Metformin enhances osteogenic differentiation of stem cells from human exfoliated deciduous teeth through AMPK pathway

Stem cells from human exfoliated deciduous teeth (SHEDs) are ideal seed cells in bone tissue engineering. As a first‐line antidiabetic drug, metformin has recently been found to promote bone formation. The purpose of this study was to investigate the effect of metformin on the osteogenic differentiation of SHEDs and its underlying mechanism. SHEDs were isolated from the dental pulp of deciduous teeth from healthy children aged 6 to 12, and their surface antigen markers of stem cells were detected by flow cytometry. The effect of metformin (10–200 μM) treatment on SHEDs cell viability, proliferation, and osteogenic differentiation was analyzed. The activation of adenosine 5′‐monophosphate‐activated protein kinase (AMPK) phosphorylation Thr172 (p‐AMPK) was determined by western blot assay. SHEDs were confirmed as mesenchymal stem cells (MSCs) on the basis of the expression of characteristic surface antigens. Metformin (10–200 μM) did not affect the viability and proliferation of SHEDs but significantly increased the expression of osteogenic genes, alkaline phosphatase activity, matrix mineralization, and p‐AMPK level expression in SHEDs. Compound C, a specific inhibitor of the AMPK pathway, abolished metformin‐induced osteogenic differentiation of SHEDs. Moreover, metformin treatment enhanced the expression of proangiogenic/osteogenic growth factors BMP2 and VEGF but reduced the osteoclastogenic factor RANKL/OPG expression in SHEDs. In conclusion, metformin could induce the osteogenic differentiation of SHEDs by activating the AMPK pathway and regulates the expression of proangiogenic/osteogenic growth factors and osteoclastogenic factors in SHEDs. Therefore, metformin‐pretreated SHEDs could be a potential source of seed cells during stem cell‐based bone tissue engineering.


| INTRODUCTION
Effective reconstruction of the severe bone defects caused by trauma, infection, and tumor resection is a great challenge in clinical practice.
Grafting autologous bone or synthetic bone substitutes are used to fill the large size bone defect. Bone grafts are the second most common tissue grafts in the world. Approximately 2,200,000 bone grafts are performed worldwide each year to repair bone defects (Bigham-Sadegh & Oryan, 2015;Ducy, Schinke, & Karsenty, 2000;Seeman, 2002). However, supplies of functional stem cells and growth factors are necessary to regenerate the de novo bone. Therefore, the development of functional stem cells with osteogenic potential is urgent to achieve ideal bone regeneration and repair.
Recently, the application of stem cell-based bone tissue engineering is at the center of attention in orthopedics. Stem cells from human exfoliated deciduous teeth (SHEDs) are characterized as mesenchymal stem cells (MSCs) with low immunogenicity. SHEDs can be differentiated into osteoblasts, odontoblasts, chondrocytes, adipocytes, nerve cells, and other cells with strong horizontal differentiation capability (Gay, Chen, & MacDougall, 2007). The potential clinical application of SHEDs is not just limited to dental diseases. A variety of tissue defects, including bone defects, could be repaired using SHEDs as progenitor seed cells (Khojasteh, Motamedian, Rad, Shahriari, & Nadjmi, 2015). The telomere length of SHEDs is twice that of the human dental pulp stem cells (DPSCs), resulting in a high degree of stemness and vitality for SHEDs (Akpinar et al., 2014). SHEDs have higher proliferative, angiogenic, and osteogenic potentials compared with DPSCs or bone marrow MSCs (BMSCs) (Hii Siew, Norhayati, Ismail Ab, & Kannan Thirumulu, 2017;Xie et al., 2019). Besides, SHEDs are conveniently available from dental practice with relatively low invasive procedures and ethical barriers. SHEDs recovered after long-term cryopreservation retained stem cells' characteristics, pluripotency, dentin/bone regeneration, and immunomodulatory function (Ma et al., 2012), which indicates the possibility of using cryopreserved autologous SHEDs for bone tissue engineering. SHEDs have thus been increasingly recognized as promising seed cell sources in tissue engineering for bone regeneration and repair.
Metformin is a safe, well-tolerated, and the first-line drug for the treatment of type 2 diabetes. Metformin has been approved for clinical use in the United Kingdom since 1958 and the United States since 1995 (Bailey & C., 2004;Tan, Alquraini, Mizokami-Stout, & MacEachern, 2016). In recent years, metformin has been found to promote osteogenic differentiation of MSCs (Orciani, Fini, Di Primio, & Mattioli-Belmonte, 2017;Vestergaard, Rejnmark, & Mosekilde, 2005), as well as improve bone repair in diabetic patients (Melton, Leibson, Achenbach, Therneau, & Khosla, 2008;Vestergaard et al., 2005). As a highly hydrophilic compound, metformin requires membrane transporters such as organic cation transporters (OCTs) to cross the cell membrane and function (Shu et al., 2007). OCTs are present in SHEDs (Aljofi, 2017), indicating that metformin may exert an effect on SHEDs activity. However, the effect of metformin on the osteogenic differentiation of SHEDs has not yet been characterized.
Adenosine 5 0 -monophosphate-activated protein kinase (AMPK) is a crucial cellular energy sensor associated with cell viability (D. G. Hardie, Ross, & Hawley, 2012). Metformin has been well characterized as an activator of AMPK. The promotion of osteogenic differentiation of MSCs and bone repair in diabetic patients by metformin has led to establishing the role of AMPK in bone metabolism. The purpose of this study was to investigate whether metformin could promote the osteogenic differentiation of SHEDs and, if so, to further evaluate the role of the AMPK pathway in the metforminmediated osteogenic effect on SHEDs.

