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Comparison of human dental follicle cells and human periodontal ligament cells for dentin tissue regeneration

Aim: To compare the odontogenic potential of human dental follicle cells (DFCs) and periodontal ligament cells (PDLCs). Materials & Methods: In vitro and in vivo characterization studies of DFCs and PDLCs were performed comparatively. DFCs and PDLCs were subcutaneously implanted into the dorsum of mice for 8 weeks after combined with treated dentin matrix scaffolds respectively. Results: Proteomic analysis identified 32 differentially expressed proteins in DFCs and PDLCs. Examination of the harvested grafts showed PDLCs could form the dentin-like tissues as DFCs did. However, the structure of dentin tissues generated by DFCs was more complete. Conclusion: PDLCs could contribute to regenerate dentin-like tissues in the inductive microenvironment of treated dentin matrix. DFCs presented more remarkable dentinogenic capability than PDLCs did. Keywords: dental follicle cells • dentin regeneration • odontogenic differentiation • periodontal ligament cells • treated dentin matrix

In the field of tooth engineering, efforts have been made to regenerate tooth structures using stem cells from different types of dental tissues, such as periodontal ligament [1] , dental follicle [2] , dental pulp [3] , root apical papilla  [4] and exfoliated deciduous teeth [5] . Although these cells have been widely investigated, it has not been acknowledged which is the optimum source of seeding cells for tooth-regeneration applications. Dental follicle is a loose ectomesenchymederived connective tissue sac surrounding the unerupted tooth germ, which plays an essential role in the development of periodontium. Previous evidences have suggested that stem cells are present in the dental follicle at different stages of tooth development [2,6] , and possess the capacity for self-renewal and the potential to differentiate into a variety of cell types, including cementoblasts/osteocytes, chondrocytes, adipocytes and neural cells  [7,8] . Our recent study manifested the feasibility of rat dental follicle cells (DFCs) used as seeding cells in tooth root construction  [9] . Additionally, DFCs are easily

10.2217/RME.15.21 © 2015 Future Medicine Ltd

accessible from clinically discarded third molar extractions and therefore a candidate source for dental tissue regeneration. Periodontal ligament is a specialized connective tissue located between the tooth root and alveolar bone, and is responsible to maintain homeostasis of the tooth-supporting structure, the periodontium. Previous studies indicate that periodontal ligament cells (PDLCs) consist of heterogeneous cell populations at different stages of differentiation and lineage commitment [10–12] . Seo et al. reported that PDLCs contain stem cells that are capable of regenerating cementum/periodontal ligament-like structure in vivo and that the application of PDLCs may be effective for periodontal regenerative therapy [1] . According to the classic tooth development theory, DFCs are the precursors of PDLCs, but the factors and mechanisms that regulate DFCs differentiation remain undefined. At the late bell stage of tooth development, DFCs come into contact with dentin after the fracture of Hertwig’s

Regen. Med. (2015) 10(4), 461–479

Ye Tian‡,1,2,3, Ding Bai‡,1,3, Weihua Guo1,2,4, Jie Li2,5, Jin Zeng1,2, Longqiang Yang1,2,4, Zongting Jiang1,2, Lian Feng1,2, Mei Yu1,2 & Weidong Tian*,1,2,6 1 State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, P.R. China 2 National Engineering Laboratory for Oral Regenerative Medicine, West China Hospital of Stomatology, Sichuan University, Chengdu, P.R. China 3 Department of Orthodontics, West China School of Stomatology, Sichuan University, Chengdu, P.R. China 4 Department of Pedodontics, West China School of Stomatology, Sichuan University, Chengdu, P.R. China 5 College of Life Science, Sichuan University, Chengdu, P.R. China 6 Department of Oral & Maxillofacial Surgery, West China School of Stomatology, Sichuan University, Chengdu, P.R. China *Author for correspondence: Tel.: +86 28 8550 3499 Fax: +86 28 8550 3499 drtwd@ sina.com ‡ Authors contributed equally

part of

ISSN 1746-0751

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Research Article  Tian, Bai, Guo et al. epithelial root sheath, initiating the development of periodontium  [13] . Hence, dentin is a pivotal microenvironment for the differentiation of DFCs. Previous studies have demonstrated the existence of the bioactive odontogenesis-associated molecules in dentin matrix [14,15] . Moreover, our recent studies found that DFCs obtained the capacity to form dentin tissues when exposed to the inductive environment of the treated dentin matrix (TDM) [16–18] . Whether the progeny cells of DFCs could also be capable of performing the dentinogenesis and what differences may exist in the differentiations of DFCs and PDLCs under the same microenvironment are interesting questions in terms of developmental biology, but have not been explored yet. Therefore, this study is designed to compare biological characteristics of DFCs and PDLCs, including cell morphology, cell proliferation, clone-forming ability, multipotential differentiation and proteome, and to investigate their odontogenic differentiation under the inductive microenvironment of dentin matrix in vitro and in vivo. These findings would expand our understanding on the odontogenic potential of PDLCs and provide evidence for the use of alternative cell sources in tooth regeneration.

