Low frequency‑pulsed electromagnetic fields promote osteogenic differentiation of bone marrow‑derived mesenchymal stem cells by regulating connexin 43 expression

Lu,Zhi-Jun; Gu,Hou-Yun; Li,Zhi-Qiang; Lin,Fei-Xiang

doi:10.3892/etm.2024.12736

Low frequency‑pulsed electromagnetic fields promote osteogenic differentiation of bone marrow‑derived mesenchymal stem cells by regulating connexin 43 expression

Authors:
- Zhi-Jun Lu
- Hou-Yun Gu
- Zhi-Qiang Li
- Fei-Xiang Lin
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Affiliations: Department of Spine Surgery, Ganzhou People's Hospital (The Affiliated Ganzhou Hospital of Jiangxi Medical College of Nanchang University, Ganzhou Hospital‑Nanfang Hospital of Southern Medical University), Ganzhou, Jiangxi 341000, P.R. China
Published online on: October 1, 2024 https://doi.org/10.3892/etm.2024.12736
Article Number: 446
Copyright: © Lu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The present study investigated the effect of connexin 43 (Cx43) on the regulation of osteogenic differentiation of rat bone marrow‑derived mesenchymal stem cells (BMSCs) using low‑frequency‑pulsed electromagnetic fields (LPEMF). The BMSCs were isolated and cultured in vitro using adherent whole‑bone marrow cultures. CCK‑8 assay was used to detect the effects of LPEMF on the proliferation ability of BMSCs and alkaline phosphatase (ALP) activity and the levels of osteogenic marker genes were detected to evaluate the osteogenic ability change following LPEMF treatment. Lentiviral vector‑mediated RNA interference was transfected into BMSCs to inhibit the expression of Cx43 and western blotting was used to detect Cx43 expression. The BMSCs showed the highest proliferation following LPEMF treatment at 80 Hz for 1 h. The results of ALP activity, osteogenic marker genes and Alizarin Red S staining showed that the osteogenic ability was notably increased following LPEMF treatment at 80 Hz for 1 h. Cx43 expression increased during the osteogenic differentiation of BMSCs following LPEMF treatment at 80 Hz. The enhanced osteogenic differentiation of the LPEMF‑treated BMSCs were partially reversed when Cx43 expression was inhibited. LPEMF may promote the osteogenic differentiation of BMSCs by regulating Cx43 expression and enhancing osteogenic ability.

