Effects of chlorhexidine, essential oils and herbal medicines (Salvia, Chamomile, Calendula) on human fibroblast in vitro

Abstract

Antiseptic rinses have been successfully used in inflammatory states of the gums and oral cavity mucosa. Antibacterial effects of chlorhexidine, essential oils and some herbs are well documented. Reaction of host tissue to these substances has much poorer documentation. The aim of the study was to analyse the influence of chlorhexidine (CHX), essential oil (EO: thymol, 0.064%; eucalyptol, 0.092%; methyl salicylate, 0.060%; menthol, 0.042%) mouth rinses and salvia, chamomile and calendula brews on fibroblast biology in vitro. The human fibroblast CCD16 line cells were cultured in incubation media which contained the examined substances. After 24 and 48 hours, the cell morphology, relative growth and apoptosis were evaluated. Exposure of fibroblasts to CHX, EO or salvia caused various changes in cell morphology. Cells cultured for 48 hours with CHX revealed a noticeably elongated shape of while cells cultured in high EO concentration or with salvia were considerably smaller and contracted with fewer projections. Chlorhexidine, EO and salvia reduced the fibroblast proliferation rate and stimulated cell death. Both reactions to EO were dose dependent. Cells exposure to chamomile or calendula brews did not change morphology or proliferation of fibroblasts. The results of this in vitro study showed that in contrast to chamomile and calendula, the brews of EO, CHX or salvia had a negative influence on fibroblast biology.

Keywords: chlorhexidine; essential oils; fibroblast; herbs; morphology; mouth rinses; proliferation.

Fig. 1

The morphology of fibroblasts line CCD16. A and B: The fibroblasts in the control group were characterised by high diversification of shapes. The centrally located nucleus revealed the granular structure of nucleic chromatin with two nucleoli. C and D: The morphology of fibroblasts after 48-hour-culture in medium with CHO 0.1%: most cells were elongated, demonstrating isolated projections with strongly condensed granular nucleic chromatin structure. E and F: The morphology of fibroblasts after 48-hour-culture in medium with essential oils: cells were considerably contracted with narrow projections; many dead cells were observed (F). G and H: The morphology of fibroblasts after 48-hour-culture in medium with salvia brew: most cells were round-shaped and had isolated narrow projections; the increasing number of apoptotic and necrotic cells was observed (H). A, C, E, G – no staining cells. B, D, F, H – cells stained with a mix of propidium iodine dye (dead cells – pink nucleus fluorescence) and Hoechst 33342 (living cells – blue nucleus fluorescence); microscope magnification 20×

Fig. 2

The effect of alcohol, chlorhexidine 0.1% (CHX), essential oils (EO) in dilution of 10% (EO 10), 50% (EO 50), 100% (EO 100) and salvia, chamomile or calendula brews on the relative changes of the number of cells in percent after 24 (A) and 48 (B) hours of culture