RESEARCH REPORTS

Biomaterials & Bioengineering

S. Yamada1*, S. Saeki1, I. Takahashi1, K. Igarashi1, H. Shinoda2, and H. Mitani1

1Division

of Orthodontics, Department of Life-Long Oral Health Science, and 2 Division of Pharmacology, Department of Oral Biology, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan; *corresponding author, dayama@mail.cc.tohoku.ac.jp J Dent Res 81(10):711-715, 2002

Diurnal Variation in the Response of the Mandible to Orthopedic Force

ABSTRACT
Bone and cartilage metabolism is known to be more active during rest than during periods of activity. The purpose of this study was to examine the hypothesis that mandibular retractive force could be more effective when applied to rats during rest. Mandibular retractive force caused a considerable reduction in the condylar length in experimental groups, and the magnitude of this reduction was greater in the Light-period (08:0020:00) group than in the Dark-period (20:0008:00) group. The differentiation and proliferation of chondrocytes were inhibited in animals in the Light-period group, compared with those in the Dark-period group. These results suggest that the orthopedic effects of mandibular retractive force vary depending on the time of day the force is applied, and that such force may be more effective while animals are resting than while they are active. KEY WORDS: diurnal variation, orthopedic effect, mandibular condylar cartilage, proliferation, collagen.

iurnal variation is an essential feature of bone and cartilage metabolism. D Various parametersincluding plasma calcium and phosphate (Markowitz et al., 1981; Calvo et al., 1991), osteocalcin (Nielsen, 1994), degraded peptides of collagen (Greenspan et al ., 1997; Ohtsuka et al ., 1998), parathyroid hormone, and growth hormone (Jubiz et al., 1972; Calvo et al., 1991)have been shown to fluctuate according to diurnal rhythms. Diurnal variations have also been reported in bone and cartilage formation (Simmons, 1962, 1992; Simmons and Nichols, 1966; Roberts et al., 1979; Petrovic et al., 1981; Stutzmann and Petrovic, 1984; Mhlbauer and Fleisch, 1990; Yosipovitch et al., 1995). In mandibular condylar cartilage, diurnal variations in cellular proliferation and collagen synthesis have been shown in growing rats (Oudet et al., 1984; Saeki, 1995). Recently, as the basic mechanisms of the "biological clock" have been explored, diurnal variations in the biological response to various therapies have been considered to enhance the treatment outcome (Lemmer, 1999). Even in the field of orthodontics, Oudet et al. (1984) demonstrated that a mandibular protrusive appliance accelerated mandibular growth in rats when the appliance was applied during rest rather than during periods of activity. Our previous studies have also shown that orthodontic tooth movement was more effectively achieved when force was applied during rest in rats (Igarashi et al., 1998; Miyoshi et al., 2001). The purpose of this study was to investigate whether there is any difference in orthopedic effect when mandibular retractive force is applied at different times of day. Overall, we tested the hypothesis that mandibular retractive force can more effectively inhibit condylar growth and the differentiation and proliferation of chondrocytes when such force is applied during rest rather than during the active period.

INTRODUCTION

MATERIALS & METHODS

Experimental Animals, Application of Mandibular Retractive Force, and Tissue Preparation
Thirty-seven five-week-old male Wistar rats were divided into 1 control and 3 experimental groups. All of the animals were adapted to a 12/12-hr light/dark illumination program (with light from 08:00 to 20:00) for 2 wks. Four animals served as a Control group (n = 4) without force application. The 3 experimental groups consisted of a Whole-day group (n = 9), a Light-period group (n = 12), and a Dark-period group (n = 12), in which animals received mandibular retractive force the whole day, from 08:00 to 20:00, and from 20:00 to 08:00, respectively. A mandibular retractive force of 300 mN was applied bilaterally directly to the mandibular condyle from the symphysis for 21 days according to the method described by Asano (1986), with some modifications. Briefly, elastic bands with a chin cup attached to the incisors were fastened to a plastic collar brace placed around the necks of the animals. All of the animals were supplied with powdered laboratory chow (CA-1, Japan CLEA, Tokyo, Japan) to facilitate

Received November 20, 2001; Last revision July 2, 2002; Accepted July 26, 2002

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were taken from the microradiographic images. Reference points for the measurements of the mandible are shown in Fig. 1.

