Periodontal ligament stem cells (PDLSCs) possess self-renewal and multilineage differentiation potential and exhibit great potential for the treatment of bone tissue defects caused by inflammation. Previous studies have indicated that static magnetic field (SMF) can enhance the proliferation and differentiation of mesenchymal stem cells (MSCs). SMF has been widely used to repair bone defects and for orthodontic and implantation treatment. In this study, we revealed that a 320 mT SMF upregulates the protein expression levels of cytokines such as MCM7 and PCNA in proliferating PDLSCs. Cell counting kit-8 results revealed that the SMF group had higher optical density values than the control group. The ratio of cells in the S phase to those in the G2/M phase was significantly increased after exposure to a 320 mT SMF. In scratch assays, the SMF-treated PDLSCs exhibited a higher migration rate than the sham-exposed group after 24 h of culture, indicating that the SMF promoted the migratory ability of PDLSCs. The activity level of the early differentiation marker alkaline phosphatase and the late marker matrix mineralization, as well as osteoblast-specific gene and protein expression, were enhanced in PDLSCs exposed to the SMF. Furthermore, AKT signaling pathway was activated by SMF. Our data demonstrated that the potential mechanism of action of SMF may enhance PDLSCs proliferation and osteogenic differentiation by activating the phosphorylated AKT pathway. The elucidation of this molecular mechanism may lead to a better understanding of bone repair responses and aid in improved stem cell-mediated regeneration.
Magnetotherapy includes pulsed electromagnetic field (PEMF), static magnetic field (SMF), and alternating electromagnetic fields. As an external physical stimulation, magnetic fields have been applied in clinical applications such as to accelerate healing of new fractures and promote bone tissue self-repair and renewal under pathological conditions [Zhang ZC et al., 2017; Naito et al., 2019]. Because of the safety, stability, compactness, and portability, SMF has also been extensively used in oral applications such as orthodontic tooth movement, dental implant restoration, and prosthesis production [Gujjalapudi et al., 2016; Shan et al., 2021]. Furthermore, it has been demonstrated that the lack of natural and weak SMF can cause insomnia, fatigue, and depression in humans, as well as increase osteoporosis risk . SMF reacts with biological systems and affects cell behavior, medium-strength SMF (1 mT to 1 T) exert a particularly high influence in these contexts [Marędziak et al., 2017]. Medium-strength SMF has been shown to affect the proliferation and differentiation of mesenchymal stem cells (MSCs), osteoblasts, human dental pulp stem cells (DPSCs), and umbilical cord-derived mesenchymal stem cells (WJMSCs) [Kim et al., 2015; Marędziak et al., 2017; Zheng et al., 2018; Chang et al., 2020; He et al., 2021]. Although SMF can enhance bone fracture healing and bone formation both in vivo and in vitro [Yun et al., 2016; Kim et al., 2017], their effects on periodontal ligament stem cells (PDLSCs) biological characteristics are not fully understood.
PDLSCs are derived from MSCs in periodontal ligament tissue. In tissue regeneration, PDLSCs have attracted extensive attention due to their self-renewal and multiple differentiation potential [Seo et al., 2004]. Studies have shown that PDLSCs can re-establish periodontal ligament tissue damaged by periodontal diseases in the oral cavity. PDLSCs-mediated regeneration is becoming one of the most attractive and productive areas in PDLSCs research and application. Physiological factors, pathological factors (an inflammatory environment, hypoxia, physical damage, etc.), and external factors (donor age, culture conditions, cytokines, etc.) cause changes in the biological characteristics of PDLSCs [Zhou et al., 2014].
AKT, also known as protein kinase B (PKB) or Rac, is a key mediator of cell proliferation and differentiation. As the downstream target of phosphoinositide-3 kinase (PI3-K), AKT plays an important role in cell survival and apoptosis [Yu and Cui, 2016; Wang et al., 2021]. Growth and survival factors such as insulin can activate the AKT signaling pathway. Activated AKT activates or inhibits cell functions by phosphorylating downstream factors such as various enzymes, kinases, and transcription factors.