Materials & methods Isolation & culture of DFCs & PDLCs

Impacted third molars and healthy premolars, which were extracted from patients (n = 10, 16–18 years of age) for orthodontic reasons in the West China Hospital of Stomatology, were used for cell isolation. All experiments were conducted in accordance with the ethical protocol approved by the Committee of Ethics of the Sichuan University and written informed consent was obtained from all the subjects and the guardians on behalf of the teenagers enrolled in this study. DFCs and PDLCs were obtained from dental follicles of impacted third molars and root scrapings of premolars, respectively [6,19] , and cultured in alpha minimal essential medium (α-MEM; Hyclone, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone, USA) in a humidified atmosphere at 37°C with 5% CO2. Cell culture medium was refreshed every two days. Cultured DFCs and PDLCs in passage numbers from two to six were used for all the experiments. Both primary cultured cells were subjected to immunofluorescent staining for cytokeratin-14 (CK14; Abcam, UK) and vimentin (Thermo Scientific, MA, USA) using the methods described in the literature [20] . Morphological observation Light microscope

DFCs and PDLCs at the third passage were seeded onto six-well plate separately, further cultured for one day, and then observed and photographed under a phase-contrast inverted microscope (Nikon, Japan). Transmission electron microscopy

DFCs and PDLCs were trypsinized and centrifuged (3000 g, 5 min, 4°C) to form pellets separately, then fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) for 1 h at room temperature and postfixed in aq. 2% (v/v) osmium tetroxide for a further 1 h. After dehydrated in an ethanol series (50, 70, 95 and 100%), the cells were embedded in Epon 812 resin. Ultrathin sections were made and stained with uranyl acetate and lead citrate. The sections were observed with a transmission electron microscopy (TEM; JEM 100 SX, Jeol, Japan). The experiment was repeated at least three times. Cell proliferation assay

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Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Tokyo, Japan) was used to quantitatively evaluate the proliferative activity of the DFCs and PDLCs. The cells were cultured in a 96-well plate at a density of 1×104 cells/ml for 7 days. 100 μl α-MEM with 10% FBS containing 10 μl CCK-8 were added to each well every day. After incubation at 37°C for 3 h, 100 μl of the above solution was taken from each sample and added to one well of a 96-well plate. Six parallel replicates were

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Comparison of human DFCs and PDLCs for dentin tissue regeneration 

prepared and the absorbance at 450 nm was determined using a spectrophotometer (Thermo Scientific, MA, USA). The test was repeated at least three times. Colony-forming assay

A total of 1×103 DFCs and PDLCs were seeded into a 100 mm cell culture dish (Costar, MA, USA) and cultured respectively. After incubation for 10 days, the cells were washed twice in phosphate buffered saline (PBS) after fixed in 4% paraformaldehyde for 10 min and stained with 1% toluidine blue. After washed with distilled water, the cells were observed and photographed under a phase-contrast inverted microscope (Leica Optical, Wetzlar, Germany). Aggregates of 50 or more cells were scored as colonies. The experiment was repeated at least three times. Flow cytometric surface marker expression analysis

DFCs and PDLCs were identified with their cell surface antigen markers by flow cytometry. Briefly, the cells at passage 3 were trypsinized and incubated with mouse monoclonal antibodies directed to the following human antigens: either conjugated with fluorescein isothiocyanate (CD44, CD90, CD31, CD34, CD45 and CD24) or with phycoerythrin (CD146, CD105 and CD29). Then the cells were washed with PBS and then suspended in PBS for analysis. All these antibodies were purchased from BD Biosciences (CA, USA). Flow-cytometry was carried out using the Beckman Coulter Cytomics FC 500 MPL system (Beckman Coulter, CA, USA). At least 1×105 cells were analyzed for each experiment. For STRO-1 staining, the cells were incubated for 30 min with antibody against STRO-1 (mouse IgM; R&D, MN, USA). The cells were then incubated with a secondary antibody (fluorescein isothiocyanate conjugated goat antimouse IgM; Vector, CA, USA) for 30 min. As anisotype control, antimouse IgM (Beckman Coulter, USA) was substituted for the primary antibody. In vitro multipotent differentiation Osteogenic differentiation

A total of 1×105 DFCs and PDLCs were seeded into each well of a six-well plate separately. At 80% confluence, the cells were cultured in osteogenic medium containing 10% FBS, 5 mM L-glycerophosphate (Sigma, USA), 100 nM dexamethasone (Sigma, USA), and 50 μg/ml ascorbic acid (Sigma, USA). Control cultures were maintained in α-MEM medium without osteogenic supplements. The medium was changed every 2 days. After 15 days of culture, cells were washed three times in PBS after fixed in 4% paraformaldehyde for 10 min and then incubated in 0.1% alizarin red solution (Sigma, USA) in TriseHCl (pH 8.3) at

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37°C for 30 min. After washed three times in PBS, cells were observed and photographed under a light microscope (Nikon, Japan). The nodule formation was quantitatively measured using image analysis system (Image-Pro Plus 5.0; Media Cybernetics, MD, USA). Adipogenic differentiation

DFCs and PDLCs were seeded in six-well plates at a density of 1×105 cells/well. At 80% confluence, the cells were cultured in adipogenic medium comprising 10% FBS, 2 mM insulin (Sigma, USA), 0.5 mM iso-butylmethylxanthine (Sigma, USA) and 10 nM dexamethasone (Sigma, USA). The medium was refreshed every 2 days. After 4 weeks of culture, the cells were washed three times in PBS after being fixed in 4% paraformaldehyde for 10 min and then incubated in 0.3% Oil Red O (Sigma, USA) solution for 15 min. After washed three times in PBS, cells were observed and photographed under a phase-contrast inverted microscope (Nikon). The lipid areas were quantitatively measured using image analysis system (Image-Pro Plus 5.0, USA). Proteomic analysis of DFCs & PDLCs 2D Electrophoresis and image analysis

