Introduction
This study aimed to investigate the effects of estrogen on root repair after orthodontically induced root resorption.
Methods
Seventy-two 6-week-old female Wistar rats were randomly divided into 3 groups: ovariectomy only (OVX), ovariectomy plus estradiol injection (OVX + E2), and sham operation (control). E2 was administrated to all the experimental animals after the establishment of the root repair model. One-way analysis of variance with the Tukey post-hoc test was used to analyze the experimental results.
Results
Micro-computed tomography and hematoxylin and eosin staining showed that the total volumes of resorption lacunae were significantly smaller in the control and OVX + E2 groups than those in the OVX group. Alkaline phosphatase and tartrate-resistant acid phosphatase stainings suggested that the cementoblastic activities and the amount of new cementum formation were inhibited while the activities of osteoclasts were obvious in the OVX group. The immunohistochemistry stainings revealed that the osteoprotegerin to receptor activator of nuclear factor-кB ligand ratio and the phosphorylated extracellular signal–regulated kinases to extracellular signal–regulated kinases ratio of the control and OVX + E2 groups were significantly greater than those of the OVX group.
Conclusions
These findings demonstrated that estrogen administration might be a solution to reduce orthodontically induced root resorption through the activation of extracellular signal–regulated kinase-1/2 pathway and enhancement of cementogenesis.
Highlights
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Estradiol (E2) had a positive effect on the regeneration of root resorption in ovariectomized rats.
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E2 promoted the function of cementoblasts and cementogenesis by activating extracellular signal–regulated kinase-1/2 pathway.
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E2 had an inhibitory effect on the cementoclasts in the late phase of root resorption.
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Estrogen therapy might help reduce orthodontically induced root resorption, especially for postmenopausal women.
With the advances in orthodontic techniques, a growing number of middle-aged and older women can seek orthodontic treatment to improve malocclusion and jaw deformities, achieving their increasing desire for better facial appearance. During clinic work, however, a high incidence of root resorption is often recorded among aged female patients, which jeopardizes their overall periodontal health and masticatory function. Therefore, it is a primary concern for orthodontists to develop methods of decelerating root resorption and promoting root repair to make orthodontic treatment more effective and safe among this high-risk population.
Estrogen is one of the most important sex hormones in human body. There are 3 major endogenous estrogens that have estrogenic hormonal activity: estrone, estradiol (E2), and estriol. Of these, E2 is the most potent and prevalent. Estrogen is responsible for the development and regulation of the female reproductive system and secondary sex characteristics. In addition, studies have found that estrogen is also the major hormonal regulator of bone metabolism. Estrogen is a requisite for growth and development of bones, proper closure of epiphyseal growth plates during bone growth, as well as bone turnover in adult bones. A decline of estrogen levels in females at menopause or males later in life leads to loss of bone mass and strength and contributes to the development of osteoporosis. , Estrogen deficiency leads to increased osteoclast formation and enhanced bone resorption. , At the cellular level, estrogen inhibits differentiation of osteoclast, probably through down-regulating the expression of osteoclastogenic cytokines from supportive cells and by directly suppressing the receptor activator of nuclear factor-кB ligand (RANKL)–induced osteoclast differentiation process. ,
As for the oral system, it has been reported that estrogen receptors (ERs) are widely found in periodontal tissues. Estrogen, acting through ERs in the cells of the periodontal ligament, has a regulatory interaction on a variety of physiological activities of the periodontium. , For female patients, especially postmenopausal women, the absence of estrogen has also been reported as a factor for promoting the loss of alveolar bone and root resorption during orthodontic therapies. , Because cementocyte resembles osteocyte in several aspects, especially the cell morphology, communication network, and cell functions, , it is reasonable to hypothesize that estrogen, as an important regulator of bone metabolism, might also play an important role during the process of root resorption repair.
Previous studies showed that estrogen regulated the phosphorylation of extracellular signal–regulated kinases (ERKs) and induced the osteogenic effects. By upgrading the expression target gene ger-1 of ERK, estrogen was able to activate the ERK1/2 pathway, thus inhibiting the apoptosis of osteoblast and increasing viability. , Recently, researchers have reported that the ERK1/2 pathway might also be an important intermediary signaling pathway for the cementogenesis and differentiation of cementoblast. , Inhibition of ERK1/2 phosphorylation inhibited the expression of cementum-related proteins. However, whether the ERK1/2 pathway is involved in the cementogenesis after root resorption, and what is the possible regulatory relationship between estrogen and the ERK1/2 pathway are largely unknown.
Our preliminary study has found that cementoblast cell line OCCM-30 expressed ERs. In addition, E2 promoted the expression of cementoblast mineralization factors and the deposition of calcium nodules. However, to date, few studies have investigated the possible anabolic effect of estrogen toward cementum formation in vivo. Therefore, this study tried to elucidate the effect of estrogen on root resorption repair after orthodontic tooth movement and the possible molecular mechanism behind it.
