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Biomaterials 32 (2011) 9696e9706

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Biomaterials

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Odontogenic differentiation of human dental pulp stem cells induced bypreameloblast-derived factors

Ji-Hyun Leea, Dong-Seol Leea, Han-Wool Chounga, Won-Jun Shonb, Byoung-Moo Seoc, Eun-Hyang Leed,Je-Yoel Chod, Joo-Cheol Parka,*aDepartment of Oral Histology-Developmental Biology & Dental Research Institute, BK21 Project, School of Dentistry, Seoul National University, 28 Yeongun-dong, Chongro-gu,Seoul 110-749, Republic of KoreabDepartment of Conservative Dentistry, School of Dentistry, Seoul National University, Seoul 110-749, Republic of KoreacDepartment of Oral and Maxillofacial Surgery, School of Dentistry, Seoul National University, Seoul 110-749, Republic of KoreadDepartment of Biochemistry, School of Dentistry, Kyungpook National University and ProtAnBio Co., Ltd, Dae-gu 700-412, Republic of Korea

a r t i c l e i n f o

Article history:Received 22 August 2011Accepted 1 September 2011Available online 16 September 2011

Keywords:Epithelial-mesenchymal interactionPreameloblast-CMHuman dental pulp stem cellOdontoblast differentiationDentin regeneration

* Corresponding author. Tel.: þ82 2 740 8668; fax:E-mail address: jcapark@snu.ac.kr (J.-C. Park).

0142-9612/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.biomaterials.2011.09.007

a b s t r a c t

The differentiation of odontoblasts is initiated by the organization of differentiating ameloblasts duringtooth formation. However, the exact roles of ameloblast-derived factors in odontoblast differentiationhave not yet been characterized. We investigated the effects of preameloblast-conditioned medium (PA-CM) on the odontogenic differentiation of human dental pulp stem cells (hDPSCs) in vitro and in vivo.Furthermore, we analyzed the PA-CM by liquid chromatography-mass spectrometry to identify novelfactors that facilitate odontoblast differentiation. In the co-culture of MDPC-23 cells or hDPSCs withmouse apical bud cells (ABCs), ABCs promoted differentiation of odontoblastic MDPC-23 cells andfacilitated odontoblast differentiation of hDPSCs. PA-CM, CM from ABCs after 3 days culture, was mosteffective in increasing the dentin sialophosphoprotein promoter activity of odontoblastic MDPC-23 cells.When PA-CM-treated hDPSCs were transplanted into immunocompromised mice, they generated pulp-like structures lined with human odontoblast-like cells showing typical odontoblast processes. However,during recombinant human bone morphogenenetic protein 2-treated hDPSCs transplantation, some ofthe cells were entrapped in mineralized matrix possessing osteocyte characteristics. After proteomicanalyses, we identified 113 types of proteins in PA-CM, of which we characterized 23. The results showthat preameloblast-derived factors induce the odontogenic differentiation of hDPSCs and promote dentinformation.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Dentin forms the bulk of the tooth. Defects in dentin arecommon due to numerous pathologies, such as dental caries,mechanical trauma, or even genetic alterations. In the last decade,great progress in tooth regeneration including regeneration ofdentin was made. However, strategies for dentin repair are mainlybased on various growth factors, transcription factors, basementmembrane components, and pulp-capping materials, such ascalcium hydroxide or mineral trioxide aggregate [1e3]. Althoughthese procedures may result in gains in pathologic reparativedentin formation, careful histological evaluation has indicated thatnone can fully restore the physiological architecture of the original

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dentin [4]. Thus, to achieve complete tissue regeneration, it isnecessary to recapitulate the process involved in the originalformation of the dentin during tooth development.

Epithelial-mesenchymal interactions are important mecha-nisms occurring during the development of various organs,including hair follicles and mammary glands [5]. Tooth develop-ment is also achieved through continuous reciprocal interactionsbetween the dental epithelium and the underlying ectomesen-chyme. Induction of ameloblasts derived from dental epithelialcells is indispensible for the differentiation of odontoblasts fromectomesenchymal cells during crown formation [6]. However, theexact roles of ameloblast-derived factors in odontoblast differen-tiation have not yet been characterized.

Human dental pulp stem cells (hDPSCs) are easy to isolate fromhuman third molars, are multipotent, and express mesenchymalstem cell markers. Human DPSCs can differentiate into varioustissues, such as odontoblasts, adipocytes, chondrocytes, and

J.-H. Lee et al. / Biomaterials 32 (2011) 9696e9706 9697

osteoblasts [7,8]. In addition, hDPSCs interact with various bioma-terials and are effective in mineralized tissue formation [9e11].Therefore, hDPSCs would be an ideal material in clinical trials andtissue engineering for bone and dentin regeneration. However,dentin regeneration using adult pulp stem cells is limited by factorssuch as epithelial shortage, because the dental epithelium,including ameloblasts, degenerates, and apoptosis occurs afterenamel formation in humans. In contrast, rodent incisors havea special epithelial structure, referred to as the ‘apical bud,’ at theapical end, which continuously grows [12]. The apical bud containsinner enamel epithelium (IEE) that differentiates into the amelo-blasts, outer enamel epithelium, and stellate reticulum [13].Therefore, murine apical bud cells (ABCs) can be used instead of thedental epithelium for the differentiation of odontoblasts andregeneration of dentin in humans.

Recently, proteomic methods are widely applied to cancer [14]and bacterial analysis [15]. Analysis of the conditioned medium(CM) of cultured cells from various diseases is ongoing [16].Although proteomic analysis makes use of mineralized tissues, suchas dentin [17] and enamel [18], secretory factors of the dentalepithelium that are essential for the odontoblast differentiationhave not yet been utilized for proteomic research.

In the present study, the effects of preameloblast-CM (PA-CM)from mouse ABCs on the odontogenic differentiation of hDPSCswere investigated in vitro and in vivo. Furthermore, we analyzed PA-CM by liquid chromatography-mass spectrometry (LC-MS/MS) toidentify factors that facilitate odontoblast differentiation anddentin formation.

