ITRM20100529A1 - BIOMEDICAL DEVICE TO TEST THE STEM CELLS - Google Patents
BIOMEDICAL DEVICE TO TEST THE STEM CELLS Download PDFInfo
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- ITRM20100529A1 ITRM20100529A1 IT000529A ITRM20100529A ITRM20100529A1 IT RM20100529 A1 ITRM20100529 A1 IT RM20100529A1 IT 000529 A IT000529 A IT 000529A IT RM20100529 A ITRM20100529 A IT RM20100529A IT RM20100529 A1 ITRM20100529 A1 IT RM20100529A1
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Description
Descrizione dell?invenzione avente per titolo: Description of the invention entitled:
?DISPOSITIVO BIOMEDICO PER TESTARE LA STAMINALIT? DELLE CELLULE? BIOMEDICAL DEVICE TO TEST STAMINALITY OF THE CELLS?
DESCRIZIONE DESCRIPTION
Settore della tecnica Technical sector
La presente domanda di brevetto per invenzione si riferisce in generale a un sistema di rilascio di farmaci, contente costrutti tridimensionali polimerici biologicamente attivi, per la valutazione della risposta biologica di colture cellulari nei confronti di differenti stimoli chimici e fattori ambientali e dei metodi di fabbricazione di detto sistema. The present patent application for invention refers in general to a drug delivery system, containing biologically active three-dimensional polymeric constructs, for the evaluation of the biological response of cell cultures towards different chemical stimuli and environmental factors and of the manufacturing methods of said system.
L?invenzione ? applicabile a qualsiasi settore, dove un tale tipo di sistema pu? essere vantaggiosamente utilizzato, ma preferibilmente questa riguarda il settore della biologia e pi? specificatamente della biologia cellulare. The invention? applicable to any sector, where such a type of system can? be advantageously used, but preferably this concerns the biology sector and more? specifically of cell biology.
Tecnica nota Known technique
Come ? noto, la terapia con le cellule staminali nonch? la medicina rigenerativa sono considerate tra i campi d?elezione della ricerca scientifica mondiale e stanno attraendo un incredibile numero di applicazioni cliniche in conseguenza del loro promettente potenziale per ci? che riguarda la rigenerazione o la sostituzione dei tessuti e degli organi danneggiati. L?interesse nei loro confronti ? dovuto, in particolar modo, agli incoraggianti risultati gi? ottenuti. How ? known, stem cell therapy as well as? regenerative medicine are considered among the elective fields of world scientific research and are attracting an incredible number of clinical applications as a result of their promising potential for this. which affects the regeneration or replacement of damaged tissues and organs. Interest in them? due, in particular, to the encouraging results already? obtained.
Tuttavia, un gran numero di interrogativi, riguardanti le migliori sorgenti di cellule staminali, il tipo, la loro sicurezza, il destino di queste cellule, le problematiche immunologiche connesse nonch? la loro armonica integrazione funzionale nell?organo bersaglio, rimangono ancora senza risposta. However, a large number of questions concerning the best sources of stem cells, the type, their safety, the fate of these cells, the related immunological issues as well as? their harmonic functional integration in the target organ, still remain unanswered.
Le questioni riguardanti la differenziazione delle cellule staminali o transdifferenziamento nelle applicazioni in vivo rappresentano ancora un tema di discussione, soprattutto per quanto riguarda l'integrazione funzionale delle cellule nell?ambiente ospite e, cosa pi? importante, la loro trasformazione in cellule maligne. Inoltre, l?individuazione costante di nuove fonti di cellule staminali o nicchie e di nuovi tipi di cellule progenitrici ? fonte di ulteriore confusione e preoccupazione per quanto riguarda l?individuazione delle cellule ottimali candidate per la medicina rigenerativa. Issues regarding stem cell differentiation or transdifferentiation in in vivo applications are still a topic of discussion, especially regarding the functional integration of cells in the host environment and, what more? important, their transformation into malignant cells. Furthermore, the constant identification of new sources of stem cells or niches and of new types of progenitor cells? source of further confusion and concern regarding the identification of optimal candidate cells for regenerative medicine.
Chiaramente, la rigenerazione degli organi richiede una complessa cascata di eventi che va ben oltre la semplice iniezione nel sito idoneo dell?appropriato tipo di cellule. In questo senso, la caratterizzazione dei fattori presenti nel microambiente ostile del tessuto danneggiato, che ostacolano la sopravvivenza e l'integrazione funzionale delle cellule trapiantate, ? oltremodo essenziale. Clearly, organ regeneration requires a complex cascade of events that goes far beyond simply injecting the appropriate cell type into the appropriate site. In this sense, the characterization of the factors present in the hostile microenvironment of the damaged tissue, which hinder the survival and functional integration of the transplanted cells,? extremely essential.
