archives of oral biology 60 (2015) 923–931

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Rho is involved in periodontal tissue remodelling with experimental tooth movement in rats Ranran Meng, Meng Song *, Jinsong Pan ** Department of Stomatology, Shanghai First People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

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abstract

Article history:

Objective: To characterise in vivo the close relationship between the Rho signalling pathway

Accepted 31 January 2015

and periodontal tissue remodelling in experimental tooth movement in rats. Materials and methods: In total, 24 male Sprague Dawley rats were divided randomly into four

Keywords:

groups. Closed-coil springs were used to create a 40-g mesial force to move the right upper

Rho signalling pathway

first molars in anaesthetised rats. The untreated contralateral side served as a control. On

ROCK

days 3, 7, 10, and 14 after force application, paraffin wax-embedded sections of the dissected

LIMK

maxilla were prepared for immunohistochemistry to localise Rho kinase (ROCK), LIM kinase

Cofilin

(LIMK), and its downstream effector (cofilin). The immunoreactivity of the molecules

Tooth movement

investigated in the periodontal ligament area was converted into grey-scale values.

Periodontal tissue remodelling

Results: The expression of Rho and its signalling were detected mainly in the disto-coronal areas of the root in the periodontal ligament area of the control and loaded teeth. In contrast, ROCK-, LIMK-, and cofilin-positive cells were rare in the mesio-coronal areas. In the experimental group, the expression of ROCK, LIMK, and cofilin on the tension side (disto-coronal areas) increased significantly on days 7, 10, and 14, compared with those of untreated control teeth. Conclusions: These data suggest that Rho is involved in periodontal tissue remodelling during orthodontic tooth movement, modulating ROCK, LIMK, and cofilin activity. # 2015 Elsevier Ltd. All rights reserved.

1.

Introduction

It is well known that mechanical forces play a fundamental role in tissue remodelling, and in vivo, many cells, including fibroblasts, osteoblasts, endothelial cells, and smooth muscle cells, are highly sensitive to mechanical stimuli. They convert such ‘‘mechanical signals’’ into biological responses and changes in their diverse cellular behaviours, such as cell proliferation and extracellular matrix gene and protein

expression, eventually resulting in remodelling and adaptive changes in living tissues.1 Orthodontic tooth movement is characterised by the concept that mechanical forces of a certain intensity applied to a tooth are transmitted to the alveolar bone and periodontal ligament (PDL), activating a series of biological signal transduction events, which subsequently initiates remodelling activities in the periodontal tissue around the tooth root.2–4 PDL cells, which are a major component of the PDL, are extremely load-sensitive and capable of restoring system

* Corresponding author at: No. 100, Haining Road, Hongkou District, Shanghai, China. Mobile: +86 13386259556. ** Corresponding author. Mobile: +86 13311986317. E-mail addresses: [email protected], [email protected] (M. Song), [email protected] (J. Pan). http://dx.doi.org/10.1016/j.archoralbio.2015.01.017 0003–9969/# 2015 Elsevier Ltd. All rights reserved.

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Fig. 1 – Intraoral photograph of the appliance for tooth movement.

homeostasis disturbed by orthodontic mechanics. This has been described from in vitro studies.5–9 The ability of a eukaryotic cell to resist deformation, to transport intracellular cargo and to change shape during movement depends on the cytoskeleton, an interconnected network of filamentous

polymers and regulatory proteins.10 Periodontal tissue is reconstructed during orthodontic force application. During the process, five micro-environments are altered by orthodontic force: extracellular matrix (ECM), cell membrane, cytoskeleton, nuclear protein matrix, and genome. ECM and PDL cells distortion initiate structural and functional changes in extracellular, cell membrane, and cytoskeletal proteins. Cytoskeletal reorganisation is one of the important changes that occur in orthodontic tooth movement. Changes in cytoskeletal protein structure and function, such as activin polymerisation and depolymerisation, continue the biological signal process.4,11–14 Previous studies have shown that mechanical stimuli can activate various intracellular signalling pathways, such as Rho family GTPases, mitogen-activated protein kinases (MAPKs), and PI3K/Akt15; however, the key signalling pathway by which orthodontic tooth movement induces remodelling activities in periodontal tissue via cytoskeletal rearrangement in PDL cells remains to be determined. In our previous research, the signal transduction pathway that mediates cytoskeletal rearrangement in human periodontal ligament cells (hPDLCs) has been characterised in vitro. It is clear that the Rho signalling pathway is responsible for cyclic strain-induced cytoskeletal rearrangement in hPDLCs.16