Material and methods
All experimental procedures were approved by the animal experiment ethics committee of the State Key Laboratory of Oral Diseases of Sichuan University in China. Seventy-two 6-week-old female Wistar rats weighing 150 ± 10 g were obtained from the university’s experimental animal center. They were kept in plastic cages with a standard 12-hour light-and-dark cycle and fed a soft diet with water ad libitum.
After 2 days of acclimatization, all the animals were randomly divided into 3 groups of 24 animals each: ovariectomy only (OVX), ovariectomy plus estradiol injection (OVX + E2), and sham-operation (control), according to the method of random number table. Thereafter, the rats of OVX and OVX + E2 groups underwent bilateral ovariectomy, and the control group underwent a sham operation.
After 2 weeks of recovery, we applied the orthodontic appliance on all experimental animals and exerted heavy force to create orthodontically induced root resorption (OIRR). An orthodontic elastic closed-coil spring (Grikin Advanced Materials, Beijing, China) was fixed between the maxillary left first molar and the incisors with ultraviolet curable resin (MB 4403; 3M Unitek, Monrovia, Calif) under anesthesia with an intraperitoneal injection of 2% ketamine hydrochloride at 2 mL per kilogram of body weight. The maxillary first molars were moved mesially with 100 g of force , ( Fig 1 ). The appliances were activated immediately on insertion, and the fit was checked daily. No reactivation was performed during the experimental period. After 2 weeks, the coil springs were removed, and orthodontic wires were fixed between the maxillary left first molars and the incisors to maintain the results of tooth movement. At this point, the root repair model was built. Six rats of each group were killed immediately after appliance removal to serve as initial controls (day 0).
Injections were administered to the rats after OIRR, during the period of root repair. The animals in the OVX + E2 group received a daily subcutaneous injection of 17β-estradiol (Sigma, St. Louis, Mo) at a dosage of 1 μg per 100 g of body weight dissolved in maize oil at a concentration of 2 μg/mL. The animals in the OVX and control groups received the same volume of vehicle (maize oil). The protocol of E2 injection was decided according to previous studies. , During the period of root repair, animals of each group were killed randomly after 7, 14, and 28 days (n = 6), and alveolar bone blocks that included the left first molar were harvested. The regions of interest consisted of the distal buccal root of the maxillary left first molar, and the adjacent periodontal ligament and alveolar bone. All experiment procedures are presented in Figure 2 .
All the samples were collected and scanned using the high-resolution micro-computed tomography (micro-CT) 50 system (Scanco Medical, Brüttisellen, Switzerland) with a voxel resolution of 10 μm, and passing through a 3-dimensional Gaussian filter (mean, 1.2; filter support, 1). The 3-dimensional stacks of images were then analyzed using VG Studio Max software (version 2.2; Volume Graphics, Heidelberg, Germany). To determine the status of alveolar bone around the roots right after the establishment of the root repair model, a 360 μm × 360 μm × 360 μm cube of trabecular bone mesial to the apical part of the distal buccal root of the maxillary left first molar was selected for analysis according to previous studies. , The distance between the cube and the root was 500 μm. Thereafter, the bone volume to total volume (BV/TV) ratio, trabecular spacing (Tb.Sp), trabecular number (Tb.N), and trabecular thickness (Tb.Th) in the cube region of the initial controls were calculated. Mimics 17.0 software was used in this study to reconstruct the 3-dimensional model of the maxillary. Three-dimensional images of the selected trabecular bone cubes were created to reflect the status of alveolar bone more directly. To judge the severity of root resorption, we separated the left first molar and calculated the total volume of the resorption pits on the mesial surface of the distal buccal root. The calculation followed the method of 3-dimensional Convex Hull Algorithm. , Assuming that the surface of the teeth was a smooth convex curve initially, we used Mimics 17.0 to measure the size of resorption lacunae by calculating the difference value between the assumed smooth surface and the pits ( Fig 3 ). The assumed surface line was required to coincide with the curvature of the root surface adjacent to the resorption lacunae.
After micro-CT scanning, the left half of the maxilla of each animal was prepared for light microscopic observation. The maxillary bone blocks were fixed in 4% paraformaldehyde in 0.1 mol/L of phosphate-buffered saline for 24 hours and decalcified in neutral 10% ethylenediaminetetraacetic acid at room temperature for at least 2 months. This solution was changed every day. After dehydration and paraffin embedding, 5-μm serial sections in a mesiodistal direction parallel to the long axis of the distal root of the first molar were cut on a microtome (HM 355S; Microm International, Walldorf, Germany) and mounted on glass slides.