Un reale approccio alla medicina rigenerativa dovrebbe considerare l'importanza della matrice extracellulare (ECM) e il forte segnale biologico da questa proveniente. L?ambiente del tessuto connettivo e il microambiente strutturale nel quale vengono immerse le cellule, esercitano un gran numero di stimoli che incidono sulle funzioni cellulari e supportano la proliferazione e il differenziamento delle cellule. A real approach to regenerative medicine should consider the importance of the extracellular matrix (ECM) and the strong biological signal coming from it. The environment of the connective tissue and the structural microenvironment in which the cells are immersed exert a large number of stimuli that affect cellular functions and support cell proliferation and differentiation.
La maggior parte dei tipi cellulari dei mammiferi hanno dimostrato di essere dipendenti dall?effettiva adesione a un supporto e quindi, per avere successo, la medicina rigenerativa non pu? ignorare l'importanza della matrice extracellulare (ECM) come principale sostegno cellulare, in grado di offrire un forte segnale biologico per la rigenerazione del tessuto. Da qui nasce l?esigenza di reperire procedure di rigenerazione dei tessuti del corpo umano mediante la semina di cellule su strutture sempre pi? innovative, dette scaffolds, create da opportuni materiali e dotate di caratteristiche opportune e quindi la loro coltivazione in appositi reattori fino alla colonizzazione degli scaffolds stessi e alla produzione di nuovo tessuto (matrice extracellulare, ECM). La fabbricazione di tali supporti (scaffolds) realizzati di materiali in grado di offrire una superficie adatta per l?adesione cellulare, la proliferazione, la differenziazione e la migrazione e in cui appropriati stimoli biochimici possano guidare la crescita di neo-tessuto funzionale, ? dunque di fondamentale importanza. Tali concetti sono alla base dell?area della cos? detta ?ingegneria tissutale?, che contempla la combinazione di cellule e scaffolds biodegradabili per produrre costrutti tridimensionali utili per un successivo impianto in vivo e la rigenerazione di organi. Most mammalian cell types have been shown to be dependent on effective adhesion to a support and therefore regenerative medicine cannot be successful. ignore the importance of the extracellular matrix (ECM) as the main cellular support, able to offer a strong biological signal for tissue regeneration. Hence the need to find regeneration procedures for the tissues of the human body by sowing cells on increasingly more structures. innovative, called scaffolds, created from suitable materials and equipped with suitable characteristics and then their cultivation in special reactors until the colonization of the scaffolds themselves and the production of new tissue (extracellular matrix, ECM). The fabrication of such scaffolds made of materials able to offer a suitable surface for cell adhesion, proliferation, differentiation and migration and in which appropriate biochemical stimuli can guide the growth of functional neo-tissue,? therefore of fundamental importance. These concepts are the basis of the area of the cos? called? tissue engineering ?, which contemplates the combination of biodegradable cells and scaffolds to produce three-dimensional constructs useful for subsequent in vivo implantation and organ regeneration.
In ambito generale la presente invenzione nasce nel campo della biologia cellulare e dell'ingegneria tissutale, dall'idea di un approccio biomimetico basato sulla simulazione della ECM e l?impiego di fattori di crescita, per guidare la differenziazione delle cellule staminali. L'idea di concentrare e organizzare spazialmente una citochina, o un altro mediatore biologico, all'interno di un ambiente tridimensionale, simulando l'istoarchitettura del tessuto nativo, ? stata recentemente messa in evidenza. Quest'approccio consente di sfruttare in pieno i vantaggi derivanti dall'uso delle cellule staminali nella medicina rigenerativa, consentendo la differenziazione delle cellule staminali in un microambiente tridimensionale preservando, al tempo stesso, le cellule dai fattori nocivi presenti nell'organo danneggiato. Oltre l'utilizzo in vivo, questo concetto ? rilevante anche per le applicazioni in vitro per quanto riguarda la comprensione dei meccanismi molecolari alla base della differenziazione delle cellule staminali. In the general context, the present invention arises in the field of cell biology and tissue engineering, from the idea of a biomimetic approach based on the simulation of the ECM and the use of growth factors, to guide the differentiation of stem cells. The idea of concentrating and spatially organizing a cytokine, or another biological mediator, within a three-dimensional environment, simulating the histarchitecture of native tissue,? was recently highlighted. This approach allows to fully exploit the advantages deriving from the use of stem cells in regenerative medicine, allowing the differentiation of stem cells in a three-dimensional microenvironment while preserving the cells from harmful factors present in the damaged organ. Besides in vivo use, this concept? also relevant for in vitro applications as regards the understanding of the molecular mechanisms underlying the differentiation of stem cells.
