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Review
. 2022 Feb 28:10:831300.
doi: 10.3389/fbioe.2022.831300. eCollection 2022.

Decellularization Strategies for Regenerating Cardiac and Skeletal Muscle Tissues

Yong How Tan  1 , Haylie R Helms  1 , Karina H Nakayama  1
Affiliations
Review

Decellularization Strategies for Regenerating Cardiac and Skeletal Muscle Tissues

Yong How Tan et al. Front Bioeng Biotechnol. .

Abstract

Cardiovascular disease is the leading cause of death worldwide and is associated with approximately 17.9 million deaths each year. Musculoskeletal conditions affect more than 1.71 billion people globally and are the leading cause of disability. These two areas represent a massive global health burden that is perpetuated by a lack of functionally restorative treatment options. The fields of regenerative medicine and tissue engineering offer great promise for the development of therapies to repair damaged or diseased tissues. Decellularized tissues and extracellular matrices are cornerstones of regenerative biomaterials and have been used clinically for decades and many have received FDA approval. In this review, we first discuss and compare methods used to produce decellularized tissues and ECMs from cardiac and skeletal muscle. We take a focused look at how different biophysical properties such as spatial topography, extracellular matrix composition, and mechanical characteristics influence cell behavior and function in the context of regenerative medicine. Lastly, we describe emerging research and forecast the future high impact applications of decellularized cardiac and skeletal muscle that will drive novel and effective regenerative therapies.

Keywords: ECM; cardiac engineered tissue; dECM; decellularized extracellular matrix; decellularized heart; decellularized muscle; extracellular matrix; skeletal muscle engineering.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Schematic overview of the donor-to-application lifecycle of decellularized tissues (A) Tissues are harvested from an animal donor (B) Whole tissues are processed with mechanical agitation to break down into smaller minced tissues (C) Whole or minced fresh tissues are exposed to a series of decellularizing agents and procedures to remove cellular materials (D) The decellularization end products are acellular tissues containing various ECM components (E) Whole acellular tissues may be seeded with cells in vitro to test for cell viability (F) A variety of dECM forms are used for the maturation of cardiomyocytes, production of injectable hydrogels, and formulation into bioink for 3D bioprinting (G) Selected forms of decellularized tissues including injectable hydrogels, 3D bioprinted materials, and whole acellular and recellularized tissues can be used for translational pre-clinical and clinical applications. Figure created using BioRender.com.
FIGURE 2
FIGURE 2
Minced tissue decellularization protocols and outcomes (A) Tissue morphology from top to bottom: H&E staining, Azan Mallory stains, Van Gieson stains, and SEM images of minced tissues before (Native, column 1) and after decellularization with SDC (Protocol 1, SDC + DNase I, column 2) and Trypsin (Protocol 2, Trypsin/EDTA + Triton X-NH4OH, column 3) protocols (B) DNA, muscle content, and connective tissue content before and after decellularization using protocol one and protocol 2. Figures A,B are adapted from “Decellularized Human Skeletal Muscle as Biologic scaffold for Reconstructive Surgery” by Porzionato et al. (2015) licensed under CC BY 4.0.
FIGURE 3
FIGURE 3
Cardiac extracellular matrix composition (A) Schematic of cardiac tissue before and after decellularization (B–C) Extracellular matrix microstructure of decellularized whole porcine hearts following four different decellularization protocols. Decell 3/4: Trypsin for 3 days followed by Triton for 4 days. Decell 1/6: Trypsin 1 day, Triton 6 days. Tryp Only: Trypsin 7 days. Trit Only: Triton 7 days. Scale bar 50 µm (B) Elastin microstructure. Two photon fluorescence (C) Collagen microstructure. Second harmonic generation imaging (D–F) Rat myocardial ECM morphology and composition before (native) and after whole heart decellularization using four different decellularization protocols. Briefly, Protocol I: SDS and Triton. Protocol II: Trypsin, EDTA, deoxycholic acid (DCA), and acetic acid. Protocol III: Glycerol, NaN3, EDTA, NaCl, DCA, SDS, and Triton. Protocol IV: SDS, DCA, NaN3, Glycerol, EDTA, Saponin, DNase I, and MgCl (D) The 15 most abundant proteins in cardiac ECM measured by liquid chromatography–mass spectrometry (LC-MS/MS) at each developmental age (E) Mean DNA content and (F) mean GAG content of the native whole rat heart before (native) and after decellularization using protocols I - IV (G) Rat myocardial ECM morphology from top to bottom: Hematoxylin and Eosin (H&E) staining, Movat Pentachrome (Movat), picrosirius red and fast green (PSR & FG), and alpha-actin of rat hearts before (Native, column 1) and after whole heart decellularization (Protocols I–IV, columns 2—5). CV, coronary vessel. ×100 magnification. Figure A is created using BioRender.com. Figures B, C are adapted with permission from Merna et al. (2013). Figure D is adapted with permission from Williams et al. (2014). Figures E–G are adapted with permission from Akhyari et al. (2011).
FIGURE 4
FIGURE 4
Differences in decellularization protocols determine dECM properties in whole tissues (A) Schematic of tissue harvest from a rat hindlimb for whole muscle decellularization (B) A breakdown of three example decellularization protocols used in whole tissue decellularization (C) Final DNA content after whole tissue decellularization using three different decellularization protocols (D) Tissue morphology from top to bottom: Macroscopic tissue images, Hematoxylin and Eosin (H&E) staining, and Scanning Election Microscope (SEM) images of whole tissues before (Fresh, first column) and after decellularization with LatB (second column), DET (third column), and SDS (fourth column) protocols (E) Massons Trichrome (MT) staining of whole tissue after decellularization (F) ECM content of collagen and GAGs remaining in decellularized whole tissues compared to fresh tissues. Figures A–F are adapted from “Decellularised skeletal muscles allow functional muscle regeneration by promoting host cell migration” by Urciuolo et al. (2018) licensed under CC BY 4.0.
FIGURE 5
FIGURE 5
Patterning and topography in dECM biomaterials (A) Material topography with a random-orientation of fibers and cells (B) Nano- and micropatterned materials with parallel orientation of fibers or channels showing differences in cell guidance and interaction (C–E) C2C12 myoblasts cultured on 3D-printed line widths of 500 μm, 1,500 μm, and 5,000 µm on (C) day 1 and (D) day 7; cells were stained green for F-actin (Phalloidin), and blue for nuclei (DAPI) (E) Quantification of % aligned cells on the varying line widths at day 1 and 7 (F) Porous and Non-Porous properties of bulk materials differentially regulate the diffusion of gases and other soluble factors (G) Quantification of average pore sizes in native skeletal muscle tissue and decellularized scaffolds treated with 0.2% SDS and 1% SDS, respectively (H) Macroscopic and SEM images of native skeletal muscle and decellularized scaffolds treated with 0.2% SDS and 1% SDS, respectively. Figures A, B, F are created using BioRender.com. Figures C–E are adapted with permission from Choi et al. (2016). Figures G, H are adapted with permission from Shapiro et al. (2019).
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