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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.

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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

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.

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). 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), and reached the maximum in day 14 (P

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.

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).

In the experimental groups, the α-SMA showed significant upregulation with time (P

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).

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).

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.

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