2. Materials and methods

2.1. Cell lines

MDPC-23 cells for odontoblasts, provided by Dr. J. E. Nör (School of DentalMedicine, University of Michigan, MI, USA), and HEK293T cells (ATCC, Rockville, MD,USA) were grown and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM,Gibco BRL, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS,Gibco BRL) and antibiotics (Penicillin-G 100 U/ml, streptomycin 100 mg/ml, fungi-zone 2.5 mg/ml, Gibco BRL) at 37 �C in a 5% CO2 humidified atmosphere. Human KBoral epithelial cells (ATCC) were grown in Minimum Essential Medium (MEM, GibcoBRL) supplemented with 10% FBS and antibiotics.

2.2. Primary cell culture

We collected human impacted third molars from 10 adults (18e22 years of age)at Seoul National University Dental Hospital (Seoul, Korea). The experimentalprotocol was approved by the hospital’s Institutional Review Board, and the patientsprovided informed consent. Human DPSCs were isolated as described previously[19]. Briefly, the teeth were cracked open to remove the pulp tissues gently withforceps. The pulp tissues were then minced into explants and placed in 60-mmculture dishes (NUNC, Rochester, NY, USA). The explants were cultured in DMEMsupplemented with 10% FBS and antibiotics.

ABCs of C57BL/6 mice were isolated and cultured as described by Fang et al. [20].Briefly, the lower incisors were separated from mice at postnatal day 7. ABCs wereenzymatically isolated from apical bud tissues, and cultured in keratinocyte serum-free medium (K-SFM, Gibco BRL) until reaching confluence. After reaching conflu-ence, the cells were cultured in DMEMwith 10% FBS and antibiotics. All experimentsusing mice were approved by Seoul National University Institutional Animal Careand Use Committee.

To culture ABCs simultaneously with hDPSCs or MDPC-23 cells, Transwell�

Permeable Supports (Corning Inc., Corning, NY, USA) were used. ABCs (8 � 104 cells/well) were seeded in the upper compartment of the Transwell�, and hDPSCs (1�105

cells/well) or MDPC-23 cells (1 � 105 cells/well) were seeded in the lowercompartment of the dish, respectively.When confluence reached 80e90%, the uppercompartment was combined with the lower compartment, and differentiation wasinduced using differentiation media, DMEM supplemented with 10% FBS, 50 mg/mlascorbic acid, and 10 mM b-glycerophosphate for 10 days.

2.3. RT-PCR and real-time PCR

Total RNA was extracted from the cells with TRIzol� reagent according to themanufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). Total RNA (2 mg) wassubjected to RT with 0.5 mg Oligo d (T) and 1 ml (50 IU) Superscript III enzyme

(Invitrogen) in a 20-ml reaction mixture at 50 �C for 1 h. The resulting mixture wasamplified by PCR. Onemicroliter of the RT product was subjected to PCR: 32 cycles at94 �C for 30 s, 55 �C for 30 s, and 72 �C for 30 s.

For RT-PCR, specific primers for ameloblastin, amelogenin, and GAPDH weresynthesized as listed in Supplementary Table 1. For the amplification of the humanAlu sequence, cellular areas in the mineralized tissue of hDPSCs transplants weredissected from the sectioned slide. Genomic DNA (2 mg) was extracted from thedissected samples using saturated sodium chloride. PCR analysis was performed forsamples containing 2 ml each of the extracted genomic DNA, 1 mg/ml human DNA asa positive control and 1 mg/ml nude mouse genomic DNA as a negative control. ThePCR products were electrophoresed on a 1.2% agarose gel, stained with ethidiumbromide, and visualized under ultraviolet light.

For real-time PCR, specific primers for DSPP, BSP, nestin, Col I, Runx2, ALP, OC,mCpne7, and GAPDH were synthesized as listed in Supplementary Table 1. Real-timePCR was performed on an ABI PRISM 7500 sequence detection system (AppliedBiosystems, Carlsbad, CA, USA) using SYBR GREEN PCR Master Mix (Takara Bio Inc.Otsu, Shiga, Japan) according to the manufacturer’s instructions. The PCR conditionswere 94 �C for 1 min followed by 95 �C for 15 s and 60 �C for 34 s for 40 cycles. Allreactions were run in triplicate and were normalized to the housekeeping gene,GAPDH. Relative differences in PCR results were calculated using the comparativecycle threshold (CT) method.

2.4. Western blot analysis

After removal of the supernatant, the pellet was resuspended in lysis buffer(100 mM Tris, pH 7.4, 350 mM NaCl, 10% glycerol, 1% Nonidet P-40, 1 mM ethyl-enediaminetetraacetic acid (EDTA), 1 mM dithiothreitol, and protease inhibitors) andincubated for 15 min on ice. Thirty micrograms of proteins were separated by 10%sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and trans-ferred onto a nitrocellulose membrane (Schleicher & Schuell, Dassel-Relliehausen,Germany). Membranes were blocked for 1 h with 5% nonfat dry milk in phos-phate buffered saline containing 0.1% Tween 20 (PBS-T) and incubated overnightwith primary antibodies against HA (COVANCE, Princeton, NJ, USA), type I collagen(SC-59772, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and GAPDH (glyceral-dehyde 3-phosphate dehydrogenase, SC-25778, Santa Cruz Biotechnology) dilutedin PBS-T buffer (1:1000) at 4 �C. Antisera against dentin sialoprotein (DSP) and bonesialoprotein (BSP) were produced as described previously [21]. After washing, themembranes were incubated with anti-rabbit or -mouse immunoglobulin G (IgG)conjugated with horseradish peroxidase (1:5000, Santa Cruz Biotechnology) for 1 h.Labeled protein bands were detected using an enhanced chemiluminescence system(Amersham Biosciences, GE Healthcare. Buckinghamshire, U.K.). Densitometry wasperformed using ImageJ software (http://rsb.info.nih.gov/ij).

