Root Resorption in Spontaneously Hypertensive Rats and Its Suppression by β2-Adrenergic Receptor Blockade

1. Introduction

Hypertension is a highly prevalent chronic disease worldwide and has been shown to affect not only the cardiovascular system but also bone metabolism [1,2]. Recent epidemiological studies and meta-analyses have revealed reduced bone mineral density and an increased risk of fracture in patients with hypertension, suggesting that hypertension may be associated with systemic abnormalities in bone metabolism [3,4]. Increased sympathetic nervous activity has attracted attention as a possible mechanism of hypertension-associated abnormalities in bone metabolism. Sympathetic neurotransmitters are known to influence bone remodeling through β2-adrenergic receptors (β2-ARs) expressed on osteoblasts and osteoclasts [5,6].

Interestingly, studies using spontaneously hypertensive rats (SHR) have shown that administration of butoxamine (BTX), a selective β2-AR antagonist, markedly suppresses alveolar bone loss and reduces tooth movement [7,8]. Kondo et al. reported that experimental tooth movement in rats increases sympathetic nerve fibers (tyrosine hydroxylase [TH] -positive nerve fibers) within the periodontal ligament tissue and enhanced osteoclast activity. In addition, sensory nerve injury or sympathectomy suppresses both tooth movement and osteoclast activity, suggesting that a neural loop involving the sensory nervous system, central nervous system, and sympathetic nervous system contributes to the regulation of tooth movement [9]. These findings suggest that hypertension-associated sympathetic hyperactivity may promote alveolar bone resorption [10,11]. However, these studies focused primarily on alveolar bone resorption and tooth movement, and did not sufficiently investigate the effects of hypertension and sympathetic hyperactivity on root resorption.

Orthodontically induced inflammatory root resorption (OIRR) is one of the major adverse effects associated with orthodontic treatment and is characterized by resorption of cementum and dentin. The development of OIRR involves impaired blood flow caused by periodontal ligament compression, hyalinization, inflammatory cytokine production, and odontoclast induction. In particular, excessive compressive stimuli that increase the intraperiodontal pressure beyond the capillary pressure have been suggested to promote odontoclast activation and root resorption [12], implying that hypertension may increase susceptibility to root resorption during orthodontic treatment [13,14]. In addition, OIRR shares osteoclast/odontoclast-dependent mechanisms with alveolar bone resorption, and root resorption and alveolar bone resorption are thought to involve common biological mechanisms [13,15]. However, it remains unclear whether hypertension-associated sympathetic hyperactivity exacerbates root resorption under orthodontic force and whether blockade of β2-AR signaling can suppress OIRR.

In this study, we established an experimental tooth movement model using continuous orthodontic force in SHR and investigated the effects of hypertension on root resorption. Furthermore, given previous findings from studies using SHR that administration of a β2-AR antagonist suppressed tooth movement and alveolar bone resorption [7,16], we analyzed the effects of β2-AR blockade on root resorption with respect to odontoclasts, inflammatory cytokines, and cementocyte dynamics.

2. Materials and Methods

2.1. Animals and Reagents

Seven-week-old male SHR/Izm and Wistar Kyoto (WKY)/Izm rats (Japan SLC, Inc., Shizuoka, Japan) were housed 3 or 4 per cage under automatically controlled environmental conditions (temperature, 23 ± 1°C; humidity, 50 ± 10%) with a 12-h light/dark cycle and were allowed free access to tap water and standard laboratory chow. All rats were acclimatized to the housing conditions for 1 week. Animal care and all experimental procedures were approved by the Animal Experimentation Committee of the School of Dentistry, Aichi Gakuin University (approval no. AGUD533; approved on 1 April 2025) and conducted in accordance with the institutional guidelines for animal experimentation. Throughout the experimental period, all rats were monitored daily for general health, body weight, respiration, appearance, coat condition, and activity. Humane euthanasia was planned if severe distress, marked hypoactivity, difficulty in feeding or drinking, or a body weight loss of more than 20% was observed. No unexpected adverse events occurred, and no animals met the humane endpoint criteria during the experimental period. BTX was purchased from Sigma-Aldrich (St. Louis, MO, USA). Within each strain, rats were randomly allocated to the control or BTX groups by a blinded staff member not involved in the study using a computer-generated random number table. Rats in the SHR(BTX) and WKY(BTX) groups received BTX orally by gastric gavage at a dose of 1.0 mg/kg once daily for 4 weeks, while rats in the SHR(control) and WKY(control) groups received an equivalent volume of saline. At the end of the experiment, the maxillae were excised. Sample sizes were determined based on previous studies using similar experimental models; no a priori sample size calculation was performed.

