Introduction
The Wnt signaling pathway acts as a key regulator of skeletal development and its homeostasis. However, the potential role of Wnt1 in the mechanotransduction machinery of orthodontic tooth movement-initiated bone remodeling is still unclear. Hence, this study focused on the regulatory dynamics of the Wnt1 expression in both the periodontal ligament (PDL) and osteocytes in vivo and in vitro.
Methods
The Wnt1 expression in the orthodontically moved maxillary first molar in mice was assessed at 0, 1, and 5 days, on both the compression and tension sides. Primary isolated human PDL (hPDL) fibroblasts, as well as murine long-bone osteocyte-Y4 (MLO-Y4) cells, were exposed to continuous compressive force and static tensile force.
Results
The relative quantification of immunodetection showed that orthodontic tooth movement significantly stimulated the Wnt1 expression in both the PDL and alveolar osteocytes on the tension side on day 5, whereas the expression on the compression side did not change. This increase in the Wnt1 expression, shown in vivo, was also noted after the application of 12% static tensile force in isolated hPDL fibroblasts and 20% in MLO-Y4 cells. In contrast, a compressive force led to the attenuation of the Wnt1 gene expression in both hPDL fibroblasts and MLO-Y4 cells in a force-dependent manner. In the osteocyte-PDL coculture system, recombinant sclerostin attenuated Wnt1 in PDL, whereas the antisclerostin antibody upregulated its gene expression, indicating that mechanically-driven Wnt1 signaling in PDL might be regulated by osteocytic sclerostin.
Conclusions
Our findings provide that Wnt1 signaling plays a vital role in tooth movement–initiated bone remodeling via innovative mechanotransduction approaches.
Highlights
- •
Wnt1 plays a part in tooth movement–initiated mechanotransduction in alveolar bone remodeling.
- •
Orthodontic tooth movement regulated the differential expression of Wnt1 in vivo.
- •
A biphasic role of Wnt1 has been shown with tension and compressive force in vitro.
Bone remodeling is highly important in the maintenance of the normal skeletal structure and is also a key factor in tooth movement. The movement of teeth through the bone requires remodeling of both periodontal tissues and alveolar bone. The controlled application of mechanical forces on teeth can activate biologic responses in periodontal tissues, which leads to the triggering of the remodeling process in the adjacent alveolar bone, resulting in tooth movement. This sequential mechanism of tooth movement–initiated bone remodeling is orchestrated by 2 types of mechanical stimuli: compression force-associated bone resorption (a catabolic process) and tension force-associated bone formation (an anabolic process). , Past evidence has demonstrated that an association exists between mechanical stimuli and tooth movement, from the viewpoints of both cellular biology and the signaling molecules that produce bone remodeling.
Wnt genes and their signaling pathways play a preferential role in most of the biologic processes of mechano-induced bone remodeling by regulating osteogenic differentiation via the augmentation of multiple osteogenic genes. , The Wnt ligand–receptor complex initially triggers a cellular response, thereby activating the intracellular Wnt signaling cascades. Intriguingly, there is genetic and clinical evidence to support that Wnt ligands are involved in the regulation of bone thickness, osteoporotic fracture, and bone strength. A past study also found that the homeostatic control of the periodontal ligament (PDL) is Wnt-dependent, whereas the elevation or suppression of Wnt signaling alters the expression of osteogenic genes.
Nineteen Wnt ligands, 10 cell-surface frizzled receptors, and several Wnt coreceptors direct signaling along 3 intracellular pathways: the canonical Wnt pathway, the noncanonical Wnt pathway, and the Wnt calcium pathway. Wnt1 is a Wnt ligand with major roles in normal skeletal development and homeostasis. This protein is mainly expressed in osteocytes rather than osteoblasts or osteoclasts. Several lines of evidence have indicated that a severe form of recessive osteogenesis imperfecta occurs in early childhood because of homozygous mutations of WNT1, whereas early-onset osteoporosis in adolescents or young adults was shown to be led by heterozygous mutations. Either the overexpression or inactivation of WNT1 production by osteocytes impacts bone formation and bone mass, indicating an essential role of WNT1 in bone development and homeostasis.