Cells (3 × 107) were lysed in 1 ml lysis buffer, containing 7 M urea (Bio-Rad, CA, USA), 2 M thiourea (Sigma, USA), 4% 3-[(3-cholamidopropyl) dimethylammonio]-1- propanesulfonate (CHAPS; Bio-Rad, USA), 100 mM Dithiothreitol (DTT; Bio-Rad, USA), 0.2% ampholyte (pH 3–10; Bio-Rad, USA) and protease inhibitor cocktail 8340 (Sigma, USA). Samples were kept on ice and sonicated in six cycles, each consist of 5 s sonication, followed by a 10 s break. After centrifugation (14,000 rpm, 30 min, 4°C), supernatants were collected and the protein concentrations were determined using the RC DC Protein Assay Kit (Bio-Rad, USA). Protein samples (2.5 mg, 350 μl) were applied to immobilized pH gradient (IPG) strip (17 cm, pH 3–10; Bio-Rad, USA) using a passive rehydration method. After 16 h of rehydration, the strips were transferred to a PROTEAN® IEF Cell (Bio-Rad, USA). Isoelectric Focusing (IEF) was performed as follows: 250 V for 30 min, linear; 1000 V for 1 h, rapid; linear ramping to 10,000 V for 5 h, and finally 10,000 V for 6 h. Then the strips were equilibrated in equilibration buffer (25 mM Tris-HCl, pH 8.8, 6 M urea, 20% glycerol, 2% SDS and 130 mM DTT) for 15 min, followed by another 15 min in the equilibrium buffer while DTT was replaced with 200 mM iodoacetamide. The second dimension was performed using 12% SDS-PAGE at 30 mA constant current per gel. The resultant gels were stained with Coomassie Brilliant Blue R-250 (Merck, Darmstadt, Germany) and scanned using GS-800 scanner (Bio-Rad, USA). The maps were analyzed by

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Research Article  Tian, Bai, Guo et al. PD-Quest software Version 8.0 (Bio-Rad, USA). Spots of each gel was normalized as percentage of total spots and evaluated in terms of O.D. Only those changed consistently and significantly (>1.5-fold) were selected for mass spectrometry (MS) analysis. In-gel digestion

In-gel digestion was performed using MS grade trypsin gold (Promega, WI, USA) according to the manufacturer’s instructions. Briefly, spots were cut out of the gel (1–2 mm diameter) using a razor blade, and destained twice with 100 mM NH4HCO3/50% acetonitrile (ACN; Sigma, USA) at 37°C for 45 min in each treatment. After dehydration with 100% ACN and drying, the gels were preincubated in 10–20 μl trypsin solution (10 ng/μl) for 1 h. Then adequate digestion buffer (40 mM NH4HCO3/10% ACN) was added to cover the gels, followed by incubation at 37°C overnight (12–14 h). The tryptic peptides were extracted with Milli-Q water, followed by twice extraction with 50% ACN/5% trifluoroacetic acid (Sigma, USA) for 1 h each time. The combined extracts were dried in a speed-VAC concentrator (Thermo Scientific, USA) at 4°C. The samples were then subjected to MS. Matrix-assisted laser desorption ionization timeof-flight mass spectrometry

Matrix-assisted laser desorption ionization time-offlight mass spectrometry (MALDI-TOF-MS) were performed on Bruker ultrafleXtreme™ MALDI-TOF/ TOF instrument (Bruker Daltonics, Bremen, Germany). Combined mass and mass/mass spectra were used to interrogate human sequences in the Swiss-Prot database using the Mascot database search algorithms. The MS/MS data were retrieved against the Homo sapiens subset of the sequences with the parameters set as follows: enzyme, trypsin; peptide mass tolerance, ±100 ppm; and fragment mass tolerance, ±0.5 Da; max missed cleavages, 1. Protein with a statistically significant protein score (p < 0.05) and best ion score were considered as confident protein. Redundancy of proteins with different names and accession numbers in protein data base was eliminated. Proteins with the highest protein score were pick out from single spot with multiple proteins. The molecular weights and isoelectic points of most proteins from the MS result were consistent with the spots presented in the gel regions.

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Time (TaKaRa Biotechnology, Dalian, China). Target mRNA was amplified by SYBR® Premix Ex TaqTM (Perfect Real Time) (TaKaRa Biotechnology, Dalian, China) using an ABI Prism 7300 System (Applied Biosystems, USA) according to the manufacturer’s instructions. The PCR were conducted as follow: 1 cycle at 95°C for 30 seconds followed by 40 cycles of 95°C for 5 seconds and 60°C for 31 s, then one cycle of 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. The primer sequences were listed in Table 1. Quantifications were performed independently in triplicate. In vitro differentiation of DFCs & PDLCs induced by TDM

The human TDM, used as inductive scaffold, was fabricated according to a well-established protocol [17] . Scanning electron microscope (SEM) was used to observe the growth of cells on TDM. To evaluate the odontogenic differentiation capacity of DFCs and PDLCs induced by TDM, quantitative real-time PCR were performed to detect genes expressed by DFCs and PDLCs induced by TDM for 7 days. Scanning electron microscopy

DFCs and PDLCs (5×104) were respectively seeded onto TDMs in a six-well plate. After 3 days of culture, they were washed in PBS three times, fixed with 2.5% glutaraldehyde at 0 °C dehydrated and dried in a critical-point dryer and finally observed under a SEM (Inspect F, FEI, Netherlands). The experiment was repeated at least three times. Real-time PCR analysis

After seeded onto TDMs at a concentration of 2×104 cells/ml and cultured for 7 days, DFCs and PDLCs were digested and used for RNA extraction. The treatments were the same for the control group as the test group except cells were seeded onto a six-well plate instead of TDMs. RNA extraction, cDNA synthesis and PCR procedures were performed as described previously. We monitored the expression of the following genes: DSPP,  DMP1, BGN, DCN, OPN and GAPDH. Primer sequences for those genes are listed in Table 1. Relative expression levels were calculated using the 2−ΔΔCTmethod [21] , and normalized to the reference GAPDH gene. This experiment was repeated three times.

