Phosphorus (P) is a dietary macronutrient essential for several physiological processes. It participates in cellular metabolism and cell signalling, it is a constituent of phospholipids, of nuclei acids and of highly active intracellular compounds such as ATP. In vertebrates, P together with calcium is crucial for mineralisation of the skeleton. Adequate dietary P levels are required to prevent P deficiency- related effects, i.e. growth retardation, osteomalacia and bone deformities, as well as P-excess- related consequences, i.e. soft tissue calcification and bone hypermineralisation. The present thesis examines the effects of different dietary P levels on the skeleton using zebrafish as a model organism in skeletal research. The aims are i) to obtain insights into the mechanisms underlying osteomalacia and excess mineralisation, related both to inadequate P intake and to disease conditions, and ii) to better elucidate the processes of bone formation and mineralisation. The zebrafish skeletal elements, mainly the vertebral column, but also the dermal fin rays and the dentition, are analysed using a wide range of complementary techniques, ranging from histological procedures to different radiographic techniques, molecular analysis and mechanical testing. The first major finding of this thesis is that bone formation and bone mineralisation are uncoupled processes. When treating zebrafish with a reduced dietary P supply, bone matrix deposition, i.e. bone formation, continues uninterrupted. New bone matrix is formed, simply this bone does not mineralise due to the lack of P. The deposition of new bone matrix is even further stimulated under low P conditions. The increased production of bone matrix does not lead to the development of skeletal malformations in zebrafish. Conversely, increased dietary P supply leads to bone hypermineralisation, increases bone stiffness and promotes fusion of vertebral bodies. Interestingly, the large amount of non-mineralised bone matrix produced under low P conditions resumes mineralisation as soon as sufficient P is provided with the diet. These late mineralised bone structures appear normal, but the volume of the newly mineralised bone is dramatically increased. It is thus possible to increase the bone volume in zebrafish, first by stimulating bone matrix formation under low P conditions, and subsequently inducing the mineralisation of the newly formed bone by providing adequate dietary P. The low dietary P conditions also reduce the mineral content of the bone already present in zebrafish, enhancing its biomechanical properties by increasing toughness and reducing stiffness. This approach has been applied to partially rescue the bone hypermineralisation of osteogenesis imperfecta, a rare bone disorder mainly characterised by bone fragility and severe bone deformities. In the osteogenesis imperfecta mutant zebrafish called Chihuahua, the data obtained from the reduced dietary P treatment suggest a reduction in the mineral content of bone, resulting in a partial improvement of the severe bone phenotype typical of the disease. The low dietary P zebrafish model presented here allows to elucidate the effects of dietary P levels on bone development and to better understand the relationship between bone formation and bone mineralisation. Moreover, the experimental evidence linking dietary low P levels with bone quality improvement, represents a promising novel approach for the treatment of disease conditions characterised by bone hypermineralisation and bone loss. Furthermore, the alternation of low dietary P/sufficient dietary P can possibly contribute to reverse bone loss due to aging or osteoporosis conditions.
Il fosforo è un macronutriente essenziale per diversi processi fisiologici, tra cui la mineralizzazione dello scheletro, e deve essere introdotto tramite l’alimentazione. È necessario assumere adeguati livelli di fosforo tramite l’alimentazione, per prevenire gli effetti correlati sia alla carenza, ovvero ritardo della crescita, ipomineralizzazione e deformità ossee, sia all'eccesso di fosforo, ovvero calcificazione dei tessuti molli e ipermineralizzazione ossea. Lo scopo di questa tesi è analizzare che conseguenze hanno diversi apporti di fosforo sullo scheletro attraverso l’alimentazione, utilizzando lo zebrafish come organismo modello. Gli obiettivi sono i) ottenere maggiori informazioni sui meccanismi alla base dell'ipo- e dell'ipermineralizzazione ossea, dovuti sia all'assunzione inadeguata di fosforo, sia a condizioni di malattia, e ii) chiarire entrambi i processi di formazione e mineralizzazione ossea. La morfologia e la mineralizzazione di diverse strutture dello scheletro di zebrafish sono analizzate utilizzando un'ampia gamma di tecniche complementari, che vanno da procedure istologiche a diverse tecniche radiografiche, analisi molecolari e test meccanici. Il primo importante risultato di questa tesi è la dimostrazione che la formazione di osso e la sua mineralizzazione sono processi indipendenti. Quando zebrafish è sottoposto ad un apporto ridotto di fosforo, la deposizione di matrice ossea non subisce alcun effetto. Anzi, la formazione di nuova matrice ossea è particolarmente stimolata in queste condizioni e avviene senza la sua conseguente mineralizzazione. Tale aumento della produzione di osso non porta allo sviluppo di malformazioni scheletriche in zebrafish. Al contrario, l'aumento dell'apporto nutrizionale di fosforo causa ipermineralizzazione e aumentata rigidità ossea, favorendo la fusione delle vertebre. È interessante notare come la grande quantità di matrice ossea non mineralizzata, prodotta in condizioni di ridotto apporto di fosforo, mineralizza non appena viene fornito fosforo in quantità sufficienti tramite l’alimentazione. Queste strutture ossee appaiono normali, ma la mineralizzazione tardiva di tali strutture ha come risultato