In this study, we introduced a 320 mT SMF (online suppl. Fig. 1; for online suppl. material, see www.karger.com/doi/10.1159/524291) to enhance PDLSCs proliferation, migration, and osteogenic differentiation. In addition, we demonstrated that this SMF regulated PDLSC proliferation and osteogenic differentiation properties by mediating the AKT signaling pathway.
Materials and Methods
SMF Exposure System
To generate an SMF stimulation in vitro, we designed a stable SMF outside the cell culture plate (Fig. 1a). The magnetic field-generating device was manufactured by Li Tian Magnetoelectric Technology Co., Ltd (https://myltem.dzsc.com/; Sichuan, China). We measured the SMF intensity with a Gauss meter (TM-701, Kanetec, Tokyo, Japan). Cell culture plates were placed in the SMF, and the sham-exposed cells were cultured in an incubator without exposure to the SMF.
Isolation and Culture of PDLSCs
PDLSCs obtained from healthy humans (H-PDLSCs) were harvested from normal impacted third molars (18–20 years old). The teeth samples had been removed for treatment purposes and were obtained from the Kunming Medical University-affiliated Stomatological Hospital under the approved guidelines set by the Research Ethical Committee of Kunming Medical University, China. PDLSCs were isolated and cultured as previously described [Liu et al., 2011].
Cell Cycle and Cell Proliferation Detection
Flow cytometry analyses were used to determine the cell cycle of the PDLSCs. A periodic test kit was used according to the protocol suggested by the manufacturer. Then, the cells were analyzed with a Cytomics FC500 Flow Analyzer v.2.2 (Beckman Coulter, Miami, FL, USA). A Cell Counting Kit-8 (CCK-8) test kit (Meilune, Dalian, China) was utilized to evaluate cell proliferation according to the manufacturer’s protocol. PDLSCs were seeded in a 4-well plate, and proliferation-related detection measures were performed after 24 h of cell culture with or without SMF exposure.
Alizarin Red Staining
Alizarin red staining (ARS) was performed as described previously [Liu et al., 2011]. After osteogenic induction for 14 days with and without SMF exposure, cells were fixed in 4% paraformaldehyde and rinsed with 1× phosphate-buffered saline (PBS). The fixed cells were stained with 0.1% Alizarin red for 5 min (pH 4.2; Sigma-Aldrich).
Alkaline Phosphatase Staining
Alkaline phosphatase (ALP) staining was performed following the protocol provided with a BCIP/NBT staining kit (Beyotime Biotechnology, Shanghai, China). After osteogenic induction for 3 days, cultured cells were fixed in 4% paraformaldehyde and then incubated in alkaline solution for 30 min.
Cell Migration Experiments
Scratch assays were performed to investigate the migratory behavior of the SMF-treated and sham-exposed cells. PDLSCs were cultured in a 6-well plate at a density of 5 × 105 to drive them to confluence overnight. The tip of a 200 μL pipette was used to scratch a line diametrically across the center of the culture dishes. The dishes were washed with PBS to remove dead and loose cells, and then, the cells in the plates were cultured with serum-free medium in a 320 mT SMF environment or sham-exposed environment. After 0, 12, and 24 h of treatment, the pictures and records were taken at the same part under an inverted phase-contrast microscope.