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1	El-Rashidy AA, Roether JA, Harhaus L, Kneser U and Boccaccini AR: Regenerating bone with bioactive glass scaffolds: A review of in vivo studies in bone defect models. Acta Biomater. 62:1–28. 2017.PubMed/NCBI View Article : Google Scholar
2	Schindeler A, McDonald MM, Bokko P and Little DG: Bone remodeling during fracture repair: The cellular picture. Semin Cell Dev Biol. 19:459–466. 2008.PubMed/NCBI View Article : Google Scholar
3	Jing D, Li F, Jiang M, Cai J, Wu Y, Xie K, Wu X, Tang C, Liu J, Guo W, et al: Pulsed electromagnetic fields improve bone microstructure and strength in ovariectomized rats through a Wnt/Lrp5/β-catenin signaling-associated mechanism. PLoS One. 8(e79377)2013.PubMed/NCBI View Article : Google Scholar
4	Hannemann PFW, Mommers EHH, Schots JPM, Brink PRG and Poeze M: The effects of low-intensity pulsed ultrasound and pulsed electromagnetic fields bone growth stimulation in acute fractures: A systematic review and meta-analysis of randomized controlled trials. Arch Orthop Trauma Surg. 134:1093–1106. 2014.PubMed/NCBI View Article : Google Scholar
5	Chang K, Chang WHS, Tsai MT and Shih C: Pulsed electromagnetic fields accelerate apoptotic rate in osteoclasts. Connect Tissue Res. 47:222–228. 2006.PubMed/NCBI View Article : Google Scholar
6	He Z, Selvamurugan N, Warshaw J and Partridge NC: Pulsed electromagnetic fields inhibit human osteoclast formation and gene expression via osteoblasts. Bone. 106:194–203. 2018.PubMed/NCBI View Article : Google Scholar
7	Huo SC and Yue B: Approaches to promoting bone marrow mesenchymal stem cell osteogenesis on orthopedic implant surface. World J Stem Cells. 12:545–561. 2020.PubMed/NCBI View Article : Google Scholar
8	Liang J, Li W, Zhuang N, Wen S, Huang S, Lu W, Zhou Y, Liao G, Zhang B and Liu C: Experimental study on bone defect repair by BMSCs combined with a light-sensitive material: g-C₃N₄/rGO. J Biomater Sci Polym Ed. 32:248–265. 2021.PubMed/NCBI View Article : Google Scholar
9	Donahue HJ: Gap junctions and biophysical regulation of bone cell differentiation. Bone. 26:417–422. 2000.PubMed/NCBI View Article : Google Scholar
10	Batra N, Kar R and Jiang JX: Gap junctions and hemichannels in signal transmission, function and development of bone. Biochim Biophys Acta. 1818:1909–1918. 2012.PubMed/NCBI View Article : Google Scholar
11	Unger VM, Kumar NM, Gilula NB and Yeager M: Three-dimensional structure of a recombinant gap junction membrane channel. Science. 283:1176–1180. 1999.PubMed/NCBI View Article : Google Scholar
12	Saez JC, Berthoud VM, Branes MC, Martinez AD and Beyer EC: Plasma membrane channels formed by connexins: Their regulation and functions. Physiol Rev. 83:1359–1400. 2003.PubMed/NCBI View Article : Google Scholar
13	Liao Y, Day KH, Damon DN and Duling BR: Endothelial cell-specific knockout of connexin 43 causes hypotension and bradycardia in mice. Proc Natl Acad Sci USA. 98:9989–9994. 2001.PubMed/NCBI View Article : Google Scholar
14	Plotkin LI: Connexin 43 hemichannels and intracellular signaling in bone cells. Front Physiol. 5(131)2014.PubMed/NCBI View Article : Google Scholar
15	Lecanda F, Warlow PM, Sheikh S, Furlan F, Steinberg TH and Civitelli R: Connexin43 deficiency causes delayed ossification, craniofacial abnormalities, and osteoblast dysfunction. J Cell Biol. 151:931–944. 2000.PubMed/NCBI View Article : Google Scholar
16	Tonon R and D'Andrea P: The functional expression of connexin 43 in articular chondrocytes is increased by interleukin 1beta: Evidence for a Ca2+-dependent mechanism. Biorheology. 39:153–160. 2002.PubMed/NCBI
17	Ransjö M, Sahli J and Lie A: Expression of connexin 43 mRNA in microisolated murine osteoclasts and regulation of bone resorption in vitro by gap junction inhibitors. Biochem Biophys Res Commun. 303:1179–1185. 2003.PubMed/NCBI View Article : Google Scholar
18	Loiselle AE, Paul EM, Lewis GS and Donahue HJ: Osteoblast and osteocyte-specific loss of Connexin43 results in delayed bone formation and healing during murine fracture healing. J Orthop Res. 31:147–154. 2013.PubMed/NCBI View Article : Google Scholar
19	Meirelles Lda S and Nardi NB: Murine marrow-derived mesenchymal stem cell: Isolation, in vitro expansion, and characterization. Br J Haematol. 123:702–711. 2003.PubMed/NCBI View Article : Google Scholar
20	Wennberg C, Hessle L, Lundberg P, Mauro S, Narisawa S, Lerner UH and Millán JL: Functional characterization of osteoblasts and osteoclasts from alkaline phosphatase knockout mice. J Bone Miner Res. 15:1879–1888. 2000.PubMed/NCBI View Article : Google Scholar
21	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001.PubMed/NCBI View Article : Google Scholar
22	Phinney DG, Kopen G, Isaacson RL and Prockop DJ: Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: Variations in yield, growth, and differentiation. J Cell Biochem. 72:570–585. 1999.PubMed/NCBI
