Gusuibu (D. fortunei) was grown in Chengdu, Sichuan Province, China. The herb was provided by the Brion Research Institute, Sun Ten Group (Taipei, Taiwan), and identified by institutional experts according to macroscopic and microscopic approaches. In addition, the chemical and physical characteristics of D. fortunei are routinely analyzed, and the product was commercialized (CAS no. 6005, Sun Ten Pharmaceutical, Sun Ten Group). Dry pieces of D. fortunei (Figure 1A) were ground into a fine powder. WEG was prepared by decocting 50 g of herb powder with 400 mL water for 1 hour, as previously described.¹⁶ After filtration, the filtrate was frozen at −70°C and then concentrated by a Freeze Dryer FD24 (Kingmech, Taiwan) into a dry powder. WEG was stored at room temperature and protected from light and moisture.

Nanoparticles of WEG (nWEG) were prepared following a previously described method.²⁰ Briefly, WEG was carefully ground up using an auto grinder-polisher (EcoMet^® 250, Buehler, Lake Bluff, IL) for 10 minutes in an aluminum oxide mortar. Fractions of particles were passed through a series of sieves. The mass percentage of each particle size range was detected. The distribution of particles of different sizes was observed and analyzed using an atomic force microscope (Mobile S, Nanosurf, Liestal District, Switzerland).

Rat osteoblasts were prepared from 3-day-old Wistar rat calvarias according to a previously described method.²¹ All procedures were performed according to the National Institutes of Health Guidelines for the Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Taipei Medical University (Taipei, Taiwan). Rat osteoblasts were seeded in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum, L-glutamine, penicillin (100 IU/mL), and streptomycin (100 μg/mL) in 75-cm² flasks at 37°C in a humidified atmosphere of 5% CO₂. These bone cells were grown to confluence prior to drug treatment. Only the first passage of rat osteoblasts was used in this study. WEG and nWEG were dissolved in deionized distilled water and filtrated through 0.25-mm filters. Rat osteoblasts were treated with different concentrations of WEG and nWEG for various time intervals in independent experiments. In protection assays, rat osteoblasts were exposed to 100 μM hydrogen peroxide, 10 μg/mL WEG, 10 μg/mL nWEG, and a combination of hydrogen peroxide with WEG or nWEG for 24 hours. Cell viability, DNA fragmentation, and cell apoptosis were quantified. Sodium nitroprusside was administered to rat osteoblasts, and cell viability was assayed.

Toxicities of WEG and nWEG were evaluated by observing cell morphologies using a light microscope and assaying cell viability by a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay, as per a previously described method.¹³ After drug treatment, morphologies of osteoblasts were observed and photographed using a light microscope. Rat osteoblasts (2 × 10⁴ cells per well) were seeded in 96-well tissue culture plates overnight. After treatment with WEG and nWEG, rat osteoblasts were cultured with new medium containing 0.5 mg/mL MTT for a further 3 hours. The blue formazan products in osteoblasts were dissolved in dimethyl sulfoxide and spectrophotometrically measured at a wavelength of 550 nm.

DNA fragmentation in rat osteoblasts was quantified by the cellular DNA fragmentation enzyme-linked immunosorbent assay kit (Boehringer Mannheim, Indianapolis, IN) as described previously.²² Briefly, rat osteoblasts (2 × 10⁵ cells) were subcultured in 24-well tissue culture plates and labeled with BrdU overnight. Cells were harvested and suspended in the culture medium. One hundred microliters of cell suspension was added to each well of 96-well tissue culture plates. Rat osteoblasts were cocultured with WEG or nWEG for another 8 hours at 37°C in a humidified atmosphere of 5% CO₂. Amounts of BrdU-labeled DNA in the cytoplasm were quantified using an Anthos 2010 microplate photometer (Anthos Labtec Instruments, Lagerhausstrasse, Wals/Salzburg, Austria) at a wavelength of 450 nm.

Apoptotic cells were determined using propidium iodide to detect DNA fragments in nuclei, according to a previously described method.²³ After drug treatment, medium containing floating cells was collected, and attached cells were washed and trypsinized. Floating and trypsinized osteoblasts were collected in the same centrifuge tubes. After centrifugation and washing, cell pellets were fixed in cold 80% ethanol. Fixed cells were stained with propidium iodide. Apoptotic cells were quantified by detecting the proportion of rat osteoblasts arrested at the sub-G1 phase using flow cytometry (FACS Calibur, Becton Dickinson, San Jose, CA).

Osteoblast maturation was determined by evaluating cell mineralization using the von Kossa and Alizarin red S dye-staining protocols.²⁴,²⁵ Rat osteoblasts were treated with 10 μg/mL of WEG, nWEG, or a combination of 10 nM dexamethasone, 100 μg/mL ascorbic acid, and 10 mM β-glycerophosphate for 21 days. After drug treatment, rat osteoblasts were washed with ice-cold phosphate-based saline buffer (0.14 M NaCl, 2.6 mM KCl, 8 mM Na₂HPO₄, and 1.5 mM KH₂PO₄) and then fixed in ice-cold 10% formalin for 20 minutes. For the von Kossa protocol, mineralized matrix was detected by treating fixed cells with 5% silver nitrate for 30 minutes, followed by subsequent washes with 5% sodium carbonate in 10% formalin for 1 minute and 5% sodium thiosulfate for 5 minutes. The reaction was stopped by washing cells twice with deionized water. For the Alizarin red S dye staining protocol, fixed osteoblasts were thoroughly rinsed and then incubated in 1% alcian blue (pH 2.5; Fisher Scientific, Pittsburgh, PA) for 12 hours. Sections were then incubated in Alizarin red S (Fisher Scientific) for 8 minutes, dehydrated briefly in xylene and covered with a coverslip in Permount (Fisher Scientific). Mineralized nodules were visualized and counted under an inverted microscope. Each experiment was performed in duplicate wells and repeated 3 times.