Fig. 2 – Immunohistochemical staining for ROCK (A, D), LIMK (B, E) and cofilin (C, F) in the PDL of the distal root of the maxillary first molar in the control group (T400). ROCK-, LIMK-, and cofilin-positive cells (arrows) were distributed primarily in the disto-coronal areas (A–C) and were rare in the mesio-coronal areas (D–F). AB indicates alveolar bone, D, dentine, PDL, periodontal ligament.

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Table 1 – The mean grey-scale values of ROCK immunoreactivity on the tension side. Group Mean grey-scale values

Control

3 days

7 days

10 days

14 days

120.06 (4.33)

126.30 (5.60)

131.31 (4.89)

156.26 (8.30)

152.40 (7.70)

Table 2 – The mean grey-scale values of LIMK immunoreactivity on the tension side. Group Mean grey-scale values

Control

3 days

7 days

10 days

14 days

123.81 (2.18)

128.53 (4.54)

133.27 (2.25)

156.01 (4.79)

154.94 (6.38)

Table 3 – The mean grey-scale values of cofilin immunoreactivity on the tension side. Group Mean grey-scale values

Control

3 days

7 days

10 days

14 days

124.45 (3.83)

128.84 (3.31)

135.00 (2.71)

157.78 (6.91)

154.74 (9.21)

Rho, a major organiser of the cytoskeleton,17 regulates the formation of actin stress fibres by activating ROCK, a prominent member of the Rho signalling pathway, which phosphorylates LIM kinase (LIMK), which, in turn, phosphorylates cofilin.18 Phosphorylated LIMK and cofilin act downstream of ROCK both in vitro and in vivo.19 Phospho-cofilin then

promotes actin polymerisation, which is responsible for proliferation and cytoskeletal rearrangement.20 These observations caused us to hypothesise a close relationship between the Rho signalling pathway and periodontal tissue remodelling during orthodontic tooth movement. In this in vivo study we examined the role of Rho and its

Fig. 3 – Immunohistochemical staining for ROCK (A, D), LIMK (B, E) and cofilin (C, F) in the PDL of the distal root of the maxillary first molar on day 3 (T400). ROCK-, LIMK-, and cofilin-positive cells (arrows) were distributed primarily in the disto-coronal areas (tension side) (A–C) and were rare in the mesio-coronal areas (compression side) (D–F). AB indicates alveolar bone, D, dentine, PDL, periodontal ligament.

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signalling in periodontal tissue remodelling during orthodontic tooth movement.

2.

Materials and methods

2.1.

Animals

All experimental procedures were approved by the Ethics Committee, Shanghai Jiao Tong University, China. 24 sixweek-old male Sprague Dawley rats, weighing 200–250 g, were used. They were divided randomly into four groups of six animals each. The animals were kept in standard clear plastic cages and fed a soft diet and water ad libitum.

2.2.

Experimental tooth movement

An orthodontic nickel–titanium closed-coil spring appliance (Grikin Advanced Materials, Beijing, China) was fixed between the maxillary right first molar and the upper incisors with a composite resin (MB 4403; 3M Unitek, Monrovia, CA, USA) under anaesthesia (intraperitoneal injection of sodium pentobarbital at 40 mg/kg body weight). A stainless-steel ligature

with a diameter of 0.2-mm (Dentaurum, Pforzheim, Germany) was threaded through the contact between the first and second right maxillary molars and attached to the closed-coil spring. A second 0.2-mm stainless-steel ligature was placed around the incisors and attached to the closed-coil spring. The untreated contralateral side served as a control. The closedcoil spring was activated for 3 mm and the maxillary right first molar was moved mesially by applying 40 g of force.20,21 with the closed-coil spring (Fig. 1). The force magnitude was measured with a force gauge (Teclock, Nagano, Japan). The orthodontic appliance was checked daily and no reactivation was performed during the experimental period.