| Cell culture
The Medical Ethical Committee of the Affiliated Stomatology Hospital of Guangzhou Medical University approved this study (KY2019008).
Retained deciduous teeth were extracted from healthy children aged 6-12 years. Informed written consents were obtained from the parents of the patients. The teeth were cleaned in sterile conditions with phosphate-buffered saline (PBS) (Gibco, USA) containing 4% penicillin/streptomycin (Gibco, USA). Then, pulp tissues were separated, cut into pieces, digested with 3 g/L collagenase I (Gibco, USA) and 4 g/L dispase (Gibco, USA) for 30 min, and centrifuged for 5 min.
The collected cells were cultured in 60 mm Petri dishes with Dulbecco's modified Eagle medium (DMEM) (Gibco, USA) containing 10% of fetal bovine serum (FBS) (Gibco, USA) and 1% of penicillin/streptomycin (Gibco, USA). When the monolayer of adherent cells reached 80% of confluence, they were trypsinized and subcultured at 5 × 10 3 cells/cm 2 . For subsequent culturing, the original culture medium in the culture plate was discarded under aseptic conditions, the plate washed with PBS for two to three times, and the pre-prepared culture medium added. Surface antigens (CD105, CD90, CD73, CD34, and CD45) were detected by flow cytometry (FCM).

| Cell viability staining
SHEDs were seeded in six-well culture plates at a density of 5 × 10 4 cells/well and incubated in a humidified incubator at 37 C under 5%

| Cell proliferation assay
Cells were seeded in 96-well plates at a density of 2 × 10 3 cells/well and cultured for 24 h. Then, the old medium was replaced with the medium containing 0, 10, 50, 100, and 200 μM metformin respectively (six repeats per concentration). At 1, 3, 5, or 7 days, the medium was discarded, and the cells were washed with PBS twice. In each well, 90-μl medium and 10-μl CCK8 reagent were added and incubated for 2 h in the incubator after adding (Dojindo, Japan). The absorbance was measured at 450 nm. The absorbance value of each time point was calculated, with the detection time as the abscissa and the absorbance value as the ordinate, and the histogram and line graph were drawn for statistical analysis.

| Osteogenic gene expression analysis
SHEDs (1.0 × 10 5 cells/well) were seeded in six-well culture plates. At 4 and 7 days after induction for SHEDs by metformin, total RNA was extracted using Trizol® (Invitrogen, Carlsbad, CA), and 1,000 ng of total RNA was used for the RT reaction using the PrimeScript RT reagent kit (Takara, Japan). Real-time quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR) was performed using SYBR Green PCR Master Mix (Thermo Fisher, USA) to detect the gene expression of runt-related transcription factor 2 (Runx2), type I collagen (COL-I), alkaline phosphatase (ALP), osteocalcin (OCN), bone morphogenetic protein 2 (BMP2), vascular endothelial growth factor A (VEGFA), receptor activator of nuclear factor-κB ligand (RANKL), and osteoprotegerin (OPG). The primer sequences for the genes are listed in Table 1. PCR conditions were as follows: 2 min at 50 C, 10 min at 95 C, then 15 s at 95 C for 40 cycles, and 60 C for 1 min in 96-well culture plates using the ViiA™ 7 q-PCR System. The data were normalized to the internal control, GAPDH. The final expression level of the gene of interest relative to controls was reported by the 2-ΔΔ Ct method.

