hpdlscs hpdlscs 1 12 ~ h 10 PBS 3 ~ 4 1 / 2 Rg1 ginsenoside Rg1 GS Rg1 GS Rg r /min 2 min 1 ml Ⅰ 1 ml DispaseⅡ ~

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1 ISSN CN / Q http / /cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology ~ 111 DOI /j cnki cjbmb Rg1 * Rg1 GS Rg1 GS human periodontal ligament stem cells hpdlscs GS Rg mol /L GS Rg1 hpdlscs MTT GS Rg d GS Rg1 ALP RUNX2 Collagen I OPN OCN GS Rg1 GS Rg1 hpdlscs Rg1 R Elevated Proliferative and Osteogenic Potential of Human Periodontal Ligament Stem Cells by Ginsenoside Rg1 GAO Peng CHENG Wen-Xiao WANG Kai-Juan SUN Ke-Mo ZHONG Mei QIN Zi-Shun YIN Li-Hua YU Zhan-Hai * Department of Prosthodontics Medicial College of Stomatology Lanzhou University Lanzhou China Abstract Ginsenoside Rg1 GS Rg1 is the main pharmacological active ingredients of ginseng GS Rg1 is not only a stimulating factor of hematopoietic stem cells as well as of bone marrow mesenchymal stem cells Human periodontal ligament stem cells hpdlscs have the potential to transform into different cells for regeneration This study we found that mol /L GS Rg1 significantly promoted the proliferation and differentiation of hpdlscs Treatment with GS Rg1 for days showed better proliferation in MTT assays The expression of ALP RUNX 2 Collagen I OPN and OCN were also increased implying an improvement in osteogenic differentiation More osteoid tissue was observed by SEM and immunohistochemistry in GS Rg1- nhac scaffolds seeded with hpdlscs when transplanted into mice Ginsenoside Rg1 could be an effective bioactive agent for periodontal tissue engineering Key words ginsenoside Rg1 human periodontal ligament stem cells proliferation osteogenic differentiation nano-hap-collagen nhac No No GZK * Tel qinzsh12@ lzu edu cn Received July Accepted August Supported by National Natural Science Foundation of China No Gansu Province Traditional Chinese Medicine Scientific Research Subject No GZK * Corresponding author Tel qinzsh12@ lzu edu cn

2 hpdlscs hpdlscs 1 12 ~ h 10 PBS 3 ~ 4 1 / 2 Rg1 ginsenoside Rg1 GS Rg1 GS Rg r /min 2 min 1 ml Ⅰ 1 ml DispaseⅡ ~ 50 min 3 5 min r /min 5 min 2 ml ml 37 5% CO 2 5 d human periodontal ligament stem cells hpdlscs 3 d % hpdlscs hpdlscs r /min 10 min 0 22 μm GS Rg1 hpdlscs 10 ~ 15 /ml GS Rg μl / 12 h 200 μl / 5 d 1 / α-mem I- 30 min SABC STRO-1 CD146 Rg1 PBS ALP 1 4 GS Rg1 hpdlscs MTT MTT GS Rg1 hpdlscs -1 3 hpdlscs STOR-1 CD146-FITC /ml 5 96 Beckmen Coulter β- 100 μl 24 h mol /L GS Rg1 DMSO - 0 mol /L GS Rg1 α-mem nano-hap-collagen nhac d CO 2 Heraeau Olympus 3 10 PBS hpdlscs PBS 3 5 min / 0 3% Triton-100 3% 15 min MTT 5 g /L 20 μl 4 h YJ μl 490 nm S-520 A 490 A 490