23	Gurusamy N, Alsayari A, Rajasingh S and Rajasingh J: Adult stem cells for regenerative therapy. Prog Mol Biol Transl Sci. 160:1–22. 2018.PubMed/NCBI View Article : Google Scholar
24	Muruganandan S and Sinal CJ: The impact of bone marrow adipocytes on osteoblast and osteoclast differentiation. IUBMB Life. 66:147–155. 2014.PubMed/NCBI View Article : Google Scholar
25	Wang T, Yang L, Jiang J, Liu Y, Fan Z, Zhong C and He C: Pulsed electromagnetic fields: Promising treatment for osteoporosis. Osteoporos Int. 30:267–276. 2019.PubMed/NCBI View Article : Google Scholar
26	Weber C, Thai V, Neuheuser K, Groover K and Christ O: Efficacy of physical therapy for the treatment of lateral epicondylitis: A meta-analysis. BMC Musculoskelet Disord. 16(223)2015.PubMed/NCBI View Article : Google Scholar
27	Ibiwoye MO, Powell KA, Grabiner MD, Patterson TE, Sakai Y, Zborowski M, Wolfman A and Midura RJ: Bone mass is preserved in a critical-sized osteotomy by low energy pulsed electromagnetic fields as quantitated by in vivo micro-computed tomography. J Orthop Res. 22:1086–1093. 2004.PubMed/NCBI View Article : Google Scholar
28	Yildiz M, Cicek E, Cerci SS, Cerci C, Oral B and Koyu A: Influence of electromagnetic fields and protective effect of CAPE on bone mineral density in rats. Arch Med Res. 37:818–821. 2006.PubMed/NCBI View Article : Google Scholar
29	Lohmann CH, Schwartz Z, Liu Y, Li Z, Simon BJ, Sylvia VL, Dean DD, Bonewald LF, Donahue HJ and Boyan BD: Pulsed electromagnetic fields affect phenotype and connexin 43 protein expression in MLO-Y4 osteocyte-like cells and ROS 17/2.8 osteoblast-like cells. J Orthop Res. 21:326–334. 2003.PubMed/NCBI View Article : Google Scholar
30	Nelson FRT, Brighton CT, Ryaby J, Simon BJ, Nielson JH, Lorich DG, Bolander M and Seelig J: Use of physical forces in bone healing. J Am Acad Orthop Surg. 11:344–354. 2003.PubMed/NCBI View Article : Google Scholar
31	Coleman JE: Structure and mechanism of alkaline phosphatase. Annu Rev Biophys Biomol Struct. 21:441–483. 1992.PubMed/NCBI View Article : Google Scholar
32	Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC, Rosewell IR, Stamp GW, Beddington RS, Mundlos S, Olsen BR, et al: Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell. 89:765–771. 1997.PubMed/NCBI View Article : Google Scholar
33	Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao YH, Inada M, et al: Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell. 89:755–764. 1997.PubMed/NCBI View Article : Google Scholar
34	Franceschi RT and Xiao G: Regulation of the osteoblast-specific transcription factor, Runx2: Responsiveness to multiple signal transduction pathways. J Cell Biochem. 88:446–454. 2003.PubMed/NCBI View Article : Google Scholar
35	Zou L, Zou X, Li H, Mygind T, Zeng Y, Lü N and Bünger C: Molecular mechanism of osteochondroprogenitor fate determination during bone formation. Adv Exp Med Biol. 585:431–441. 2006.PubMed/NCBI View Article : Google Scholar
36	Gordon JA, Tye CE, Sampaio AV, Underhill TM, Hunter GK and Goldberg HA: Bone sialoprotein expression enhances osteoblast differentiation and matrix mineralization in vitro. Bone. 41:462–473. 2007.PubMed/NCBI View Article : Google Scholar
37	Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR and de Crombrugghe B: The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell. 108:17–29. 2002.PubMed/NCBI View Article : Google Scholar
38	Oldknow KJ, Macrae VE and Farquharson C: Endocrine role of bone: Recent and emerging perspectives beyond osteocalcin. J Endocrinol. 225:R1–R19. 2015.PubMed/NCBI View Article : Google Scholar
39	Gregory CA, Gunn WG, Peister A and Prockop DJ: An Alizarin red-based assay of mineralization by adherent cells in culture: Comparison with cetylpyridinium chloride extraction. Anal Biochem. 329:77–84. 2004.PubMed/NCBI View Article : Google Scholar
40	Gramsch B, Gabriel HD, Wiemann M, Grümmer R, Winterhager E, Bingmann D and Schirrmacher K: Enhancement of connexin 43 expression increases proliferation and differentiation of an osteoblast-like cell line. Exp Cell Res. 264:397–407. 2001.PubMed/NCBI View Article : Google Scholar
41	Merrifield PA and Laird DW: Connexins in skeletal muscle development and disease. Semin Cell Dev Biol. 50:67–73. 2016.PubMed/NCBI View Article : Google Scholar
42	Plotkin LI, Laird DW and Amedee J: Role of connexins and pannexins during ontogeny, regeneration, and pathologies of bone. BMC Cell Biol. 17 (Suppl 1)(S19)2016.PubMed/NCBI View Article : Google Scholar
43	Rosselló RA, Wang Z, Kizana E, Krebsbach PH and Kohn DH: Connexin 43 as a signaling platform for increasing the volume and spatial distribution of regenerated tissue. Proc Natl Acad Sci USA. 106:13219–13224. 2009.PubMed/NCBI View Article : Google Scholar