Real-time PCR

In vivo differentiation of DFCs & PDLCs induced by TDM

Total cellular RNA was extracted from DFCs and PDLCs using RNAiso Plus (TaKaRa Biotechnology, Dalian, China) according to the manufacturer’s instruction. cDNAs were synthesized from extracted RNA with PrimeScript® RT reagent Kit Perfect Real

In order to compare the odontogentic capacity of DFCs and PDLCs in vivo, we transplanted the cells combined with TDM into the dorsum of immunodeficient mice. All animal experiments were conducted in accordance with the ethical protocol approved by the Committee

Regen. Med. (2015) 10(4)

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Comparison of human DFCs and PDLCs for dentin tissue regeneration 

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Table 1. Oligonucleotide primer sequences. Target cDNA

Primer sequence (5′-3′)  

Product size (bp)

NCBI No.

DSPP

F:ctgttgggaagagccaagataag

129

NM_014208.3

 

R:ccaagatcattccatgttgtcct

 

 

DMP-1

F:gtgagtgagtccaggggagataa

111

NM_004407.3

 

R:ttttgagtgggagagtgtgtgc

 

 

BGN

F:ggagaacagtggctttgaacct

112

NM_001711.4

 

R:attcagggtctcagggaggtct

 

 

DCN

F:gtcatagaactgggcaccaatc

140

BT019800.1

 

R:gtaagggaaggaggaagaccttg

 

 

OPN

F:cagttgtccccacagtagacac

127

J04765.1

 

R: gtgatgtcctcgtctgtagcatc

 

 

CNN2

F: accggctcctgtccaaatatg

151

NM_004368.2

 

R: cccggctgtagcttgttca

 

 

TBCA

F: cctcgcgtgagacagatcaag

219

NM_004607.2

 

R: caaatatgcggcttccaacct

 

 

CSTB

F: acctgcgagtgttccaatct

120

NM_000100.3

 

R: gccttgtccaaagtcaggat

 

 

GAPDH

F: ctttggtatcgtggaaggactc

132

NM_008084

 

R: gtagaggcagggatgatgttct

 

 

F: Forward primer; R: Reverse primer.

of Ethics of the Sichuan University for animal experiments. Six immunodeficient mice were divided into two groups: DFCs group (TDM combined with DFCs) and PDLCs group (TDM combined with PDLCs). DFCs and PDLCs were seeded onto TDM scaffolds separately at a density of 5×104 cells/scaffold and incubated at 37°C for 3 days. Implantation of cell/TDM constructs was performed under deep anesthesia. Eight weeks later, implants were obtained from the immunodeficient mice under deep anesthesia. A natural mandibular third molar was used as control. Samples were fixed with 4% paraformaldehyde overnight at 4°C, demineralized with 10% EDTA (pH 8.0) and embedded in paraffin. Paraffin sections were prepared and subjected to hematoxylin and eosin and Masson’s staining. The amount of newly formed dentin matrix was expressed as the average thickness of the newly formed dentin, which was calculated by dividing the area of the new dentin by the length of the distinct line of demarcation between TDM scaffold and the newly formed dentin matrix. The analyses were quantified with Image-pro plus 5.0 software (Media Cybernetics, USA). Immunohistochemistry was used to identify the newly formed tissues and detect the presence of proteins related to dentinogenesis. Immunohistochemical antibodies included dentin sialoprotein (DSP, 1:100 dilution; Santa Cruz, USA), COL-I (1:200 dilution; Abcam, UK), BGN (1:100 dilution; Abcam, UK), DCN (1:200

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dilution; Santa Cruz, USA), and human mitochondria (1:100 dilution; Millipore, USA). All antibodies were used according to the manufacturers’ protocol. PBS was used as a negative control instead of the primary antibody. Biotinylated secondary antibodies (1:1000) were purchased from Dako (Dako, USA). The images were captured using microscopy (Leica Optical, Germany). Statistical analysis

Data were gathered at least in triplicate, and expressed as mean ±SD. Statistical analyses of the data were performed by paired t-test using SPSS 11.5 software (SPSS, USA). A value of p < 0.05 was considered statistically significant. Results Morphological & growth characteristics

The primary cells were grown from dental follicle of wisdom tooth germ and periodontal ligament of premolars, respectively (Figure 1A) . Both cell phenotypes were negative for the epithelial cell marker, CK-14, and positive for vimentin, a mesenchymal cell marker (Figure 1A) . After subculturing, DFCs and PDLCs showed similar morphologies, as observed to be the typical morphology of mesenchymal cells with spindle shape. However, DFCs were smaller in size, and PDLCs exhibited relatively flat and elongated compared with

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A

C

PDLCs

DFCs

PDLCs

Vimentin

CK-14

The 3rd passage cells

Primary cells

DFCs

B

2.0

DFCs PDLCs

OD-value

1.5

**

**

3 4 5 Time (days)