2.3.

Tissue preparation

On days 3, 7, 10, and 14 after orthodontic tooth movement, anaesthetised animals from each group were sacrificed. Then, the maxilla of each animal was dissected and divided into right and left halves. The soft tissues around the maxilla, except the gingiva, were removed. The specimens were fixed in 4% paraformaldehyde in 0.1 M PBS for 24 h and decalcified in 10% (pH 7.4) ethylenediaminetetraacetic acid (EDTA) at room temperature for at least 4 weeks. This solution was changed

Fig. 4 – Immunohistochemical staining for ROCK (A, D), LIMK (B, E) and cofilin (C, F) in the PDL of the distal root of the maxillary first molar on day 7 (T400). ROCK-, LIMK-, and cofilin-positive cells (arrows) were distributed mainly in the distocoronal areas (tension side) (A–C) and were rare in the mesio-coronal areas (compression side) (D–F). AB indicates alveolar bone, D, dentine, PDL, periodontal ligament.

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every other day. After dehydration and paraffin wax embedding, specimens were cut mesiodistally with a microtome into 5-mm serial sections (HM 355S; Microm International, Walldorf, Germany) and mounted on glass slides. Selected sections were prepared for haematoxylin and eosin and immunohistochemistry staining for Rho-associated protein kinase (ROCK), LIM kinase (LIMK), and cofilin.

2.4.

Immunohistochemistry

After deparaffinisation and rehydration, the tissue sections were rinsed with Tris-buffered saline solution (TBS) at pH 7.4 for 10 min and then soaked in methanol/hydrogen peroxide for 10 min in the dark to block endogenous peroxidase activity. Then, sections were washed in TBS and preincubated with TBS containing 4% bovine serum albumin (TBS/BSA) for 20 min to prevent non-specific background staining. Antibodies were diluted in 4% BSA. Subsequently, sections were incubated with a monoclonal primary antibody of rabbit origin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in a 1:100 working solution (ROCK), 1:200 working solution (LIMK), or 1:100 working solution (cofilin) at 4 8C overnight in a humidified chamber. The sections were rinsed in TBS and incubated with

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a rabbit anti-goat immunoglobulin diluted 1:100 in TBS/BSA (Santa Cruz Biotechnology), as a secondary antibody for 30 min in a humidity chamber at RT. After another rinse, the sections were developed with diaminobenzidine (DAB) for 5 min, which yields a brown staining product, rinsed, and then counterstained with Mayer’s haematoxylin for 5 s, dehydrated, and cover-slipped for light microscopy analysis. To demonstrate the specificity of the immunoreactions, negative controls were performed omitting the primary antibody and replacing it with non-immune immunoglobulin G at the same concentration. To establish representative regions of the periodontal tissue, the light microscopic images were captured per specimen in the mesiocoronal and distocoronal regions of the distal root of the maxillary first molar at 400 magnification. In those sections, three regions were selected at 30-mm intervals for immunohistochemical examination under a Zeiss Axioskop 2 plus microscope (Carl Zeiss, Gottingen, Germany). The obtained images were analyzed using an image-analysis software (ImageJ 1.440, National Institute of Health, USA), and the immunoreactive intensity for all proteins investigated was converted to grey-scale values.

Fig. 5 – Immunohistochemical staining for ROCK (A ,D), LIMK (B, E) and cofilin (C, F) in the PDL of the distal root of the maxillary first molar on day 10 (T400). ROCK-, LIMK-, and cofilin-positive cells (arrows) were distributed primarily in the disto-coronal areas (tension side) (A–C) and were rare in the mesio-coronal areas (compression side) (D–F). AB indicates alveolar bone, D, dentine, PDL, periodontal ligament.

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2.5.

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Statistical analysis

Data were analysed using the SPSS software (ver. 17; SPSS, Chicago, IL, USA). All results are expressed as means and standard deviations. The immunoreactivity of the molecules investigated between groups was evaluated by one-way analysis of variance. The significance level was set at P < 0.05.

3.

