Abstract: BACKGROUND: Terpinen-4-ol has been found to have a significant antibacterial activity against pathogenic bacteria associated with peri-implantitis and can reduce the formation of surface biofilm and the occurrence of peri-implantitis when applied to the surface of titanium implants; however, the studies regarding its biosafety are lacking.
OBJECTIVE: To explore the antibacterial effects of terpinen-4-ol on Staphylococcus aureus and Streptococcus mutans and its effect on the growth and osteogenic differentiation of MC3T3-E1 cells.
METHODS: Different concentrations of terpinen-4-ol were dissolved in dimethyl sulfoxide. The minimum inhibitory concentration and minimum bactericidal concentration of pineolene 4 alcohol solution against Staphylococcus aureus and Streptococcus mutans were tested. The 1/2 minimum inhibitory concentration, minimum inhibitory concentration, and minimum bactericidal concentration of pineolene 4 alcohol were tested by bacteriostatic zone test. MC3T3-E1 cells were treated with different concentrations (0%, 0.3%, 0.2%, 0.1%, v/v) of terpinen-4-ol in medium or calcification-inducing solution. The proliferation activity of cells was detected by CCK-8 assay. Cells were stained with rhodamine-phalloidin to observe cytoskeleton. Differentiation ability of cells was detected by alkaline phosphatase activity assay. The formation of mineralized nodules was observed by alizarin red staining. The mRNA expression of Runx2 and osteocalcin was detected by real-time reverse transcription-polymerase chain reaction.
RESULTS AND CONCLUSION: (1) Minimum inhibitory concentration and minimum bactericidal concentration of terpinen-4-ol against Streptococcus mutans were 0.1% (v/v) and 0.4% (v/v) respectively and against Staphylococcus aureus were 0.03% (v/v) and 0.125% (v/v) respectively. With the increase of the concentration of terpinen-4-ol, the diameter of bacteriostatic zone was significantly increased (P < 0.05). (2) CCK-8 assay showed that terpinen-4-ol promoted the proliferation of MC3T3-E1 cells, with the most obvious effects in the 0.2% group. (3) Rhodamine-phalloidin staining showed that terpinen-4-ol promoted the adhesion of MC3T3-E1 cells. (4) Alkaline phosphatase activity assay and alizarin red staining showed that terpinen-4-ol promoted the early differentiation and mineralization of MC3T3-E1 cells, with the most obvious effects in the 0.2% group. (5) Real-time reverse transcription-polymerase chain reaction showed that the mRNA expression of Runx2 was significantly higher in the 0.3%, 0.2%, and 0.1% groups than in the 0% group at 1 day of calcification induction (P < 0.05), and the mRNA expression of Runx2 and osteocalcin was significantly higher in the 0.2% and 0.1% groups than in the 0.3% and 0% groups at 7 and 14 days of calcification induction (P < 0.05). (6) These results indicate that terpinen-4-ol can inhibit the growth of Staphylococcus aureus and Streptococcus mutans and promote the proliferation, differentiation and mineralization of MC3T3-E1 cells.

Key words: terpinen-4-ol, Staphylococcus aureus, Streptococcus mutans, MC3T3-E1 cells, osteocalcin, Runx2