Introduction: New implantable drug-delivery scaffolds, which combine bone substitutes and anti-cancer molecules, are emerging as an alternative therapy for bone tissue repair and treatment of bone cancer, including osteosarcoma (OS). Innovative ceramic scaffolds composed of strontium-substituted nanostructured calcium-deficient hydroxyapatite (CD-HA 2%Sr) with drugs methotrexate (CD-HA 2%Sr-MTX) and doxorubicin (CD-HA 2%Sr-DOX) may represent an innovative delivery system for a novel therapeutic strategy, both for bone regeneration and OS treatment. OS is an aggressive malignant neoplasm of the bone, which mainly affects pediatric and young adult patients. Despite the several disadvantages, treatments consist of surgery and chemotherapy using high-dose methotrexate and doxorubicin drugs. In this study, for the first time, these innovative biomaterials were tested in vitro for their efficacy. Aims. The aims of this study were to evaluate on human adipose-derived mesenchymal stem cells (hASCs) (i) the cytocompatibility and (ii) the osteoinductivity of CD-HA 2%Sr scaffolds and (iii) to assess the cytotoxic effect of CD-HA 2%Sr-MTX and CD-HA 2%Sr-DOX drug-delivery scaffolds on OS cells proliferation. Material and methods. The cytocompatibility and osteoinductivity properties of CD-HA 2%Sr were assessed in hASCs grown on the scaffold, up to day 14. Cytocompatibility was investigated using Alamar Blue and Live/Dead assays, cytoskeleton morphology and human extracellular matrix PCR Array, whereas osteoinductivity was evaluated using human osteogenesis PCR Array, ELISA test specific for osteocalcin (OCN) and mineral matrix deposition analysis. The anti-OS cell proliferation activity of CD-HA 2%Sr-MTX (45μg/mL) and CD-HA 2%Sr-DOX (5μg/mL) was assessed employing the fluorescent engineered human osteosarcoma cell line SAOS-eGFP grown on biomaterials, up to day 7. The effects of released drugs were evaluated in terms of cell numbers and fluorescence intensity rate reductions in SAOS-eGFP cells grown on scaffolds. In addition, the structure of CD-HA 2%Sr scaffolds with both hASCs and SAOS-eGFP cells, and the structure of CD-HA 2%Sr-MTX and CD-HA 2%Sr-DOX scaffolds with SAOS-eGFP cells were analysed by scanning electron (SEM) and confocal microscopes (CM). Results. The increasing number of hASCs, the well-organised cytoskeleton architecture alongside the up-regulation of extracellular matrix genes including integrins, cadherins, collagens and MMPs suggested that CD-HA 2%Sr scaffold owns in vitro cytocompatibility. In hASC cultures, the increased OCN protein expression and matrix mineralization, alongside the up-regulation of genes involved in skeletal development, demonstrated CD-HA 2%Sr scaffold displays in vitro osteoinductivity. In addition, decreasing cell numbers, SEM and CM analyses, alongside fluorescence intensity measurement indicated that CD-HA 2%Sr-MTX and CD-HA 2%Sr-DOX scaffolds displayed a cell-killing effect on SAOS-eGFP cells compared to CD-HA 2%Sr, thus validating the in vitro anti-proliferative properties of these scaffolds. Conclusion. Overall, these in vitro results demonstrated the cytocompatibility and osteoinductivity properties of CD-HA 2%Sr scaffold. In addition, experimental data with functionalized CD-HA 2%Sr-MTX and CD-HA 2%Sr-DOX scaffolds indicate that these innovative biomaterials could represent a good delivery system for methotrexate and doxorubicin, which are anti-tumour drugs, for OS therapy. At the same time, since these innovative scaffolds, employed in vitro as with drug-delivery system, own a good osteoinductive properties too, they could be used as a novel therapeutic strategy for bone tissue regeneration.

