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J Dent Res 83(4): 338-342, 2004
© 2004 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Effect of Nitric Oxide on the Recovery of the Hypofunctional Periodontal Ligament

H. Watarai*, H. Warita, and K. Soma

Orthodontic Science, Department of Orofacial Development and Function, Division of Oral Health Sciences, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan;

* corresponding author, watarai.orts{at}tmd.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The relationship between occlusal stimuli and a hypofunctional periodontal ligament (PDL) structure has been reported, though changes in occlusal recovery conditions were still unclear. Nitric oxide (NO) produced by NO synthase (NOS) is considered a factor for vascular and immune system control, and it increases according to mechanical stimuli. The objective of this study was to examine the relationship between NOS and occlusal stimuli in PDL by comparing hypofunction with occlusal recovery. The study focused on the expression of endothelial NOS (eNOS) and inducible NOS (iNOS). Their expression significantly decreased in occlusal hypofunction compared with the control group and increased close to normal in an occlusal recovery group. The change in the immunopositive area was more dramatic than the immunopositive cell number. Moreover, the rate of iNOS increase was higher than that of eNOS. This study suggests that NO plays an important role in the recovery of the hypofunctional PDL.

KEY WORDS: nitric oxide • occlusal recovery • periodontal ligament • blood vessel


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The structure and function of the periodontal ligament (PDL) are intimately related to occlusal function. The loss of normal occlusal function leads to atrophic changes in the PDL, such as narrowing of the periodontal space, disorientation of collagen fibers, and vascular constriction (Amemiya et al., 1980; Kaneko et al., 2001). There is evidence that periodontal blood vessels are intimately related to occlusal stimuli. This evidence includes a reduction in the diameter and the number of periodontal blood vessels (Tanaka et al., 1998). In addtion, an increased expression of endothelin has been related to hypofunction (Hayashi et al., 2001). Several studies using occlusal recovery models revealed widening blood vessels in the PDL after the application of occlusal stimuli (Saeki, 1959; Koike, 1996; Suhr et al., 2002). However, there are many ambiguities in the relationship between occlusal stimuli and the change in blood vessels.

The function and integrity of blood vessels are regulated by the nervous system and several local factors, one of which is nitric oxide (NO). Since NO was discovered as an endothelial-derived relaxing factor in 1987, it has been recognized as a biologically active molecule that has various functions (Moncada et al., 1991). It is synthesized from L-arginine in a process catalyzed by nitric oxide synthase (NOS) (Marletta et al., 1998; Stuehr, 1999). There are many kinds of cells that produce NOS and are classified into three types: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). Their production appears to be tissue-specific. Endothelial NOS is produced in endothelial cells and osteoclasts (Rubin et al., 2003), and iNOS is produced in vascular smooth-muscle cells, fibroblasts, and macrophages (Sasu et al., 2001; Connelly et al., 2003). The expression of NOS in the PDL and dental pulp of rats, cats, and dogs has been reported (Kerezoudis et al., 1993; Lohinai et al., 1997). These findings suggest the existence of NO in physiologically normal dental pulp and periodontal tissues, and a possible regulatory role in these tissues.

