| Sign In to gain access to subscriptions and/or personal tools. |
Early Responses of Periodontal Ligament Cells to Mechanical Stimulus in vivoDepartment of Orthodontics, Dental Clinic, University of Bonn, Welschnonnenstr. 17, D-53111 Bonn, Germany; Correspondence: * corresponding author, bourauel{at}uni-bonn.de
Previous studies have indicated that human periodontal ligament cells undergo osteoblastic differentiation via the ERK pathway under mechanical stress in vitro. This study aimed to verify this principle in vivo. The right upper first molars of 25 anesthetized rats were loaded with constant forces of 0.1 N for up to 8 hrs. The untreated contralateral side served as a control. Paraffin-embedded sections were analyzed by immunohistochemistry for proliferating cell nuclear antigen (PCNA), runt-related transcription factor 2 (Runx2/Cbfa1), and phosphorylated extracellular signal-regulated kinases 1/2 (pERK1/2). In selected areas under tension, the proportions of Runx2-positive and pERK1/2-positive cells increased within 8 hrs of loading, whereas these proportions in selected areas under pressure were significantly lower than those in control teeth. Moreover, there were no significant changes in the number of PCNA-positive cells. Thus, mechanical stimulus up-regulates Runx2, and this regulation may be achieved via the ERK pathway.
Key Words: periodontal ligament • experimental tooth movement • rat molar • MAPK • Runx2.
A mechanical force applied to a tooth is transmitted to the root-surrounding tissues of the periodontium and initiates remodeling activities that allow for movement of the tooth through alveolar bone. The traditional simplified explanation for these processes, the theory of "pressure and tension" of the periodontal ligament, has been questioned by many researchers, but can still be found in recent orthodontic textbooks (Graber and Vanarsdall, 1994). Some authors have followed the ideas of biomechanical orthopedics and focused on the bending of alveolar bone as the missing link between the mechanical stimulus and the biological response (Melsen, 1999), and, as a consequence, the role of the periodontal ligament was claimed to be of minor importance. Recent research, applying modern methods of cellular and molecular biology, has again focused on the role played by periodontal ligament cells (Sandy et al., 1993). Periodontal ligament fibroblasts have been shown to possess osteogenic potential and to undergo osteoblastic differentiation in response to various stimuli (Basdra and Komposch, 1997; Matsuda et al., 1998). The ability of these cells to respond to mechanical stimuli could be the key factor in mediating bone-remodeling processes. However, little is known about these responses, and this reflects the deficiency in our understanding of mechanotransduction mechanisms. In vitro studies of cell cultures of different osteoblastic cell lines have demonstrated that the initial reaction of these cells to mechanical strain is, at least partly, mediated by deformation of the cytoskeleton via physical interaction of collagen I and receptors of the integrin family (Carvalho et al., 1996). A key link between these membrane-bound receptors and changes in the pattern of gene expression has been shown to be the mitogen-activated protein (MAP) kinase pathways (Matsuda et al., 1998; Xiao et al., 2000; Lai et al., 2001). Extracellular signal-regulated kinases (ERKs), members of the MAPK family, have been shown to participate in a diverse array of cellular programs, including cell differentiation, cell proliferation, and apoptosis. The modus of these physiological responses is cell-type-specific. Mechanical stimuli have been shown to activate ERK1/2 in mechanosensitive cells, such as endothelial cells (Tseng et al., 1995) and smooth-muscle cells (Reusch et al., 1997) in vitro. In osteoblastic cells, the ERK1/2 signal pathway is involved in different cellular responses induced by mechanical stimuli such as collagen synthesis (Chaudhary and Avioli, 2000), cyclo-oxygenase expression (Wadhwa et al., 2002), and osteopontin production (You et al., 2001). Ziros et al.(2002) have shown, in vitro, that mechanical stimuli lead to increased expression of runt-related transcription factor 2 (Runx2), also referred to as core-binding factor 1 (Cbfa1), a transcription factor that is a pivotal regulator of osteoblast differentiation, via the ERK1/2 pathway. The bone-specific transcription factor Runx2 stimulates transcription of osteoblastic markers such as osteocalcin, type I collagen, osteopontin, alkaline phosphatase, and matrix metalloproteinase-13 (MMP13), which are regulated in the course of osteoblastic maturation (Komori, 2002; Lian et al., 2004). In the present study, the in vitro results were corroborated in vivo by the application of a precisely calibrated load to the upper first molars of rats, and by quantitative analysis of immunohistochemical detection of ERK1/2 and Runx2 in regions presenting the classic tension and pressure zones around the mesial root. In addition, we examined the relationship between cell differentiation and cell proliferation in the periodontal ligament during the early phase of tooth movement.
Animals Twenty-five male Wistar rats, 12 wks of age and weighing 300–350 g each (Harlan Winkelmann, Borchen, Germany) were used. They were provided with food and water ad libitum. The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the local district government and the Animal Care Commissioner of the University of Bonn (Germany).
