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To obtain PSLs, PS, phosphatidylcholine and cholesterol at a 2:1:1 molar ratio were dissolved in chloroform methanol (9:1 v/v). The solvent was evaporated in the presence of nitrogen gas in a glass tube and was further removed in a vacuum chamber for 2 h. The lipid film was subsequently hydrated in phosphate-buffered saline (PBS) (pH 7.4) and vigorously vortexed to obtain a 0.25-mM suspension of total lipid. The lipid suspension was filtered through a 0.1-μm membrane filter. PS was purchased from Avanti Polar Lipids (Alabaster, AL, USA), and other chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

HDPCs for primary culture were obtained from premolars that were removed from patients for orthodontic purposes under the guidelines approved by the Institutional Review Board of Seoul National University Dental Hospital (CRI06015). The teeth were cut at the apex, and the dental pulp was removed from the pulp cavity and immersed in minimal essential medium (MEM) that contained 20% fetal bovine serum (FBS) and antibiotic solution (100 U/ml of penicillin-G and 100 mg/ml of streptomycin). The pulp was minced into pieces and incubated at 37℃ in humidified atmosphere (5% CO₂/95% air) for 20 days with media exchanges at 3-day intervals. The dental pulp cell were subcultured to the fourth passage in MEM that contained 10% FBS for use. Cell culture reagents and antibiotics were purchased from GIBCO-BRL (Carlsbad, MD, USA).

CM from PSL-treated macrophages was obtained after culturing treated THP-1 cells. To obtain CM, M0 macrophages were treated with PSLs for 6 and 24 h. After centrifuging at 10000 rpm for 5min, the supernatants were stored at −80℃ until use. The CM aliquots were labeled as CM-6 h and CM-24 h.

In this study, the effect of CM from PSL-treated macrophages on the differentiation of HDPCs was investigated by measuring the ALP activity. The ALP activity was assessed using the following two methods: (a) 4-nitrophenyl phosphate colorimetric assay and (b) ALP direct staining assay. For 4-nitrophenyl phosphate colorimetric assay, HDPCs were exposed to CM from macrophages for 6 days in growth media (GM, MEM containing 10% FBS) and differentiation media (DM, MEM containing 10% FBS, 50 mg/mL L-ascorbic acid, 10^-8 M dexamethasone and 2mM β-glycerophosphate). Cells in 96-well plates were incubated in a mixture of 140 μL of alkaline buffer solution and 10 μL of 1.5 M MgCl₂ solution that contained 67 mM 4-nitrophenyl phosphate (Fluka, Buchs, Switzerland) for 30 min at 37℃. The reaction was discontinued by adding 0.5 M NaOH, and the optical density was measured at 405 nm. ALP enzyme activity was normalized to total protein amount. Cell protein was quantitated using a bicinchoninic acid protein assay kit (iNtRon Biotechnology; Sungnam, Korea). During the treatment, CM was exchanged by replacing the culture medium that contained CM every 3 days. For ALP direct staining assay, a culture insert (ibidi, Munich, Germany) was used. The culture insert was placed on a culture dish, and 100 μL of THP-1 cell suspension was added in one side of the insert. THP-1 cells were incubated at 37℃ for 1 day and were differentiated to non-polarized M0 state by exposure to 100 nM phorbol 12-myristate 13-acetate (Sigma-Aldrich) for 2 days; the media was replaced with fresh media that contained PSLs for 1 day. HDPCs were incubated in the other side of the insert. Following PSL treatment, the culture insert was carefully removed, and the cells were cultured with GM and DM. After 6 days of culture, HDPCs and macrophages were stained with an ALP staining kit (SBI, California, USA) (Takahashi et al., 2006).

To observe the effect of PSL-treated macrophages on the odontogenic differentiation of HDPCs, mRNA expressions of dentin sialophosphoprotein (DSPP) and osteocalcin (OCN) were investigated. HDPC was exposed to CM for 10 days in DM to measure DSPP and OCN mRNA expressions. Total RNA was then extracted from cells using an RNA extraction reagent (WelPrep Total RNA Isolation Reagent; Welgene, Daegu, Korea). cDNA was then prepared using a cDNA synthesis kit (Power cDNA Synthesis kit, iNtRON Biotechnology). Real-time polymerase chain reaction was performed with a 20-μL mixture that contained 10 μL of SYBR Premix Ex Taq (Takara Bio, Otsu, Japan), 0.4 μL of ROX Reference Dye II (Takara Bio), cDNA, and primers. The reaction was performed using the ABI PRISM 7500 Sequence Detection System Thermal Cycler (Applied Biosystems, Foster City, CA). The primer sequences were as follows: DSPP,

forward 5'-TGACTCAAAAGGAGAGAAGATGAT-3',

reverse 5'-ATTTACCTTTGCCACTGTCTGATTT-3'; OCN,

forward 5'-CTGACGAGTTGGCTGACC-3',

reverse 5'-CAAGGGGAAGAGGAAAGAAGG-3'; GAPDH,

forward 5'-GTCGGAGTCAACGGATTTGG-3',

reverse 5'-GGGTGGAATCATATTGGAACATG-3'.

