Fetal bovine serum (FBS) was purchased from Gibco-BRL (Gaithersburg, MD, USA). Anti-COLIA, OPN, OCN, OSX, RUNX2, LXRα, LXRβ, Smo, Gli1, β-actin, goat anti-mouse, and goat anti-rabbit antibodies were supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Unless otherwise specified, chemicals and laboratory wares were from the Sigma Chemical Company (St Louis, MO, USA) and Falcon Labware (Becton-Dickinson, Franklin Lakes, NJ, USA), respectively.
Periodontal ligament stem cell culture
Periodontal ligaments were obtained from extracted human molars donated by the Kyung Hee University Department of Oral and Maxillofacial Surgery. All subjects involved in this study were informed about its purpose and procedures, and the study was approved by the Kyung Hee University Review Board. Written informed consent was obtained from all donors or their guardians on behalf of minor participants.
Periodontal ligaments were collected from the middle thirds of roots and cultured in α-minimal essential medium (α-MEM; Gibco-BRL) containing 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml; Gibco-BRL), according to a method described previously [20, 21]. After two passages, the cells were subjected to magnetic isolation using magnetic beads (Miltenyi Biotec, Germany) and antibodies to detect the STRO-1 antigen (mesenchymal stem cell marker; Millipore, Billerica, MA, USA). The resulting STRO-1(+) cell population was cultured in α-MEM plus 10% FBS at 37 °C with a humidified gas mixture of 5% CO2/95% air. All experiments were carried out with passage 4–7 cells. To induce osteogenic differentiation, cells were maintained in an osteogenic medium comprised of α-MEM containing 5% FBS, 50 μg/ml ascorbic acid, 1 μM dexamethasone, and 3 mM β-glycerophosphate for 1 day before the application of oxysterols (22(S)-hydroxycholesterol and 20(S)-hydroxycholesterol (SS)). Different concentrations of oxysterols (0.5–5 μM) were supplemented into the osteogenic medium, which was changed every other day. Oxysterols were dissolved in dimethyl sulfoxide (DMSO) immediately before use, and the final concentration of DMSO did not exceed 0.1% (v/v) in any of the experiments. DMSO at 0.1% was used as a control.
Alkaline phosphatase activity
Alkaline phosphatase (ALP) activity was assayed as described previously . Briefly, the cells were washed twice with phosphate-buffered saline (PBS) and lysed in 50 mM Tris–HCl buffer (pH 7.0) containing 1% (v/v) Triton™ X-100 and 1 mM phenylmethylsulfonyl fluoride (PMSF). The total protein concentration was then quantified according to the Bradford method . The entire cell lysate was assayed by adding 200 μl p-nitrophenylphosphate (Sigma Chemical Company) as a substrate for 30 min at 37 °C. The reaction was stopped by the addition of 3 M NaOH and the absorbance was read on a spectrophotometer at 405 nm. The enzyme activity was expressed as millimoles per 100 μg of protein.
Intracellular calcium assay
The cells were washed three times with PBS and lysed in 50 mM Tris–HCl buffer (pH 7.0) containing 1% (v/v) Triton™ X-100 and 1 mM PMSF without EDTA. The protein content was then quantified according to the Bradford method . The intracellular calcium content was measured using a calcium assay kit according to the manufacturer’s instructions (BioAssay Systems, Hayward, CA, USA.), and the absorbance was read on a spectrophotometer at 602 nm. The calcium content was expressed as milligrams per 100 mg of protein.
Alizarin Red staining
Alizarin Red staining was performed as described in our previous publication . Briefly, the culture media were discarded and cells were fixed for 20 min in 4% paraformaldehyde, washed three times with ice-cold PBS, and stained for 20 min with Alizarin Red (pH 4.2; Sigma). Finally, the solution was aspirated, and the cells were washed with deionized water and then observed under a light microscope. To quantify mineralization, bound dye was extracted in 10 mM sodium phosphate containing 10% cetylpyridinium chloride and quantified spectrophotometrically at 562 nm.
