Early effects of orthodontic forces on osteoblast differentiation in a novel mouse organ culture model

Abstract

Objective: To develop a mouse orthodontic organ culture model and examine early-induced changes in osteoblast differentiation markers within the periodontal ligament (PDL) and alveolar bone.

Methods: Mandibles from 4- to 12-week-old transgenic mice were dissected and hemisected. A conventional superelastic orthodontic spring (25 grams) was bonded to the incisor and first molar on one side of the mandible; the other side served as a control. Dissected mandibles were cultured for 6 hours and then were histologically analyzed for proliferation (BrdU immunostaining) and fluorescent protein expression. Additionally, an in vivo model using the same methods was applied to 3.6 Col1-GFP transgenic mice.

Results: In vitro, after 6 hours of orthodontic loading, a significant increase was noted in 3.6Col1-GFP- and BSP-GFP-positive cells within the tension side of the PDL compared with unloaded controls. On the compression side, a significant decrease in positive cells in 3.6Col1-GFP mice was observed in the PDL compared with unloaded controls. In vivo, the same tendencies were found.

Conclusion: This novel in vitro mandibular tooth movement organ culture model coupled with transgenic mouse technology provides a powerful tool for delineating initial cellular and molecular events of orthodontic tooth movement.

Figure 1

(A) Spring appliance bonded to hemisected mouse mandibles. (B and C) Hematoxylin/eosin-stained sagittal cross sections of unloaded and loaded molars (D and E) after 6 hours. Arrow shows the direction of the orthodontic force.

Figure 2

BrdU-stained sections showing the proliferating cells (black arrow) on tension (A and B) and compression (C and D) sides of loaded (B and D) and unloaded molars (A and C).

Figure 3

In vitro BrdU labeling index. Analysis of proliferation by BrdU staining was performed in the furcation of the first molar in areas exposed to compression or tension. Points represent the mean and SEM (n  =  3) for mice of loaded and unloaded controls. * P < .05.

Figure 4

Fluorescent images (A through F) and hematoxylin/eosin stainings (G through L) of sagittal sections of organ cultured hemisected mandibles from 3.6Col1-GFP and BSP-GFP transgenic mice (unloaded [A, C, and E] and loaded for 6 hours [B, D, and F] first molars). Arrows signify the direction of force.

Figure 5

In vitro 3.6Col1-GFP labeling index. Expression of 3.6Col1-GFP was analyzed in the furcation of the first molar in areas exposed to compression or tension. Points represent the mean and SEM (n  =  3) for mice of loaded and unloaded controls. * P < .05.

Figure 6

In vitro BSP-GFP labeling index. BSP-GFP expression was analyzed in the furcation of the first molar in areas exposed to compression and tension. Points represent the mean and SEM (n  =  3) for mice of loaded and unloaded controls. * P < .05.

Figure 7

BrdU stained sections show proliferating cells (black arrows) on tension (A and B) and compression (C and D) sides of loaded (B and D) and unloaded molars (A and C).

Figure 8

In vivo BrdU labeling index. Proliferation by BrdU staining was analyzed in the furcation of the first molar in areas exposed to compression or tension. Points represent the mean and SEM (n  =  3) for mice. * P < .05.

Figure 9

In vivo fluorescent images (A, B, C, and D) and hematoxylin/eosin stainings (E, F, G, and H) of sagittal sections of mandibles from 3.6Col1-GFP transgenic mice exposed to orthodontic force (C, D, G, and H) or serving as controls (A, B, E, and F). Arrows depict direction of force.

Figure 10

In vivo 3.6Col1-GFP labeling index. Expression of 3.6 Col1-GFP was analyzed in the furcation of the first molar in areas exposed to compression or tension. Points represent the mean and SEM (n  =  3) for mice. * P < .05.

Figure 11

(A) Fibroblastic-like cells in the PDL (white arrows) in the tension side of in vivo 3.6Col1-GFP mice. (B) Corresponding hematoxylin/eosin stainings show the alveolar bone, the PDL, and dentin.