Recent studies have focused on the relationship between mechanical stimuli and NO (Nakago-Matsuo et al., 2000). NO production increased in response to cyclic tension force application in vitro (Kikuiri et al., 2000), whereas administration of NO inhibitor reduced experimental tooth movement in vivo (Hayashi et al., 2002; Shirazi et al., 2002). Few studies are available on the recovery process of atrophic PDL with NO. The objective of this study was to examine the changes in the PDL structure and the association of eNOS and iNOS using hypofunction/recovery models.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Experimental Model
Thirty-six seven-week old male Sprague-Dawley rats, mean weight 230 ± 15 g (mean ± SD), were divided into normal (n = 12) and experimental (n = 24) groups. The latter group was additionally divided into hypofunction (n = 12) and recovery (n = 12) groups. According to the method developed by Suhr et al.(2002), an anterior bite plate and a metal cap constructed from band material (0.180 x 0.005 inch; Rocky Mountain Morita, Tokyo, Japan) were attached to the maxillary and mandibular incisors, respectively (Fig. 1Go) by means of light-curing composite resin (Clearfil Liner Bond II, Kuraray Co. Ltd., Okayama, Japan) to induce a hypofunctional condition in the molar region. According to previous studies, atrophic changes in PDL, such as narrowing of the periodontal space and disarrangement of periodontal fibers, became noticeable after 7 days of occlusal contact loss (Koike, 1996; Tanaka et al., 1998). A recent study (Suhr et al., 2002) also revealed complete recovery of the PDL after 7 days of occlusal function recovery. This study therefore established an observation period 7 days after bite plate attachment, and an additional 7 days following appliance removal. Twelve rats were killed at 7 days for the hypofunction group. The appliances were removed from another 12 rats to allow for occlusal recovery for 7 days before death. A preliminary study showed that the eNOS and iNOS immunoreactivities of the PDL were not age-dependent in normal rats at an age of 7–9 wks. All animals were fed ad libitum with a powder diet (CE-2, Clea Japan Inc, Shizuoka, Japan), and had free access to drinking water. The experimental procedures were approved by the Animal Ethics Committee of Tokyo Medical and Dental University.



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  		Figure 1. Experimental method. An anterior bite plate and a metal cap were attached to the maxillary and mandibular incisors, respectively, with a light-curing resin used to produce occlusal hypofunction in the molar region.

 
Tissue Preparation
After administration of inhalant anesthesia with diethyl ether (Wako Pure Chemical Industries, Osaka, Japan), the animals were deeply anesthetized by injection of ketamine hydrochloride (40 mg/kg, i.p.; Veterinary Ketalar 50®, Sankyo, Tokyo, Japan) containing 20% xylazine hydrochloride (4.0 mg/kg, i.p.; Celactal®, 2% injection, Bayer-Japan, Tokyo, Japan) as a muscle relaxant to facilitate the operation. Animals were perfused intracardially through the left ventricle with 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. Then the maxilla were removed en bloc and immersed in the same fixative at 4°C for 2 hrs, and decalcified in 4% EDTA solution at 4°C for 6–8 wks. Decalcified tissues were immersed overnight in 30% sucrose solution at 4°C and embedded in optimal cutting temperature (OCT) compound (Sakura Finetek Europe, Zoeterwoude, the Netherlands). Twenty-µm-thick serial sections were cut horizontally by means of a cryostat (Leica CM3000, Nussloch, Germany), and mounted on poly-L-lysine-coated glass slides (Matsunami, Osaka, Japan). The observation site was at the level of 500–600 µm from the furcation of the disto-palatal root of the maxillary first molars (Fig. 2Go).



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  		Figure 2. Observation site for histomorphometry. (A) Cross-sections were obtained at the level of 500–600 µm from the furcation of the disto-palatal root of the maxillary first molars. (B) The cell number and total area of NOS-immunoreactivity were recorded at a 200 x 200 µm2 square in the center of the distal periodontal area. {alpha} = Mesiodistal line that passed through the central point of the root. P = The intersection of the {alpha}-line and the borderline between the distal PDL and alveolar bone. ß = The line that passed through point P and perpendicular to the {alpha}-line. M, mesial; D, distal; Bu, buccal; L, lingual; PDL, periodontal ligament; R, root.

