Impact of the dystrophic environment in conditioning 3D skeletal muscle model production.

Duchenne Muscular Dystrophy (DMD) is a genetic disorder characterized by progressive muscle wasting. It is one of the most common forms of muscular dystrophy, affecting primarily males due to its X-linked recessive inheritance pattern. It results from mutations in the DMD gene, which is responsible for producing a protein known as dystrophin. Dystrophin is crucial for muscle function since it helps strengthen muscle fibers and protect them from injury as muscles contract and relax. The absence of or defects in dystrophin production leads to muscle damage and inflammation, causing progressive muscle weakness and degeneration. Unfortunately, for this type of disease there is currently no cure. This study aims to generate and characterize a 3D in vitro model for studying DMD and possibly drug testing. The model we aim to produce is a fully diseased model and is produced starting from decellularized diaphragm from dystrophic mouse (Mdx model), which serves as a scaffold and immortalized human myoblasts derived from patients affected by DMD. The two important phases that lead to the production of the 3D model are decellularization of the mouse diaphragm, which is obtained with 3 steps of a detergent-enzymatic treatment through which the cellular components are effectively removed, and the subsequent recellularization with cells derived from a dystrophic patient (immortalized myoblasts). Previously, other diaphragmatic models were generated using diaphragms from wild-type (Wt) mice and healthy cells. To re-create a fully diseased 3D model, two different conditions were tested: DMD cells in Wt diaphragms and DMD cells in Mdx diaphragms; both conditions were analyzed after 3 and 7 days of culture. Results showed that DMD cells engraft and repopulate the Wt scaffold, but they do not migrate inside the dystrophic one, neither at 3 nor at 7 days. To better understand the underlying mechanisms preventing DMD cells from migrating within the Mdx environment, we performed a scratch test and a wound healing assay in a 2D setting. With these assays, we demonstrated that the DMD cells can spontaneously migrate and close the formed gap. We then proceeded with the determination of the Mdx diaphragm perfusion capacity comparing it with both Wt diaphragm and an internal control. The results showed that there is no statistical difference between the Wt and the Mdx diaphragm, but the Wt is uniform in terms of permeability, while the Mdx is variable depending on the animal and the area considered. Subsequently, we carried out a multiple cytokine array through which two cytokines were found to be more present in the Mdx diaphragm compared to the Wt: Endostatin and Cxcl4. Given these results, through a 2D wound healing assay, we tested the migratory ability of DMD cells using cell media supplemented with Endostatin, Cxcl4, and a combination of the two. We hypothesized that cells should exhibit a reduced migratory capacity in the presence of both cytokines (Cxcl4 and Endostatin), if these factors are involved in cell migration, and indeed we found a trend of reduced migratory capacity only when both cytokines were present. To further confirm our hypothesis, we tested cell migration in 3D setting performing a recellularization using Wt diaphragms with the addition of the cytokines in the culture media. Also in this case, the condition in which both cytokines were present demonstrated less cell migration. In this study we observed that the production of 3D in vitro model with the use of DMD cells and Mdx diaphragm is challenging since cells do not migrate inside the dystrophic environment, as instead happens in the Wt diaphragm. Furthermore, the discovery of cytokines directly involved in inhibition of cell migration opens future strategy to deeper investigate the dystrophic environment in search of new therapeutic targets.