Fig 1.
(A) The experiment schedule. Gingival massage was performed on the gingiva on the mesial aspect of the maxillary left first molar twice a week for 4 weeks, for a total of eight sessions. The blood flow velocity in the same location was measured 3 days after the gingival massage using a laser Doppler flowmeter. After completing 4 weeks of blood measurements, hematoxylin and eosin + Indian ink staining specimens and resin injected blood vessel mold specimens were prepared. (B) The typical change in gingival blood flow during reactive hyperemia. Base blood flow (dotted line: Base Flow), the difference between the Base Flow and the peak blood flow* (white two-way arrow: Peak), time required until the peak blood flow halves (black two-way arrow: T1/2), and total increase in blood flow (gray shaded area: Mass).
Fig 2.
Changes in age-related reactive hyperemia.
(A) Base Flow, (B) Peak, (C) T1/2, (D) Mass. The 1-year-old rats showed reduction in Base Flow compared with the 7-week-old rats as well as a shortening of T1/2 compared with the 7-week-old and 6-month-old rats. The data are expressed as mean ± standard error of the mean (n = 8–10). *P<0.05 vs. 7 weeks, #P<0.05 vs. 6 months.
Fig 3.
Effect of gingival massage on age-related decline in reactive hyperemia.
(A) Base Flow, (B) Peak, (C) T1/2, (D) Mass. Compared with the control group, 4 weeks of massage in the gingival massage group lengthened the T1/2 in the 7-week-old rats and increased the mass in the 6-month-old and 1-year-old rats. The data are expressed as mean ± standard error of the mean (n = 6). *P<0.05 vs. each control groups.
Fig 4.
Morphological analysis (hematoxylin and eosin + Indian ink staining specimens).
(A–C) Control group, (A) 7-week-old, (B) 6-month-old, (C) 1-year-old, (D–F) Gingival massage (MSG) group, (D) 7-week-old, (E) 6-month-old, (F) 1-year-old. Looped blood vessels were observed corresponding to the lamina propria under the outer epidermis of the gingival surface layer. In the control group, the height of the looped blood vessels (white arrowheads) reduced with age. There was interruption of blood flow in some blood vessels; hence, the ink was unable to enter these vessels (black arrowheads). Compared with the control group, most of the blood vessels in the MSG group maintained an ink-injected circulation (white arrowheads). The total area of blood vessels stained with ink within a certain range of images (200×200 μm) was increased in the MSG group compared to the control group for all three ages (A: 4177.0±1199.9, B: 2180.0±527.3, C: 1858.2±781.3, D: 5285.4±602.2, E: 3759.8±1592.0, and F: 3878.2±657.5 μm2, n = 3 in each group) (A: 4177.0±1199.9, B: 2180.0±527.3, C: 1858.2±781.3, D: 5285.4±602.2, E: 3759.8±1592.0, and F: 3878.2±657.5 μm2, n = 3 in each group). Regarding the one-year-old rats, there was a significantly higher amount of blood vessel staining with ink in the gingival massage group compared to the control group.
Fig 5.
Morphological analysis (vascular corrosion casting model of gingival circulation).
(A–C) Control group, (A) 7-week-old, (B) 6-month-old, (C) 1-year-old, (D–F) Gingival massage (MSG) group, (D) 7-week-old, (E) 6-month-old, (F) 1-year-old. This photograph shows the synthetic resin injected into the blood vessel. Therefore, where there is blood flow (or vasculature), there is resin, and where there is no blood flow, there is no resin. The 7-week-old control group (A) has blood flow (vasculature) in the looped capillaries around the teeth, indicating that blood flow decreases with age. In the massage group, there was an increase in blood flow (vasculature) compared to the control group at each period. In the control group, the loops (white arrowheads) had age-related reduction in height and exhibited loss of loop morphology. Part of the surface of the blood vessels was rough; the flow of blood in some blood vessels was interrupted (black arrowheads). The MSG group had a dense vascular network including loops (white arrowheads) compared with the control group.