Abstract
To detect myofibroblast formation on the tension side during orthodontic tooth movement in vivo and myofibroblast expression of alpha-smooth muscle actin (α-SMA) induced by tension both in vivo and in vitro.
Fifty 6-week male rats were used in this in vivo study, and the right maxillary first molar was moved mesially, which served as the experimental group, and the left maxillary first molar served as the control. Rats were sacrificed at days 0, 3, 5, 7, and 14 after force loading. Myofibroblasts, identified with α-SMA, were examined through immunohistochemistry. For the in vitro study, human periodontal ligament (PDL) fibroblasts were obtained. Cyclic mechanical tension was applied to the fibroblasts for 0, 1, 3, 6, and 12 hours. Transmission electron microscopy was used to detect the ultrastructure of myofibroblasts. α-SMA mRNA gene expression was quantified by real-time quantitative PCR. The expression of α-SMA was detected by immunofluorescence and quantified by Western blotting.
In vivo, the myofibroblasts expressing α-SMA were identified both in the experimental group and in the control group. The expressions of α-SMA were increased in the tension areas of the experimental group over time, and reached the maximum in day 14. In vitro, fibronexus junctions and actin microfilaments in the cells could be found with transmission electron microscopy. Cyclic mechanical tension could significantly induce α-SMA expression at 12 hours (P < .01) than the controls.
Myofibroblasts existed in the PDL. The expressions of α-SMA in the myofibroblasts were significantly up regulated under tension both in vivo and in vitro.
INTRODUCTION
Periodontal ligament (PDL) under tension results in bone remodeling during orthodontic tooth movement.1,2 The viscoelasticity of collagen fibers suggests that the fibers in PDL cannot sustain the force alone under the stress and strain over a long period of time.3,4 PDL is composed of fibers and cells, and the majority of cells are fibroblasts which cannot generate force themselves. Recent researches in many other fields have found that fibroblasts could differentiate into myofibroblasts under mechanical stimulation.5
Myofibroblasts have the appearance of fibroblasts and neo-express smooth muscle cells' marker alpha-smooth muscle actin (α-SMA).6 α-SMA is a mechano-sensitive protein that locates in the stress fibers under mechanical load.7 Transforming growth factor-beta1 (TGF-β1) can induce fibroblast activation and α-SMA expression.8,9 The characteristics of myofibroblasts are their cytoskeletons which contain α-SMA and the stress fibers.10,11 The synergistic reaction of the α-SMA and the stress fibers provide the myofibroblasts ability with contraction force,12 and myofibroblasts can apply this force to the local extracellular matrix by the interaction between the cell's cytoskeleton network and the connection of these stress fibers to the extracellular matrix. These can make the whole extracellular matrix and cell unit contractile, which results in tissue contraction.13 The characteristics of myofibroblasts suggest that the cells have a role in the production of the contractile force in the sites in which high tension exists.14–16
During orthodontic tooth movement, the PDL is stretched, inflammated, and remodelled in the tension side. Cytokines are released17 and progenitor cells are recruited and differentiated. Since fibroblasts cannot sustain force, we hypothesize that myofibroblasts are activated in the mechanical tension side. To our best knowledge, no studies have investigated the activation of myofibroblasts during orthodontic tooth movement.
Therefore, the aim of the present study was to investigate the pattern of α-SMA expression in the myofibroblasts from the tension area during experimental tooth movements in vivo, and the effects of cyclic mechanical tension on α-SMA expression in myofibroblasts in vitro.
MATERIALS AND METHODS
Animals
Fifty 6-week-old 200–300 g male Sprague-Dawley rats were used in this experiment. They were maintained in a temperature-controlled room (25–28°C) with a 12/12-hour light-dark cycle. All procedures on the experimental animals were approved by the Institutional Committee for Animal Care, Sichuan University, China.
Appliance for Experimental Tooth Movement
In each rat, a fixed, unilateral appliance was placed for mesial movement of the right maxillary first molar; the right served as the experimental teeth, the left as the control.18 The appliance consisted of a 6-mm length of closed-coil spring (3M, Monrovia, Calif) affixed with 0.010-inch steel ligature (Shangchi, Shanghai, China). The maxillary incisors were used as the anchorage and were fixed with the coil spring by a ligature. After an intraperitoneal injection of sodium pentobarbital, 40-g force was applied on the experimental teeth for 0, 3, 5, 7, 14 days, accordingly.