 
Immunohistochemistry
Immunohistochemical staining was performed by use of eNOS and iNOS monoclonal antibodies (Transduction Laboratories, Lexington, KY, USA) (Heeringa et al., 2001). After being washed in 0.01 M phosphate-buffered saline (PBS) for 15 min, the prepared sections were treated with 0.3% H2O2 in absolute methanol for 30 min to inactivate endogenous peroxidase, and pre-incubated in 2% normal horse serum (Vector, Burlingame, CA, USA) in 0.01 M PBS for 30 min. The sections were then incubated with eNOS or iNOS rat monoclonal antibody and diluted at 1:500 with 2% normal horse serum for 30 min at 37°C humid atmosphere. This was followed by 2 consecutive incubations with biotinylated anti-mouse IgG (1:200) and avidin-biotin-peroxidase (ABC) complex, respectively (ABC-PO kit, Vector, Burlingame, CA, USA), for 30 min. All incubations were followed by 15 min of washing with PBS. Immunoreactivity was visualized with the use of 0.02% 3,3-diaminobenzidine tetrahydrochloride (Dojin Chemical, Kumamoto, Japan) and 0.01% H2O2 in 0.05 M Tris-HCl buffer (pH 7.6). The immunostained sections were finally mounted with 70% glycerin.

We confirmed the specificity of immunostaining by omitting anti-mouse IgG or ABC complex, or by replacing primary antibody with PBS. Immunostaining was not observed in sections of the negative control. Sections of lung tissue from the normal rats were used as a positive control (Monica et al., 2002).

Quantitative Analysis
The cross-sections at the level of 500–600 µm from the furcation of the disto-palatal roots of the maxillary first molars were chosen for quantitative analysis. The blood vessel diameter, the number of NOS-immunopositive cells, and the total NOS-immunopositive area were measured in a square area of interest (200 µm x 200 µm, Fig. 2Go). We drew the mesiodistal line that passed through the central point of root (which is line {alpha}). We determined point P that was the intersection of the line {alpha} and the borderline between the distal PDL and alveolar bone. We drew the line that passed through point P and perpendicular to the line {alpha} (which is line ß). We made a square as Fig. 2Go.

The immunostained specimens were observed and photographed by a light microscope (Nikon Microphoto-FXA, Nikon, Tokyo, Japan) equipped with a digital camera (DXm1200, Nikon, Tokyo, Japan), and stored in a 24-bit true-color TIFF format. Measurement was performed 3 times in the representative section obtained from the 12 samples of each group by means of image analysis software (Image-Pro, Media Cybernetics, Silver Spring, MD, USA) (O’Donnell et al., 1995; Hoang et al., 1997). The blood vessel diameter was measured by a scale provided by the software. The number of NOS-immunopositive cells was counted manually. Analogue microscopic images were converted into digital images, and the image analysis program was used to establish a threshold and measure the immunopositive area. A threshold that defined the cell margin was established with the software, and the total immunostained area in the digital images measured. Twelve rats in each group were used to measure NOS-immunopositive cells and the summary of the NOS-immunopositive area. Each section was counted on 3 different days, and 5 consecutive sections per animal were counted to correct differences in observation. The size of a cell was estimated as NOS-immunopositive area/NOS-immunopositive cell number in each group, and the cell sizes of the 3 groups compared. The number of NOS-immunopositive cells and the total immunopositive area were analyzed by ANOVA followed by Scheffé’s post hoc test (p < 0.05), with the use of Statview 5.0J software (SAS Institute, Cary, NC, USA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The body weight in normal, occlusal hypofunction, and recovery groups increased during the experimental period. There was no significant difference in the mean body weight among the groups. In the seven-day occlusal hypofunction group, the PDL thickness was thinner and blood vessels were smaller compared with those in the control group. In the seven-day occlusal recovery group, the PDL thickness and blood vessel diameter recovered to levels comparable with those in the control group. The average diameter of blood vessels was 17.36 ± 2.18 µm (mean ± SD) in the normal group, 8.29 ± 1.42 µm (mean ± SD) in the hypofunctional group, and 15.64 ± 1.89 µm (mean ± SD) in the recovery group. The differences between blood vessel diameters of the hypofunction group and those of the other two groups were statistically significant (p < 0.01).

