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Compressive Force Induces Osteoblast Apoptosis via Caspase-8

¹ Division of Orthodontics and Dentofacial Orthopedics and
² Division of Oral Dysfunction Science, Department of Oral Health and Development Sciences, School of Dentistry, Tohoku University, 4-1 Seiryo-cho, Aoba-ku, Sendai 980-8575, Japan; and
³ Shimizu Orthodontic Clinic, 6-1, 2F ESTA-Build., Sakae-cho, Fukushima 960-8031, Japan

^* corresponding author, goga{at}mail.tains.tohoku.ac.jp

	ABSTRACT

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Periodontal remodeling during orthodontic tooth movement is a result of mechanical stresses. The application of excessive orthodontic force induces cell death. However, the nature of compressive force-induced cell death is unclear. We examined whether the in vitro application of continuous compressive force would induce apoptosis in human osteoblast-like cells (MG-63 cells), and investigated the mechanism by which apoptosis was initiated. The cells became aligned irregularly, and cell viability decreased, indicating that the compressive force caused cell death. According to the TUNEL analysis, the number of apoptotic cells increased significantly in a time-and force-dependent manner. Caspase-3 activity increased with the magnitude of the compressive force, and this effect was reduced significantly by a caspase-8 inhibitor, whereas a caspase-9 inhibitor had no such effect. We conclude that the in vitro application of compressive force can induce apoptosis in MG-63 cells through the activation of caspase-3 via the caspase-8 signaling cascade.

KEY WORDS: apoptosis • mechanical force • osteoblast • caspase • tooth movement

	INTRODUCTION

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Mechanical stresses, such as compressive and tension forces, are loaded on the periodontium during orthodontic tooth movement, and periodontal remodeling takes place in response to these stresses. During periodontal remodeling, many kinds of cells in the periodontium—including periodontal ligament (PDL) fibroblasts, osteoblasts, cementoblasts, vascular cells, and hematopoietic cells—play varied and important roles. Osteocytes and osteoblasts are particularly important as mechano-sensing cells (Pavalko et al., 2003). The application of light orthodontic force causes direct resorption of alveolar bone, while the application of excessive orthodontic force results in excessive compressive force, which induces local ischemia, tissue hyalinization, and cell death in the periodontal ligament (Reitan and Rygh, 1994).

Earlier studies revealed that the pressure applied during orthodontic tooth movement caused tissue necrosis (Rygh, 1974). In addition to necrosis, apoptosis is another mode of cell death (Kerr et al., 1972) and is characterized by changes in the morphology of the cell, including membrane blebbing, volume loss (shrinkage), and the condensation of chromatin. Apoptosis involves the proteolytic activation of caspases and is induced by initiator caspases such as caspase-8 and -9, which, in turn, trigger an amplifying cascade of effector caspases, including caspase-3, -6, and -7. The effector caspases are responsible for the alteration of cell morphology (Earnshaw et al.second pathway is activated by a set of molecules, such as cytochrome c, secreted from the mitochondria. They induce oxidative stress, stimulus of radiation, and the loss of survival signals by growth factors and cytokines that activate caspase-9. Both of the pathways activate executor caspases (Hengartner, 2000). Recent studies have demonstrated that, during orthodontic tooth movement, the application of pressure caused an increase in the number of apoptotic cells at the pressure site (Hatai et al., 2001; Rana et al., 2001; Mabuchi et al., 2002). However, little is known about how mechanical force induces apoptotic cell death.

During bone remodeling, osteoblasts play a crucial role in the formation of bone by synthesizing bone matrix proteins and differentiating into osteocytes. Osteoblasts also regulate the maturation of osteoclasts and thereby cause bone resorption (Ducy et al., 2000). In the present study, we examined whether the in vitro application of a continuous compressive force would induce apoptosis in MG-63 human osteoblast-like cells. Having established that this was the case, we examined the mechanism by which compressive force initiated apoptosis.

	MATERIALS & METHODS

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Cell Culture
We used the human osteoblast-like cell line MG-63, which was provided by the Cancer Cell Repository of the Institute of Development of Tohoku University, Japan, and has been shown to have an osteoblast-like phenotype (Shui and Scutt, 2001). MG-63 cells were incubated in a minimum essential medium (Flow Laboratories, McLean, VA, USA) containing 10% fetal bovine serum (Flow Laboratories) and 100 units/mL each of penicillin and streptomycin (Wako Chemical, Osaka, Japan). Cells were seeded onto cover glasses (diameter, 30 mm; Matsunami, Osaka, Japan) at a density of 1 x 10⁵ cells/cover glass. The cover glasses were then incubated with 2 mL of culture media in six-well culture plates (Iwaki, Chiba, Japan). The cells were grown to confluence in a humidified 5% CO₂ incubator at 37°C.