Immunohistochemistry
The rats were killed on days 0, 3, 5, 7, and 14, respectively (n = 10 each time), and fixed by cardiac perfusion with freshly made 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. They were then demineralized in 17% EDTA (pH 7.4) for 6–8 weeks. The demineralized tissues were dehydrated and embedded in paraffin. The tissue blocks were cut into 6-mm thick mesiodistal serial sections. The mouse anti-α-SMA (Abcam, Cambridge, UK) was used as primary antibody. A PV-9000 kit (Zhongshan Biotechnology Company, Beijing, China) was adopted to localize the secondary antibody. Staining was visualized with DAB. Simultaneously, negative control was performed without the primary antibody.
Image Analysis
The study area was the coronal one third of the palatal root of the teeth under 400× magnification with Nikon E600 microscopy (Nikon, Tokyo, Japan). Because the size of the study area at each micrograph varied, α-SMA protein expressions were measured by the mean optical density (MOD). Both the size of the study area, selected manually, and the integrated optical density (IOD) of the positive stains were measured by Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, Md) (MOD = IOD/study area). The procedures were repeated three times for each sample.
Cell Culture
Human PDL fibroblasts were isolated from healthy PDL of premolar teeth of individuals (12–17 years old, n = 5) for orthodontic reasons in accordance with the methods of Palmon et al.19 All patients gave their informed consent before providing the samples. The periodontal tissues were detached from the central one third of the root surface. The cells from explants were cultured for 7 days. After a confluent monolayer of migrating cells had formed, the cells were passaged. Experiments were carried out with cells from the third or fourth passage.
Application of Cyclic Mechanical Tension in Vitro
The fibroblasts were seeded onto 10 cm2 (5 × 2 cm) cell culture plates and cultured. When the cells grew up to 80% of the plates, they were subjected to 4000 microstrains using a uniaxial four-point bending system20 in the incubator at a frequency of 0.5 Hz for 0, 1, 3, 6, or 12 hours. The loading procedures were repeated three times for each time point. In the control groups, fibroblasts were cultured in the same conditions but without mechanical tension loading.
Transmission Electron Microscopy
After application of mechanical tension for 12 hours, the cells were harvested and fixed in 3% glutaraldehyde at 4°C for 2 hours. The fixed cells were washed with PBS, and post-fixed in 1% osmium tetroxide for 1 hour at 4°C. The fixed cells were dehydrated and embedded in Araldite. Ultrathin sections (60 nm) were cut and stained with uranyl acetate and lead citrate. These stained sections were examined under a transmission electron microscope (Hitachi H-7650, Tokyo, Japan).
Immunofluorescence
After application of mechanical tension for 12 hours, the cells were fixed in 3% paraformaldehyde and permeabilized in 0.25% Triton-X 100. After incubation with mouse anti-α-SMA (Abcam) for 2 hours, cells were incubated with goat anti-mouse-TRITC (Zhongshan Biotechnology) for 1 hour. Coverslips were mounted with slow fade reagent with DAPI (Invitrogen). Subsequently, cells were embedded in 50% glycerol and viewed by fluorescence microscope (Bio-Rad, Hercules, Calif).
Real-time Fluorescence Quantitative RT-PCR
α-SMA mRNA levels were quantified by RT FQ-PCR based on the TaqMan technique. Trizol reagent cDNA was synthesized from 2 µL of total RNA as a template, with oligo (dT20) primer and reverse transcriptase, using the Takara RT-PCR kit (Takara, Japan) for RT-PCR. Then, 5 µL of the cDNA mixture was subjected to PCR amplification using specific primers and TaqMan probes (Table 1).
Oligonucleotide primers and TaqMan probes for α-SMA were designed from the GenBank databases. The amplification profile (temperature [°C]/time [s]) was 94/120 (initial denaturation), 94/20 (denaturation), 58/20 (optimal annealing condition), and 60/30 (extension), all for 40 cycles. The PCR products were quantified with GAPDH as reference gene and all qRT-PCR reactions were performed three times with comparable results.
Western Blotting
Cells were washed and then lysed and sonicated in a lysis buffer (KeyGen total protein extraction kit, KeyGen Biotech, Nanjing, Jiangsu, China). The cytosolic fraction was collected as the supernatant after centrifugation and assayed quantitatively with the BCA method. After boiling for 5 minutes, 25 µL of the lysate was applied to SDS–10% PAGE at 120 V for 5 hours, and the proteins in the gel were transferred to a PVDF membrane (Millipore). Membranes were incubated in blocking agent for 1 hour at RT, followed by incubation with mouse anti-α-SMA (Abcam) overnight at 4°C. Membranes were then incubated with goat anti-mouse secondary antibody for 1 hour at RT (Zhongshan Biotechnology), followed by streptavidin-HRP (Millipore). Band intensities were determined using the ChemiDoc XRS Gel documentation system and Quantity One software (Bio-Rad). The band intensity ratio was analyzed, respectively.
