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Zinc Chloride for Odontogenesis of Dental Pulp Stem Cells viaMetallothionein Up-regulationChia-Yung Lin, DDS, PhD,*Hsin-Hua Lin, MA,Mong-Hsun Tsai, PhD,k Shau-Ping Lin, PhD,k

and Min-Huey Chen, DDS, PhD*

Abstract

Introduction:Previous studies have shown that zincchloride (ZnCl2) can induce metallthionein (MT) in theliver and kidney to protect tissues against toxicantsand shows a better corneal wound healing than conven-tional drugs do. We hypothesized that ZnCl2 canpromote odontogenesis of dental pulp stem cells(DPSCs) via MT. The purpose of this study was to inves-tigate the effects of ZnCl2 on human DPSCs and theexpression of MT. Methods: DPSCs were isolated by

flow cytometry with selective surface marker CD146and STRO-1. After they grew into confluence, DPSCswere induced into odontoblasts with or without ZnCl2supplemented in the culture medium for 21 days. Theeffect of ZnCl2on DPSCs differentiation was examinedfollowed by alkaline phosphatase staining/activity andquantitative real-time polymerase chain reaction anal-ysis. Results: By treating DPSCs with ZnCl2, the durationof mineralization was shortened and expressions ofdifferentiation markers into odontoblasts were moresignificant than those without ZnCl2 stimulation.Besides, the MT gene expression was increased withthe increasing expressions of odontoblasts markers

after treated with ZnCl2.Conclusion:This was the firstreport that ZnCl2could promote odontoblastic differen-tiation of DPSCs through the up-regulation of gene MT.(J Endod 2011;37:211216)

Key WordsHuman dental pulp stem cells, metallothionein, odonto-blastic differentiation, odontogenesis, zinc chloride

Odontoblasts can generate reparative dentin when teeth are irritated(1). Althoughcalcium hydroxide is widely used for conservative pulp therapy, its cytotoxicity anddifficult manipulation are still problems(2). Many efforts are made to develop newmaterials(35). In addition, mineral trioxide aggregate, bioaggreate(6), and calciumphosphate cement with its modifications(7)are all under investigations to be bettercandidate materials. Dental pulp stem cells (DPSCs)(8)are maintained undifferenti-ated in the niche provided by the pulp(9, 10). DPSCs can be induced into odontoblastsin vitroand characterized with expressions of dentinsialophosphoprotine (DSPP) anddentin matrix acidic phosphoprotein (DMP-1)(11, 12).

Zinc (Zn) is an important regulator involved with various enzymes and proteinsand plays an important role in bone metabolism(13). Low serum Zn and low boneturnover were reported in small for their gestational age preterm infants(14), andlowZnintakeisassociatedwithlowbonemassinadultwomen (15). Inadequate dietaryintake of Zn causesa decrease in thenumber of osteoblasts(16), and ZnaugmentsDNAsynthesis in murine osteoblast-like cells in a dose-dependent manner(17). It is alsoinvolved in the stimulation of collagen production in rat calvaria(18) and has an inhib-itory effect on bone resorption and a stimulatory effect on bone formation/mineraliza-tion(19). Cellular trafficking of Zn is controlled by storage proteins and transporters.Metallothioneins (MTs) are cellular metal storage proteins induced by essential traceelementssuch as Zn andCu (20). Thisubiquitous heavy metal binding and cysteine-richprotein reacts with free radicals and organic electrophiles and can be induced by zincchloride (ZnCl2) treatment(21, 22). MT levels may vary with age and type of tissue and

depend on nutritional and physiological factors. ZnCl2 is used as an additive tointravenous solutions for total parenteral nutrition. ZnCl2 not only prevents thekidney and liver from mercury and acetaminophen toxicities but also enhancescorneal wound healing, and these effects were regulated via the MTs(23).

For promoting odontogenesis, novel genes such as hemeoxygenase-1 stimulatedby simvastatin have been identified(24, 25). To our knowledge, no information isavailable regarding the effects of MT simulated by Zn on DPSCs. We hypothesizedthat ZnCl2can promote DPSCs differentiation via MT up-regulation.

