ICA was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China); Dickkopf-1 (DKK-1) was purchased from PeproTech, Inc. (Rocky Hill, NJ, USA); foetal bovine serum (FBS) was obtained from Gibco, USA; high glucose Dulbecco's modified Eagle medium (H-DMEM), penicillin, streptomycin and trypsin were purchased from Hangzhou Jinuo Biotechnology Co., Ltd. (Hangzhou, China); all the plates and flasks used for cell culture were purchased from Corning, USA; an alkaline phosphatase (ALP) staining kit, Cell Counting kit-8 (CCK-8) and ALP activity detection kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

A new born C57 rat was sacrificed and immersed in 70% ethanol for 10 min; then, the mandible body was isolated from the soft tissue, washed with phosphate-buffered saline (PBS; containing 200 IU/ml penicillin and 200 µg/ml streptomycin) three times and cut into small pieces with diameters between 1 and 2 mm. Two millilitres of 0.25% trypsin with ethylenediaminetetraacetic acid was added to the tube containing the mandible pieces, which were incubated for 30 min in a 37°C water bath and then centrifuged at 1,100 r/min. The pellet was collected, and PBS was added for 5 additional centrifugations. The pellet was finally resolved in H-DMEM containing 15% FBS, 200 IU/ml penicillin and 200 µg/ml streptomycin and cultured in an incubator (37°C, 5% CO₂) for 20 min. Rarefied osteoblastic cells were obtained after the fibroblasts with differential attachment were removed. These cells were maintained in H-DMEM with 15% FBS, 200 IU/ml penicillin, and 200 µg/ml streptomycin, and 5 mM β-glycerophosphate, 100 mM L-ascorbic acid, and 0.1 mM dexamethasone were added for differentiation experiments. The growth situation of the osteoblastic cells was checked by cytometry. ALP staining and alizarin red staining were used to check the differentiation and mineralization of the isolated osteoblastic cells afterwards.

The present study was carried out in accordance with the recommendations of the National Institutes of Health Guide for the Care and Use of Experimental Animals. The protocol was approved by the Ethics Committee of Beijing Stomatological Hospital (Beijing, China).

Twenty micrograms of ICA (powder) was dissolved in 1 ml of dimethyl sulfoxide (DMSO) and stored at −20°C. Later, a series of working concentrations (0, 0.0015, 0.015, 0.15, 1.5, 15, 30, 60 and 100 µM) was obtained from this stock. Cells in the logarithmic growth phase were digested by trypsin.

For CCK-8 measurement, cells were seeded in 96-well plates at 4,000 cells per well. Forty-eight h later, different working concentrations of ICA were added to the well, and the control sample was treated with DMSO. All the cells were then divided into three groups according to the incubation period, namely, 24, 48 and 72 h. After a certain incubation time, the medium was aspirated, and 100 µl of DMEM with 10 µl of CCK-8 solution was added. According to the manufacturer's instructions, after 2 h, the optical density (OD) value of each sample was determined by a microplate reader at a wavelength of 450 nm.

For the ALP activity measurement, cells were seeded into 12-well plates at 50,000 cells per well. After a certain incubation period with ICA, the cells were harvested into 1.5-ml Eppendorf (EP) tubes and lysed by 1% Triton X-100 for 30 min. The supernatant was then collected after centrifugation at 12,000 r/min for 5 min, the concentration of the total protein was determined using the bicinchoninic acid (BCA) method, and the ALP activity was measured using the ALP activity detection kit according to the manufacturer's procedure.