| Western blotting
Cell lysate from SHEDs culture was extracted using RIPA buffer. The lysate was centrifuged at 12,000 rpm for 15 min to collect the supernatant. Total protein concentration was measured using bicinchoninic acid (BCA)-based protein analysis kit (BestBio, China). Total protein (30 μg) was loaded in 10% SDS-PAGE gel (Beyotime, China) for protein separation and transferred into polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% nonfat milk powder for 1 h at 37 C. The membrane was then incubated at 4 C overnight with primary antibodies, which included rabbit anti-human AMPKα (Cell Signaling Technology, USA), rabbit anti-human p-AMPK (Thr172) (Cell Signaling Technology, USA), and rabbit anti-human GAPDH (Cell Signaling Technology, USA). HRP-conjugated Affinipure Goat Anti-Rabbit IgG (Proteintech, USA) was incubated at room temperature for 1 h. Protein bands were detected using an Enhanced Chemical Luminescence kit (Millipore, USA). The ImageJ software was used to semiquantify band intensity.

| ALP activity and staining
SHEDs (1.0 × l0 4 cells/well) were inoculated in 48-well culture plates, and a 250-μl complete medium was added to each well. After 24 h, different concentrations of metformin were added to the medium. On Day 4, the original medium was removed, and the cells were washed with PBS twice. The cell lysis buffer containing 0.1% Triton x-100 was added to each well. The cells were lysed on ice for 30 min and transferred into 1.8-ml tubes, which were centrifuged (12,000 rpm) at 4 C for 15 min. The supernatant was absorbed for subsequent operation.
The protein concentration was determined by the BCA method, as T A B L E 1 List of the primers used for the reverse transcription and polymerase chain reaction (q-PCR) Genes Primer sequence

| Mineralization assays
SHEDs were seeded into 48-well culture plates with a density of 2 × 10 4 cells/well. After 24 h, the cell culture medium was replaced with osteogenic medium (50 g/ml ascorbic acid, 10 mM

| AMPK inhibition by compound C
On the basis of the results of the above-mentioned experiments, 100 μM of metformin was selected as the most effective concentration to continue the further experiment. Compound C, an AMPK inhibitor, was dissolved in dimethyl sulfoxide (DMSO) following the manufacturer's instruction to obtain a 1 mM stock solution. A working concentration of 10 μM was added in the culture 1 h before the addition of metformin. The equivalent concentration of DMSO was added to the mock control group. We divided the SHEDs culture into four groups: the control group, the 10-μM compound C group, the 100-μM metformin group, and the 10-μM compound C mixed with the 100-μM metformin group. q-PCR, western blotting, and matrix mineralization assay were performed in these cultures.

| No effect of metformin treatment on SHEDs viability
To ascertain whether metformin treatment would have an impact on the growth of SHEDs, we compared the cell viability and proliferation between with and without metformin treatment. Figure

| Effect of metformin treatment on osteogenic gene expression in SHEDs
To study the impact of metformin treatment on the osteogenic differ- Furthermore, bone healing involves bone formation by osteoblast and bone resorption by osteoclast and is tightly controlled by the RANKL/RANK/OPG molecular triplet. RANKL/OPG is the key to maintain the dynamic balance between bone resorption and bone formation in bone remodeling (Liu et al., 2010). Consistent with the above upregulation of genes in bone formation, the expression of RANKL/OPG in metformin-treated cells was significantly lower than that in vehicle-treated control cells (Figure 3).

| Effects of metformin treatment on matrix mineralization in SHEDs' culture
To further assess the effects of metformin treatment on osteogenic differentiation of SHEDs, we examined matrix mineralization of SHEDs culture by using Alizarin Red staining (ARS). ARS was performed at Days 14 and 21 after osteogenic induction. The dense staining in Figure 4a indicates the higher degree of matrix mineralization in the metformin-treated SHEDs than those in the control cells at Day 14 (Figure 4a, left panels). By quantitative analysis on the dye extract with 10% CPC, we found that metformin treatment for 14 days significantly increased the mineralization in SHEDs as compared with the vehicle-treated controls (Figure 4b). However, there were no differences between metformin treatment and vehicle at Day 21 (Figure 4a, right panels; Figure 4c), possibly due to an overriding effect by the osteogenic medium over time.
We also analyzed the ALP expression and activity. ALP is a marker for early osteogenesis. Consistent results on ALP expression and activity were obtained. ALP activity in metformin-treated SHEDs was significantly higher than that of the control cells (Figure 4d). At Day 4, metformin-treated SHEDs showed enhanced ALP staining as compared with the control (Figure 4e).

