In Vitro Cell.Dev.Biol.—Animal DOI 10.1007/s11626-014-9824-4

Zanthoxylum schinifolium enhances the osteogenic potential of periodontal ligament stem cells So Yeon Kim & Seong Yeong An & Jeong Seok Lee & Jung Sun Heo

Received: 17 July 2014 / Accepted: 16 September 2014 / Editor: T. Okamoto # The Society for In Vitro Biology 2014

Abstract The present study demonstrates the osteogenic effect of Zanthoxylum schinifolium on periodontal ligament stem cells (PDLSCs). The dried herb of Z. schinifolium was first extracted with 70% ethanol and subsequently fractionated into five parts: n-hexane, methylene chloride (MC), ethyl acetate (EA), n-butanol (BuOH), and water fractions. The proliferation of PDLSCs was first assessed and increased by hexane, EA, or BuOH fraction of Z. schinifolium. We evaluated the osteogenic differentiation of PDLSCs by alkaline phosphatase (ALP) activity, messenger RNA (mRNA) expression of runt-related transcription factor 2 (RUNX2), osterix (OSX), FOSB, and FRA-1 as osteogenic transcription factors, and protein levels of osteopontin (OPN) and RUNX2 in response to each hexane, MC, EA, BuOH, or water fraction of Z. schinifolium. The significant ALP activity appeared in PDLSCs treated with hexane, EA, or BuOH fraction. The mRNA expression of osteogenic transcription factors was also increased by hexane, EA, or BuOH fraction with doses of 5, 10, 25, and 50 μg/ml compared to control group. We further assessed immunofluorescence staining with OPN and RUNX2 confirmed that the treatment of hexane, EA, or BuOH fraction enhances PDLSC osteogenic differentiation. In conclusion, these data suggest that fractions from Z. schinifolium differentially regulate PDLSC function. Among them, proliferation and osteogenic potential of PDLSCs were enhanced by hexane, EA, or BuOH fraction. Keywords Zanthoxylum schinifolium . Periodontal ligament stem cells . Proliferation . Osteogenic differentiation So Yeon Kim and Seong Yeong An contributed equally to this study. S. Y. Kim : S. Y. An : J. S. Lee : J. S. Heo (*) Department of Maxillofacial Biomedical Engineering and Institute of Oral Biology, School of Dentistry, Kyung Hee University, 26 Kyunghee-daero, Dongdaemun-gu, Seoul 130-701, South Korea e-mail: [email protected]

Introduction The current research in periodontal regeneration has harmonized with tissue engineering and supply of appropriate cells. In order to process this multidisciplinary strategy, the understanding of biomaterials and stem cell biology needs to precede the clinical challenges. In particular, stem cell research has become an important branch to comprehend the regenerative medicine. Since multipotential stem cells have been identified in oral tissues (Gronthos et al. 2000; Miura et al. 2003; Seo et al. 2004), which are plentiful and easy to collect from tissue and have been shown to be able to differentiate into bone, dental tissue, cartilage, and even neural tissue (Nakashima et al. 2009; Singhatanadgit et al. 2009; Zainal Ariffin et al. 2012), they can be widely employed in not only periodontal regenerative medicine but also basic stem cell science. Among these dental stem cells, periodontal ligament stem cells (PDLSCs) have the potential to differentiate into osteoblasts, cementoblasts, and periodontal ligament fibroblasts and eventually form a typical alveolar bone/ periodontium-like structure under appropriate conditions (Seo et al. 2004; Gay et al. 2007). Thus, PDLSCs are suggested as a practical regenerative resource, and such strategy can be extended for a clinical treatment. Natural herbs have been extensively utilized for a medicinal purpose in various diseases. Among miscellaneous herbs, Zanthoxylum schinifolium is an aromatic plant that is widely used as a pungent condiment and seasoning in Korea and other East Asian countries (Paik et al. 2005). This herb has been known to have medicinal activities including anti-tumor (Kim et al. 2006), anti-oxidant (Kim et al. 2002), inhibition of inflammation (Tsai et al. 2000), and increase of cardiac contraction (Cui et al. 2009). Previous study reported that the extract of Z. schinifolium contains various compounds such as alkaloids, furans, benzenoids, triterpenoids, diterpenoids, sesquiterpenoids, steroids, flavonoids, schinilenol,

KIM ET AL.

schinindiol, and coumarins (Chen et al. 1995; Cheng et al. 2002; Min et al. 2011). However, it has not been investigated whether the extract of Z. schinifolium can be a promising bioactive compound in dental medicine. Thus, this study examined the effect of Z. schinifolium on periodontal regenerative capacity using PDLSC model system, which was assessed by cellular proliferation and the differentiation of PDLSCs into an osteogenic lineage.