In vitro Application of Continuous Compressive Force
Our previously established experimental model (Kanzaki et al., 2002) was used. We applied force to the confluent cells in the six-well plates (30 mm in diameter) by inserting glass wells (20 mm in diameter) that contained lead pellets into the wells of the plate. The amount of compressive force was controlled by adjustments in the mass of the lead pellets.

Cell Viability Assay
The viability of the cells was examined by use of the Alamar Blue^TM assay system (BioSource, Camarillo, CA, USA) (Ahmed et al., 1994). After the application of compressive force, 0.2 mL of Alamar Blue^TM reagent was immediately added to 2 mL of culture medium. The cells were then incubated (CO₂ atmosphere, 37°C) for 3 hrs. The absorbance of the medium was monitored at 570 and 600 nm by means of a spectrophotometer (Shimazu, Kyoto, Japan). Cell viability was represented as the ratio of the number of viable cells vs. the control, which was standardized as 100%.

Apoptosis Assays
    TUNEL Assay
Apoptotic cells were detected with the TUNEL assay. In apoptotic cells, the chromatin DNA is cut by endonucleases at linker DNA sites between nucleosomes. In the TUNEL method, 3'-OH DNA ends generated by DNA fragmentation are nick-end-labeled with fluorescein-dUTP, mediated by terminal deoxynucleotidyl transferase. After the application of compressive force, the cells were washed twice with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (Wako) in PBS at 4°C for 15 min. The cells were washed with PBS and treated with 0.5% Tween 20 containing 0.2% bovine serum albumin (Wako) in PBS at room temperature for 15 min. The cells were again washed with PBS and then incubated with 30 µL of terminal transferase and fluorescein (FITC)-labeled dUTP with the MEBSTAIN Apoptosis Kit Direct (MBL, Nagoya, Japan) for 1 hr at 37°C. The cells were washed with PBS again, and apoptotic cells were visualized by fluorescence microscopy (Leitz DMRBE, Leica, Wetzlar, Germany).

    Caspase-3 Activity
Caspase-3 activity was measured with the use of a caspase-3 colorimetric assay kit (BioVision Research Products, Mountain View, CA, USA), which detects the chromophore p-nitroanilide after cleavage from the labeled substrate DEVD-pNA. After the application of compressive force, the cells were washed with PBS and suspended in a lysis buffer for 10 min on ice. The cell suspension was homogenized by trituration with a syringe before being centrifuged. The supernatant was harvested for analysis. The protein content of the supernatant was quantified with the Micro BCA^TM protein assay reagent kit (Pierce, Rockford, IL, USA). Protein (100 µg) was added to 50 µL of 2x reaction buffer containing 0.5 µL each of 10 mM DTT and 4 mM DEVD-pNA, and the mixture was incubated at 37°C for 1 hr. The absorbance was measured at 405 nm via a microtiter plate reader (Model 550, Bio-Rad Lab., Richmond, CA, USA).

    Apoptosis Signaling Pathway
The cultured cells were treated with either 30 µM LETD-CHO (a specific inhibitor of caspase-8) or 30 µM LEHD-CHO (a specific inhibitor of caspase-9) (Thorburn et al., 2003) (Calbiochem, San Diego, CA, USA) before compressive force was applied. After 24 hrs of compressive force application, caspase-3 activity was measured as described above.

Statistical Analysis
Data are presented as the mean ± standard deviation (SD). Statistical analyses were carried out by a two-way analysis of variance (ANOVA) and a Bonferroni/Dunn post hoc test.

	RESULTS

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Compressive Force Altered the Morphology and Reduced the Viability of MG-63 Cells
The in vitro application of continuous compressive force altered the morphology of MG-63 cells (Figs. 1A–1E). After the application of compressive force, the arrangement of the cells was irregular, and the spaces among cells were greater than those in the control. Atrophic cells were observed after the application of greater magnitudes of compressive force.

View larger version (75K): [in this window] [in a new window]   Figure 1. Morphology and viability were changed by compressive force. Phase-contrast microscopic appearance of MG-63 cells under compressive force for 24 hrs. Bar = 100 µm. The compressive force resulted in a change of the morphology of MG-63 cells. The arrangement of the cells became irregular, and several spaces were observed between cells, as compared with control cells. As the compressive force became greater, some atrophic cells began to appear. (A) Control; (B) 1.02 x 10–4 N/cm2; (C) 2.04 x 10–4 N/cm2; (D) 4.08 x 10–4 N/cm2; and (E) 6.12 x 10–4 N/cm2. (F) Viability of MG-63 cells under compressive force. Values represent the mean ± SD of the relative absorbance of 3 experimental samples (exp.) per control sample (cont.). Cell viability was decreased at all levels of compressive force applied for 12 hrs, and there was no significant difference between cell viability at 12 and that at 24 hrs. Greater compressive force tended to inhibit cell viability to a greater extent. Significantly different from controls: * p < 0.05 (Bonferroni/Dunn method); a, vs. control; b, vs. 6.12 x 10–4 N/cm2 (n = 3).

