37,530 research outputs found
Inhibition of growth, morphological and morphometrical changes induced by amido-anthraquinone derivatives in NCTC 2544 an HeLa cells.
No full textIn this work, we have studied cytotoxicity induced in NCTC 2544 and HeLa cell lines by two newly-synthesised potential anticancer drugs, the amido-anthraquinone derivatives 8-diethylaminopropionamido-1,4,5-trihydroxy-9,10-anthracenedione bromide (A888) and 5,8-bisdiethylaminopropionamido-1,4-dihydroxy-9,10-anthracenedione dibromide (A890). The results have been compared to the results obtained with mitoxantrone, a well-known anticancer agent. Using the neutral red uptake assay, A888 showed less cytotoxic action compared to mitoxantrone and A890. According to the results of morphological analysis after exposure for five hours, anthraquinone derivatives induced the formation of cytoplasmic vacuoles and changes in the arrangement of nucleus in both cell lines. On the basis of morphometrical analysis, we have observed an increase in total cellular area with a decrease in the nuclear/cytoplasmic ratio. We have found that the increase in total cellular area was higher in NCTC 2544 cells than in HeLa cells. From these data, we can conclude that NCTC cells are more sensitive to the compounds tested than HeLa cells. However, the growth inhibition assay and morphological analysis did not confirm these results
Electrospun polyphosphazene nanofibers for in vitro osteoblast culture
No full textSince 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 m. The in vitro degradation carried out in phosphate buffer, pH 7.4, at 37°C, 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±300 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
Bioartificial system composed of muscle acellular matrix, myoblasts, and VEGF-loaded porous silica gel for the treatment of congenital diaphragmatic hernia
No full textUtilizzo di precursori muscolari scheletrici e precursori derivati da sistema neuroenterico nella correzione di difetti sfinteriali
No full textThis project rises from the hypothesis of using cell therapy and tissue engineering in the gastrointestinal system. The clinical environment the project borne is the Pediatric Surgery. However the faced topics embrace all age population. In particular we explored the possible correction of sphincter defect by using skeletal muscle precursors. In this view we studied the capability of skeletal muscle precursors (committed stem cells) of integrating into contractile tissue of gut. In vitro and in vivo experiments have been done. The most data have been obtained from the murine model of cryo-injury of gastroesophageal junction. We studied the regeneration after injection of cultured or freshly isolated satellite cell (obtained by the single fibre technique) compared with other cell populations. The candidate personally managed several technique to obtain skeletal muscle precursors, primary cell culture, co-culture between various cell-type; analysis techniques ( conventional histology, immunofluorescent analysis of cryo-sections, western-blotting, cytofluorimetric anlysis, statistical analysis, microscopy imaging). Further micro-surgery techniques have been used in the in vivo experiments on small animals. Results have been presented in international meetings or publications:
- Murine Muscle Precursor Cells survived and integrated in a Cryoinjured Gastroesophageal Junction.
Fascetti Leon F, Malerba A, Boldrin L, Leone E, Betalli P, Pasut A, Zanon GF, Gamba PG, Vitiello L, DeCoppi P. J Surg Res. Epub 2007 Jun 19
- Muscle precursors injection in the gastroesophageal junction: further experience.
Fascetti Leon F, Malerba A, Boldrin L, Zanon GF, Gamba PG, Pierro A, De Coppi P. Oral presentation at the 8th European Congress of Paediatric Surgery (EUPSA) Turin, Italy 2007
In vitro enhancement of muscle precursors cells differentiation enhanced by co-cultures with neurogenic cells. A Malerba, F Fascetti Leon, L Boldrin, C Caldwell, N, Thapar, A Pierro, P De Coppi British Association of Paediatric Surgeons (BAPS) annual meeting Edimburgh, UK 2007
- Further experience with the injection of muscle precursors in the gastroesophageal junction
Fascetti Leon F, Malerba A, Boldrin L, Vitello L, Talenti E, Zanon GF, Gamba PG, Pierro A, De Coppi P. presentation at the American Paediatric Surgeons Association (APSA) Orlando, Florida 2007
- Long term integration of muscle precursor cells injected in the lower esophageal sphincter.
Fascetti Leon F, Malerba A, Boldrin L, Betalli P, Gamba PG, Vitiello L, A. Pierro, DeCoppi P. submitted.
