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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2016, Vol. 10 Issue (3) : 286-296     DOI: 10.1007/s11684-016-0456-9
RESEARCH ARTICLE |
Influence of the intensity and loading time of direct current electric field on the directional migration of rat bone marrow mesenchymal stem cells
Xiaoyu Wang1,Yuxuan Gao1,Haigang Shi2,Na Liu1,Wei Zhang2,Hongbo Li1,*()
1. Department of Stomatology, Chinese PLA General Hospital, Beijing 100853, China
2. Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, China
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Abstract  

Exogenic electric fields can effectively accelerate bone healing and remodeling through the enhanced migration of bone marrow mesenchymal stem cells (BMSCs) toward the injured area. This study aimed to determine the following: (1) the direction of rat BMSC (rBMSC) migration upon exposure to a direct current electric field (DCEF), (2) the optimal DCEF intensity and duration, and (3) the possible regulatory role of SDF-1/CXCR4 axis in rBMSC migration as induced by DCEF. Results showed that rBMSCs migrated to the positive electrode of the DCEF, and that the DCEF of 200 mV/mm for 4 h was found to be optimal in enhancing rBMSC migration. This DCEF strength and duration also upregulated the expression of osteoblastic genes, including ALP and OCN, and upregulated the expression of ALP and Runx2 proteins. Moreover, when CXCR4 was inhibited, rBMSC migration due to DCEF was partially blocked. These findings indicated that DCEF can effectively induce rBMSC migration. A DCEF of 200 mV/mm for 4 h was recommended because of its ability to promote rBMSC migration, proliferation, and osteogenic differentiation. The SDF-1/CXCR4 signaling pathway may play an important role in regulating the DCEF-induced migration of rBMSCs.

Keywords DCEF      migration      osteogenesis differentiation      rBMSCs      SDF-1/CXCR-4     
Corresponding Authors: Hongbo Li   
Just Accepted Date: 25 May 2016   Online First Date: 17 June 2016    Issue Date: 30 August 2016
URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-016-0456-9     OR     http://academic.hep.com.cn/fmd/EN/Y2016/V10/I3/286
Fig.1  Experimental design and process.
Direction Group
A1 A2 A3
Top Positive electrode Negative electrode
Bottom Negative electrode Positive electrode
Tab.1  Direction of rBMSCs migration
Group B1 B2 B3
Upper chamber rBMSCs rBMSCs rBMSCs
Lower chamber DMEM DMEM+ SDF-1 DMEM+ SDF-1+ AMD3100
Group B4 B5 B6
Upper chamber rBMSCs+ DCEF rBMSCs+ DCEF rBMSCs+ DCEF
Lower chamber DMEM DMEM+ SDF-1 DMEM+ SDF-1+ AMD3100
Tab.2  Electric field has enhanced effect of SDF-1
Factor Group
C1 C2 C3 C4
OIF - - + +
DCEF - + - +
Tab.3  Grouping of osteogenic differentiation
Gene Primer sequences
CXCR4 Forward: 5′-TGACGGACAAGTACAGGCTGC-3′Reverse: 5′-CCAGAAGGGAAGCGTGATGA-3′
β-actin Forward: 5′-ATATCGCTGCGCTCGTCGTC-3′Reverse: 5′-CCTTGGGTCAGGTTTAGAG-3′
RUNX2 Forward: 5′-CCCGTGGCCTTCAAGGT-3′Reverse: 5′-CGTTACCCGCCATGACAGTA-3′
ALP Forward: 5′-CTGCCTACTTGTGTGGCGTGA-3′Reverse: 5′-CCACCCATGATCACGTGCATA-3′
Tab.4  The primer sequences used for real-time PCR
Fig.2  rBMSCs culture, differentiation, and characterization. (A) The third generation of rBMSCs displayed in spindle shape (100×). (B and C) The third generation of rBMSCs differentiated into osteogenic and adipogenic lineages by oil red O staining and Alizarin Red S. (D) Flow cytometric analysis showed that rBMCSs were positive for CD44, CD105, CD29, CD146, and Stro-1 but negative for CD45.
Fig.3  Direct current electric field stimulation. (A) Effects of DCEF on rBMSCs migration direction. The amounts of rBMSCs migrating to the lower chamber in DCEFs of different polar patterns. (B) Results of intensity and loading time screening. Data was the number of experimental migrated cells relative to the control group. (C) Migration of rBMSCs in response to DCEF of different intensities and loading durations. The range of loaded time is from 0 to 6?h, and the range of intensity is from 0 to 300?mV/mm. The nonmigrating rBMSCs were removed, and the migrated cells were stained with DAPI followed by observation under a fluorescence microscope. Transwell chamber assay showed that DCEF promoted rBMSCs migration (20×). (D) Relative percentage of experimental migrated cells in the experimental as compared with the normal group. (E) Growth curve showed the effect of the DCEF on the proliferation of rBMSCs. (F) rBMSCs were exposed in DCEF after which cell cycle analysis was performed with flow cytometer. The control group: the result of cell cycle and proliferation index without the DCEF. G1, 91.97%; G2, 3.04%; S, 4.98%. The DCEF group: the result of cell cycle and proliferation index with the DCEF. G1, 63.91%; G2, 10.37%; S, 25.72%. (G) Percentage of cell cycle analysis by flow cytometry. *P?<?0.05 vs. non-group, **P?<?0.01 vs. non-group, Student’s t test.
Fig.4  Protein expression and gene expression in rBMSCs under DCEF. (A) Two kinds of influential factors were added to the culture of rBMSCs. Western blot analysis and scanning densitometer of ALP and RUNX2 expression in rBMSCs under DCEF. (B) Relative expression change of ALP and RUNX2. (C) ALP activation indicated differences in osteogenesis. (D) Relative density change of osteogenesis. (E) Real-time PCR analysis of OCN and BSP expression in rBMSCs under DCEF. *P?<?0.05 vs. control group, **P?<?0.01 vs. control group, Student’s t test. +, this variable was applied; −, this variable was not applied.
Fig.5  SDF-1/CXCR4 axis influences rBMSCs migration. The nonmigrating rBMSCs were removed and the migrated cells were stained with DAPI followed by observation under a fluorescence microscope. (A) Transwell chamber assay showed that SDF-1 promoted rBMSCs migration, which could be inhibited by AMD3100 in DCEF (100×). (B) Relative percentage of experimental migrated cells in experimental as compared with control group. Result is the summary of the six separate experiments. (C) Western blot analysis and scanning densitometer of CXCR4 expression in rBMSCs. (D) Relative expression of CXCR4. *P?<?0.05 vs. non-group, **P?<?0.01 vs. non-group, Student’s t test. +, this variable was applied; −, this variable was not applied.
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