3 1 Rg ALP ICA hpdlscs 3 hpdlscs RT-PCR GS Rg1 hpdlscs 10 5 /ml h 3 hpdlscs /ml h 10-5 mol /L GS Rg1 0 mol /L GS Rg1 α-mem % α-mem mol /L GS Rg1 0 mol /L GS Rg1 α- 10 nmol /L 0 05 mmol /L MEM 10% α-mem 10 mmol /L β nmol /L 0 05 mmol /l 5 7 d PBS 3 10 mmol /L β- 7 d 0 1% TritonX μl 4 RNA cdna PCR RUNX2 Collagen1 OPN OCN β 520 nm ALP Table 1 Table 1 Primer sequences used in this study Gene GenBank accession No Primer sequences RUNX2 Hs F 5'- CACTGGCGCTGCAACAAGA-3' R 5' - CATTCCGGAGCTCAGCAGAATAA-3' COL1A2 Hs F 5' -GAGGGCAACAGCAGGTTCACTTA-3' R 5' - TGGGCCAATGTCCACAAAGA-3' OPN NM F 5' - GATGAATCTGATGAACTGGTCACTG-3' R 5' - GGTGATGTCCTCGTCTGTAGCA-3' OCN NM F 5' - GACGAGTTGGCTGACCACA-3' 3 hpdlscs /ml h mol /L GS Rg1 0 mol /L GS Rg1 R 5' -CAAGGGGAAGAGGAAAGAAGG-3' GS Rg1 hpdlscs nhac α-mem 10% α-mem 72 h 10 nmol /L 0 05 mmol /L BALB 6 10 mmol /L β ~ 22 g d 1 PBS 2 0 1% 1 5 hpdlscs nhac mol /L nhac 24 h 2 ~ 3 mm 4 μm HE nhac 1 7 nhac 24 x 珋 ± s SPSS ml 24 h 1 72 h 3% GS Rg1 hpdlscs mol /L GS Rg1 0 mol /L GS Rg1 α-mem 10% α-mem 10 nmol /L 0 05 mmol /L 10 mmol /L β- 30 d P < hpdlscs nhac nhac ~ 3 mm hpdlscs 3 ~ 7 d nhac

106 31 1 ~ 2 10 ~ 14 d Fig 1 14 d 2 2 21 d 80% 1 2 STOR-1 CD146 5 5 ~ 7 d 6 ~ 12 Fig 2 Fig 1 Condition of cells Periodontal ligament stem cells PDLSCs maintained in α-minimum essential medium α-mem

abnormally shaped Another 7 days later cells began to gather in one place and grew radiatedly or spirally as a whole Cells grew on nearly 80% area of the wells Fig 2 Fluorescent staining of

4 ~ 2 10 ~ 14 d Fig 1 14 d d 80% 1 2 STOR-1 CD ~ 7 d 6 ~ 12 Fig 2 Fig 1 Condition of cells Periodontal ligament stem cells PDLSCs maintained in α-minimum essential medium α-mem Gibco USA containing 15% foetal bovine serum FBS Gibco USA for 7 days A 14 days B and 21 days C were observed by phase-contrast microscopy 7 days after culturing PDLSCs were spindle-like or abnormally shaped Another 7 days later cells began to gather in one place and grew radiatedly or spirally as a whole Cells grew on nearly 80% area of the wells Fig 2 Fluorescent staining of periodontal ligament colonies Fluorescent staining of periodontal ligament colonies which had been cultured for 21 days with Hoechest Periodontal ligament colonies were cultured for 21 days and immunostained with and without CD146 The same colonies were cultured for 21 days and immunostained with and without Stro-1

1 Rg1 107 2 3 hpdlscs GS Rg1 1 10-5 mol /L hpdlscs 1 2 3 4 5 d hpdlscs 1 d P > 0 05 2 d Table 2 GS Rg1 hpdlscs P < 0 05 1 ~ 2 d GS Rg1 GS Rg1 A 570 0 21 ± 0 01 Table Proliferation of hpdlscs

0 01 0 57 ± 0 01 0 58 ± 0 01 Compared with the control group P < 0 05 x 珋 ± s Fig 3 Cell proliferation of hpdlscs stimulated by GS Rg1 MTT assays were performed over 5 days The number of PDLSCs