Cell culture have been conduced at the Regenerative Medicine Lab of Della Città della Speranza (c/o Dipartimento di Pediatria e Istituto VIMM). Part of the analyses and the co-culture have been conduced at the Institute of Child Health - Great Ormond Street Hospital of London. The in vivo section have been done at The work allows the conclusion that adult skeletal muscle precursors could be used in the regeneration of digestive tract contractile tissue. In particular good engrafting and data regarding the possible trans-differentiation through smooth muscle lineage led to follow this approach (correction of sphincter defect). Few data have been obtained from the preliminary experiments on anal sphincter. Furthermore, the capability of integrating and interacting in vitro between ganglion derived cells and muscle cells, let us think to the possible wilder application to gastro-intestinal contraction defects.Il progetto di ricerca nasce dall’ipotesi dell’utilizzo di approcci di terapia cellulare ed ingegneria tissutale nel tratto digerente. L’ambiente clinico da cui nasce lo spunto per questo progetto di ricerca è la Chirurgia Pediatrica. Tuttavia le tematiche affrontate abbracciano trasversalmente ogni fascia di età. Ci si è interessati in particolare alla possibilità di correggere difetti “sfinteriali” tramite l’utilizzo di precursori derivati dal muscolo scheletrico.
In tale ottica sono state esplorate le capacità di integrazione nel tessuto contrattile del tubo digerente di precursori cellulari del muscolo scheletrico (cellule staminali committed). Sono stati eseguiti esperimenti in vivo e in vitro. La maggior parte dei dati sono stati ottenuti utilizzando un modello di danno da congelamento a carico della giunzione gastro-esofagea di topo. Sono state esplorate le possibilità di rigenerazione dopo iniezione di cellule satellite coltivate o a fresco ottenute con la tecnica della singola fibra.
Il candidato ha personalmente utilizzato diverse tecniche per l’ottenimento di precursori staminali da muscolo scheletrico, colture cellulari primarie, coculture tra vari tipi cellulari, nonché varie tecniche di analisi (istologia convenzionale, analisi d’immunofluorescenza delle criosezioni, western-blotting, citofluorimetria, analisi statistica e d’immagine microscopica). Per gli esperimenti in vivo,inoltre, sono state utilizzate tecniche di microchirurgia sul piccolo animale.
Risultati del lavoro svolto sono stati oggetto di presentazioni in congressi nazionali ed internazionali:
- Murine Muscle Precursor Cells survived and integrated in a Cryoinjured Gastroesophageal Junction.
Fascetti Leon F, Malerba A, Boldrin L, Leone E, Betalli P, Pasut A, Zanon GF, Gamba PG, Vitiello L, DeCoppi P. J Surg Res. Epub 2007 Jun 19
- Muscle precursors injection in the gastroesophageal junction: further experience.
Fascetti Leon F, Malerba A, Boldrin L, Zanon GF, Gamba PG, Pierro A, De Coppi P. Oral presentation at the 8th European Congress of Paediatric Surgery (EUPSA) Turin, Italy 2007
In vitro enhancement of muscle precursors cells differentiation enhanced by co-cultures with neurogenic cells. A Malerba, F Fascetti Leon, L Boldrin, C Caldwell, N, Thapar, A Pierro, P De Coppi British Association of Paediatric Surgeons (BAPS) annual meeting Edimburgh, UK 2007
- Further experience with the injection of muscle precursors in the gastroesophageal junction
Fascetti Leon F, Malerba A, Boldrin L, Vitello L, Talenti E, Zanon GF, Gamba PG, Pierro A, De Coppi P. presentation at the American Paediatric Surgeons Association (APSA) Orlando, Florida 2007
- Long term integration of muscle precursor cells injected in the lower esophageal sphincter.
Fascetti Leon F, Malerba A, Boldrin L, Betalli P, Gamba PG, Vitiello L, A. Pierro, DeCoppi P. submitted.
Gli esperimenti “in vivo” e ‘analisi istologica sono stati condotti presso il Centro inter-dipartimentale Vallisneri dell’Unioversità di Padova.
Le colture cellulari sono state eseguite presso il Laboratorio di Medicina Rigenerativa Della Città della Speranza (c/o Dipartimento di Pediatria e Istituto VIMM).
Parte dell’analisi e delle co-culture sono state eseguite presso l’Institute of Child Health - Great Ormond Street Hospital di Londra.
