Bone remodeling is crucial to the success of many dental procedures and is tightly regulated. Vascular endothelial growth factor (VEGF), a key cytokine for angiogenesis, is also an important regulator of bone remodeling. We aimed to examine the mechanisms by which VEGF induces bone remodeling by studying its effects on cultured osteoblasts and osteoclasts.
Preosteoblastic MC3T3-E1 cells were treated with vehicle or VEGF-A165. Cell proliferation, migration, and invasion potentials were assessed. Preosteoclastic RAW264.7 cells were treated with vehicle or VEGF with or without the receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoclast formation was measured with tartrate-resistant acid phosphatase staining. Conditioned media from vehicle-treated or VEGF-treated MC3T3-E1 cells were tested for the levels of RANKL and osteoprotegerin (OPG) and were used to treat RAW264.7 cells to observe osteoclast formation.
VEGF significantly induced MC3T3-E1 cell proliferation, migration, and invasion. VEGF did not directly induce osteoclastogenesis but significantly increased the RANKL/OPG ratio in the conditioned media from the MC3T3-E1 cultures; this significantly increased osteoclast formation.
VEGF stimulates osteoclast differentiation by increasing the osteoblastic RANKL/OPG ratio but has no direct effect on osteoclast precursor cells, and it induces osteoblast proliferation, migration, and invasion potentials in vitro.
We examined the effects of vascular endothelial growth factor (VEGF) in cell cultures.
VEGF stimulated osteoblast proliferation, migration, and invasion potentials.
VEGF did not induce osteoclast differentiation directly.
VEGF enhanced osteoclast differentiation indirectly.
Bone remodeling is the lifelong continuous replacement of old bone tissue with new bone and is crucial to the success of a variety of dental procedures, including dental implants, periodontal disease treatment, and orthodontic tooth movement. It is a tightly coupled local process starting with bone resorption, followed by reversal and bone formation phases. Osteoclasts and osteoblasts are organized as basic multicellular units and cooperate to perform the resorption-reversal-formation sequence of the bone remodeling process. The level of bone remodeling is closely regulated by hormones, cytokines, prostaglandins, and mechanical loading. Receptor activator of nuclear factor-kappa B ligand (RANKL), an osteoblastic cell-derived factor, is crucial for osteoclast differentiation, survival, and function by binding to osteoclast cell-surface receptor, its receptor on preosteoclasts. Osteoprotegerin (OPG), another osteoblastic cell-derived factor, interrupts the RANKL/osteoclast cell-surface receptor binding as a decoy receptor of RANKL, inhibiting osteoclastogenesis. Therefore, the RANKL/OPG ratio largely determines the formation of functional osteoclasts and the activation of the resorption phase of bone remodeling. The expression of RANKL and OPG by osteoblastic cells is regulated by various systemic hormones and local factors.
Orthodontic treatment duration is a major concern for patients and is closely associated with various side effects, including external root resorption, carious lesions, and open gingival embrasures. According to the American Association of Orthodontists, about 4 million people in this country receive orthodontic treatment each year. Accelerating tooth movement can therefore benefit millions of patients by reducing their treatment durations and side effects. The rate of orthodontic tooth movement is largely determined by bone remodeling. A full-thickness mucoperiosteal flap alone was sufficient to induce de novo regional acceleratory phenomenon, a significantly increased bone remodeling activity as part of the trauma healing process. Vascular endothelial growth factor (VEGF) was shown to play an essential role in the postflap healing procedure that led to activated bone remodeling. Local injections of VEGF in mice significantly increased the numbers of osteoclasts and the rate of orthodontic tooth movement, whereas neutralizing the anti-VEGF antibody had the opposite effect. A previous study showed that VEGF promotes bone remodeling primarily by reducing OPG, but it had no effect on RANKL in osteoblastic cell cultures up to 72 hours. The long-term effects of VEGF on osteoclast and osteoblast formation and activities, and on bone remodeling, are yet to be elucidated.
The aim of this study was to examine the mechanisms by which VEGF induces bone remodeling by studying its effects on osteoblasts and osteoclasts in vitro.
Material and methods
MC3T3-E1 subclone 4 cells, subcloned by Dr Renny Franceschi at the University of Michigan from the original immortalized neonatal murine calvarial cells, were purchased (CRL-2593; American Type Culture Collection, Manassas, Va). The cells were grown in alpha minimum essential medium (A1049001; Gibco BRL, Gaithersburg, Md); 10% heat-inactivated fetal calf serum (Gibco BRL), penicillin (100 U/mL), and streptomycin (100 μg/mL) were added to the media. Cells (5000/cm 2 ) were plated in 6-well dishes in a humidified atmosphere of 5% carbon dioxide at 37°C and grown until confluent.
RAW264.7 cells, a tumor cell line induced by Abelson murine leukemia virus, were purchased (TIB-71; American Type Culture Collection). Cells were grown in Dulbecco’s Modified Eagle’s Medium (30-2002; American Type Culture Collection). We added 10% heat-inactivated fetal calf serum, penicillin (100 U/mL), and streptomycin (100 μg/mL) to the media. The cells were plated in 96-well plates in a humidified atmosphere of 5% carbon dioxide at 37°C. Formation of tartrate-resistant acid phosphatase positive multinucleated cells (TRAP+ MNC) was assessed with a commercial kit (F4523; Sigma-Aldrich, St Louis, Mo).