2.2. Experimental Tooth Movement

Experimental tooth movement was initiated 1 week after the start of drug administration. The rats were anesthetized by intraperitoneal administration of a mixture of medetomidine hydrochloride (Meiji Seika Pharma Co., Ltd., Tokyo, Japan), midazolam (Astellas Pharma Inc., Tokyo, Japan), and butorphanol tartrate (Meiji Seika Pharma Co., Ltd., Tokyo, Japan). In accordance with the methods described by Dunn et al. [17] and Sato et al. [7], a Ni-Ti closed-coil spring delivering a force of 50 gf (Sentalloy®, Tomy, Japan) was attached between the maxillary incisors and the maxillary left first molar using a 0.020-inch ligature wire to make the left first molar move mesially for 3 weeks. Composite resin (TRANSBOND; 3M Unitek Japan) was applied over the ligature wire on the incisors to prevent slippage and pulpal irritation from exposed dentin (Figure 1(a), b).

2.3. Analysis of Tooth Movement Distance and Root Morphology Using Micro-Computed Tomography

Three weeks after the initiation of experimental tooth movement, the maxillae were excised and subjected to three-dimensional (3D) structural analysis using micro-computed tomography (μCT; Rigaku, Tokyo, Japan). The scanning parameters were as follows: tube voltage, 90 kV; tube current, 150 μA; scanning time, 2 min; and voxel size, 20 × 20 × 20 μm. The distance of tooth movement was evaluated by measuring the narrowest distance between the maxillary left first and second molars. In addition, the volume of the distopalatal root of the maxillary first molar was measured by defining the region from the furcation to the root apex as the region of interest (ROI), segmenting the root hard tissue from the surrounding tissues on 3D reconstructed images, and measuring the segmented root volume using dedicated software (TRI/3D-BON; Ratoc System, Tokyo, Japan).

2.4. Blood Pressure Measurement

Blood pressure was measured using the tail-cuff method with a noninvasive automatic blood pressure monitoring system for rats and mice (BP-98A-L; Natsume, Tokyo, Japan). The warming chamber was set to 39°C, and after 5 min of warming, the cuff was placed between the proximal and middle portions of the tail while the rat was in a stable condition. Measurements were started once the pulse had stabilized, and the mean of 3 measurements was used for analysis. BTX was administered immediately after blood pressure measurement (Figure 1c).

2.5. Histopathological Examination

The harvested maxillae were fixed in 10% neutral-buffered formalin and decalcified in 10% EDTA (pH 7.2) at 4°C for 4 weeks. The specimens were embedded in paraffin and cut into serial mesiodistal sections of 5 μm in thickness. The observation plane was selected so that all roots in the molar region could be examined.

To measure the number of odontoclasts per root surface (Oc.N/BS) and odontoclast surface per root surface (Oc.S/BS), tartrate-resistant acid phosphatase (TRAP) staining was performed using a TRAP staining kit (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Based on the method of Mavragani et al. [15], a rectangular ROI measuring 2,000 × 500 μm2 was defined on the mesial side of the distopalatal root of the maxillary first molar in the TRAP-stained sections. Within the ROI, TRAP-positive multinucleated cells in contact with the root surface or located within resorption lacunae were defined as odontoclasts. The number of odontoclasts and the proportion of the root surface covered by odontoclasts were measured using ImageJ (Figure 1d).