un notevole aumento del volume osseo. Di conseguenza, si può dedurre che è possibile aumentare il volume delle ossa in zebrafish, stimolando prima la formazione di matrice ossea in condizioni di ridotto apporto di fosforo, e successivamente inducendone la mineralizzazione fornendo adeguati livelli di fosforo con l’alimentazione. In condizioni di ridotto apporto di fosforo, le ossa di zebrafish hanno anche un ridotto contenuto di minerale, caratteristica che ne migliora le proprietà biomeccaniche, aumentandone la resistenza e riducendone la rigidità. Questo approccio ha rivelato risultati promettenti nel moderare, sebbene in modo parziale, l'eccessiva mineralizzazione delle ossa che caratterizza l'osteogenesi imperfetta, una rara malattia genetica che causa fragilità e gravi deformità ossee. Il ridotto apporto di fosforo tramite l’alimentazione nel modello zebrafish chiamato Chihuahua, un valido modello per lo studio dell’osteogenesi imperfetta, ha dimostrato risultati incoraggianti nel modulare parzialmente il grave fenotipo osseo della malattia. Il nuovo modello zebrafish presentato in questa tesi è utile sia per chiarire gli effetti dei diversi apporti di fosforo sullo sviluppo osseo, sia per comprendere meglio la relazione tra formazione di matrice ossea e mineralizzazione ossea. Inoltre, l’evidenza sperimentale che lega il ridotto apporto di fosforo con la migliorata qualità dell’osso rappresenta un nuovo e promettente approccio per il trattamento di patologie caratterizzate da ipermineralizzazione ossea e/o riduzione del volume osseo. Inoltre, l'alternanza tra ridotto e aumentato apporto di fosforo può contribuire a contrastare la perdita di volume osseo dovuta all'invecchiamento o legata all’osteoporosi.
"Dietary phosphorus: effects on skeletal development and mineralisation in zebrafish (Danio rerio)"
COTTI, SILVIA
2023-01-30
Abstract
Phosphorus (P) is a dietary macronutrient essential for several physiological processes. It participates in cellular metabolism and cell signalling, it is a constituent of phospholipids, of nuclei acids and of highly active intracellular compounds such as ATP. In vertebrates, P together with calcium is crucial for mineralisation of the skeleton. Adequate dietary P levels are required to prevent P deficiency- related effects, i.e. growth retardation, osteomalacia and bone deformities, as well as P-excess- related consequences, i.e. soft tissue calcification and bone hypermineralisation. The present thesis examines the effects of different dietary P levels on the skeleton using zebrafish as a model organism in skeletal research. The aims are i) to obtain insights into the mechanisms underlying osteomalacia and excess mineralisation, related both to inadequate P intake and to disease conditions, and ii) to better elucidate the processes of bone formation and mineralisation. The zebrafish skeletal elements, mainly the vertebral column, but also the dermal fin rays and the dentition, are analysed using a wide range of complementary techniques, ranging from histological procedures to different radiographic techniques, molecular analysis and mechanical testing. The first major finding of this thesis is that bone formation and bone mineralisation are uncoupled processes. When treating zebrafish with a reduced dietary P supply, bone matrix deposition, i.e. bone formation, continues uninterrupted. New bone matrix is formed, simply this bone does not mineralise due to the lack of P. The deposition of new bone matrix is even further stimulated under low P conditions. The increased production of bone matrix does not lead to the development of skeletal malformations in zebrafish. Conversely, increased dietary P supply leads to bone hypermineralisation, increases bone stiffness and promotes fusion of vertebral bodies. Interestingly, the large amount of non-mineralised bone matrix produced under low P conditions resumes mineralisation as soon as sufficient P is provided with the diet. These late mineralised bone structures appear normal, but the volume of the newly mineralised bone is dramatically increased. It is thus possible to increase the bone volume in zebrafish, first by stimulating bone matrix formation under low P conditions, and subsequently inducing the mineralisation of the newly formed bone by providing adequate dietary P. The low dietary P conditions also reduce the mineral content of the bone already present in zebrafish, enhancing its biomechanical properties by increasing toughness and reducing stiffness. This approach has been applied to partially rescue the bone hypermineralisation of osteogenesis imperfecta, a rare bone disorder mainly characterised by bone fragility and severe bone deformities. In the osteogenesis imperfecta mutant zebrafish called Chihuahua, the data obtained from the reduced dietary P treatment suggest a reduction in the mineral content of bone, resulting in a partial improvement of the severe bone phenotype typical of the disease. The low dietary P zebrafish model presented here allows to elucidate the effects of dietary P levels on bone development and to better understand the relationship between bone formation and bone mineralisation. Moreover, the experimental evidence linking dietary low P levels with bone quality improvement, represents a promising novel approach for the treatment of disease conditions characterised by bone hypermineralisation and bone loss. Furthermore, the alternation of low dietary P/sufficient dietary P can possibly contribute to reverse bone loss due to aging or osteoporosis conditions.File | Dimensione | Formato | |
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Silvia PhD thesis_revised_coverESSE3.pdf
Open Access dal 05/08/2023
Descrizione: PhD thesis of Silvia Cotti
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Tesi di dottorato
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