Basic Study
Copyright ©The Author(s) 2025. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Aug 26, 2025; 17(8): 107124
Published online Aug 26, 2025. doi: 10.4252/wjsc.v17.i8.107124
Aligned nanofiber scaffolds combined with cyclic stretch facilitate mesenchymal stem cell differentiation for ligament engineering
Cheng-Wei Yang, Ya-Qiang Zhang, Hong Chang, Rui Gao, Dan Chen, Hao Yao
Cheng-Wei Yang, Ya-Qiang Zhang, Department of Orthopaedics, The 940th Hospital of Joint Logistics Support Force of PLA, Lanzhou 730050, Gansu Province, China
Hong Chang, Department of Ultrasonography, The 940th Hospital of Joint Logistics Support Force of PLA, Lanzhou 730050, Gansu Province, China
Rui Gao, Department of Orthopaedics, Shanghai Changzheng Hospital, Naval Medical University, Shanghai 200003, China
Dan Chen, Hao Yao, Department of Hematology, The General Hospital of Western Theater Command PLA, Chengdu 610083, Sichuan Province, China
Co-first authors: Cheng-Wei Yang and Ya-Qiang Zhang.
Co-corresponding authors: Dan Chen and Hao Yao.
Author contributions: Yang CW and Zhang YQ contributed equally to this study and are co-first authors of this article. Yang CW contributed to the conceptual design of the study and supervised data collection and analysis; Zhang YQ performed data acquisition and laboratory experiments; Chang H, Gao R, and Chen D analyzed the data and contributed to interpretation; Yao H assisted with data interpretation and manuscript drafting; Yao H and Chen D supervised the entire project, coordinated all phases of the study, and finalized the manuscript for submission, they contributed equally to this manuscript and are co-corresponding authors of this study. All authors reviewed and approved the final version of the manuscript.
Supported by Sichuan Province Science and Technology Support Program, No. 2024NSFSC1292; the Program of General Hospital of Western Theater Command, No. 2021-XZYG-C45 and No. 2021-XZYG-B32; the Natural Science Foundation of Gansu Province, No. 23JRRA538; and the National Natural Science Foundation of China, No. 81601905.
Institutional animal care and use committee statement: All procedures involving animals were approved by the Ethics Committee of the General Hospital of Western Theater Command PLA (No. 2024EC4-ky001) and were performed in accordance with relevant regulations to ensure animal welfare and ethical considerations.
Conflict-of-interest statement: The authors report no relevant conflicts of interest for this article.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Data sharing statement: Data will be available upon request from the authors. The data that support the findings of this study are available from the corresponding author upon reasonable request.
Open Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Hao Yao, PhD, Department of Hematology, The General Hospital of Western Theater Command PLA, No. 270 Rongdu Avenue, Jinniu District, Chengdu 610083, Sichuan Province, China. yaohao9001@163.com
Received: March 25, 2025
Revised: May 11, 2025
Accepted: July 4, 2025
Published online: August 26, 2025
Processing time: 149 Days and 23.8 Hours
Abstract
BACKGROUND

Tendon tissue engineering requires biomimetic scaffolds and mechanical cues to direct mesenchymal stem cell differentiation toward tenogenic lineages. Bone marrow-derived mesenchymal stem cells (BMSCs), aligned nanofiber scaffolds, and cyclic uniaxial stretching can be used to create a functional engineered ligament tissue.

AIM

To investigate the effects of aligned nanofiber scaffolds and cyclic stretch on BMSC tenogenesis for ligament engineering.

METHODS

BMSCs were cultured on aligned and random poly-lactic acid nanofiber scaffolds under static and cyclic tensile conditions (0.5 Hz, 2% strain, 2 hours/day) for 7 days using a mechanical loading system (CFILLOAD-300). The Ras homolog gene family (Rho)-associated coiled coil-containing kinase (ROCK) inhibitor Y27632 was applied to explore its role in tenogenic differentiation. Scaffold morphology was assessed by scanning electron microscopy, while cell morphology, viability, and alignment were evaluated via confocal microscopy with F-actin and 4’,6-diamidino-2-phenylindole staining. Tenogenic gene expression (collagen type I alpha 2, collagen type III alpha 1, tenascin C, and tenomodulin) was quantified by quantitative polymerase chain reaction, and ligament-related protein levels (collagen I, collagen III, tenascin C, and tenomodulin) were analyzed by western blot.

RESULTS

Scanning electron microscopy revealed that aligned scaffolds provided consistent directional structure, whereas random scaffolds displayed a disordered fiber arrangement. Confocal microscopy showed that under static conditions, BMSCs on aligned scaffolds grew parallel to fiber alignment, while those on random scaffolds grew randomly. Under cyclic tensile strain, BMSCs on both scaffold types exhibited elongation along the direction of strain, adopting a spindle-shaped morphology. Cyclic uniaxial strain enhanced cell viability and metabolic activity based on CCK-8 assay results and upregulated ligament-specific gene and protein expression on aligned scaffolds compared to static conditions. BMSCs on aligned scaffolds under tensile strain showed the highest expression of tenogenic markers, suggesting a synergistic effect of scaffold alignment and mechanical loading. ROCK inhibition with Y27632 upregulated alternative signaling pathways (focal adhesion kinase and runt-related transcription factor 2), further promoting tenogenic differentiation.

CONCLUSION

Aligned nanofiber scaffolds combined with cyclic tensile strain provide an optimal environment for guiding BMSC differentiation toward ligamentous lineages, as assessed by increased expression of ligament-specific markers. Mechanical stimulation (uniaxial stretching) significantly influences BMSC tenogenic differentiation, and the combined use of aligned nanofibers and tensile strain further enhances this effect. The ROCK pathway plays a regulatory role in this process, though its precise mechanisms require further investigation.

Keywords: Tendon tissue engineering; Bone marrow-derived mesenchymal stem cells; Nanofiber scaffold; Cyclic tensile strain; Tenogenic differentiation; Ras homolog gene family (Rho)-associated coiled coil-containing kinase inhibition; Y27632

Core Tip: This study investigated the role of aligned nanofiber scaffolds and cyclic stretch in promoting Bone marrow-derived mesenchymal stem cell (BMSC) differentiation towards ligamentous tissue. We demonstrate that cyclic tensile strain, when applied to BMSCs on aligned nanofiber scaffolds, enhances tenogenic differentiation, as demonstrated by increased expression of collagen and tenogenic markers. These findings suggest that mechanical cues and scaffold alignment are critical in guiding BMSC differentiation for ligament tissue engineering, providing valuable insights for developing strategies to improve ligament regeneration.