For the western blot analysis, protein from MC3T3-E1 cell cultures was extracted with radioimmunoprecipitation assay buffer following the manufacturer’s instructions (Santa Cruz Biotechnology, Santa Cruz, Calif). Protein concentrations were measured by bicinchoninic acid assay (Thermo Fisher Scientific, Waltham, Mass). Conditioned media from MC3T3-E1 cells were filtered with a 0.44-μm filter under sterile conditions and stored at –80°C. The same experimental procedures were followed as published previously. Briefly, equal amounts (100 μg) of protein (for VEGF receptor-1 [VEGRF-1] and VEGF receptor-2 [VEGFR-2]) or equal volumes (25 μL) of media (for RANKL and OPG) were used for 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, Calif). The total protein on the nitrocellulose membrane was stained with a protein stain kit (24580; Thermo Scientific, Rockford, Ill), which has been used in previous studies. Membranes were washed with tris-buffered saline solution (pH 7.6) before blocking with 5% weight per volume of nonfat dry milk in 1 × tris-buffered saline solution containing 0.05% Tween 20 (Sigma-Aldrich) for 60 minutes. Then the membranes were incubated with primary antibody of VEGFR-1 and VEGFR-2 for cell extracts and RANKL or OPG for media samples, washed with tris-buffered saline solution containing 0.05% Tween 20, and incubated with horseradish peroxidase-conjugated secondary antibody (1:1000). The signal was detected with LumiGLO chemiluminescent reagent (Cell Signaling Technology, Danvers, Mass). Primary antibodies, including VEGFR-1 (AF471; R&D Systems, Minneapolis, Minn), VEGFR-2 (AF644; R&D Systems), RANKL (AF462; R&D Systems), or OPG (AF459; R&D Systems), were incubated at 0.1 μg per milliliter overnight at 4°C.
Cell proliferation was tested with the 5-bromo-2′-deoxyuridine (BrdU) assay. MC3T3-E1 cells (1 × 10 3 cells per well) were plated in 96-well plates in the media described above. Vehicle or VEGF (10, 20, or 40 ng/mL) was added at the beginning of cell plating and treated for 24 hours. The BrdU assay followed the same procedures as published previously. Briefly, the cells were fixed, and the DNA was denatured after removing the labeling medium. Then anti-BrdU-peroxidase antibody was added. Substrate reactions to detect the immune complexes were quantified by measuring the absorbance with a spectrophotometer.
For cell migration and invasion assessment, we used 8.0-μm-pore BD BioCoat Control Insert 24-well plates (354578; BD Bioscience, San Jose, Calif) and Matrigel Invasion Chamber 24-well plates (354480; BD Biosciences) to measure migration and invasion of MC3T3-E1 cells, respectively. We plated 2 × 10 5 cells in 0.4% fetal bovine serum medium in the top chamber after it was collected and washed. Vehicle or VEGF (20 ng/mL) was added to the bottom chamber. Cell migration and invasion assessment followed the same experimental procedures as published before. Briefly, cells on the top surface were removed after 22 hours. Cells on the lower surface were stained with 0.5% crystal violet after being fixed. Then the stained cells were photographed under bright field microscopy. Ten random high-power fields per insert were used to count migrated or invaded cells, and averages were calculated. Fold changes of the numbers of migrated or invaded cells to the untreated controls in triplicate experiments were determined.
For VEGF stimulated osteoblastic MC3T3-E1 cell proliferation, migration, and invasion, the expression of VEGF receptors by MC3T3-E1 cells was assessed by western immunoblotting. These cells express VEGFR-1 and VEGFR-2 (data not shown). Proliferation analysis by the BrdU assay showed that VEGF at 10 ng per milliliter did not affect MC3T3-E1 cell proliferation ( Fig 1 , A ; P >0.05), whereas VEGF at 20 or 40 ng per milliliter significantly stimulated MC3T3-E1 cell proliferation ( Fig 1 , A ; 3.60- and 3.98-fold, respectively; P <0.01). In addition, VEGF (20 ng/mL) significantly increased MC3T3-E1 cell migration ( Fig 1 , B ; 2.29-fold; P <0.01) and invasion ( Fig 1 , C ; 15.74-fold; P <0.001) compared with the vehicle-treated cells.
VEGF did not directly affect osteoclast formation in RAW264.7 cell cultures. To test the direct effects of VEGF on osteoclast formation, we first treated osteoclastic precursor RAW264.7 cells directly with VEGF (20 ng/mL). RAW264.7 cells can differentiate into mature osteoclasts by RANKL without macrophage colony-stimulating factor. As expected, RANKL induced TRAP+ MNC formation used here as the control. VEGF treatment alone did not stimulate any osteoclast formation, nor did VEGF increase osteoclast formation induced by RANKL ( Fig 2 ). TRAP+ MNC counts are shown in the Table .
|Day 3||Day 4||Day 5|
|RANKL||61.3 ± 7.8 ∗||71.7 ± 7.2 ∗||23.6 ± 4.0 ∗|
|RANKL + VEGF||54.7 ± 4.2 ∗||79.3 ± 4.7 ∗||20.4 ± 6.1 ∗|
Conditioned media from VEGF-treated MC3T3-E1 cells stimulated osteoclast formation in RAW264.7 cell cultures. Osteoclast formation in RAW264.7 cells was also assessed with conditioned media from osteoblastic MC3T3-E1 cells previously treated with VEGF (20 ng/mL) or vehicle for 4, 7, 14, and 21 days ( Fig 3 , A ). Conditioned media from MC3T3-E1 cells treated with VEGF at all time points significantly increased TRAP+ MNC formation, whereas those from the control group had a minimum effect ( Fig 3 , B ; P <0.001) at all time points. Among the RAW264.7 cultures treated with conditioned media from VEGF-treated MC3T3-E1 cells, osteoclast formation was significantly higher at the earlier times of days 4 and 7 compared with the later times of days 14 and 21 ( Fig 3 , B ; P <0.01).