Il lavoro svolto porta alla conclusione che cellule precursori muscolari derivati da tessuto muscolare scheletrico adulto sono potenzialmente utilizzabili nella rigenerazione delle strutture contrattili del tubo digerente. In particolare la buona capacità di integrazione e dati sulla possibile trans-differenziazione verso la linea cellulare muscolare liscia stimolano a proseguire nella direzione di una terapia cellulare dei difetti sfinteriali. Pochi dati sono derivati dagli esperimenti condotti sullo sfintere anale interno, che vanno pertanto considerati preliminari. Inoltre la capacità di integrazione e interazione in vitro tra cellule nervose gangliari e muscolari derivate da muscolo scheletrico fanno pensare ad un più ampio utilizzo nei difetti di contrattilità gastro-intestinali
Endothelial cells from human cerebral aneurysm and arteriovenous malformation release ET-1 in response to vessel rupture
No full textCerebral aneurysms and arteriovenous malformations (AVM) are a common cause of stroke and cerebral hemorrage. Both are often discovered when they rupture, causing subarachnoid hemorrhage (SAH). SAH-induced vasospasm is mediated by enhanced vasoconstriction due to endothelin-1 (ET-1). We investigated whether endothelial cells (ECs) obtained from aneurysm and AVM express phenotypic and genotypic alterations contributing to the development of vasospasm after SAH. We isolated ECs from human AVM and aneurysm and then confirmed their EC origin by polymerase chain reaction and immunocytochemistry with endothelial markers. Experiments were also carried out with human cerebral microvascular and umbilical vein ECs (HCECs and HUVECs respectively) for comparison. We tested EC proliferation ability and microtubule formation in Matrigel at different cell passages. Five aneurysm (3 ruptured, 2 unruptured) and :3 AVM (2 ruptured, 1 unruptured) ECs were tested for ET-1 release in the culture medium. Aneurysm and AVM ECs expressed von Willebrand factor, Adrenomedullin, and exhibited a progressive reduction of proliferation and in vitro angiogenic ability after the V passage. Significantly higher levels of ET-1 have been detected in ECs from ruptured aneurysms and AVMs. We report the first successful isolation and characterization of primary EC lines from human cerebral vascular lesions. Augmented release of ET-1 is correlated with the rupture of the abnormal vessel confirming its role in vasospasm after SAH. Furthermore, ECs obtained from these vascular malformations can be used as an experimental model to study SAH-induced vasoconstriction
Ruolo della via di segnale di Wnt5a nel fenotipo dei miofibroblasti sub-epiteliali intestinali in corso di Colite Ulcerosa
No full textABSTRACT
Background: Wnts are a highly conserved family of secreted glicoproteins involved in several physiological and pathological processes. Intestinal subepithelial myofibroblasts (ISEMFs), a cellular population releasing growth factors and cytokines involved in tissue remodelling, have been recently identified as a source of Wnts ligands. Since Wnt5a, the main ligand of the non-canonical pathway, plays a pivotal role in intestinal epithelial repair and is up-regulated in chronic inflammatory disorders such as rheumatoid arthritis, the aim of our study was to evaluate, in ISEMFs derived from normal subjects and ulcerative colitis (UC) patients, the expression of Wnt5a and its receptors and determine its autocrine role on ISEMFs. Methods: ISEMFs were isolated from colonic mucosa of healthy subjects (n=6) or from patients with UC (n=10). Wnt5a and Frzd1, 2, 4, 5 expressions was evaluated by quantitative RT-Time PCR and Western Blot. To determine the autocrine effect of Wnt5a, we performed functional studies using Wnt5a KO by shRNA or exposing ISEMFs to human recombinant Wnt5a. Following 96 hours culture, cell proliferation was quantitated by 3H-tymidine incorporation and cell cycle analysis. To evaluate anti-apoptotic role of Wnt5a, we performed functional studies exposing ISEMFs to human recombinant Wnt5a and staurosporin. Following 24 hours culture Annexin V and caspase-3 assay was estimated. To identify pathway induced by Wnt5a signal, functional studies using β-catenin KO by shRNA or exposing ISEMFs to Ca2+ inhibitors was performed and CamKII protein levels was measured by Western blot. Results: UC-derived ISEMFs showed a significantly higher Wnt5a expression at mRNA and protein level (p<0.05) in basal condition as compared to controls. Furthermore, as compared to controls UC-derived ISEMFs produced significantly higher amounts of Wnt5a following exposure to LPS or TGFβ (p<0.05). ISEMFs derived both normal and UC patients showed a similar pattern of Wnts receptors. Wnt5a silencing in UC-derived ISEMFs and in control ISEMFs, caused a significant reduction in cell proliferation (p<0.05). Indeed, rWnt5a supplementation stimulated cell proliferation and resistance to staurosporin induced-apoptosis. Wnt5a signal is β-catenin independent because supplementation of rWnt5a in KO β-catenin ISEMFs led to a significative increase of cell proliferation (p<0.05). On the contrary,Wnt5a signal seems to be mediated by CamKII, promoting an increase of its phosphorilation. Conclusions: ISEMFs from UC patients produce an excess of Wnt5a that is able to stimulate cellular proliferation, resistance to apoptosis and that acts by CamKII activation. We speculate that Wnt5a plays a key role to expand the ISEMFs population in UC patients and might contribute to tissue damage and remodeling during chronic inflammation.Wnt5a rappresenta il ligando prototipo della via non canonica di Wnt, via di segnale coinvolta nei processi di embriogenesi, riparazione tissutale e carcinogenesi dell’epitelio intestinale. Fisiologicamente, Wnt5a agisce sulla migrazione e proliferazione di tipi cellulari diversi coinvolti nel mantenimento dell’omeostasi epiteliale, fungendo da mediatore nel dialogo tra epitelio e mesenchima. Numerosi dati sperimentali suggeriscono inoltre che l’aumento di Wnt5a possa essere un meccanismo comune dell’infiammazione cronica in quanto è sovra-espresso nell’artrite reumatoide, nell’aterosclerosi e nella psoriasi. Abbiamo pertanto iniziato ad esaminare il ruolo di Wnt5a nell’infiammazione cronica intestinale.