2.5. Preparation of preameloblast-conditioned medium (PA-CM)

ABCs were seeded at 7.5 � 105 cells on 100-mm collagen-coated dishes. Whenconfluence reached 90%, the cells were cultured in DMEM differentiation media asdescribed above. After 3 days of differentiation, the cells were washed twice withPBS, differentiation media without FBS and EGF, and incubated for additional 8 hbefore the supernatant was collected. After filtration using a 0.2-mm pore filter(Nalgene, Rochester, NY, USA), 200 ml of harvested CM were concentrated usingammonium sulfate precipitation and dialyzed against PBS at 4 �C. KB cells CM (KB-CM) was prepared as for PA-CM.

2.6. Luciferase assay

MDPC-23 cells were seeded on a 24-well plate at a density of 5 � 104 cells/well.After 24 h, the cells were transfected with Lipofectamine Plus� reagent (Invitrogen)according to the manufacturer’s instructions. For each transfection, 0.4 mg luciferasereporter plasmid pGL3basic (control), the dentin sialophosphoprotein (DSPP)promoter expression vector (pGL3LUC DSPP -791w þ 54), and the bone sialoprotein(BSP) promoter (pGL3LUC �2478w þ 60) were used as described previously [21].Transfected cells were treated with or without PA-CM or KB-CM for 48 h. After 48 hof transfection, cells were lysed for luciferase activity assessment using the lucif-erase reporter gene assay system (Roche Applied Science, Indianapolis, IN, USA)according to the manufacturer’s instructions. The measurements were performedwith a luminometer (FLUOStar OPTIMA, BMC Laboratory, Offenburg, Germany), andthe experiments were performed in triplicate.

2.7. Alizarin red S staining

MDPC-23 cells were seeded on 35-mm dishes at a density of 1 � 105 cells/welland cultured in differentiation media for 2 weeks with or without PA-CM. Afterculture, the dishes were washed three times with PBS (pH 7.4) and fixed in a 4%paraformaldehyde solution for 20 min. Cells were stained with a 1% alizarin red S(SigmaeAldrich, St Louis, MO, USA) solution in 0.1% NH4OH at pH 4.2 for 20 min atroom temperature.

J.-H. Lee et al. / Biomaterials 32 (2011) 9696e97069698

2.8. Antibody production

Antibody against mCpne7 was produced by immunization of rabbit with thesynthetic peptides NH2-CVNAKYKQKKRNYKNSG-COOH (amino acid residues264e280 of Cpne7).

2.9. In vivo transplantation and histological analysis

HumanDPSCs (1�107 cells) weremixedwith 100mg hydroxyapatite/tricalciumphosphate (HA/TCP) ceramic powder (Zimmer Inc.,Warsaw, IN, USA)with orwithoutPA-CM (50 mg) on 0.5% fibrin-gel and were then transplanted subcutaneously intoimmunocompromised mice (NIH-bg-nu/nu-xid, Harlan Sprague Dawley, Indian-apolis, IN, USA). As a positive control, recombinant human bone morphogeneticprotein 2 (rhBMP-2, 5 mg, Cowellmedi, Busan, Korea) was used. Samples were ob-tained after 6 and 12 weeks, respectively, fixed in 4% paraformaldehyde, and decal-cified in a 10% EDTA (pH 7.4) solution at 4 �C. The samples were embedded in paraffinand stained with hematoxylin-eosin (H-E). The mandibles of mice at postnatal day 1were also decalcified in 10% EDTA (pH 7.4) and processed for immunohistochemistry.

For immunohistochemistry, the sections were incubated overnight at 4 �C withrabbit polyclonal DSP [21], BSP [21], type I collagen (SC-59772, Santa CruzBiotechnology), Nestin (MAB353, Millipore, Billerica, MA, USA), mCpne7, andhuman nuclei monoclonal antibodies (MAB1281, Millipore) at a dilution of 1:100.Secondary anti-rabbit or -mouse IgG antibody was incubated with the sections atroom temperature for 30 min, which were then reacted with the avidin-biotin-peroxidase complex (Vector Laboratory, Burlingame, CA, USA). Signals were con-verted using a diaminobenzidine kit (Vector Laboratory). Nuclei were stained withhematoxylin.

To measure the quantity of newly formed mineralized tissues in vivo, theanalysis LS starter program (OLYMPUS Soft Imaging Solution, Müster, Germany)

Fig. 1. Effects of ABCs on the expression of DSPP and BSP mRNA and protein in MDPC-23 an10 days, analyzed by quantitative real-time PCR. *p < 0.05. (B) Expression of DSPP mRNA du*p < 0.05. (C) Expression of BSP mRNA during the differentiation of MDPC-23 cells for 10differentiation of hDPSCs for 10 days, analyzed by quantitative real-time PCR. (E) DSP and Bby western blot. GAPDH was used as a control. (F) DSP and BSP protein expression during tha control.

was used. The rate was calculated as the percentage of mineralized tissue per totalarea.

For scanning electron microscopy (SEM), samples were fixed in 0.1 M cacodylatebuffer (pH 7.3) containing 2.5% glutaraldehyde for 30 min and in 0.1 M cacodylatebuffer (pH 7.4) containing 1% osmium tetroxide for 1 h. After rapid dehydrationthrough an ethanol gradient, critical point drying, and sputter coating with gold,cells were observed under SEM (S-4700, HITACHI, Tokyo, Japan).

2.10. Proteomic analysis of PA-CM

LC-MS/MS analysis was performed as previously reported [17]. Briefly, ABC-CM(day 0) and PA-CM (day 3) were stained with Coomassie blue. Protein bands wereexcised from Coomassie-stained gels and destained in 75 mM ammonium bicar-bonate/40% ethanol (1:1). The tryptic peptide mixture was eluted from the gels with0.1% formic acid. LC-MS/MS analysis was carried out using a Thermo Finnigan’sProteome Xworkstation LTQ linear ion trapMS (Thermo Electron, San Jose, CA, USA)equipped with NSI sources (San Jose, CA, USA). All MS/MS samples were analyzedusing Sequest version v.27, rev. 11 (ThermoFinnigan, San Jose, CA, USA), which wasset to search for the ipiMOUSE 3.49 database (IPI ver.3.49, 55,309 entries) assumingsemiTrypsin as the digestion enzyme. Scaffold (version Scaffold-01_07_00, Pro-teome Software Inc., Portland, OR, USA) was used to validate MS/MS-based peptideand protein identifications. After identifying the proteins, each dataset was used ina subtractive analysis by ProtAn X, an in-house analysis program.

