Abstract
Background/Purpose: Bone remodeling is regulated by the replacement of old bone with new bone through sequential osteoclastic resorption and osteoblastic bone formation. In this study, we compare the functional difference in osteoclastogenesis of murine marcophage when applied to the receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) produced by osteoblasts in response to tensile stress in a co-culture system.
Materials and methods: We examine the secretion of RANKL, and OPG at different time-points when the osteoblast cells are being cultured under tensile force. In order to confirm whether the media produced by the osteoblast cells’ treatment with tensile force induces osteoclastogenesis or not, and murine marcophage (RAW264.7 cells) were then co-cultured with osteoblasts.
Results: In both the mono-culture and co-culture systems, the two cell types cultured under tensile force and normal environment showed no significant differences (p > 0.05) at any time. However, in the co-culture system, a significant (p < 0.05) increase of 2.27-, 3.00-, or 3.27-fold production of tartrate-resistant acid phosphatase (TRAP) in RAW264.7 cells under tensile force was found when compared with the corresponding TRAP activity in RAW264.7 cells under normal environment at day 3, 7, or 15, respectively. a significant (p < 0.05) increase of 2.27-, 3.00-, and 3.27-time was found for tartrate-resistant acid phosphatase (TRAP) synthesis in RAW264.7 cultured tensile force when compared with the control group at 3, 7, and 15 days, respectively. Moreover, the results indicate that the tensile force up-regulated the secretion of RANKL and inhibited OPG synthesis. Therefore, RAW264.7 cells appear to increase their production of TRAP
in the media of osteoblasts under tensile force, promoting TRAP activity nearly 2.8 times higher than in media of osteoblasts under normal environment for 3 days.
Conclusion: These results suggest that osteoblasts influence the secretion of RANKL more than OPG when stimulated with osteoclastgenesis via RANKL under tensile force.
Introduction
Orthodontic tooth movement combines both physiological and pathological responses to externally applied forces. Efficient tooth movement may be accomplished by mechanical, biomechanical, or biostimulatory methods. Bone remodeling is regulated by the replacement of old bone with new bone through sequential osteoclastic resorption and osteoblastic bone formation.1 The balance between bone resorption and formation is eliminated when a force is loaded onto a tooth, resulting in more bone formation than resorption on the tension side and more bone resorption than formation on the pressure side in the alveolar bone. This process causes the tooth to move in a specified direction.2-4 Recently, several studies have investigated whether osteoblast cells constitutively express osteogenesis cytokines and growth factors, many of which are mechanoresponsive, such as collagen I, osteopontin, alkaline phosphatase, osteocalcin, and anti-inflammatory cytokines.5 These cytokines are beneficial in regard to mediating the cellular and molecular responses in orthodontic tooth movement and contribute to the remodeling of alveolar bone tissue.6,7 Mature osteoblast-related cells produce osteoprotegerin (OPG) and the receptor activator of nuclear factor kappa B ligand (RANKL), which has opposing effects on osteoclasts.8,29
Mechanical forces contain several different forces, such as tensile, compressive and shear stresses. The mechanical microenvironment around the tissue affects the phenotype and function of a cell. Several studies have verified mechanical force can effectively change gene expression, protein secretion and cell behavior.9-11 Accumulating evidence indicates that different mechanical forces stimulate cellular messages and results through similar mechanoreceptors and intracellular biochemical cascade signaling
effectors in many types of cells.10,11 Pro-inflammatory cytokines secreted by osteoblasts are immediately up-regulated under tensile force and remain up-regulated in the presence of force.12 In addition, osteoclast formation and differentiation are regulated by the balance among the receptor activators RANKL, OPG, and macrophage colony-stimulating factor (M-CSF).13 At least two studies have reported that osteoblasts express RANKL in response to mechanical stress,12,14 whereas another study reports that they do not.15 The anchylosed teeth can be moved by mechanical stress, suggesting that osteoblasts play a major role in alveolar bone resorption under tensile force.
This study investigates and compares macrophages and osteoblasts in vitro co-cultures under tensile force to elucidate and distinguish cytokine signaling between these cell types and determine more representative in vitro cellular responses to in vivo models, or provide evidence for confounding in vitro cell behavior.