The canonical Wnt pathway acts as a regulator of bone mass that comes through the study of mutations in the low-density lipoprotein receptor-related protein (LRP) 5 Wnt coreceptor. Wnt1 ligands bind the first beta-propeller of the LRP5/6 Wnt coreceptors. , The sclerostin (Scl) binds the same first beta-propeller, which impair the binding of Wnt ligands, thereby antagonizing the canonical Wnt pathway. , In fact, antisclerostin antibodies have been currently developed in a clinical trial as an anabolic strategy in osteoporosis and other low bone mass conditions. , These previous findings lead to requiring further consideration to elucidate the interactions between Scl and Wnt1 ligands in the canonical Wnt signaling pathway that contribute to the alveolar bone remodeling process.
We previously reported that orthodontic tooth movement (OTM) stimulates Scl in alveolar osteocytes, whereas PDL-derived Scl acts as a principal mediator of the bone remodeling process. However, the relationship between tooth movement–initiated bone remodeling and the expression of Wnt1 remains unclear. Further study is therefore essential to investigate the mechanisms of the actions of Wnt1 during tooth movement as well as to elucidate the interactions between Scl and Wnt1 in the alveolar bone remodeling process.
We hypothesized that Wnt1 might act as an integral component of the mechanotransduction machinery of tooth movement. To address this hypothesis, we investigated the regulatory dynamics of the Wnt1 expression in both osteocytes and the PDL using OTM model mice. In addition, we explored the role of Wnt1 ligands on tension-induced bone formation and compression-induced bone resorption in vitro. The current study will help to gain insight into the molecular level of bone remodeling in OTM, which will be useful in the development of new therapeutic approaches and techniques for improving orthodontic treatment.
Material and methods
All animal procedures were performed in accordance with the regulations of Okayama University’s Committee on Use and Care of Animals (OKU-2018687). Eighteen male, 8-week-old Institute of Cancer Research (ICR) mice were purchased from CREA Japan (Tokyo, Japan). There were 6 mice at each time point (0, 1, and 5 days). After anesthetizing the mice with an intraperitoneal injection of isoflurane (10 g/kg), the OTM appliances were set at the right side and activated to deliver 10 g of force at 0, 1, and 5 days as shown in Figure 1 , A . On day 0, mice without tooth movement were used as a control group. Serial sections of the distobuccal root of the maxillary first molars were prepared. The mesial surface of the distal-buccal root was considered to be the compression side, whereas the distal surface was considered to be the tension side, according to the direction of tooth movement.
Formalin-fixed and paraffin-embedded tissue sections were blocked in universal blocking reagent (Nacalai Tesque, Kyoto, Japan), then incubated overnight at 4°C in a humidified container with anti-Wnt1 rabbit polyclonal antibody (Abcam, Cambridge, United Kingdom) diluted 1:100 in phosphate-buffered saline with 1% bovine serum albumin (Sigma-Aldrich, St Louis, Mo) followed by Alexa Flour 488 rabbit antigoat IgG (Thermo Fisher Scientific KK, Yokohama, Japan) secondary antibody. Nuclei were stained with Hoechst 33342 (Sigma-Aldrich). The slides were then washed with phosphate-buffered saline with tween 20 and embedded in fluorescence mounting medium (Dako, Carpinteria, Calif). Images were captured with a DP72 microscope (Olympus, Tokyo, Japan) with the same settings. Image processing was uniformly performed using ImageJ/Fiji for the quantification of Wnt1 immunoreactivity.
Human PDL (hPDL) fibroblasts were isolated from human extracted premolars, as described previously. The cells were seeded onto plastic cell culture dishes (Greiner Bio-One, Frickenhausen, Germany) in 10% heat-inactivated fetal bovine serum (HyClone Laboratories, Logan, Utah) with antibiotics. MLO-Y4 cells (an osteocyte-like cell line) were cultured onto 2-dimensional (2D) collagen-coated cell culture plates (AGC Techno Glass, Shizuoka, Japan) or 3-dimensional (3D) collagen gel (1.5 × 10 6 cells/mL; Nitta Gelatin Co, Ltd, Osaka, Japan), as described previously. The cells were maintained at approximately 60% confluency throughout the culture period, and the medium was changed after 24 hours and every 2 or 3 days.