2.11. Plasmid construction

Full-length mouse copine 7 (mCpne7, NM_170684) cDNAwas generated by PCR,subcloned into the PCR�2.1 T vector (Invitrogen), and ligated into the EcoRV and ApaIsites of the pCMV-tag2B over-expression vector or Hind III and EcoR V sites of the

d hDPSCs. (A) Expression of DSPP mRNA during the differentiation of MDPC-23 cells forring the differentiation of hDPSCs for 10 days, analyzed by quantitative real-time PCR.days, analyzed by quantitative real-time PCR. (D) Expression of BSP mRNA during theSP protein expression during the differentiation of MDPC-23 cells for 10 days, analyzede differentiation of hDPSCs for 10 days, analyzed by western blot. GAPDH was used as

J.-H. Lee et al. / Biomaterials 32 (2011) 9696e9706 9699

pcDNA3-HA vector. Hsp90ab1 (NM_008302), Hspa8 (NM_031165), and Lgals3bp(NM_011150) cDNAs were also cloned into pCMV-tag2B similar to mCpne7.

siRNAwas synthesized (Integrated DNATechnologies, San Diego, CA, USA) basedon the chosen 19-nt of mCpne7 (50-AGG GTG TTC TGA AAG AAA T-30). This siRNA-expression plasmid was prepared using the pSUPER-retro-neo-GFP retro virussiRNA-expression vector (OligoEngine, Seattle, WA, USA), according to the manu-facturer’s instructions.

2.12. Retroviral production and virus infection

Platinum-E cells were transfected with a pSUPER-retro-neo-GFP retrovirusvector that encodes mCpne7 siRNA, or an empty vector using Lipofectamine Plus�reagent (Invitrogen). After 48 h, viral supernatants were harvested and filtered usinga 0.45-mm syringe filter. MDPC-23 cells were infected with virus supernatant in thepresence of polybrene (10 mg/ml, SigmaeAldrich).

2.13. Immunofluorescence

To identify secreted mCpne7 transfer from ameloblasts to odontoblasts, ABCs(8 � 104 cells/well) after transfection with pcDNA.3.1-HA-mCpne7 were co-cultured with MDPC-23 cells (1 � 105 cells/well) in Transwell� dishes (CorningInc) for 1 day. After co-culture, HA-tagged mCpne7 transfected ABCs wereremoved and MDPC-23 cells were fixed with 4% paraformaldehyde for immu-nofluorescence staining. Cells were incubated overnight at 4 �C with primaryanti-HA antibody (COVANCE, Princeton, NJ, USA) and fluorescent-labeledsecondary anti-mouse antibody (Invitrogen). DAPI (40 , 6 diamidino-2-phenylindole, SigmaeAldrich) was used to identify cell nuclei (1:1000 dilu-tion). After washing, the cells were visualized using a confocal laser scanningmicroscope (Olympus, Tokyo, Japan).

To investigate the stemness of isolated hDPSCs, hDPSCs were also immuno-stained with -CD-44 (BD Pharmigen, San Jose, CA, USA) and STRO-1 primary anti-bodies (R&D Systems, Minneapolis, MN, USA) as described above.

Fig. 2. Effects of PA-CM on the DSPP promoter activity and mineralized tissue formation oamelogenin mRNAs in ABCs after 3 days in culture, analyzed by RT-PCR. GAPDH was used aswith or without ABCs-CM or KB-CM. Promoter activity was determined as luciferase light unempty expression vector) mean � S.D. of three separated experiments. *p < 0.05, **p < 0.0analyzed by alizarin red S staining. MDPC-23 cells were cultured for 2 weeks with or withouformed mineralized tissue in 6 and 12 weeks was analyzed by the LS starter program. (For intweb version of this article.)

2.14. Statistical analysis

The data were analyzed for statistical significance using a non-parametricManneWhitney test.

3. Results

3.1. Effects of ABCs on differentiation of MDPC-23 and hDPSCsin vitro

To investigate whether dental epithelial cells promote differ-entiation of odontoblastic MDPC-23 cells and induce odontoblastdifferentiation of hDPSCs, we co-cultured MDPC-23 cells or hDPSCswith ABCs for 10 days and analyzed the expression levels ofodontoblast differentiation markers by real-time PCR and westernblot. The expression levels ofDSPPmRNA, amarker of differentiatedodontoblasts, increased significantly in co-cultured MDPC-23 cellsor hDPSCs with ABCs compared to cells cultured alone from days3e10 (Fig. 1A,B). However, the expression levels of BSP mRNAdecreased in co-cultured MDPC-23 cells or hDPSCs with ABCscompared to cells cultured alone from days 3e10 (Fig. 1C,D). Theexpression levels of DSP protein increased from days 7e10, whileBSP protein decreased from day 7 in co-cultured MDPC-23 cells orhDPSCs with ABCs compared to cells cultured alone (Fig. 1E,F).Nestin expression increased approximately 4-fold in MDPC-23 cellsco-cultured with ABCs compared to MDPC-23 cells cultured aloneon day 7 (Suppl. Fig. 1A). Expression levels of type I collagen

f hDPSCs. (A) A microphotograph of isolated ABCs. (B) Expression of ameloblastin anda control. (C) DSPP promoter activity. Promoter activity was evaluated in MDPC-23 cellsits/protein and expressed as fold activation compared to that of control (transfection of1. (D) Effects of PA-CM on the mineralized nodule formation of MDPC-23 cells in vitrot PA-CM. (E) Effects of PA-CM on the mineralized tissue formation in vivo. Total area oferpretation of the references to colour in this figure legend, the reader is referred to the

J.-H. Lee et al. / Biomaterials 32 (2011) 9696e97069700

increased approximately 2-fold in MDPC-23 cells co-cultured withABCs compared toMDPC-23 cells cultured alone from days 3e7 anddecreased on day 10 (Suppl. Fig. 1B). These findings suggest thatABCs facilitate odontoblast differentiation of MDPC-23 cells andhDPSCs in vitro.

