Decellularization of Extracellular Matrix from Equine Skeletal Muscle

Abstract

Equine represents an attractive animal model for musculoskeletal tissue diseases, exhibiting much similarity to the injuries that occur in humans. Cell therapy and tissue bioengineering have been widely used as a therapeutic alternative by regenerative medicine in musculoskeletal diseases. Thus, the aim of this study was to produce an acellular biomaterial of equine skeletal muscle and to evaluate its effectiveness in supporting the in vitro culture of equine induced pluripotency stem cells (iPSCs). Biceps femoris samples were frozen at -20°C for 4 days and incubated in 1% sodium dodecyl sulfate (SDS), 5 mM EDTA + 50 mM Tris and 1% Triton X-100; the effectiveness of the decellularization was evaluated by the absence of remnant nuclei (histological and 4',6-diamidino-2-phenylindole [DAPI] analysis), preservation of extracellular matrix (ECM) proteins (immunofluorescence and immunohistochemistry) and organization of ECM ultrastructure (scanning electron microscopy). Decellularized samples were recellularized with iPSCs at the concentration of 50,000 cells/cm² and cultured in vitro for 9 days, and the presence of the cells in the biomaterial was evaluated by histological analysis and presence of nuclei. Decellularized biomaterial showed absence of remnant nuclei and muscle fibers, as well as the preservation of ECM architecture, vascular network and proteins, laminin, fibronectin, elastin, collagen III and IV. After cellularization, iPSC nuclei were present at 9 days after incubation, indicating the decellularized biomaterial-supported iPSC survival. It is concluded that the ECM biomaterial produced from the decellularized equine skeletal muscle has potential for iPSC adhesion, representing a promising biomaterial for regenerative medicine in the therapy of musculoskeletal diseases.

Keywords: Biomaterial; Decellularized extracellular matrix; Regenerative medicine; Skeletal muscle; iPSC.