This study aimed to investigate the effect of EphB4/ephrinB2 signaling on orthodontically-induced root resorption repair and the possible molecular mechanism behind it.
Seventy-two 6-week-old male Wistar rats were randomly divided into 3 groups: blank control group, physiological regeneration group (PHY), and EphB4 inhibitor local injection group (INH). A root repair model was built on experimental rats of the PHY and INH groups. The animals in the INH groups received a daily periodontal local injection of EphB4 inhibitor NVP-BHG712, whereas the blank control group and PHY groups received only the vehicle.
Histologic staining and microcomputed tomography analysis showed that root regeneration was inhibited in the INH group compared with the PHY group with a greater number of osteoclasts. Immunohistochemical staining showed active EphB4/ephrinB2 signaling activities during root regeneration. The cementogenesis-related factors cementum attachment protein, alkaline phosphatase, osteopontin, and runt-related transcription factor 2, and osteoclastic-related factors RANKL and osteoprotegerin were affected by regulated EphB4/ephrinB2 signaling.
These findings demonstrated that the EphB4/ephrinB2 signaling might be a promising therapeutic target for novel therapeutic approaches to reduce orthodontically-induced root resorption through enhancement of cementogenesis.
EphB4/ephrinB2 signaling activity was found during physiological root regeneration.
Inhibition of signaling hindered the formation of new cementum after heavy forces in rats.
- • EphB4/ephrinB2
signaling is essential for the regeneration of orthodontically-induced root resorption.
EphB4/ephrinB2 signaling helps speed the repair of orthodontically-induced root resorption.
One of the most common undesirable complications of orthodontic treatment is orthodontically-induced root resorption (OIRR). Characterized by the loss of apical root material, such as mineralized cementum and dentin, this clinical problem can be affected by the type of tooth movement, force magnitude, and duration of treatment. If handled improperly, OIRR may occasionally lead to severe root shortening that threatens the health of the tooth and even the stability of the treatment results. Therefore, investigating the underlying mechanisms of OIRR and developing novel therapeutic approaches to accelerate its regeneration has been a popular research subject in recent years.
As a specialized calcified substance covering the root of a tooth, cementum is the part of the periodontium that attaches the teeth to the alveolar bone by anchoring the periodontal ligament (PDL). Under physiological conditions, cementum is fundamental in maintaining periodontal homeostasis and long-term stability of teeth, protecting root from resorption caused by infection, trauma, or orthodontic treatment. , Its ability to maintain its shape and repair during tooth movement is considered an important biological foundation of orthodontic treatment. Meanwhile, external root resorption tends to begin with the resorption of cementum when subjected to undesirable local orthodontic force. , Therefore, many studies recently have focused on cementum regeneration under a pathologic state. Among them, some have shown that resorption craters might be restored through stimulation of cementogenesis. This suggests that promoting cementogenesis could be a practical way of solving OIRR.
Eph receptors belong to a subfamily of receptor tyrosine kinases activated by ligands called Eph receptor-interacting proteins (ephrins). Ephs and ephrins are both divided into 2 A and B groups. Generally, EphA receptors (EphA1-A8, A10) interact with ephrinA (ephrinA1-A5), whereas EphB receptors (EphB1-B6) interact with ephrinB ligands (ephrinB1-B3). The Eph/ephrin signaling participates in a wide spectrum of developmental processes, including spatial organization of different cell populations, axon guidance, formation of synaptic connections between neurons, and blood vessel remodeling. Its cross-regulation with other communication pathways guarantees the proper function of the adult body, including the skeletal system. , , In terms of bone biology, the bidirectional EphB4/ephrinB2 signaling is found to be particularly important in the maintenance of bone homeostasis. The reverse signaling through ephrinB2 into osteoclast precursors suppresses osteoclast differentiation by inhibiting the osteoclastogenic cellular oncogene-Fos (c-Fos)-nuclear factor of activated T cells c1 (NFATc1) cascade, whereas forward signaling through EphB4 into osteoblasts enhances osteogenic differentiation, and overexpression of EphB4 in osteoblasts increases bone mass in transgenic mice. ,
Recent studies suggested that EphB4/ephrinB2 pathway might also play a vital role in the remodeling of alveolar bone. One study reported that transgenic expression of ephrinB2 in PDL stem cells could promote osteogenic differentiation via stimulation of the phosphorylation of ephrinB2 and EphB4. During orthodontic tooth movement, upregulation of ephrinB2-EphB4 signaling between fibroblasts within the PDLs and osteoblasts of the alveolar bone might contribute to osteogenesis at tension sites. Stimulated with ephrinB2, osteoblasts increased their osteoblastogenic genes runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) expression and showed functional signs of osteoblastic differentiation. In compression areas, EphB4 and ephrinB2 expression, was significantly decreased, which contributed to alveolar bone resorption through regulating transcription factors like Runx2 and Sp7. Although much research progress has been made on the regulatory role of EphB4/ephrinB2 signaling in alveolar bone remodeling, studies are lacking on its biological functions on other oral and maxillofacial tissues.
