In vitro study on the effects of mechanical load on the behavior and signal pathway regulation of pediatric hip cartilage cells

  • Yan Liu Department of Sports Medicine,The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, China; postal code 510120
  • Peng Yang Department of Sports Medicine,The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, China; postal code 510120
  • Zhijian Pan Department of Orthopedics, The Second Clinical Medical College, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong , China.postal code 510000
  • Guangxi Chen Department of Sports Medicine,The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, China; postal code 510120
  • Hongliang Liu Department of Sports Medicine,The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, China; postal code 510120
  • Jiangfa Huang Department of Sports Medicine,The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, China; postal code 510120
Keywords: pediatric hip cartilage cells; mechanical load; signaling pathways; dose-response relationship
Article ID: 1240

Abstract

Objective: To explore the effects of mechanical load on the behavior and related signaling pathways of pediatric hip cartilage cells, providing experimental evidence for optimizing cartilage regeneration and repair strategies. Methods: Human-derived pediatric hip cartilage cells were cultured in vitro and subjected to uniaxial tensile stress at a specific frequency (1 Hz) with varying strain amplitudes (0%, 2%, 5%, 10%). Cell proliferation curves were assessed using the CCK-8 assay, while qPCR was used to detect changes in the expression of COL2A1, ACAN, and genes such as ITGB1, TGF-β1, WNT3A, MAPK1, among others. Western Blot analysis was conducted to measure protein levels of Collagen II, Aggrecan, MMP-13, as well as p-ERK, p-p38, β-catenin, MAPK1, and MAPK14. The relationship between strain amplitude and cellular biological effects was also analyzed. Results: After mechanical strain application, cell proliferation significantly increased on days 5 and 7 (p < 0.05), and COL2A1 and ACAN gene expression levels were upregulated (p < 0.05). Protein levels of Collagen II and Aggrecan significantly increased, while MMP-13 levels decreased (p < 0.05). Upstream signaling molecules such as Integrin β1, TGF-β1, and WNT3A were upregulated (p < 0.05), while TGFBR2 showed no significant changes (p > 0.05). Downstream molecules p-ERK, MAPK1, and MAPK14 were significantly upregulated (p < 0.05), whereas p-p38 and β-catenin showed no significant differences (p > 0.05). ACAN and p-ERK expression levels exhibited a dose-response relationship with increasing strain amplitude (p < 0.05). Conclusion: Mechanical load promotes the proliferation and matrix synthesis of pediatric hip cartilage cells through specific regulation of upstream and downstream signaling pathway molecules, showing strain intensity-dependent molecular responses. This study lays the foundation for precision regulation strategies in cartilage development and repair.

References

1. Fu S, Thompson CL, Ali A, et al. Mechanical loading inhibits cartilage inflammatory signalling via an HDAC6 and IFT-dependent mechanism regulating primary cilia elongation. Osteoarthritis and Cartilage. 2019; 27(7): 1064-1074. doi: 10.1016/j.joca.2019.03.003

2. Fang T, Zhou X, Jin M, et al. Molecular mechanisms of mechanical load-induced osteoarthritis. International Orthopaedics. 2021; 45(5): 1125-1136. doi: 10.1007/s00264-021-04938-1

3. Cai X, Warburton C, Perez OF, et al. Hippo Signaling Modulates the Inflammatory Response of Chondrocytes to Mechanical Compressive Loading. bioRxiv. 2023. doi: 10.1101/2023.06.09.544419

4. Bloks NG, Harissa Z, Mazzini G, et al. A Damaging COL6A3 Variant Alters the MIR31HG‐Regulated Response of Chondrocytes in Neocartilage Organoids to Hyperphysiologic Mechanical Loading. Advanced Science. 2024. doi: 10.1002/advs.202400720

5. He Z, Leong DJ, Xu L, et al. CITED2 mediates the cross‐talk between mechanical loading and IL‐4 to promote chondroprotection. Annals of the New York Academy of Sciences. 2019; 1442(1): 128-137. doi: 10.1111/nyas.14021

6. Gilbert SJ, Bonnet CS, Blain EJ. Mechanical Cues: Bidirectional Reciprocity in the Extracellular Matrix Drives Mechano-Signalling in Articular Cartilage. International Journal of Molecular Sciences. 2021; 22(24): 13595. doi: 10.3390/ijms222413595

7. Qi H, Zhang Y, Xu L, et al. Loss of RAP2A Aggravates Cartilage Degradation in TMJOA via YAP Signaling. Journal of Dental Research. 2022; 102(3): 302-312. doi: 10.1177/00220345221132213

8. Stampoultzis T, Guo Y, Nasrollahzadeh N, et al. Mimicking Loading-Induced Cartilage Self-Heating in Vitro Promotes Matrix Formation in Chondrocyte-Laden Constructs with Different Mechanical Properties. ACS Biomaterials Science & Engineering. 2023; 9(2): 651-661. doi: 10.1021/acsbiomaterials.2c00723

