Suppression of osteogenic differentiation and mitochondrial function change in human periodontal ligament stem cells by melatonin at physiological levels

Isolation and culture of hPDLSCs

The study protocol was approved by the Medical Ethics Committee of the Hospital of Stomatology Sun Yat-sen University, China (KQEC-2019-10). We extracted and collected 15 healthy human premolars from 15 healthy adult patients aged 18–25 years undergoing orthodontic therapy at the Hospital of Stomatology, Sun Yat-sen University. All participating patients signed a written informed consent form. hPDLSCs were isolated and cultured as previously described (Seo, Miura & Gronthos, 2004). In our study, cells grown in alpha modified Eagle’s medium (α-MEM) containing 10% fetal bovine serum (FBS) were used at passage 3–5 for subsequent experiments. hPDLSCs were cultured in osteogenic medium (OS, 10% FBS, 0.1 mM dexamethasone, 0.2 mM ascorbic acid, and 10 mM β-glycerophosphate; Sigma-Aldrich, St. Louis, MO, USA). The hPDLSCs were cultured in basal growth medium or OS with MT at various concentrations (0, 1 pM, 0.1 nM, 10 nM and 1 µM, Sigma-Aldrich) for 3, 7 or 21 days. All above media and reagents were purchased from Gibco (Grand Island, NY, USA) unless otherwise noted.

Multipotential differentiation

hPDLSCs were induced in OS, adipogenic medium (Cyagen Biosciences, Santa Clara, CA, USA), and chondrogenic medium (Cyagen Biosciences) for 21 days. Next, the cells were fixed in 4% paraformaldehyde and stained with 1% Alizarin red staining (ARS) solution (Cyagen Biosciences) for the osteogenesis assay. Additionally, 3 mg/mL oil red O (Sigma-Aldrich) was used for adipogenesis analysis and Alcian blue staining for the chondrogenic assay. The images were viewed using an inverted microscope (Zeiss AG, Oberkochen, Germany).

Cell surface marker expression of hPDLSCs

Passage 3 hPDLSCs were prepared for flow cytometric analysis to detect stem cell surface makers. The cells were incubated in TrypLE buffer (Gibco) to obtain single-cell suspensions and then resuspended in phosphate-buffered saline (PBS) containing 2% FBS. The cells were incubated with the mesenchymal markers CD34, CD45, CD73, CD90, CD164 and CD166 (BD Biosciences, Franklin Lakes, NJ, USA) for 30 min at 4 °C. Cell samples were analyzed by flow cytometry (Beckman Coulter, Brea, CA, USA).

MT affects hPDLSCs viability

The hPDLSCs were treated with different concentrations of MT (0, 1 pM, 0.1 nM, 10 nM, and 1 µM). Cell viability was tested with the cell counting kit-8 (CCK8, Dojindo, Kumamoto, Japan) after 0, 24, 48, 72 and 96 h according to the manufacturer’s instructions. The absorbance was determined at a wavelength of 450 nm.

Alkaline phosphatase (ALP) activity assay

hPDLSCs were cultured in OS supplemented with different concentrations of MT for 3 days. Next, a commercial ALP kit (Jiancheng, Nanjing, China) was used to measure ALP activity following the manufacturer’s instructions. The generation of p-nitrophenol in the presence of ALP was determined by measuring the OD at a wavelength of 520 nm. The OD was normalized to the protein concentration.

Quantitative assay of ARS

hPDLSCs were exposed to MT at various concentrations in OS for 21 days. The cells were stained with ARS and images were acquired with a camera (Nikon, Tokyo, Japan). Next, 500 µL of 0.1 M hexadecylpyridinium chloride monohydrate (Sigma) was added to each well to dissolve the contents at 37 °C for 30 min. Subsequently, 100 µL of samples was transferred to the wells of a 96-well plate, and the absorbance of the supernatant was measured at 562 nm.

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) analysis

The hPDLSCs were cultured for 7 days, and total mRNA was extracted using a RNA-Quick purification kit (YISHAN Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Next, first-strand cDNA was synthesized using PrimeScript™ reverse transcription (RT) Master Mix (TaKaRa, Shiga, Japan). PCR was performed with SYBR Green I Master Mix (Roche Applied Science, Basel, Switzerland) following the manufacturer’s protocol. Gene-specific primers used in this study were commercially synthesized, and their sequences are listed in Table 1.

Western blotting

The hPDLSCs were lysed for 30 min at 7 days post osteogenic induction. Cell extracts containing 40 µg total protein were loaded and separated by 4–20% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SurePAGE, Ubiotechnology, Guangdong, China) and transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membranes were blocked in Tris-buffered saline +0.1% Tween-20 (TBST) containing 5% bovine serum albumin (BioFroxx, Einhausen, Germany) for 2 h at 20 °C–25 °C after transfer. The membranes were immunoblotted with the following primary antibodies at a 1:1,000 dilution: polyclonal rabbit anti-OPN (Novus Biologicals, Littleton, CO, USA); polyclonal rabbit anti-OCN (Novus Biologicals), mitochondrial dynamics antibody sampler kit (Cell Signalling Technology, Danvers, MA, USA); and monoclonal mouse anti-β-actin (Beyotime, Shanghai, China) overnight at 4 ° C. This was followed by incubation with secondary antibodies at 20 °C–25 °C for 1 h. Immunoreactive bands were visualized by chemiluminescence detection reagents (Millipore, Temecula, CA) according to the manufacturer’s instructions. Band intensities were quantified using ImageJ 1.36b (NIH, Bethesda, MD, USA).