Histological Analysis
Samples were further rinsed with 0.01 M PBS, decalcified in 10% EDTA in 0.01 M PBS, pH 7.4, for 10 wks, rinsed with 0.01 M PBS, dehydrated in a graded series of ethanol, and embedded in paraffin. Three-micrometer-thick standardized sagittal serial sections were cut, deparaffinized, rehydrated in a graded series of ethanol, and stained with hematoxylin and eosin.
Figure 1. Reference points for linear and angular measurements. Da: antero-superior edge of the mandibular alveolar bone. Cd: most posterior point on the condyle. In: root apex of the mandibular incisor. Go: posterior tip of the angular process. aGo: deepest point on the antegonial notch. The length of the mandibular process (Cd-aGo), length of the condylar process (Cd-In), mandibular length (Da-Cd and Da-Go), and height of the mandibular ramus (Cd-Go) were measured. Angular measurements used in the present study were Da-Cd-aGo and Cd-Go-Da, which reflect the form of the ramus process.

Immunohistochemistry for Proliferating Cell Nuclear Antigen (PCNA) and Type II and Type X Collagen
Double immunohistochemical-cytochemical staining for PCNA and diamidino phenylindole dihydrochloride (DAPI) was performed with monoclonal antibody for PCNA (PC10, DAKO Japan, Kyoto, Japan) according to the manufacturer's instructions. Briefly, after deparaffinization, the sections were rehydrated, microwaved in 10 mM citrate buffer, pH 6.0, for 70 sec, and left in the same buffer for 30 min at 80C. They were incubated overnight at 4C with primary antibody and then incubated with secondary antibody conjugated with fluorescein (FITC) (Bio Source International, Camarillo, CA, USA). Nuclear counterstaining was performed with DAPI diluted in the secondary antibody solution. The central part of the condylar cartilage was photographed, and the numbers of DAPI-positive nuclei as the total cell number and PCNA-positive nuclei as proliferating cells in the proliferative and transitional cell layers were counted. The ratio of proliferating cells (PCNA-positive cells/DAPI-positive cells) was also calculated. Immunohistochemistry for type II and type X collagen was performed as described previously (Takahashi et al., 1995; Saitoh et al., 2000). Briefly, after the sections were deparaffinized, they were treated with hyaluronidase for 30 min at room temperature and incubated overnight at 4C with rabbit anti-bovine type II collagen antibody or rabbit anti-rat type X collagen antibody (LSL, Tokyo, Japan) diluted in 0.5% BSA, 0.01 M PBS, pH 7.4. Sections

food intake. They were weighed every day during the experimental period. At the end of the experimental period, they were killed under pentobarbital anesthesia. Bilateral mandibles with articular discs were dissected, divided into left and right halves, and fixed overnight at 4C in 4% paraformaldehyde in 0.01 M phosphatebuffered saline (PBS), pH 7.4. The present animal experiment was conducted with the approval of the Animal Care and Use Committee of Tohoku University, whose guidelines for the use of experimental animals are based on the Principles of Laboratory Animal Care from the National Institutes of Health.

Morphological Analysis of Mandibles

After fixation, the mandibles were rinsed several times with 0.01 M PBS, and soft x-ray microradiographs were taken at 30 kVp and 5 mA for 20 sec (Softex, type CMB, Tokyo, Japan) as described previously (Furuta et al., 1999). Linear and angular measurements
Table 1. Linear and Angular Measurements (mean + SD) Control Group (C) (N = 4) Linear measurement (mm) Mandibular length Da-Go 23.2 + 0.4 Da-Cd 22.8 + 0.4 Ramus height Cd-Go 8.9 + 0.4 Cd-aGo 16.2 + 1.0 Condylar length Cd-In Angular measurement () Da-Cd-aGo Cd-aGo-Da Whole-day Group (W) (N = 9)

Light-period Group (L) (N = 12)

Dark-period Group (D) (N = 12)

19.5 + 0.4 20.5 + 0.2 8.2 + 0.2 14.3 + 0.5

(** vs. C,L,D)a (** vs. C,L,D) (** vs. C) (** vs. C,L,D)

21.4 + 0.4 21.4 + 0.2 8.2 + 0.5 15.0 + 0.3

(** vs. C,W) (** vs. C,W) (** vs. C) (** vs. C,W) (* vs. D) (** vs. C,W,D)

21.3 + 0.4 21.3 + 0.3 8.3 + 0.3 15.3 + 0.4

(** vs. C,W) (** vs. C,W) (** vs. C) (** vs. C,W) (* vs. L) (** vs. C,W,L)

9.3 + 0.3

6.9 + 0.2

(** vs. C,L,D)

7.6 + 0.3

8.1 + 0.3

26.8 + 1.0 109.9 + 1.2

29.1 + 1.1 109.0 + 2.3

(** vs. C,D) (** vs. D)

31.3 + 2.4 107.3 + 1.6

(** vs. C,D) (* vs. D) (* vs. D)

29.9 + 1.1 108.8 + 2.2

(** vs. C,W) (* vs. L) (** vs. C) (* vs. W,L)

Groups which showed significantly different values are indicated by capitalized initials of group names in parentheses. N, number of samples. **P < 0.01. *P < 0.05.
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were further incubated with FITCconjugated secondary anti-rabbit IgG antibody (Bio Source International, Camarillo, CA, USA) at room temperature for 2 hrs, mounted, and observed by fluorescent microscopy (Leitz DMRD, Leica Microsystems AG, Wetzlar, Germany).