Studi precedenti hanno dimostrato l'induzione del processo di differenziazione delle cellule staminali all'interno di scaffolds funzionalizzati con fattori di crescita. L?idrossiapatite per la differenziazione osteocondrale e i glicosaminoglicani vaso-specifici, come l'eparina per il differenziamento in senso endoteliale, sono stati utilizzati in scaffolds tridimensionali in grado di mimare la ECM, fornendo con ci? una prova della possibilit? di produrre uno scaffold adatto alla semina di cellule staminali contenente gli appositi fattori per la differenziazione verso i fenotipi desiderati. Inoltre, i membri della superfamiglia TGF-B hanno dimostrato di essere efficaci nell'indurre la differenziazione muscolare. Previous studies have demonstrated the induction of the differentiation process of stem cells within scaffolds functionalized with growth factors. Hydroxyapatite for osteochondral differentiation and vaso-specific glycosaminoglycans, such as heparin for endothelial differentiation, have been used in three-dimensional scaffolds capable of mimicking the ECM, providing with this a proof of the possibility? to produce a scaffold suitable for stem cell seeding containing the appropriate factors for differentiation towards the desired phenotypes. Furthermore, members of the TGF-B superfamily have been shown to be effective in inducing muscle differentiation.
Il brevetto KR20060127399 di Melvin S. Schindler et al., si riferisce a una struttura nanofibrillare per la coltura cellulare, ma non comprende n? i composti bioattivi n? i farmaci impiegati nella presente invenzione e non ? destinato allo screening delle capacit? differenziative delle cellule o della loro risposta biologica. Patent KR20060127399 by Melvin S. Schindler et al., Refers to a nanofibrillar structure for cell culture, but does not include n? the bioactive compounds n? the drugs used in the present invention and not? intended for screening of capacities? differentiation of cells or their biological response.
Il brevetto EP1989295 si riferisce a un metodo per indurre la differenziazione osteogenica nelle cellule staminali mesenchimali, tuttavia non considera l'impiego di idrossiapatite e di bifosfonati per indurre la differenziazione osteogenica. Patent EP1989295 refers to a method for inducing osteogenic differentiation in mesenchymal stem cells, however it does not consider the use of hydroxyapatite and bisphosphonates to induce osteogenic differentiation.
Il brevetto US2003026786, da Pittenger MF et al., descrive un metodo per indurre la differenziazione condrogenica nelle cellule staminali mesenchimali, tuttavia, la presente invenzione si riferisce all'uso di ulteriori e diversi composti bioattivi. US2003026786, from Pittenger MF et al., Describes a method for inducing chondrogenic differentiation in mesenchymal stem cells, however, the present invention refers to the use of further and different bioactive compounds.
Il brevetto AU2003234740 B2, da Pittenger et al., descrive un metodo per indurre la differenziazione adipogenica di cellule staminali mesenchimali, ma non considera l'aggiunta di ulteriori composti. Patent AU2003234740 B2, from Pittenger et al., Describes a method for inducing adipogenic differentiation of mesenchymal stem cells, but does not consider the addition of further compounds.
Il brevetto WO9639035, da Caplan et al., si riferisce a un metodo per indurre il differenziamento miogenico, ma non considera l'utilizzo di ulteriori composti. Il brevetto US2006003311, da Fulde et al., descrive un metodo per lo screening in vitro di eventi cellulari. Tuttavia questo fa riferimento a condizioni di coltura in vitro di cellule, in cui le cellule trasfettate contenenti costrutti promoterreporter sono coltivate in un ambiente tridimensionale simile ad un tessuto. Patent WO9639035, from Caplan et al., Refers to a method for inducing myogenic differentiation, but does not consider the use of further compounds. US2006003311, from Fulde et al., Describes a method for in vitro screening of cellular events. However this refers to in vitro cell culture conditions, in which transfected cells containing promoterreporter constructs are grown in a three-dimensional tissue-like environment.
Descrizione dell?invenzione Description of the invention
Il concetto innovativo alla base della presente invenzione, che rappresenta un evidente strumento utile nelle mani del ricercatore, consiste nel prevedere la possibilit? di attuare uno screening rapido della staminalit? di un tipo cellulare e delle sue capacit? differenziative. The innovative concept at the base of the present invention, which represents an evident useful tool in the hands of the researcher, consists in foreseeing the possibility? to implement a rapid screening of the stem? of a cellular type and its capabilities? differentiation.
Pi? specificatamente la presente invenzione riferisce di un dispositivo, basato su costrutti polimerici tridimensionali biologicamente attivi, in grado di testare la staminalit? di colture o co-colture cellulari, di valutare la loro capacit? di differenziazione osteogenica, condrogenica, adipogenica, endoteliale e muscolare, di analizzare la risposta biologica ai composti chimici e infine ai metodi impiegati per la fabbricazione dello stesso. Pi? specifically, the present invention relates to a device, based on biologically active three-dimensional polymeric constructs, capable of testing the staminality? of cultures or cell co-cultures, to evaluate their ability? of osteogenic, chondrogenic, adipogenic, endothelial and muscular differentiation, to analyze the biological response to chemical compounds and finally to the methods used for its manufacture.