Experimental Protocol
Upon completion of experiments, the anesthetized animals were killed by an intravenous application of T61 (embutramid mebezonium iodide, 2 mL; Intervet, Unterschleissheim, Germany) and decapitated. The maxillae were removed and dissected into right and left segments. The soft tissues around the jaw bone, except for the gingiva, were removed. The specimens were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 24 hrs and decalcified in 10% ethylene diamine tetraacetic acid (EDTA) at room temperature for 5 wks. After being dehydrated in ascending grades of alcohol, cleared in xylene, and paraffin-embedded, 5-µm serial sections were cut parasagittally on a microtome (HM 355s; Microm Int., Walldorf, Germany) and mounted on glass slides (K. Roth, Karlsruhe, Germany). To establish representative regions of the periodontium and the adjacent alveolar bone, those sections were chosen that showed the mesial root with complete radicular pulp. From these, three sections at 50-µm intervals were taken for each quantitative analysis. Immunohistochemical staining was carried out with anti-PCNA mouse monoclonal antibody (diluted at 1:500, ZYMED Laboratories, South San Francisco CA, USA), Runx2 goat polyclonal antibody (diluted at 1:35, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and pERK1/2 mouse monoclonal antibody (diluted at 1:100, US Biological, Swampscott, MA, USA). The sections were deparaffinized and rehydrated, rinsed with tris-hydroxymethyl aminomethane-buffered saline solution (TBS) at pH 7.4 for 10 min, and then soaked in methanol/H2O2 for 45 min in the dark, to block endogenous peroxidase activity. In TBS/BSA (4% bovine serum albumin)-diluted anti-PCNA, Runx2 and pERK1/2 antibodies were used overnight at 4°C in a humidity chamber. Subsequently, sections were washed in TBS and incubated with Envision+/HRP anti-mouse or anti-goat immune globulin/HRP (DakoCytomation, Hamburg, Germany), as secondary antibodies for 30 min in a humidity chamber at RT. Antibody complexes were visualized after the addition of AEC+ (DakoCytomation, Hamburg, Germany) substrate for 10 min. Thereafter, slides were rinsed, counterstained with Mayer’s hematoxylin for 5 sec, rinsed again, and mounted. Negative controls were prepared by omission of the primary antibodies from the staining procedures. All chemicals were purchased from Sigma (Deisenhofen, Germany). The specificity of the antibodies used had been confirmed by immunoblotting analysis (Ogata et al., 1985) or by the manufacturer (US Biological, Swampscott, MA, USA), respectively.
Counting of immunohistochemically positive cells was performed in two separate areas in each selected section (Fig. 1A Values were calculated as the mean and standard deviation for each group. We performed Student’s t tests to determine differences between groups and with regard to the localization of counted positive cells. To evaluate the accuracy of the method, we calculated an interobserver error of 6.9% (CV) and an intra-observer error of 2.2% (CV) after the author and an investigator blinded to the study double-counted 20 randomly chosen sections.
Histology and Immunohistochemistry Orthodontic loading of the upper right first molar resulted in a mesial tipping of the molar. Hence, zones of pressure and tension were formed (Kawarizadeh et al., 2004). In accordance with this assumption, zones of pressure and tension adjacent to the mesial root of the first upper molar could be clearly identified. Tension zones with typically stretched periodontal ligament fibers were located on the disto-coronal aspects of the mesial root (Figs. 2D  , 2H). The pressure zones were located on the mesio-coronal aspects of the mesial root (Figs. 2C, 2G).
Application of orthodontic loading simultaneously induced a significant increase of the number of periodontal ligament cells positive for phosphorylated ERK1/2 and, at the same time, for Runx2 in the tension zones (Figs. 3B , 3D) in a time-dependent manner (p < 0.001). In the untreated control teeth, the mean proportions of positive cells were as follows: for pERK, 27.1% on the disto-coronal aspects (Fig. 3B) and 44.5% on the mesio-coronal aspects (Fig. 3A); and for Runx2, 22.0% on the disto-coronal aspects (Fig. 3D) and 18.8% on the mesio-coronal aspects (Fig. 3C), respectively. In pressure zones (mesio-coronal aspects), a comparison of the data showed that the mean values of pERK1/2-positive cells were significantly decreased compared with those of the control (Fig. 3A). With respect to Runx2-positive cells, the results were similar, but not significantly, in these zones (Fig. 3C). In addition, no differences among all treated groups in these zones were detected with regard to the mean values for pERK1/2-positive and Runx2-positive cells. No statistically significant differences in proportions of PCNA-positive nuclei were observed between experimental pressure and tension zones and the respective areas in the control group (data not shown).
