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Giatsidis, Giorgio (2018) Effetti biologici delle forze meccaniche esterne sui tessuti molli: ottimizzazione preclinica per l'applicazione translazionale in chirurgia rigenerativa.
Biological effects of external mechanical forces on soft tissues: preclinical optimization for translational application in regenerative surgery.
[Ph.D. thesis]

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Abstract (english)

In reconstructive surgery, tissues are routinely transferred to repair a defect caused by trauma, cancer, chronic diseases, or congenital malformations. Surgical transfer intrinsically impairs metabolic supply to tissues placing a risk for ischemic complications such as necrosis, impaired healing, or infection. Pre-surgical induction of angiogenesis in tissues (preconditioning) limits ischemic complications and improves outcomes but very few preconditioning strategies have successfully been translated to clinical practice.
The first goal of our research was to improve current standard of care in reconstructive surgery by developing a translational technique that can effectively and safely increase the vascularization of soft tissues. To achieve this goal, we optimized, using preclinical animal models resembling clinical needs and scenarios in a controlled setting, a method that adopts non-invasive external suction (External Volume Expansion, EVE) to precondition tissues through the induction of hypoxia-mediated angiogenesis. Using a sequential approach in a rodent model we determined the parameters of application (frequency, suction levels, duration, and interfaces) that fine-tune the balance of enhanced angiogenesis, attenuation of hypoxic tissue damage, and length of treatment. The optimized parameters of application (short, cyclical stimulations at moderate suction) almost doubled tissue vascular density after only 5 days of treatment. Our outcomes also showed that the use of micro-deformational interfaces of treatment retain the biological effectiveness of EVE while further reducing the cutaneous damage by distributing forces across the stimulated tissue. Our model confirmed that the optimized technique significantly improves the survival of transferred soft tissues (+20-30%), such as adipose tissue grafts, and can achieve the same beneficial outcomes in animal models of pathologic cutaneous vascularization, such as the one occurring in the skin of patients affected type-2 diabetes. We assessed that EVE retains a beneficial effect on the vascularization and proliferation (adipogenesis) of soft tissues when used both as a pre-conditioning method (before surgeries) and as a post-conditioning method (after surgeries) As a second goal of our research we integrated the knowledge on the application of EVE on soft tissues, to the use of a shelf-ready, bio-mimetic, decellularized allograft adipose matrix (AAM) with the aim of developing an innovative and minimally-invasive strategy for in vivo regeneration of soft tissues. In an animal model we tested the potential of a human-derived, injectable AAM to regenerate soft tissues when used in combination with EVE. This strategy significantly improved long-term volume retention (50-80% higher) and histological quality of reconstructed tissues compared to current standard of care (adipose grafts). The AAM induced both adipogenesis and angiogenesis. Combined use of the AAM and adipose grafts mitigated efficacy.
Our studies suggest that EVE can improve the outcomes of reconstructive surgeries by safely and promptly enhance vascularity of soft tissues, in addition to its edema-/mechanically-induced adipogenic effect (confirmed by our study). EVE's use with an AAM, instead, can synergistically and effectively induce in vivo soft tissue regeneration. These translational principles are ready to be translated to clinical trials and, if outcomes will be confirmed, they could establish the basis for a novel therapeutic paradigm in reconstructive and regenerative surgery for the benefit of a large number of patients.

Abstract (italian)