Materials and MethodsCell Culture

Isolation of DPSCs. All experimental procedures were performed according to

protocols approved by the Review Committee of College of Medicine, National TaiwanUniversity. Dental pulp cells were isolated from normal human third molars. Eight thirdmolars were collected from two healthy adults (18-year-old woman and a 25-year-oldman) in National Taiwan University Hospital. Teeth surfaces were cleaned with phos-phate buffered saline (PBS) and cut by sterilized diamond burs. The pulp tissue wasgently separated by forceps and digested in a solution of 3 mg/mL collagenase type Iand 4 mg/mL dispase for 1 hour at 37C. Single-cell suspensions were obtained bypassing through a 70-mm strainer (Pharmingen; BD, San Jose, CA) and then culturedin 6-cm dishes (Costar, Cambridge, MA) with Dulbeccos Modified Eagle Media (lowglucose) (Gibco Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10%fetal bovine serum (FBS) (HyClone, Logan, UT)/100 U/mL penicillin + 100 mg/mLstreptomycin (Biological Technologies, Beit Haemek, Israel)/2 mmol/L L-glutamine(Gibco, Grand Island, NY).

From the Graduate Institute of *Clinical Dentistry and OralBiology,School of Dentistry, National Taiwan University, Taipei,Taiwan ROC; National Taiwan University Hospital, National

Taiwan University, Taipei, Taiwan;

Institute of Biotechnology,College of Bio-Resources and Agriculture, National TaiwanUniversity, Taipei, Taiwan ROC; and kAgricultural Biotech-nology Research Center, Academia Sinica, Taiwan ROC.

Address requests for reprints to Dr. Shau-Ping Lin, Instituteof Biotechnology, College of Bio-Resources and Agriculture,National Taiwan University, Taipei, Taiwan ROC., and Dr. Min-Huey Chen, Graduate Institute of Clinical Dentistry, Schoolof Dentistry, National Taiwan University, Taipei, Taiwan ROC.E-mail address: shaupinglin@ntu.edu.tw; minhueychen@ntu.edu.tw.0099-2399/$ - see front matter

Copyright 2011 American Association of Endodontists.doi:10.1016/j.joen.2010.11.009

Basic ResearchBiology

JOEVolume 37, Number 2, February 2011 ZnCl2for Odontogenesis of DPSCs via MT Up-regulation 211

mailto:shaupinglin@ntu.edu.twmailto:minhueychen@ntu.edu.twmailto:minhueychen@ntu.edu.twhttp://dx.doi.org/10.1016/j.joen.2010.11.009http://dx.doi.org/10.1016/j.joen.2010.11.009mailto:minhueychen@ntu.edu.twmailto:minhueychen@ntu.edu.twmailto:shaupinglin@ntu.edu.tw

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After 1 week, dental pulp cells were observed to grow at approx-imately 90% confluence; then, cells were subcultured in 10-cm dishesfor another 2 weeks. Approximately 107 cells could be derived fromeach tooth. All cells from eight teeth were mixed together to acquirean average situation for the selection of DPSCs. Adherent cells weredetached by 0.25% trypsin-EDTA, centrifuged for 5 minutes at 500 g,and resuspended in PBS. Aliquots containing 107 cells were incubatedwith mouse antihuman CD146 conjugated with phycoerythrin (Phar-mingen) (maximum absorption at 495 nm) and mouse antihumanSTRO-1 (Invitogen, Carlsbad, CA) for 30 minutes at 4C; cells were

washed with PBS with 2% FBS followed by staining with 1mgFITCconju-gate goat antimouse immunoglobulin G for 30 minutes at 4C. All cellswere then passed through a 70-mm strainer and collected by BectonDickinson FACS Aria fluorescence activated cell-sorting system..

Characterization of DPSCsFor characterization, odontoblastic differentiation was performed

by using odontoinduction medium (OM) containing Dulbeccos Modi-fied Eagle Media (low glucose), 10%FBS, 100 U/mL penicillin, 100mg/mL streptomycin, 0.1 mmol/L ascorbic acid, 100 mmol/L b-glycero-phosphate (Fluka, Buchs, Switzerland), and 108 mmol/L dexametha-sone (Sigma-Aldrich, St. Louis, MO). When cells grew into 90%confluence, the original medium was replaced by OM and cultured

for the following 21 days. The differentiation potentials were identifiedby reverse-transcription polymerase chain reaction (RT-PCR) forexpressions of DSPP, DMP-1, and OCN. The oligonucleotide RT-PCRprimers were listed inTable 1. DPSCs in the third passage were usedfor subsequent experiments.

Preparation of Induction Medium With ZnCl2To evaluate effects of ZnCl2, 500 mL OM was added with 0.0034 g,

0.0068 g, and 0.0136 g ZnCl2(Sigma-Aldrich), respectively, at concen-trations of 50 mmol/L, 100 mmol/L, and 200 mmol/L. Those withoutZnCl2were used as controls.

MTT Assay

For each experiment, DPSCs were seeded at a density of 1 104/mL in 24-well plates. Experimental groups were those cultured in OMwith 50 mmol/L, 100 mmol/L, and 200 mmol/L ZnCl2; those in OMwithout ZnCl2served as controls. After 1, 4, and 7-day of cells culturedin OM with different concentrations of ZnCl2, media were removed and100 mL of MTT (3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyltetrazol-ium bromide, Sigma) solution (2 mg/mL in PBS) was added intoeach well and following procedures were performed as describedpreviously (26).