Ancora pi? specificatamente la presente invenzione riferisce di un dispositivo biomedico costituito da gruppi di supporti polimerici tridimensionali funzionalizzati, in cui ciascun gruppo ? caratterizzato da una differente composizione o funzionalizzazione (secondo quanto riportato in seguito) ed ? in grado di promuovere il differenziamento cellulare verso uno specifico fenotipo (tra cui quello osteogenico, condrogenico, endoteliale, adipogenico e muscolare) oppure di permettere la valutazione della risposta biologica nei confronti della composizione chimica utilizzata nella funzionalizzazione. Even more? specifically, the present invention relates to a biomedical device consisting of groups of functionalized three-dimensional polymeric supports, in which each group? characterized by a different composition or functionalization (as reported below) and? able to promote cell differentiation towards a specific phenotype (including osteogenic, chondrogenic, endothelial, adipogenic and muscular) or to allow the assessment of the biological response to the chemical composition used in functionalization.
L'unicit? e la non-ovviet? di tale invenzione si basa sull'uso della combinazione di agenti bioattivi (cocktails), rivelati per la prima volta in questa invenzione, dispersi nelle fibre degli scaffold polimerici tridimensionali, al fine di realizzare un test di differenziazione in grado di indurre il differenziamento delle cellule verso gli specifici e predetti fenotipi osteogenici, condrogenici, endoteliali, adipogenici e muscolari. The uniqueness? and the non-obvious? of this invention is based on the use of the combination of bioactive agents (cocktails), revealed for the first time in this invention, dispersed in the fibers of the three-dimensional polymeric scaffolds, in order to carry out a differentiation test capable of inducing cell differentiation towards the specific and predicted osteogenic, chondrogenic, endothelial, adipogenic and muscular phenotypes.
Il miglioramento rispetto ai test di differenziamento sviluppati nell?arte nota, si basa, principalmente, sulla presenza di costrutti polimerici tridimensionali, la cui struttura ? realizzata in modo da costituire una matrice in grado di mimare quella extracellulare (ECM), esaltando la somiglianza biologica dell'ambiente di coltura con quello nativo, in cui le cellule si trovano normalmente, rispetto ai substrati bidimensionali comunemente utilizzati in vitro. La capacit? di valutare la staminalit? delle colture senza la necessit? di impiegare terreni di coltura appositamente realizzati ? dovuta alla presenza di fattori differenziativi all?interno dei supporti stessi. The improvement with respect to the differentiation tests developed in the known art is mainly based on the presence of three-dimensional polymeric constructs, whose structure? realized in such a way as to constitute a matrix capable of mimicking the extracellular one (ECM), enhancing the biological similarity of the culture environment with the native one, in which the cells are normally found, compared to the two-dimensional substrates commonly used in vitro. The capacity to evaluate the staminality? of crops without the need? to use specially developed culture media? due to the presence of differentiating factors inside the supports themselves.
L'invenzione ? quindi utile per valutare la risposta biologica di vari tipi di cellule e pu? trovare impiego in diverse applicazioni, tra cui: l?identificazione e la caratterizzazione di staminali e cellule progenitrici; la valutazione della risposta a composti farmacologici di cellule cancerose differenziate o staminali; la valutazione di resistenza ai farmaci da parte di cellule staminali, di cellule differenziate, di batteri o di virus. The invention? therefore useful for evaluating the biological response of various types of cells and pu? find use in various applications, including: identification and characterization of stem and progenitor cells; the evaluation of the response to pharmacological compounds of differentiated cancer cells or stem cells; the assessment of drug resistance by stem cells, differentiated cells, bacteria or viruses.
Nella realizzazione della presente invenzione, la semplicit? d?impiego e la migliorata adesione e sopravvivenza cellulare mostrata dai supporti oggetto dell?invenzione stessa, rispetto alle condizioni di coltura dei comuni supporti non tridimensionali, permettono l'applicazione diretta di biopsie tissutali contenenti cellule staminali o di escissioni tumorali intraoperatorie al sistema oggetto dell?invenzione, in seguito ad una lieve dissociazione enzimatica tissutale, al fine di ottenere un rapido screening delle capacit? differenziative di cellule staminali potenziali o della resistenza a composti farmacologici di cellule tumorali. In the realization of the present invention, the simplicity? use and the improved adhesion and cell survival shown by the supports object of the invention itself, compared to the culture conditions of common non-three-dimensional supports, allow the direct application of tissue biopsies containing stem cells or intraoperative tumor excisions to the system object of the ? invention, following a slight tissue enzymatic dissociation, in order to obtain a rapid screening of the capacities? potential stem cell differentiation or resistance to drug compounds of cancer cells.