Materials and Methods Materials. Fetal bovine serum (FBS) was purchased from Gibco-BRL (Gaithersburg, MD). The osteopontin, runtrelated transcription factor 2 (RUNX2), goat-anti-mouse, and goat-anti-rabbit antibodies were supplied by Santa Cruz Biotechnology (Delaware, CA). Unless otherwise specified, chemicals and laboratory wares were purchased from Sigma Chemical Company (St. Louis, MO) and Falcon Labware (Becton-Dickinson, Franklin Lakes, NJ), respectively. Periodontal ligament stem cell culture. Periodontal ligaments were obtained from extracted human molars. All the subjects involved in this study were informed about the purpose and procedures of this study, which were approved guidelines by the Review Board of Kyung Hee University. Written informed consent was obtained from all donors. Explants, obtained from the middle third of the root, were cultured in α-MEM (Invitrogen, Carlsbad, CA) containing 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml) (Sigma) according to a previously described method (Seo et al. 2004). After two passages, cells were subjected to magnetic isolation with antibodies to detect STRO-1 antigen (Millipore, Billerica, MA) and magnetic beads (Miltenyi Biotec, Cologne, Germany). The resulting cell populations STRO-1 (+) was cultured in α-MEM plus 10% FBS at 37°C with a humidified gas mixture of 5% CO2/95% air. All the experiments were carried out with passage 4–7 cells. Extraction of Z. schinifolium. The dried herb of Z. schinifolium (1 kg) was soaked with 2 L of 70% ethanol for 3 d at room temperature. The extraction was repeated three times. The extract was then filtered through Whatman no. 3 filter paper and concentrated using rotary evaporator. Ethanol extracts of Z. schinifolium were obtained after lyophilization. The ethanol extract was then dissolved in 100 ml of water and sequentially extracted with 100 ml of n-hexane, methylene chloride (MC), ethyl acetate (EA), and water-saturated nbutanol using liquid–liquid partition. The extraction was performed three times per solvent. After removal of the solvents, five fractions were obtained: hexane fraction (1.9 g), MC fraction (182.3 g), EA fraction (23.7 g), n-butanol (BuOH) fraction (60.3 g), and water fraction (72.8 g). Each fraction

was dissolved in dimethyl sulfoxide (DMSO) immediately before use, and the final concentration of DMSO did not exceed 0.1% (v/v) in any of the experiments. DMSO at 0.1% was used as a control. Cell viability analysis. Cytotoxicity was evaluated by using the MTT [3-(4,5-dimethylthiazol-2yl)-2.5-diphenyltetrazolium bromide; Sigma] (5 mg/ml in PBS as stock solution) assay, which was based on the reduction of the dye MTT to formazan crystals, an insoluble intracellular blue product, by cellular dehydrogenases (Huang et al. 2004). Briefly, cells were seeded on 96-well culture plates at a density of 1×105 cells/ml and treated with various concentrations of hexane, MC, EA, BuOH, and water fractions (0, 10, 25, 50, and 100 μg/ml) for 48 h. After exposure, 20 μl MTT was added to the cells in each well and incubated at 37°C for 4 h, then the medium and MTT were removed and 150 μl dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The plates were agitated to ensure complete dissolution of the purple formazan crystals, and the optical density was measured at the wavelength of 492 nm using an ELISA reader (Packard, Instrument Co., Downers Grove, IL). Alkaline phosphatase activity. Cells were washed twice with PBS and lysed in a 50 mM Tris–HCl buffer (pH 7.0) containing 1% (v/v) Triton X-100 and 1 mM PMSF. The total protein was then quantified using the Bradford procedure (Bradford 1976). The entire cell lysate was assayed by adding 200 μl of p-nitrophenylphosphate (pNPP) as a substrate (Sigma) for 30 min at 37°C. The reaction was stopped by adding 3 N NaOH, and the absorbance was read spectrophotometrically at 405 nm. The enzyme activity was expressed as millimolar per 100 μg of protein. Alizarin red staining. The culture media was discarded, and the cells were fixed for 30 min in 4% paraformaldehyde fluid, washed three times with ice-cold phosphate-buffered saline (PBS), stained for 5 min with alizarin red (Sigma), and observed under light microscope. RNA isolation and real-time RT-PCR. The total RNA was extracted from the cells using TRIzol reagent (Invitrogen), following the manufacturer’s instructions. The real-time quantification of RNA targets was then performed in the RotorGene 2000 real-time thermal cycling system (Corbett Research, Sydney, Australia) using a QuantiTect SYBR Green RT-PCR kit (QIAGEN, Alameda, CA). The reaction mixture (20 μl) contained 200 ng of the total RNA and 0.5 μM of each primer, the appropriate amounts of enzymes and fluorescent dyes, as recommended by the supplier. The Rotor-Gene 2000 cycler was programmed as follows: 30 min at 50°C for reverse transcription; 15 min at 95°C for DNA polymerase activation; 15 s at 95°C for denaturing; and

ZANTHOXYLUM SCHINIFOLIUM-INDUCED OSTEOGENESIS OF PDLSCS

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Figure 1. Effects of Z. schinifolium extracts on cell viability. Cells were treated with different concentrations of hexane, MC, EA, BuOH, or water fraction (0, 5, 10, 25, 50, and 100 μg/ml) for 48 h, then cell viability was

assessed as described in “Materials and Methods.” The values reported are the mean±SD of five independent experiments. *P

Zanthoxylum schinifolium enhances the osteogenic potential of periodontal ligament stem cells.

The present study demonstrates the osteogenic effect of Zanthoxylum schinifolium on periodontal ligament stem cells (PDLSCs). The dried herb of Z. sch...
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