Statistics
Two-way analysis of variance (ANOVA) tests were used for comparisons across time between groups in vivo. One-way ANOVA tests were used for comparisons within groups in vivo and between groups in vitro. Differences were considered significant when P < .05.
RESULTS
Immunohistochemistry and Image Analysis
In the experimental tooth movement, the fibroblast-like cells expressing α-SMA were identified both in the experimental groups and in the control groups (Figure 1). However, the expression intensities of α-SMA in the control groups (Figure 1a) and in the compression areas of the experimental groups (Figure 1b) were significantly lower than those in the tension areas of the experimental groups (Figure 1c) at 3 days (P < .05), 5 days, 7 days, and 14 days (P < .01; Figure 1d). The expressions of α-SMA in the compression areas of experimental groups were a little lower than those in the control groups, but there was no significant statistical difference (P > .05). In the tension areas of experimental groups, the expressions of α-SMA were increased over time: the expressions increased from day 0 to day 5 (P < .05), kept level between day 5 and day 7 (P > .05), and reached the maximum in day 14 (P < .05).
Immunohistochemical staining of α-SMA in the periodontal ligament (PDL). Myofibroblasts expressing α-SMA (arrows) were identified in the groups. The expression intensities of α-SMA in the tension area of the experimental group (c) were significantly higher than those in the control group (a) and the compression area of the experimental group (b). The mean optical density (MOD) of α-SMA expressions over time in the PDL (mean ± SD) (d). B indicates alveolar bone; T, tooth.
*** Comparisons between groups.
††† Comparisons between the tension areas of experimental groups over time.
***, ††† P < .001.
Immunohistochemical staining of α-SMA in the periodontal ligament (PDL). Myofibroblasts expressing α-SMA (arrows) were identified in the groups. The expression intensities of α-SMA in the tension area of the experimental group (c) were significantly higher than those in the control group (a) and the compression area of the experimental group (b). The mean optical density (MOD) of α-SMA expressions over time in the PDL (mean ± SD) (d). B indicates alveolar bone; T, tooth.
*** Comparisons between groups.
††† Comparisons between the tension areas of experimental groups over time.
***, ††† P < .001.
Transmission Electron Microscopy
Transmission electron microscopy showed that some structural characteristics of myofibroblasts, such as fibronexus junctions (FJs), which were roughly parallel with extracellular fibrils and diverged into the outside matrix, actin microfilaments, rough endoplasmic reticulum, lysosomes, and pinosome, could be found (Figure 2).
Transmission electron microscopy showing ultrastructure of myofibroblasts after application of mechanical tension for 12 hours. m indicates actin microfilaments; RER, rough endoplasmic reticulum; LYS, lysosomes; v, pinosome; c, arrow, fibronexus junction. Magnification 1000×.
Transmission electron microscopy showing ultrastructure of myofibroblasts after application of mechanical tension for 12 hours. m indicates actin microfilaments; RER, rough endoplasmic reticulum; LYS, lysosomes; v, pinosome; c, arrow, fibronexus junction. Magnification 1000×.
α-SMA Expressions After Application of Cyclic Mechanical Tension in Vitro
Cyclic mechanical tension triggered an upregulation of α-SMA expression in vitro. Immunofluorescent detection indicated that the tension stimulation could induce the expression of stress fibers, and the stress fibers contained with α-SMA in cells (Figure 3).
Immunofluorescence showing stress fibers, which contained with α-SMA, expression in myofibroblasts.
Immunofluorescence showing stress fibers, which contained with α-SMA, expression in myofibroblasts.
In the experimental groups, the α-SMA showed significant upregulation with time (P < .01) and showed a peak expression at 12 hours. The expression of α-SMA mRNA was an eight-fold increase compared with the level before mechanical loading. The α-SMA expressions did not change with time in the control groups. Western analysis showed that α-SMA was expressed in cells. At 3, 6, and 12 hours, the α-SMA mRNA and protein expressions at the mechanical loading groups were significantly higher than the nonloading groups (P < .01 at 3 hours; P < .001 at 6 and 12 hours) (Figures 4 and 5).
Effects of mechanical tension on α-SMA mRNA expression in vitro. The α-SMA levels were presented as the ratio of its expression at each time point to the baseline level at time point 0 (comparisons between the mechanical loading group and the 0 hour group).
** P < .01; *** P < .001.
Effects of mechanical tension on α-SMA mRNA expression in vitro. The α-SMA levels were presented as the ratio of its expression at each time point to the baseline level at time point 0 (comparisons between the mechanical loading group and the 0 hour group).
** P < .01; *** P < .001.
Western analysis showing α-SMA expression. At 3, 6, and 12 hours, the α-SMA expressions at the mechanical loading groups were significantly higher than the nonloading groups (comparisons between the mechanical loading groups and the 0 hour group).