La chirurgia ricostruttiva si basa sul trasferimento di tessuti da un distretto corporeo ad un altro al fine di riparare un difetto tissutale causato da un trauma, un tumore, una malattia cronica, o una malformaizoen congenita. Questo trasferimento chirurgico compromette la vascolarizzazione (e quindi il support metabolico) dei tessuti trasferiti, mettendoli a rischio per complicanze ischemiche quali la necrosi, laguarigione inefficace delle ferite, o la sovrainfezione batterica. L'induzione di fenomeni angiogenici nei tessuti prima della chirurgia (pre-condizionamento) limita le complicanze ischemiche e migliora I risultati chirurgici; tuttavia, pochissime strategie di pre-condizionamento sono oggi disponibili nella pratica clinica.
Il primo obiettivo di questa ricerca era di migliorare gli attuali standard in chirurgia ricostruttiva attraverso lo sviluppo di tecniche traslazionali in grado di aumentare la vascolarizzazione dei tessuti in maniera efficace e sicura. Al fine di raggiungere tale obiettivo abbiamo ottimizzato, usando modelli preclinici animali rappresentativi di condizioni cliniche controllate, un metodo che adopera una stimolazione meccanica esterna non invasiva tramite pressione negativa (Espansione Volumetrica Esterna, EVE) per precondizionare I tessuti attraverso l'induzione di fenomeni angiogenici causati da una ischemia transitoria. Tramite questa strategia di ottimizzazione sequenziale in un modello murino abbiamo definite i parametri di trattamento ottimali di EVE (frequenza, livelli di pressione, durata, interfaccia di trattamento) in grado di bilanciare l'induzione di angiogenesis con l'attenuazione del danno ischemico causato ai tessuti, e con la durata di trattamento. L'ottimizzazione di EVE (brevi, cicliche stimulazioni a suzione moderata) ha dimostrato la capacità di raddoppiare la densità vascolare dei tessuti stimulati dopo solo 5 giorni di trattamento. I nostri risultati hanno anche dimostrato che l'uso di interfacce di trattamento a micro-deformazione garantisce il mantenitmento degli stessi effetti biologici di EVE ma allo stesso tempo reduce il danno cutaneo causato ai tessuti tramite la distribuzione delle forze meccaniche su tutto il tessuto stimulato. I nostri modelli sperimentali hanno confermato che l'ottimizzazione di EVE permette di aumentare significativamente (+20-30%) la sopravvivenza dei tessuti trasferiti (ad esempio il tessuto adiposo), e che gli stessi effetti possono essere osservati in modelli di vascolarizzazione cutanea patologica (ad esempio la cute di soggetti affetti da diabete di tipo 2). Inoltre, abbiamo confermato che EVE induce la vascolarizzazione e la proliferazione (adipogenesi) dei tessuti molli sia quando utilizzara come metodo di pre-condizionamento (prima della chirurgia) dei tessuti sia quando utilizzata come metodo di post-condizionamento (dopo la chirurgia).
Come secondo obiettivo di questa ricerca abbiamo integrato le conoscenze acquisite sull'applicazione di EVE ai tessuto molli all'uso di una matrice adiposa allogenica (AAM) -ottenuta tramite decellularizzazione di tessuto adipose umano, caratterizzata da proprietà bio-mimetiche, e realizzata in una formulazione iniettiabile "pronta all'uso" - con lo scopo di sviluppare una strategia innovativa e mini-invasiva per la rigenerazione in vivo di tessuto molli. In un modello animale abbiamo testato il potenziale della AAM di rigenerare i tessuti molli quando utilizzata in combinazione con EVE. Questa strategia ha portato ad un significativo aumento volumetrico (+50-80% a 12 settimane) ed un miglioramento della struttura istologica dei tessuti molli ricostruiti in comparazione ai risultati ottenuti con le terapie standard attuali (innesti di tessuto adiposo). Abbiamo evidenziato come la AAM sia in grado di indurre sia fenomeni adipogenici che fenomeni angiogenici: l'applicazione combinate di AAM e innesti di tessuto adiposo, invece, mitigano I risultati ottenibili con l'uso esclusivo della AAM.
In conclusion, i nostril studi suggeriscono che EVE è in grado di migliorare i risultati ottenibili in chirurgia ricostruttiva attraverso un incremento, sicuro e rapido, della vascolarizzazione dei tessuto molli, in aggiunta all'efftto adipogenico (mediato da stimolazione meccanica diretta ed edema dei tessuti) gia descritto nella precedente letteratura e qui confermato dai nostril risultati. L'utilizzo di EVE con l'AAM, invece, può, efficacemente e sinergisticamente, indurre fenomeni rigenerativi dei tessuto molli in vivo. Questi principi traslazionali sono pronti per essere validati in trial clinici e, qualora I loro risultati venissero confermati, potrebbero porre le basi per lo sviluppo di nuovi paradigm terapeutici in chirurgia ricostruttiva e in chirurgia rigenerativa, per il beneficio di un grande numero di pazienti

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EPrint type:Ph.D. thesis
Tutor:Bassetto, Franco
Ph.D. course:Ciclo 30 > Corsi 30 > MEDICINA MOLECOLARE
Data di deposito della tesi:09 January 2018
Anno di Pubblicazione:09 January 2018
Key Words:regenerative medicine; regenerative surgery; mechanical forces; angiogenesis; preconditioning; scaffolds; adipose tissue
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/19 Chirurgia plastica
Struttura di riferimento:Dipartimenti > Dipartimento di Medicina Molecolare
Codice ID:10593
Depositato il:26 Oct 2018 08:34
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