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Statistical Analysis
All the numerical data were analyzed by one-way analysis of variance (ANOVA) followed by the TukeyKramer multiple-comparison test. P < 0.05 was considered to be a significant difference.

RESULTS
Growth of the Animal and Morphometric Analysis of Mandibles The experimental animals weighed less than the control animals throughout the experimental period. At the beginning of the experiment, the mean (+ SD) body weight of all of the animals was 116 + 5 g. At the end of the experimental period, the mean body weight was 174 + 7 g for the Control group, 108 + 14 g for the Light-period group, 110 + 7 g for the Dark-period group, and 98 + 6 g for the Whole-day group. There were no statistically significant differences in body weight among the experimental groups. The results of linear and angular measurements are shown in Table 1. While there were no significant Figure 2. Histological specimens stained with H-E of central area of the mandibular condylar cartilage differences in most of the linear (A1 to D1) and photomicrographs of immunoreactivity for type II (A2 to D2) and type X collagen (A3 to measurements between the LightD3) antibody in the central area of the mandibular condylar cartilage. (A) Control group, (B) Wholeand Dark-period groups, the Cdday group, (C) Light-period group, and (D) Dark-period group. F, fibrous layer; P, proliferative cell layer; T, transitional layer; M, mature cell layer; H, hypertrophic cell layer. Scale bar = 200 m. aGo and Cd-In lengths in the Lightperiod group were significantly thickness was mainly due to the presence of fewer or no smaller than those in the Dark-period group (P < 0.05). With mature or hypertrophic cells. The effect in the proliregard to angular measurements, Cd-aGo-Da in the Light-period ferative and transitional cell layers was not as obvious as group was significantly smaller than that in the Dark-period that in the mature and hypertrophic layers, although the group (P < 0.05). cell density in these layers seemed to be lower in the Histological Observation experimental groups than in the Control group. These The mandibular condylar cartilage can be divided into 5 cell changes appeared to be greater in the Light-period group layers, as described by Mizoguchi et al. (1990) and Luder et than in the Dark-period group. al. (1988), i.e., fibrous, proliferative, transitional, mature, and Proliferative Activity of Cartilage Cells hypertrophic layers (Fig. 2A1). In the present study, we focused on these layers in the central area, where condylar PCNA-positive cells were observed in the proliferative and transitional cell layers in all of the groups (data not shown). cartilage articulates with the thinnest part of the articular disc. Significantly lower proliferating activity was found in the In the central area of the condyle, the thickness of the whole cartilage was obviously decreased in all of the Whole-day and Light-period groups than in the other groups (Table 2). Cell proliferative activity was inhibited in the Lightexperimental groups (Figs. 2B1, 2C1, 2D1) compared with period group by about 50% (P < 0.01) and 60% (P < 0.01) that in the Control group (Fig. 2A1). This decrease in
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Table 2. Proliferating Activity of Cartilage Cells (mean + SD) Control Group (C) (N = 3) PCNA Total cell PCNA/Total cell (%)
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Whole-day Group (W) (N = 3) 12.3 + 4.6 137.0 + 29.5 9.0 + 3.3 (** vs. C,D)a (** vs. C) (** vs. C,D)

Light-period Group (L) (N = 7) 23.7 + 5.9 158.0 + 30.8 15.0 + 2.8 (** vs. C,D) (** vs. C) (** vs. C,D)

Dark-period Group (D) (N = 7) 52.3 + 6.9 185.4 + 28.1 28.6 + 5.3 (** vs. C,W,L) NS (** vs. W,L)

81.3 + 15.6 243.0 + 46.1 33.5 + 1.3

Groups which showed significantly different values are indicated by capitalized initials of group names in parentheses. N, number of samples. NS, not significant. **P < 0.01.

compared with that in the Dark-period and Control groups, respectively. In the Whole-day group, cell proliferation was inhibited by about 80% (P < 0.01) compared with that in the Control group.

