Antibacterial And Cytotoxic Effects of Cysteamine Alone And in Combination With Various Intracanal Medications (In Vitro Study)

Abstract

Summary The microbiological ecosystem is considered the main obstacle in endodontics. Due to its variability and defending mechanisms especially E. faecalis biofilm. Instrumentation in combination with chemical disinfection is of great importance to decrease microbiological load to the level that allows the immune mechanism to deal with many challenges related to the anatomy of the root canal system and microbiological virulence factors. Intracanal medications are one of the foundation stones in root canal disinfection and they showed variability in their antibacterial effectivity and biocompatibility.

Our study aimed to assess and compare the antibacterial effect of Cysteamine material and its combinations with chlorhexidine, triple antibiotic paste, and calcium hydroxide on E. faecalis biofilm inoculated in root canals ex-vivo. As well, the biocompatibility was assessed in vitro using BHK-21 fibroblast by MTT reduction assay.

Preparations of tested medications were made in fresh solutions to avoid the instability of Cysteamine. Methylcellulose was added to the solutions to obtain gel consistency for the antibacterial test. The plain gel was prepared using methylcellulose and water for control groups. Preparations used for cytotoxicity were used in liquid form without the addition of methylcellulose as it’s considered inert material and to allow filtrations of solutions before testing.

Forty-four Human extracted single-rooted teeth were used for antibacterial effect testing. They were decoronated, standardized to 15 mm in length, and shaped with a combination of rotary Fanta F one files and manual files till size 50. After that, the smear layer was removed by sodium hypochlorite followed by saline then EDTA. The final wash was done by saline, then dried by paper points. Apical foramina were sealed using flowable composite. Longitudinal grooves were done on the outer surface of four teeth, acting as an observational group, planned for biofilm monitoring using the scan electron microscope. After that, all specimens were autoclaved.

Specimens were inoculated in suspension infected by E. faecalis and incubated for 30 days at 37℃ in 100% humidity to ensure biofilm formation. Negative control samples were inoculated in sterile saline. They followed the same sequence except for the inoculation of E. faecalis in the broth.

After the incubation period, biofilm was verified using the field emission scanning electron microscope on the observational group. The remaining Specimens were randomly assigned into two groups, experimental and control groups. These groups are subdivided into subgroups according to the intracanal medication used. Experimental group subgrouping was as follows: subgroup A: Cysteamine gel, subgroup B: CHX Cysteamine combination gel, subgroup C: CaOH Cysteamine combination gel, and subgroup D: TAP Cysteamine combination gel. The Control group was subdivided into positive and negative and medicated with plain gel. All samples were medicated similarly for seven days and sealed by a temporary filling. After seven days, roots were reassessed under strict aseptic conditions and intracanal samples were obtained using k files and paper points. Samples were collected in test tubes and undergo culture-based analysis by calculating CFU.

Cytotoxicity and biocompatibility were assessed using MTT assay on BHK cells seeded in well microtiter plates at an initial cell density of approximately 2.5 × 104 cells/1 ml and allowed to attach overnight. BHK cells were transferred to 12 cell culture titer plates (each plate contains 12 wells). The cell culture medium was then removed and test materials suspension was dispensed to the BHK cell line precultured well plates in a descending order to achieve serial dilution.

The testing materials were sterile filtrated using a 0.22 μm syringe filter. BHK-21 cells precultured well plates (Nunc, USA) were treated with descending 12-fold serially diluted medications at 37 °C for 24 h. MTT solutions were added to each well and incubated for a further four hours in the same culture conditions. Plates were read at 570 nm using a microplate reader. Cell viability % was calculated and (IC50) was calculated from curves constructed by plotting cell survival percent versus drug concentration.

Results showed that the CHX Cysteamine combination had the highest antibacterial effect with the highest cytotoxicity compared to other tested medications in all concentrations. TAP Cysteamine combination is the second-highest drug in antibacterial effect with the lowest cytotoxicity in higher concentrations. Cysteamine had the lowest antibacterial results with an acceptable cytotoxicity level compared to other medications. CaOH Cysteamine combination showed a non-significant difference in antibacterial effect to TAP Cysteamine combination with higher cytotoxicity in higher concentrations and lower cytotoxicity level in lower concentrations.