Hematoxylin and eosin staining was performed to determine the number of cementocytes per unit area of cellular cementum and the empty lacunae ratio on the compression side of the distopalatal root of the maxillary first molar. The empty lacunae ratio was calculated as the number of empty lacunae divided by the sum of the numbers of empty lacunae and cementocytes within the cellular cementum and expressed as a percentage (Figure 1e-g).

Next, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining was performed using a TUNEL Assay Kit–HRP-DAB (ab66110; Abcam, Cambridge, UK) to detect apoptotic cells. TUNEL-positive cells were counted within the cellular cementum on the mesial side of the distopalatal root of the maxillary first molar and expressed as the number of cells per unit area.

Immunohistochemical staining for tumor necrosis factor (TNF)-α was also performed using Histofine Simple Stain Rat MAX-PO and Histofine Simple Stain DAB solution (Nichirei Bioscience Inc., Tokyo, Japan), along with an anti-TNF-α antibody (ab6671; 1:250; Abcam Inc.). In accordance with the method described by Rogers et al. [18], the proportion of the positively stained area within the periodontal ligament space between the first molar and the alveolar bone was determined and classified into four categories: 0–20%, 21–40%, 41–60%, and ≥61%. These categories were assigned scores of 1, 2, 3, and 4, respectively. The stained images were scored independently by two evaluators.

2.6. Statistical Processing

The experimental data are presented as the mean ± standard deviation. Statistical analyses were performed using two-way analysis of variance, with strain (WKY/SHR) and treatment (control/BTX) as factors, followed by Šídák’s multiple-comparison test. Comparisons between two independent groups in the supplementary analyses were performed using an unpaired Student’s t-test. All statistical analyses were performed using IBM SPSS Statistics Ver. 30 (IBM, Armonk, NY, USA). A P value of <0.05 was considered statistically significant.

3. Results

3.1. Changes in Body Weight

There were no significant differences in body weight among the groups at the start of the experiment. All groups showed similar increases in body weight during the experimental period, with no significant differences among the groups (Figure 2a).

3.2. Changes in Blood Pressure

Blood pressure was measured at the start of experimental tooth movement and 21 days later. On days 0 and 21 of tooth movement, the SHR(control) group showed significantly higher blood pressure values than the WKY(control) group. The SHR(BTX) group also showed significantly higher blood pressure values than the WKY(BTX) group. However, no significant differences in blood pressure were observed between days 0 and 21 of tooth movement within any group (Figure 2b).

3.3. Tooth Movement Distance

The mesial movement distance of the first molar was significantly greater in the SHR(control) group than in the WKY(control) group. In both the WKY and SHR groups, BTX-treated animals showed significantly lower values for the mesial movement distance of the first molar than control animals (Figure 3a-c).

3.4. Root Length and Root Volume

On the non-loaded side, there were no significant differences in root length or root volume between the WKY and SHR groups (Figure S1). The SHR(control) group showed significantly lower values for root length and root volume than the WKY(control) group. No significant differences in root length or root volume were observed between the WKY(BTX) and WKY(control) groups, whereas the SHR(BTX) group showed significantly higher values for both root length and root volume compared with the SHR(control) group (Figure 4b, c). Furthermore, even under conditions in which the distance of tooth movement was adjusted to a comparable level, the SHR group still showed significantly lower values for root length and root volume compared with the WKY group (Figure S2).

3.5. Odontoclast Number and Odontoclast Surface

Compared with the WKY(control) group, the SHR(control) group showed significantly higher values for odontoclast number and odontoclast surface. No significant differences were observed between the WKY(BTX) and WKY(control) groups. In contrast, the SHR(BTX) group showed significantly lower values for odontoclast number and odontoclast surface compared with the SHR(control) group (Figure 5a-c).

3.6. Inflammatory Cytokines

The TNF-α immunostaining score was significantly higher in the SHR(control) group than in the WKY(control) group. No significant difference was observed between the WKY(BTX) and WKY(control) groups. In contrast, the SHR(BTX) group showed a significantly lower TNF-α immunostaining score compared with the SHR(control) group (Figure 6a, b).