Collagen Immunohistochemistry The results of immunohistochemical staining for type II and type X collagen in the central area are shown in Fig. 2. In the Control group, well-organized cartilaginous tissue, including mature and hypertrophic cell layers, showed a positive reaction to type II collagen antibody (Fig. 2A2), and the hypertrophic cell layer showed a positive reaction to type X collagen antibody (Fig. 2A3). In the Whole-day group, type II collagenpositive cell layers were thin, and type X collagen-positive layers were not observed (Figs. 2B2, 2B3). Type X collagen was localized in a thin single-cell layered area at the lower borders of cartilaginous layers in the Light-period group (Fig. 2C3), while thicker multi-cell layered cartilaginous tissue reacted to type X collagen antibody in the Dark-period group (Fig. 2D3).

Growth of the Condyle With regard to the differences in the Light- and Dark-period groups, inhibition of the growth of the condylar process was more effective in the Light-period group than in the Dark-period group. Furthermore, the mandibular ramus was more perpendicularly upright in the Light-period group than in the Dark-period group, based on angular measurements, although these changes could also have been due to remodeling in the gonial area or the alveolar process. Taken together, these results suggest that the use of a chincap while animals are resting is possibly more effective than that while they are active. Proliferation of Chondrocytes Simmons (1962) demonstrated the existence of diurnal rhythm in mitotic activity in epiphysial growth cartilage. As described earlier, many different organs show diurnal rhythm in mitotic activity (Roberts et al ., 1979). Some investigators have reported that the proliferating activity is up to 30% higher in the light period (resting period of animals) than in the dark period (active period of animals) in normal rats (Oudet et al., 1984). The present immunohistochemical findings for PCNA clearly indicate that many more cells were proliferating in the Dark-period group than in the Light-period group. It is possible that the active phase of cell proliferation may be more sensitive to environmental stimulation such as mechanical stress. Differentiation of Chondrocytes We demonstrated that both the maturation and terminal differentiation of chondrocytes were inhibited by the application of mandibular retractive force, especially in the Light-period group. In the present study, while the thickness of the type II collagen-positive cell layer in the Light-period group was similar to that in the Dark-period group, the type X collagen-positive cell layer was apparently thinner in the Lightperiod group. Thus, there may be a diurnal variation in the inhibition of the terminal differentiation of chondrocytes by the application of mandibular retractive force. Our previous study (Saeki, 1995) showed that collagen synthesis in the mandibular condylar cartilage cells showed clear diurnal variation. When evaluated by autoradiography with 3H-proline, collagen synthetic activity in the light period was twice as high as that in the dark period. Considering that the thickness of the type II and type X collagen-positive layers represents the quantity of deposited molecules, the deposition of type X collagen may be inhibited more than the deposition of type II collagen in the Light-period group. Since the synthesis and deposition of the collagenous matrix occur during the light period rather than during the dark period, the

DISCUSSION
Experimental Procedures In the present experiment, we used a chincap appliance as a mandibular retractive appliance to evaluate the orthopedic effect on mandibular growth and condylar cartilage cell proliferation and differentiation. The application of a chincap in the mandibular incisor area induced bone remodeling, and therefore we did not define any reference point in this area. The observed differences in body weight might have been due to the reduced intake of food in the experimental groups. Since no statistical differences in body weight were observed among the experimental groups, we mainly compared the differences among these groups. It has been reported that type II collagen is a marker of differentiated chondrocytes (Silbermann et al., 1987; Luder et al., 1988; Mizoguchi et al., 1990; Takahashi et al., 1995) and is expressed mainly in the mature and hypertrophic cell layers, but not in the fibrous and proliferative cell layers. Type X collagen is a marker of hypertrophic chondrocytes (Kielty et al ., 1985) and is selectively expressed in the hypertrophic cell layer (Ohashi et al ., 1997). PCNA is known to be one of the cell-cycle markers expressed throughout the G1 to S phases (Takasaki et al., 1981). Thus, we believe that the present experimental design was able to demonstrate diurnal variations in response to orthopedic effects in the mandible and condylar cartilage in rats.

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inhibition of active collagen synthesis and secretion during the light period appears to inhibit condylar growth more effectively. In the present study, we clearly demonstrated that the orthopedic effects generated by a mandibular retraction appliance were more effective when applied during the resting period of the animals. The morphological changes observed in the mandible may have been mainly caused by the inhibition of both cell proliferation and matrix deposition in the condylar cartilage. Further investigation is needed to clarify the mechanism underlying the observed variation.

ACKNOWLEDGMENTS
We are grateful to Prof. Manabu Kagayama, Dr. Yasuyuki Sasano, and Dr. Hirotoshi Akita, Division of Oral Molecular Biology, Department of Oral Biology, and Dr. Mie Ohtsuka, Division of Pharmacology, Department of Oral Biology, Tohoku University Graduate School of Dentistry, for their valuable advice. This research was supported in part by Grantsin-Aid (Nos. 12557180, 13672138, and 14771173) for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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