5 1 Rg hpdlscs GS Rg mol /L hpdlscs d hpdlscs 1 d P > d Table 2 GS Rg1 hpdlscs P < ~ 2 d GS Rg1 GS Rg1 A ± 0 01 Table Proliferation of hpdlscs stimulated by GS Rg1 2 Fig 3 n = 6 Group 1 day 2 day 3 day 4 day 5 day E-M5 Rg ± ± 0 03 * 0 68 ± 0 01 * 0 73 ± 0 01 * 0 77 ± 0 01 * Negative control ± ± ± ± ± 0 01 Compared with the control group P < 0 05 x 珋 ± s Fig 3 Cell proliferation of hpdlscs stimulated by GS Rg1 MTT assays were performed over 5 days The number of PDLSCs increase with the culturing days adding in absorbance at 570 nm There are no significant difference between GS Rg-1 group and negative control group in the first day n = 6 P > 0 05 A statistically significant difference was observed between the 1 day group and 2 day group which means mol /L GS Rg1 have the strongest effect on cell proliferation at this time point The data are expressed as the mean ± SD n = 6 compared with the control group * P < 0 05 Fig 4 The expression of ALP After the PDLSCs reached confluence the normal culture medium was replaced with an osteogenic medium containing and not containing mol /L GS Rg1 The cells were cultured for an additional 3 5 and 7 days The ALP activity of PDLSCs was significantly enhanced in osteogenic medium with mol /L GS Rg1 n = 6 compared with the control group P < 0 05 A most significant difference was observed between the 3 day group and 5 day group The data are expressed as the mean ± SD n = 6 compared with the control group * P < hpdlscs 2 5 RT-PCR mol /L GS Rg1 hpdlscs mol /L GS Rg1 hpdlscs d ALP d 7 d GS Rg1 RUNX2 ALP P < Collagen1 OPN OCN P < GS Rg1 3-5 d ALP Collagen I 2 9 ± 0 03 Table ± 0 03 Table 3 Fig 4 n = 6 Fig 5 n = Table 3 The expression of ALP mol /L GS Rg1 hpdlscs Group 3 day 5 day 7 day d E-M5 Rg ± 0 04 * 1 78 ± 0 06 * 2 36 ± 0 04 * Negative control 0 41 ± ± ± ~ * Compared with the control group P < 0 05 x 珋 ± s 28 d Fig 6

108 31 Table 4 RT-PCR for the detection of osteoblastic marker expression Group RUNX2 Collagen1 OPN OCN E-M5 Rg-1 1 5 ± 0 03 * 2 3 ± 0 02 * 1 7 ± 0 03 * 1 81 ± 0 03 * Negative control 1 1 1 1 *

6 Table 4 RT-PCR for the detection of osteoblastic marker expression Group RUNX2 Collagen1 OPN OCN E-M5 Rg ± 0 03 * 2 3 ± 0 02 * 1 7 ± 0 03 * 1 81 ± 0 03 * Negative control * Compared with the control group P < 0 05 x 珋 ± s Fig 5 RT-PCR for the detection of osteoblastic marker expression The gene expression of osteoblastic marker in hpdlscs obtained from either the E-M5 Rg-1 group or negative control group After 7-days induction with osteogenic medium containing and not containing mol /L GS Rg1 runtrelated transcription factor-2 RUNX2 collagen I osteopontin OPN and osteocalcin OCN mrna expression were determined by real-time PCR The data are expressed as the mean ± SD n = 6 compared with the control group P < 0 05 The relative mrna PDLSCs was significantly enhanced in osteogenic medium with mol /L GS Rg1 A most significant difference was observed between the GS Rg-1 group and negative control group of collagen I 2 7 hpdlscs nhac 7 PDLSCs 8 9 Rg1 GS Rg mol /L GS Rg1 1 GS Rg1 hpdlscs nhac Fig d nhac nhac mol /L GS Rg1 hpdlscs 1 d GS Rg1 hpdlscs Fig 8 P > d hpdlscs 3 P < ~ 2 d GS Rg1 hpdlscs 80% ~ 90% 0 21 ± mol /L GS Rg1 hpdlscs 1 ~ 2 d hpdlscs GS Rg1