3.2. Effects of ABCs-CM on DSPP promoter activity in MDPC-23 cells

Initially, the primarily isolated ABCs showed a round epithelialcell-like appearance (Fig. 2A). ABCs after 3 days in cultureexpressed ameloblastin and amelogenin mRNA (Fig. 2B). Based onthese findings, we defined the cells from ABCs after 3 days inculture as preameloblasts (PA).

Generally, DSPP is regarded as a specific marker of odontoblastdifferentiation [22]. To select the most effective ABCs-CM forodontogenic differentiation, we evaluated the effects of variousABCs-CM (days 0, 3, and 7) on DSPP promoter activity in odonto-blastic MDPC-23 cells. DSPP promoter activity was increased in allABCs-CM compared to control (MDPC-23 cells alone). Interestingly,PA-CM, ABCs-CM after 3 days in culture, was most effective inincreasing DSPP promoter activity (Fig. 2C).

To confirm whether CM of other epithelial cells, other thandental epithelial cells (ABCs), promotes odontoblast differentiation,DSPP promoter activity was measured after treatment of MDPC-23cells with human KB-CM. However, KB-CM had no influence onDSPP promoter activity (Fig. 2C). These findings suggest that

Fig. 3. Histological finding of dentin/pulp-like tissue formation in hDPSCs. Human DPSCs wa 0.5% fibrin-gel and transplanted subcutaneously into immunocompromised mice. (A) Pulp-black boxed A. (C) Higher magnification of dotted boxed A. (D) Mineralized tissue formationboxed D. Arrows indicate osteocytes in mineralized matrix. (F) Higher magnification of dohDPSCs group. (H) Higher magnification of black boxed G. Arrows indicate odontoblast pArrows indicate odontoblast processes of differentiating odontoblasts. (AeI): H-E staining.hDPSCs group in 12 weeks. White arrows indicate odontoblast processes of differentiating

odontoblast differentiation is promoted only by substancessecreted by the dental epithelium.

3.3. Effects of PA-CM on mineralization of MDPC-23 cells in vitroand mineralized tissue formation of hDPSCs in vivo

To identify the effects of PA-CM onmineralized nodule formation,odontoblastic MDPC-23 cells were cultured in differentiationmedium for 2 weeks with or without PA-CM treatment, and theformation of mineralized nodules was evaluated by alizarin red Sstaining. In microscopic observations, mineralized nodules weredetected after day 3 in the PA-CM-treated group and was markedlyincreased until day 14, while the MDPC-23 cells only group began todemonstrate mineralized nodules on day 7 (Fig. 2D). These datasuggest that PA-CM promotes mineralization of odontoblasts in vitro.

To ascertain the stemness of isolated hDPSCs by explant culture,the cells were immunostained with STRO-1 and CD-44, both stemcell markers. Isolated hDPSCs expressed both STRO-1 and CD-44(Suppl. Fig. 2).

Based on the findings from these in vitro experiments, wetransplanted hDSPCs into the subcutaneous tissues of immuno-compromised mice with or without PA-CM in order to investigatethe effects of PA-CM on the differentiation of hDPSCs and miner-alized tissue formation in vivo. Recombinant hBMP-2 was used asa positive control, because it is well known that BMP-2 inducesmineralized tissue formation of hDPSCs [23,24]. Twelveweeks after

ere mixed with 100 mg HA/TCP with or without PA-CM (50 mg) or rhBMP-2 (5 mg) onlike tissue formation in 12 weeks in the hDPSCs only group. (B) Higher magnification ofat 12 weeks in the rhBMP-2-treated hDPSCs group. (E) Higher magnification of blacktted boxed D. (G) Dentin/pulp complex formation in 12 weeks in the PA-CM-treatedrocesses of differentiating odontoblasts. (I) Higher magnification of dotted boxed G.(J) Scanning electron micrograph of differentiated odontoblast in the PA-CM-treatedodontoblasts. Scale bar ¼ 20 mm.

J.-H. Lee et al. / Biomaterials 32 (2011) 9696e9706 9701

transplantation, all three groups generated mineralized tissues onthe border of HA/TCP. When areas of formed mineralized tissue ofeach groupweremeasured, rhBMP-2 induced themostmineralizedtissue formation 6 weeks after transplantation, and PA-CM formedapproximately one-half the levels of mineralized tissue comparedto rhBMP-2 did. However, 12 weeks after transplantation, the PA-CM-treated group formed similar amounts of mineralized tissuesas the rhBMP-2-treated group did (Fig. 2E).

3.4. Effects of PA-CM on odontogenic differentiation of hDPSCsin vivo

Odontoblasts form mineralized dentin and a palisade layerbetween the pulpedentin interface [25]. Ex vivo expanded adultdental pulp stem cells can generate a dentin/pulp-like complex byin vivo transplantation without any other treatment [7]. Consistent

Fig. 4. Immunohistochemical phenotype of the cells. (A) Dentin sialoprotein (DSP) expressweakly in differentiating odontoblasts. (B) DSP expression in 12 weeks in the rhBMP-2-treexpression in 12 weeks in the PA-CM-treated hDPSCs group. DSP protein (arrows) was strongroup. (E) Nestin expression in the rhBMP-2-treated hDPSCs group. (F) Nestin expression in tformed dentin matrix near odontoblasts. (G) Type I collagen (Col I) expression in the hDPexpression in the PA-CM-treated hDPSCs group. (J) BSP expression in the hDPSCs only groexpressed in mineralized matrix and adjacent cells. (L) BSP expression in the PA-CM-treatedstained with hematoxylin. (N) Immunohistochemistry for human nuclei monoclonal antibod(O) PCR for human Alu sequence amplification. White arrowhead indicates amplified humgenomic DNA, H: positive control using human genomic DNA, P: pulp genomic DNA, M: M

with these results, hDPSCs generated a dentin/pulp-like structurelined with odontoblast-like cells that surround a pulp-like tissue inthe hDPSCs only group (Fig. 3AeC). On the other hand, a few cells inthe rhBMP-2-treated group were entrapped in the mineralizedmatrix, possessing osteocyte characteristics (Fig. 3DeF). In the PA-CM-treated group, however, the cells exhibited odontoblast char-acteristics with palisade arrangement along the pulp and typicalodontoblast processes in the dentin matrix (Fig. 3GeI). Under SEM,the odontoblast-like cells appeared to have been elongated and hadodontoblast processes (Fig. 3J). These results suggest that incontrast to the hDPSCs only group, dentin/pulp-like complexformation was more extensive in the PA-CM-treated group and thehistological architecture of the tissue generated in the PA-CM-treated group resembles the morphology of the normal den-tinepulp complex more closely than tissues generated in the othergroups.