| Activation of AMPK by metformin treatment in SHEDs
After treatment with metformin (10, 50, 100, and 200 μM) for 24 h, the expression of p-AMPK (Thr172) was significantly increased as F I G U R E 3 Effect of metformin treatment on osteogenic gene expression in stem cells from human exfoliated deciduous teeth (SHEDs). P4 SHEDs were treated with the indicated concentrations of metformin for 4 or 7 days. The expression of genes (OCN, COL-I, RUNX2, ALP, BMP2, VEGFA, and RANKL/OPG) was detected by reverse transcription and polymerase chain reaction (q-PCR). The data are presented as mean ± SD (n = 3). ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 as compared with the control compared with the control treatment ( Figure 5). Interestingly, consistent with the above osteogenic effects by metformin, the activation of AMPK seemed to be most significant at a metformin concentration of 100 μM. A dose-dependent effect on AMPK activation by metformin treatment was observed at clinically relevant concentrations; however, the effect might begin to reduce at a higher concentration of metformin such as 200 μM tested in the present study. In the subsequent experiments, 100 μM of metformin was employed.

| Metformin promotes osteogenic differentiation of SHEDs via AMPK signaling
To determine the role of AMPK signaling in the osteogenic effects of metformin in SHEDs, we added AMPK inhibitor in the cultures. Compound C is widely used as a cell-permeable AMPK inhibitor (X. Liu, Chhipa, Nakano, & Dasgupta, 2014). Compound C treatment dramatically decreased the expression of p-AMPK (Thr172) (Figure 6a). Notably, compound C treatment not only abolished the activation of F I G U R E 4 Effect of metformin treatment on osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHEDs). SHEDs were cultured in an osteogenic induction medium for 14 or 21 days. The degree of mineralization was assessed by Alizarin Red staining (ARS) (scale bar = 2.5 mm).
(a). The Semi-quantitative result of the mineralized matrix (b, c). SHEDs were treated with the indicated concentrations of metformin for 4 days. Alkaline phosphatase (ALP) activity (d)and ALP staining (e) of SHEDs. The data are presented as mean ± SD (n = 4) ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 as compared with the control [Colour figure can be viewed at wileyonlinelibrary.com] F I G U R E 5 Effect of metformin treatment on adenosine 5 0 -monophosphate-activated protein kinase (AMPK) activation in stem cells from human exfoliated deciduous teeth (SHEDs). SHEDs were treated with the indicated concentrations of metformin for 24 h. The expression of AMPK α Thr172 was detected by western blot. The data are presented as mean ± SD (n = 3). ****p < 0.0001 as compared with the control AMPK by metformin but also reduced the constitutive level of active AMPK in SHEDs. The matrix mineralization enhanced by metformin treatment was fully abolished by compound C treatment (Figure 6b).
Consistent with its inhibition toward constitutive AMPK activation, compound C treatment alone also led to a decreased matrix mineralization in SHEDs. Furthermore, as compared with metformin-treated cells and vehicle-treated control cells, the expressions of osteogenic genes, including COL-I, ALP, RUNX2, and OCN, were significantly reduced in the presence of compound C (Figure 6c). These results indicated that AMPK activation was required in the promotion of osteogenic differentiation by metformin in SHEDs.