1 Rg1 109 Fig 6 Differention of periodontal ligament colonies related to different culture time PDLSCs were seeded at density of 2 10 5 /ml per well in three 6-well plate Every three wells in a plate

mol /L GS Rg1 The other group D E F was treated with α-minimum essential medium containing 15% foetal bovine serum and osteogenic differentiation medium Alizarin red staining the wells after 14 days

7 1 Rg1 109 Fig 6 Differention of periodontal ligament colonies related to different culture time PDLSCs were seeded at density of /ml per well in three 6-well plate Every three wells in a plate were taken as a group One group A B C was treated with α-minimum essential medium α-mem Gibco USA containing 15% foetal bovine serum FBS Gibco USA and osteogenic differentiation medium with mol /L GS Rg1 The other group D E F was treated with α-minimum essential medium containing 15% foetal bovine serum and osteogenic differentiation medium Alizarin red staining the wells after 14 days A D 21days B E 28 days C F culturing Less staining can be seen in the controls not subjected to mol /L GS Rg1 D E F The amount of deposition of mineralization increase as the duration of GS Rg mol /L incubation was increased from 14 days A to 21 days B and to 28 days C ALP RUNX2 Collagen1 OPN OCN ALP ALP mol /L GS 10-5 mol /L GS Rg1 hpdlscs Rg1 hpdlscs 7 d d ALP d RUNX2 Collagen1 OPN OCN ALP P < 0 05 GS Rg1 3-5 d Collagen ± ALP 10-5 mol /L GS Rg1 hpdlscs 0 95 ± mol /L GS Rg mol /L GS Rg1 hpdlscs hpdlscs d Fig 8 Observation of experiment in vivo Scaffolds seeded with cells were subjected to α-minimum essential medium containing 15% foetal bovine serum and osteogenic differentiation medium without B or with A GS Rg1 at mol /L for 3 days The aforementioned two kinds of scaffolds were implanted into dorsal subcutaneous pockets of three 6-week-old immunocompromised beige mice respectively 30 days later the mice were killed The operation area were submitted for section and HE-staining Osteoid-like structure and few fibrous bone could be found in the control nhac without cells A In contrast numerous fibrous bone were formed in the nhac implant with cells B Osteoblasts newly generated bone tissue and blood vessels can be observed around bone trabeculae

110 31 Fig 7 Observation of hpdlscs on nhac Incise nhac scaffolds into 2-3 mm diameter particulate Then scaffolds were seeded with 5 10 5 /ml cell suspension solution A following negative pressure

8 Fig 7 Observation of hpdlscs on nhac Incise nhac scaffolds into 2-3 mm diameter particulate Then scaffolds were seeded with /ml cell suspension solution A following negative pressure suction process made sure hpdlscs entering pores of scaffold well After being cultured for 3 days with α-minimum essential medium α-mem Gibco USA containing 15% foetal bovine serum FBS Gibco USA scaffolds were fixed dehydrated dried and observed by SEM 450 Scaffolds seeded with none cells went through the same process to make a negative control The morphology of nhac scaffold with B and without A cells was taken by S- 520 SEM Japan PDLSCs attached and proliferated well on nhac scaffold Red arrow in B 14 ~ 28 d 1 References 10-5 mol /L GS Rg1 hpdlscs Yang Q Yu Z H Du J D 19 nhac the expressions of interleukin-6 tissues in periodontitis rats J technique for bone tissue engineering J 3 Liu Q Kou J P Yu B Y NF-κB activation J nhac hpdlscs nhac hpdlscs nhac 30 d HE nhac 5 Shuai C Gao C Feng P et al nhac mol /L GS Rg1 hpdlscs hpdlscs mol /L GS Rg1 hpdlscs GS Rg Rg-1 6 J et al Efects of ginsenoside Rg-1 on bone gla protein in periodontal J Pract Stomatol Ariani M D Matsuura A Hirata I et al New development of carbonate apatite-chitosan scaffold based on lyophilization Dent Mater J 2013 Ginsenoside Rg1 protects against hydrogen peroxide-induced cell death in PC12 cells via inhibiting Neurochem Int Shuai C J Yang B Peng S P et al Development of composite porous scaffolds based on poly lactide-co-glycolide /nanohydroxyapatite via selective laser sintering J Int J Adv Manuf Tech Grain growth associates mechanical properties in nano- hydroxyapatite bone scaffolds J J Nanosci Nanotechnol Park J C Lee S M Kim J C et al Effect of humoral factors from hpdlscs on the biologic activity of habcs J Oral Dis Gardner O F Archer C W Alini M et al Chondrogenesis of mesenchymal stem cells for cartilage tissue engineering J Histol Histopathol Wang L Xia J Liu Q et al Effect of lipopolysaccharide on proliferation and inflammatory factors expression of human periodontal ligament stem cells J West China J Stomatol