6

**

* 1.0

*

0.5 0.0

0

1

2

7

Figure 1. Morphological and growth characteristics of dental follicle cell and periodontal ligament cell cultures. (A) Primary cells and the third passage cells grown from human dental follicle tissue and periodontal ligament tissue. Immunocytochemical staining showed both cells were positive for vimentin, but negative for CK-14. Bar: 100 μm. (B) Growth curves of DFCs and PDLCs analyzed by CCK-8 assay. DFCs exhibited higher proliferative activity than PDLCs. *p < 0.05. **p < 0.01. (C) Ultrastructural observation of DFCs and PDLCs. The red arrows indicate the homogeneous electron-dense granules, which are regarded as an identification mark of DFCs. Lots of lysosomes (blue arrows) were observed in the cytoplasm of DFCs. There were more rough endoplasmic reticula (red rectangle) and mitochondria (green arrows) in PDLCs. DFC: Dental follicle cell; OD: Optical density; PDLC: Periodontal ligament cell. For color images please see online www.futuremedicine.com/doi/full/10.2217/RME.15.21

DFCs. CCK-8 detection showed that DFCs had stronger proliferation ability than PDLCs (Figure 1B) . Ultrastructural comparison

TEM evaluations showed DFCs and PDLCs with phenotypes of mesenchymal fibroblast ultrastructure (Figure 1C)  [22,23] . Homogeneous electron-dense granules, an identifying marker of DFCs, were observed

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in DFCs, but not in PDLCs. In the nucleus, less heterochromatin was observed in DFCs than that in PDLCs. There are more abundant organelles, such as mitochondria and rough endoplasmic reticula, in the cytoplasm of PDLCs than that of DFCs. Besides, bundles of microfilaments could be observed in the cytoplasm of PDLCs, while DFCs demonstrated a large number of lysosomes and free ribosomes.

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Comparison of human DFCs and PDLCs for dentin tissue regeneration 

0 100 101 102 103 104 PE 256

0

STRO-1 12.6% M1

0 100 101 102 103 104 FITC 256

100 101 102 103 104 PE

0 100 101 102 103 104 PE 256

CD29 99.9% M1

0 100 101 102 103 104 PE 256

STRO-1 7.6% M1

0 100 101 102 103 104 FITC

256

CD105 99.6% M1

0 100 101 102 103 104 PE 256

CD105 99.0% M1

Events

CD44 M1

0 100 101 102 103 104 FITC 256

CD29 M1

Events

99.9%

Events

CD146 24.5% M1

Events

PDLCs

256

Events

Events

CD146 35.6% M1

99.8%

CD45 M1 2.1%

Events

100 101 102 103 104 FITC

256 DFCs

0 100 101 102 103 104 FITC

Events

Events

CD90 99.8% M1

256

0 100 101 102 103 104 PE 256 3.4%

CD34 M1

Events

256

0

CD44 99.9% M1

Events

0 100 101 102 103 104 FITC

PDLCs

256 Events

Events

DFCs

CD90 99.9% M1

0 100 101 102 103 104 FITC 256 1.5%

CD45 M1

Events

256

After the induction in adipogenic and osteogenic media, differentiated cells were respectively analyzed for the accumulation of lipid clusters and formation of mineralized nodules (Figure 3B) . Formation of calcium mineralized nodules as indicated by Alizarin red stain-

0 100 101 102 103 104 FITC

256 2.7%

0 100 101 102 103 104 FITC 512

CD31 M1

Events

Toluidine blue staining revealed that all cultures contained subpopulation of cells that were capable of generating new fibroblast colonies from single

Adipogenic & osteogenic differentiation

CD34 3.0% M1

0 100 101 102 103 104 FITC 256 2.2%

CD31 M1

Events

Colony forming

cells (Figure 3A) . Cells from the dental follicle tissue established multiple and larger new colonies, whereas PDLCs established fewer colonies. The colony-forming assay showed that DFCs displayed stronger clone formation ability than PDLCs. At day 10, DFCs showed a colony-forming efficiency of 17.1 ±2.7%, whereas colony-forming efficiency for PDLCs was 10.9 ±1.9% (p < 0.05).

Events

The results of flow cytometric analysis of cell surface antigens on DFCs and PDLCs at passage 3 are presented in Figure 2. Expression levels of mesenchymal stem cell markers CD146 and STRO-1 were higher in DFCs than those of PDLCs. Both DFCs and PDLCs were stained positive for mesenchymal cell markers (CD105, CD90, CD44 and CD29), and negative for hematopoietic lineage markers (CD34 and CD45) and endothelium antigen (CD31).

Events

Immunophenotypic characterization

0 100 101 102 103 104 FITC

0 100 101 102 103 104 FITC

Figure 2. Analysis of cell surface antigens. Flow cytometric analysis indicated that DFCs and PDLCs have similar surface molecule phenotype. They were positive for STRO-1, CD146, CD105, CD90, CD44 and CD29, but negative for CD34, CD31 and CD45. DFC: Dental follicle cell; FITC: Fluorescein isothiocyanate; PDLC: Periodontal ligament cell; PE: Phycoerythrin.

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A *

DFCs

Colony-forming efficiency (%)

20 15 10 5 0

PDLCs

PDLCs

DFCs

B

DFCs

PDLCs *

Nodule area (%)

Osteo

30 20 10 0

DFCs

Lipid area (%)

5

Adipo

PDLCs *

4 3 2 1 0 DFCs

PDLCs

Figure 3. Colony-forming and multipotential differentiation capacity of dental follicle cells and periodontal ligament cells. (A) Cells were plated at 1×103 cells/100 mm dish. After incubation for 10 days, the colonies were stained with Toluidine Blue. The analysis result of colony-formation rate indicated that DFCs were more highly clonogenic than PDLCs. (B) Osteogenic differentiation in DFCs and PDLCs was displayed by the formation of Alizarin red-positive calcified deposits. Adipogenic differentiation in DFCs and PDLCs was shown by the formation of Oil red O-positive lipid vacuoles. The quantitative analysis result of the area of mineralized nodules and lipid droplets showed DFCs out-performed PDLCs in their ability to undergo adipogenic differentiation, but PDLCs were more mineralized compared with DFCs. Bar: 100 μm. * p < 0.05. Adipo: Adipogenic differentiation; DFC: Dental follicle cell; Osteo: Osteogenic differentiation; PDLC: Periodontal ligament cell.

ing was observed in adherent cell cultures of DFCs and PDLCs. Intercellular formation of lipid droplets stained with Oil red O solution were clearly exhibited in DFCs and PDLCs. The quantitative assay showed DFCs formed more lipid droplets but less mineralized nodules comparing to PDLCs.