Periodontal ligament fibroblasts comprise an osteoblast-like population which plays a pivotal role in remodeling and repair (Lekic and McCulloch, 1996). In addition, mechanical stimuli evoke a variety of biological events, including gene expression in periodontal ligament cells. The mechanisms that activate biochemical responses to mechanical stimuli are still not well-understood. We performed the present study to investigate the responses of periodontal ligament cells to short-term orthodontic loading. We demonstrated that application of precise short-term loading activates ERK1/2 and Runx2, respectively. It has been demonstrated that the ERK pathway plays an important role in down-regulation of type I collagen gene expression in osteoblastic cells in vitro (Chaudhary and Avioli, 2000). In MC3T3-E1 osteoblastic cells, mechanical stress has been shown to induce COX-2 gene expression (Wadhwa et al., 2002), increase of osteopontin (OPN) mRNA levels (You et al., 2001), and immediate early gene expression of c-Fos (Hatton et al., 2003) via the ERK signaling pathway. Continuous mechanical stretching of human periodontal ligament cells has been shown to enhance ERK activity, resulting, concurrently, in a marked increase in activator protein 1 (AP-1) activity, a transcription factor, which plays a pivotal role in controlling gene expression in osteoblast differentiation (Peverali et al., 2001). Moreover, when a specific ERK inhibitor was used, a causal link between the ERK and AP-1 constituents, c-Fos and c-Jun, was demonstrated (Kletsas et al., 2002). There are complex mechanisms for interaction between AP-1 and Runx2, including direct physical interactions (Franceschi and Xiao, 2003). In another study with human periodontal ligament cells stimulated by mechanical stretching, Runx2 activation was shown to be mediated by phosphorylation of ERKs (Ziros et al., 2002). Furthermore, u0126 treatment, a selective inhibitor of ERK1/2, produced a clear down-regulation of Runx2 binding activity in mechanically stimulated human periodontal ligament cells (Ziros et al., 2002). These findings strongly implicated the ERK cascades in the stretch-elicited up-regulation of Runx2 binding activity. This cited study provided novel information on a molecular link between mechanical stimulation and osteoblast differentiation through the induction of expression and DNA binding potential of Runx2. In our study, a rat molar was loaded orthodontically with a high-resolution 3D force/torque transducer for up to 8 hrs while the animal was under anesthesia. We have not found any reports about studies using any similar well-controlled animal model. We found an obvious correlation between periodontal ligament pERK1/2-positive and Runx2-positive cells. The proportion of Runx2-positive cells is similar to the proportion of pERK1/2-positive cells within the same time interval. These proportions were increased in the selected area under tension, suggesting that periodontal ligament cells undergo osteoblastic differentiation via the ERK pathway in these zones. In addition, due to the mechanical loading, we observed an increase in pERK- and Runx2-positive cells which occurred in a time-dependent manner, suggesting that periodontal ligament cells are the source of osteoblasts after prolonged mechanical stimulus. Whereas in in vitro studies it has been shown that mechanical stress induces DNA synthesis in human periodontal ligament cells following 6 hrs of stretching (Kletsas et al., 1998), in our study no significant proliferation could be observed after 8 hrs of mechanical stimulus. One possible explanation for the discrepancy between those results and the ones presented here might be the total incubation period of 48 hrs used after mechanical stretching in vitro. Studies on cell kinetics of orthodontically stimulated periodontal ligament in the rat indicated a notable increase in DNA synthesis at about 20 hrs after application of the orthodontic force (Roberts and Jee, 1974). Altogether, our findings show that short-term mechanical stimulus in the first instance induces differentiation of periodontal ligament cells toward osteoblasts via the ERK cascade and does not influence the proliferation of these cells. Thus, our results confirmed the hypothesis of Roberts et al.(1982), that new osteoblasts are derived from periodontal ligament cells during orthodontically induced osteogenesis. Furthermore, the results of this study are in accordance with the results of various studies where the finite element method was used, suggesting the dominant role of the periodontal ligament in remodeling processes of alveolar bone (Bourauel et al., 2000; Kawarizadeh et al., 2003). Activation of ERK by orthodontic loading is a promising approach to explain the regulation of osteogenesis by mechanical loading. Further studies that investigate changes in ERK activity and expression of Runx2 when teeth are subjected to higher levels of force or exposed for a longer time under well-controlled experimental conditions should be very helpful for furthering our understanding of remodeling processes following orthodontic tooth movement.
This study was supported by the Medical Faculty of the University of Bonn (BONFOR project, O-135.0007). The authors thank Ms. Inka Bay for her technical assistance. Received for publication September 3, 2004. Revision received June 13, 2005. Accepted for publication June 18, 2005.
Journal of Dental Research, Vol. 84, No. 10,
902-906 (2005) This article has been cited by other articles:
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
|
 |
|
Sign In to gain access to subscriptions and/or personal tools.
TOP
ABSTRACT
INTRODUCTION