Il modello preso in esame è rappresentato da miofibroblasti subepiteliali intestinali (ISEMFs) da Rettocolite Ulcerosa (RCU). Le ISEMFs sono cellule coinvolte nell’omeostasi intestinale: nell’intestino rappresentato il maggior sito di produzione del collagene e di altre proteine della matrice extracellulare, promuovendo dunque il rimodellamento tessutale. Inoltre interagiscono con le cellule epiteliali, le staminali e i macrofagi promuovendo e la restituzione epiteliale e la secrezione di citochine e chemochine. Si è osservato che nella mucosa dei pazienti affetti da IBD, il numero di queste cellule è significativamente aumentato rispetto a quello della mucosa sana, e che un’alterazione della loro funzionalità è coinvolto nella fisiopatologia delle IBD.
L’aumentata espressione di Wnt5a a livello trascrizionale e proteico è stata verificata in soggetti affetti da RCU, con correlazione diretta con il grado di attività di malattia. Studi di immunoistochimica hanno permesso, mediante co-localizzazione con α-SMA, di osservare la distribuzione di Wnt5a nel tessuto e la sua preponderante espressione in miofibroblasti sub epiteliali. La caratterizzazione di Wnt5a è stata dunque effettuata su miofibroblasti primari estratti da mucosa di Rettocolite Ulcerosa e tessuto controllo. Cellule derivate da tessuto infiammato hanno mostrato maggiori livelli di espressione proteica di Wnt5a. Dosaggi del trascritto e western blotting hanno permesso di osservare che il TGFβ e LPS sono in grado promuovere una sovra-espressione di Wnt5a nei miofibroblasti intestinali. Abbiamo quindi condotto dei saggi per verificare se Wnt5a regolasse caratteristiche fisiologiche e fenotipiche nelle ISEMFs stesse. A tal fine,dopo aver verificato la presenza dei recettori per Wnt5a, abbiamo iniziato degli studi di tipo funzionale mediante RNA interference (RNAi) per lo spegnimento genico (loss of function); perciò abbiamo messo a punto la metodica per il Knockdown di Wnt5a in colture primarie di ISEMFs con vettori adenovirali codificanti shRNA. Viceversa sono stati condotti degli studi di gain of function supplementando al terreno di coltura cellulare la proteina purificata rWnt5a. Sono stati effettuati dei saggi proliferazione rilevata mediante il metodo dell’incorporazione della timidina triziata da cui emerso che nelle ISEMFs Wnt5a promuove la proliferazione. Inoltre sono stati effettuati dei saggi sul ciclo cellulare mediante colorazione di ioduro di propidio e di resistenza all’apoptosi mediante anessina V e test di attivazione della caspasi-3, da cui emerge che questa glicoproteina promuove la sopravvivenza cellulare.
Complessivamente questi dati sembrano indicare che Wnt5a ha il ruolo di promuovere l’espansione e il mantenimento di questo tipo cellulare e quindi di perpetuare il danno tissutale e l’infiammazione cronica intestinale in RCU
Effect of synthetic peptides on osteoblast adhesion
No full textThe 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
Growth and Differentiation of Circulating Stem Cells After Extensive Ex Vivo Expansion
No full textBackground:: 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
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