Programa de Pós-Graduação em Anatomia dos Animais Domésticos e Silvestres, Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia, Departamento de Cirurgia, Disciplina de Anatomia, Av. Prof. Dr. Orlando Marques de Paiva, 87, Cidade Universitária, 05508-270 São Paulo, SP, Brazil.
Graduação em Ciências Biológicas, Universidade São Judas Tadeu, Av. Pereira Barreto, 1479, Baeta Neves, 09751-000 São Bernardo do Campo, SP, Brazil.
An Acad Bras Cienc. 2021 Jun 28;93(2):e20190942. doi: 10.1590/0001-3765202120190942. eCollection 2021.
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.
体积性肌肉损失导致功能减弱，常采用肌肉移植物或生物材料植入进行治疗。通过组织脱细胞化获得的细胞外基质已广泛用作组织工程中的生物生物材料。最佳的脱细胞化方法因组织而异，对基质的质量有重大影响。本研究旨在比较四种方案的效果，这些方案根据组织储存温度和化学试剂顺序而有所不同，旨在脱细胞化鼠类骨骼肌。从小鼠中取出胫骨前肌，并在-20°C 下冷冻或在室温下储存，然后用含有 EDTA+Tris、SDS 和 Triton X-100 的溶液按不同顺序进行脱细胞化。对样本进行宏观方面、细胞去除、DNA 含量减少、蛋白质保留和基质三维结构的分析。与起始于 EDTA+Tris 的方案相比，以 SDS 溶液孵育开始的处理方案优化了细胞和 DNA 含量的去除，并保留了基质的超微结构和组成。在脱细胞化之前冷冻样本有利于细胞去除，无论化学试剂的顺序如何。因此，冷冻骨骼肌并以 1% SDS 溶液开始脱细胞化显示出最佳效果。