The normal and occlusal hypofunction groups had identifiable eNOS only in blood vessels (Figs. 3AGo, 3BGo). In the occlusal recovery group, eNOS was detected in blood vessels, and in mononuclear phagocyte lineage at the border of the PDL and alveolar bone (Fig. 3CGo). After 7 days of induced hypofunction, the number of eNOS-immunopositive cells and the eNOS-immunopositive area were significantly decreased (p < 0.05) when compared with that of the normal group. In contrast, following 7 days of occlusal recovery, there were significant (p < 0.05) increases in the number of eNOS-immunopositive cells (1.58 times) and the eNOS-immunopositive area (2.13 times). These levels were not significantly different from those found in control conditions (Figs. 4AGo, 4CGo).



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  		Figure 3. NOS immunoreactivity in the distal side of the disto-palatal root of the maxillary first molar. Endothelial NOS immunostaining sections in normal, hypofunction, and recovery groups (A,B,C). (A) Blood vessels in PDL were observed clearly in general. Endothelial NOS was noted only in blood vessels. (B) The diameters of periodontal blood vessels appeared to be smaller, and the number of eNOS-immunopositive cells decreased. (C) The recovered periodontal blood vessels increased in diameter and number compared with the hypofunction group. In addition, the number of eNOS-immunopositive cells increased close to that of the normal periodontal group. Mononuclear phagocyte lineages were observed at the border of the PDL and alveolar bone.

Inducible NOS immunostaining sections in normal, hypofunction, and recovery groups (D,E,F). (D) Inducible NOS-immunopositive cells were observed in vascular smooth-muscle cells, fibroblasts, and mononuclear phagocyte lineage. (E) The PDL displayed a decrease in thickness and disorientation of PDL fibers. The diameters of periodontal blood vessels appeared to be smaller, and the number of iNOS-immunopositive cells decreased. (F) Vascular smooth-muscle cells and fibroblasts recovered to close-to-normal levels. Mononuclear phagocyte lineage was observed at the border of the PDL and alveolar bone, especially in the rugged profile area. , blood vessel; ->, fibroblast; , mononuclear phagocyte lineage. Bar: 100 µm.

 


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  		Figure 4. Quantitative analysis of the numbers of NOS-immunopositive cells and the total NOS-immunopositive area. We used image analysis software to count immunopositive cell numbers and to measure the immunopositive area in the unit area (200 µm x 200 µm). In comparison with the normal group, the numbers of eNOS- and iNOS-immunopositive cells (A,B) significantly decreased in the hypofunction group, and they increased to close-to-normal levels after the recovery period. eNOS cell numbers: normal, 17.41 ± 3.11 cells/unit; hypofunction, 9.05 ± 2.17 cells/unit; and recovery, 15.75 ± 2.92 cells/unit. iNOS cell numbers: normal, 74.75 ± 5.47 cells/unit; hypofunction, 30.58 ± 4.18 cells/unit; and recovery, 70.58 ± 6.11 cells/unit. Immunopositive area (%) means the percentage of immunopositive cells in the unit area. The pattern changes in the immunopositive area were similar to those in the cell number (C,D). The change in the NOS-immunopositive area was greater than the NOS-immunopositive cell number in both eNOS and iNOS. eNOS-immunopositive area: normal, 1.71 ± 0.29%; hypofunction, 0.72 ± 0.19%; and recovery, 1.62 ± 0.19%. iNOS-immunopositive area: normal, 6.00 ± 0.56%; hypofunction, 1.81 ± 0.32%; and recovery, 5.64 ± 0.57%. The data are means ± SD. N = 12 for each group. *p < 0.05. **p < 0.01.

 
The normal, occlusal hypofunction, and recovery groups had identifiable iNOS in blood vessels, fibroblasts, and mononuclear phagocyte lineage (Figs. 3DGo, 3EGo, 3FGo). Mononuclear phagocyte lineage cells were found at the border of the PDL and alveolar bone. After 7 days of induced hypofunction, the number of iNOS-immunopositive cells and the iNOS-immunopositive area were significantly decreased (p < 0.01) when compared with those of the normal group. In contrast, following 7 days of occlusal recovery, there were significant (p < 0.01) increases in the number of iNOS-immunopositive cells (2.37 times) and iNOS-immunopositive area (3.08 times). These levels were not significantly different from those found in control conditions (Figs. 3DGo, 3FGo).