3.7. Cementocyte Number and Empty Lacunae Ratio

The SHR(control) group showed a significantly lower number of cementocytes per unit area and a significantly higher empty lacunae ratio than the WKY(control) group. No significant differences in cementocyte number or empty lacunae ratio were observed between the WKY(BTX) and WKY(control) groups. In contrast, the SHR(BTX) group showed a significantly greater cementocyte number and a significantly lower empty lacunae ratio compared with the SHR(control) group (Figure 7a, b).

3.8. Apoptotic Cells

The SHR(control) group showed a significantly higher number of TUNEL-positive cells than the WKY(control) group. No significant difference was observed between the WKY(BTX) and WKY(control) groups. In contrast, the SHR(BTX) group showed a significantly lower number of TUNEL-positive cells than the SHR(control) group (Figure 8a, b).

4. Discussion

The involvement of the sympathetic nervous system in bone metabolism and orthodontic tooth movement has recently attracted growing attention. Sato et al. reported that the distance of tooth movement was increased in SHR and that administration of BTX, a selective β2-AR antagonist, suppressed both tooth movement and alveolar bone resorption [7]. Kondo et al. also demonstrated that TH-positive nerve fibers in the periodontal ligament increased during tooth movement and that sympathetic nervous signaling contributed to osteoclast activation and enhanced tooth movement [9]. In the present study, we used the experimental tooth movement model described by Dunn et al. [17] and Sato et al. [7] to investigate the impact of hypertension on OIRR and the effects of β2-AR antagonists. The results showed that SHR exhibited a greater distance of tooth movement and reduced root length and root volume compared with WKY rats. On the other hand, BTX administration suppressed the increase in tooth movement distance and the changes in root morphology observed in SHR. These findings suggest that hypertension-associated sympathetic hyperactivity may be involved not only in tooth movement under orthodontic force but also in the progression of root resorption.

Root resorption is known to occur as a result of sterile inflammation in the periodontal ligament on the compression side [13,19]. Zhong et al. have described that orthodontic force increases compressive hydrostatic pressure within the periodontal ligament and that root resorption becomes more likely to occur when this pressure exceeds capillary pressure [12]. In the present study, SHR showed reductions in root length and root volume, suggesting that hypertension may increase susceptibility to root resorption in response to orthodontic force. In addition, the observation that BTX administration suppressed the reductions in root length and root volume in SHR suggests that blockade of β2-AR signaling may inhibit OIRR.

We then evaluated changes in odontoclasts on the root surface on the compression side. During the early stage of tooth movement, periodontal ligament compression causes impaired blood flow and hyalinization, thereby contributing to inflammatory cell infiltration and odontoclast induction [13,19]. Regarding the relationship between the sympathetic nervous system and bone metabolism, Togari has reported that β2-ARs are expressed on osteoblasts and osteoclasts and that sympathetic neurotransmitters such as noradrenaline may promote osteoclastogenesis and bone-resorptive activity through β2-AR signaling [20]. In the present study, SHR showed increases in odontoclast number and odontoclast surface, and these increases were suppressed by BTX administration. Therefore, hypertension-associated sympathetic hyperactivity may have promoted odontoclast induction or activation on the root surface through β2-AR signaling, thereby contributing to the progression of OIRR.

Inflammatory cytokines are also considered to play an important role in the progression of root resorption. Rogers et al. found, in an LPS-induced periodontitis model, an increased expression of TNF-α, interleukin (IL)-1β, and IL-6 and an increase in TRAP-positive cells, accompanied by enhanced bone resorption [18]. Takeguchi et al. also reported that, in an experimental periodontitis model using SHR, BTX administration reduced the expression of TNF-α, IL-1β, and IL-6, decreased the number of osteoclasts, and suppressed alveolar bone resorption [11]. Furthermore, Muramatsu et al. demonstrated that administration of guanabenz, which reduces sympathetic nervous activity, decreased the expression of TNF-α, IL-1β and IL-6, decreased the number of TH-positive cells, and suppressed alveolar bone resorption in an experimental periodontitis model using SHR [21]. In the present study, SHR likewise showed an increased TNF-α immunostaining score in the periodontal ligament space, and this increase was suppressed by BTX administration. These findings suggest that β2-AR-mediated sympathetic signaling may enhance inflammatory cytokine expression in the periodontal ligament under orthodontic force and thereby promote OIRR through odontoclast induction.