The statistical significance of differences between the control and drug-treated groups was evaluated using Student’s t-test, and differences were considered statistically significant at P values of <0.05. Differences between drug-treated groups were considered significant when the P value of Duncan’s multiple-range test was <0.05. Statistical analysis between groups over time was carried out by a 2-way analysis of variance (ANOVA).

Dry pieces of Gusuibu (D. fortunei) (Figure 1A) were ground into a fine powder, and the WEG was prepared (Figure 1B). nWEG was prepared, and the distribution of the particles with different sizes was analyzed (Figure 1C). Results revealed that >90% of nWEG particles were <1000 nm.

Cell morphologies, cell viability, and apoptotic cells were analyzed to determine the cytotoxicity of nWEG to rat osteoblasts. Treatment of rat osteoblasts with 1, 10, 100, and 1000 μg/mL nWEG for 72 hours did not affect cell morphologies (data not shown). The results of the viability analyses also showed that exposure to 1, 10, 100, and 1000 μg/mL nWEG for 72 hours did not cause the death of rat osteoblasts (Figure 2A). Furthermore, after treatment for 72 hours, nWEG at 1, 10, 100, and 1000 μg/mL nWEG did not induce osteoblast apoptosis (Figure 2B).

Cell viability was analyzed carried out to compare the toxicities of WEG and nWEG to primary rat osteoblasts (Figure 3). Exposure of rat osteoblasts to 1000 μg/mL WEG for 24 and 48 hours did not influence cell viability (Figure 3A). Meanwhile, when the treatment time intervals reached 72 hours, WEG caused a significant 31% decrease in osteoblast viability. In comparison, exposure to 1000 μg/mL nWEG for 24, 48, and 72 hours did not affect the viability of rat osteoblasts (Figure 3B).

DNA fragmentation and apoptotic cells were analyzed to evaluate the mechanism of WEG-induced death of rat osteoblasts (Figure 4). Treatment of rat osteoblasts with 1000 μg/mL WEG for 72 hours induced DNA fragmentation by 2.4-fold (Figure 4A). Following exposure to 1000 μg/mL nWEG for 72 hours, the genomic DNA of rat osteoblasts was not damaged. Treatment with 1000 μg/mL WEG for 72 hours induced apoptosis of rat osteoblasts by 20% (Figure 4B, C). nWEG did not cause osteoblast apoptosis. Sodium nitroprusside was administered to rat osteoblasts as a positive control (Figure 4). Exposure of rat osteoblasts to sodium nitroprusside caused significant 9.1-fold and 89% increases in DNA fragmentation and cell apoptosis, respectively (Figure 4A–C).

The effects of WEG and nWEG on stress-induced insults to primary rat osteoblasts were determined (Figure 5). Exposure of rat osteoblasts to 100 μM hydrogen peroxide for 24 hours caused a significant 40% reduction in cell viability (Figure 5A). After treatment with 10 μg/mL WEG or nWEG alone, viability of rat osteoblasts did not change. Exposure to WEG decreased hydrogen-caused death of rat osteoblasts by 49%. However, nWEG completely alleviated the hydrogen peroxide-caused decrease in cell viability (Figure 5A). Treatment of rat osteoblasts with 100 μM hydrogen peroxide for 24 hours induced DNA fragmentation and cell apoptosis by 4.7-fold and 54%, respectively (Figure 5B, C). Neither WEG nor nWEG caused genomic DNA injury or cell apoptosis. Treatment of rat osteoblasts with WEG caused significant 57% and 45% decreases in hydrogen peroxide-induced DNA fragmentation and cell apoptosis, respectively (Figure 5B, C). In comparison, nWEG completely lowered hydrogen peroxide-induced DNA damage and caused a 65% attenuation of oxidative stress-triggered apoptosis (Figure 5B, C).

The defense by WEG and nWEG against nitrosative stress-induced insults to rat osteoblasts was further evaluated (Figure 5D). Exposure of rat osteoblasts to 2 mM sodium nitroprusside for 24 hours caused a 78% reduction in cell viability. Treatment with WEG or nWEG alone at 10 μg/mL did not influence the viability of rat osteoblasts. However, exposure to WEG and nWEG significantly attenuated sodium nitroprusside-induced death of rat osteoblasts by 36% and 56%, respectively (Figure 5D).

Effects of WEG and nWEG on osteoblast maturation were determined by analyzing cell mineralization (Figure 6). Results of von Kossa staining revealed that no mineralized nodules were observed in control osteoblasts (Figure 6A, left-top panel). After repeated treatment of primary rat osteoblasts with 10 μg/mL WEG for 21 days, mineralized nodules were observed (right-top panel). Exposure of rat osteoblasts to nWEG at the same concentration of 10 μg/mL for 21 days stimulated more mineralized nodules (left-bottom panel). Analysis with the Alizarin red S dye-staining protocol showed similar results as with the von Kossa method; that is, nWEG had better potential to promote osteoblast mineralization than WEG (Figure 6B, right-top and left-bottom panels). A combination of dexamethasone, ascorbic acid, and β-glycerophosphate was applied to rat osteoblasts as a positive control, and the results revealed such treatment massively stimulated osteoblast maturation (Figure 6A, 6B, right-bottom panels).