During the hypofunctional period, the cell size was 0.75 times in eNOS-immunopositive cells and 0.79 times in iNOS-immunopositive cells compared with that of the controls. In contrast, during the recovery period, the cell size was 1.35 times in eNOS-immunopositive cells and 1.30 times in iNOS-immunopositive cells compared with that of the hypofunctional groups. The difference in cell size between the control and the occlusal recovery groups was not significant.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study found that eNOS and iNOS were present in PDL during phases of both occlusal hypofunction and recovery. In both the normal group and the hypofunction group, immunoreactivity for eNOS was noted only in blood vessels. In the recovery group, eNOS immunoreactivity was found in both blood vessels and mononuclear phagocyte lineage. This is the first report of eNOS in PDL. Our findings are consistent with those of previous research reporting eNOS expression in blood vessels in other organs (Ermert et al., 2002). In addition, our study identified changes in the levels of eNOS in association with changes in occlusal function. This is consistent with a previous report of eNOS secretion in response to increased shear stress (Wedgwood et al., 2001). The diameters of blood vessels are reduced in occlusal hypofunction (Tanaka et al., 1998; Hayashi et al., 2001). In occlusal hypofunction, the decrease of eNOS is thought to be the result of the decrease in shear stress, caused by decreased blood flow resulting from the decreased need for blood supply. In contrast, the increase in eNOS in occlusal recovery may be caused by increased shear stress due to increased blood flow resulting from an increased need for blood supply.

Our observation of inducible NOS in blood vessels, mononuclear phagocyte lineage, and fibroblasts has not been reported previously for PDL, but has been reported in other organs (Warner et al., 1995). Following 7 days of hypofunction, iNOS-immunoreactivity significantly decreased in comparison with normal control. It returned to near-control levels after recovery. NO production has been shown to be associated to frequency of stimuli and degree of stretching force (Kikuiri et al., 2000). Although the effect of compression is unknown, these results suggest that PDL cells may produce NO by many types of mechanical stress. Although inducible NOS was not affected by shear stress in a previous study (Wagner et al., 1997), a second study had demonstrated that cyclic tension force enhanced the production of interleukin-1-beta (IL-1ß) (Shirazi et al., 2002). IL-1 is a chemical mediator that is known to promote synthesis of iNOS in vascular smooth-muscle cells. Therefore, the increase in iNOS level may be the indirect effect of IL-1 activation as a result of altered occlusal stimuli (Shirazi et al., 2002).

The change in the NOS-immunopositive area was greater than the NOS-immunopositive cell number in both eNOS and iNOS. These findings indicate that the cell size as well as the cell number decreased in hypofunction and increased nearly to control in the recovery period. The change in iNOS expression in the occlusal recovery period was greater than that of eNOS. After activation of cells by different inducers (bacterial, cytokines), iNOS is expressed and active for hours to days as a "high-output" enzyme (MacMicking et al., 1997). The result of our study, in terms of change in iNOS, is consistent with this study.

In summary, this study demonstrated that the production of eNOS and iNOS decreased in hypofunctional PDL and increased following occlusal function recovery. If an increase in NO is assumed to accompany the increase in eNOS and iNOS in occlusal recovery, NO in PDL may be partially regulated by occlusal stimuli. This mediator may play an important regulatory role for blood vessel expansion and as a mediator of mechanical stress, maintaining the integrity of periodontal tissues under physiological conditions.


   ACKNOWLEDGMENTS
 
This research was supported by Grants-in-Aid for Scientific Research (Nos. 14571941, 14370688) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Part of this study was presented at the Diamond Anniversary Commemorative Meeting of the Japanese Orthodontic Society, Tokyo, Japan, October 8–11, 2001 (Watarai et al., 2001).

Received February 24, 2003; Last revision February 2, 2004; Accepted February 3, 2004


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 ABSTRACT
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 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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