BACKGROUND: Skeletal growth is influenced by multiple factors, both local and systemic. Among the local factors, it appears that the restraint provided by the perichondrium-periosteum complex exerts negative feedback on proliferating chondrocytes, thus negatively modulating bone growth. Elastic fibers within the perichondrium-periosteum may play a significant role in this process. Interestingly, individuals with Marfan syndrome, characterized by a deficiency in the formation of these fibers, typically exhibit increased height and exaggerated growth of long bones. AIMS: Investigate the elastic fibers' role in bone growth using the enzyme elastase MATERIALS AND METHODS: A two-stage study, consisting of an in vitro and an in vivo component, was designed. The first stage involved a novel experimental model of organ cultures using rat tibiae, while the second stage involved Wistar rats. In the in vitro study, each pair of tibiae was taken, and one randomly selected tibia from each pair was treated with elastase (30U in 0.7ml) through incubation for 1 hour. The contralateral tibia was used as a control. All samples were cultured for 14 days, and measurements were taken at regular intervals (T0, T7, T14) to detect any growth differences. Histochemical, immunohistochemical, and ultrastructural analyses were also performed to assess the enzymatic digestion of fibers in the treated samples compared to the controls and to ensure the absence of any damage to surrounding tissues. In the in vivo study, each animal received a local injection of elastase (0.1ml) near the diaphyseal mid third of the right medial tibia, while the contralateral tibia served as a control. After 35 days, the animals were euthanized, and the tibias were collected, measured, and subjected to radiographic, histochemical, and immunohistochemical analyses. RESULTS: In both studies, the following findings were reported: - Histochemical, immunohistochemical, and ultrastructural analysis revealed a significant reduction in the abundance of elastic fibers in the treated samples compared to the controls. - The integrity of collagen fibers and the surrounding tissues was preserved. - Statistically significant differences in growth were observed between the treated samples and the control samples. CONCLUSIONS: The enzyme used demonstrated effectiveness and selectivity in its action. The data substantiate the initial hypothesis regarding the role of elastic fibers in skeletal growth, as the treated group exhibited significantly greater differences in growth compared to the control group.
INTRODUZIONE: L’accrescimento scheletrico è un processo modulato da molteplici fattori, sia locali che sistemici. Tra i fattori locali, la contenzione pericondrio-periostale sembrerebbe esercitare un feedback negativo sui condrociti in proliferazione, andando a modulare negativamente la crescita ossea. In questo contesto un ruolo potrebbe essere svolto dalle fibre elastiche che si trovano a questo livello, infatti individui affetti da Sindrome di Marfan, caratterizzata da un deficit nella formazione di queste fibre, risultano essere tipicamente molto alti e con un’esagerata crescita delle ossa lunghe. OBIETTIVO: Indagare il ruolo delle fibre elastiche nell’accrescimento osseo tramite l’impiego dell’enzima elastasi MATERIALI E METODI: è stato disegnato uno studio in due fasi, in vitro e in vivo; per la prima è stato impiegato un nuovo modello sperimentale di colture d’organo (tibie di ratto), mentre la seconda ha previsto l’impiego di ratti Wistar. Studio in vitro: di ogni coppia di tibie prelevata ne è stata trattata una, scelta in modo randomizzato, tramite incubazione per 1h con l’enzima elastasi (30U in 0.7ml), mentre la controlaterale è stata presa come controllo. Tutti i campioni sono stati messi in coltura per 14 giorni. A intervalli di tempo regolari (T0, T7, T14) sono stati misurati per evidenziare eventuali differenze di crescita. Sono state anche effettuate analisi istochimiche, immuno-istochimiche e ultrastrutturali per identificare la digestione enzimatica delle fibre nei trattati rispetto ai controlli ed escludere eventuali danni ai tessuti limitrofi. Studio in vivo: in ogni animale è stata eseguita un’iniezione locale di elastasi (0.1ml) in prossimità del terzo medio diafisario della tibia mediale destra, lasciando la tibia controlaterale come controllo. Dopo 35 giorni, gli animali sono stati sacrificati, le tibie prelevate, misurate e analizzate tramite radiografia, analisi istochimica e immunoistochimica. RISULTATI: In entrambi gli studi si è registrato: - Sostanziale riduzione del patrimonio delle fibre elastiche nei trattati rispetto ai controlli all’analisi istochimica, immuno-istochimica e ultrastrutturale - Conservazione del patrimonio delle fibre collagene e dell’integrità dei tessuti limitrofi - Differenze di crescita statisticamente significative tra trattati rispetto ai controlli CONCLUSIONI: L’enzima utilizzato si è mostrato efficace e selettivo nella sua azione. I dati confermano l’ipotesi di partenza sul ruolo delle fibre elastiche nell’accrescimento scheletrico; infatti, i trattati hanno avuto differenze di crescita significativamente maggiori rispetto ai controlli.
Fibre elastiche: studio sperimentale in vitro e in vivo sul ruolo nell'accrescimento osseo e nelle malattie correlate
BONI, ELENA
2022/2023
Abstract
BACKGROUND: Skeletal growth is influenced by multiple factors, both local and systemic. Among the local factors, it appears that the restraint provided by the perichondrium-periosteum complex exerts negative feedback on proliferating chondrocytes, thus negatively modulating bone growth. Elastic fibers within the perichondrium-periosteum may play a significant role in this process. Interestingly, individuals with Marfan syndrome, characterized by a deficiency in the formation of these fibers, typically exhibit increased height and exaggerated growth of long bones. AIMS: Investigate the elastic fibers' role in bone growth using the enzyme elastase MATERIALS AND METHODS: A two-stage study, consisting of an in vitro and an in vivo component, was designed. The first stage involved a novel experimental model of organ cultures using rat tibiae, while the second stage involved Wistar rats. In the in vitro study, each pair of tibiae was taken, and one randomly selected tibia from each pair was treated with elastase (30U in 0.7ml) through incubation for 1 hour. The contralateral tibia was used as a control. All samples were cultured for 14 days, and measurements were taken at regular intervals (T0, T7, T14) to detect any growth differences. Histochemical, immunohistochemical, and ultrastructural analyses were also performed to assess the enzymatic digestion of fibers in the treated samples compared to the controls and to ensure the absence of any damage to surrounding tissues. In the in vivo study, each animal received a local injection of elastase (0.1ml) near the diaphyseal mid third of the right medial tibia, while the contralateral tibia served as a control. After 35 days, the animals were euthanized, and the tibias were collected, measured, and subjected to radiographic, histochemical, and immunohistochemical analyses. RESULTS: In both studies, the following findings were reported: - Histochemical, immunohistochemical, and ultrastructural analysis revealed a significant reduction in the abundance of elastic fibers in the treated samples compared to the controls. - The integrity of collagen fibers and the surrounding tissues was preserved. - Statistically significant differences in growth were observed between the treated samples and the control samples. CONCLUSIONS: The enzyme used demonstrated effectiveness and selectivity in its action. The data substantiate the initial hypothesis regarding the role of elastic fibers in skeletal growth, as the treated group exhibited significantly greater differences in growth compared to the control group.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12075/13468