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Introduction. The employment of recellularized autologous extracellular matrices (ECM) as scaffold for the reconstruction of complex tissues like skeletal muscle has been recognized as a successful strategy for regenerative medicine. Compared to the application of synthetic patches, which represent the current standard of care for congenital neonatal malformations like omphalocele and gastroschisis, the implantation of a naturally derived bioengineered construct has great advantages such as providing biochemical and mechanical stimuli driving cell growth and differentiation. However, the decellularization method chosen to produce the scaffold deeply influences its properties and the recellularization efficiency. Therefore, the aim of this work was to study the best method to decellularize human muscle and to prove the recellularization efficiency.
Metodology. Two detergents (sodium deoxycholate and sodium dodecyl sulphate) and latrunculin B were used in cycles of decellularization together with DNase I. At the end of each cycle, the samples were analyzed for residual DNA content, ECM component preservation (collagen network, laminin, glycosaminoglycans), and mechanical properties. Finally, in the matrices produced, human muscle precursor cells were injected to evaluate the recellularization efficiency.
Results. The usage of detergents allowed the obtainment of an 85% reduction in DNA content, but muscle ECM architecture and composition was more impaired with detergents with respect to latruculin B. In addition, a higher amount of residual sulphated glycosaminoglycans was found after treatment with latrunculin B, while the detergents clearly compromised the glycocalyx. Furthermore, in the ECM treated with detergents, cells accumulated only at the border of the samples, while in latrunculin B-treated matrices cells were more evenly distributed, with high proliferative and myogenic capacity.
Conclusions. These results indicate the latrunculin B protocol as the best method for the obtainment of a human skeletal muscle ECM compared with the usage of detergents. The efficient decellularization did not impair native tissue architecture, mechanical properties, and composition. The high recellularization yield achieved makes it a tool for the in vitro production of a bioengineered skeletal muscle construct, with the final goal to produce implantable tissues for regenerative medicine purposes.
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