Modi preferiti di realizzazione dell?invenzione Preferred ways of carrying out the invention
Si vuole sottolineare come nel seguito saranno illustrate, a titolo esemplificativo ma non limitativo, soltanto alcune delle possibili forme di realizzazione della presente invenzione, essendo possibile descriverne molte altre sulla base delle particolari soluzioni tecniche individuate. It should be emphasized that in the following, only some of the possible embodiments of the present invention will be illustrated, by way of non-limiting example, it being possible to describe many others on the basis of the particular technical solutions identified.
In un aspetto pi? specifico la presente invenzione si riferisce a un sistema a rilascio controllato di farmaco in grado di testare la staminalit? di colture cellulari o co-colture valutandone, simultaneamente, la capacit? di differenziazione osteogenica, condrogenica, adipogenica, endoteliale e muscolare unitamente alla risposta biologica nei confronti di diversi composti chimici. In one aspect more? Specifically, the present invention refers to a controlled drug release system capable of testing the staminality? of cell cultures or co-cultures by evaluating, simultaneously, the capacity? of osteogenic, chondrogenic, adipogenic, endothelial and muscular differentiation together with the biological response to different chemical compounds.
In un aspetto ancora pi? specifico, l'invenzione si riferisce a un supporto tridimensionale e biologicamente attivo, in cui il materiale ? rappresentato da un polimero sintetico o da una miscela di polimeri contenenti almeno uno tra i seguenti: acido polilattico (PLA), acido poliglicolico (PGA), policaprolattone (PCL), polistirene o una loro combinazione e in cui il PLA rappresenta la composizione preferita; oppure da un polimero naturale contenente almeno uno tra collagene, chitosano, alginato o una loro combinazione e in cui il collagene rappresenta la composizione preferita. Si riferisce anche alle miscele contenenti i polimeri derivati sinteticamente o naturalmente dai materiali sopra descritti. In an even more? specific, the invention refers to a three-dimensional and biologically active support, in which the material? represented by a synthetic polymer or a mixture of polymers containing at least one of the following: polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), polystyrene or a combination thereof and in which PLA is the preferred composition; or from a natural polymer containing at least one of collagen, chitosan, alginate or a combination thereof and in which collagen is the preferred composition. It also refers to blends containing the polymers derived synthetically or naturally from the materials described above.
Nella realizzazione preferita la presente invenzione si riferisce a scaffold tridimensionali e realizzati con il metodo dell?elettrofilatura, in cui il supporto tridimensionale biologicamente attivo include fibre non tessute che presentano un diametro medio compreso tra 30 nanometri e 500 micron e pi? preferibilmente tra 0,5 e 5 micron. In ulteriori realizzazioni della presente invenzione, il supporto tridimensionale ? ottenuto con altre tecniche di fabbricazione, comprendenti l?utilizzazione di porogeni, di separazione di fase, di liofilizzazione e di tecniche di stereo litografia (es. modellazione per deposizione da fuso). In the preferred embodiment, the present invention relates to three-dimensional scaffolds made with the electrospinning method, in which the biologically active three-dimensional support includes non-woven fibers having an average diameter between 30 nanometers and 500 microns and more. preferably between 0.5 and 5 microns. In further embodiments of the present invention, the three-dimensional support? obtained with other manufacturing techniques, including the use of porogens, phase separation, lyophilization and stereo lithography techniques (eg modeling by melt deposition).
In ulteriori aspetti, la presente invenzione si riferisce alla sopra menzionata piattaforma polimerica tridimensionale in combinazione con miscele preformate di composti bioattivi in grado di testare selettivamente le capacit? di differenziazione delle cellule verso i fenotipi osteogenici, condrogenici, adipogenici, endoteliali e muscolari o alla valutazione della risposta biologica delle cellule nei confronti di altri composti farmacologici. In alcune realizzazioni, detti supporti sono combinati con tali composti bioattivi per mezzo di almeno una delle successive tecniche: In further aspects, the present invention relates to the aforementioned three-dimensional polymer platform in combination with preformed mixtures of bioactive compounds capable of selectively testing the capacities. differentiation of cells towards osteogenic, chondrogenic, adipogenic, endothelial and muscle phenotypes or the evaluation of the biological response of the cells towards other pharmacological compounds. In some embodiments, said supports are combined with such bioactive compounds by means of at least one of the following techniques:
- legame chimico che coinvolge gruppi amminici primari, gruppi carbossilici, gruppi tiolici sulla superficie dei polimeri forniti con gruppi amminici primari, gruppi carbossilici, gruppi tiolici appartenenti a dette molecole bioattive, in presenza o in assenza di catalizzatori; - chemical bond involving primary amino groups, carboxylic groups, thiol groups on the surface of the polymers supplied with primary amino groups, carboxyl groups, thiol groups belonging to said bioactive molecules, in the presence or absence of catalysts;
- miscelazione normotermica in soluzioni dei polimeri utilizzando solventi organici o inorganici, come diclorometano, tetraidrofurano, cloroformio, acido acetico, acido trifluoroacetico, acido lattico, esafluoropropanolo, etanolo, metanolo, o miscele dei suddetti, e in cui la miscela di clorometano/metanolo (80:20 v/v) rappresenta il solvente preferito; - normothermic mixing in polymer solutions using organic or inorganic solvents, such as dichloromethane, tetrahydrofuran, chloroform, acetic acid, trifluoroacetic acid, lactic acid, hexafluoropropanol, ethanol, methanol, or mixtures of the above, and in which the chloromethane / methanol mixture ( 80:20 v / v) represents the preferred solvent;
- assorbimento di dette molecole sul polimero per mezzo di microspray o elettrospray con o senza successivo legame chimico. A differenza del brevetto KR20060127399, citato in precedenza, il dispositivo qui riferito include l?impiego di composti bioattivi e medicinali ed ? finalizzato alla verifica delle abilit? differenziative di cellule o della risposta biologica delle cellule. - absorption of said molecules on the polymer by means of microspray or electrospray with or without subsequent chemical bond. Unlike the patent KR20060127399, cited above, the device referred to here includes the use of bioactive and medicinal compounds and? aimed at verifying the skills? differentiation of cells or the biological response of cells.