ion in 12 weeks in the hDPSCs only group. DSP protein (open arrowheads) expressedated hDPSCs group. DSP protein was hardly detected in differentiating cells. (C) DSPgly expressed in differentiating odontoblasts. (D) Nestin expression in the hDPSCs onlyhe PA-CM-treated hDPSCs group. Nestin protein (arrows) was clearly detected in newlySCs only group. (H) Col I expression in the rhBMP-2-treated hDPSCs group. (I) Col Iup. (K) BSP expression in the rhBMP-2-treated hDPSCs group. BSP (arrowheads) washDPSCs group. (M) Pre-immune serum control showed negative staining. Nuclei wereies. Black arrows indicate human nuclei immunostaining of hDPSCs. Scale bar ¼ 50 mman Alu sequence in human dental pulp stem cells (Ne: negative control using mouseolecular marker).

J.-H. Lee et al. / Biomaterials 32 (2011) 9696e97069702

To inspect the dentin-like mineralized tissue identified bymorphology and observe the expression of various dentin matrixproteins, we performed immunohistochemistry. BSP and DSP areregarded as specific markers of osteoblast and odontoblast differ-entiation, respectively [22]. DSP expressionwas observedweakly inthe hDPSCs only group but barely in the rhBMP-2-treated group(Fig. 4A,B). However, in the PA-CM-treated group, the expression ofDSP protein was clearly detected in odontoblast-like cells thatcontact the formed matrix (Fig. 4C). Nestin is expressed in theodontoblast processes of fully differentiated odontoblasts [26]. Inthe hDPSCs only and rhBMP-2-treated groups, nestin was stainedweakly in the mineralized matrix (Fig. 4D,E). In contrast, nestinwasclearly observed in dentin-like mineralized tissue containingodontoblast processes in the PA-CM-treated group (Fig. 4F). In boneand dentin, type I collagen is the most abundant collagenousprotein [22]. As expected, type I collagenwas expressed in the cellsand matrix of all groups (Fig. 4GeI). In the hDPSCs only and PA-CM-treated groups, BSP was hardly expressed, whereas in the rhBMP-2-treated group, BSP was detected in both the cells and matrix(Fig. 4JeL). These findings indicate that the cells in the PA-CM-treated group resemble the characteristics of normal odontoblastsmore closely than other groups and that consequently PA-CMinduces the odontoblast differentiation of hDPSCs in vivo.

Anti-human nuclei monoclonal antibody was used to confirmthe origin of mineralized tissue by immunohistochemistry. Humannuclei specifically immunostained in cells embedded in formed

Fig. 5. Proteomic analysis of PA-CM. (A) Ascertainment of two CMs by Coomassie blue stainin3 (PA-CM). (M, molecular marker). (B) Classification of overlapping proteins between ABCs-CCellular distributions of total 117 proteins. (D) Functional classification of total 117 proteins. (to the web version of this article.)

mineralized tissue (Fig. 4N). In addition, it was confirmed that thehuman-specific Alu sequence was amplified in genomic DNA ofmineralized tissues on the slide (Fig. 4O), thereby suggesting thatmineralized tissue is formed by hDPSCs.

3.5. Proteomic analysis of PA-CM

PA-CM was found to promote odontoblast differentiation andmineralized tissue formation of hDPSCs in vitro and in vivo.Therefore, to investigate what protein in PA-CM induces odonto-blast differentiation and promotes mineralized tissue formation,we analyzed ABCs-CM on day 0 and day 3 (PA-CM) using LC-MS/MS. First, proteins of various sizes were detected in ABCs- andPA-CM by SDS-PAGE (Fig. 5A). Second, 113 proteins were found: 69on day 0 and 71 on day 3. Among them, 23 proteins were known toco-exist on day 0 and day 3 (Fig. 5B and Supplementary Table 2.).Because of protein localization analysis, 18% of proteins were foundto exist in the cytoplasm and 15% in the extracellular matrix(Fig. 5C). Additionally, from functional analysis, 20% of proteinswere found to regulatemetabolic process and 18% participate in cellgrowth (Fig. 5D).

3.6. Functional characterization of Cpne7 protein

Among the 23 proteins found between day 0 and day 3 CM, fourfactors with peptide numbers greater than 1.0 by LC MS/MS

g. Thirty mg of CM were loaded for the staining in each lane. 1: day 0 (ABCs-CM), 2: dayM (day 0) and PA-CM (day 3). Twenty three proteins co-existed on day 0 and day 3. (C)For interpretation of the references to colour in this figure legend, the reader is referred

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analysis were chosen. To confirm their expression in ABCs,expression levels ofmCpne7, Hspa8, Lgals3bp, and Hsp90ab1mRNAswere investigated by RT-PCR. As a result, four genes were expressedin ABCs (data not shown). To investigate whether these genesaffected odontoblast differentiation, we cloned the genes, co-transfected them with DSPP promoter in MDPC-23 cells, andmeasured DSPP promoter activity. Interestingly, only mCpne7increased DSPP promoter activity approximately by 3.5-foldcompared to the control (MDPC-23 cells only), whereas the othergenes did not show any effect (Fig. 6A).

mCpne7 protein was readily detected in intracellular compart-ments and the CM collected from serum-free cultures in HEK293 Tcells after transfection of a HA-tagged mCpne7 by western blotusing anti-HA antibody (Fig. 6B). Next, to examine whether themCpne7 secreted by preameloblasts concentrated in differentiatingodontoblasts, expression ofmCpne7 in odontoblastic MDPC-23 cellswas investigated by immunofluorescence using an anti-HA anti-body after co-culture with ABCs that were transfected with a HA-tagged mCpne7 construct. As expected, ABCs-secreted diffusiblemCpne7 was concentrated in MDPC-23 cells (Fig. 6C). On day 1, themandibular first molar was at the advanced bell stage. mCpne7protein was localized in differentiating odontoblasts (Fig. 6D).

