Preclinical Development of Bioengineered Allografts Derived from Decellularized Human Diaphragm

Volumetric muscle loss (VML) is the traumatic/surgical loss of skeletal muscle, causing aesthetic damage and functional impairment. Suboptimal current surgical treatments are driving research towards the development of optimised regenerative therapies. The grafting of bioengineered scaffolds derived from decellularized skeletal muscle may be a valid option to promote structural and functional healing. In this work, a cellular human diaphragm was considered as a scaffold material for VML treatment. Decellularization occurred through four detergent-enzymatic protocols involving (1) sodium dodecyl sulfate (SDS), (2) SDS + Tergitol^TM, (3) sodium deoxycholate, and (4) Tergitol^TM. After decellularization, cells, DNA (≤50 ng/mg of tissue), and muscle fibres were efficiently removed, with the preservation of collagen/elastin and 60%-70% of the glycosaminoglycan component. The detergent-enzymatic treatments did not affect the expression of specific extracellular matrix markers (Collagen I and IV, Laminin), while causing the loss of HLA-DR expression to produce non-immunogenic grafts. Adipose-derived stem cells grown by indirect co-culture with decellularized samples maintained 80%-90% viability, demonstrating the biosafety of the scaffolds. Overall, the tested protocols were quite equivalent, with the patches treated by SDS + Tergitol^TM showing better collagen preservation. After subcutaneous implant in Balb/c mice, these acellular diaphragmatic grafts did not elicit a severe immune reaction, integrating with the host tissue.