An Acad Bras Cienc (2021) 93(2): e20190942 DOI 10.1590/0001-3765202120190942 Anais da Academia Brasileira de Ciências | Annals of the Brazilian Academy of Sciences Printed ISSN 0001-3765 I Online ISSN 1678-2690 www.scielo.br/aabc | www.fb.com/aabcjournal CELLULAR AND MOLECULAR BIOLOGY Effects of chemical and physical methods on Running title: decellularization of murine skeletal muscles DECELLULARIZATION OF MURINE SKELETAL MUSCLES CARLA M.F.C. MIRANDA, LUCIANO C.P.C. LEONEL, RAFAEL R. CAÑADA, DURVANEI A. MARIA, MARIA ANGÉLICA MIGLINO, MARIANO DEL SOL & SONJA E. LOBO Academy Section: CELLULAR Abstract: Volumetric muscle loss causes functional weakness and is often treated with AND MOLECULAR BIOLOGY 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 e20190942 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 93 harvested from rats and were frozen at -20°C or stored at room temperature, followed (2) by decellularization in solutions containing EDTA + Tris, SDS and Triton X-100, applied 93(2) in different sequences. Samples were analyzed for macroscopic aspects, cell removal, DOI decrease of DNA content, preservation of proteins and three-dimensional structure 10.1590/0001-3765202120190942 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. Key words: extracellular matrix, skeletal muscle, decellularization, biomaterial. INTRODUCTION injuries, loss of function and high morbidity at the donor site (Jana et al. 2016). Alternatively, Skeletal muscles correspond to 50% of the body implant of biological biomaterials, composed mass and present high regenerative capacity of tissue’s extracellular matrix (ECM), has been (Juhas & Bursac 2013). However, their self- investigated. Decellularization protocols are renewal ability is decreased after orthopedic and tissue and organs dependent and directly impact peripheral nerve damages, irreversible muscle the quality of the resulting ECM (Leonel et al. atrophy and volumetric muscle loss (VML) (Wu 2017). Ideal ECM-based scaffolds should mimic et al. 2012). VML is the immediate loss of muscle the structure, biochemical and biomechanical fibers and can lead to inadequate mechanical cues of the tissue to be reconstructed (Lee et and functional performances of the remaining al. 2017). musculature. Decellularized tissues have been used alone Reconstruction of VML commonly requires or loaded with stem and/or progenitor cells transplant of autologous muscle grafts, which (Parmaksiz et al. 2016), in several applications, present several drawbacks, including insufficient including regeneration of skin (Chen et al. 2004), donor tissue, particularly in case of severe ligaments (Endress et al. 2012) and tendons An Acad Bras Cienc (2021) 93(2) CARLA M.F.C. MIRANDA et al. DECELLULARIZATION OF MURINE SKELETAL MUSCLES (Yin et al. 2013). In the case of skeletal muscles, MATERIALS AND METHODS allografts (ECM from donors of the same species as the recipient) and xenografts (from donors This study was approved by the Animal Care and of species other than the recipient) have been Use Committees of the Faculdade de Medicina analyzed in vitro, in vivo and clinically (Sicari et Veterinária e Zootecnia da Universidade de São al. 2014, Porzionato et al. 2015). Paulo (CEUA n° 9553300114) and the Instituto Tissue decellularization can be performed Butantan (CEUA n° 2989051015), Brazil. using chemical, physical, enzymatic methods, or Fifty male Wistar rats, weighing 350 grams, 3 with a combination of them. Chemical methods months-old, were used as donors of the right and comprehend the use of ionic (sodium dodecyl left tibialis anterior muscles (TA) (n= 100). Animals sulfate; SDS), nonionic (Triton X-100) or zwitterionic were subjected to euthanasia: anesthesia was (3-[(3-chola-midopropyl) dimethylammonio]- performed through intraperitoneal injection of 1- propanesulfonate; CHAPS) surfactants, acid Ketamine and Xylazine (100mg/Kg and 10mg/ (peracetic acid) and basic solutions (ammonium Kg, respectively), followed by an overdose of the before mentioned drugs (3x de concentration). hydroxide; NH4OH), to disrupt and to eliminate cells (Gilpin & Yang 2017). Physical methods This was followed by intravenous injection of include freeze-thaw cycles, which reduce the 10% potassium chloride. After absence of heart content of vital cells (Burk et al. 2014). Enzymatic beating, thoracic and respiratory