Nuovi biomateriali impiantabili con sistema di rilascio di farmaci anti-tumorali stanno emergendo come terapia alternativa per la riparazione del tessuto osseo e il trattamento del cancro osseo, compreso l'osteosarcoma (OS). Biomateriali composti da idrossiapatite nanostrutturata, con stronzio (CD-HA2%Sr) funzionalizzati con i farmaci metotrexato (CD-HA2%Sr-MTX) e doxorubicina (CD-HA2%Sr-DOX), potrebbero rappresentare una nuova strategia terapeutica per la rigenerazione ossea e il trattamento dell’OS. L’OS è una neoplasia maligna dell’osso che colpisce pazienti pediatrici. Nonostante gli svantaggi, la linea terapeutica attuale consiste in chirurgia associata a chemioterapia con alte dosi di metotrexato e doxorubicina. Per la prima volta in questo studio, l’efficacia dei suddetti biomateriali è stata testata in vitro. Gli obiettivi dello studio erano la valutazione delle (1)citocompatibilità e (2)osteoinduttività del biomateriale CD-HA2%Sr con l’impiego di cellule staminali mesenchimali da tessuto adiposo umano (hASCs) e (3)dell’effetto citotossico dei biomateriali funzionalizzati CD-HA2%Sr-MTX e CD-HA2%Sr-DOX su cellule tumorali di OS. La citocompatibilità e osteoinduttività del biomateriale CD-HA2%Sr sono state analizzate nelle hASCs cresciute a contatto con i biomateriali fino a 14 giorni. La citocompatibilità è stata investigata tramite i saggi di vitalità cellulare Alamar Blue e Live/Dead, morfologia del citoscheletro e PCR Array per l’analisi dei geni coinvolti nella deposizione della matrice extracellulare. L’osteoinduttività è stata valutata tramite PCR Array per l’analisi dei geni coinvolti nel processo di osteogenesi, test ELISA specifico per l’osteocalcina (OCN) e l’analisi della deposizione di fosfati di calcio. L’attività citotossica dei biomateriali CD-HA2%Sr-MTX (45μg/mL) e CD-HA2%Sr-DOX (5μg/mL) è stata valutata utilizzando cellule umane di osteosarcoma ingegnerizzate con la proteina verde fluorescente (SAOS-eGFP) cresciute a contatto con i biomateriali fino a 7 giorni. L’effetto citotossico dei farmaci rilasciati è stato analizzato mediante la valutazione del numero di cellule e la misurazione dell’intensità della fluorescenza emessa dalle cellule cresciute a contatto con i biomateriali. Inoltre, la struttura dei biomateriale CD-HA2%Sr a contatto con hASCs e SAOS-eGFP, e CD-HA2%Sr-MTX e CD-HA2%Sr-DOX a contatto con SAOS-eGFP sono state analizzate con il microscopio elettronico a scansione (SEM) e confocale (CM). La proliferazione delle hASCs a contatto con il biomateriale, l’architettura del citoscheletro ben organizzata e l’up-regolazione dei geni coinvolti nella deposizione della matrice extracellulare come integrine, caderine, collagene e metalloproteinasi hanno dimostrato che il biomateriale CD-HA2%Sr presenta citocompatibilità in vitro. L’aumentata espressione di OCN e mineralizzazione della matrice nelle colture di hASCs, insieme all’up-regolazione dei geni coinvolti nell’osteogenesi, hanno confermato l’osteoinduttività in vitro del biomateriale CD-HA2%Sr. D’altra parte, i risultati delle analisi Alamar Blue, SEM e CM, insieme alla misurazione della fluorescenza, hanno mostrato una diminuzione del numero di cellule SAOS-eGFP a contatto con i biomateriali funzionalizzati, CD-HA2%Sr-MTX e CD-HA2%Sr-DOX, rispetto a quello non funzionalizzato, CD-HA2%Sr, confermando la loro proprietà citotossica nei confronti delle cellule tumorali di OS. I risultati in vitro hanno dimostrato le proprietà di citocompatibilità e osteoinduttività del biomateriale CD-HA2%Sr e che i biomateriali funzionalizzati, CD-HA2%Sr-MTX e CD-HA2%Sr-DOX, potrebbero rappresentare un buon sistema di rilascio dei farmaci metotrexato e doxorubicina per la cura dell’OS. Poiché questi biomateriali possiedono buone proprietà osteoinduttive, potrebbero rappresentare una nuova strategia terapeutica per la rigenerazione dell’osso.