RNA isolation and real-time reverse-transcriptase polymerase chain reaction
Real-time reverse-transcriptase polymerase chain reaction (RT-qPCR) was performed as described in our previous study . Total RNA was extracted from the cells using the TRIzol™ reagent (Invitrogen, USA) following the manufacturer’s protocol. Real-time quantification of RNA targets was then performed using a Rotor-Gene 2000 real-time thermal cycling system (Corbett Research, Australia) with a QuantiTect SYBR® Green reverse-transcriptase polymerase chain reaction (RT-PCR) kit (Qiagen, CA, USA). The reaction mix (20 μl) contained 200 ng of total RNA, 0.5 μM of each primer, and appropriate amounts of enzymes and fluorescent dyes, as recommended by the supplier. The Rotor-Gene 2000 cycler was programmed as follows: 30 min at 50 °C for reverse transcription, 15 min at 95 °C for DNA polymerase activation, 15 s at 95 °C for denaturing, followed by 45 cycles of 15 s at 94 °C, 30 s at 55 °C, and 30 s at 72 °C. Data were collected during the extension step (30 s at 72 °C). The PCR reaction was followed by melting curve analysis to verify the specificity and identity of the RT-qPCR products, which distinguished specific PCR products from nonspecific PCR products resulting from primer dimer formation. The temperature of the PCR products was increased from 65 °C to 99 °C at a rate of 1 °C/5 s, and the resulting data were analyzed using the manufacturer’s software. The primers used were 5′-TGA AAC GAG TCA GCT CTG GAT G-3′ (forward) and 5′-TGA AAT TCA TGG CTG TGG AA-3′ (reverse) for OPN; 5′-TGA GGA GGA AGT TCA CTA TGG-3′ (forward) and 5′-TTC TTT GTG CCT GCT TTG C-3′ (reverse) for OSX; 5′-ATG AGA GCC CTC ACA CTC CTC-3′ (forward) and 5′-GCC GTA GAA GCG CCG ATA GGC-3′ (reverse) for OCN; 5′-AAG CCC ATG CCT ACG T-3′ (forward) and 5′-TGC AGA CGC AGT GCA AAC A-3′ (reverse) for LXRα; 5′-TCG TGG ACT TCG CTA AGC AA-3′ (forward) and 5′-GCA GCA TGA TCT CGA TAG TGG A-3′ (reverse) for LXRβ; 5′-CCC TGT GGA ATG TAC CTA TGT G-3′ (forward) and 5′-GAG GTG TCC CAA AGA TGC AA-3′ (reverse) for ABCA1; and 5′-GCT CTC CAG AAC ATC ATC C-3′ (forward) and 5′-TGC TTC ACC ACC TTC TTG-3′ (reverse) for GAPDH.
Western blotting analysis
Western blotting analysis was conducted as reported previously . Protein extract samples (20 μg) were separated by 8–10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membranes. The blots were washed with TBST (10 mM Tris–HCl (pH 7.6), 150 mM NaCl, 0.05% Tween-20), blocked with 5% skim milk for 1 h, and incubated with the appropriate primary antibodies (anti-OCN, anti-OSX, anti-Runx2, anti-COLA1, anti-ALP, anti-LXR α, anti-LXR β, anti-SMO, anti-Gli1, or anti-β-actin; Santa Cruz Biotechnology) at the dilutions recommended by the supplier. The membranes were then washed and the primary antibodies were detected with goat anti-rabbit immunoglobulin G (IgG) or goat anti-mouse IgG conjugated to horseradish peroxidase. The blots were developed with enhanced chemiluminescence (Bio-Rad Laboratories, Hercules, CA, USA) and exposed to X-ray film (Eastman-Kodak, Rochester, NY, USA).
In the experiments involving animal tissues, the specimens containing sockets were acquired from the maxilla using a trephine bur (inner diameter 3.3 mm, outer diameter 4.0 mm) (XTP3404; Dentium, Seoul, Korea) after the rats were euthanized. The specimens were immediately stored at −80 °C and ground with a mortar and pestle. The ground specimens were lysed in ice-cold radioimmunoprecipitation assay buffer (RIPA buffer; Cell Signaling, Danvers, MA, USA) containing 1 mM PMSF and clarified by centrifugation at 12,000 rpm for 20 min. Protein concentrations were determined using a protein assay kit (Bio-Rad Laboratories). Western blotting analysis was then performed as already described.
The cells were washed with PBS, fixed for 5 min using 4% paraformaldehyde, and permeated for 20 min using 0.1% Triton™ X-100 at room temperature. The cells were then washed three times and blocked for 1 h using 4% BSA in PBS at room temperature. The cells were treated with primary antibodies (1:100; rabbit anti-OCN, rabbit anti-OSX, rabbit anti-Runx2) and incubated overnight at 4 °C. Subsequently, the cells were treated with Alexa Fluor® 488 or 594 goat anti-rabbit IgG (1:500; Invitrogen Life Technologies, USA) for 2 h at room temperature. Fluorescence images were obtained under a fluorescence microscope (Fluoview 300; Olympus, Japan).