** P < .01; *** P < .001.
Western analysis showing α-SMA expression. At 3, 6, and 12 hours, the α-SMA expressions at the mechanical loading groups were significantly higher than the nonloading groups (comparisons between the mechanical loading groups and the 0 hour group).
** P < .01; *** P < .001.
DISCUSSION
The present results found that myofibroblasts existed in the PDL. As previously described, a key phenotypic feature of the myofibroblasts, namely α-SMA, was examined as a marker of myofibroblasts.21,22 Many researches have proved that myofibroblasts could be observed in the tension-generating tissues.14,23 Normal PDL is the tissue which is under mechanical tension, and much higher tension exists in the orthodontic periodontium than in the normal PDL. Therefore, myofibroblasts were suggested to be in the PDL, especially in the orthodontic periodontium. In the present study, this suggestion was proved. It is detected that some specialized fibroblasts were positive to α-SMA antibody in the PDL both in the experimental groups and in the control groups, while some other fibroblasts were negative.
In order to test the structural characteristics of the PDL myofibroblasts under tension, the PDL fibroblasts were cultured and cyclic mechanical force was applied. The ultrastructure of the cells was checked. The myofibroblast phenotypes are stress fibers8 which promise myofibroblasts exert high contractile force, and FJs which are adhesion complex that use transmembrane integrins to link intracellular actin with extracellular and transmit the force generation by stress to the surrounding extracellular matrix.10,24 This study showed neo-expression of α-SMA in stress fibers in the fibroblast-like cells. And, transmission electron microscopy found that bundles of actin microfilaments and FJs existed in the cells. Therefore, the results indicated that the structure of loaded PDL fibroblast-like cells coincided with the normal structural characteristics of myofibroblasts.
Our results demonstrated that orthodontic mechanical tension could induce PDL fibroblasts to myofibroblasts. Mechanical tension can cause the alignment of individual microfilaments and recruitment filaments to stress fibers, as well as formation of FJs. FJs control myofibroblast differentiation. Supermaturation FJs require high extracellular tension.10 The results confirmed the findings that the tension was essential for myofibroblast differentiation and mechanical force could upregulate the expression of α-SMA.12,14 In vivo, with little tension, the α-SMA expressions were quite weak25,26 in the control groups and in the compression areas of experimental groups. However, in the tension areas of experimental groups, the expressions of α-SMA were greatly upgraded over time. In vitro, mechanical force could induce the expression of the α-SMA.27 Therefore, as the neo-expression of α-SMA is the most reliable marker of the myofibroblasts,10 it could be proved that the mechanical tension force could induce the PDL myofibroblast differentiation from fibroblasts, which are the majority of cells in the periodontium. Furthermore, this might support the bold assumption that abundant myofibroblasts could ensure enough contractile force to keep the bone remodeling during orthodontic treatment.
The characteristics of the myofibroblasts existing in the tension area of the PDL and the capability of generating contraction force may provide myofibroblasts an opportunity to play an important role in the orthodontic tooth movement: sustaining the tension of PDL to finish the bone remodeling and taking part in periodontium remodeling. What is more significant is that the presence of TGF-β1 in the orthodontic periodentium28 is crucial to provoking and upgrading the expression of myofibroblasts.29 TGF-β1 induces fibroblast activation and α-SMA expression mainly via the Smad3 pathway.30 TGF-β1 receptor complex leads Smad3 association with Smad3 translocation into the nucleus, and Smad3 binding in the promoter area to regulate α-SMA transcription.31 As myofibroblasts have many properties above, one would expect that the myofibroblasts may be involved in the force transmission and remolding of periodontium during orthodontic tooth movement, which however needs further investigation.
CONCLUSIONS
Myofibroblasts, which neo-expressed α-SMA in stress fibers and fibronexus junctions, existed in the PDL.
Orthodontic mechanical tension could induce and upgrade expression of α-SMA in the PDL myofibroblasts. The expressions of α-SMA were upgraded over time under tension.
Acknowledgments
National Nature Science Foundation of China funded the study (grant 30970705). The experiment was performed in State Key Laboratory of Oral Disease (Sichuan University), West China Stomatology Hospital.
REFERENCES
Author notes
PhD student-graduate, State Key Laboratory of Oral Diseases, Department of Orthodontics, West China Stomatological Hospital, Sichuan University, Sichuan, China
Post doctoral researcher, State Key Laboratory of Oral Diseases, West China Stomatological Hospital, Sichuan University, Sichuan, China
Professor, Department of Orthodontics, West China Stomatological Hospital, State Key Laboratory of Oral Diseases, Sichuan University, Sichuan, China
MS student-graduate, West China Stomatological Hospital, State Key Laboratory of Oral Diseases, Sichuan University, Sichuan, China