Measurement of intracellular ATP content

The hPDLSCs were cultured for 7 days, after which the ATP level was measured with an enhanced ATP assay kit (Beyotime) according to the manufacturer’s instructions. Absorbance was measured with a luminometer (GloMax Multiplus plate reader, Promega, Madison, WI, USA). Luminescence intensity was divided by the total number of cells.

Measurement of intracellular ROS

hPDLSCs were cultured for 7 days in TrypLE buffer to obtain single-cell suspensions, resuspended in α-MEM containing 2% FBS, incubated with 5 µM CellROX^® green reagent (Invitrogen, Carlsbad, CA, USA) at 37 °C for 30 min, and then washed with PBS three times. The fluorescence intensity of 1 × 10⁴ cells was recorded by flow cytometry.

NAD⁺/NADH assay

hPDLSCs were cultured for 7 days. Intracellular NAD⁺ and NADH levels were measured using an NAD⁺ and NADH assay kit (Beyotime) according to the manufacturer’s instructions. The absorbance of the supernatant was measured at 450 nm, and the NAD⁺/NADH ratio was calculated as follows: [NAD⁺ − NADH]/NADH.

Statistical analysis

All data are expressed as the means ± S.D. One-way analysis of variance was used to compare groups, and Fisher’s least significant difference (LSD) was used for post-hoc analysis. P < 0.05 was considered to indicate statistical significance. All experiments were performed in triplicate and SPSS 22.0 software (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis.

Isolation and characterization of PDLSCs

hPDLSCs were successfully obtained and exhibited a homogeneous, large, fibroblast-like morphology (Fig. 1A). hPDLSCs showed morphological changes and osteogenic/adipogenic/chondrogenic differentiation potential after 3 weeks of induction (Figs. 1B–1D). The mesenchymal stem cell (MSC) properties of the hPDLSCs were characterized by identifying cell surface markers. hPDLSCs were positive for the mesenchymal markers cluster of differentiation 73 (CD73, 96.54%), CD90 (99.78%), CD146 (71.30%), and CD166 (93.74%) but negative for the hematopoietic markers CD34 and CD45 in flow cytometric analysis (Figs. 1E–1J). These results suggest that hPDLSCs were successfully isolated and had good osteogenic differentiation ability.

MT does NOT affect the viability of hPDLSCs

There was no significant difference between the MT groups and basal growth medium group (P > 0.05) (Fig. 1K). The cell growth rate was relatively stable, and the growth of each group relative to the previous time point was significantly different (data shows in Dataset S3). These results indicate that MT has no apparent cytotoxicity.

Physiological concentrations of MT affect hPDLSCs osteogenic differentiation

The ARS experiments indicated that 1 pM–10 nM MT reduced the effect of the OS (P < 0.05, Figs. 2A, 2B). We also found that treatment with MT at 1 pM, 0.1 nM, and 10 nM caused a greater decrease in ALP activity than OS alone. Particularly, the highest decrease in ALP activity was observed in the 0.1 nM MT-treated group (P < 0.05, Fig. 2C). In contrast, treatment with the pharmacological MT concentration of 1 µM increased ALP activity, but there was no significant difference compared to OS alone.

Next, we detected the expression levels of OPN and OCN, which are considered as closely associated with osteogenic differentiation, by qRT-PCR and western blotting after day 7 of osteogenic induction. The analysis showed that physiological MT treatment downregulated the osteogenic markers OCN and OPN (Figs. 2D, 2E–2H). In the differentiated groups, 0.1 nM MT showed the highest reduction compared to OS alone (P < 0.05), and the suppressive effect was abrogated at the lowest pharmacological concentration (1 µM). These results indicate that physiological concentrations of MT but not 1 µM reduced the differentiation-promoting effect of OS on hPDLSCs.

Changes in mitochondrial respiratory function of hPDLSCs following osteogenic induction and MT treatment

We detected the intracellular ROS activity of hPDLSCs by flow cytometry. We found that ROS generation in the osteogenic induction group was lower than that in basal growth medium group, whereas the 0.1 nM MT-treated group showed higher levels than any other MT-treated group with OS (P < 0.05, Figs. 3A, 3B). Furthermore, measurement of the intracellular ATP and NAD⁺/NADH levels showed that osteogenic differentiation increased the production of intracellular ATP, whereas 1 pM, 0.1 nM and 10 nM MT reduced this effect (P > 0.05, Fig. 3C). We also found that the NAD⁺/NADH ratio was reduced in the OS group (P <0.001, Fig. 3D). Notably, the increase in the ratio was not concentration-dependent at physiological concentrations of MT. Furthermore, the 1 pM and 0.1 nM MT-treated groups showed significant differences from the OS alone group (P <0.05).

Effects of MT on mitochondrial dynamics during osteogenesis

The protein levels of mitochondrial dynamics were assessed by western blotting. The expression of MFF and P-MFF was initially increased and then decreased with increasing MT concentrations (Figs. 4A, 4C, 4D). In addition, the expression levels of MFN-2 and TOM20 first decreased and then increased with increasing MT concentrations (Figs. 4A, 4B, 4E). The expression of MFN-2 and TOM20 was significantly decreased at 0.1 nM MT compared to in the OS alone group (P <0.01, P <0.001, respectively, Figs. 4B, 4E). We also found that at MT concentrations >0.1 µM, the expression of MFN-2 and TOM20 was gradually increased, which continued up until 1 µM. Furthermore, the expression levels of MFN-2 and TOM20 at 1 µM MT were comparable to or even higher than those in the OS alone group (Figs. 4B, 4E).