Diaphragm muscle diseases in three-dimensional model

The diaphragm is a unique mammalian respiratory skeletal muscle (SkM), that can be severely affected by a plentiful number of myopathies; of note are congenital diaphragmatic hernia (CDH) and Duchenne muscular dystrophy (DMD). In recent years, tissue engineering offered several new insights for the generation of implantable organs and tissues, or the modelling of diseases in 3D models. To recapitulate the complexity of SkM tissue, various scaffold formulations and cells have been used, what remains to be investigated is the possibility to create ad hoc 3D models that can rely on the specific characteristics of the different muscles of the human-body. In this context, the use of the recellularization technique, based on decellularized scaffold, together with multiple human cell mixtures and external mechanical stimuli, represents a promising option to reach the major goals of SkM tissue engineering. The aim of this work is to offer and validate an advanced humanized tissue engineering approach for the in vitro generation of 3D diaphragmatic-like tissues, as proof of concept of a new option for the in vivo surgical treatment of CDH and the in vitro development of a dystrophic 3D diaphragmatic model to investigate patient specific DMD phenotypes. We recellularized healthy decellularized murine diaphragms (wt dECM) injecting a mixed population of primary human SkM cells and fibroblasts (hFb); in parallel we developed a custom-made bioreactor able to dynamically stimulate the 3D constructs, leading to the proper myofibers alignment inside the scaffolds. The recellularized constructs were analyzed by means of different techniques to test cell engraftment, proliferation, and differentiation. We observed the amelioration of cell disposition in the dynamically stimulated construct when compared with the static counterpart, at multiple culture time points. In parallel, the Fb number was maintained at physiological levels, suggesting that it is possible to avoid the production of fibrotic 3D diaphragm-like constructs by using the bioreactor. By a myogenic point of view, analyses demonstrated an upper expression of myogenic genes and an increased expression of late myogenic proteins in dynamic condition. Furthermore, the dynamically stimulated constructs displayed a general healthy and more organized structure, with mature myofibers radially organized. Finally, we implanted in a surgical model of CDH the dynamically stimulated constructs, and we observed the benefits in terms of host cell recruitments, and construct’s thickness maintenance. Adapting the techniques, we setup the decellularization protocol for dystrophic diaphragms (mdx dECM). We recellularized mdx and wt dECM using healthy and DMD SkM cells, evaluating the efficiency of the protocol in terms of cell growth, migration, and differentiation. The study of the mdx dECM revealed a highly fibrotic, non-homogeneous and differently bioactive environment when comparted with the wt dECM. We observed that the best match to obtain the DMD diaphragmatic-like model was that given by mixing wt dECM and DMD SkM cells. In this mix, the use of the bioreactor promoted the achievement of properly engrafted and mature 3D DMD diaphragm-like model. Given the positive outcomes, with this thesis we demonstrated that a tissue engineering approach based on the dynamic stimulation, throughout the use of a specific bioreactor, of recellularized dECM, is a valid solution for the generation of in vivo implantable 3D diaphragmatic constructs. Moreover, we highlighted the great differences among healthy and pathologic dECM. We hope that this study can lead to move the next steps to generate clinically relevant constructs for CDH treatment, and will allow the generation of much more complex 3D micro-environments, mimicking the different chemo-attractive factors among the various myopathies, putting the focus not only on the cell type used, but also on the importance of using organ specific ECM.