Deve altres? riferirsi, che la presente invenzione prevede di seguito alcune ulteriori forme di realizzazione in corrispondenza delle seguenti varianti qualitative e quantitative riguardanti i costituenti del cocktail di funzionalizzazione dei predetti scaffolds tridimensionali bioattivi. It must also? to refer, that the present invention provides hereinafter some further embodiments in correspondence with the following qualitative and quantitative variants concerning the constituents of the functionalization cocktail of the aforesaid three-dimensional bioactive scaffolds.
In una realizzazione del processo di produzione della presente invenzione i sopra menzionati scaffolds tridimensionali bioattivi sono funzionalizzati utilizzando un cocktail di funzionalizzazione composto di: 1-20% di FBS (con la composizione preferita pari al 10%), 3,5-10 mM di b-glicerofosfato (con la composizione preferita pari a 3,5 mM), 0,1-10 ?M di desametasone (con la composizione preferita pari a 0,1 ?M), 50 ?M di ascorbat-2-fosfato, 1-25% di idrossiapatite (con la composizione preferita pari al 10%), bifosfonati azotati e non azotati per indurre il differenziamento osteogenico (con l?impiego di 10-100 ?M di zoleandronato nella composizione preferita). Differentemente dall?EP1989285, riferito sopra, il dispositivo qui riferito considera l?impiego di idrossiapatite e bifosfonati per l?induzione del differenziamento osteogenico. In one embodiment of the manufacturing process of the present invention the aforementioned bioactive three-dimensional scaffolds are functionalized using a functionalization cocktail composed of: 1-20% of FBS (with the preferred composition equal to 10%), 3.5-10 mM of b-glycerophosphate (with preferred composition 3.5 mM), 0.1-10 μM dexamethasone (with preferred composition 0.1 μM), 50 μM ascorbat-2-phosphate, 1 -25% hydroxyapatite (with the preferred composition equal to 10%), nitrogenous and non-nitrogenous bisphosphonates to induce osteogenic differentiation (with the use of 10-100? M of zoleandronate in the preferred composition). Differently from EP1989285, referred to above, the device referred to here considers the use of hydroxyapatite and bisphosphonates for the induction of osteogenic differentiation.