Materials and Methods Cell culture
RAW 264.7 macrophage cells and MC3T3-E1 osteoblast cells (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco's Modified Eagle Medium (DMEM, Caisson Laboratories, North Logan, UT) containing 10% fetal bovine serum (GeneDireX, Las Vegas, NV), 100 U/mL penicillin/100 μg/mL streptomycin (Caisson) at 37°C in a humidified atmosphere of 5% CO2 in air. The culture medium was changed every 3 days. Cells (5 x 104 cell/mL) were seeded on covered glasses for 24 h, and
transferred to 6-well plates (GeneDireX) with CO2 independent medium (Gibco, Langley, OK) supplemented with 10% FBS and 1% PS. Before cell experiments, the cover glasses were sterilized by immersion in 75% ethanol followed by exposure to ultraviolet (UV) light for 6 h. The 6-well plates contained cover glasses (mono-culture: MC3T3-E1 or RAW264.7 on cover glass; co-culture: two cover glasses, one MC3T3-E1, and the other RAW264.7) were in a tension incubator (TI, Model. 3618P, Lab-Line Instrument, Thermolyne Co., IL) or normal incubator. The inside of the tensile incubator was kept at -100 kPa (1 Pa = 1/100,000 kg/cm2, equal to a negative force of 101g/mm2) at 37oC. A full description of the culture process implemented for each signaling condition is shown in Fig. 1.
Proliferation Assays
After the various predetermined culture times, cell proliferation was evaluated using the PrestoBlue® assay (Invitrogen, Grand Island, NY). Briefly, at the end of the appointed time, the cover glasses were transferred to new 24-well plate (GeneDireX) and
washed with PBS three times. Each well was then filled with 400 µL solution (PrestoBlue®: DMEM = 1: 9) and incubated at 37°C for 30 min after which the solution in each well was transferred to a new 96-well plate (GeneDireX). Plates were read in a multiwell spectrophotometer (Hitachi, Tokyo, Japan) at 570 nm with a reference wavelength of 600 nm. The results were obtained in triplicate from three separate experiments for each test.
ELISA analysis
RANKL and OPG protein are secretions from MC3T3-E1 cells cultured under a tensile or normal incubator for various numbers of days and collected the cultured medium. A RANKL ELISA assay kit (Abcam, Cambridge, MA) and OPG ELISA assay kit (Sigma-Aldrich, St. Louis, MO) were used to determine the protein concentration following the manufacturer’s instructions. The protein concentration was measured by correlation with a standard curve. Analyses of blank disks served as controls. All experiments were done in triplicate.
Tartrate-resistant acid phosphatase (TRAP) Activity
After being cultured for different numbers of days, cells on the cover glasses were fixed with 4% formalin for 15 min and 95% EtOH for 3 min in order to measure TRAP activity. Later, the cover glasses were immersed in 10 mM citrate buffer (pH 4.6) containing 10 mM sodium tratrate and p-nitrophenylphosphate. After incubation for 1 h, the reaction mixtures were transferred to a new 24-well. The reaction was stopped by the
addition of 5 N NaOH and quantified by absorbance at 405 nm. All experiments were done in triplicate from three separate experiments for each test.
In addition, to accurately addressing the roles of RANKL in osteoclastgenesis, the RAW264.7 cells were incubated with 10 µg/mL RANK antibody (GeneTex, San Antonio, TX) for 30 min on ice. Following this procedure, the RAW 264.7 were suspended in various test media (a. 10 ng/mL RANKL; b. 10 ng/mL OPG; c. 10 ng/mL RANKL mixed 10 ng/mL OPG; d. medium from MC3T3-E1 on normal incubator; e. medium from MC3T3-E1 on tensile incubator) for 15 days, after which TRAP activity was measured. Cells cultured with DMEM were used as a control (Ctl). All experiments were done in triplicate in three separate experiments for each test.
Statistical analysis
One-way analysis of variance (ANOVA) statistical analysis was used to evaluate the significance of the differences between the mean values in comparison with those of the controls. Scheffe’s multiple comparison testing was used to determine the significance of the deviations in the data for each specimen. In all cases, the results were considered statistically significant at a p value < 0.05.
Results
Cell proliferation
The proliferation of the cells cultivated as a mono-culture as well as a co-culture under tensile force was calculated by PrestoBlue® assay and the results are shown in Fig. 2. In the mono-culture, the two cells cultured under tensile force and normal environment for all time-points show no significant effects (p > 0.05). Interestingly, the normal environment and the tensile force do not affect the conditions of cell growth in the case of the co-culture model.