RNA Isolation and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis
Total RNA was extracted using TRIzol reagent (Invitrogen, CA, USA). Reverse transcription was performed with a prime script RT reagent kit (TaKaRa, Japan). The primers were purchased from RiBo Bio Co., Ltd. (Guangzhou, China)
Western Blot Analysis
Whole cell lysates used for Western blotting were extracted with lysis buffer (Meilune, Dalian, China). Protein estimation was performed with a protein assay kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s protocol. The total cell lysate was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then the proteins in the gels were transferred to polyvinylidene fluoride (PVDF) membranes (BioRad, Richmond, CA, USA). The membranes were blocked in 5% non-fat dried milk in PBS +0.1% Tween-20 (PBST) (Gibco, USA). The membranes were then probed overnight with the following primary antibodies against runx family transcription factor 2 (RUNX2) (1:1,000, ab236639; Abcam, UK), ALP (1:1,000, ab229126; Abcam, UK), bone sialoprotein (BSP) (1:1,000, DF7738; Affinity Biosciences, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:20,000, 10494-1-AP; Proteintech, USA), β-actin (1:3,000, 20536-1-AP; Proteintech, USA), protein kinase B (AKT) (1:1,000, #9272; Cell Signaling Technology, USA), phosphorylated (p-)Akt (Ser473) (1:1,000, #15116; Cell Signaling Technology, USA), minichromosome maintenance complex component 7 (MCM7) (1:3,000, 11225-1-AP; Proteintech, USA), and proliferating cell nuclear antigen (PCNA) (1:5,000, 10205-2-AP; Proteintech, USA).
Statistical analyses were performed using SPSS v.26.0. The significance was assessed by independent two-tailed Student’s test or two-way analysis of variance (ANOVA). Statistical significance was set at p < 0.05.
SMF Promoted PDLSCs Proliferation
To confirm the effect of the 320 mT SMF on PDLSCs proliferation, the protein expression levels of PCNA and MCM7 were measured by Western blotting. The results indicated that, compared to that in the control group, the protein expression levels of PCNA and MCM7 were increased in the SMF group (Fig. 1b). The CCK-8 results revealed that the optical density (OD) values gradually increased with time and that the SMF group exhibited higher OD values than the control group (Fig. 1c). Furthermore, flow cytometry analysis revealed that the ratio of cells in the S phase to cells in the G2/M phase was significantly increased after exposure to the 320 mT SMF (Fig. 1d).
SMF Promoted PDLSCs Migratory Ability
The application of the 320 mT SMF enhanced PDLSCs migration into the scratch wounded area (Fig. 2a). Microscopic images revealed that SMF and sham-exposed cells at the edge of the scratch wound started to migrate 12 h after the scratch wound was made (Fig. 2a, 3, 4). However, a greater number of leading cells were noted at the scratch edge of the SMF-treated group than at that of the sham-exposed group after 24 h (Fig. 2a, 5, 6). Furthermore, the SMF-treated PDLSCs had a higher migration rate than the sham-exposed group after 24 h (Fig. 2b).
SMF Promoted PDLSCs Osteogenic Differentiation
To determine the effect of the 320 mT SMF on osteogenesis, PDLSCs were cultured with osteogenesis-specific induction. After 3 days of induction, ALP staining and activity were increased in the SMF-exposed cells (Fig. 3a). After 14 days of induction, matrix mineralization, as revealed by ARS, was enhanced in the SMF group (Fig. 3b). The expression of osteogenic markers was detected 7 days after osteogenic induction. SMF significantly upregulated the mRNA levels of ALP, RUNX2, and BSP (Fig. 4a). The Western blot analysis indicated the same results. After 7 and 14 days of osteogenic induction, the protein expression levels of ALP, RUNX2, and BSP were increased in SMF-exposed cells (Fig. 4b).
SMF Induced Activation of the AKT Signaling Pathway
Previous studies have indicated that the AKT signaling pathway is related to cell proliferation and differentiation. To explore the specific mechanism by which SMF regulates PDLSCs proliferation, the AKT level in the PDLSCs was examined by Western blotting. Compared to that in the control group, the p-AKT protein level was significantly upregulated in the SMF group after 24 h of culture (Fig. 5a). After 14 days of osteogenic induction, the p-AKT protein level was evaluated. The results indicated that SMF significantly upregulated the p-AKT protein level (Fig. 5b). SMF may promote PDLSC proliferation and osteogenic differentiation via the AKT signaling pathway.
