Minimum essential medium α (α-MEM), fetal bovine serum (FBS) and trypsin/EDTA solution were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Unless otherwise stated, all other chemicals and reagents were obtained from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany).

Dry roots of Cimicifuga heracleifolia Komarov (500 g) were obtained from Chungju Hospital of Korean Medicine, College of Korean Medicine, Semyung University (Chungju, Korea) were immersed in distilled water and boiled under reflux for 150 min, and the resulting extract was centrifuged at 5,000 × g for 10 min at room temperature. The supernatant was concentrated to a volume of 300 ml using a rotary evaporator under reduced pressure (Eyela NE-1001; Tokya Rikakikai Co., Ltd., Tokyo, Japan). The concentrates were then freeze-dried in a lyophilizer (Labconco, Kansas, MO, USA) to obtain 92.8 g solid residue, corresponding to a yield of 18.6% (w/w).

Healthy gingival tissues were obtained from healthy patients undergoing crown-lengthening procedures. This study was reviewed and approved by the Institutional Review Board of Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, (Seoul, Republic of Korea; KC11SISI0348), and informed consent was obtained from all patients.

The tissues were immediately placed into sterile phosphate-buffered saline (PBS; Welgene, Daegu, South Korea) with 100 U/ml penicillin and 100 µg/ml streptomycin at 4°C. The gingival tissue was de-epithelialized, minced, digested with collagenase IV and incubated at 37°C in a humidified incubator with 5% CO₂ and 95% O₂. The non-adherent cells were washed with PBS after 24 h, supplied with α-MEM (Gibco) containing 15% FBS (Gibco), 100 U/ml penicillin and 100 µg/ml streptomycin (both Sigma-Aldrich), 200 mM L-glutamine (Sigma-Aldrich) and 10 mM ascorbic acid 2-phosphate (Sigma-Aldrich), and fed every 2–3 days.

The stem cells were plated at a density of 1.0×10⁴ cells/well. The cells were incubated in α-MEM, which included 15% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin and 1 mM ascorbic acid 2-phosphate, in the presence of Cimicifugae Rhizoma at final concentrations ranging from 0.1 to 10 µg/ml, specifically: 0 (untreated control), 0.1, 1 and 10 µg/ml.

Cell proliferation analyses were performed at day 15. A solution of WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] from Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Tokyo, Japan) was added to the cultures, and the spheres were incubated for 30 min at 37°C. The CCK-8 assay identifies viable cells via the formation of a formazan dye from WST-8 by the oxidative action of mitochondrial dehydrogenases in living cells. The spectrophotometric absorbance of the samples at 450 nm was measured using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).

The stem cells were plated at a density of 1.6×10⁴ cells/chamber in an 8-chamber slide and then incubated in α-MEM, containing 15% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 mM β-glycerophosphate, 1 mM ascorbic acid 2-phosphate and 100 µM dexamethasone in the presence of Cimicifugae Rhizoma at final concentrations of 0.1, 1 and 10 µg/ml, respectively. The medium was replaced with fresh induction medium every 3 days. At day 21, the cells were fixed with 4% paraformaldehyde by incubation for 30 min at room temperature and the cells were washed with sterile PBS three times. The cells were then treated with 1% Triton X-100 for 5 min, and washed with PBS three times. Blocking was conducted with 1% bovine serum albumin (Sigma-Aldrich) in PBS for 30 min at 4°C in the dark. The cells were incubated with a primary antibody against osteocalcin (sc-30044; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 1:100 dilution for 1 h at 4°C in the dark. The cells were washed with sterile PBS three times and then incubated with secondary antibodies (sc-53805; Santa Cruz Biotechnology, Inc.) at 1:200 dilution for 1 h at 4°C in the dark. Coverslips were mounted with mounting media (Vector Laboratories, Inc., Burlingame, CA, USA) containing 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI). The cells were observed under a fluorescence microscope (Axiovert 200; Zeiss GmbH, Jena, Germany). Relative osteogenesis was determined with the aid of image analysis software (ImageJ; National Institutes of Health, Bethesda, MD, USA).

The stem cells were plated at a density of 1.6×10⁴ cells/chamber in an 8-chamber slide and then incubated with adipogenic induction medium (StemPro^® Adipogenesis Differentiation kit; Gibco) in the presence of the Cimicifugae Rhizoma at final concentrations of 0.1, 1 and 10 µg/ml, respectively. The medium was replaced with fresh induction medium every 3–4 days. At day 14, the cells were fixed with 4% paraformaldehyde by incubation for 30 min at room temperature, and permeabilized in 1% Triton X-100 with PBS for 5 min. Blocking was conducted with 1% bovine serum albumin (Sigma-Aldrich) in PBS at 4°C for 30 min. The cells were incubated with primary antibody against adipocyte fatty acid-binding protein (sc-271529; Santa Cruz Biotechnology, Inc.) for 1 h at 4°C and then incubated with secondary antibodies [goat anti-mouse IgG1 heavy chain (fluorescein isothiocyanate) ab97239; Abcam, Cambridge, UK)] at 1:200 dilution for 1 h in the dark. The cells were mounted with mounting medium and DAPI. The cells were observed under a fluorescence microscope. Relative adipogenesis was determined with the aid of ImageJ analysis software.

Results are presented as the means ± standard deviations. One-way analysis of variance with Tukey's post hoc test were performed to determine the differences between the groups using commercially available software (SPSS 12 for Windows; SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The cell proliferation results at day 15 are shown in Fig. 1. The relative values for the cells treated with 0.1, 1 and 10 µg/ml Cimicifugae Rhizoma were 112.5±6.3, 111.3±5.7 and 105.8±10.0%, respectively, when the CCK-8 result of the untreated control group on day 21 was considered to be 100% (100.0±5.3%). The relative value in the CCK-8 assay of 0.1 µg/ml Cimicifugae Rhizoma was significantly different when compared with the control (P<0.05).

Images showing the immunofluorescence staining of osteocalcin in cells treated with Cimicifugae Rhizoma at final concentrations of 0, 0.1, 1 and 10 µg/ml for 21 days are shown in Fig. 2. The relative osteogenic differentiation percentages of the 0.1, 1 and 10 µg/ml groups were calculated to be 171.5±13.7, 125.6±28.7 and 150.5±9.0%, respectively, when the osteogenic differentiation of the untreated control group on day 21 was considered to be 100% (P>0.05; Fig. 3).

Images showing the immunofluorescence staining of adipocyte fatty acid-binding protein in cells treated with Cimicifugae Rhizoma at final concentrations of 0, 0.1, 1 and 10 µg/ml at day 14 are shown in Fig. 4. The relative adipogenic differentiation rates of the of 0.1, 1 and 10 µg/ml groups were calculated to be 97.5±15.0, 102.9±12.8 and 87.0±6.8%, respectively, when the adipogenic differentiation of the untreated control group on day 14 was considered to be 100% (100.0±6.5%; P>0.05; Fig. 5).