Customised dynamic bioreactor system enables the production of radially aligned 3D diaphragmatic muscle constructs promoting ECM remodelling and inhibiting fibroblasts overgrowth.

The production of skeletal muscle constructs useful for in vivo large defects replacement, such as congenital diaphragmatic hernia (CDH), is still considered a challenge. The standard application of prosthetic material presents strong limitations, like hernia recurrences in a remarkable number of CDH patients. With this work, we developed a tissue engineering approach based on decellularized diaphragmatic muscle and human cells for the generation in vitro of diaphragmatic-like tissues as proof-of-concept of a new option for the surgical treatment of large diaphragm defects. A specific bioreactor for diaphragmatic muscle was achieved to control mechanical stimulation during the construct generation. In vitro tests demonstrated the increased maturation of mechanically stimulated constructs, with the formation of new oriented and aligned muscle fibres. Moreover, after in vivo orthotopic implantation in a CDH mouse model, mechanically stimulated muscles maintained the presence of human cells within myofibers, supporting the idea of a new therapeutic option for this dramatic congenital malformation. Production of diaphragmatic-like tissues using mouse decellularized extracellular matrix, human cells and mechanical stimulation with bioreactor Gene expression was measured using Affymetrix platform in 12 diaphragm-like tissues (6 STATIC and 6 DYNAMIC samples)