Periodic Reporting for period 1 - GAMBBa (Gene-activated AntiMicrobial Biomaterials for Bone regeneration)
Período documentado: 2020-09-01 hasta 2022-08-31
The overarching research aim of this proposal is to address the clinical problem of osteomyelitis, i.e. bone infection, by developing a biomaterial scaffold that eliminates bacteria while regenerating bone simultaneously. For that, the biomaterial scaffold combines, for the first time, the controlled delivery of osteoanabolic genetic cargo for bone healing with non-antibiotic antimicrobial nanoparticles (a-NPs). During the project, I assessed the effect of various metal ions, such as zinc and copper, on the toxicity of most common models of bacteria, gram-positive Staphylococcus aureus (S.aureus) and gram-negative Escherichia coli (E.coli) and viability of human mesenchymal stem cells (hMSCs) in 2D cultures showing that the dosages higher than 5 mM result in bacterial lysis. These antimicrobial ions were then incorporated into inorganic materials: nanohydroxyapatite (a-nHA) and bioglass (a-BG). I showed that the incorporation of Cu-BG into collagen scaffolds significantly reduces the attachment of S.aureus equipping the biomaterial scaffold with antimicrobial properties. During the project, I evaluated various genetic cargoes showing that antagomiR-138 exhibits favourable osteogenic properties stimulating human mesenchymal stem cells to differentiate into bone cells. I have also assessed the osteogenic potential of miR-26a and antagomiR-133a in vitro. The genetic cargoes were further incorporated into collagen scaffolds and assessed in vivo. The antagomiR-138 was incorporated into antimicrobial scaffolds, which contained Cu-BG. Its osteogenic and angiogenic potential was confirmed using the ex ovo embryo chick model. Based on the results, the scaffolds combining antimicrobial nanoparticles with osteogenic genetic cargoes might be a potential solution for treating infected bone tissue.
GAMBBa is organised with four key scientific objectives:
Objective 1: Develop and evaluate antimicrobial nanoparticles (a-NPs) via substitution with antimicrobial metal ions. In my first objective, I assessed the effect of metal ions, zinc, copper and magnesium on the toxicity of most common models of bacteria, gram-positive Staphylococcus aureus, Staphylococcus epidermis and gram-negative Escherichia coli. performed a dose-dependent study showing that concentrations above 5 mM and 10 mM induce bacterial lysis. I evaluated the effect of the same ions on the behaviour of human mesenchymal stem cells assessing their metabolic activity and DNA content. I incorporated relevant ions into the structure of nanohydroxyapatite and bioglass which was confirmed through colourimetric methods.
Objective 2: Incorporate antimicrobial a-NPs into regenerative scaffolds and test physiochemical and biological features. In my second objective, I incorporated the optimised nanoparticles into collagen-based scaffolds. I confirmed the effective incorporation of relevant nanoparticles using colourimetric methods. I assessed the effect of scaffolds containing either Zn-nHA or Cu-BG on human mesenchymal stem cells. I showed that the ionic products of both types of scaffolds did not affect the metabolic activity and DNA content of hMSCs, showing no toxic effects. I confirmed these results by visualising the morphology of hMSCs through fluoresce microscope.
Objective 3: Incorporate therapeutic genetic cargo into the scaffold; evaluate co-delivery of antimicrobial and osteogenic agents in vitro. I choose genetic cargoes with proven osteogenic features to be incorporated into antimicrobial scaffolds. I showed that the incorporation of genetic cargoes did not influence the antimicrobial properties of the scaffolds. The scaffolds containing miR mimics and antagomiR reduced the attachment of S.aureus and E.coli to their surface compared to the control. This demonstrates their anti-fouling properties. I also assessed the interaction of the scaffolds with human mesenchymal stem cells showing that the delivery of genetic cargoes enhances the production of calcium and mineralisation.
Objective 4: Evaluate in vivo healing and antimicrobial efficacy of the scaffold in a rat infection model. During my fourth objective, I evaluated the regenerative efficacy of the scaffolds containing genetic cargoes using two in vivo models. The miR-26a-activated scaffolds and antagomiR-133a-activated scaffolds were assessed in vivo in a calvarial defect model. The antagomiR-138-activated scaffold was assessed in the femoral defect model. Finally, I incorporated antagomiR-138 into scaffolds containing Cu-BG nanoparticles and assessed their osteogenic and angiogenic potential using the ex ovo chick embryo model.
Objective 1: Develop and evaluate antimicrobial nanoparticles (a-NPs) via substitution with antimicrobial metal ions. In my first objective, I assessed the effect of metal ions, zinc, copper and magnesium on the toxicity of most common models of bacteria, gram-positive Staphylococcus aureus, Staphylococcus epidermis and gram-negative Escherichia coli. performed a dose-dependent study showing that concentrations above 5 mM and 10 mM induce bacterial lysis. I evaluated the effect of the same ions on the behaviour of human mesenchymal stem cells assessing their metabolic activity and DNA content. I incorporated relevant ions into the structure of nanohydroxyapatite and bioglass which was confirmed through colourimetric methods.
Objective 2: Incorporate antimicrobial a-NPs into regenerative scaffolds and test physiochemical and biological features. In my second objective, I incorporated the optimised nanoparticles into collagen-based scaffolds. I confirmed the effective incorporation of relevant nanoparticles using colourimetric methods. I assessed the effect of scaffolds containing either Zn-nHA or Cu-BG on human mesenchymal stem cells. I showed that the ionic products of both types of scaffolds did not affect the metabolic activity and DNA content of hMSCs, showing no toxic effects. I confirmed these results by visualising the morphology of hMSCs through fluoresce microscope.