9 1 Rg glutamate J Brain Res Dlx5 Msx2 J 16 Leung K W Ng H M Tang M K et al Ginsenoside-Rg1 Sun C X Zhang L K Feng X X et al Changes in the mediates a hypoxia-independent upregulation of hypoxia-inducible expression of Dlx5 Msx2 and Dlx5 /Msx2 in hpdlscs loaded with cyclic tensile stress J J Sichuan Univ Med Sci 2011 factor-1α to promote angiogenesis J Angiogenesis Du J Cheng B Zhu X et al Ginsenoside Rg1 a novel glucocorticoid receptor agonist of plant origin maintains glucocorticoid efficacy with reduced side effects J J Immunol Fang F Chen X Huang T et al Multi-faced neuroprotective effects of Ginsenoside Rg1 in an Alzheimer mouse model J Biochim Biophys Acta Yin H Liu Z Li F et al Ginsenoside-Rg1 enhances angiogenesis and ameliorates ventricular remodeling in a rat model of myocardial infarction J J Mol Med Jiang B Xiong Z Yang J et al Antidepressant-like effects of ginsenoside Rg1 are due to activation of the BDNF signalling pathway and neurogenesis in the hippocampus J Br J Pharmacol Wang X D Gu T X Shi E Y et al Effect and mechanism of panaxoside Rg1 on neovascularization in myocardial infarction rats J Chin J Integr Med Radad K Gille G Moldzio R et al Ginsenosides Rb1 and Rg 1 effects on mesencephalic dopaminergic cells stressed with 17 Wei F Qu C Song T et al Vitamin C treatment promotes mesenchymal stem cell sheet formation and tissue regeneration by elevating telomerase activity J J Cell Physiol J Yang K Cui X X Deng C et al Effects of advanced glycation end products on the osteogenic differentiation of human bone marrow mesenchymal stem cells and human periodontal ligament stem cells J Chin J Conservative Dentistry Bhamidipati M Scurto A M Detamore M S The future of carbon dioxide for polymer processing in tissue engineering J Tissue Eng Part B Rev Rg1 J Wang P Zhou Y Wang Y P et al Promotion of ginsenosidee Rg1 on proliferation and osteogenic potential of human periodontal ligament stem cells J J Third Mil Med Univ

TGF-β AngⅡ B SD ~ 220g SPF. SCXK No SYXK ~ 25 40% ~ 70% OR37G-C

TGF-β AngⅡ B SD ~ 220g SPF. SCXK No SYXK ~ 25 40% ~ 70% OR37G-C 2017 7 43 4 Journal of Southwest Minzu University Natural Science Edition Jul. 2017 Vol. 43 No. 4 doi10. 11920 /xnmdzk. 2017. 04. 008 1 1 1 2 1 1 3 1. 610212 2. 6100413. 610031 AngⅡ TGF-β. SD 60 180 ~