Development of a Murine Model for the Exploration of the Biological Effects of External Volume Expansion.Sviluppo di un modello murino per l'esplorazione degli effetti biologici dell'espansione volumetrica esterna

Background: External Volume Expansion (EVE) refers to a class of devices that non-invasively stretch and expand tissue compartments by external application of suction. EVE has been suggested to increase compartments volume and stimulate the formation of a more developed vascular network, leading to less stiff and better vascularized tissues. It is proposed to patients as a method to prepare recipient sites, in particular breasts, in view of a fat grafting procedure, basing on the theory that fat grafts will better survive and retain volume if the recipient site is more vascularized and provides less compression. However, the method requires high patient compliance and no experimental validation for it has been attempted. Aims: basing on our group's previous experience in downsizing and testing in animal models clinical devices for wound healing, in particular in settings requiring the application of mechanical forces to soft tissues, we proposed to design an animal model for EVE in which to test the validity of the hypothesis of its being beneficial to fat grafting and explore its mechanisms and potentials. Methods: we designed and built a miniaturized EVE device to be applied to the dorsum of mice. We then designed a series of stepwise incremental studies. We tested the capacity of EVE of inducing angiogenesis and cell proliferation with 28 days long continuous stimulation. We analyzed its effects on tissues in terms of mechanical stretch, hypoxia and ischemia, edema, inflammation, cell proliferation and angiogenesis after a single 2 hours stimulation. We produced a mathematical modeling for the effects of EVE on tissues in relation to fat grafting. We tested if EVE is beneficial to fat grafting and if beneficial effects are maintained also in the setting of chronic radiation damage. We tested if EVE can stimulate adipogenesis and what role inflammation can play in it. Results: in our series of studies, we successfully designed a miniaturized animal model in which to test External Volume Expansion. We demonstrated that the hypotheses of stimulation of cell proliferation, angiogenesis, and expansion of tissue compartments on which it is proposed as a preparatory method to fat grafting is confirmed in experimental settings. We showed how mechanical stretch of tissues, hypoxia and ischemia, edema, and inflammation are all intervening factors that can contribute to these effects. Our results suggest that pre-stimulation with EVE is successful in achieving increased fat graft weight and volume retention, and that its beneficial effects are maintained also in the setting of recipient sites having sustained radiation injury. We also demonstrated that EVE has a potential for direct stimulation of adipogenesis, and gathered supportive results to a role for macrophages in this. Discussion: our results validate the technique for its use in the preparatory phase to fat grafting, and can help moving towards making fat grafting a more effective and reliable procedure with improved outcomes for patients. We gathered evidence that help increasing our understanding of how EVE works and what it implies for tissues. This is the basis for optimizing the technique, make it safer, and increase patients' compliance. For example, stimulation patterns can be improved, duration of treatment can be reduced, and practices such as continuation of EVE after fat grafting should be abandoned as detrimental. Our unexpected observations on adipogenesis also open interesting opportunities, such as that of re-starting EVE after fat grafting when this is at the peak of its remodeling phase. And linking this effect with the understanding of the similarity to other conditions in which adipogenesis is seen and desired, such as tissue engineering, or pathological, such as lymphedema, can expand the potential of our animal model to alternative broader fields.