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RESEARCH REPORT |
Department of Orthodontics, Hiroshima University Faculty of Dentistry, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan;
*corresponding author, mkaku{at}hiroshima-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: VEGF • osteoclast • osteoblast • bone remodeling • experimental tooth movement
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Orthodontic tooth movement and the related tissue remodeling are achieved by osteoclastic bone resorption and osteoblastic new bone formation. It was also reported that osteoblasts produce VEGF (Harada et al., 1994), and it is well-understood from these findings that experimental tooth movement is a useful in vivo model for investigating the expression and role of VEGF in the periodontal tissues with active remodeling.
Meanwhile, in a recent study, recombinant human (rh)VEGF induced many osteoclasts in osteopetrotic (op/op) mice via Flt-1 as well as macrophage-colony-stimulating factor (M-CSF) (Niida et al., 1999). In the following study, the number of osteoclasts induced by local administration of rhM-CSF was substantially greater than that induced by rhVEGF in op/op mice (Kaku et al., 2000). In addition, we have demonstrated that the local administration of rhVEGF significantly enhances the number of osteoclasts induced by experimental tooth movement (Kaku et al., 2001). However, it was still unknown whether rhVEGF injection and mechanical stimuli might increase the amount of tooth movement.
Thus, we conducted this study to investigate the expression of VEGF within periodontal tissues during experimental tooth movement using an immunohistochemical approach. Furthermore, we investigated the effect of rhVEGF injection on the rate of tooth movement and the comparison of the numbers of osteoclasts induced by the injection of rhVEGF and rhM-CSF.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental tooth movement of the upper incisors was continued for 10 days. The mice were killed under general anesthesia with sodium pentobarbital. They were fixed in 4% paraformaldehyde and rinsed in distilled water. Then, the specimens were decalcified in 14% EDTA (pH 7.4) for 14 days and embedded in paraffin. The premaxillary bones, including the upper incisors, were cut into frontal sections of 4 µm thickness. The expression of VEGF on the tension side was examined on these sections immunohistochemically stained with rabbit anti-mouse VEGF polyclonal antibody (LAB VISION, Fremont, CA, USA), by means of a Vectastain ABC-GO KIT (Vector Laboratories, Burlingame, CA, USA), and counterstained with methyl green. Normal rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as the control for the polyclonal antibody.
Effects of VEGF on the Amount of Experimental Tooth Movement
Seventy 30-day-old C57BL/6J mice were divided into two groups. In the first group, which served as the controls (n = 5), phosphate-buffered saline (PBS) was injected immediately at the beginning of experimental tooth movement. The second group (n = 5) underwent the same activation with an injection of 0.5 µg rhVEGF (Pepro Tech, London, UK) into the buccal gingival groove around the incisors every other week. In our previous study (Kaku et al., 2001), we demonstrated the dose of VEGF to be sufficient for enhancing the appearance of osteoclasts induced by experimental tooth movement. Experimental tooth movement was continued for 21 days in both groups. The distances between the left and right appliances’ tips, bonded onto the upper incisors, were measured every three days on the dorsoventral cephalograms. A rat-and-mouse cephalometric x-ray apparatus (Asahi Roentgen Ind. Co., Kyoto, Japan) was used at 20-25 kV and 6 mA with an exposure time of 3.0 sec for Kodak Dental Ultra-speed film (Eastman Kodak Co., Rochester, NY, USA). We then measured the width of the midpalatal suture every three days on the frontal sections (n = 5) and subtracted it from the distance of tooth movement according to as previous study (Stark and Sinclair, 1987).
On days 3, 7, 14, and 21 after initiating the experiment, we investigated the changes in the numbers of osteoclasts in the groups (n = 5). The histological sections were stained with tartrate-resistant acid phosphatase (TRAP) and counterstained with hematoxylin. Osteoclasts were identified as TRAP-positive, multi-nucleated cells located on the bone surface. The number of osteoclasts that appeared in the PDL space on the pressure side of the incisors was counted on 5 sections at 35-µm intervals for each specimen.
Comparison of the Number of Osteoclasts Induced by the Injection of rhVEGF and rhM-CSF
Sixty-four 30-day-old C57BL/6J mice were divided into 8 groups with 8 animals in each. The first group received PBS injections without any tooth movement, serving as the controls (group N). The second and third groups without experimental tooth movement underwent injection of 0.5 µg rhVEGF and 0.5 µg rhM-CSF, respectively (groups V and M). Both 0.5 µg rhVEGF and 0.5 µg rhM-CSF were injected into the fourth group (group VM). PBS was injected into the fifth group with experimental tooth movement (group T). The remaining three groups underwent the same activation with injection of 0.5 µg rhVEGF, 0.5 µg rhM-CSF, and both 0.5 µg rhVEGF and 0.5 µg rhM-CSF, respectively (groups VT, MT, and VMT). The experimental tooth movement was continued for three days, because the number of osteoclasts induced by rhVEGF and rhM-CSF peaked three days after the single injection, as was demonstrated in our previous report (Kaku et al., 2000). The number of osteoclasts was counted as described above.
Statistical Treatment
To examine differences in the numbers of osteoclasts and the amount of tooth movement among multiple groups, we performed analysis of variance (ANOVA) and multiple-comparison tests (Fisher) using Statview® (Abacus Concepts, Inc., Berkeley, CA, USA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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shows the expression of VEGF within the periodontal tissues 10 days after the experimental tooth movement was initiated.