Fig. 6. Cellular localization and migration of mCpne7 in ameloblasts and odontoblasts. (A) Dand heat shock cognate 71-kDa protein (Hspa8) and Lgals3bp were cloned into the pCMV-tagpromoter activity was measured. *p < 0.05, **p < 0.01. (B) Expression of HA-tagged mCpne7Empty vector, WCL: Whole cell lysate, CM: Conditioned medium. (C) Migration of mCpne7mCpne7 was transfected into ABCs and detected in MDPC-23 using an anti-HA antibody dpostnatal day 0. Higher magnification (lower panel, immunohistochemistry) of boxed up(arrows). Am: ameloblast, Od: odontoblast, P: pulp.

These results suggest that mCpne7 may act as a diffusible signalingmolecule that regulates odontoblast differentiation through epi-thelialemesenchymal interactions.

To determine whether mCpne7 have important roles in odon-toblast differentiation, we measured mRNA levels of odontoblastdifferentiation markers by real-time PCR after either over-expres-sion or inactivation ofmCpne7 inMDPC-23 cells. Mouse Cpne7 over-expression increased the expression levels of odontoblast differ-entiation markers, DSPP, nestin, Col I, Runx2, ALP, and OC, comparedto control (Fig. 7A,B). In contrast, mRNA expression levels ofodontoblast differentiation markers were decreased after mCpne7inactivation using shRNA (Fig. 7C,D). Collectively, these resultssuggest the mCpne7 as a new candidate gene that may haveimportant roles in odontoblast differentiation.

4. Discussion

The differentiation of odontoblasts from the undifferentiatedectomesenchyme of the dental papilla is initiated by an organiza-tion of the cells of the inner dental epithelium during toothformation [6]. Tissue culture experiments have established thatsuch an odontogenic inductive interaction could take place across

SPP promoter activity. Mouse Cpne7 (mCpne7), heat shock protein 90ab1 (Hsp90ab1),2B vector and co-transfected them with DSPP promoters into MDPC-23 cells, and DSPPprotein in HEK293T cells after transfection with mCpne7, analyzed by western blot. EV:protein from ABCs to MDPC-23 cells, examined by immunofluorescence. HA-tagged

uring the co-culture. (D) Expression of mCpne7 during mouse tooth development onper panel (H-E staining). Cpne7 protein was localized in differentiating odontoblasts

Fig. 7. Effects of mCpne7 over-expression and inactivation on the expression of odontoblast differentiation markers. (A) Confirmation of mCpne7 over-expression in MDPC-23 cells.mCpne7 was transfected into MDPC-23 cells and its expression levels were analyzed by quantitative real-time PCR. (B) Effects of mCpne7 over-expression on the expression ofodontoblast differentiation markers in MDPC-23 cells analyzed by quantitative real-time PCR. (C) Confirmation of mCpne7 inactivation in MDPC-23 cells. mCpne7 shRNA wastransfected into MDPC-23 cells and its expression level was analyzed by quantitative real-time PCR. (D) Effects of mCpne7 inactivation on the expression of odontoblast differ-entiation markers in MDPC-23 cells analyzed by quantitative real-time PCR.

J.-H. Lee et al. / Biomaterials 32 (2011) 9696e97069704

a thin, porous filter between developing ameloblasts and odonto-blasts [6,27]. Therefore, the search for diffusible soluble factors inameloblast-lineage cells responsible for inducing the differentia-tion of odontoblasts became the mission for dental researchers.

Postnatal DPSCs in the tooth pulp contain undifferentiatedectomesenchymal cells that can differentiate into odontoblasts [7].To date, several studies [28e30] have shown that various factorssuch as rat incisor tooth germ cells and acid-soluble tooth proteinsregulate the fate of DPSCs. It was also reported that mesenchymalstem cells can be reprogrammed into odontoblast lineages and re-exhibit the embryonic events of tooth development under ABCsinduction [31]. However, two fundamental issues must still bedetermined to better understand odontoblast differentiation bydental epithelial factors. First, which developmental stage of dentalepithelial cells secretes the most effective factors for odontogenicdifferentiation of DPSCs? Second, which factors are released fromdental epithelial cells and which are responsible for odontogenicdifferentiation? Answers to these questions are important tounderstand normal odontoblast differentiation but also to repairpathologic dentin defects using postnatal DPSCs. In the presentstudy, we evaluated the effects of dental ABCs-CM in variousdevelopmental stages of ameloblast differentiation (days 0, 3, and7) and non-dental KB-CM on odontoblast differentiation throughevaluation of DSPP promoter activity in odontoblastic MDPC-23cells. PA-CM, CM from ABCs after 3 days in culture, induced theodontoblast differentiation of hDPSCs in vivo and in vitro, but KB-CM from non-dental epithelial cells did not have any effects onodontoblast differentiation. After proteomic analysis, PA-CM wasfound to contain 71 proteins. Of the identified proteins, copine 7was discovered as a new candidate gene involved in odontoblastdifferentiation and dentin regeneration.

Human DPSCs can be isolated from the pulp by variousmethods, including enzyme dissociation [32] and explant culture[33]. In the present study, we used explant culture because it

allows the recovery of a population of dental mesenchymal stemcells, which show notable proliferation potential, multipotency,and a long lifespan [33]. Consistent with our data, it was also re-ported that explant culture comprises homogenous cell pop-ulations expressing stem cell markers, such as STRO-1, CD146,CD34, and CD45.

Previous data demonstrated that odontoblast differentiation isregulated by various factors such as DMP-4 [34], nerve growthfactor [35], and BMP-2 [36]. hDPSCs transfected with BMP-2especially showed enhanced DSPP expression in vitro, and miner-alized tissue formation increased in vivo [24,36]. In the presentstudy, we considered the possibility that small amounts of BMP inPA-CM could affect hDPSCs differentiation. Therefore, we blockedBMP activity in PA-CM with the extracellular BMP antagonist,noggin [37], and evaluated DSPP promoter activity. There were nochanges in DSPP promoter activity after noggin treatment in PA-CM(Suppl. Fig. 3A), suggesting that PA-CM affects DSPP promoteractivity by factors that are irrelevant to BMP-2.