Results

In this study, immunoreactivity for all the proteins investigated was visible throughout the PDL in both the control and experimental groups, but tended to be mainly localised on the distal side of the root. There was no immunostaining under the negative control conditions. As a reaction to mechanical stimuli, changes in immunoreactivity for all investigated proteins occurred in the experimental group. The mean grey-scale values of ROCK, LIMK, and cofilin immunoreactivity on the tension side (distocoronal) are summarised in Tables 1–3. The expression of ROCK on the tension side (disto-coronal) was enhanced significantly on days 7, 10, and 14 in comparison

with the controls (Figs. 2A, 4A, 5A and 6A). The expression of ROCK on day 3 did not change significantly compared with the controls (Figs. 2A, 3A). The expression of ROCK on days 10 and 14 was enhanced significantly compared with that of days 3 and 7, respectively (Fig. 7A). The expression of LIMK increased significantly on days 7, 10, and 14 compared with that of the untreated control teeth (Figs. 2B, 4B, 5B and 6B). The expression of LIMK on day 3 did not change significantly compared with that for the untreated control teeth (Figs. 2B, 3B). The expression of LIMK on days 10 and 14 increased significantly compared with that on days 3 and 7 (Fig. 7B). Regarding cofilin immunoreactivity, the results were similar to those for ROCK and LIMK (Figs. 2C, 3C, 4C, 5C, 6C and 7C).

4.

Discussion

In this study, we showed for the first time that the Rho-cofilin signalling pathway plays an important role in periodontal tissue remodelling via cytoskeletal rearrangement in PDL cells during orthodontic tooth movement. To assess the relationship

Fig. 6 – Immunohistochemical staining for ROCK (A, D), LIMK (B, E) and cofilin (C, F) in the PDL of the distal root of the maxillary first molar on day 14 (T400). ROCK-, LIMK-, and cofilin-positive cells (arrows) were distributed primarily in the disto-coronal areas (tension side) (A–C) and were rare in the mesio-coronal areas (compression side) (D–F). AB indicates alveolar bone, D, dentine, PDL, periodontal ligament.

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Fig. 7 – Mean grey-scale values of ROCK (A), LIMK (B), and cofilin (C) immunoreactivity in the disto-coronal areas. The symbols indicate significant differences ( p < 0.05) compared with the control group (*) or among the experimental groups (^).

between the Rho signalling pathway and periodontal tissue remodelling, we established a model for experimental tooth movement in rats. We next showed the distribution and changes of ROCK, LIMK, and cofilin in the PDL using immunohistochemistry.

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It is known that Rho family proteins play important roles in cytoskeletal rearrangement. Cofilin phosphorylation can be activated by Rho GTPases, blocking actin depolymerisation, and thus leading to cytoskeletal rearrangement.19,22,23 Furthermore, cytoskeletal reorganisation is one of the important changes that occur in orthodontic tooth movement. The present results showed that the application of orthodontic force on teeth in rats enhanced the expression of ROCK, LIMK, and cofilin on the tension side, indicating that the biological actions of the factors investigated in periodontal tissue were stimulated under this condition of force application. This observation is consistent with previous studies, which suggested that Rho family proteins play important roles in cytoskeletal rearrangement and tissue remodelling. Bhowmick et al.18 showed that the small GTPase Rho and its downstream effector Rho kinase (ROCK) mediate TGF-b1induced remodelling of mammary epithelial cell-to-cell contact. Qi et al.24 found that Rho and its downstream effector could mediate cyclic strain-induced migration of vascular smooth muscle cells and promote muscular tissue remodelling. In TGF-b1-stimulated hPDLCs, ROCK increases the phosphorylation of LIMK and cofilin. The phosphorylation of cofilin then promotes actin polymerisation, leading to cytoskeletal rearrangement.25 Thus, it seems that upregulation of ROCK may mediate cytoskeletal rearrangement in PDL cells. Then, the rearrangement will promote periodontal tissue remodelling, leading to the acceleration of orthodontic tooth movement. The expression of the factors investigated on days 10 and 14 showed significant differences compared with those of days 3 and 7, indicating that periodontal tissue adaptation to orthodontic force depends on correctly expressing needed proteins at the right times and places. Additionally, the mesial movement of the right upper first molars on days 10 and 14 was more clearly visible compared with those on days 3 and 7, suggesting that the remodelling of periodontal tissue was more active on days 10 and 14. The new PDL that was built on the tension side accelerated periodontal tissue remodelling via enhancing the numbers of PDL cells. As a result, the expression of ROCK, LIMK, and cofilin on the tension side was remarkably increased on days 10 and 14. In contrast to the tension side, we found that the expression levels of the three molecules were negligible on the pressure side. This may have been caused by necrotic changes in PDL cells or because PDL cells subjected to tensional and compressive forces are able to perceive two different forms of mechanical stimuli and respond in different a manner.26 Furthermore, recent studies have elucidated that RhoE has been described as ROCK inhibitor27 and RhoE, which expression was induced in human PDL cells under compressive force, is actively linked to the cytoskeletal rearrangement on the pressure side.28 In our previous research, RhoA is involved in cyclic strain-induced cytoskeletal rearrangement of human periodontal ligament cells. Therefore, RhoE seems to function antagonistically to RhoA in cytoskeletal regulation. This may suggest that there is a link between RhoE and the pressure side lack of ROCK, LIMK, and cofilin immunoreactivity. In summary, this study demonstrated that Rho plays a key role in the remodelling of periodontal tissue via cytoskeletal

