Culture Growth Phase-Dependent Influence of Extracellular Vesicles Derived from Stem Cells from Human Exfoliated Deciduous Teeth on Oral Mucosa Cells Proliferation in Paracrine Co-Culture with Urethral Epithelium - Implication for Urethral Reconstruction

Sep 30, 2026

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Urethral stricture causes fibrotic narrowing that impairs urethral mucosa and spongiosum function. Oral mucosal graft urethroplasty can work well but remains limited by procedural demands and specialized centers, motivating interest in endoscopic oral mucosa approaches. This study tests whether extracellular vesicles from stem cells of human exfoliated deciduous teeth (SHED-EVs) enhance oral mucosal fibroblast growth under urethramimetic paracrine co-culture, and whether culture growth phase shapes EV activity. SHED-EVs collected in logarithmic versus stationary phases produced distinct proliferation kinetics and phase-dependent miRNA cargo profiles, supporting phase-matched adjunct formulations.

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CategoryHealthcare

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Article Culture Growth Phase-Dependent Influence of Extracellular Vesicles Derived from Stem Cells from Human Exfoliated Deciduous Teeth on Oral Mucosa Cells Proliferation in Paracrine Co-Culture with Urethral Epithelium: Implication for Urethral Reconstruction Tsuyoshi Kawaharada 1,2, Daisuke Watanabe 1,2,3, *, Kazuki Yanagida 1,3,4, Kashia Goto 2, Ailing Hu 2, Yuhei Segawa 5, Madoka Higuchi 5, Masayuki Shinchi 5, Akio Horiguchi 4, Tatsuya Takagi 2 and Akio Mizushima 1,2 Academic Editor: Dmitry B. Zorov Received: 14 November 2025 Revised: 11 December 2025 Accepted: 26 December 2025 Published: 27 December 2025 Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license. 1 Department of Molecular and Cellular Therapeutics, Juntendo University Graduate School of Medicine, Tokyo 113-8421, Japan 2 Department of Palliative Medicine, Juntendo University Graduate School of Medicine, Tokyo 113-8421, Japan 3 Department of Urology, Koto Hospital, Tokyo 136-0072, Japan 4 Division of Reconstruction Center for Trauma, Burn and Tactical Medicine, National Defense Medical College Hospital, Saitama 359-8513, Japan 5 Department of Urology, National Defense Medical College, Saitama 359-8513, Japan * Correspondence: [da-watanabe@juntendo.ac.jp](da-watanabe@juntendo.ac.jp); Tel.: +81-3-3813-3111; Fax: +81-3-3813-0861 Abstract Urethral stricture is a disease of fibrotic narrowing that compromises the urethral mucosa and spongiosum. Oral mucosal graft urethroplasty delivers excellent outcomes in complex cases, yet its procedural demands restrict availability beyond specialized centers. Endoscopic transplantation of oral mucosa has been proposed; while feasibility is shown, clinical efficacy remains suboptimal. We asked whether extracellular vesicles from stem cells of human exfoliated deciduous teeth (SHED-EVs) promote oral mucosa fibroblast (OMF) growth under urethramimetic paracrine conditions and whether culture growth phase tunes EV function. SHED-EVs were collected during logarithmic (SHED-EV-L) or stationary (SHED-EV-S) phases under xeno-free conditions, isolated by a standardized workflow, and characterized by nanoparticle tracking analysis. miRNA cargo was profiled with a human miRNA microarray platform and normalized for comparative analyses. OMF proliferation was quantified in a horizontal indirect co-culture with urethral epithelial cells using incubator-based time-lapse imaging. SHED-EV-L produced a sustained pro-proliferative effect across 24–96 h, whereas SHED-EV-S showed a weaker early effect with a late catch-up; both exceeded vehicle at 96 h. Fibrosis-related miRNA heat maps showed culture growth phase-dependent patterns: SHED-EV-L displayed relatively higher signals for miR-31-3p, miR-146b-3p, several let-7 members, and selected miR-181 isoforms, whereas SHED-EV-S showed a marked relative increase of miR-486-3p; miR-21, miR-99/100, and miR-205 were broadly comparable between phases. These findings indicate that culture growth phase is a practical design lever that orients SHED-EV cargo and function, supporting phase-matched formulations for adjunctive transurethral applications and motivating in vivo validation and manufacturing-oriented quality controls. Keywords: cell culture growth phase; extracellular vesicles; stem cells from human exfoliated deciduous teeth (SHED); oral mucosa fibroblasts; indirect co-culture; urethral reconstruction; microRNA; oral mucosal engraftment; urethral stricture; regenerative medicine 1. Introduction Urethral stricture is commonly managed along a therapeutic spectrum ranging from minimally invasive endoscopic procedures, including dilation and direct visual internal urethrotomy (DVIU), to definitive urethroplasty, with oral mucosal urethroplasty widely considered the gold standard for complex or recurrent cases [1,2]

FAQs

What is the main objective of this study?

To determine whether SHED-derived extracellular vesicles promote oral mucosal fibroblast growth in a urethral-epithelium paracrine co-culture model, and whether EV function is tuned by the culture growth phase.

How were the SHED-derived extracellular vesicles prepared and compared?

EVs were collected during logarithmic (SHED-EV-L) or stationary (SHED-EV-S) phases under xeno-free conditions, isolated using a standardized workflow, and characterized with nanoparticle tracking analysis.

How did the two EV preparations differ in their effects on proliferation?

SHED-EV-L produced a sustained pro-proliferative effect from 24–96 hours, while SHED-EV-S showed a weaker early effect followed by a late catch-up; both surpassed vehicle at 96 hours.

Sep 30, 2026

Uploaded by Angel