DSP has previously been known to be expressed mainly in thedentinal tubule of peritubular dentin rather than in the dentinmatrix [38]. In contrast, BSP is a non-collagenous protein in bone,dentin, cementum, and calcified cartilage tissues. BSP is approxi-mately 10-folds more abundant in bone than in dentin, whereasDSPP is 400-fold more abundant in dentin than in bone [22]. In thepresent study, BSP levels significantly higher than DSP in themineralized tissue generated in the rhBMP-2-treated group, whilemineralized tissue formed in the PA-CM-treated group displayedthe opposite tendency. In essence, these data suggest that PA-CMpromotes the induction of more odontogenic characteristics ofhDPSCs than do rhBMP-2. However, it was demonstrated thatnatural dentin releases inducers of odontoblast differentiation,including BMP-2, which enhance the expression of markers ofdifferentiation in stem cells from exfoliated deciduous teeth [39].Therefore, further investigation is needed to clarify the exact roles

J.-H. Lee et al. / Biomaterials 32 (2011) 9696e9706 9705

of dentin-derived BMP-2 and preameloblast-derived factors onodontoblast differentiation of hDPSCs.

Previously, differentiation of hDPSCs into specific cell lineageswas thought to be determined by the local microenvironmentincluding growth factors, receptor molecules, signaling molecules,transcription factors, and extracellular matrix proteins [28,40].Accordingly, hDPSCs in an abnormal pulp environment, such asreparative dentinogenesis by dental carries and injury mightdifferentiate into osteoblast-like cells and produce osteodentin thatexhibits similar characteristics as bone [41]. In such cases, we arereminded that undifferentiated ectomesenchymal cells in adultpulp are exposed to all factors required for odontoblast differenti-ation except for the final epithelial influence. Conversely, this factsuggests that dental epithelial induction such as by PA-CM in thepresent study, for instance, is indispensible for hDPSCs odontoblastdifferentiation.

Because of proteomic analysis using LC-MS/MS, various proteinswere identified in PA-CM. Among proteins that were identifiedboth at proliferation (day 0) and early ameloblast differentiation(day 3), titin, alpha-actinin, and transketolase that take part incellular structure and metabolism, increased at the early amelo-blast differentiation stage. The giant sarcomeric protein titin isgenerally known as a provider of elasticity to striated muscles andnuclear protein involved in the control of chromosome dynamics,gene expression, signal transduction, and cell proliferation inhuman, Drosophila melanogaster, and Caenorhabditis elegans non-muscle cells [42]. Alpha actinin-1 is an actin-binding and cross-linking protein that is expressed in virtually all cells and regulatesacid-sensing ion channels [43]. In addition, thrombospondin1, oneof the proteins that decrease in the early ameloblast differentiationstage, is known to inhibit bone mineralization by osteoblasts tomaintain bone homeostasis [44]. Our analyzed results show thatlevels of secreted proteins related tometabolic processes increased,but those related to bone decreased according to ameloblastdifferentiation.

In the present study, periostinwas identified in PA-CM. Periostinis expressed first in the teeth and periodontal ligament and isknown to take part in integrity maintenance of the periodontalligament [45,46]. Recently, it was reported that periostin-null micedisplayed an increase in dentin mass. In addition, the null pulpspace gradually became narrower and narrower with age [47]. Toinvestigate the effects of periostin on odontoblast differentiationand dentin formation in hDPSCs, we evaluated the effects of peri-ostin neutralization using its antibody on the expression of DSPprotein that regulates the mineralized matrix formation of dentin.Consistent with a previous report [47], DSP protein was increasedslightly in the periostin-neutralized PA-CM during MDPC-23 cellculture (Suppl. Fig. 3B). These findings suggest that dentin forma-tion is promoted by substances in PA-CM rather than periostin.

Generally, copines are evolutionarily conserved proteins presentin protozoa, plants, nematodes, and mammals. They were firstidentified in preparations of Ca2þ-dependent, phospholipid-binding proteins in Paramecium [48]. Copine proteins havedistinct domains with well-studied structural homology, althoughthe biochemical activities utilized for their biological roles are notwell studied. The copine proteins are characterized by two C2domains at the N terminus and a VonWillebrand A domain at the Cterminus [49]. Copine7 was first identified and characterized insporadic breast cancer [50]. In addition, when Ca2þ influx exists inthe cell, cell membrane-localized copine7 shifts to the cytoplasmand nucleus [51]. The role of Ca2þ is important for the differentia-tion of odontoblasts and dentin mineralization. In the presentstudy, copine7 was secreted by preameloblasts and concentrated indifferentiating odontoblasts. With the onset of dentin formation,copine7 is no longer detectable in the differentiating ameloblasts.

Mouse Cpne7 over-expression increased the expression levels ofodontoblast differentiation markers in MDPC-23 cells, while inac-tivation of mCpne7 decreased them. These results suggest Cpne7have important roles as a diffusible signaling molecule regulatesodontoblast differentiation through epithelialemesenchymalinteractions. However, further investigation is needed to clarify themechanisms by which Cpne7 modulates odontoblastdifferentiation.

5. Conclusions

Our findings suggest that PA-CM induces the odontogenicdifferentiation of hDPSCs and promotes dentin formation in vivoand in vitro. In addition, we analyzed PA-CM by proteomicmethods,by finding and characterizing 113 types of proteins. Of the identifiedproteins, Cpne7 is a new candidate that is involved in odontoblastdifferentiation. Conclusively, hDPSCs in combination with PA-CMcould be valuable in not only odontoblast differentiation but alsorepair and regeneration of the dentinepulp complex.

Acknowledgments

This study was supported by a grant of the Korea HealthcareTechnology R&D Project, Ministry of Health & Welfare, Republic ofKorea (A101578)

Appendix. Supplementary material

Supplementary material associated with this article can befound, in the online version, at doi:10.1016/j.biomaterials.2011.09.007.

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