Alteration of Oral and Perioral Soft Tissue in Mice following Incisor Tooth Extraction

Abstract

Oral and perioral soft tissues cooperate with other oral and pharyngeal organs to facilitate mastication and swallowing. It is essential for these tissues to maintain their morphology for efficient function. Recently, it was reported that the morphology of oral and perioral soft tissue can be altered by aging or orthodontic treatment. However, it remains unclear whether tooth loss can alter these tissues' morphology. This study examined whether tooth loss could alter lip morphology. First, an analysis of human anatomy suggested that tooth loss altered lip morphology. Next, a murine model of tooth loss was established by extracting an incisor; micro-computed tomography revealed that a new bone replaced the extraction socket. Body weight was significantly lower in the tooth loss (UH) group than in the non-extraction control (NH) group. The upper lip showed a greater degree of morphological variation in the UH group. Proteomic analysis and immunohistochemical staining of the upper lip illustrated that S100A8/9 expression was higher in the UH group, suggesting that tooth loss induced lip inflammation. Finally, soft-diet feeding improved lip deformity associated with tooth loss, but not inflammation. Therefore, soft-diet feeding is essential for preventing lip morphological changes after tooth loss.

Keywords: anatomy; human; lip morphology; mouse; tooth loss.

Figure 1

The morphology of the upper lip in humans: Patients and cadavers were divided into two groups based on the presence (Group A) or absence of anterior teeth (Group B). (A) Magnetic resonance imaging findings: The lip bent toward the oral cavity side in Group B compared with the findings in Group A (Panels a–d). The angle between the α-line and the Su–In line was smaller in Group B than in Group A (Panels e and f; * p < 0.05). The distance from Su to In, or N–Su, was not significantly different between the groups. (B) Cadaver findings: The lip bent toward the oral cavity side in Group B compared with the findings in Group A (Panels b and c). The length of line b revealed greater thickening of the oral mucosa in Group B than in Group A (Panels a and e; ** p < 0.01). The lengths of line a and line c did not differ between the groups (Panels a, d, and f). Su: subnasal point; In: inferior tip of the upper lip.

Figure 2

The establishment of incisor-deficient mice: (A) Before or after anterior tooth extraction. After tooth extraction, new bone formation was identified in the extraction sockets (arrowheads). (B) Physical description of the tooth loss (UH) and non-extraction control groups (NH). Body size was smaller in the UH group than in the NH group. Body weight was significantly lower in the UH group than in the NH group (* p < 0.05, ** p < 0.01, *** p < 0.01). The total protein expression was lower in the UH group than in the NH group (* p < 0.05). (C) Micro-computed tomography. Upper incisors were identified in the tooth socket (yellow arrows). New bone formation was identified in the extraction sockets (purple arrows). The lower incisor tooth was extended in UH mice compared with NH mice (red arrowheads). (D,E) Alteration of the lip and tongue morphology after tooth loss. The width of the area between the upper and lower lips was significantly different between the NH and UH groups (* p < 0.05), but there was no change in the major axis of the area between the groups. The width and major axis of the tongue did not differ between the groups.

Figure 3

Lapping behavior in mice: (A) Electrophysiological single units during lapping behavior. Autocorrelogram of the lapping events revealed the temporally precise rhythmicity of the behavior at a periodicity of approximately 8 Hz. The upper row presents the first half, and the lower row presents the second half. Lapping counts in the second half were smaller in the tooth loss (UH) group than in the non-extraction control (NH) group (* p < 0.05). (B) Tongue movement during lapping behavior. Recorded tongue movements revealed that NH mice protruded their tongues anteroinferiorly, whereas UH mice protruded their tongues anterosuperiorly.

Figure 4

(A) Experimental procedure for the proteomic investigation. Histogram of protein counts grouped according to log2 protein ratios. In total, 1473 proteins were detected in the two groups using high-sensitivity liquid chromatography–tandem mass spectrometry. (B) Volcano plot of the statistical significance of differences (Student’s t-test p-value) as a function of the average protein ratios. Fifteen proteins were enriched in UH mouse lips (orange), while 14 were enriched in NH mouse lips (green). Heatmap illustrating quantitative alterations of representative proteins. Enrichment of S100a8, S100a9, keratin 6a (KRT6a), keratin 6b (KRT6b), keratin 8 (KRT8), and keratin 16 (KRT16) expression was detected in UH mouse lips.

Figure 5

An upper lip comparison between the non-extraction control (NH) and tooth loss (UH) groups: (A) Histological studies of upper lips. The upper lip was more strongly protruded into the oral cavity in UH mice than in NH mice (Panels c–g). (B) High-magnification views of the upper lips. After extracting an incisor, the thickness of the upper lip’s epithelium was increased (Panels a and g) (Panel m; ** p < 0.01). The cross-sectional area of the orbicularis oris muscle (arrowheads) was reduced (Panels b and h) (Panel n; ** p < 0.01). KRT6A/6B fluorescence intensity in the granular layer was lower in UH mice than in NH mice (Panels c, d, i, j, o, and p). S100A8/A9 density was significantly higher in UH mice than in NH mice (Panels e, f, k, and l) (Panels q and r; ** p < 0.01).

Figure 6

Soft-diet feeding affects lip morphology: (A) Body weight was significantly higher in the soft-diet feeding (US) group than in the hard-diet feeding (UH) group (Panel b; * p < 0.05, *** p < 0.001). Because the thickness and area of the lamina propria were smaller in US mice than in UH mice (Panels c and d) (Panels e and f; * p < 0.05), lip protrusion into the oral cavity was less severe in US mice. (B) The thickness of the epithelium and area of the muscle did not differ between the groups (Panels a, f, k, and l). The fluorescence intensity of KRT6A/6B in the granular layer did not differ between UH and US mice (Panels m and n). The density of S100A8/A9 was not different between the groups (Panels d, e, i, j, o, and p).