| DISCUSSION
MSCs are basal cells that can adhere to grow and express certain signature surface marker proteins such as CD73, CD90, and CD105 but not hematopoietic cell markers CD45, CD14, and CD34 and endothelial cell marker CD31 (Dominici et al., 2006). Metformin has been widely reported to promote osteogenic differentiation of MSCs derived from different origins such as those derived from bone marrow, placenta, umbilical cord blood, adipose tissue, and muscle (Al Jofi et al., 2018;Gu, Gu, Yang, & Shi, 2017;Marycz et al., 2016;Wen-Qi Ma, Wang, Zhu, Han, & Liu, 2019). However, although the great clinical potential of SHEDs in tissue engineering has been increasingly recognized, it was unknown whether metformin treatment could induce the osteogenic differentiation of SHEDs. In this study, we have demonstrated that metformin treatment can significantly increase the expression of osteogenic genes and matrix mineralization in SHEDs.
Moreover, the osteogenic effect of metformin in SHEDs was found to be via AMPK activation.
Our results first confirmed that SHEDs are MSCs. Metformin treatment did not affect the viability and proliferation of SHEDs at up to 200 μM, which is in line with the fact that metformin is a safe drug F I G U R E 6 Adenosine 5 0 -monophosphate-activated protein kinase (AMPK) activation was required in the promotion of osteogenic differentiation by metformin in stem cells from human exfoliated deciduous teeth (SHEDs). SHEDs were treated with the AMPK inhibitor compound C (10 μM) and/or metformin(100 μM)for 24 h. The expression of AMPK α Thr172 was detected by western blot (a). SHEDs were cultured in an osteogenic induction medium for 17 days. The mineralized nodule was assessed by ARS (scale bar = 2.5 mm). (b). SHEDs were treated with compound C and/or metformin for 4 days. The expression of osteogenic genes was detected by reverse transcription and polymerase chain reaction (q-PCR) (c). The data are presented as mean ± SD (n = 3). *p < 0.05 vs. control group，# p < 0.05 vs. Met group，@p < 0.05 vs. Comp. C group. Con, control; Met+Comp.C, metformin + compound C; Comp. C, compound C; Met, metformin [Colour figure can be viewed at wileyonlinelibrary.com] widely used in diabetic patients. However, in a previous report, metformin treatment at 100 μM showed moderate cytotoxicity in human chorionic villous MSCs (Gu et al., 2017). These different observations may be due to the differences in experimental conditions and/or cell types. Alternatively, there could be subtle cytotoxicity associated with the high concentration of metformin treatment in SHEDs that was undetectable with our assays yet. Actually, the effects of metformin on osteogenic gene expression, matrix mineralization, and AMPK activation were maximized at 100 μM. In clinical patients, the plasma concentration of metformin is around 10 μM (Smolders et al., 2017). Our data indicated that metformin treatment could significantly promote osteogenic differentiation of SHEDs at the low concentrations relevant to these clinical levels. When directly used at a local site such as tissue engineering for specific bone regeneration and repair, a concentration higher than those in the plasma may be employed to achieve optimal effects.
Ischemia is one of the major risk factors for reduced bone healing.
Blood vessels not only provide oxygen but also serve as conduits for additional osteoblasts, which play a positive role in promoting cell dif- AMPK is a serine/threonine-protein kinase that acts as a sensor for cellular energy and nutrition (D. G. Hardie, 2007). The protein complex consists of one catalytic subunit (α) and two regulatory subunits (β and γ) (D. G. Hardie, Carling, & Carlson, 1998 (Pierotti et al., 2013), inhibits adipogenesis, promotes the blood vessel formation, and increases osteogenesis but reduces bone resorption (Gao, Li, Xue, Jia, & Hu, 2010;Gu et al., 2017;Martin, Hayward, Viros, & Marais, 2012). All of these effects by metformin seem to be related to its action as an AMPK activator. In the present study, we demonstrated that metformin could also increase the activation (i.e., phosphorylation at Thr172) of AMPK in SHEDs. AMPK inhibitor compound C abolished the metformin-induced osteogenic differentiation of SHEDs. Our data and abundant literature evidence indicate osteogenic effects of metformin, but Jeyabalan and colleagues reported no effects of metformin on osteogenesis in vivo (Jeyabalan et al., 2013;Lin et al., 2017). It is likely that the seed cells used in some studies did not have sufficient osteogenic potential or that the animal models were not close enough to the clinical situation of bone regeneration. SHEDs have multiple advantages, such as low immunogenicity, high viability, and abundant clinical sources. Our findings have thus warranted further studies, particularly preclinical evaluation in animals, to explore the clinical potential of SHEDs in combination with metformin in the areas of bone regeneration and repair.
The molecular mechanism underlying the role of AMPK activation in osteogenic differentiation and bone regeneration remains unclear.
AMPK activation may promote bone formation by enhancing autophagy (Li, Su, Sun, Cai, & Deng, 2018). AMPK controls the osteogenic differentiation of human MSCs through early mTOR inhibitionmediated autophagy and late activation of the Akt/mTOR signaling axis (Aleksandar Pantovic et al., 2013). On the other hand, as a direct substrate of AMPK, RUNX2 can stimulate osteoblast differentiation and bone formation (Chava, Chennakesavulu, Gayatri, & Reddy, 2018). In addition to promoting bone formation, the activation of the AMPK pathway can inhibit bone resorption (Yamaguchi et al., 2008). Interestingly, we found that metformin can not only promote osteogenesis but also increase the expression of vascular genes and decrease the expression of osteoclast genes in SHEDs. Future mechanistic studies on osteogenic effects of metformin in SHEDs may reveal important insights into osteogenic differentiation of stem cells and bone metabolism.
In conclusion, metformin treatment has no adverse effect on

DATA AVAILABILITY STATEMENT
Some or all data, models, or code generated or used during the study are available from the corresponding author by request.