Decellularized Human Diaphragm Muscle as a Biologic Scaffold for Reconstructive Surgery

Therapeutic options available in the clinical practice to restore severe loss of muscle mass after trauma or tumor excision offer limited functional and aesthetic results and they are limited by several drawbacks, such as donor site morbidities and increased risk of complication. Thus, tissue engineering is focusing on the development of different strategies to induce and favor tissue regeneration limiting scar formation and fibrosis. Among those, the creation of 3-D scaffold has shown favorable results thanks to its tissue-specificity and ability to stimulate cell differentiation toward the tissue of origin. In this project, we aimed to create a decellularized human-derived scaffold from diaphragm muscle. Four different detergent-enzymatic protocols were compared to identify the ideal treatment to remove immunogenic signals maintaining structural and functional properties to allow scaffold implantation and cell seeding. Together with DNase I and Trypsin, four different detergents (sodium dodecyl sulfate, SDS + TergitolTM, sodium deoxycholate and TergitolTM) were compared. All protocols were able to remove nuclei, DNA and muscle fibers preserving collagen, elastin and glycosaminoglycans. In addition while HLA-DR was no more detectable, Collagen I and IV, Laminin (key components of the ECM) were expressed. Macroscopic evaluation and tensile strength confirmed the preservation of functionality and structure without differences among protocols. Adipose-derived mesenchymal stem cells co-culture and seeding showed absence of cytotoxicity and ability of the graft to support cell proliferation after all decellularization. Protocol n. 2 (SDS + TergitolTM) exhibited the higher preservation of collagen structure and fibers orientation. For this reason scaffolds treated with this protocol were implanted in mice for 14 days during the in vivo biocompatibility test, which demonstrated favorable integration with VEGF positivity and without signs of severe immunological response from the host.