Controlli di qualità e rappresentazione 3D delle microtomografie a raggi X applicate allo studio dei modelli murini per la progeria

La progeria è una malattia genetica rarissima che genera un invecchiamento precoce, con una incidenza di 1 caso su 4 milioni di nascite e circa 100 casi noti attualmente in tutto il mondo. L’età media di sopravvivenza è di circa 13.5 anni, con una aspettativa di vita tra gli 8 e i 21 anni. La massa ossea ridotta e le anomalie scheletriche sono tra i principali disordini dell’invecchiamento precoce. Al fine di sviluppare una migliore comprensione della patogenesi e della progressione della sindrome, oltre che per progredire nelle possibili terapie, sono stati sviluppati modelli animali che replicano in-vivo ed in condizioni controllate la genesi e l’evoluzione della malattia. Uno dei modelli animali più diffusi è quello murino, il cui sistema scheletrico può essere studiato mediante microtomografia a raggi X. Attualmente considerata come il gold standard, grazie alla sua elevata risoluzione spaziale, la micro-CT fornisce importanti indicazioni sulla struttura trabecolare e corticale del tessuto osseo. Le misure microtomografiche dei segmenti ossei murini sono affette da variabilità intrinseca legata ai parametri di scansione, al posizionamento del campione da parte dell'operatore, alla selezione manuale o automatizzata della ROI, alla scelta del filtro e dei parametri di segmentazione. Questo lavoro di tesi, svolto presso il Laboratorio di Tecnologia Medica (LTM) dell’Istituto Ortopedico Rizzoli (IOR) di Bologna, parte da una approfondita ricerca bibliografica in merito ad accuratezza e ripetibilità delle misure ossee morfometriche e densitometriche del modello murino analizzato mediante micro-CT. Sono stati quindi definiti e applicati opportuni protocolli di controllo qualità, di scansione e di rendering 3D al fine di avere la possibilità di confronto riguardo i modelli murini analizzati, in particolare per il femore e il cranio, e opportunità di indagine più approfondita tramite la visualizzazione tridimensionale dei campioni in analisi

The accuracy of cone beam computed tomography (CBCT) to determine newly formed bone within grafted maxillary sinus in sheep

Grafting of the maxillary sinus floor has become a common surgical intervention to increase bone volume for implant placement (Wallace and Froum, 2003); the procedure can be performed either as an 1-stage procedure with simultaneous implant placement or as a 2-stage procedure before implant placement (Bruggenkate and Bergh, 1998). In a 2-stage procedure, the chosen graft material is placed into the sinus floor and the graft material is left to consolidate with newly formed bone. This consolidation should preferably occur before implant placement. However, currently there is no clinical tool to assess healing within the grafted sinus. A trephine bone biopsy can be harvested for histological assessment but this is invasive and not clinically useful. The current clinical guideline is to wait between six to twelve months after maxillary sinus grafting before implant placement (Rodriguez et al., 2003) CBCT (Cone beam computed tomography) is a clinical 3-D (three-dimensional) radiographic tool for assessment of mineralised tissue (Ehrhart et al., 2008; Estrela et al., 2008) and may be used for assessment of graft healing within maxillary sinus. However, there are a limited number of studies looking at the use of CBCT in bone-density measurements (Benavides et al., 2012). Micro-computed tomography (µCT) is a 3-D radiographic tool mainly used for in vitro studies. Specimens with a volume of approximately 5cm3 can be scanned with up to a 1µm voxel resolution producing high-resolution radiographic images for mineralised tissues. There is growing evidence to suggest that µCT can be used as a substitute method for histology to measure mineralised tissue, particularly trabecular bone (Thomsen et al., 2000; Thomsen et al., 2005). With no clinical tool available for assessment of graft healing within the sinus, the purpose of this study was to assess whether CBCT can be used to measure the amount of newly formed bone in grafted maxillary sinus in sheep. To validate this, CBCT was compared with two reference standards; micro-computed tomography (µCT) and histology. Aim: To assess the effectiveness of CBCT for quantifying newly formed bone within grafted sinus sites, using an animal model. Method: Maxillary sinus grafting in six sheep with bovine xenograft (Endobon®) was evaluated after a sixteen-week healing period. Specimens from each animal were analysed using three imaging techniques: CBCT, µCT and resin-embedded histological sections. Two-dimensional "virtual" CBCT sections were matched with corresponding 2-D µCT sections and digitised histological sections. µCT and CBCT images were calibrated using known-density radiographic calibration standards. Using image analysis software (Image J, NIH, USA), % new bone (%NB), % residual graft (%RG), % mineralised tissue (%MT) were measured for matched regions of interest across each imaging technique and compared statistically (p<0.05). Results: CBCT measured %NB and %RG significantly higher than µCT and histology. µCT measured %NB significantly higher than histology. %RG measurements of µCT and histology were not significantly different. CBCT measured %MT significantly higher than both µCT and histology. %MT measurements of µCT and histology were statistically different but were very similar. Conclusion: Micro-computed tomography (µCT) measurements of residual graft and new bone were affected as the radiodensities of residual graft (Endobon®) and new bone were similar. µCT however appeared to be capable of measuring the combined area of graft and new bone (i.e., mineralised tissue) similar to histomorphometry. Cone-beam computerised tomography (CBCT) markedly overestimated new bone, residual graft and the total mineralised tissue. CBCT lacks the resolution to accurately determine newly formed bone after maxillary sinus grafting, an important step before definitive implant placement