Fabrication of Adipose-Derived Stem Cell-Based Self-Assembled Scaffold under Hypoxia and Mechanical Stimulation for Urethral Tissue Engineering

Abstract

Long urethral strictures are often treated with autologous genital skin and buccal mucosa grafts; however, risk of hair ingrowth and donor site morbidity, restrict their application. To overcome this, we introduced a tissue-engineered human urethra comprising adipose-derived stem cell (ASC)-based self-assembled scaffold, human urothelial cells (UCs) and smooth muscle cells (SMCs). ASCs were cultured with ascorbic acid to stimulate extracellular matrix (ECM) production. The scaffold (ECM) was stained with collagen type-I antibody and the thickness was measured under a confocal microscope. Results showed that the thickest scaffold (28.06 ± 0.59 μm) was achieved with 3 × 104 cells/cm2 seeding density, 100 μg/mL ascorbic acid concentration under hypoxic and dynamic culture condition. The biocompatibility assessment showed that UCs and SMCs seeded on the scaffold could proliferate and maintain the expression of their markers (CK7, CK20, UPIa, and UPII) and (α-SMA, MHC and Smootheline), respectively, after 14 days of in vitro culture. ECM gene expression analysis showed that the ASC and dermal fibroblast-based scaffolds (control) were comparable. The ASC-based scaffold can be handled and removed from the plate. This suggests that multiple layers of scaffold can be stacked to form the urothelium (seeded with UCs), submucosal layer (ASCs only), and smooth muscle layer (seeded with SMCs) and has the potential to be developed into a fully functional human urethra for urethral reconstructive surgeries.

Ascorbic Acid 2-Phosphate-Releasing Supercritical Carbon Dioxide-Foamed Poly(L-Lactide-Co-epsilon-Caprolactone) Scaffolds Support Urothelial Cell Growth and Enhance Human Adipose-Derived Stromal Cell Proliferation and Collagen Production

“…Terefore, as 90% of produced collagen is COL I [50], the efect of A2P in scPLCL A2P seems to be associated with the COL I maturation rather than in the mRNA expression. Previously, supplemented AA or its derivatives have been shown to increase the collagen production in hASCs and bone marrow-derived stem cells [53,55,58,81], and similarly to our fndings, COL III mRNA but not COL I mRNA expression was increased in A2P-supplemented osteoblasts [97]. However, Yu et al reported an increase in both total collagen and COL I mRNA amounts in hASCs cultured with supplemented A2P [53].…”

“…Even though A2P enhanced the hASC proliferation in our study, too high concentrations of AA or A2P in cell culture have been suggested to lead to cytotoxicity and reduced viability [58,81,91], although contradictory reports have been published. For hASCs, cytotoxicity of supplemented A2P appears to be dose-dependent and could be prevented by increasing the cell density.…”

“…In addition, electrospun PLA scafolds were shown to release 50% of the incorporated A2P in distilled water within the frst 24 h [60]. Previously, 100 µg/ml AA concentration was reported to be most favourable for hASCs whereas concentrations above 300 µg/ml showed cytotoxic efects [81].…”

Tetrahexyldecyl Ascorbate (THDC) Degrades Rapidly under Oxidative Stress but Can Be Stabilized by Acetyl Zingerone to Enhance Collagen Production and Antioxidant Effects

“…A nutrient mixture containing AA also inhibited secretion of MMP-9 monomer and dimer in a broad set of cancer cell lines [75], and similar MMP inhibitory activity has been observed in other human and animal models [76,77]. The effects of AZ-stabilized THDC observed in this study thus appear consistent with prior work demonstrating links between AA release, collagen synthesis, and MMP inhibition [4,5,[71][72][73][74][75][76][77][78].…”

Therapies Based on Adipose-Derived Stem Cells for Lower Urinary Tract Dysfunction: A Narrative Review

“…From this point of view, the ASC-assembly scaffold is more advantageous in repairing urethral tissue [ 36 ]. Rashidbenam et al [ 37 ] co-cultured ASCs with vitamin C to stimulate ECM production. Then, the ASC sheet seeded with urothelial and smooth muscle cells was used as a scaffold.…”