ICAM1 promotes the proliferation, migration, and odontoblast differentiation of human dental pulp stem cells by activating the TGF-β1/Smad pathway

Background: Intercellular adhesion molecule 1 (ICAM1) plays an important role in regulating cellular processes associated with tissue repair. Dental pulp stem cells (DPSCs) are essential for dental tissue regeneration, particularly in response to pulp injury. However, the regulatory mechanisms by which ICAM1 influences DPSC behavior remain to be fully elucidated. Methods: ICAM1 was overexpressed or silenced in DPSCs using viral transduction, and Transforming Growth Factor (TGF)-β1 expression was also knocked down to explore downstream effects. Cell proliferation was evaluated by Cell Counting Kit (CCK)-8 assay, while cell migration was evaluated via Transwell assays and Western blotting for Matrix Metalloproteinase (MMP)-2 and MMP-9. Odontoblastic differentiation was determined by Alkaline Phosphatase (ALP) activity assays, Alizarin Red S staining, and Western blot analysis of odontogenic markers including Dentin Sialophosphoprotein (DSPP), Osteocalcin (OCN), Runt-related transcription factor 2 (RUNX2), and Osterix (OSX). Activation of the TGF-β1/Mothers Against Decapentaplegic Homolog (Smad) pathway was examined through Western blotting of TGF-β1, Smad2, phosphorylated Smad2 (p-Smad2), Smad3, and phosphorylated Smad3 (p-Smad3). Results: Overexpression of ICAM1 enhanced DPSC proliferation, migration, and odontoblastic differentiation, which was accompanied by upregulation of the TGF-β1/Smad signaling pathway. Conversely, ICAM1 knockdown suppressed these cellular activities and attenuated pathway activation. Furthermore, TGF-β1 knockdown reversed the promotive effect of ICAM1 overexpression on odontoblastic differentiation, suggesting that ICAM1 exerts its effects in a TGF-β1-dependent manner. Conclusions: ICAM1 facilitates the proliferation, migration, and odontoblastic differentiation of DPSCs by activating the TGF-β1/Smad signaling pathway. These findings highlight the potential of ICAM1 as a regulatory target for enhancing dental pulp regeneration.

ICAM1; Dental pulp stem cell; TGF-β1/Smad pathway; Odontoblastic differentiation; Proliferation; Migration

[1] Xie Z, Shen Z, Zhan P, Yang J, Huang Q, Huang S, et al. Functional dental pulp regeneration: basic research and clinical translation. International Journal of Molecular Sciences. 2021; 22: 8991.

[2] Igna A, Mircioaga D, Boariu M, Stratul SI. A diagnostic insight of dental pulp testing methods in pediatric dentistry. Medicina. 2022; 58: 665.

[3] Goyal V. Pediatric endodontics. International Journal of Clinical Pediatric Dentistry. 2022; 15: S1–S2.

[4] Hassan R, Kouchaje C, Almattit H, Alnassar I. Evaluation of the onset of pulpal, soft tissue anesthesia, and pain using buffered lidocaine in children: a randomized-controlled trial. Signa Vitae. 2024; 20: 33–39.

[5] Elnawam H, Abdelmougod M, Mobarak A, Hussein M, Aboualmakarem H, Girgis M, et al. Regenerative endodontics and minimally invasive dentistry: intertwining paths crossing over into clinical translation. Frontiers in Bioengineering and Biotechnology. 2022; 10: 837639.

[6] Qiao X, Tang J, Dou L, Yang S, Sun Y, Mao H, et al. Dental pulp stem cell-derived exosomes regulate anti-inflammatory and osteogenesis in periodontal ligament stem cells and promote the repair of experimental periodontitis in rats. International Journal of Nanomedicine. 2023; 18: 4683–4703.

[7] Katata C, Sasaki JI, Li A, Abe GL, Nor JE, Hayashi M, et al. Fabrication of vascularized DPSC constructs for efficient pulp regeneration. Journal of Dental Research. 2021; 100: 1351–1358.

[8] Sui B, Wu D, Xiang L, Fu Y, Kou X, Shi S. Dental pulp stem cells: from discovery to clinical application. Journal of Endodontics. 2020; 46: S46–S55.

[9] Singh M, Thakur M, Mishra M, Yadav M, Vibhuti R, Menon AM, et al. Gene regulation of intracellular adhesion molecule-1 (ICAM-1): a molecule with multiple functions. Immunology Letters. 2021; 240: 123–136.

[10] Bui TM, Wiesolek HL, Sumagin R. ICAM-1: a master regulator of cellular responses in inflammation, injury resolution, and tumorigenesis. Journal of Leukocyte Biology. 2020; 108: 787–799.

[11] Makino Y, Fujikawa K, Matsuki-Fukushima M, Inoue S, Nakamura M. Role of innate inflammation in the regulation of tissue remodeling during tooth eruption. Dentistry Journal. 2021; 9: 7.