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Comparative proteomic analysis of DFCs & PDLCs

The protein expression profiles of DFCs and PDLCs were acquired by 2-DE. Representative 2-DE maps for a subsample of three pairs of samples that were matched by the PD-Quest software are shown in Figure 4A . Differentially expressions of proteins were

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Comparison of human DFCs and PDLCs for dentin tissue regeneration 

considered to be statistically significant (p < 0.05) when the intensity alterations were over 1.5-fold and at the same time recurred more than two times. By applying these criteria, a total of 35 spots were identified as differentially expressed; among these, 12 proteins were higher expressed in DFCs (spots 1–12) while the rest were higher expressed in PDLCs (Figure 4A) . Thirteen mostly differentially expressed proteins (2 higher expressed in DFCs and 11 higher expressed in PDLCs) are shown in figure, and their corresponding spots are boxed and enlarged in the surrounding area (Figure 4D) . Thirty-five differentially expressed protein spots were subsequently subjected to MS/MS analysis. The MS/MS data were queried using the search algorithm Mascot against the ExPASy protein sequence database. Proteins were identified based on these criteria including pI, MW, the number of matched-peptides and MOWSE score. Details of the protein information were listed in Table 2. The identified proteins were

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classified into different groups according to subcellular localization (Figure 4B) . Of these, the majority (26 of 35 proteins) were located in cytoplasm (77%), 11% in mitochondrion, 6% were endoplasmic reticulum, and the rest were situated in nucleus. The most predominate proteins which are expressed differentially in these two cells include heat shock protein β -1(HSPB1), superoxide dismutase [Cu-Zn] (SOD1), Calponin-2 (CNN2), Cystatin-B (CSTB), Tubulin-specific chaperone A (TBCA). The intrinsic interaction of the 35 proteins was analyzed using the web-based tool STRING (Figure 4C) . To examine whether the proteomics identification of these proteins corresponded to changes at the transcriptional level, three proteins (CNN2, TBCA and CSTB) with significant expression changes were chosen for validation by real-time PCR. As shown in Figure 4E, CNN2, TBCA and CSTB were found to be consistent with the observations in 2-DE analysis.

A

B PDLCs

DFCs

80 KDa

Nucleus Endoplasmic reticulum 6% 6% Mitochondrion 11%

Cytoplasm 77%

pH 3–10 NL

pH 3–10 NL

C

D HSPB1 SOD1 ENO1 CAPG PDLIM1 CNN2 ERP29 PARK7 PRDX2

DBI D

TBCA T

C CSTB

ERP29 E ERP2 9

DFCs

CNN2 C

UQCRFS1 U UQCR FS1 S100A11

PDLCs

COTL1 C COTL 1

PARK7 PARK PAR PA P AR A R 7

S100A6 S S100 A6 6 ECHS1

SOD1 S

S100A4 S S100 A4

LASP1 LASP

SOD2

STMN1 STMN S N1

TPI1

GPX1 G

PDLIM1 P PDLI M1 ENO1 EN ENO E N NO O

PRDX2 PRDX PRD PR P RD 2

ESD E PCBP1 P PCBP 1

PAFAH1B2

PPP1CA P PPP1 C

PRDX3 PRDX P 3

CAPG C

EIF4H BAG2

STMN1 TBCA CSTB S100A11

HSPB1 H HSPB

PSME1 P PSME 1

DFCs PDLCs

E

Relative mRNA expression

10 KDa

5 4 3 2 1 0

DFCs PDLCs

*

** *

CNN2 TBCA CSTB

Figure 4. Proteomics analysis of differentially expressed proteins in human dental follicle cells and periodontal ligament cells. (A) Representative 2-DE images of DFCs and PDLCs. Thirty-five differentially expressed spots (12 higher expressed in DFCs and 23 higher expressed in PDLCs) were identified (as numbered). (B) Identified proteins were classified into four groups. These included cytoplasm (77%), mitochondrion (11%), endoplasmic reticulum (6%) and nucleus (6%). (C) Protein–protein interaction network generated by STRING. (D) Expression profile of the 13 related proteins. The selected area was symmetrically boxed, and arrows indicated each protein spot or its theoretical location. (E) Validation of three significantly differentially expressed protein at mRNA level via real-time PCR. * p < 0.05. ** p < 0.01. DFC: Dental follicle cell; NL: Nonlinear gradient; PDLC: Periodontal ligament cell.