In the present study, we also evaluated the number of cementocytes, the empty lacunae ratio, and the number of TUNEL-positive cells in cellular cementum. On the compression side under orthodontic force, impaired blood flow and hyalinization are known to occur and induce cell death [19,22]. Osteocytes respond to mechanical stimuli and changes in the local environment, and apoptotic death of these cells has been suggested to be involved in tissue remodeling [23]. Hamaya et al. noted the presence of TUNEL-positive cells and apoptotic changes in alveolar bone osteocytes adjacent to the hyalinized periodontal ligament during tooth movement [24]. Moin et al. also found an increased number of TUNEL-positive osteocytes in a tooth movement model, followed by an increase in TRAP-positive osteoclast formation, suggesting the possible involvement of osteocyte death in the induction of bone resorption [25].

Cementocytes share morphological characteristics with osteocytes and may respond to changes in the mechanical environment [26]. Matsuzawa et al. found that the numbers of cleaved caspase-3-positive and ssDNA-positive cementocytes increased in the cellular cementum on the compression side during tooth movement, followed by root resorption mediated by TRAP-positive cells [22]. An increase in empty lacunae is also thought to reflect cementocyte loss and impaired maintenance of cementum homeostasis [22,26,27]. In the present study, SHR showed a decreased number of cementocytes, an increased empty lacunae ratio, and an increased number of TUNEL-positive cells in cellular cementum. However, these changes were suppressed by BTX administration. Thus, hypertension-associated sympathetic hyperactivity may impair cementum homeostasis not only through induction of odontoclasts but also through cementocyte damage and apoptosis, thereby contributing to the progression of OIRR.

Taken together, the present study demonstrated that exacerbation of OIRR in SHR was accompanied by increased inflammatory cytokine expression in the periodontal ligament space, increased odontoclasts on the root surface, and decreased cementocytes and increased TUNEL-positive cells in cellular cementum. These findings suggest that hypertension-associated sympathetic hyperactivity may promote OIRR through enhanced inflammatory cytokine expression, odontoclast induction, and cementocyte damage. In addition, the selective β2-AR antagonist BTX may alleviate OIRR by suppressing these responses.

The novelty of this study lies in demonstrating an association between sympathetic hyperactivity and OIRR. Given that hypertension is a highly prevalent chronic disease and that the number of adult orthodontic patients is increasing, the present findings, which suggest that the systemic condition associated with hypertension may influence the risk of root resorption during orthodontic treatment, are considered clinically important. However, some limitations should be kept in mind. Because this was an animal study, caution is required when directly extrapolating these findings to human patients with hypertension. In addition, the present study did not directly evaluate β2-AR expression in cementocytes, leaving it unclear whether sympathetic signaling acted directly on cementocytes or affected them indirectly through inflammatory cytokines or other mediators. Another limitation of this study is that sympathetic nervous activity itself was not directly measured. Further studies are therefore needed to investigate β2-AR expression in cementocytes and the direct relationship between sympathetic signaling and cementocyte damage.

Taken together, these findings suggest that hypertension-associated sympathetic hyperactivity may promote OIRR through enhanced inflammatory cytokine expression, odontoclast induction, and cementocyte damage under orthodontic force. In addition, β2-AR signaling may represent a novel biological target for controlling root resorption during orthodontic treatment.

5. Conclusions

This study demonstrated increased root resorption associated with tooth movement in an experimental tooth movement model using SHRs. SHR showed reduced root length and root volume, increased odontoclast numbers, enhanced inflammatory cytokine expression, decreased cementocyte numbers, and an increased empty lacunae ratio. On the other hand, these changes were suppressed by administration of butoxamine, a β2-AR antagonist.

These findings suggest that hypertension-associated sympathetic hyperactivity may promote OIRR through enhanced inflammatory responses, odontoclast induction, and cementocyte damage. In addition, β2-AR signaling may represent a novel biological target for controlling root resorption associated with orthodontic treatment.