Ricerc@Sapienza

Regenerative medicine is an emerging field of research whose aim is to restore the impaired function of a defective tissue or organs, using different approach as cell therapy and Tissue Engineering (TE). Traditional strategies of cell delivery are based on cell injection in the site of injury. Different cells have been used in this therapeutic approach (Sicari B. et al 2014) but a significant number of cells is required owing to the low capability to migrate from the site of injection (Rando T.A. et al, 1995). To overcome this issue, different studies approached ex vivo cultures. These cultures show a reduced ability to repair skeletal muscle after in vivo transplantation; in fact, expanded cells show a regenerative potential decreased compared to freshly isolated cells (Montarass D. al, 2005). The development of an engineered tissue is recognized as a valid alternative for the replacement and repair of damaged skeletal muscles and to cell therapy in skeletal muscle regeneration. As reported by Langer and Vacanti in 1993 `TE is an interdisciplinary field that applies the principles of engineering and the life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function¿ (Lager and Vacanti,1993). The aim of TE is to mimic the structure and function of a tissue in vitro and in vivo, to obtain a functional construct to implant as a therapeutic device and to study physiological and pathological conditions (Lager and Vacanti, 1993). It requires the interaction among three main components: cells, a matrix that support cell growth and all the mechanical and molecular signals that recapitulate the in vivo niche in which cells reside. In general, tissue engineering uses a scaffold to mimic the ECM, to support proliferation and differentiation of progenitor cells (Kamelger F.S. et al., 2004; Koning M. et al., 2009). An ideal skeletal muscle scaffold requires high porosity and an adequate pore size necessary to facilitate cell seeding and diffusion of nutrients. The matrix should be biocompatible to block the immune response in the host tissue. In addition, biodegradability is another essential property necessary to promote host tissue integration (Rossi C.A. et al, 2010). The restrictions of synthetic scaffolds or extracellular matrix-based materials required alternative tissue-engineering solutions. Scaffold-free approaches seek to create tissues by mimicking developmental processes: cell condensation, proliferation, cell differentiation, ECM production, and tissue maturation (DuRaine GD et al., 2015). In general, the constructs realized without any scaffolds are the main method for translation in clinical of the engineered tissue. As previously described, the X-MET shows morphological and biomechanical properties like muscle and it is generated in absence of scaffold. Furthermore, the generation of 3D construct allows the measurement of contractile force (Dennis and Kosnik, 2000; Vandenburgh et al., 2008; Carosio et al., 2013), and can be used as a model for anti-drug tests (Vandenburgh et al., 2008) and to study various diseases that affect the muscle (Lee and Vandenburgh, 2013). Besides its therapeutically utility, another important issue concerning the generation of a 3-dimentional (3-D) tissue, is its versatility to be used as an in vitro tool to study complex processes such as tissue homeostasis, differentiation and regeneration.3-dimentional construct shows advantages in studying such processes in vitro since conventional 2-D system displays some important limitations. First, the classical 2D culture doesn¿t reproduce the complex network of cell-cell interactions. Moreover, in the context of skeletal muscle, the adherence of muscle cells to a rigid surface limits the possibility to analyse muscle cell contractility and all other functional properties. The 3D cell cultures, compared to the 2D cell system, better reflect the physiological conditions in which the cells of the body are acting, reproducing more faithfully the complex system of cell-cell interaction. In addition, 3D cultures yield to a reduction in the use of animals. In view of all these features, the X-MET can be used as a three-dimensional model of in vitro skeletal muscle, simplifying the study of complex cellular processes. It has been previously shown that 3D X-MET structure survives in culture for an extended period of time if compared to the classical 2D structure; also a reduction of size has been observed for the X-MET after 40-days in culture (Carosio et al, 2013). Furthermore, preliminary data shown that a depletion of growth factors from the culture medium (as occurs for example in cancer) leads to a pronounced atrophy of myotubes with a consequent alteration in the contractile properties of X-MET. X-MET is therefore an ideal in vitro muscle model to study atrophy, loss of muscle mass and alterations in contractile properties associated with conditions mimicking cachexia.