TRAP Activity
The differentiation of RAW264.7 into osteoclasts has been estimated through the detection of TRAP activity in the mono-culture as well as in the co-culture with MC3T3-E1 under different environment (Fig. 3). In the mono-culture (Fig. 3, left, down), the TRAP activity of the RAW264.7 cells increased from day 7 to day 15 under tensile force and the cells increased significantly more than in the control environment (p < 0.05), which stimulated the differentiation to form osteoclasts. In the co-culture system (Fig. 3, right, down), there is a time-dependent course as well as for the comparison of tensile force and normal environment. A significant (p < 0.05) increase of 2.27-, 3.00-, and 3.27-time was found for TRAP synthesis in RAW264.7 cells’ cultured tensile force in comparision with the control group at 3, 7, and 15 days, respectively. However, there was no TRAP secretion from MC3T3-E1 cells present in either the mono- or co-culture under different environments for any of the measured culture times (Fig. 3, up).
RANKL and OPG secretion
The secretion of RANKL and OPG was also detected, and the results are shown in Figs. 4A and B. RANKL protein secretion in MC3T3-E1 cells cultured under tensile force was nearly 1.5 times higher than in the control cells cultured under normal environment for 3 days (Fig. 4A). In addition, the tensile force elicited a significant (p < 0.05) decrease of 20%, 28%, and 41% in OPG synthesis compared with those cultured in the normal environment on days 3, 7 and 15, respectively (Fig. 4B). These results indicate that the tensile force up-regulated the secretion of RANKL and inhibited OPG synthesis.
Effects of RANKL and OPG on TRAP secretion
OPG, as a secreted stromal cell-derived ‘‘decoy’’ receptor, specifically binds RANKL and thereby effects osteoclast differentiation. In order to determine the differentiation state of the RAW 264.7 cells with various conditions of DMEM, the established osteoclast marker protein TRAP was analyzed (Fig. 5A). The data shows that each time it was measured it was found that the TRAP activity had increased strongly and significantly (p < 0.05) after incubation of the cultures with RANKL. The quantities were from 14 to 20-times more than Ctl during the 15-day incubation period; the OPG did not result in a further promotion (Fig. 5A). However, the OPG caused an approximately 40% reduction of the TRAP activity when RANKL stimulation. Interestingly, the RAW264.7 cells increased their production of TRAP in the presence of MC3T3 E1-conditioned media compared to Ctl and OPG, and the media of MC3T3-E1 under tensile force led to
TRAP activity nearly 2.8 times higher than in the media of MC3T3-E1 after it had been kept in the normal environment for 3 days.
Blocking RANK significantly (p < 0.05) attenuated RAW264.7 cells synthesis TRAP activity. For example, in the case of RAW264.7 cells with neutralizing RANK, the RANKL had a reduction of 68% and 71% in the TRAP activity after 7 and 15 days, respectively, compared to 31% and 19% for the OPG group. In addition, the reduction of TRAP activity was stimulated by the media of MC3T3-E1 under tensile force that was similar to the RANKL+OPG group.
Discussion
Orthodontists are interested in alveolar bone changes which occur under orthodontic force. The relative extent to which these two competing processes occur controls the proportion of the various cell types and osteoblasts, reflecting the specific biomechanics of osteogenesis.6,7,16,17 Mechanical stimulation is a necessary physiological factor modulating bone structure and function. The mechanical response of bone is mainly dependent on the balance between osteoclastic bone resorption and osteoblastic bone formation, and the biological behavior of osteoblasts and osteoclasts can be influenced by many types of mechanical stimuli, such as stretch,18 compressive stress,19 tensile stress,20 and fluid shear stress.21 In a previous study, our group worked on the biomechanical analysis of osteogensis and mainly focused on the effects of tensile force on osteoblast cells and periodontal ligament cells (PDL), and our findings have shown that tensile force can profoundly affect the differentiation and function of PDL and regulate the process of remodeling. In this study, we use cell co-culture systems incorporating osteoblasts and macrophages, two primary effector cell types in the foreign body response, to determine if they might better reflect actual inflammatory information than their respective mono-cultures. To achieve this, osteoblasts and macrophage cells were not only co-cultured in the same plates, but also with different communication media. Both cell types were co-cultured together in contact to mimic juxtacrine signaling. As part of this comparison process, macrophages were treated with osteoblast-conditioned medium and vice versa in order to determine if cytokine presence alone, without direct feedback from the other cell type at once, produced clearly different responses from direct contact in co-cultures and mono- cultures.
