Human premolars planned to be extracted for orthodontic treatment were used. The institutional Review Board of Gangnam Severance Hospital, Yensei University approved both the protocol and informed consent. Written informed consent was obtained from each patient (No 3-2009-0069). After extraction, the teeth were stored in 50 ml polyethylene tubes containing 10 ml alpha minimum essential medium (α-MEM)(Gibco/BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone, Loga, UT, USA) and 1% penicillin. After washing 3 times with α-MEM (3% antibiotics/antimycotics), the tooth was held using sterilized gauze. The soft tissue on the middle 1/3 of the root was obtained by curettage with a No.15 blade. The tissue collected was kept in a 100 mm² culture dish containing α-MEM and the culture plate was washed three times with the same culture media. A new culture dish was used to apply the tissue, which was kept in the incubator at 37℃ in a humidified atmosphere containing 5% CO₂ in air until the plate became full with growing cells. The remaining tooth was split after making bucco-linugal grooves. The pulp tissue was removed using a sterilized spoon excavator and cultured in a similar manner as periodontal tissue.

White MTA (ProRoot) and Bioaggregate were mixed according to the manufacturer's instructions. A 6-well tissue culture dish was coated with the MTA paste prepared with sterile water (10 mg cement/ml of H₂O per well). PDL and pulp cells were seeded on culture dishes coated with the freshly mixed cements to determine if the initial toxicity of the freshly mixed material can affect cell attachment and growth.

The cells were plated on either an MTA coated or plastic surface (5.0×10⁵ cells/well in a 6-well plate), and cultured for 1, 6, 48 and 72 hours before being examined by phase microscopy (Nikon TMS, Nikon Inc. Melville, NY, USA). Cells grown on cement coated dishes could not be visualized by this method since both cements used do not transmit light. Thus, the cells grown on plastic surface around cements were examined under the microscope and photos were taken.

The cells were grown for 3 days on a 12-well culture plate coated with each type of cement. After 1×10⁵ cells were seeded, the cells grown on a regular plastic plate were used as the control. An EZ-Cytox cell viability assay kit (DAEILL LAB Service Co., Seoul, Korea), which measures the cell dehydrogenase activity was used. This kit is similar to a MTT assay measuring the mitochondrial activity in viable cells. After adding 100 µL of the assay reagent, cell culture plate was kept in a 37℃ incubator for 1 hour. Subsequently, 100 µL of supernatant was taken and added to a 96-well plate. The color density at 450 nm was read using an ELISA reader (Molecular Devices Inc/E-max, Washington, DC, USA). The data is presented as a percentage when the density of the control was considered to be 100. The average of the triplicates was used for data analysis.

The data from the cell viability tests were analyzed using a Student's t-test and one-way ANOVA. The control value was set to 100, and the results are expressed as the mean% ± standard deviation. A p value < 0.05 was considered significant.

The effects of Bioaggregate cement on human pulp and PDL cell growth were examined by observing the cells grown on this cement using a phase microscope (Figure 1). The effects of MTA and Bioaggregate on cell growth were determined by examining the cells around the materials because cells grown on top of the material could not be visualized. Both types of cells on Bioaggregate showed no inhibition zones or gaps around the material at any time intervals. However, although cells were fully grown around MTA, an inhibition zone was detected in the human pulp and PDL cell culture grown with MTA.

Before performing the viability tests, each cell culture plate was observed by optical microscopy to determine if the cell were growing healthily without dead floating cells.

There were no significant differences in the human pulp and PDL cell cultures between control and experimental groups (Figure 2). There were 6.3 ± 8.02% and 9.25 ± 9.74% less viable cells in human PDL cells when cultured with MTA and Bioaggregate. The similar result was shown in pulp cells.