Root resorption repair is a process in which dynamic cementum restoration and periodontal tissue remodeling take place. Considering the similar biochemical composition and molecular biological properties between cementum and bone, it is reasonable to hypothesize that the EphB4/ephrinB2 pathway, as a crucial regulator, could also involve in cementum formation during root regeneration. Applying in vivo approaches, we aimed to elucidate the effect of EphB4/ephrinB2 signaling on OIRR and the possible molecular mechanism behind it.
Material and methods
All experimental procedures were approved by the Ethics Committee of West China Hospital of Stomatology. The number of experimental animals used in this study was determined on the basis of the sample size calculation using the resource equation approach according to a previous study. For group comparison using 1-way analysis of variance, 6 was an appropriate number of animals per group, and the total sample size was calculated as 72 for this study. Accordingly, 72 6-week-old male Wistar rats weighing 200 ± 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 with a soft diet and water ad libitum.
After 2 days of acclimatization, an orthodontic appliance was randomly applied on 48 experimental animals, and 100 g of heavy force was exerted to create OIRR using an orthodontic elastic closed-coil spring (Grikin Advanced Materials, Beijing, China) fixed between the maxillary left first molar and the incisors. , Same orthodontic appliance was applied on the rest 24 rats without activation to serve as a blank control group (CON). After 2 weeks, coil springs were removed, and orthodontic wires were fixed between the maxillary left first molars and the incisors passively to maintain the results of tooth movement. At this point, the molar roots of experimental rats started to regenerate from OIRR. Six rats of each group were killed immediately by an overdose of pentobarbital to investigate the original state right after root resorption (day 0).
Experimental animals were randomly divided into 2 groups of 24 animals: physiological regeneration group (PHY) and EphB4 inhibitor local injection group (INH). The animals in the INH groups received daily periodontal local injection of 20 μL EphB4 inhibitor NVP-BHG712 (A8683; APExBIO, Houston, Tex) on the attached gingiva on both the buccal and palatal side of the maxillary left first molar with a concentration of 4 μmol/L. The CON group and PHY group received only the vehicle (0.1% acetic acid) of the same volume. During the next 28 days of root repair period, animals of each group received injections daily until they were killed randomly after 7, 14, and 28 days (n = 6), and alveolar bone blocks that included the maxillary left first molar were harvested. The regions of interest consisted of the distal buccal root of the molar and the adjacent PDLs. All experiment procedures and the intraoral picture of the animal models were presented in Figure 1 , A and B .
Then, all the samples were scanned using the high-resolution microcomputed tomography 50 system (Scanco Medical, Brüttisellen, Switzerland) with a voxel resolution of 10 μm, passing through a 3-dimensional (3D) Gaussian filter (mean, 1.2; filter support, 1). Mimics 21.0 software was employed to reconstruct the 3D model of the maxillary left first molar ( Fig 1 , C ). To evaluate the degree and extent of root resorption, we separated the distal buccal root of each sample, and the total volume of the resorption pits on the mesial surface of the distal buccal root was calculated. The calculation followed the method of the 3D Convex Hull Algorithm. , Considering the surface of the teeth was roughly a smooth convex curve initially, Mimics 21.0 was used to draw the assumed surface line around the resorption lacunae by the same technician ( Fig 1 , D ). By calculating the difference value of the root volume with and without the assumed surface, the size of the resorption lacunae can be determined. For each sample, the total volume of resorption lacunae was divided by its root length and expressed as resorption lacunae per millimeter of root length.