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Regen. Med. (2015) 10(4)

Q06323

P68402

P30084

P60174

P60174

P30048

P47985

P04792

P00441

Q14019

P06733

P40121

Q15365

3

4

5

6

7

8

9

10

11

12

13

14

15

Poly(rC)-binding protein 1

Macrophage-capping protein

Alpha-enolase

Coactosin-like protein

mRNA splicing, via spliceosome

Barbed-end actin filament capping

Glycolysis; plasminogen activation; transcription

Defense response to fungus

Activation of MAPK activity

Cell death

Heat shock protein β-1

Hydrogen peroxide catabolic process

Embryo development; gluconeogenesis

Embryo development; gluconeogenesis

Fatty acid β-oxidation

Respiratory electron transport chain; response to antibiotic

Superoxide dismutase [Cu-Zn]

Cytoplasm

Cytoplasm

Cytoplasm

Subcellular location

‡

†

PAFAH1B2

PSME1

ESD

PPP1CA

Gene name

TPI1

TPI1

Nucleus

Cytoplasm

Cytoplasm

Cytoplasm

Cytoplasm

Cytoplasm

PCBP1

CAPG

ENO1

COTL1

SOD1

HSPB1

Mitochondrion UQCRFS1

Mitochondrion PRDX3

Cytoplasm

Cytoplasm

Mitochondrion ECHS1

Lipid degradation; brain Cytoplasm development

DNA damage response

Formaldehyde catabolic process

Branching morphogenesis of a tube; cell cycle

Biological process

Cytochrome b-c1 complex subunit Rieske, mitochondrial

Thioredoxin-dependent peroxide reductase, mitochondrial

Triosephosphate isomerase

Triosephosphate isomerase

Enoyl-CoA hydratase, mitochondrial

Platelet-activating factor acetylhydrolase IB subunit β

Proteasome activator complex subunit 1

S-formylglutathione hydrolase

Serine/threonine-protein phosphatase PP1-alpha catalytic subunit

Protein Name

Accession numbers were obtained from the ExPASy database. Theoretical molecular weight (kDa) and pI from the ExPASy database. § Probability-based MOWSE (molecular weight search) scores. All protein spots were identified by Bruker ultrafleXtreme matrix assisted laser desorption ionization time-of-flight mass spectrometry. Mr: Molecular weight; pl: Isoelectric point.

P10768

P62136

1

2

Accession No.†

Spot No.

Table 2. Proteins identified by matrix assisted laser desorption ionization time-of-flight mass spectrometry.

37987

38760

47481

16049

16154

22826

29934

28023

31057

31057

31823

25724

28876

31956

38229

Theoretical Mr‡

6.66

5.82

7.01

5.51

5.70

5.98

8.55

5.77

5.65

5.65

5.88

5.57

5.78

6.54

5.94

Theoretical pI‡

232

176

34

119

104

334

185

189

62

220

176

125

98

284

57

Score § 

Research Article  Tian, Bai, Guo et al.

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Q15056

P04179

Q99497

Q99497

23

24

25

26

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Actomyosin structure organization

Regulation of transcription, DNAdependent; response to hypoxia

Ion transport

Biological process

Protein folding

Protein folding

Embryo development; gluconeogenesis

Tubulin-specific chaperone A

Stathmin

Peroxiredoxin-2

Glutathione peroxidase 1

Protein DJ-1

Protein DJ-1

Superoxide dismutase [Mn]

‘De novo ‘posttranslational protein folding

Axonogenesis

Hydrogen peroxide catabolic process

§

‡

†

EIF4H

BAG2

BAG2

TPI1

ERP29

CNN2

PDLIM1

LASP1

Gene name

Cytoplasm

Cytoplasm

STMN1 TBCA

Cytoplasm

PRDX2 Cytoplasm

Cytoplasm

GPX1

PARK7

PARK7

Mitochondrion SOD2

Cytoplasm

Cytoplasm

Cytoplasm

Cytoplasm

Endoplasmic reticulum

Cytoplasm

Cytoplasm

Cytoplasm

Subcellular location

Angiogenesis involved in Cytoplasm wound healing

Adult locomotory behavior; autophagy

Adult locomotory behavior; autophagy

Age-dependent response to reactive oxygen species

Eukaryotic translation initiation Regulation of factor 4H translational initiation; virus-host interaction

BAG family molecular chaperone regulator 2

BAG family molecular chaperone regulator 2

Triosephosphate isomerase

Endoplasmic reticulum resident Intracellular protein protein 29 transport

Calponin-2

PDZ and LIM domain protein 1

LIM and SH3 domain protein 1

Protein Name

Accession numbers were obtained from the ExPASy database. Theoretical molecular weight (kDa) and pI from the ExPASy database. Probability-based MOWSE (molecular weight search) scores. All protein spots were identified by Bruker ultrafleXtreme matrix assisted laser desorption ionization time-of-flight mass spectrometry. Mr: Molecular weight; pl: Isoelectric point.

O75347

O95816

22

30

O95816

21

P16949

P60174

20

29

P30040

19

P32119

Q99439

18

28

O00151

17

P07203

Q14847

16

27

Accession No.†

Spot No.

12904

17292

22049

22360

20050

20050

24878

27425

23928

23928

31057

29033

34074

36505

30097

Theoretical Mr‡

Table 2. Proteins identified by matrix assisted laser desorption ionization time-of-flight mass spectrometry (cont.).

5.25

5.75

5.67

6.15

6.32

6.32

8.35

6.66

6.25

6.25

5.65

6.77

6.92

6.55

6.61

Theoretical pI‡

99

285

329

283

71

53

34

27

32

87

540

182

172

180

110

Score § 

Comparison of human DFCs and PDLCs for dentin tissue regeneration 

Research Article

471

472

123 5.32 10231 S100A6 Nucleus Axonogenesis Protein S100-A6 P06703

Accession numbers were obtained from the ExPASy database. ‡ Theoretical molecular weight (kDa) and pI from the ExPASy database. § Probability-based MOWSE (molecular weight search) scores. All protein spots were identified by Bruker ultrafleXtreme matrix assisted laser desorption ionization time-of-flight mass spectrometry. Mr: Molecular weight; pl: Isoelectric point.

