Effect of T-type Calcium Channel on Osteogenic Differentiation of Human Dental Follicle Cells

doi:10.13701/j.cnki.kqyxyj.2021.10.008

Abstract

Abstract: Objective: To investigate the effect of Ca²⁺ on osteogenic differentiation of human dental follicle cells (hDFCs) and to explore its molecular mechanism. Methods: hDFCs were isolated and cultured. Its source was verified by flow cytometry and immunofluorescence staining. The gene expression of L-type calcium channel, T-type calcium channel, inositol triphosphate receptor, and ryanodine receptor of hDFCs and mRNA expression of osteogenic differentiation related gene were detected by reverse transcription polymerase chain reaction (RT-PCR). Alizarin red staining was used to evaluate the formation of calcium nodules after the removal of extracellular Ca²⁺, intracellular Ca²⁺, and the application of voltage-gated calcium channel specific blockers. Results: The osteogenic ability of hDFCs was significantly decreased after removal of extracellular Ca²⁺ or intracellular Ca²⁺. RT-PCR results showed that hDFCs expressed L-type calcium channel, T-type calcium channel, inositol triphosphate receptor, and ryanodine receptor. Nifedipine, an L-type calcium channel blocker, had no effect on the osteogenic mineralization of the cells, while amlioride, a T-type calcium channel blockers significantly inhibited the osteogenic mineralization of the cells. Elevation of extracellular Ca²⁺ concentration (5 mmol/L) significantly promoted osteogenic differentiation of hDFCs, and the effect was inhibited by application of KN-93 (10 μmol/L, a Ca²⁺/CaMK Ⅱ signaling pathway specific blocker). Conclusion: T-type calcium channel mediated calcium influx is involved in the osteogenic differentiation of hDFCs.

Key words: human dental follicle cells, Ca²⁺, T-type calcium channel, osteogenic differentiation

YANG Jingjing, ZUO Dongchuan, XIE Yihang, CAI Xin, YUAN Xiaoping, ZENG Jin. Effect of T-type Calcium Channel on Osteogenic Differentiation of Human Dental Follicle Cells[J]. Journal of Oral Science Research, 2021, 37(10): 900-905.

References

[1] Zhou T, Pan J, Wu P, et al. Dental Follicle Cells: Roles in Development and Beyond [J]. Stem Cells International, 2019, 2019: 9159605.
[2] Morsczeck C, Reichert TE. Dental stem cells in tooth regeneration and repair in the future [J]. Expert Opinion on Biological Therapy, 2018, 18(2):187-196.
[3] Carafoli, Ernesto Krebs, Joachim. Why Calcium? How Calcium Became the Best Communicator [J]. Journal of Biological Chemistry,2016,291(40):20849-20857.
[4] Annette C Dolphin.Functions of Presynaptic Voltage-gated Calcium Channels [J]. Function (Oxford, England),2021,2(1):zqaa027.
[5] Barradas A, Fernandes H, Groen N, et al. A calcium-induced signaling cascade leading to osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells [J]. Biomaterials, 2012, 33(11):3205-3215.
[6] 何梦婷,白丁.牙囊在牙萌出的作用及其调控机制研究进展[J].口腔医学研究,2020,36(2):107-110.
[7] 赵娴,曾锦,左东川,等.钙离子对人牙囊细胞增殖、迁移和成骨分化的影响[J].上海口腔医学,2019, 28(6):572-577.
[8] Kawano S, Otsu K, Kuruma A, et al. ATP autocrine/paracrine signaling induces calcium oscillations and NFAT activation in human mesenchymal stem cells [J]. Cell Calcium, 2006, 39(4):313-324.
[9] Koori K, Maeda H, Fujii S, et al. The roles of calcium-sensing receptor and calcium channel in osteogenic differentiation of undifferentiated periodontal ligament cells [J]. Cell Tissue Res, 2014, 357(3):707-718.
[10] Nelso P, Tran Z, Zhang H, et al. Transient receptor potential melastatin 4 channel controls calcium signals and dental follicle stem cell differentiation [J]. Stem Cells, 2013, 31(1):167-177.
[11] Heubach JF, Graf EM, Leutheuser J, et al. Electrophysiological properties of human mesenchymal stem cells [J]. J Physiol, 2004, 554(Pt 3):659-672.
[12] Zahanich I, Graf EM, Heubach JF, et al. Molecular and functional expression of voltage-operated calcium channels during osteogenic differentiation of human mesenchymal stem cells [J]. Bone Miner Res, 2010, 20(9):1637-1646.
[13] Wen L, Wang Y, Wang H, et al. L-type calcium channels play a crucial role in the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells [J]. Biochem Biophys Res Comm, 2012, 424(3):439-445.
[14] Ma Z, Liao J, Zhao C, et al. Effects of the 1, 4-dihydropyridine L-type calcium channel blocker benidipine on bone marrow stromal cells [J]. Cell Tissue Res, 2015, 361(2):467-476.
[15] Kim J, Oh H, Lee S, et al. T-type calcium channels are required to maintain viability of neural progenitor cells [J]. Biomol Ther, 2018, 26(5):439-445.
[16] 张熙,娄淼,周乐,等.脑外伤后T型钙通道对神经干细胞增殖的诱导作用[J].西安交通大学学报:医学版,2016,37(4):484-488.
[17] Qu F, Zhao Z, Yuan B, et al. CaMK Ⅱ plays a part in the chondrogenesis of bone marrow-derived mesenchymal stem cells [J]. Int J Clin Exp Pathol, 2015, 8(5):5981-5987.
[18] Shin MK, Kim MK, Bae YS, et al. A novel collagen-binding peptide promotes osteogenic differentiation via Ca²⁺/calmodulin-dependent protein kinase Ⅱ/ERK/AP-1 signaling pathway in human bone marrow-derived mesenchymal stem cells[J]. Cell Signal, 2008, 20(4):613-624.