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Hypoxia-preconditioned adipose-derived stem cell exosomes promote osteogenic differentiation and bone formation in bone marrow mesenchymal stem cells through the Piezo1/Ca2+ signaling axis
Wumiti Taxi, Jie Sun, Yi-Hua Zhu, Tian-Chi Zhang, Yang Guo, Ya-Lan Pan, Meng-Min Liu, Xi Chen, Chun-Lei Zhang, Kai Tong, Li-Ning Wang, Tao Jiang, Yong Ma
Wumiti Taxi, Jie Sun, Yi-Hua Zhu, Tian-Chi Zhang, Yang Guo, Ya-Lan Pan, Meng-Min Liu, Xi Chen, Li-Ning Wang, Yong Ma, Laboratory of New Techniques of Restoration & Reconstruction of Orthopedics and Traumatology, Nanjing University of Chinese Medicine, Nanjing 210046, Jiangsu Province, China
Yang Guo, Jiangsu CM Clinical Innovation Center of Degenerative Bone & Joint Disease, Nanjing University of Chinese Medicine, Wuxi 210022, Jiangsu Province, China
Chun-Lei Zhang, Kai Tong, Department of Orthopedic ang Traumatology, Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine, Nanjing 210046, Jiangsu Province, China
Tao Jiang, Department of Orthopedic ang Traumatology, Changzhou TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Changzhou 213300, Jiangsu Province, China
Co-first authors: Wumiti Taxi and Jie Sun.
Co-corresponding authors: Tao Jiang and Yong Ma.
Author contributions: Taxi W and Sun J contributed to the writing - original draft, they contributed equally to this manuscript and are co-first authors; Zhu YH, Zhang TC, and Chen X contributed to data curation and formal analysis; Guo Y, Pan YL, and Liu MM contributed to data curation; Zhang CL, Tong K, and Jiang T contributed to writing - review and editing; Guo Y, Pan YL, Liu MM, Zhang CL, Tong K, and Jiang T contributed to methodology; Taxi W, Sun J, and Wang LN contributed to the conceptualization; Ma Y and Jiang T contributed to acquisition and funding, they contributed equally to this manuscript and are co-corresponding authors.
AI contribution statement: We used the “Home for research AI Intelligent Polishing tool 2.0” for language polishing of the manuscript. We confirm that the” Home for research AI Intelligent Polishing 2.0 tool” was only used for language polishing and did not influence the scientific content, interpretation, or conclusions of the manuscript.
Supported by National Natural Science Foundation of China, No. 82474549, No. 82074458, No. 82174411, and No. 82505718; the Natural Science Foundation of Jiangsu Province, No. BK20221351, No. BK20220470, and No. BK20240745; the Jiangsu CM Clinical Innovation Center of Degenerative Bone and Joint Disease (Jiangsu Science and Education of Traditional Chinese Medicine)[2021], No. 4; the Natural Science Foundation of Jiangsu Higher Education Institutions, No. 22KJB360012; and Yancheng Basic Research Program General Project, No. YCBK2024087.
Institutional animal care and use committee statement: All experimental procedures were strictly performed in accordance with animal ethical welfare guidelines and were approved by the Experimental Animal Ethics Committee of Nanjing University of Chinese Medicine (Approval No. 202312A044).
Conflict-of-interest statement: All 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: The datasets and
Supplementary materials from this study are accessible upon reasonable request from the corresponding author.
Corresponding author: Yong Ma, Laboratory of New Techniques of Restoration & Recon
struction of Orthopedics and Traumatology, Nanjing University of Chinese Medicine, No. 138 Xianlin Road, Nanjing 210046, Jiangsu Province, China.
mayong@njucm.edu.cn
Received: November 28, 2025
Revised: December 26, 2025
Accepted: March 2, 2026
Published online: May 26, 2026
Processing time: 179 Days and 4.7 Hours
BACKGROUND
Stem cell-derived exosomes represent a promising cell-free strategy for bone regeneration, yet the mechanisms underlying their osteogenic effects remain incompletely defined. Hypoxic preconditioning has been reported to enhance exosome bioactivity, but its potential interaction with mechanosensitive signaling pathways during osteogenesis is poorly understood.
AIM
To evaluate the osteogenic effects of hypoxia-preconditioned adipose-derived stem cell exosomes (HY-Exos) and to examine the possible involvement of Piezo1-dependent calcium signaling.
METHODS
Bone marrow mesenchymal stem cells were treated in vitro with exosomes derived from adipose-derived stem cells cultured under normoxic or hypoxic conditions. Osteogenic differentiation was assessed by alkaline phosphatase and alizarin red S staining and quantitative polymerase chain reaction. Piezo1 expression and intracellular calcium dynamics were analyzed by western blotting and Fluo-4 AM imaging, with GsMTx4 used for functional inhibition. In vivo, HY-Exos were systemically administered to ovariectomized rats, and bone microarchitecture was evaluated by micro-computed tomography. Statistical analyses were performed using Student’s t-test or one-way ANOVA.
RESULTS
HY-Exos showed higher cellular uptake and more strongly promoted osteogenic differentiation than normoxic exosomes, as evidenced by increased alkaline phosphatase activity (P < 0.01), mineralized nodule formation (P < 0.001), and upregulated expression of runt-related transcription factor 2 and osterix. HY-Exos were associated with elevated Piezo1 protein expression (P < 0.01) and sustained calcium oscillations, which were suppressed by GsMTx4 (P < 0.001). In ovariectomized rats, HY-Exos improved bone mineral density (P < 0.01) and bone volume/total volume (P < 0.001).
CONCLUSION
HY-Exos enhance osteogenic differentiation and bone formation and are associated with changes in Piezo1-related calcium signaling. These findings support the therapeutic potential of hypoxia-conditioned exosomes for osteoporosis while indicating a mechanosensitive signaling pathway that warrants further investigation.
Core Tip: This study reveals that exosomes derived from hypoxia-preconditioned adipose-derived stem cells significantly enhance osteogenic differentiation and bone formation through the Piezo1/calcium signaling axis. Hypoxia-preconditioned adipose-derived stem cells promote bone marrow mesenchymal stem cell mineralization and improve bone microarchitecture in an ovariectomized rat model. These effects are abolished by the Piezo1 inhibitor GsMTx4, identifying Piezo1 as a novel mechanistic target. Our findings propose a promising exosome-based strategy for the treatment of osteoporosis.
