Effect of Calcium Hydroxide Associated with Chlorhexidine on Cultured Cells

Objectives: evaluate the calcium hydroxide-based paste (Calen®), associated or not to 0.4% chlorexidine digluconate, in culture of RAW 264.7 macrophages lineage and primary culture of osteoblastic lineage.

Methods: In RAW 264.7 macrophages culture we evaluated: cellular viability (MTT assay), immunestimulatory (nitric oxide detection) and anti-inflammatory (nitric oxide, TNF-a and IL-1a detection) properties. In primary cell culture of osteoblastic lineage we evaluated the cellular viability (3, 7 and 10 days), total protein content (Lowry modified method for 7 and 10 days), alkalinic phosphatase activity (thymolphthalein liberation for 7 and 10 days, and in situ by Fast red method), extracellular matrix non-collagen protein (stained by indirect immunofluorescence and direct fluorescence by phalloidin and DAPI for 3, 7 and 14 days), and mineralized matrix nodular formation (stained by Alizarin red for 3, 7 and 14 days). The data were statistically analyzed by Kruskal-Wallis test, followed by Dunn's post test (significance of 5%).

Results: We observed that occurs low stimullatory activity for the different concentrations evaluated of calcium hydroxide-based paste (Calen®), associated or not to 0.4% chlorexidine digluconate. Also, we could observe anti-inflammatory activity with inhibition of nitric oxide and cytokines quantification (TNF-a e IL1-a), after the use of 25µg/ml calcium hydroxide associated to chlorhexidine (25µg/ml). Same results to the control were observed after evaluation of Calen® paste associated or not to 0.4% chlorexidine digluconate, because the data of total protein content, alkaline phosphatase, and mineralized matrix nodular formation were not statistically different (p>0.05). Related to the morphological characterization of primary culture, the results obtained were the same after comparison of Calen® paste, associated or not to 0.4% chlorexidine digluconate.

Conclusions: we could conclude that the addition of chlorexidine showed no additional benefits to the calcium hydroxide-based paste.