9. Chang SH, Mori D, Kobayashi H, et al. Excessive mechanical loading promotes osteoarthritis through the gremlin-1–NF-κB pathway. Nature Communications. 2019; 10(1). doi: 10.1038/s41467-019-09491-5

10. Savadipour A, Nims RJ, Katz DB, et al. Regulation of chondrocyte biosynthetic activity by dynamic hydrostatic pressure: the role of TRP channels. Connective Tissue Research. 2021; 63(1): 69-81. doi: 10.1080/03008207.2020.1871475

11. Li X, Han Y, Li G, et al. Role of Wnt signaling pathway in joint development and cartilage degeneration. Frontiers in Cell and Developmental Biology. 2023; 11. doi: 10.3389/fcell.2023.1181619

12. Jia S, Liu W, Zhang M, et al. Insufficient Mechanical Loading Downregulates Piezo1 in Chondrocytes and Impairs Fracture Healing Through ApoE‐Induced Senescence. Advanced Science. 2024; 11(46). doi: 10.1002/advs.202400502

13. Jiang W, Liu H, Wan R, et al. Mechanisms linking mitochondrial mechanotransduction and chondrocyte biology in the pathogenesis of osteoarthritis. Ageing Research Reviews. 2021; 67: 101315. doi: 10.1016/j.arr.2021.101315

14. Zhao D, Li H, Liu S. TIMP3/TGF‑β1 axis regulates mechanical loading‑induced chondrocyte degeneration and angiogenesis. Molecular Medicine Reports. 2020. doi: 10.3892/mmr.2020.11386

15. Lee W, Nims RJ, Savadipour A, et al. Inflammatory signaling sensitizes Piezo1 mechanotransduction in articular chondrocytes as a pathogenic feed-forward mechanism in osteoarthritis. Proceedings of the National Academy of Sciences. 2021; 118(13). doi: 10.1073/pnas.2001611118

16. Zhang M, Xiong S, Gao D, et al. Tension regulates the cartilage phenotypic expression of endplate chondrocytes through the α‐catenin/actin skeleton/Hippo pathway. Journal of Cellular and Molecular Medicine. 2024; 28(4). doi: 10.1111/jcmm.18133

17. Dietmar HF, Weidmann PA, Alberton P, et al. Load activated FGFR and beta1 integrins target distinct chondrocyte mechano-response genes. bioRxiv. 2024. doi: 10.1101/2024.10.11.617817

18. Aeppli T, Zhang Z, Zaman F, et al. SAT190 The Effect Of Mechanical Loading On Bone Growth Ex Vivo. Journal of the Endocrine Society. 2023; 7(Supplement_1). doi: 10.1210/jendso/bvad114.487

19. Somemura S, Kumai T, Yatabe K, et al. Physiologic Mechanical Stress Directly Induces Bone Formation by Activating Glucose Transporter 1 (Glut 1) in Osteoblasts, Inducing Signaling via NAD+-Dependent Deacetylase (Sirtuin 1) and Runt-Related Transcription Factor 2 (Runx2). International Journal of Molecular Sciences. 2021; 22(16): 9070. doi: 10.3390/ijms22169070

20. Song CX, Liu SY, Zhu WT, et al. Excessive mechanical stretch‑mediated osteoblasts promote the catabolism and apoptosis of chondrocytes via the Wnt/β‑catenin signaling pathway. Molecular Medicine Reports. 2021; 24(2). doi: 10.3892/mmr.2021.12232

21. Loke C, & Mukherjee N. Finite element modeling of cells in response to loading: Effect of cytoskeleton. 2023.

22. Higgins SM, Jones RC, Lawrence KM, et al. Investigation of the chondrocyte response to varying mechanical load: assessment of impact stress. Osteoarthritis and Cartilage. 2020; 28: S179. doi: 10.1016/j.joca.2020.02.290

23. Yan M, Sun Z, Wang J, et al. Single-cell RNA sequencing reveals distinct chondrocyte states in femoral cartilage under weight-bearing load in Rheumatoid arthritis. Frontiers in Immunology. 2023; 14. doi: 10.3389/fimmu.2023.1247355

24. Kotelsky A, Carrier JS, Buckley MR. Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants. Journal of Visualized Experiments. 2019; (143). doi: 10.3791/58487

Published
2025-03-24
How to Cite
Liu, Y., Yang, P., Pan, Z., Chen, G., Liu, H., & Huang, J. (2025). In vitro study on the effects of mechanical load on the behavior and signal pathway regulation of pediatric hip cartilage cells. Molecular & Cellular Biomechanics, 22(5), 1240. https://doi.org/10.62617/mcb1240
Section
Article