Compressive Force Induced Apoptosis in MG-63 Cells
The TUNEL assay revealed that significantly more of the cells that were subjected to continuous compressive force had a strongly stained nucleus compared with that of control cells (Figs. 2A–2C); this indicated that the application of continuous compressive force induced apoptosis in the MG-63 cells. The percentage of apoptotic cells increased in a time- and force-dependent manner. Specifically, a compressive force of 1.02 x 10^–4 N/cm² caused a slight increase in the percentage of apoptotic cells, but this was not statistically significant (Fig. 2D). By contrast, 2.04 x 10^–4 or 4.08 x 10^–4 N/cm² of compressive force significantly increased the percentage of apoptotic cells (Fig. 2D). Our results indicate that a compressive force of 2.04 x 10^–4 or 4.08 x 10^–4 N/cm² for 12 or 24 hrs can increase the number of apoptotic cells, whereas a force of 1.02 x 10^–4 N/cm² is insufficient to induce apoptosis. Therefore, we conclude that compressive forces greater than 2.04 x 10^–4 N/cm² represent excessive force in vitro. This premise was demonstrated experimentally by the application of a force of 6.12 x 10^–4 N/cm², which resulted in a significant decrease in cell viability and an increase in cell detachment. Based on these results, forces between 1.02 x 10^–4 and 4.08 x 10^–4 N/cm² were used in subsequent apoptosis experiments.

View larger version (47K): [in this window] [in a new window]   Figure 2. Apoptosis detection by the TUNEL method with compressive force application for 24 hrs in MG-63 cells. The apoptotic cells among MG-63 cells under compressive force were detected by the TUNEL method. Stained nuclei were observed in some cells under compressive force, but no stained nuclei were seen in control cells. (A) Control, magnification, x200; (B) cells under 2.04 x 10–4 N/cm2 of compressive force, x200; (C) cells under 2.04 x 10–4 N/cm2 of compressive force, x400. Bar = 100 µm. (D) Percentage of apoptotic cells induced by compressive force in MG-63 cells. Values represent the mean ± SD of the relative absorbance of 3 experimental samples (exp.) per control sample (cont.). Percentages were significantly different from controls at all forces except 1.02 x 10–4 N/cm2. At 2.04 x 10–4 and 4.08 x 10–4 N/cm2 of compressive force, the percentage of apoptotic cells increased between 12 and 24 hrs. The percentage of apoptotic cells increased in a force-dependent manner. Significantly different from controls: * p < 0.05 (Bonferroni/Dunn method); a, vs. control; b, vs. 1.02 x 10–4 N/cm2; c, between 12 hrs and 24 hrs (n = 3).

View larger version (15K): [in this window] [in a new window]   Figure 3. Caspase-3 activity increased via the caspase-8 signaling cascade. (A) Force-dependent caspase-3 induction. Relative absorbance of caspase-3 is shown following the application of a compressive force of 2.04 x 10–4 or 4.08 x 10–4 N/cm2 for 24 hrs. Values represent the mean ± SD of the relative absorbance of 3 experimental samples (exp.) per control sample (cont.). Caspase-3 activity in the cells increased markedly under continuous compressive forces of 2.04 x 10–4 and 4.08 x 10–4 N/cm2. Caspase-3 activity increased with greater compressive force. Significantly different from controls at each weight: ** p < 0.01 (Bonferroni/Dunn method) (n = 3). (B) Effects of caspase-8 and -9 inhibitors on caspase-3 activity. Relative absorbance of caspase-3 activity is shown following the application of a compressive force of 4.08 x 10–4 N/cm2 for 24 hrs. To examine the signal pathway of the compressive-force-induced caspase-3 activation, we treated the cells with an inhibitor specific for caspase-8 (LETD-CHO) or caspase-9 (LEHD-CHO) before applying the compressive force, and then measured caspase-3 activity in the cells. The caspase-8 inhibitor significantly reduced the compressive-force-induced caspase-3 activation, while the caspase-9 inhibitor did not. Values represent the mean ± SD of the relative absorbance of 3 experimental samples (exp.) per control sample (cont.). ** p < 0.01 (Student’s t test). White columns, non-compressive force; black columns, the application of a compressive force of 4.08 x 10–4 N/cm2 (n = 3).