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Fig. 8 – The flow chart outlining strain-induced periodontal tissue remodelling during orthodontic tooth movement via the Rho-cofilin signalling pathway.

rearrangement in PDL cells and also suggests a potential role for Rho in mechanotransduction in PDL cells (Fig. 8). These observations may provide new insight into understanding orthodontic tooth movement. More research on their physiological roles is necessary to clearly elucidate the remodelling mechanisms of periodontal tissue during orthodontic tooth movement.

Funding This study was funded by the National Natural Science Foundation of China (No. 11172177 and 91229103).

Competing interest No.

Ethical approval All experimental procedures were approved by Ethics Committee, Shanghai Jiao Tong University, China.

Acknowledgements This study was supported by the Laboratory of Oral Tumour Biology, Department of Oral and Maxillofacial Surgery, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine. The authors thank Dr. Wantao Chen at this laboratory, Zonglai Jiang, Qingping Yao and Yue Han at Institute of Mechanobiology&Medical Engineering, Shanghai Jiao Tong University for their valuable suggestions and technical assistance.

references

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22. Sumi T, Matsumoto K, Takai Y, et al. Cofilin phosphorylation and actin cytoskeletal dynamics regulated by rho- and Cdc42activated LIM-kinase 2. J Cell Biol 1999;147(7):1519–32. 23. Vardouli L, Moustakas A, Stournaras C. LIM-kinase 2 and cofilin phosphorylation mediate actin cytoskeleton reorganization induced by transforming growth factor-beta. J Biol Chem 2005;280(12):11448–57. 24. Qi YX, Qu MJ, Long DK, et al. Rho-GDP dissociation inhibitor alpha downregulated by low shear stress promotes vascular smooth muscle cell migration and apoptosis: a proteomic analysis. Cardiovasc Res 2008;80(1):114–22. 25. Wang L, Wang T, Song M, et al. Rho plays a key role in TGFb1-induced proliferation and cytoskeleton rearrangement of

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human periodontal ligament cells. Arch Oral Biol 2014;59(2):149–57. 26. He Y, Macarak EJ, Korostoff JM, et al. Compression and tension: differential effects on matrix accumulation by periodontal ligament fibroblasts in vitro. Connect Tissue Res 2004;45(1):28–39. 27. Riento K, Ridley AJ. ROCKs: multifunctional kinases in cell behaviour. Nat Rev Mol Cell Biol 2003;4:446–56. 28. de Araujo RM, Oba Y, Kuroda S, et al. RhoE regulates actin cytoskeleton organization in human periodontal ligament cells under mechanical stress. Arch Oral Biol 2014;59(February (2)):187–92. http://dx.doi.org/10.1016/ j.archoralbio.2013.11.010. [Epub 2013 Nov 25].

Rho is involved in periodontal tissue remodelling with experimental tooth movement in rats.

To characterise in vivo the close relationship between the Rho signalling pathway and periodontal tissue remodelling in experimental tooth movement in...
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