The dynamic remodeling process of bone formation is finely regulated at the microenvironmental level where a molecule is able to affect the activation of the bone-resorbing osteoclasts and the extracellular matrix-producing osteoblasts.1 Osteoblasts secrete RANKL and OPG with opposing effects on osteoclasts. RANKL can interact alternatively with two receptors, both cytokines of the tumor necrosis factor (TNF) receptor superfamily.22 RANK is a transmembrane protein expressed on osteoclast precursors, while the second one, OPG, is soluble and can act as a decoy receptor for RANKL.23 RANKL interacts with RANK on macrophage to promote osteoclast differentiation. However, the soluble decoy receptor OPG will bind with RANKL, but doing so precludes RANK-RANKL interaction. Therefore, the RANKL/ OPG ratio determines the degree of the coupling of bone formation to bone resorption.24 We have shown for the first time that osteoblasts regulate the secretion of RANKL and OPG proteins under tensile force. Analysis of our RANKL ELISA data shows similar up-regulation by tensile stimulation (Fig. 4A). Many cytokines and chemical mediators regulate the expression of RANKL, which is positively regulated by 1,25-(OH)2D3, IL-1β, TNF-α, and PGE2.25,26 We previously reported that osteoblast cells promote several immune marker secretions after treatment with tensile force, such as iNOS, TNF-α, and IL-1β. Thus, we have suggested that IL-1β and TNF-α cytokines are essential molecules in RANKL signaling. In addition, OPG production is negatively affected by glucocorticoids, PGE2, and PTH, while it is positively regulated by TGF-β.27 OPG cytokine secretion decreases significantly when there is tensile stress on days 3–15. We suggest that TGF-β expression is reduced in osteoblast cells by a tensile-force load, and the tensile-force-stimulated down-regulation of OPG in osteoblast cells.28 Chung et al.28
asserts that OPG is reduced in a magnitude-dependant manner following 3%, 5%, and 10% elongation by cyclic tensile stress. Therefore, our data suggests that RANKL and OPG regulation are influenced significantly by tensile stress.
Moreover, osteoblastic cells in vitro not only produce less OPG but also more osteoclast-activating RANKL secretion, which promote the RANKL-RANK interaction on osteoclasts under tensile force. In addition, anti-RANK antibodies inhibit more TRAP expression when exposed to the media treatment with osteoblast under tensile force. Concordantly, the osteoclasts differentiated from RAW264.7 cells are markedly restrained by conditioned medium from MC3T3-E1, which had been pre-incubated under tensile force. Therefore we conclude that tensile force not only stimulates the differentiation of the macrophage but also affects the interaction between osteoclasts and osteoblasts, leading to an inhibited number of bone-resorbing osteoclasts.
Acknowledgements
The authors acknowledge receipt of a grant from the Chung Shan Medical University Hospital under the project CSH-2013-C-017 and the National Science Council grants (NSC 102-2314-B-040-007-MY3) of Taiwan. The authors declare that they have no conflicts of interest.
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Figure Legends
Figure 1. Depiction of the cell signaling feedback culture scenarios and signaling condition utilized in this in vitro study.
Figure 2. The PrestoBlue® assay performed for viability of MC3T3-E1 and Raw264.7 cells mono- and co-cultured under tensile force or normal incubator. In the two culture systems, the two cells cultured under tensile force and normal environment for all time-points show no significant effects.
Figure 3. TRAP activity of MC3T3-E1 and Raw264.7 cells mono- and co-cultured under tensile force or normal incubator. There was no TRAP secretion from MC3T3-E1 cells present in either the mono- or co-culture under different environments for any of the measured culture times. When Raw264.7 cell cultured in the co-culture system, there is a time-dependent course as well as for the comparison of tensile force and normal environment. *p < 0.05, compared with normal incubator.
Figure 4. (A) RANKL and (B) OPG protein secreted from MC3T3-E1 cells cultured under tensile force incubator and analyzed by ELISA. RANKL protein secretion in MC3T3-E1 cells cultured under tensile force was higher than in the control cells cultured under normal environment. In addition, the tensile force elicited decrease of OPG synthesis compared with those cultured in the normal environment. *p < 0.05, compared with normal incubator.
Figure 5. (A) TRAP activity of Raw264.7 cells treated with different cytokine (RANKL, OPG, and RANKL+OPG) and the medium pre-cultured with MC3T3-E1 under tensile force or normal incubator. (B) Effect of anti-RANK antibody on the TRAP secretion of culture different condition medium. *p < 0.05, compared with normal incubator.
