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Comparative Study
. 2025 Aug:197:117512.
doi: 10.1016/j.bone.2025.117512. Epub 2025 May 3.

Comparison of the bone remodeling in the midpalatal suture during maxillary expansion between young and middle-aged mice

Hyeran Helen Jeon  1 , Mary Cruz Contreras Salas  2 , Kyungjoon Park  3 , Lindsay Fisher  3 , Sara Ha  3 , Caroline Palmer  3 , Fionna Chan  3 , Dana T Graves  4
Affiliations
Comparative Study

Comparison of the bone remodeling in the midpalatal suture during maxillary expansion between young and middle-aged mice

Hyeran Helen Jeon et al. Bone. 2025 Aug.

Abstract

Maxillary expansion is a common orthodontic procedure for treating maxillary transverse deficiency. However, the cell responses to mechanical force may vary across different age groups, suggesting the need for age-specific treatment protocols. To compare the age-related responses to the mechanical force, we examined the 6-week- and 12-month-old mice undergoing maxillary expansion with 0.012-in. stainless steel orthodontic wire bonded to the maxillary first and second molars (25 g force). Mice were euthanized on days 0, 3, 7, and 14 for analysis. MicroCT analysis, tartrate-resistant acid phosphatase (TRAP) stain, and immunofluorescence/immunohistochemistry stain using antibodies to RUNX2, alkaline phosphatase (ALP), Gli1 and Ki67 along with the TUNEL assay, were conducted to evaluate suture width, osteoclast activity, new bone formation and mesenchymal stem cell (MSC) proliferation and apoptosis. Both 6-week- and 12-month-old mice exhibited successful midpalatal suture opening, but young mice demonstrated earlier and more intense osteoclast activity, along with higher expression of RUNX2 and ALP. Young mice also exhibited a higher percentage of Gli1+Ki67+ immunopositive cells, while middle-aged mice showed a higher percentage of Gli1+TUNEL+ positive cells on day 3 after maxillary expansion. Our findings suggest that aging negatively impacts mechanical force-induced bone remodeling by reducing osteoclastogenesis, osteogenesis, and MSC proliferation while increasing MSC apoptosis.

Keywords: Aging; Bone remodeling; Maxillary expansion; Mesenchymal stem cell (MSC); Osteoclastogenesis; Osteogenesis.

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Conflict of interest statement

Declaration of competing interest The authors state no conflict of interest.

Figures

Fig. 1.
Fig. 1.. Mouse maxillary expansion model.
An 0.012-in. stainless steel orthodontic wire forming an opening loop was bonded to the 1st and 2nd maxillary molars bilaterally using light-cured adhesive. The initial width of the expander is set to 2.5 mm, and it is activated to 2 mm to exert a force of 25 g. Bar, 1 mm.
Fig. 2.
Fig. 2.. MicroCT analysis.
(A) Osirix analysis on a young D3 sample using a coronal slice at the palatal roots of the maxillary 1st molars. Top to bottom: a) intermolar width (crown), b) intermolar width (furcation), suture width: c) oral third,d) middle third,e) nasal third, f) intermolar width (root apices). Bar, 1 mm. (B) Bone volume fraction analysis. A region of interest (ROI) measuring 0.5 mm (width) x 0.1 mm (height) was drawn in the coronal plane, encompassing the midpalatal suture and bilateral bones at the level of the maxillary first molars bilaterally. The purple area represents g) the suture gap, and the brown represents h) the bony edges of the mid-palatal suture. Bar, 1 mm. (C) The ROI spanned from the mesial margin of the palatal root to the distal margin of the distobuccal root, with an average total volume of 0.05 mm3. Bar, 1 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3.
Fig. 3.. MicroCT measurements.
(A) 3D constructed microCT images. Bar, 1 mm. (B—C) Suture width at oral third, (D-E) Suture width at middle third, (F-G) Suture width at nasal third, (H—I) Intermolar width at furcation, (J-K) Bone volume fraction. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Y: young mice and A: middle-aged mice.
Fig. 3.
Fig. 3.. MicroCT measurements.
(A) 3D constructed microCT images. Bar, 1 mm. (B—C) Suture width at oral third, (D-E) Suture width at middle third, (F-G) Suture width at nasal third, (H—I) Intermolar width at furcation, (J-K) Bone volume fraction. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Y: young mice and A: middle-aged mice.
Fig. 4.
Fig. 4.. Osteoclast formation.
(A) TRAP-stained images (x20). Bar, 50 μm. (B—C) The number of osteoclasts in the bone marrow area. **P < 0.01. Y: young mice and A: middle-aged mice.
Fig. 5.
Fig. 5.. Expression of Osteogenic Markers.
(A) Representative images of RUNX2 immunofluorescence stain in the midpalatal suture. B: bone and S: midpalatal suture. 40×. Bar, 50 μm. (B—C) The percentage of RUNX2+ cells per area. (D-E) The percentage of ALP matrix. *P < 0.05, ***P < 0.001. Y: young mice and A: middle-aged mice.
Fig. 6.
Fig. 6.. Gli1/Ki67 Immunofluorescence stain.
(A) Representative images in the midpalatal suture. 40×. Bar, 25 μm. (B—C) The percentage of Gli1-immunopositive cells. (D-E) The percentage of Ki67-immunopositive cells. (F-G) The percentage of Gli1/Ki67-immunopositive cells. *P < 0.05, **P < 0.01. Y: young mice and A: middle-aged mice.
Fig. 6.
Fig. 6.. Gli1/Ki67 Immunofluorescence stain.
(A) Representative images in the midpalatal suture. 40×. Bar, 25 μm. (B—C) The percentage of Gli1-immunopositive cells. (D-E) The percentage of Ki67-immunopositive cells. (F-G) The percentage of Gli1/Ki67-immunopositive cells. *P < 0.05, **P < 0.01. Y: young mice and A: middle-aged mice.
Fig. 7.
Fig. 7.. Gli1/TUNEL Immunofluorescence stain.
(A) Representative images in the midpalatal suture. 40×. Bar, 25 μm. (B—C) The percentage of TUNEL-positive cells. (D-E) The percentage of Gli1/TUNEL-immunopositive cells. *P < 0.05, **P < 0.01. Y: young mice and A: middle-aged mice.
See this image and copyright information in PMC

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