all’impianto del dispositivo stesso. Per tale scopo particolare attenzione è rivolta alla definizione e alla caratterizzazione di specifiche leghe di Mg, in particolare in ambito ortopedico ed endovascolare. Tuttavia, come noto, il processo di biodegradazione comporta un decremento delle proprietà meccaniche e, questione ancor più delicata, il rilascio di prodotti di corrosione potenzialmente citotossici, derivanti dagli elementi alliganti. Il presente lavoro intende quindi mostrare i risultati dello studio di corrosione e citocompatibilità della lega AZ91D, selezionata per l’elevato contenuto di Al e ritenuto un possibile fattore di rischio nell’insorgenza di malattie neurodegenerative, danni muscolari e ridotta vitalità di cellule del tessuto osseo. Sono stati eseguiti test di immersione (ASTM G31) su campioni di AZ91D in soluzione tampone fosfato (PBS) in condizioni standard di incubatore (37 °C, 5% CO2, 95% HR) per 7, 14 e 28 giorni. Al termine dei periodi indicati i campioni sono stati decapati (ASTM G1) e la microstruttura superficiale e la composizione chimica sono state investigate tramite microscopia elettronica a scansione e analisi di spettroscopia a dispersione d’energia. Per valutare la resistenza alla corrosione della lega sono stati acquisiti spettri di impedenza elettrochimica e curve di polarizzazione, nonché è stata misurata la velocità di perdita di massa ai diversi tempi di immersione. Sono state inoltre eseguite misure di assorbimento atomico sulle soluzioni di PBS per valutare la concentrazione di ioni di Mg, Al e Zn. Infine, sono stati condotti test indiretti di citotossicità mediante saggio MTS su cellule di neuroblastoma umane SH-SY5Y esposte a PBS condizionato con la lega. Dalle misure di perdita di peso e dai test elettrochimici è stata riscontrata una diminuzione della velocità di dissoluzione del materiale ed un aumento del potenziale e della resistenza alla corrosione con il tempo di immersione. Da ciò si deduce la formazione di un film stabile sulla superficie dei campioni dopo un certo tempo di esposizione alla soluzione. Infine, gli esperimenti di citotossicità hanno dimostrato che i prodotti di degradazione della lega AZ91D non riducono in maniera significativa la vitalità cellulare rispetto alla coltura di cellule SH-SY5Y con diluizioni progressive del terreno in PBS. I risultati preliminari riportati non evidenziano particolari effetti citotossici della lega AZ91D, tuttavia sono necessarie analisi supplementari che riproducano il più possibile le reali condizioni fisiologiche al fine di completare il profilo tossicologico del biomateriale in esame

Effect of synthetic peptides on osteoblast adhesion

The quality of the early cell/rnaterial interactions is responsible for the long-term functional properties of ally implanted device. Accordingly, "next generation" dental/orthopedic biomaterials should be able to promote osteoblast adhesion thus improving the integration process between Surgically placed implants and biological tissues. Recent Studies have identified a wide range of biochemical signals that call be exploited to promote adhesion, migration, proliferation and differentiation of cells. The clinical use of natural factors to promote osteoblast adhesion is complicated because those are often insoluble and unstable macromolecules and, in addition, it is difficult to obtain them in high quantities, with good purity grade and at low cost. A valid alternative could be the use of short peptides carrying the minimum active sequence of the natural macromolecular factor. This paper describes the properties of two classes of peptides, promoting different adhesion mechanisms, to enhance rat bone marrow osteoblast adhesion both to polystyrene and to acellular bone matri