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). Meanwhile, no immunoreactivity was detected in the negative control (Fig. 1B).
Changes in the Numbers of Osteoclasts and the Amount of Tooth Movement
Changes in the amount of tooth movement every three days and changes in the numbers of osteoclasts on days 3, 7, 14, and 21 are shown in Fig. 2A.
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). In contrast, about 5 osteoclasts were found in the controls on the same days. Significant differences in the numbers of osteoclasts were found between the two groups on days 3, 7, and 14 (Fig. 2A).
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).
Throughout the experimental period, the width of the midpalatal suture was quite small, and the mean amount of sutural expansion in the rhVEGF injection group and controls was about 0.045 mm (Figs. 2A, 2B).
Changes in the Numbers of Osteoclasts Expressed by rhVEGF or rhM-CSF Injection
In control group N, osteoclasts were rarely detected, although about 7 osteoclasts per area were induced on the pressure side of the PDL space in groups V and M, showing significant differences in the numbers of osteoclasts from group N. However, no significant differences in the numbers of osteoclasts were found between groups V and M. In addition, the number of osteoclasts was significantly greater in group VM than in groups N (p < 0.01), V, and M (p < 0.05) (Fig. 4A).
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).
Furthermore, the numbers of osteoclasts in the experimental tooth movement groups were significantly greater than in the groups without experimental tooth movement (VT vs. V, MT vs. M, p < 0.05). However, no significant differences in the numbers of osteoclasts were found between groups VM and VMT (Fig. 4C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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VEGF has various biological functions, such as vascular permeability and migration of human monocytes (Senger et al., 1983; Barleon et al., 1996; Clauss et al., 1996). A recent study reported that VEGF had an osteoclast differentiation function in op/op mice, and the responses of osteoclast precursor cells to VEGF were directly mediated via Flt-1 (Niida et al., 1999).
Very recently, we demonstrated that local administration of rhVEGF enhanced the number of osteoclasts during experimental tooth movement, and the number of nuclei per osteoclast in rhVEGF injection mice was significantly larger than that in control mice (Kaku et al., 2001). In this study, the amount of tooth movement in the rhVEGF injection group was larger than that in controls. Especially, from 15 to 18 days, the rate of tooth movement showed a significant increase. The reason may be because a large number of osteoclasts induced by rhVEGF appeared at 14 days and produced a large amount of bone resorption, leading to a substantial increase in tooth movement from 15 to 18 days after the beginning of the experiments. Meanwhile, the number of osteoclasts in group VT decreased at 21 days. We hypothesized that this was because (1) of the insufficient number of precursor cells of osteoclasts in the PDL space, and (2) apotosis of osteoclasts occurred in the experimental group to maintain homeostasis. Considering that VEGF may play a role in removing necrotic tissue by accelerating the angiogenesis in the compression area, we can conclude that a combination of rhVEGF injection and orthodontic force application is more efficient for tooth movement.
The previous study demonstrated that a severe deficiency of osteoclasts in op/op mice can be cured by injections of rhM-CSF (Felix et al., 1990; Kodama et al., 1991a; Wiktor-Jedrzejczak et al., 1991; Sundquist et al., 1995). Direct action of M-CSF on osteoclast lineage cells was exhibited by the expression of the receptor for M-CSF, c-fms, in osteoclasts both in vitro (Kodama et al., 1991b) and in vivo (Hofstetter et al., 1992). From these findings, it was indicated that M-CSF plays an essential role in the differentiation of osteoclasts. In addition, it became clear that M-CSF supports osteoclast differentiation in cooperation with osteoclast differentiation factor (ODF)/osteoprotegerin ligand (OPGL)/TNF-related activation-induced cytokine (TRANCE)/RANKL (Lacey et al., 1998; Yasuda et al., 1998). The present study showed that the number of osteoclasts induced by rhVEGF was almost equivalent to that induced by rhM-CSF, regardless of mechanical force application. However, it was reported that the number of osteoclasts recruited by rhVEGF was less than that by rhM-CSF in op/op mice (Kaku et al., 2000). From these findings, it is suggested that an excess of exogenous rhM-CSF injection is not required for differentiation of osteoclasts in normal mice with sufficent endogenous M-CSF. The number of osteoclasts was significantly greater in group VM than in groups V and M. The number of osteoclasts in group VMT was significantly larger than that in groups VT and MT. These results suggested that VEGF and M-CSF might function independently for the recruitment of osteoclasts. Thus, we expect that simultaneous injection of rhVEGF and rhM-CSF is more efficient for orthodontic tooth movement. The number of osteoclasts in groups VT and MT was significantly larger than that in groups V and M, respectively. However, there was no significant difference in the numbers of osteoclasts between groups VM and VMT. For this reason, we hypothesized that there were insufficient numbers of precursor cells of osteoclasts in the PDL space of group VMT.
Several chemical mediators such as prostaglandin and 1,25-(OH)2D3 can accelerate the rate of tooth movement by means of similar mechanisms to increase the number of osteoclasts (Yamasaki et al., 1984; Collins and Sinclair, 1988; Takano-Yamamoto et al., 1992). In comparison with these factors, VEGF is suggested to be more appropriate in terms of wound-healing function through induction of angiogenesis. Therefore, VEGF has possible clinical applications for achieving efficient orthodontic treatment.
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ACKNOWLEDGMENTS |
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Received February 12, 2002; Last revision October 30, 2002; Accepted November 22, 2002
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