Page 82 - IJB-10-5

International Journal of Bioprinting dECM bioink for 3D musculoskeletal tissue reg.

In NTIRE, small electrical pulses are applied to the entire with other chemical decellularization protocols to achieve
tissue and cells. This process induces potential instability efficient decellularization while preserving biochemical
and micropore formation in the cell membrane, leading to and biomechanical properties. 96,110
the release of intracellular components. While NTIRE is
103
effective in preserving the morphology and integrity of the 4.3. Enzymatic methods
ECM network, its limited probe size restricts its suitability Enzymatic decellularization involves using proteases (e.g.,
for decellularizing large tissues or organs. trypsin), nucleases (e.g., RNase and DNase), lipases, and
99
collagenases to selectively cleave cell adhesion proteins,
4.2. Chemical methods detach and lyse cells from the surrounding matrix, and
Chemical decellularization is commonly achieved by using disrupt cell adhesion to the ECM. 92,115 Nucleases catalyze
chemical agents, such as acids, bases, ions, or non-ionic the hydrolytic cleavage of deoxyribonucleotides and
detergents. These agents promote the hydrolytic degradation ribonucleotide chains, fragment RNA or DNA sequences,
of biomolecules and destroy the cell membrane. 96,104 The and facilitate the removal of nucleotides after cell lysis. 92,99
effectiveness of chemical decellularization depends on Although enzymatic treatments are effective in
factors such as chemical concentration, exposure time, and removing cells or cells, they tend to destroy ECM structures,
tissue type.
105
thereby affecting their inherent mechanical strength.
4.2.1. Acids and bases Additionally, it is challenging to completely remove cellular
Acid–base treatments decellularize the ECM by dissolving components with enzymes. Any residual enzymes in the
cytoplasmic components and degrading nucleic acids dECM can hinder cell revascularization, leading to adverse
99
(e.g., RNA and DNA). Additionally, acid–base treatments immune responses, such as inflammation and apoptosis.
have sterilizing capabilities. 92,96 Acid decellularization Therefore, enzymes are often used in combination with
agents mainly include acetic acid, peroxyacetic acid chemical methods to enhance decellularization efficiency.
(PAA), sulfuric acid, hydrochloric acid, and deoxycholic Yang et al. used a combination of 0.25% trypsin-EDTA and
acid. 92,106,107 Acid decellularization is typically employed collagenase type II for the initial treatment of cartilage.
to denature ECM proteins, including GAG, collagen, and The treated cartilage was then immersed in a hypotonic
GFs, resulting in reduced ECM strength. Conversely, the buffer, containing 0.5% Triton X-100 and ammonium
commonly used alkaline reagents are sodium hydroxide, hydroxide, for effective decellularization, while preserving
ammonium hydroxide, sodium sulfide, and calcium the bioactive factors. 118
hydroxide. 92,108 These reagents can denature chromosomal
and plasmid DNAs, disrupt the crosslinking of collagen 5. Strategies for preparing decellularized
fibers, and completely remove essential biomolecules (e.g., extracellular matrix-based bioinks
GFs) from the matrix during decellularization, resulting
in a significant reduction in mechanical properties The preparation of dECM-based bioinks involves a
of ECM. 20,109 three-step process: (i) tissue preparation, (ii) tissue
decellularizing, and (iii) post-decellularization processing.
4.2.2. Detergents Tissue preparation involves removing connective
Detergents can be characterized into ionic, nonionic, tissue, large blood vessels, fat, and other impurities and
and zwitterionic types. Ionic detergents efficiently dividing the tissue into smaller portions. Thereafter,
lyse cytoplasmic and nuclear membranes and DNA decellularization methods are selected based on tissue
by disrupting protein–protein junctions. They are characteristics to remove native cells, while preserving the
107
commonly used for the decellularization of tissues and ECM’s internal structure and bioactive components. After
organs, including heart, liver, kidney, ovary, and decellularization, the remaining toxic decellularization
110
111
113
112
skeletal muscle, with sodium dodecyl sulfate (SDS) agents were removed, and the dECM was subjected to
114
being the most frequently used. Although SDS is a very component analysis to verify the absence of cellular
effective decellularizing agent, it may also disrupt the components. Subsequently, sterilization was performed to
internal structure of the ECM, reduce the content of GAG prevent adverse immune reactions. 116,138 Depending on the
and essential GFs, and disrupt collagen integrity. 115,116 In biochemical and physical properties of dECMs, a variety
contrast, non-ionic detergents (e.g., Triton X-100) destroy of sterilization methods can be employed. Peracetic acid
inter-lipid and inter-lipid–protein connections while and/or ethanol are commonly used for sterilizing dECMs.
maintaining the integrity of protein–protein connections, Alternatively, ultraviolet (UV) light, ethylene oxide, and
making them suitable for maintaining the internal gamma radiation can also be employed, but they may alter
structure of the ECM. Nonionic detergents are more the structure and properties of the dECM. Once the
139
99
suitable for thinner tissues, and they are often combined residual reagents are removed and the dECM is sterilized,
Volume 10 Issue 5 (2024) 74 doi: 10.36922/ijb.3418