Nosocomial infections are an ever growing medical and hospital problem. These infections have a significant clinical and economic impact: according to the World Health Organization (WHO), nosocomial infections cause prolonged length of stay, long-term disability, increased resistance of microorganisms to antibiotics, an additional economic burden on health care systems, and significant excess mortality. In Italy, 10.780 people die each year from hospital infections due to antibiotic resistance. By 2050 then there will be about 450.000 people dying, and this phenomenon will have cost our country at least 12 billion euros. Hence the purpose of this thesis was to evaluate the antibacterial, antiviral and cell proliferative effects of copper-doped calcium phosphate bioglasses (CPG_Cu) compared to undoped bioglasses (CPG) for application in medical implantations. These bioglasses are made of inorganic calcium-phosphate and showed in previous works to have good mechanical, thermal and optic properties and also to be bioresorbable. Thanks to these characteristics, they could have a potential for biomedical applications. Hence, a valid approach to make these calcium-phosphate bioglasses antibacterial, could be the doping with ions such as copper (Cu2+). Three strains of Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae) as well as two strains of Gram-positive bacteria (Staphylococcus aureus and the MRSA strain) were used to evaluate the antibacterial activity. After direct incubation onto both types of bioglasses (Cu2+-doped and undoped), cell viability (MTT assay and CLSM) and morphology (SEM observations) were performed; the results showed that both the antibacterial and the anti-adhesive properties were present in the CPG_Cu bioglasses, especially against Gram-negative bacteria; meanwhile the activities against Gram-positive bacteria were lower. Next, CPG and CPG_Cu bioglasses were at first treated in a time-dependent manner with SARS-CoV-2 virus particles; the supernatant was then used to infect Vero E6 cells to test their survival. The results showed that CPG and CPG_Cu bioglasses are able to reduce the infectivity of SARS-CoV-2 in a short period of time. Finally, the biocompatibility of both types of bioglasses was assessed by using the NIH-3T3 cell line, derived from mouse embryonic fibroblasts. In particular, the cell viability obtained by using MTT and Alamar blue assay was supported by CLSM and SEM observations. Furthermore, collagen and fibronectin deposition was quantified by ELISA assay. The results showed the biocompatibility of both Cu-doped and undoped bioglasses with NIH-3T3 cells. In conclusion, based on the previous results, calcium phosphate bioglasses with the addition of CuO seem suitable for use in various application contexts, including those aimed at reducing bacterial and viral activity and promote cellular proliferation, e.g. orthopaedic field, surgery probes, drug delivery, photodynamic therapy.
Le infezioni nosocomiali sono un problema medico e ospedaliero in continua crescita. Queste infezioni hanno un impatto clinico ed economico significativo: secondo l'Organizzazione Mondiale della Sanità (OMS), le infezioni nosocomiali causano un prolungamento della durata di degenza, disabilità a lungo termine, aumento della resistenza dei microrganismi agli antibiotici, un onere economico aggiuntivo per i sistemi sanitari e un significativo eccesso di mortalità. In Italia muoiono ogni anno 10.780 persone per infezioni ospedaliere da antibiotico-resistenza. Entro il 2050 i decessi saranno circa 450.000 e questo fenomeno sarà costato al nostro Paese almeno 12 miliardi di euro. Lo scopo di questo lavoro di tesi è stato quindi quello di valutare gli effetti antibatterici, antivirali e proliferativi dei biovetri di fosfato di calcio drogati con rame (CPG_Cu) rispetto ai vetri non drogati (CPG), in modo da trovare applicazione come impianti medici. Questi biovetri sono costituiti da fosfato di calcio inorganico e hanno dimostrato in precedenti studi di avere buone proprietà meccaniche, termiche e ottiche e di essere anche bio-riassorbibili. Grazie a queste caratteristiche, potrebbero avere un buon potenziale in applicazioni biomediche. Pertanto, un valido approccio per rendere questi biovetri di fosfato di calcio antibatterici potrebbe essere il loro drogaggio con ioni come il rame (Cu2+). Per valutare l'attività antibatterica sono stati utilizzati tre ceppi di batteri Gram-negativi (Escherichia coli, Pseudomonas aeruginosa e Klebsiella pneumoniae) e due ceppi di batteri Gram-positivi (Staphylococcus aureus e ceppo MRSA). Dopo l'incubazione diretta su entrambi i tipi di biovetri (Cu2+ drogato e non drogato), sono state