In un?ulteriore realizzazione del processo di produzione della presente invenzione i sopra menzionati scaffolds tridimensionali bioattivi sono funzionalizzati utilizzando un cocktail di funzionalizzazione composto di: IMDM ad alto glucosio, 1% di FBS, 0,1-10 ?M di desametasone (con la composizione preferita pari a 0,1 ?M), 50-200 ?g/ml di acido ascorbico (con la composizione preferita pari a 50 ?g/ml), 100 ?g/ml di sodio piruvato, 40 ?g/ml di prolina, 100-700 ng/ml di proteina morfogenica dell?osso-6 (con la composizione preferita pari a 500 ng/ml), 0,1-10 ng/ml di TGF-?3 (con la composizione preferita pari a 10 ng/ml) (oppure 10 ng/ml di TGF-?1), 10-150 mg/ml di ITS (con la composizione preferita pari a 50 mg/ml) premix (6,25 ?g/ml di insulina, 6,25 ?g/ml di transferrina, 6,25 ng/ml di acido selenioso, 1,25 mg/ml di albumina da siero bovino e 5,35 mg/ml di acido linoleico), 1-20% di acido ialuronico (con la composizione preferita pari al 10%), 1-10% di condroitina solfato (con la composizione preferita pari al 5%), 0,25-12,5 mg/ml di bevacizumab (con la composizione preferita pari a 5 mg/ml) per indurre il differenziamento condrogenico. A differenza di US2003026786, riferito sopra, la presente invenzione fa riferimento all?impiego di differenti composti bioattivi (in particolare 1-20% di acido ialuronico (con la composizione preferita pari al 10%), 1-10% di condroitina solfato (con la composizione preferita pari al 5%), 0,25-12,5 mg/ml di bevacizumab (con la composizione preferita pari a 5 mg/ml). In ancora un'ulteriore realizzazione del processo di produzione della presente invenzione i sopra menzionati scaffolds tridimensionali bioattivi sono funzionalizzati utilizzando un cocktail di funzionalizzazione composto di: 1-15% di FBS (con la composizione preferita pari al 10%), 1-20% di siero di coniglio (con la composizione preferita pari al 5%), 0,1-10 ?M di desametasone (con la composizione preferita pari a 1 ?M), 50-300 mM di indometacina (con la composizione preferita pari a 200 mM), 1-20 ?M di insulina (con la composizione preferita pari a 10 ?M), tiazolidinedione, agonista dei recettori attivati dalla proliferazione dei perossisomi (PPAR) (preferibilmente 1-100 ?M di troglitazone), 0,5 mM di 3-isobutil-1-metilxantina per indurre il differenziamento adipogenico. A differenza di AU2003234740B2, riferito sopra, il dispositivo qui riferito considera l?aggiunta di differenti composti, in particolare tiazolidinedioni, per indurre i recettori attivati dalla proliferazione perossisomiale (PPAR) e stimolare le vie differenziative mediate dalla leptina. In ancora un?ulteriore realizzazione del processo di produzione della presente invenzione i sopra menzionati scaffolds tridimensionali bioattivi sono funzionalizzati utilizzando un cocktail di funzionalizzazione composto di: 20-200 UI/ml di eparina, 50 ng/ml di VEGF, 0,01-100 ?g/ml di eritropoietina, 1-50 ng/ml di estratto cerebrale bovino, 1-100 ?M di un fibrato (specialmente clofibrato) per indurre il differenziamento endoteliale. A differenza dell?EP2188370, riferito sopra, la presente invenzione fa riferimento all?impiego di differenti composti, in particolare di un fibrato (preferibilmente clofibrato) per stimolare le vie differenziative mediate dall?ossido nitrico attraverso la Proteina-Kinasi-C. In a further embodiment of the manufacturing process of the present invention the aforementioned bioactive three-dimensional scaffolds are functionalized using a functionalization cocktail composed of: high glucose IMDM, 1% FBS, 0.1-10? M dexamethasone (with the preferred composition of 0.1? M), 50-200? g / ml of ascorbic acid (with the preferred composition equal to 50? g / ml), 100? g / ml of sodium pyruvate, 40? g / ml of proline, 100-700 ng / ml of bone morphogenic protein-6 (with the preferred composition equal to 500 ng / ml), 0.1-10 ng / ml of TGF-? 3 (with the preferred composition equal to 10 ng / ml) (or 10 ng / ml of TGF-? 1), 10-150 mg / ml of ITS (with the preferred composition equal to 50 mg / ml) premix (6.25? g / ml of insulin, 6 , 25? G / ml of transferrin, 6.25 ng / ml of selenious acid, 1.25 mg / ml of bovine serum albumin and 5.35 mg / ml of linoleic acid), 1-20% hyaluronic acid ( with the preferred composition equal to 10%), 1-10% of chondroitin sulfate (with the com preferred position equal to 5%), 0.25-12.5 mg / ml of bevacizumab (with the preferred composition equal to 5 mg / ml) to induce chondrogenic differentiation. Unlike US2003026786, referred to above, the present invention refers to the use of different bioactive compounds (in particular 1-20% of hyaluronic acid (with the preferred composition equal to 10%), 1-10% of chondroitin sulphate (with the preferred composition equal to 5%), 0.25-12.5 mg / ml of bevacizumab (with the preferred composition equal to 5 mg / ml). In still a further embodiment of the manufacturing process of the present invention the aforementioned bioactive three-dimensional scaffolds are functionalized using a functionalization cocktail consisting of: 1-15% FBS (with the preferred composition equal to 10%), 1-20% rabbit serum (with the preferred composition equal to 5%), 0 , 1-10 μM dexamethasone (with preferred composition equal to 1 μM), 50-300 mM indomethacin (with preferred composition equal to 200 mM), 1-20 μM insulin (with preferred composition equal a 10? M), thiazolidinedione, agonist of receptors activated by proliferation ne of peroxisomes (PPAR) (preferably 1-100 µM of troglitazone), 0.5 mM of 3-isobutyl-1-methylxanthine to induce adipogenic differentiation. Unlike AU2003234740B2, reported above, the device reported here considers the addition of different compounds, in particular thiazolidinediones, to induce peroxisomal proliferation-activated receptors (PPARs) and stimulate leptin-mediated differentiation pathways. In still a further embodiment of the production process of the present invention the above mentioned bioactive three-dimensional scaffolds are functionalized using a functionalization cocktail composed of: 20-200 IU / ml of heparin, 50 ng / ml of VEGF, 0.01-100 ? g / ml of erythropoietin, 1-50 ng / ml of bovine brain extract, 1-100? M of a fibrate (especially clofibrate) to induce endothelial differentiation. Unlike EP2188370, referred to above, the present invention refers to the use of different compounds, in particular of a fibrate (preferably clofibrate) to stimulate the differentiation pathways mediated by nitric oxide through Protein-Kinase-C.
