Effects of chemical and physical methods on decellularization of murine skeletal muscles.

Carla M.F.C Miranda,Rafael R Cañada,Sonja E Lobo,Mariano Del Sol,Luciano C.P.C Leonel,Durvanei A Maria,Maria Angélica Miglino

doi:10.1590/0001-3765202120190942

Abstract

Volumetric muscle loss causes functional weakness and is often treated with muscle grafts or implant of biomaterials. Extracellular matrices, obtained through tissue decellularization, have been widely used as biological biomaterials in tissue engineering. Optimal decellularization method varies among tissues and have significant impact on the quality of the matrix. This study aimed at comparing the efficacy of four protocols, that varied according to the temperature of tissue storage and the sequence of chemical reagents, to decellularize murine skeletal muscles. Tibialis anterior muscles were harvested from rats and were frozen at -20°C or stored at room temperature, followed by decellularization in solutions containing EDTA + Tris, SDS and Triton X-100, applied in different sequences. Samples were analyzed for macroscopic aspects, cell removal, decrease of DNA content, preservation of proteins and three-dimensional structure of the matrices. Processing protocols that started with incubation in SDS solution optimized removal of cells and DNA content and preserved the matrix ultrastructure and composition, compared to those that were initiated with EDTA + Tris. Freezing the samples before decellularization favored cell removal, regardless of the sequence of chemical reagents. Thus, to freeze skeletal muscles and to start decellularization with 1% SDS solution showed the best results.

Highlights

Skeletal muscles correspond to 50% of the body mass and present high regenerative capacity (Juhas & Bursac 2013)
This study aimed at comparing the effects of physicochemical methods on decellularization of murine skeletal muscles, which are an important tool for investigations in tissue engineering and translational medicine
Decellularized muscles were evaluated according to the following aspects: macroscopic features of the samples, removal of cells, concentration of double-stranded DNA (dsDNA), the extracellular matrix (ECM) 3D structure and preservation of proteins

Summary

Introduction

Skeletal muscles correspond to 50% of the body mass and present high regenerative capacity (Juhas & Bursac 2013). Their selfrenewal ability is decreased after orthopedic and peripheral nerve damages, irreversible muscle atrophy and volumetric muscle loss (VML) (Wu et al 2012). Reconstruction of VML commonly requires transplant of autologous muscle grafts, which present several drawbacks, including insufficient donor tissue, in case of severe injuries, loss of function and high morbidity at the donor site (Jana et al 2016). Ideal ECM-based scaffolds should mimic the structure, biochemical and biomechanical cues of the tissue to be reconstructed (Lee et al 2017)

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Conclusion