PCR conditions were 95℃ for 30 s followed by 40 cycles of denaturation at 95℃ for 15 s and annealing at 60℃ for 34 s. Gene expressions were evaluated using the CT (threshold cycle) value and were reported as the average expression ratio of the target gene to GAPDH to normalize the expression.

Alizarin red S staining was used to determine the extent of cell matrix mineralization. HDPCs were exposed to CM for 20 days in DM. To stain calcified nodules, cell cultures were fixed with 3.7% formaldehyde for 24 h, washed with PBS, and then stained with 20 mg/mL alizarin red S for 10 min. The amount of alizarin red S that bound to the cell matrix was quantified by extraction with 10% cetylpyridinium chloride, as previously described (Lee DH et al., 2006).

One-way ANOVA was performed to determine the significance of the effects of CM PSL-treated macrophages on ALP activity, DSPP and OCN mRNA expressions, and ECM mineralization of HDPCs. p values of < 0.05 were considered to be statistically significant.

ALP is an early marker for cell differentiation (Stucki et al., 2001). The ALP activity was assessed using two methods: (a) 4-nitrophenyl phosphate colorimetric assay and (b) ALP direct staining assay. To investigate the effects of CM from PSL-treated macrophages on the ALP activity of HDPCs, 4-nitrophenyl phosphate colorimetric assay was used. HDPCs used in this experiment had an ALP value of 31.9 (units/mg protein) in GM and 83.4 (units/mg protein) in DM, which confirmed sufficient differentiation potency of these cells (Figure 1). HDPC was cultured in diluted CM for 6 days, and the amount of CM in total media was increased from 5% to 50%. In GM, no statistically significant differences were observed after measuring the ALP activity of HDPC using CM-6 h, but in DM, a statistically significant increase by 10% was observed, with similar values until 50%. The experiment conducted with CM-24h showed similar results using CM-6h.

Figure 1. 
The ALP activity of HDPCs cultured with CM from PSL-treated macrophages. HDPCs were cultured with CM from PSL-treated macrophages in GM and DM for 6 days for the ALP activity. Each value represents the mean ± SD of the triplicate experiments. *p < 0.05 vs. untreated control group.

The ALP activity of HDPCs co-cultured with PSL-treated macrophages is shown in Figure 2. There was no difference in the intensity of ALP staining between PSL-treated and non-treated groups.

Figure 2. 
The ALP staining of HDPC cultured with PSL-treated macrophage. HDPCs were co-cultured with PSL-treated macrophages in GM and DM. The blue color shows the staining for ALP of HDPCs.

DSPP and OCN mRNA expressions were observed with GM, DM, CM-6h, and CM-24h. DSPP mRNA expressions increased in DM compared with those in GM. Average values in DM, CM-6h, and CM-24h decreased but were not statistically significant. In contrast, OCN mRNA expressions did not increase in GM and DM. There was no change in the average value of OCN expression, no statistically significant differences were observed in the correlation with to the results obtained for DSPP (Figure 3).

Figure 3. 
Effects of CM from PSL-treated macrophages on DSPP and OCN mRNA expressions in HDPCs. HDPCs were cultured in GM, DM, and DM that contained 10% CM from PSL-treated macrophages for 10 days to assess DSPP and OCN mRNA expressions. Each value represents the mean ± SD of triplicate experiments.

The effect of CM on mineralization was investigated by alizarin red S staining after 20 days of incubation in GM, DM, CM-6h, and CM-24h. When mineralization was quantified, DM showed a 4-fold increase than GM, whereas no significant changes were noted in CM-6h and CM-24h (Figure 4).

Figure 4. 
The matrix mineralization of HDPC cultured with CM from PSL-treated macrophages. HDPCs were cultured with DM that contained 10% CM from PSL-treated macrophage for 20 days and were then stained with alizarin red S (A). The mineralization was quantified by measuring the amount of alizarin red S (B). Each value represents the mean ± SD of triplicate experiments. *p < 0.05 vs. control group (GM).