Objective 3: Incorporate therapeutic genetic cargo into the scaffold; evaluate co-delivery of antimicrobial and osteogenic agents in vitro. I choose genetic cargoes with proven osteogenic features to be incorporated into antimicrobial scaffolds. I showed that the incorporation of genetic cargoes did not influence the antimicrobial properties of the scaffolds. The scaffolds containing miR mimics and antagomiR reduced the attachment of S.aureus and E.coli to their surface compared to the control. This demonstrates their anti-fouling properties. I also assessed the interaction of the scaffolds with human mesenchymal stem cells showing that the delivery of genetic cargoes enhances the production of calcium and mineralisation.
Objective 4: Evaluate in vivo healing and antimicrobial efficacy of the scaffold in a rat infection model. During my fourth objective, I evaluated the regenerative efficacy of the scaffolds containing genetic cargoes using two in vivo models. The miR-26a-activated scaffolds and antagomiR-133a-activated scaffolds were assessed in vivo in a calvarial defect model. The antagomiR-138-activated scaffold was assessed in the femoral defect model. Finally, I incorporated antagomiR-138 into scaffolds containing Cu-BG nanoparticles and assessed their osteogenic and angiogenic potential using the ex ovo chick embryo model.
The project GAMBBa aimed to address the clinical problem of osteomyelitis, i.e. bone infection, by developing a biomaterial scaffold that eliminates bacteria while regenerating bone simultaneously. The biomaterial scaffold combined for the first time the controlled delivery of osteoanabolic genetic cargo for bone healing with non-antibiotic antimicrobial nanoparticles (a-NPs). The use of antimicrobials alternative to antibiotics was particularly advantageous as it addressed the rising problem of antimicrobial resistance to antibiotics. Overall, the project generated scientific, economic and societal impacts described below:
SCIENTIFIC IMPACTS: The treatment of musculoskeletal infections and the ever-growing antimicrobial resistance represent overarching health problems. European institutions must develop the next generation of therapies to overcome the handicaps of current treatments to fight the AMR. The project GAMBBa made remarkable scientific progress in developing more effective therapies with lesser late and long-term effects for the treatment of resistant antimicrobial infections and by investigating antibiotic-free therapies based on the delivery of metal ions. This strategy is particularly advantageous as metal ions induce multiple killing mechanisms in bacteria, making it challenging to develop protective mechanisms against the toxic effects of metal ions. The performance of the scaffolds was assessed in a small animal model in the pilot study providing information regarding the pre-clinical performance of materials.
HEALTH AND WELLBEING IMPACTS: Nearly 50% of the health burden of AMR is caused by infections with bacteria resistant to last-line antibiotics, such as carbapenems and colistin, making it extremely difficult or impossible to treat infected patients. Numbers are expected to rise due to the over-prescription of antibiotics and the emergence of new highly-resistant strains. The new scaffold-based technology developed within the GAMBBa would have significant advantages over existing clinical treatments of bone infections. The project explored the use of metal ions as an alternative to antibiotics addressing the problem of antimicrobial resistance to antibiotics that might save lives and lessen the strains on society, health services, families and carers. Of note, these therapeutics could be potentially applied to myriad applications, such as wound infections. The diversification of tissue applications could be investigated through follow on grants, including ERC Starting and Wellcome Trust grants or through collaboration with the industry partners sought through the Advanced Materials and BioEngineering Research (AMBER) centre. The investigation of the delivery of osteogenic microRNAs progressed the development of gene therapies in the regenerative medicine.
SCIENTIFIC IMPACTS: The treatment of musculoskeletal infections and the ever-growing antimicrobial resistance represent overarching health problems. European institutions must develop the next generation of therapies to overcome the handicaps of current treatments to fight the AMR. The project GAMBBa made remarkable scientific progress in developing more effective therapies with lesser late and long-term effects for the treatment of resistant antimicrobial infections and by investigating antibiotic-free therapies based on the delivery of metal ions. This strategy is particularly advantageous as metal ions induce multiple killing mechanisms in bacteria, making it challenging to develop protective mechanisms against the toxic effects of metal ions. The performance of the scaffolds was assessed in a small animal model in the pilot study providing information regarding the pre-clinical performance of materials.
HEALTH AND WELLBEING IMPACTS: Nearly 50% of the health burden of AMR is caused by infections with bacteria resistant to last-line antibiotics, such as carbapenems and colistin, making it extremely difficult or impossible to treat infected patients. Numbers are expected to rise due to the over-prescription of antibiotics and the emergence of new highly-resistant strains. The new scaffold-based technology developed within the GAMBBa would have significant advantages over existing clinical treatments of bone infections. The project explored the use of metal ions as an alternative to antibiotics addressing the problem of antimicrobial resistance to antibiotics that might save lives and lessen the strains on society, health services, families and carers. Of note, these therapeutics could be potentially applied to myriad applications, such as wound infections. The diversification of tissue applications could be investigated through follow on grants, including ERC Starting and Wellcome Trust grants or through collaboration with the industry partners sought through the Advanced Materials and BioEngineering Research (AMBER) centre. The investigation of the delivery of osteogenic microRNAs progressed the development of gene therapies in the regenerative medicine.