Acellular Urethra Bioscaffold: Decellularization of Whole Urethras for Tissue Engineering Applications

Abstract

Patients with stress urinary incontinence mainly suffer from malfunction of the urethra closure mechanism. We established the decellularization of porcine urethras to produce acellular urethra bioscaffolds for future tissue engineering applications, using bioscaffolds or bioscaffold-derived soluble products. Cellular removal was evaluated by H&E, DAPI and DNA quantification. The presence of specific ECM proteins was assessed through immunofluorescence staining and colorimetric assay kits. Human skeletal muscle myoblasts, muscle progenitor cells and adipose-derived stromal vascular fractions were used to evaluate the recellularization of the acellular urethra bioscaffolds. The mechanochemical decellularization system removed ~93% of tissue’s DNA, generally preserving ECM’s components and microarchitecture. Recellularization was achieved, though methodological advances are required regarding cell seeding strategies and functional assessment. Through microdissection and partial digestion, different urethra ECM-derived coating substrates were formulated (i.e. containing smooth or skeletal muscle ECM) and used to culture MPCs in vitro. The skeletal muscle ECM substrates enhanced fiber formation leading to the expression of the main skeletal muscle-related proteins and genes, as confirmed by immunofluorescence and RT-qPCR. The described methodology produced a urethra bioscaffold that retained vital ECM proteins and was liable to cell repopulation, a crucial first step towards the generation of urethra bioscaffold-based Tissue Engineering products.

Decellularized human ovarian scaffold based on a sodium lauryl ester sulfate (SLES)-treated protocol, as a natural three-dimensional scaffold for construction of bioengineered ovaries

“…Another application of decellularized ECM is improvement in the biological properties of synthetic materials [ 11 ] or fabrication of hydrogels [ 12 – 15 ]. Decellularization techniques have been successfully used for a variety of organs including the heart [ 16 ], blood vessels [ 17 ], urethra [ 18 ], urinary bladder [ 19 ], kidney [ 20 , 21 ], trachea [ 22 ], lung [ 23 ], skeletal muscle [ 24 ], liver [ 25 ], and gastrointestinal tract [ 26 , 27 ].…”

Tissue and organ decellularization in regenerative medicine

“…In some studies, Triton X‐100 has been reported to be the best decellularizing agent compared with other detergents and that for many tissues and organs including ligaments, small intestine, annulus fibrosus, liver and aortic valves . On the other hand, some studies demonstrated that Triton X‐100 had poor cell removal capacity for many tissues and organs such as veins, cornea, urethra, heart and kidneys …”

“…SDS incorporates into the outer membrane layer by increasing its curvature, creating a stress which results in the membrane disruption . SDS proved to be superior when compared with other detergents in removing cells and genetic materials in many tissues and organs including aorta, veins, heart, kidneys and urethra . Triton X‐100 and SDS have also shown negative influence on tissues and organs used for decellularization but, on the other hand, when these two detergents were used in combination, the mixture allowed efficient decellularization and preservation of ECM structures and this has been tested on tendons, kidneys, SIS, and liver …”

“…42,[86][87][88][89] On the other hand, some studies demonstrated that Triton X-100 had poor cell removal capacity for many tissues and organs such as veins, cornea, urethra, heart and kidneys. 18,[90][91][92][93] A detergent commonly used in decellularization is the SDS. SDS is an anionic detergent, used in commercial applications such as in cleaning and in the making of cosmetics.…”

“…95 SDS proved to be superior when compared with other detergents in removing cells and genetic materials in many tissues and organs including aorta, veins, heart, kidneys and urethra. 18,90,92,93,96 Triton X-100 and SDS have also shown negative influence on tissues and organs used for decellularization but, on the other hand, when these two detergents were used in combination, the mixture allowed efficient decellularization and preservation of ECM structures and this has been tested on tendons, kidneys, SIS, and liver. 11,43,97,98 Another ionic detergent/bile salt frequently used in decellularization is sodium deoxycholate (SDC).…”