To evaluate the role of AKT in regulating the behavior of PDLSCs exposed to the 320 mT SMF, the AKT inhibitor LY294002 was incorporated into the complete medium used in the cell proliferation experiment. For both the SMF-treated group and the sham-exposed group, the Western blot results showed that the LY294002-incorporated cells exhibited lower protein expression levels of PCNA and MCM7 (Fig. 6a, b). The CCK-8 results led to the same conclusion (Fig. 6c, 7a, b). AKT phosphorylation enhances cell proliferation to a certain extent.
As very promising seed cells for use in regenerative medicine and tissue engineering, PDLSCs, which are derived from oral periodontal tissue, have received extensive attention in recent years. Driving PDLSCs differentiation in a specific direction is an important outcome for the effective application of PDLSCs. Physiological, pathological, and external factors cause changes in PDLSCs’ biological characteristics. Studies have shown that applying appropriate physical stimuli, such as mechanical stress, to PDLSCs can promote their various capabilities [Zhang et al., 2016]. A SMF is a physical stimulus that has been proven useful in bone defect repair, orthodontics, and implants [Gujjalapudi et al., 2016; Kim et al., 2017; Shan et al., 2021]. A constant magnetic field with specific parameters can promote the growth and secretion function of rat osteoblasts and inhibit the growth and osteoclast function of osteoclasts, thereby improving bone density and bone strength and showing effective intervention in osteoporosis [Zhang ZC et al., 2017; Yang et al., 2018; Hao et al., 2019; Tasić et al., 2020]. In this study, we focused on the effects of a SMF on cell proliferation, migration, and differentiation and the specific molecular mechanisms were explored to evaluate the possibility of using SMF as a therapeutic agent in bone regeneration.
Previous studies have revealed that moderately intense SMF can enhance the proliferation of different cell types, such as human bone marrow-derived MSCs (HBMSCs), tissue-derived MSCs, and DPSCs [Kim et al., 2015; Marędziak et al., 2017; Lew et al., 2018; Zheng et al., 2018; Wu et al., 2020]. In the present study, 320 mT SMF exposure promoted PDLSCs proliferation, which was consistent with the conclusions of previously published studies. However, the exact effect of a SMF on cell proliferation and viability has been debated. Some studies have shown conclusions contrary to those of our research. A SMF significantly inhibited the proliferation of human osteoblasts [Javani Jouni et al., 2013]. In addition, SMF have been proven to play negative roles in the proliferation of BMSCs [Denaro et al., 2008; Marędziak et al., 2014]. The different effects of SMF on cells may be related to different culture conditions, cell types, field intensity, and/or SMF exposure time or type [Zafari et al., 2015; Marycz et al., 2018]. The loss of cell expansion capacity in vitro restricts their clinical application; therefore, increasing the number of high-quality stem cells is a relatively important in tissue engineering and regenerative protocols.
As a basic cellular activity, migration plays an important role in the regeneration of injured tissues [Howard et al., 2010]. The application of a SMF modulates the migration of the exposed cells [Wu et al., 2020]. The rate of migration induced by a SMF depends on cell type. Studies have indicated that 30 and 120 mT SMF enhances the migration in neuroblastoma and fibroblastoma cells. A SMF promoted DPSC migration to dental tissue wounds, promoting wound healing [Lew et al., 2019]. In this study, we observed that a 320 mT SMF enhanced the migratory ability of PDLSCs. In diseases that cause bone defects, SMF may induce PDLSCs to migrate to bone defects at a higher rate and thereby enhance bone regeneration.