After microCT scanning, the left half of the maxilla of each animal was fixed and decalcified for paraffin embedding. Five-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. Selected sections were treated with hematoxylin and eosin (G1120; Solarbio, Beijing, China) and tartrate-resistant acid phosphatase (TRAP) (Sigma, St Louis, Mo) staining and examined under a light microscope (Eclipse 80i microscope; Nikon, Toyko, Japan). The number of TRAP-positive cells on the compression side of the periodontal area was counted and expressed as cell numbers per millimeter of root length.
For immunohistochemical staining, 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. Then sections were incubated with primary antibodies diluted in blocking solution with different dilution rates: EphB4 (20883-1-ap, 1:200; Proteintech, Wuhan, China); ephrinB2 (ET1705-33, 1:200; Huabio, Hangzhou, China); cementum attachment protein (CAP dilution 1:50, SC-53947; Santa Cruz, Shanghai, China); ALP (ET1601-21, 1:400; Huabio); osteopontin (OPN, 0806-6, 1:200; Huabio); Runx2 (ET1612-47, 1:400; Huabio); osteoprotegerin (OPG, R1608-4, 1:250; Huabio); RANKL (ab169966, 1:200; Abcam, Shanghai, China) and sclerostin (SOST, 21933-1-AP, 1:200; Proteintech), at 4°C overnight and then 37 °C for 1 hour. After a rinse, slides were incubated with goat antirabbit or goat antimouse IgG secondary antibody conjugated to horseradish peroxidase (SP-9000; Zhongshan Bio-Tech, Beijing, China) for 30 min at 37°C. The immunoreaction was visualized by using a 3.3’-diaminobenzidine kit (ZLI-9017; Zhongshan Bio-Tech, Beijing, China) according to the manufacturers’ instructions. The regions of interest were defined as the cementum in an apical third area of the compression side of the distal buccal root and the layer of cells (presume cementoblasts) lining on it. Within the regions of interest, areas with the proper stained color, which reflected positive immunoreactivities, were first selected. Integrated optical density of selected areas was then calculated by Image-Pro Plus (version 6.0; Media Cybernetics, Bethesda, Md). Finally, the means of integrated optical density in each group were calculated to reflect the average intensity of immunohistochemical staining.
Statistical analyses were performed using the SPSS (version 22; IBM Corporation, Armonk, NY). Data are presented as mean ± standard deviation from at least 3 independent experiments. Statistical comparison was performed using a 1-way analysis of variance. P <0.05 was considered statistically significant.
The process of root regeneration was first examined by hematoxylin and eosin staining ( Fig 2 , A ). Compared with the CON group, periodontal fibers of the PHY and INH groups lined irregularly, and large resorption lacunae were seen on the root surface on day 0. Along with the root repair process, force compressed PDLs lined more regularly, and the root surface became smoother. At the end of the experiment, the root surface of the PHY and INH groups appeared smoother.
To compare the root regeneration process between groups quantitatively, we used 3D models of all the samples that were reconstructed using Mimics 21.0 software ( Fig 2 , B ). In general, total volumes of resorption lacunae decreased over time, and the roughness of the reconstructed root surface also reduced. On day 0, no significant difference was found between PHY and INH groups. After that, faster cementum repair was found in the PHY group, with significantly smaller resorption volume than that of the INH group from day 7 to 28 ( Fig 3 , A ).
TRAP-positive cells resembling osteoclast were mainly observed on the compression side of the periodontal membrane ( Fig 4 ) . Few positive cells were found in the CON group throughout the experimental period under physiological conditions. On day 0, right after heavy force-induced root resorption, a considerable number of TRAP-positive cells appeared in both PHY and INH groups, which decreased along with the root regeneration process. On days 7 and 14, the positive cell number was significantly smaller in the PHY group than in the INH group. No significant difference was found between groups on day 28 ( Fig 3 , B ).