Cells were transfected for 24 h with a Stealth small interfering RNA (siRNA) specific to LXRα, LXRβ (100 nM; Bioneer, Korea), or an unrelated control siRNA targeting the green fluorescent protein. The sequences of the siRNAs were as follows: 5′-GAG ACA UCU CGG AGG UAC A-3′ (forward) and, 5′-UGU ACC UCC GAG AUG UCU C-3′ (reverse) for siRNA-LXRα; and 5′-CGA GCU UUG CCG UGU CUG U-3′ (forward) and 5′-ACA GAC ACG GCA AAG CUC G-3′ (reverse) for siRNA-LXRβ. Briefly, the cells were seeded and grown in 60-mm culture dishes until they reached 70% confluence. The serum was then exchanged with antibiotic-free media and incubation continued for 24 h. The cells were transfected for 24 h with either an siRNA specific to LXR or a negative control siRNA (scrambled) targeting a site that is absent from the human, mouse, and rat genomes using the Lipofectamine® RNAiMAX transfection reagent (Invitrogen, USA) according to the manufacturer’s instructions, before being subjected to chemical treatments.
Alveolar bone defect model and study design
Eighteen 8-week-old male Sprague–Dawley rats weighing 250–300 g were used for this study. The rats were divided into three groups: the control, BMP-2, and SS groups. Each group contained six rats, three for western blotting analysis and the other three for micro-computed tomography (μCT) analysis. After extraction of the left and right maxillary first molars from each rat, the extraction sockets were left untreated for natural healing in the control group. Recombinant human bone morphogenetic protein-2 (rhBMP-2; Cowellmedi Co., Ltd, Seoul, Korea) was injected into both extraction sockets on the third day after the extraction in the BMP-2 group. Also, SS was injected twice, on the third and fifth days after the extraction, in the SS group (Fig.
). All rats were provided food and water ad libitum, and were housed under standardized environmental conditions. The rats were euthanized 15 days after extraction via asphyxiation in a CO
chamber. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Kyung Hee University Hospital at Gangdong (KHNMC AP 2016-002).
Critical steps in the animal experiment. Timeline for establishment of the alveolar bone defect model and the injection of each agent. β-APN β-aminopropionitrile, BMP‐2 bone morphogenetic protein‐2, SS 22(S)-hydroxycholesterol and 20(S)-hydroxycholesterol
For 5 days before the extraction procedure, the rats were fed a diet containing β-aminopropionitrile (β-APN; Sigma-Aldrich, St Louis, MO, USA) at a rate of 0.4% β-APN per gram of chow to facilitate subsequent extraction of the maxillary first molars. Under general anesthetic via an intramuscular injection of alfaxalone (Alfaxan®, 0.1 ml/100 g), the left and right maxillary first molars of all rats (n = 48) were extracted, and bleeding was controlled. An intramuscular injection of gentamicin (3 mg/kg; DaeSung Microbiological Labs, Uiwang, Korea) and a subcutaneous injection of 1% ketoprofen (0.3 ml/kg; Uni Biotech, Chungnam, Korea) were administered to prevent infection and to relieve pain, respectively, for 3 days after the extraction.
Injection of oxysterols and rhBMP-2
The oxysterol combination (SS) was prepared by mixing equal amounts of 100 μM 20S-hydroxycholesterol (Sigma-Aldrich) with 100 μM 22S-hydroxycholesterol (Sigma-Aldrich) dissolved in 1% DMSO in PBS. The other test agent, rhBMP-2, was diluted to a saturated concentration of 1.5 mg/ml with injectable saline. A 50-μl Hamilton syringe (no. 705; Hamilton Company, Reno, NV, USA) with a 32-gauge needle was used to administer an injection into the extraction sockets. Under general anesthesia, 10 μl of SS and 10 μl of rhBMP-2 were injected slowly into the extraction sockets of the rats in each group.
All specimens were fixed in 10% formalin for 7 days and then imaged using a high-resolution μCT system (Skyscan 1173; Skyscan, Kontich, Belgium) at 90 kV and 88 μA, with an image pixel size of 9.94 μm. The regions of interest (ROI) were defined as five-root sockets of each tooth from the alveolar crest to the apex. The scanned data for the ROI were reconstructed and analyzed using three-dimensional analysis software (NRecon software; Skyscan). Newly formed bone volume (%) was calculated by multiplying the quotient of the bone volume divided by the total volume of the ROI by 100.
All data are expressed as the mean ± standard deviation (SD). One-way ANOVA was used for multiple comparisons (Duncan’s multiple range test), using SPSS software version 10.0. P < 0.05 was considered statistically significant. The animal study data were analyzed using the nonparametric Kruskal–Wallis test, and by the Tukey HSD test for multiple comparisons (SAS for Windows version 9.2). P < 0.05 was considered statistically significant.