Innovative drug delivery scaffolds as novel therapeutic strategy for bone tissue regeneration and treatment of osteosarcoma

LANZILLOTTI, CARMEN
2022

Abstract

Introduction: New implantable drug-delivery scaffolds, which combine bone substitutes and anti-cancer molecules, are emerging as an alternative therapy for bone tissue repair and treatment of bone cancer, including osteosarcoma (OS). Innovative ceramic scaffolds composed of strontium-substituted nanostructured calcium-deficient hydroxyapatite (CD-HA 2%Sr) with drugs methotrexate (CD-HA 2%Sr-MTX) and doxorubicin (CD-HA 2%Sr-DOX) may represent an innovative delivery system for a novel therapeutic strategy, both for bone regeneration and OS treatment. OS is an aggressive malignant neoplasm of the bone, which mainly affects pediatric and young adult patients. Despite the several disadvantages, treatments consist of surgery and chemotherapy using high-dose methotrexate and doxorubicin drugs. In this study, for the first time, these innovative biomaterials were tested in vitro for their efficacy. Aims. The aims of this study were to evaluate on human adipose-derived mesenchymal stem cells (hASCs) (i) the cytocompatibility and (ii) the osteoinductivity of CD-HA 2%Sr scaffolds and (iii) to assess the cytotoxic effect of CD-HA 2%Sr-MTX and CD-HA 2%Sr-DOX drug-delivery scaffolds on OS cells proliferation. Material and methods. The cytocompatibility and osteoinductivity properties of CD-HA 2%Sr were assessed in hASCs grown on the scaffold, up to day 14. Cytocompatibility was investigated using Alamar Blue and Live/Dead assays, cytoskeleton morphology and human extracellular matrix PCR Array, whereas osteoinductivity was evaluated using human osteogenesis PCR Array, ELISA test specific for osteocalcin (OCN) and mineral matrix deposition analysis. The anti-OS cell proliferation activity of CD-HA 2%Sr-MTX (45μg/mL) and CD-HA 2%Sr-DOX (5μg/mL) was assessed employing the fluorescent engineered human osteosarcoma cell line SAOS-eGFP grown on biomaterials, up to day 7. The effects of released drugs were evaluated in terms of cell numbers and fluorescence intensity rate reductions in SAOS-eGFP cells grown on scaffolds. In addition, the structure of CD-HA 2%Sr scaffolds with both hASCs and SAOS-eGFP cells, and the structure of CD-HA 2%Sr-MTX and CD-HA 2%Sr-DOX scaffolds with SAOS-eGFP cells were analysed by scanning electron (SEM) and confocal microscopes (CM). Results. The increasing number of hASCs, the well-organised cytoskeleton architecture alongside the up-regulation of extracellular matrix genes including integrins, cadherins, collagens and MMPs suggested that CD-HA 2%Sr scaffold owns in vitro cytocompatibility. In hASC cultures, the increased OCN protein expression and matrix mineralization, alongside the up-regulation of genes involved in skeletal development, demonstrated CD-HA 2%Sr scaffold displays in vitro osteoinductivity. In addition, decreasing cell numbers, SEM and CM analyses, alongside fluorescence intensity measurement indicated that CD-HA 2%Sr-MTX and CD-HA 2%Sr-DOX scaffolds displayed a cell-killing effect on SAOS-eGFP cells compared to CD-HA 2%Sr, thus validating the in vitro anti-proliferative properties of these scaffolds. Conclusion. Overall, these in vitro results demonstrated the cytocompatibility and osteoinductivity properties of CD-HA 2%Sr scaffold. In addition, experimental data with functionalized CD-HA 2%Sr-MTX and CD-HA 2%Sr-DOX scaffolds indicate that these innovative biomaterials could represent a good delivery system for methotrexate and doxorubicin, which are anti-tumour drugs, for OS therapy. At the same time, since these innovative scaffolds, employed in vitro as with drug-delivery system, own a good osteoinductive properties too, they could be used as a novel therapeutic strategy for bone tissue regeneration.
MARTINI, Fernanda
DI VIRGILIO, Francesco
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Descrizione: PhD Thesis Lanzillotti Carmen
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/2482878
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