movements processes often apply the proteolytic enzyme and loss of mucous membranes coloring were Trypsin and the nucleases DNase and RNase to observed, TA muscles were dissected and facilitate nucleotides elimination after cell lysis harvested. (Boccafoschi et al. 2017). Murine skeletal muscle decellularization All methods have pros and cons. For instance, SDS can lead to complete cell removal and Harvested muscles were initially washed in eliminates up to 90% of DNA, but it may decrease running water and incubated in phosphate the ECM glycosaminoglycans and growth factors buffered saline (PBS) 1x + 0.5% penicillin and content; Triton X-100 is less aggressive than SDS, streptomycin (LGC Biotechnology, Brazil), for 3 but it does not eliminate cellular components times of 5 minutes each, under agitation on a that remain attached to the matrix (Gilpin & shaker TS-2000A VDRL (Biomixer, USA). Yang 2017). Therefore, distinct solutions and Four protocols were tested. They varied methods have been combined to guarantee cell according to (1) the sequence of chemical removal and to minimize deleterious effects to reagents that were used and (2) the storage the ECM physical structure and composition. temperature. i.e. frozen at -20ºC or kept at room This study aimed at comparing the effects of temperature (RT). physicochemical methods on decellularization Protocols were the following: of murine skeletal muscles, which are an 1- EDTA+Tris RT group: samples were important tool for investigations in tissue incubated in 5mM ethylenediamine tetraacetic engineering and translational medicine. acid (EDTA; LGC Biotechnology, Brazil) associated with 50 mM Tris (LGC Biotechnology, Brazil) for 2 days; rinsed in 1x PBS + 0.5% antibiotics (Penicillin-Streptomycin, 3x, 5 minutes each), followed by incubation in 1% sodium dodecyl An Acad Bras Cienc (2021) 93(2) e20190942 2 | 11 CARLA M.F.C. MIRANDA et al. DECELLULARIZATION OF MURINE SKELETAL MUSCLES sulfate (SDS; LGC Biotechnology, Brazil) for 4 quantification of remaining DNA and days. New washing was carried out using 1x PBS immunohistochemistry were also performed. + 0.5% antibiotics (3x, 5 minutes) and incubation in 1% Triton X-100 (LGC Biotechnology, Brazil) for Histology 2 days. Throughout this process, samples were Decellularized and control (non-processed) kept at room temperature (RT) (n= 25). skeletal muscles (n= 4) were fixed in 4% PFA for 2- EDTA+Tris -20°C group: after the initial 48 hours, incubated in alcohol 70% overnight, washing and before decellularization, samples dehydrated in ethanol (80%, 90%, 95%, 100% were frozen at -20°C for 4 days (n= 25). Then, twice, 30 min), incubated in xylene (2x of 30 they were thawed, incubated in 1x PBS for 20 min) (Dynamic, Brazil), included in paraffin (ERV- minutes at room temperature and decellularized PLAST, EasyPath, Brazil) for 3h and sectioned according to the sequence of solutions described into 5 μm thickness slices (microtome Leica above. RM 2125RT, Germany). Before staining, slices 3- SDS RT group: samples were incubated were incubated in xylene (2x, 15 minutes) and in 1% SDS for 4 days; rinsed in 1x PBS + 0.5% rehydrated in decreasing series of alcohol (100%, antibiotics (3x, 5 minutes each); incubated in 5 100%, 95%, 70%, 3 minutes) and in running water mM EDTA + 50 mM Tris for 2 days; rinsed again (10 minutes). in 1x PBS + 0.5% antibiotics (3x, 5 minutes each) Hematoxylin and Eosin (H&E), Masson’s and, incubated in 1% Triton X-100 for 2 days. Trichrome and Picrosirius staining were Samples were kept at room temperature (RT) performed to allow observation of muscle throughout the process (n= 25). fibers and cell nuclei, and the organization and 4- SDS -20°C group: after harvesting, preservation of collagen fibers. samples were washed and frozen at -20°C (n= Sections were analyzed under a Nikon 80i 25). After 4 days, they were thawed, incubated light microscope (Japan) in a 50x objective in 1x PBS for 20 minutes at room temperature (for H&E and Masson’s Trichrome) and under and decellularized according to the sequence of polarized light, using a Carl Zeiss microscope solutions described for protocol SDS RT. (Germany) with 16x objective (for Picrosirius Throughout these processes, samples staining). remained immersed in 25 ml of solutions, under agitation, at room temperature, for 10 h, during DNA quantification the