Alkaline Phosphatase StainDPSCs were cultured in OM with different concentrations of ZnCl2

(0, 50, 100, and 200 mmol/L) in six 12-well plates with the density of 1

104 cells/mL (n= 6). On days 7, 10, and 14, each well of cells witha different concentration of ZnCl2were fixed by 4% paraformaldehyde.After this, the alkaline phosphatase (ALP) stain kit (BioPioneer, SanDiego, CA) was applied for alkaline phosphatase staining by manufac-turers instructions.

ALP ActivityDPSCs were cultured in OM with different concentrations of ZnCl2

as described. ALP activity was expressed as micromoles of reaction

product (p-nitrophenol) per 30 minutes from milligram of cellularprotein as described(27).

Quantitative RT-PCRDPSCs cultured in OM with 100 mmol/L ZnCl2were used for quan-

titative RT-PCR (qRT-PCR) analysis at 0, 7, and 14 days. Those in OMwithout ZnCl2 were used as controls. Reactions were prepared withthe TaqMan (Applied Biosystems by Life Technologies, Foster City,CA) method according to the manufacturers instructions. The productwas aggregated on the bottom of the well by a 2,000-rpm centrifugalforce for 1 minute, and the plate was put in ABI Prism 7900HT (AppliedBiosystems by Life Technologies) for PCR reaction and data collection.

The GAPDH (Hs99999905_ml), DSPP (Hs00171962_ml), OCN(Hs00758162_ml), and MT (Hs00744661_sH) were standardizedby the TaqMan gene expression assays and quantified by SequenceDetector Software 2.21 (Applied Biosystems by Life Technologies).

Statistical AnalysisOne-way analysis of variance was used to estimate the difference

that resulted from the addition of ZnCl2, and a Scheffe test was usedfor further analysis. Statistical significance was defined as p < 0.05.

ResultsDPSCs

After a 1-week culture of isolated pulp cells, colony formation wasfound. About 1.22% STRO-1+ CD146+ cells from isolated cells weresorted as DPSCs. Expressions of DSPP, DMP-1, and OCN after inductionwere also confirmed by RT-PCR (Fig. 1AandB).

MTT AssayMTT assay indicates the activity of vital cells.Figure 1Cshowed the

ratio of MTT conversion of cells in OM with 50, 100, and 200 mmol/LZnCl2relative to that of cells in OM without ZnCl2(controls) after incu-bation for1, 4, and7 days. At alldata points, theratioof MTT conversionof DPSCs in ZnCl2showed no significant difference with that of controls(p> 0.05). According to this result, ZnCl2had no toxicity to DPSCs.

TABLE 1. Oligonucleotide Primer Sequences Used in the Reverse-Transcription PCR To Identify Dental Pulp Stem Cells

Gene name Sequence Tm Size

DSPP F:5GCC ATT CCA GTT CCT CAA GC 3R:5 CAT GCA CCA GGA CAC CAC TT 3

59C 144bp

DMP1 F:5 GAT CAG CAT CCT GCT CAT GTT C 3R:5 GAG CCA AAT GAC CTT CCA TT 3

59C 109bp

OCN F:5 ATG AGA GCC CTC AGA CTC CTC 3R:5 CGG GCC GTA GAA GCG CCG ATA 3

60C 297bp

GAPDH F:5 GTC TTC ACC ACC ATG GAG AAG GCT 3

R:5 CAT GCC AGT GAG CTT CCC GTT 3

58C 392bp

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Figure 1. (A) The isolation of DPSCs was performed by the Fluorescence Activated Cell Sorting System. About 1.22% STRO-1+ CD146+ cells from the mixtureisolated cells of eight teeth were sorted as DPSCs. (B) By the reverse-transcription PCR, the DPSCs were examined to show the DSPP, DMP-1, and OCN expression.(C) The MTT of DPSCs was cultured in the odontogenic induction medium with different concentration of ZnCl2for 1, 7, and 14 days. No significant differences wereobserved between DPSCs within or without ZnCl2. (D) The ALP staining of these DPSCs showed that with 100mmol/L ZnCl2the ALP staining was more evident infastening the mineralization at 10 days than that with other concentrations of ZnCl2. (E) The ALP activities of DPSCs cultured in the odontogenic induction medium

with different concentrations of ZnCl2after 7, 10, and 14 days showed that the ALP activity of DPSCs cultured in 100 mmol/L ZnCl2at 10 day was the highest (*p