Ten micrograms of DKK-1 was dissolved in 5 ml of PBS (containing 10% FBS), and the working concentration of DKK-1 was 0.1 µg/ml. Osteoblastic cells were seeded in 35-mm dishes at 1×10⁵/ml and cultured for 3 days. Then, the cells were treated with 1.5 µM ICA, 0.1 µg/ml DKK-1 or their mixture, and DMSO-treated cells were used as a control. The cells were again divided into three groups with incubation times of 24, 48 and 72 h. After each incubation period, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to determine the gene expression level of the marker proteins, including β-catenin, cyclin D1, runt-related transcription factor 2 (RUNX2) and ALP. Total RNA was isolated using TRIzol reagent (Sunbio, Beijing, China) according to the manufacturer's instructions. cDNA was synthesized using a cDNA synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) following the manufacturer's protocol. The cDNA was then amplified by RT-qPCR with the SYBR Premix Ex Taq kit (Takara Biotechnology, Otsu, Japan) and the following thermocycling conditions: 95°C for 10 min, followed by 45 cycles of 95°C for 10 sec, 60°C for 30 sec and 72°C for 30 sec. The primers used are shown in Table I. Each sample was assessed in triplicate and analysed using the 2^−ΔΔCq method (10).

The data were analysed using GraphPad 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). Every type of experiment was repeated 4 times, and the results were presented as the mean ± standard deviation. The results were first checked for normality and homogeneity using a Kolmogorov-Smirnov test. A t-test was used to compare two independent groups. For experiments divided into several groups, statistical analysis was performed by one-way analysis of variance with Tukey's post hoc pairwise comparisons. P<0.05 was considered to indicate a statistically significant difference.

The growth curve of osteoblastic cells isolated from a rat mandible was obtained (Fig. 1). The curve met the criteria for a classic cell growth curve, which contains four phases: lag phase, log phase, stationary phase and death phase. The cells were in the log phase between day 2 and day 6; therefore, this period was selected for cell treatments.

We performed ALP staining by using the ALP activity detection kit. Over 95% of the osteoblasts were stained by the ALP marker (Fig. 2), indicating that these cells were healthy enough to express ALP. The osteoblastic cells showed a typical morphology with a fibroblast appearance: Most showed an irregular fusiform shape. These cells also firmly adhered to the plate.

The mineralization of the isolated osteoblastic cells was checked by alizarin red staining. After 21 days of culture, many red dots were visible under a microscope (Fig. 3). Those dots were the mineralized calcium nodules, suggesting that the osteoblasts can grow well until mineralization and maturation.

The proliferation level of osteoblastic cells in groups with different stimuli and treatment times was determined by the CCK-8 method (Fig. 4). The cells treated with DMSO were used as a blank control under this circumstance. ICA just can promote the proliferation of osteoblastic cells modestly after treatment for 24 h at concentrations between 0.0015 and 60 µM. In regard to 48 h of incubation, 0.0015 µM ICA promoted the proliferation of osteoblastic cells significantly. With continued culture to 72 h, ICA at concentrations between 0.15 and 15 µM significantly promoted the proliferation of osteoblastic cells. However, 100 µM ICA significantly suppressed the proliferation of osteoblastic cells at all time points.

The ALP activity was also determined to evaluate the effect of ICA on osteoblast differentiation (Fig. 5). ICA at certain concentrations significantly improved the ALP activity after the osteoblastic cells were treated for 24 or 48 h, and ICA at concentrations between 0.015 and 15 µM increased the ALP activity significantly after the cells were treated for 72 h.

To verify the role of the Wnt/β-catenin signalling pathway in the effect of ICA, we determined the mRNA level of several marker proteins, including β-catenin, RUNX2, cyclin D1 and ALP (Fig. 6). The concentration of ICA applied was 1.5 µM because of its significant proliferation and differentiation effect, as shown above (Fig. 4). DKK-1 is an inhibitor of the Wnt/β-catenin signalling pathway and was used as a negative control. Compared with the mRNA levels of all the marker proteins in the control group, those in the ICA group were significantly increased at all time points of incubation; the one exception was the mRNA level of cyclin D1, which was slightly but not significantly increased at 24 h. The mRNA levels of all the marker proteins in the control group were significantly suppressed by DKK-1 treatment, except the mRNA level of cyclin D1, which was slightly decreased at 24 h. The elevating effects of ICA on the mRNA level of all the marker proteins were inhibited significantly in the ICA and DKK-1 mixture group at all time points, except the mRNA level of cyclin D1 at 24 h.