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.
Highlights
Regeneration and proper function[9]
Derived matrices can be obtained through organ/tissue decellularization to produce bioscaffolds that retain a similar composition, microstructure and biomechanical properties as the native tissue/organ while preserving most of the signaling cues required for organ development, repair and physiological regeneration[12]
Female urethras had an average length of 4.7 ± 1.6 cm, while male urethras had 6.6 ± 1.8 cm
Summary
Regeneration and proper function[9]. To do so, naturally-derived and tissue-specific ECM can serve as a basis to develop tissue engineering products for SUI therapies. In 2009, Zhang and colleagues demonstrated the dramatic effect of tissue-specific ECM compounds on the enhancement of cell proliferation and differentiation from each specific tissue[10] Such matrices can provide pivotal information about tissue-specific niches and be used to develop a reliable in vitro human model of SUI to test new therapies and drugs, since most work has been performed in vivo, using animal models[11]. Derived matrices can be obtained through organ/tissue decellularization to produce bioscaffolds that retain a similar composition, microstructure and biomechanical properties as the native tissue/organ while preserving most of the signaling cues required for organ development, repair and physiological regeneration[12]. In 2015, De Filippo and colleagues used acellular collagen matrices derived from decellularized porcine bladders to create seeded tubularized grafts for urethra repair[23] These structures were seeded with autologous smooth muscle cells and transplanted into 3 cm urethral defects in rabbits.
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