valutate la vitalità cellulare (saggio MTT e CLSM) e la morfologia (osservazioni al SEM); i risultati hanno mostrato che sia le proprietà antibatteriche che quelle antiadesive erano presenti nei biovetri CPG_Cu, soprattutto contro i batteri Gram-negativi; mentre le attività contro i batteri Gram-positivi erano inferiori. Successivamente, i biovetri CPG e CPG_Cu sono stati trattati in un primo momento, in modo dipendente dal tempo, con particelle virali di SARS-CoV-2; il surnatante è stato poi utilizzato per infettare cellule Vero E6 per testarne la sopravvivenza. I risultati hanno dimostrato che i biovetri CPG e CPG_Cu sono in grado di ridurre l'infettività del SARS-CoV-2 in breve tempo. Infine, la biocompatibilità di entrambi i tipi di biovetri è stata valutata utilizzando la linea cellulare NIH-3T3, derivata da fibroblasti embrionali di topo. In particolare, la vitalità cellulare ottenuta con i saggi MTT e Alamar blue è stata supportata da osservazioni CLSM e SEM. Inoltre, la deposizione di collagene e fibronectina è stata quantificata mediante saggio ELISA. I risultati hanno dimostrato la biocompatibilità con le cellule NIH-3T3 di entrambi i biovetri, drogati e non drogati. In conclusione, sulla base dei risultati precedenti, i biovetri di fosfato di calcio con l'aggiunta di CuO sembrano adatti all'uso in vari contesti applicativi, tra cui quelli volti a ridurre l'attività batterica e virale e a promuovere la proliferazione cellulare, ad esempio in campo ortopedico, nella somministrazione di farmaci, nella terapia fotodinamica e come sonde chirurgiche.
PROMETTENTI VETRI DI FOSFATO DI CALCIO PER APPLICAZIONI BIOMEDICHE
MARASCO, ALESSANDRA
2021/2022
Abstract
Nosocomial infections are an ever growing medical and hospital problem. These infections have a significant clinical and economic impact: according to the World Health Organization (WHO), nosocomial infections cause prolonged length of stay, long-term disability, increased resistance of microorganisms to antibiotics, an additional economic burden on health care systems, and significant excess mortality. In Italy, 10.780 people die each year from hospital infections due to antibiotic resistance. By 2050 then there will be about 450.000 people dying, and this phenomenon will have cost our country at least 12 billion euros. Hence the purpose of this thesis was to evaluate the antibacterial, antiviral and cell proliferative effects of copper-doped calcium phosphate bioglasses (CPG_Cu) compared to undoped bioglasses (CPG) for application in medical implantations. These bioglasses are made of inorganic calcium-phosphate and showed in previous works to have good mechanical, thermal and optic properties and also to be bioresorbable. Thanks to these characteristics, they could have a potential for biomedical applications. Hence, a valid approach to make these calcium-phosphate bioglasses antibacterial, could be the doping with ions such as copper (Cu2+). Three strains of Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae) as well as two strains of Gram-positive bacteria (Staphylococcus aureus and the MRSA strain) were used to evaluate the antibacterial activity. After direct incubation onto both types of bioglasses (Cu2+-doped and undoped), cell viability (MTT assay and CLSM) and morphology (SEM observations) were performed; the results showed that both the antibacterial and the anti-adhesive properties were present in the CPG_Cu bioglasses, especially against Gram-negative bacteria; meanwhile the activities against Gram-positive bacteria were lower. Next, CPG and CPG_Cu bioglasses were at first treated in a time-dependent manner with SARS-CoV-2 virus particles; the supernatant was then used to infect Vero E6 cells to test their survival. The results showed that CPG and CPG_Cu bioglasses are able to reduce the infectivity of SARS-CoV-2 in a short period of time. Finally, the biocompatibility of both types of bioglasses was assessed by using the NIH-3T3 cell line, derived from mouse embryonic fibroblasts. In particular, the cell viability obtained by using MTT and Alamar blue assay was supported by CLSM and SEM observations. Furthermore, collagen and fibronectin deposition was quantified by ELISA assay. The results showed the biocompatibility of both Cu-doped and undoped bioglasses with NIH-3T3 cells. In conclusion, based on the previous results, calcium phosphate bioglasses with the addition of CuO seem suitable for use in various application contexts, including those aimed at reducing bacterial and viral activity and promote cellular proliferation, e.g. orthopaedic field, surgery probes, drug delivery, photodynamic therapy.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/14909