In ancora un?ulteriore realizzazione del processo di produzione della presente invenzione i sopra menzionati scaffolds tridimensionali bioattivi sono funzionalizzati utilizzando un cocktail di funzionalizzazione composto di: 1-5 ng/ml di TGF-?1 (con la composizione preferita pari a 2,5 ng/ml), 1-5 ng/ml di PDGFBB (con la composizione preferita pari a 5 ng/ml), 1-3 ?mol/l di 5-azacitidina (con la composizione preferita pari a 3 ?mol/l) per indurre il differenziamento miogenico. In still a further embodiment of the production process of the present invention the above mentioned bioactive three-dimensional scaffolds are functionalized using a functionalization cocktail composed of: 1-5 ng / ml of TGF-? 1 (with the preferred composition equal to 2.5 ng / ml), 1-5 ng / ml of PDGFBB (with the preferred composition equal to 5 ng / ml), 1-3? mol / l of 5-azacytidine (with the preferred composition equal to 3? mol / l) to induce myogenic differentiation.
In ancora un'ulteriore realizzazione della presente invenzione si fa riferimento a supporti polimerici tridimensionali allocati all?interno di almeno un contenitore plastico compartimentalizzato per la valutazione, simultanea ma isolata, delle risposte biologiche delle cellule nei confronti di differenti composizioni chimiche, della multi potenzialit? delle cellule candidate e del grado di staminalit?, cio? la capacit? di differenziare verso differenti linee cellulari. A differenza del US2006003311, riferito sopra, il dispositivo della presente invenzione contiene fattori in grado di indurre nelle cellule la risposta biologica oggetto dell?analisi e non richiede l?aggiunta di composti farmacologici nel mezzo di coltura, dato che le caratteristiche intrinseche dei supporti funzionalizzati sono in grado di indurre, da sole, le volute risposte biologiche. Inoltre, il dispositivo biomedico qui rivelato non richiede la trasfezione delle cellule con geni reporter. In still a further embodiment of the present invention reference is made to three-dimensional polymeric supports located inside at least one compartmentalized plastic container for the simultaneous but isolated evaluation of the biological responses of the cells towards different chemical compositions, of the multi-potentiality. of the candidate cells and the degree of staminality ?, that is? the capacity? to differentiate towards different cell lines. Unlike US2006003311, referred to above, the device of the present invention contains factors capable of inducing the biological response under analysis in the cells and does not require the addition of pharmacological compounds in the culture medium, given that the intrinsic characteristics of the functionalized supports they are able to induce, by themselves, the desired biological responses. Furthermore, the biomedical device revealed here does not require transfection of cells with reporter genes.
La presente invenzione ? stata descritta a titolo illustrativo, ma non limitativo, secondo alcune forme di realizzazione preferite, ma ? da intendersi che eventuali variazioni e/o modifiche potranno essere apportate dagli esperti del settore senza per questo esulare dal relativo ambito di protezione, cos? come definito dalle seguenti rivendicazioni dipendenti. The present invention? has been described for illustrative, but not limitative purposes, according to some preferred embodiments, but? it is to be understood that any changes and / or modifications may be made by experts in the sector without thereby going beyond the relative scope of protection, so? as defined by the following dependent claims.
Claims (11)
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP3960848A4 (en) * | 2019-04-23 | 2023-07-26 | Cellatoz Therapeutics, Inc. | Method for regulation of selective differentiation of musculoskeletal stem cells |
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| US20050095695A1 (en) * | 2003-11-05 | 2005-05-05 | Shindler Melvin S. | Nanofibrillar structure and applications including cell and tissue culture |
| WO2006083394A2 (en) * | 2004-12-21 | 2006-08-10 | Ethicon, Inc. | Postpartum cells derived from placental tissue, and methods of making, culturing, and using the same |
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| EP3960848A4 (en) * | 2019-04-23 | 2023-07-26 | Cellatoz Therapeutics, Inc. | Method for regulation of selective differentiation of musculoskeletal stem cells |
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