Continuous compression force was applied to MLO-Y4 cells and hPDL fibroblasts, as described previously. A confluent cell layer was compressed by 0.24 g/cm 2 or 2.40 g/cm 2 using a glass cylinder for 3 hours, 6 hours, or 48 hours, according to the methods of our previous report.
Both MLO-Y4 cells and hPDL fibroblasts were grown in a 20 × 20-mm silicone hybrid chamber (SC4Ha; Menicon Life Science, Nagoya, Japan) containing culture medium at 37°C under 5% CO 2 for approximately 24 hours then statically stretched for 3 hours, 6 hours, or 48 hours with a stretch magnitude of 12% or 20% using the ShellPa mechanical stretch system (Menicon Life Science). An average tooth movement of 281 μm occurred after the application of 500 g of force to the human maxillary incisors ; this amount can be calculated to represent elongation of the PDL by approximately 23% at the level of the alveolar crest on the tension side. Hence, stretch magnitudes of 12% or 20% were chosen based on the methods of a previous study to mimic in vivo OTM. , Control cells were placed in the same way in the chambers for the same period.
Total RNA was extracted from MLO-Y4 cells or hPDL fibroblasts using Isogen (Nippon Gene, Tokyo, Japan). To synthesize complementary DNA, we used 500 μg of total RNA with a ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan) for quantitative reverse transcription-polymerase chain reactions. The mRNA levels encoding each target gene of interest were normalized with glyceraldehyde 3-phosphate dehydrogenase mRNA in the same samples as housekeeping genes. The gene expression was determined as the fold change of the gene of interest relative to the control group.
To mimic in vivo tooth movement on the compression side, we cocultured hPDL fibroblasts in Transwell inserts (Fisher Scientific, Waltham, Mass) above the 3D-cultured MLO-Y4 cell layers in 6-well plates, and subsequently subjected to a compressive force, as described previously.
Human recombinant sclerostin (rhSCL) (50 ng/mL; R&D Systems, Minneapolis, Minn) or antisclerostin antibody (Scl-Ab) (1.0 μg/mL; Bio-Rad, Hercules, Calif) were added to the medium to treat the cocultured hPDL fibroblasts and MLO-Y4 osteocytes throughout the experiment, in accordance with the methods described in previous reports. ,
Statistical analysis
Data were analyzed by a 2-tailed Student t test, paired t test, or 1-way analysis of variance with Fisher least significant difference test using the GraphPad Prism 6 software program (GraphPad Software, San Diego, Calif). The values were presented as the mean ± standard deviation. P values of <0.05 were considered to indicate statistical significance.
Results
To identify the Wnt1 expression dynamics, we constructed the experimental models of OTM, and coronal sections containing the halfway point of the distobuccal root of the maxillary first molars were chosen ( Fig 1 , A ). Immunodetection showed the expression of Wnt1 in both the PDL and the alveolar osteocytes surrounding the mesial and distal sides of the distobuccal roots, whereas the Wnt1 expression was strongly positive in the PDL area of tension side on the OTM models ( Fig 1 , B : arrowheads ). In addition, Wnt1 was also weakly expressed in the alveolar osteocytes of the tension side compared with control ( Fig 1 , B ). Relative quantification of the expression profiles showed that OTM significantly increased the Wnt1 expression on day 5 in both the PDL and the alveolar tension sides compared with the control group. Conversely, the expression on the compression side was slightly reduced in the PDL area and was not changed in the alveolar side on day 5 ( Fig 1 , C and D ). These findings indicated that tension force induced the increased expression of Wnt1 in both the PDL and alveolar osteocytes during OTM.
To confirm the mechanism of OTM-induced Wnt1 regulation, we first applied static tensile force (12% or 20%) on primary isolated hPDL fibroblasts and 2D-cultured MLO-Y4 cells for 3 hours, 6 hours, or 48 hours ( Fig 2 , A ). The expression of Wnt1 showed a significant increase in hPDL fibroblasts exposed 12% tensile force and in 2D-cultured MLO-Y4 cells exposed to 20% tensile force ( Fig 2 , B and C ).
