Age-associated changes in salivary biomarkers and periodontal ligament fibroblast senescence in vitro
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OBJECTIVES: Aging is characterized by a progressive loss of function and presents a primary risk factor for major human pathologies. There is a positive correlation between aging and periodontal destruction. Saliva is a complex fluid that contains numerous biological markers and has an important diagnostic value. D-Galactose has been used to induce senescence in multiple cell types. The effect of D-Galactose on human periodontal ligament fibroblasts (hPDLF) has not been demonstrated. The first part of this study investigated the impact of age on a wide range of salivary biomarkers of inflammation and tissue turnover. The second part of this study investigated the induced senescence effect of D-Galactose on hPDLF. METHODS: Part I included 74 healthy subjects undergoing orthodontic treatment. Whole saliva samples were collected prior to starting orthodontic treatment and were studied by multiplex immunoassay for several inflammatory and tissue turnover markers. For part II, hPDLF were cultured and passaged. At p3, cells were assigned to control and test groups. The test group received D-Galactose-supplemented media in different concentrations. Cells were incubated for 24 and 48 hrs. (SA-ß-Gal) staining, Western blotting and supernatant analysis were done to investigate senescence-associated changes in hPDLF. RESULTS: For part I, the final number of participants included 30 males and 44 females, with the age range 8-63 years old. Subjects were categorized into quartiles (<13 y.o., 13-16 y.o., 17-27 y.o., >27 y.o.). Significant difference among the four age groups was found in BMP-4, fibronectin, VEGF, IGFBP-2, IGFBP-4, IGFBP-6, IGFBP-7, IL-1, lactoferrin, MMP-2 (p<0.05). In most of these analyses, the young adults in the third quartile (17-27 y.o.) were significantly different from others (p<0.05). For part II, D-Galactose treated cells showed a dose-dependent increased expression of (SA-ß-Gal), higher expression of pMAPK and decreased expression of fibronectin (p<0.05). CONCLUSIONS: From part I, the data suggested that salivary biomarkers of inflammation and tissue turnover showed significant variation in a systemically and periodontally healthy group of individuals when different age groups were compared. From part II, the data suggested that D-Galactose induced hPDLF to express senescence-related changes including higher expression of SA-ß-Gal and increased pMAPK activity.