[12] Li Y, Feng C, Gao M, Jin M, Liu T, Yuan Y, et al. MicroRNA-92b-5p modulates melatonin-mediated osteogenic differentiation of bone marrow mesenchymal stem cells by targeting ICAM-1. Journal of Cellular and Molecular Medicine. 2019; 23: 6140–6153.

[13] Liu YF, Zhang SY, Chen YY, Shi K, Zou B, Liu J, et al. ICAM-1 deficiency in the bone marrow niche impairs quiescence and repopulation of hematopoietic stem cells. Stem Cell Reports. 2018; 11: 258–273.

[14] Zhang Y, Zhang Y, Chen Y, Wu W, Sun J, Zhang X, et al. Impact of intercellular adhesion molecule 1-positive dental pulp stem cells in deep caries progression. International Endodontic Journal. 2025; 58: 1184–1196.

[15] Liu S, Yan X, Guo J, An H, Li X, Yang L, et al. Periodontal ligament-associated protein-1 knockout mice regulate the differentiation of osteoclasts and osteoblasts through TGF-beta1/Smad signaling pathway. Journal of Cellular Physiology. 2024; 239: e31062.

[16] Chen M, Wu C, Fu Z, Liu S. ICAM1 promotes bone metastasis via integrin-mediated TGF-beta/EMT signaling in triple-negative breast cancer. Cancer Science. 2022; 113: 3751–3765.

[17] Chen J, Xu H, Xia K, Cheng S, Zhang Q. Resolvin E1 accelerates pulp repair by regulating inflammation and stimulating dentin regeneration in dental pulp stem cells. Stem Cell Research & Therapy. 2021; 12: 75.

[18] Kim Y, Kang SW, Ye JR, Kim SE, Chae YK, Nam OH. Osteogenic potential of Frondoside A in human periodontal ligament cells: an RNA-Seq analysis. Journal of Periodontal & Implant Science. 2024; 54: 393–404.

[19] Li P, Ou Q, Shi S, Shao C. Immunomodulatory properties of mesenchymal stem cells/dental stem cells and their therapeutic applications. Cellular & Molecular Immunology. 2023; 20: 558–569.

[20] Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, et al. Stem cell properties of human dental pulp stem cells. Journal of Dental Research. 2002; 81: 531–535.

[21] Yuan X, Liu M, Cao X, Yang S. Ciliary IFT80 regulates dental pulp stem cells differentiation by FGF/FGFR1 and Hh/BMP2 signaling. International Journal of Biological Sciences. 2019; 15: 2087–2099.

[22] Chang W, Kim R, Park SI, Jung YJ, Ham O, Lee J, et al. Enhanced healing of rat calvarial bone defects with hypoxic conditioned medium from mesenchymal stem cells through increased endogenous stem cell migration via regulation of ICAM-1 targeted-microRNA-221. Molecular Cell. 2015; 38: 643–650.

[23] Tsutsui TW. Dental pulp stem cells: advances to applications. Stem Cells and Cloning. 2020; 13: 33–42.

[24] Yu L, Zeng L, Zhang Z, Zhu G, Xu Z, Xia J, et al. Cannabidiol rescues TNF-alpha-inhibited proliferation, migration, and osteogenic/odontogenic differentiation of dental pulp stem cells. Biomolecules. 2023; 13: 118.

[25] Lampiasi N. The migration and the fate of dental pulp stem cells. Biology. 2023; 12: 742.

[26] Kucukkaya Eren S, Bahador Zirh E, Zirh S, Sharafi P, Zeybek ND. Combined effects of bone morphogenetic protein-7 and mineral trioxide aggregate on the proliferation, migration, and differentiation of human dental pulp stem cells. Journal of Applied Oral Science. 2022; 30: e20220086.

[27] Liu Y, Liu N, Na J, Li C, Yue G, Fan Y, et al. Wnt/beta-catenin plays a dual function in calcium hydroxide induced proliferation, migration, osteogenic differentiation and mineralization in vitro human dental pulp stem cells. International Endodontic Journal. 2023; 56: 92–102.

[28] Heydarpour F, Sajadimajd S, Mirzarazi E, Haratipour P, Joshi T, Farzaei MH, et al. Involvement of TGF-beta and autophagy pathways in pathogenesis of diabetes: a comprehensive review on biological and pharmacological insights. Frontiers in Pharmacology. 2020; 11: 498758.

[29] Shi Y, Wang S, Zhang W, Zhu Y, Fan Z, Huang Y, et al. Bone marrow mesenchymal stem cells facilitate diabetic wound healing through the restoration of epidermal cell autophagy via the HIF-1α/TGF-β1/SMAD pathway. Stem Cell Research & Therapy. 2022; 13: 314.

[30] He XY, Sun K, Xu RS, Tan JL, Pi CX, Wan M, et al. Spatial signalling mediated by the transforming growth factor-beta signalling pathway during tooth formation. International Journal of Oral Science. 2016; 8: 199–204.

[31] Gao P, Liu C, Dong H, Li Q, Chen Y. TGF-beta promotes the proliferation and osteogenic differentiation of dental pulp stem cells a systematic review and meta-analysis. European Journal of Medical Research. 2023; 28: 261.