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 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.
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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