For morphologic examination, selected sections were stained with hematoxylin and eosin (HE), following the manufacturer’s instructions (G1120; Solarbio, Beijing, China). Tartrate-resistant acid phosphatase (TRAP) staining was performed for the observation of osteoclast activities (Sigma, St. Louis, Mo). The numbers of TRAP-positive cells in the same periodontal area of the compression side of the roots were counted and subjected to statistical analysis. The stained slides were examined twice by 2 operators (T.L. and C.Z.), and any disagreement was resolved through discussion or assessment by a third investigator (Z.Z.). All the measurements were done using the single-blind method. In addition, histologic observation of the alkaline phosphatase (ALP) staining was performed in paraffin-embedded sections for the evaluation of osteoblast activities. ALP staining was performed with BCIP/NBT Alkaline Phosphatase Colour Development Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Three sections per animal at each group were stained with HE, TRAP, and ALP staining. Each stained slide was required to be selected at intervals of at least 5 sections. Slides from all the individual rats were stained and analyzed in this study. These sections were observed under a microscope (ECLIPSEE200; Nikon Instruments, Melville, NY) and a digital camera system (Penguin 600CL CCD; Pixera, San Jose, Calif), and microphotographs were taken.
For the immunohistochemistry staining of osteoprotegerin (OPG), RANKL, ERK1/2 and phosphorylated ERK1/2 (P-ERK1/2), tissue sections were placed in 3% hydrogen peroxide for 30 minutes in the dark. Subsequently, sections were blocked in a blocking solution containing 4% bovine serum albumin for 20 minutes to prevent unspecific background staining. Thereafter, sections were incubated with polyclonal primary antibodies diluted in blocking solution with different dilution rates: OPG (ab73400), 1:200; RANKL (ab62516), 1:400; ERK1/2 (ab17942), 1:200; P-ERK1/2(ab214362), 1:400, at 4°C overnight in a humidified chamber. The slides were rinsed and then incubated for 30 minutes with anti-rabbit immunoglobulin (ZSGB-Bio; Beijing, China) diluted 1:100 in phosphate-buffered saline solution as a secondary antibody. After they were rinsed, the tissue sections were stained in a 3,3′-diaminobenzidine solution for about 30 seconds, rinsed, and then counterstained with Mayer’s hematoxylin, dehydrated, and cover-slipped for light microscopic analysis. To prove the specificity of the immunoreactions, we carried out negative controls by omitting the primary antibody. The immunoreactivities of OPG, RANKL, ERK1/2 and P-ERK1/2 were evaluated using Image J software (National Institutes of Health, Bethesda, Md) according to previous studies. , First, all the images were converted to gray scale images. Then, areas with proper density, which reflected positive immunoreactivities, were selected, and the total positive areas were measured. Finally, the positive cytoplasm staining area and/or observation area ratio was calculated to reflect the immunoreactive intensity.
Statistical analysis
The data were processed with SPSS statistical software (version 11.5; SPSS, Chicago, Ill). The results were presented as means ± standard deviations. The data of the micro-CT measurements, cell counts, OPG, RANKL, ERK1/2, and P-ERK1/2 immunoreactivities were compared between groups at the same time point using one-way analysis of variance with Tukey post-hoc test. The significance level was set at P <0.05.
Results
The state of alveolar bone was detected by values of BV/TV, Tb.Sp, Tb.N, and Tb.Th of the region of interest. All the measurements of bone parameters of the initial controls (day 0) in all 3 groups had no significant difference, and the status of alveolar bone looked similar on the 3-dimensional images of the selected trabecular bone cubes ( Table and Supplementary Fig ). No significant difference was found on other experimental days either ( Supplementary Table ). Figure 4 shows the reconstructed 3-dimensional model of the distal buccal root of the left first molar by Mimics 17.0. The total volume of resorption lacunae on the mesial surface was calculated ( Fig 5 , A ). On day 0, the total volumes of resorption lacunae in all 3 groups had no significant difference. Thereafter, the total volumes of resorption lacunae decreased over time, and the reconstructed tooth models displayed that the levels of roughness on the root surface were also reduced. On day 14 and day 28, the resorption volume of control and OVX + E2 groups were significantly smaller than that of the OVX group ( P <0.05).
| Parameters | Control | OVX | OVX + E2 | P |
|---|---|---|---|---|
| BV/TV (%) | 0.40 ± 0.02 | 0.37 ± 0.03 | 0.35 ± 0.03 | NS |
| Tb.Sp (mm) | 0.25 ± 0.01 | 0.24 ± 0.02 | 0.26 ± 0.01 | NS |
| Tb.N (mm −2 ) | 2.10 ± 0.11 | 2.10 ± 0.15 | 2.12 ± 0.13 | NS |
| Tb.Th (mm) | 0.19 ± 0.01 | 0.17 ± 0.02 | 0.21 ± 0.02 | NS |
Multinucleated clastic cells appeared around the resorption lacunae on the surface of the compression side of mesial roots ( Fig 6 ). These cells, assuming cementoclasts, were supposed to form the resorption lacunae around themselves. Coinciding with the results of micro-CT analysis, the size of the resorption lacunae of all 3 groups decreased from day 0 to day 28. On day 28, the remnant resorption lacunae were larger in the surface of the roots of the OVX group than in control and OVX + E2 groups.
