Human tissue samples were collected from the clinically healthy teeth of 11–14 adolescents who had undergone teeth extraction for orthodontic treatment, no history of periodontal disease and a relatively healthy periodontium. The periodontal ligament tissues were obtained as remnants or discarded tissues following routine dental procedures at the Second Affiliated Hospital of. All protocols for the handling of human tissue were approved by the Research Ethics Committee of Harbin Medical University (Harbin, China) and written informed consent was obtained from the families of the patients. The isolation and culture of human mesenchymal stem cells from healthy periodontal ligament tissues was performed as previously described (16). Briefly, tissues were treated aseptically and incubated overnight at 4°C with 2 mg/ml dispase (Sigma-Aldrich, St. Louis, Mo, USA) and 4 mg/ml collagenase IV (Worthington Biochemical, Lakewood, NJ, USA). The dissociated cell suspension was filtered through a 70-μm cell strainer (Falcon; BD Biosciences, Franklin Lakes, NJ, USA), plated on nontreated 10-cm petri dishes (VWR International, West Sussex, UK) with complete α-minimal essential medium (Invitrogen Life Technologies, Carlsbad, CA, USA) containing 20% fetal bovine serum (Clontech Laboratories Inc., Mountain View, CA, USA), 100 U/ml penicillin, 100 μg/ml streptomycin (Invitrogen Life Technologies), 2 M L-glutamine, 100 mM nonessential amino acid and 550 μM 2-mercaptoethanol (Sigma-Aldrich). The suspension was cultured at 37°C in a humidified tissue culture incubator with 5% CO₂ and 95% O₂. After 72 h, the nonadherent cells were removed. The plastic-adherent confluent cells were passaged with 0.05% trypsin containing 1 mM EDTA and continuously subcultured and maintained in complete growth medium. Cells from the fourth to sixth passages were used in the experiments. Cells from the third passage were fixed by 10% formaldehyde solution and stained with vimentin and keratin according to standard immunohistochemical methods.

hPDLCs were seeded in a 96-well plate (2×10³ cells/well) with varied doses of ICA (0, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸ and 10⁻⁹ mol/l). After 96 h, 20 μl of MTT was added and the cells were incubated for 4 h. Following the addition of 150 μl dimethylsulfoxide, the cells were agitated for 10 min, and the concentration was analyzed by measuring the absorbance at 490 nm with an iMark microplate reader (Bio-Rad, Hercules, CA, USA).

An ALP staining kit (Sigma-Aldrich) was used. Subsequent to fixation with 70% ethanol, cells were incubated with a solution of 0.25% naphthol AS-BI phosphate and 0.75% Fast Red Violet LB Base dissolved in 0.1 M Tris buffer (pH 9.3). The ALP activity assay was conducted according to the manufacturer’s instructions and normalized on the basis of protein concentrations.

Total RNA was isolated from cultured cells undergoing osteogenic differentiation using an RNeasy Mini kit (Qiagen, Valencia, CA, USA). Adipocyte- and osteocyte-specific genes were amplified using the One-Step RT-PCR kit (Qiagen). The specific primers used were as follows: Forward, 5′-AGGGCTGTAAGGACATCG-′3 and reverse, 5′-GGAGTGCTTGTATCTCGGTT-3′ for ALP; and forward, 5′-CATTGCCGACAGGATGCA-3′ and reverse, 5-′CATCTGCTGGAAGGTGGACAG-3′ for β-actin.

Cells were lysed with buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 150 mM NaCl, 0.5% Triton X-100, 10 mM sodium fluoride, 20 mM 2-ME, 250 μM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride and complete protease inhibitor mixture (Sigma-Aldrich), and were incubated at 4°C for 1 h. The lysates were ultrasonicated (AIS92-IIDL ultrasonicator; Aismir, Beijing, China) and centrifuged at 12,000 × g for 10 min. Protein concentrations were determined using the bicinchoninic acid method. Proteins (50–100 μg) were separated on 8–10% polyacrylamide-sodium dodecyl sulfate gels and electroblotted onto nitrocellulose membranes (Hybond ECL; Amersham Pharmacia, Picastaway, NJ, USA). Subsequent to blocking with Tris-buffered saline and 5% nonfat dry milk for 2 h, the membrane was incubated overnight at 4°C with antibodies against human ALP (mouse ant-human; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) followed by incubation with a horseradish peroxidase-conjugated secondary antibody (goat anti-mouse; 1:2,000; Pierce, Rockford, IL, USA) for 45 min at room temperature, and the signals were visualized by enhanced chemiluminescence detection. As a loading control, the blots were reprobed with a specific antibody against human β-actin (mouse anti-human; dilution, 1:5,000; Santa Cruz Biotechnology, Inc.).

Statistical significance was assessed by two-tailed Student’s t-test or analysis of variance. P<0.05 and P<0.01 were considered to indicate a statistically significant difference.

Following primary culture for 4–10 days, fibroblast-like cells with a long fusiform shape emerged beside the tissue block (Fig. 1A). When subcultured to the fourth generation, cells adhered to the bottom of the culture plate and were observed to exhibit a star- or long fusiform-like shape under the microscope. Cell cytoplasm was plump with round or oval nuclei. Cell arrangement was observed to be in a gyrate or radial shape (Fig. 1B). To confirm the origin of cultured cells, various types of stains were applied. Hematoxylin and eosin-stained cell bodies showed long fusiform- or star-like shapes. Nuclei were rounded or oval-shaped and located in the center of the cell body. Cytoplasm stained with anti-vimentin polyclonal antibody appeared brown, while keratin was not stained (Fig. 1D and E). These results suggested that isolated cells had an interstitial opposed to an epithelial origin.

As shown in Table I, icariin accelerated the proliferation of hPDLCs when the concentration was between 10⁻⁵ and 10⁻⁶ mol/l (P<0.01 compared with the control group). The effect appeared to be concentration-dependent within this concentration range. Lower icariin concentrations exhibited no significant effect on hPDLC proliferation while higher concentrations inhibited the division of hPDLCs (P<0.05).

High ALP activity is a well-known index for hPDLC ossification. Previous studies have shown that LPS inhibited hPDLC differentiation and this process was associated with ALP activity decreases. When icariin (10⁻⁵ mol/l) was added to media containing LPS, the ALP activity was not changed at ~24 h; however, following incubation for 96 h, hPDLCs exhibited high ALP activity (Table II).

To identify at which level ALP activity returned to normal levels, RT-PCR and western blot analysis were used to measure the gene expression and protein levels of ALP, respectively. The RT-PCR results showed that no significant ALP expression difference was detected after 24 h. Differentiation emerged between the LPS group and icarrin-treated group after 96 h. The ALP activity of the icariin group was greater than that observed in the control group (P<0.01) (Fig. 2).

These results suggested that the ALP gene expression level that had been inhibited by LPS was recovered by icariin following prolonged exposure. These results were similar in the western blot analysis (Fig. 3). LPS suppressed ALP expression at 24 and 96 h, while icariin accelerated this inhibition at 24 h, but markedly increased the expression at 96 h, exceeding the level of the control group (P<0.01). Thus, the ALP activity fluctuation was directly correlated with the protein level of the enzyme, which was controlled by the ALP expression.