Reagents, in vitro cell cultures, and osteogenesis of human periosteum-derived mesenchymal stem cells (PO-MSCs)
All chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell culture media and fetal bovine serum were purchased from Invitrogen (Waltham, MA, USA). Human periosteal tissues were obtained from patients who granted informed consent for collection of the tissues, as required by the Ethics Committee of Gyeongsang National University Hospital (GNUH 2014-05-012). PO-MSCs were then isolated as described previously (19). Briefly, periosteal explants were harvested from mandibles during surgical extraction of impacted lower third molars. Periosteal pieces were washed and cultured at 37°C, 95% humidified air, and 5% CO2 in 100-mm culture dishes containing Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin. Resulting adherent cells were passaged by gentle trypsinization and reseeding in a fresh medium. The cell culture medium was changed every 3 days during the isolation period. For osteogenic differentiation, PO-MSCs were cultured using osteogenesis induction medium (OM) which is composed of DMEM, supplemented with 50 μg/mL L-ascorbic acid 2-phosphate, 10 nM dexamethasone, and 10 mM β-glycerophosphate (19).
Assessment of PO-MSC proliferation and viability under resveratrol treatments
Proliferation of PO-MSCs was measured by a cell counting. Briefly, 2 × 104 cells were seeded in 24‐well plates and were cultured in OM medium. Resveratrol treatment was performed by treating cells with vehicle (ethanol) or a range of resveratrol concentrations from 500 nM upto 20 µM. 5 day and 10 day cultures were trypsinized and resulting detached cells were counted with a hemocytomer. Viability of PO-MSCs treated with resveratrol was determined by MTT assays. Shortly, 2 × 104 cells were seeded in 24‐well plates and cultured in OM medium with resveratrol treatments (500 nM ~ 20 µM). 5 day and 10 day cultures were subjected to a colorimetric MTT assay.
Measurements of alkaline phosphatase (ALP) activities
ALP activities were determined colorimetrically using an ALP Assay Kit (Takara, Kusatsu, Japan) according to the manufacturer's instructions. In brief, whole cell lysates were prepared using a NP-40 lysis buffer (Thermo Scientific, Waltham, USA). Cell lysates were then incubated with p-nitrophenylphosphate, a colorless substrate for ALP, in a Tris-HCl buffer (pH 9.5) at 37 ℃ for 15 min. Upon splitting off the phosphate group of p-nitrophenylphosphate by ALP, p-nitrophenol is released and detected spectrophotometrically (absorption maximum, 405 nm). These ALP activities then were normalized to cellular protein contents determined by the Bradford method.
Measurements of calcium deposits
Mineralization of PO-MSC cultures with resveratrol treatments (500 nM and 5 µM) in OM medium was assessed by von Kossa staining [30]. Calcium contents during osteogenesis of PO-MSCs were also measured by a calcium deposition assay. In brief, at days 14 and 21 of cell cultures, PO-MSCs grown in OM medium with resveratrol treatments (500 nM and 5 µM) were decalcified with 0.6‐N HCl for 1 day at room temperature. Then, the calcium content was determined by the colorimetrical o-cresolphthalein complexone method (Calcium C-test, Wako Chemicals, Japan), whereby calcium reacts with o‐cresolphthalein to form a purple complex that absorbs light with wavelength of 570 nm. After decalcification, the total protein content in the supernatants was measured by the Bradford method and was used to normalize calcium content.
Measurements of mitochondrial mass by flow cytometry and fluorescent microscopy
Mitochondrial mass of cultured PO-MSCs were quantified by flow cytometry. Briefly, 1 × 105 cells were stained in PBS with MitoTracker Green FM dye (Invitrogen, Waltham, MA, USA) for 30 min, washed, and resuspended in 200 μl PBS with 1% fetal bovine serum. Cellular fluorescence signals were then analyzed using a FACSCalibur (BD Biosciences, San Jose, CA, USA). Resulting flow cytometry data were analyzed using FlowJo software version 8.7.3 (Ashland, OR, USA). For fluorescent microcopy, PO-MSCs cultured on chamber slides were stained with MitoTracker Green FM dye. Cellular mitochondria were visualized under a fluorescent microscope (Zeiss Axio Observer Z1, Carl Zeiss, Oberkochen, Germany) and images were analyzed using ImageJ software (NIH, Bethesda, CA, USA).
mtDNA copy number analysis by quantitative PCR
For mtDNA copy number analysis, total cellular DNA was extracted from cultured PO-MSCs. A quantitative real-time PCR method was used to determine the relative abundance of mtDNA versus nuclear 18S rDNA using corresponding mitochondrial and nuclear PCR primer sets in two parallel PCR reactions as described previously (19). Relative mtDNA copy number was calculated as the ratio of the amount of amplification obtained with mtDNA versus nuclear 18S rDNA PCR primer sets for each sample and plotted normalized to the control group. The sequence of the PCR primer pairs are as follows: the nuclear 18S rRNA fragment was amplified by the primer pair 5'-TAGAGGGACAAGTGGCGTTC-3' and 5'-CGCTGAGCCAGTCAGTGT-3'; and the mitochondrial COX1 fragment was amplified by the primer pair 5'-CACCCAAGAACAGGG TTTGT-3-3' and 5 '-TGGCCATGGGTATGTTGTTAA-3'.
Statistical analysis
All experiments were performed using at least three independent cell cultures. Error bars in all figures represent the mean ± SEM and statistical analyses were computed using Graphpad Prism 7 software (GraphPad, San Diego, CA, USA). The Student’s two-tailed t-test was used for the determination of statistical relevance between groups, and a p value of <0.05 was considered statistically significant.