	DISCUSSION

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Apoptosis, a critical process in cell proliferation and differentiation (Jacobson et al., 1997), also serves to protect an organism from extracellular stress induced by the removal of growth factors (Jilka et al., 1998), exposure to radiation (Haimovitz-Friedman et al., 1994), or the administration of anti-cancer drugs (Boesen-de Cock et al., 1998). This study demonstrates the induction of apoptosis and subsequent decrease in cell viability in the osteoblast-like cell line MG-63 following the application of excessive compressive force. Moreover, the induction of apoptosis was observed to be force-and time-dependent, suggesting that excessive mechanical force directly induced apoptosis in these cells. Further experimental studies also indicated that the signal transduction cascade controlling apoptosis in MG-63 cells involves a caspase-8-mediated induction of caspase-3. These results suggest that excessive orthodontic force within sites compressed during periodontal remodeling may also cause osteoblast apoptosis.

During orthodontic treatment, the initial changes in the periodontal tissue surrounding the pressure side of the tooth are divided into initial and secondary phases, known as hyalinization and bone resorption, respectively. The gradual compression of periodontal tissue leads to the shrinkage and disappearance of cell nuclei and the degradation of capillaries and microfibrils. The degree of hyalinization depends on the magnitude of force that is applied. Hyalinized zones that are caused by light forces are frequently found on small root surfaces (Kronfeld and Weinmann, 1940). In contrast, excessive forces lead to necrosis of the alveolar bone as well as extensive hyalinization. Hyalinized tissue (Reitan, 1962) is distinct from necrotic bone (Macapanpan et al., 1954), because it contains newly formed collagen fibrils and capillaries created by connective tissue cells within the formerly cell-free hyalinized tissue. The molecular mechanisms that underlie these differences between hyalinized and necrotic tissue are unclear, but might be related to differences in the cellular processes of necrosis and apoptosis. Recent studies have suggested that apoptotic osteocytes, adjacent to a hyalinized periodontal ligament in alveolar bone on the pressure side, exhibited characteristics of necrosis during initial tooth movement (Hamaya et al., 2002). This study indicates that cell death may be an important biological process of periodontal remodeling during orthodontic tooth movement.

It is difficult to determine the optimal force during therapeutic orthodontic tooth movement, because periodontal tissue reactions vary among individuals. A study by Schwartz (1932) reported that the optimal orthodontic force is no more than capillary pressure, while Jarabak (1960) suggested that the optimal orthodontic force is 1–4 oz. Appropriate mechanical loading may inhibit osteoblast apoptosis, resulting in increased bone mass via an increase in the number of osteoblasts at the site of new bone formation (Pavalko et al. 2002). However, this study demonstrates that excessive compressive forces lead to osteoblast apoptosis. These results suggest that the osteoblast reaction changes with differing load force. Although the number of apoptotic cells increased in a time-dependent manner (Fig. 2D), cell viability was not altered in our study. These results suggest that another cell death mechanism, such as necrosis, may be associated with the observed change in cell viability. During tooth movement in vivo, recent studies have reported an increase in the number of TUNEL-positive periodontal ligament cells at 12 hrs, reaching a maximum at 24 hrs (Hatai et al. 2001). These results indicate that the increase in apoptotic cells over time did not correlate with a decrease in cell viability. This difference in induction time suggests that the role of apoptotic osteoblasts in tissue remodeling may differ from that of osteoblasts that have died by another cell death mechanism.

Collectively, these studies have shown the effect of an in vitro compressive force on the apoptotic signaling cascade of osteoblasts. Our results have demonstrated that a compressive force is capable of inducing the activation of caspase-3 in MG-63 cells through the activation of caspase-8. This study suggests that caspase-8 may be activated through the death receptor following the application of a mechanical force. The apoptosis pathway in mouse osteoblasts includes tumor necrosis factor-alpha and a variety of growth factors, cytokines, and hormones (Hill et al., 1997). Many related signal molecules, such as Fas (Jilka et al., 1998), Bcl-2 (Tanaka et al., 2002), and Apaf-1 (Zou et al., 1997), have also been implicated. The two principal signal transduction pathways of apoptosis may be interconnected at an unknown level. In these studies, the use of compressive force was shown to lead to the induction of apoptosis through caspase-8 activation of the caspase-3 signaling cascade. Additional studies are required to delineate this signal transduction pathway further.

	ACKNOWLEDGMENTS

 
This work was supported by grants-in-aid for scientific research from the Japanese Ministry of Education, Science, Sport, and Culture (Nos. 07557283, 09771823, 12671984, and 12557179).

Received December 20, 2004; Last revision September 29, 2005; Accepted October 14, 2005

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