In the present study, SMF increased ALP activity, promoted the formation of mineralized nodules, and upregulated the mRNA and protein expression levels of ALP, BSP, and Runx2. Evidently, SMF stimulates the osteogenic differentiation of PDLSCs, which is important for bone regeneration. Our data are consistent with those reported in previous studies of MSCs [Chang et al., 2020; He et al., 2021; Wu et al., 2021; Zhang et al., 2021], human osteoblast-like MG63 cells [Rana et al., 2019], and DPSCs [Zheng et al., 2018; Hsu et al., 2020; Farzaneh et al., 2021]. The data obtained from multiple experiments strongly support the idea that a SMF confers profound pro-osteogenic properties to cells and that applying SMFs may stimulate tissue regeneration, providing a new therapy for periodontal bone defects.
Accumulating evidence has shown that multiple signaling pathways, such as the Wnt/β-catenin, p38, JNK/MAPK, and NF-κB signaling pathways, are involved in regulating proliferation and osteogenesis [Kim et al., 2017a, b]. These signaling pathways construct a complex network of cell functions. In the present study, we found that the AKT signaling pathway may be a main pathway in PDLSCs proliferation and osteogenic differentiation. The application of a 320 mT SMF significantly enhanced the PDLSCs proliferation and osteogenic differentiation via AKT. Previous studies have shown that AKT phosphorylation can promote the proliferation of adipose tissue-derived MSCs [Marędziak et al., 2017], myoblast cells [Liu et al., 2017], DPSCs [Zheng et al., 2018], etc. Furthermore, the AKT signaling pathway is also known to be involved in the progression of bone development [Liu et al., 20017] and the enhancement of the osteoblast differentiation of stem cells [Li, 2020; Lv et al., 2020]. It has been reported that the AKT signaling pathway enhances the DNA binding of Runx2 and that Runx2 upregulates the expression of AKT, Runx2 and AKT, indicating mutually dependent processes in osteoblasts [Fujita et al., 2004].
PDLSCs play a considerable role in periodontal regeneration and repair. Highly efficient PDLSCs-mediated regeneration therapy may be a result from the profound proliferative ability and directed differentiation potential of these cells. Therefore, searching for effective PDLSCs stimulation that increases their proliferative potential and directed differentiation potential seems to be a fully reasonable strategy, especially considering the potential clinical application of PDLSCs. In the present study, SMF was proven to promote the proliferation, migration, and osteogenic differentiation of PDLSCs. Moreover, some studies have reported that SMF possess anti-inflammatory properties [Hsieh et al., 2015; Shang et al., 2019]. SMF may improve the microenvironment of damaged tissues by initiating the proliferation and differentiation of stem cells, thereby facilitating tissue repair.
As indicated in this study, the effect of SMF on the biological characteristics of PDLSCs provides us with a better understanding of PDLSCs and aids in the improvement of stem cell-mediated regeneration. However, the influence of magnetic fields on PDLSCs differentiation still leads to many questions: What are the best induction conditions for inducing directed differentiation of these cells, and what is the specific mechanism by which PDLSCs respond to magnetic fields? To find the answers, further exploration is needed.
This study protocol was approved by the Research Ethical Committee of Kunming Medical University, China. The Ethics Review Number is KYKQ2021MEC028.
Written informed consent was obtained from participants or their parents to participate in the study.
Conflict of Interest Statement
The authors have no conflicts of interest to declare.
The present work was supported by the National Natural Science Foundation of China (grant numbers: 31860326 and 31301067), Joint Fund of Yunnan Provincial Science and Technology Office, Kunming Medical University [grant numbers: 2017FE468-(227) and 2018FE001-(260)], and Graduate Innovation Foundation of Kunming Medical University (grant number: 2021S028).
K.Z. significantly contributed to and performed experiments in the present study, prepared the figures, and wrote the manuscript. W.G. and S.L. helped perform experiments. Y.L. and Z.Z. conceived the idea and helped proofread the figures and manuscript. All authors read and approved the final manuscript.
All data generated or analyzed in this study are included in this article and its supplementary material files. Further inquiries can be directed to the corresponding author.