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To evaluate the odontogentic capacity of DFCs and PDLCs in vitro, we cultured the cells with TDMs for 7 days. The cells were observed by SEM after they were seeded on TDM for 3 days. The cells were polygonal, stretched and attached well on the surface of TDM, and DFCs were arranged more closely than PDLCs (Figure 5A) . On day 7, the cells were trypsinized and assessed by real-time PCR (Figure 5B) . The results showed that the expression of DSPP, BGN and OPN in DFCs was higher than that of PDLCs, whereas no difference was detected in the expression of DCN and DMP-1. After induced by TDM for 1 week, DCN and DMP-1 exhibited higher expression level in DFCs than in PDLCs, but BGN expressed in DFCs was lower. And the expression of DSPP in DFCs was still higher than that of PDLCs. OPN was upregulated in PDLCs after the induction.

†

341 6.56 11849 S100A11 Negative regulation of DNA replication Protein S100-A11 P31949 34

Hair follicle development P07108 33

Acyl-CoA-binding protein

Cytoplasm

6.12 10038 DBI

74

5.85 11953 S100A4 Epithelial to mesenchymal transition P26447 32

Protein S100-A4

Endoplasmic reticulum.

6.96 11190 CSTB Adult locomotory behavior; regulation of apoptotic process P04080 31

Cystatin-B

Cytoplasm

Cytoplasm

Theoretical pI‡ Theoretical Mr‡ Biological process Accession No.†

Protein Name

Subcellular location

Gene name

253

240

In vitro differentiation of DFCs & PDLCs induced by TDM

Spot No.

Table 2. Proteins identified by matrix assisted laser desorption ionization time-of-flight mass spectrometry (cont.).

Score § 

Research Article  Tian, Bai, Guo et al.

In vivo dentin regeneration by DFCs & PDLCs

Histological examination of the grafts displayed significant dentin formation from both DFCs and PDLCs after induced by TDM under the dorsal surface of immunocompromised mice for 8 weeks. Hematoxylin and eosin staining showed that DFCs regenerated complete dentinlike tissues, including dentin with calcospherites, predentin and odontoblast-like cell layer, while in the dentin generated by PDLCs, the boundary between dentin and predentin was barely visible that seemed only a single layer (Figure 6). The quantitative analysis indicated that there was no significant difference in amount of newly formed dentin matrix of the two groups (Figure 6). Immunohistochemistry analysis showed that the regenerated tissues were positive for DSP and DMP-1, which are regarded as marker proteins for the identification of dentin. Besides, these two types of newly generated tissues were positive for BGN. As for DCN, the dentin regenerated by DFCs exhibited a positive staining, but in PDLCs-formed dentin, no positive staining for DCN was detected (Figure 7). Both the newly regenerated tissues of the two groups were positive for human mitochondria, indicating that exogenous DFCs or PDLCs participated in the regeneration of new tissue [17] . Discussion Recently, stem cells from various dental tissues have received extensive attention in the field of tissue engineering that enables us to contemplate new and promising therapy for lost or injured teeth [24] . These dental stem cells play a vital role in tooth development and provide an attractive cell source for tissue engineering and regenerative medicine. Specifically, it has been reported that mesenchymal stem cells exist in dental

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Comparison of human DFCs and PDLCs for dentin tissue regeneration 

A

4 3 2 1 0

DSPP *

**

Control

Induced

DMP-1 * * *

Control

PDLCs DFCs

Relative gene expression

5 4 3 2 1 0

Induced

PDLCs DFCs

Relative gene expression

Relative gene expression

Relative gene expression

B

PDLCs

4 3 2 1 0

*

BGN ** **

Control

Induced

DCN * ** **

1.5 1.0

PDLCs DFCs

Relative gene expression

DFCs

Research Article

8 6 4 2 0

*

OPN **

Control

*

PDLCs DFCs

Induced

PDLCs DFCs

0.5 0.0

Control

Induced

Figure 5. The expression of odontogenic related genes in dental follicle cells and periodontal ligament cells. (A) Scanning electron microscopy examination of the growth of DFCs and PDLCs on treated dentin matrix (TDM). The cells were stretched and attached well on the surface of TDM after cultured for 3 days, and DFCs were arranged more closely than PDLCs. (B) The expression of DSPP, DMP-1, BGN, DCN and OPN in DFCs and PDLCs cultured with or without TDM was examined by real-time PCR method. The data are expressed as mean ±SD of triplicates of each experiment. * p < 0.05. ** p < 0.01. DFC: Dental follicle cell; PDLC: Periodontal ligament cell.

follicle and periodontal ligament [1,2] , which are from periodontium of different developmental stages [25] . However, the characterization and differences in odontogenic potential of those cells are not fully understood. In this comparative study, we attempted to shed light on the distinct biological properties of DFCs and PDLCs, and to analyze their odontogenic differentiation under the same inductive environment of TDM scaffolds, in order to provide evidence for the suitability of each of these two cell populations for future application in dental-tissue regeneration protocols. The observation of morphologic and growth characteristics showed notable differences between DFCs and PDLCs. DFCs contained more smaller cells shaped as short fusiform or polygon, with a higher growth rate, probably due to the fact that they were from a developing tissue. Compared with DFCs, PDLCs were mainly comprised of slower growing cells that were relatively elongated. Thus, the proliferative potential of these fibroblast-like cells was shown to be

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inversely dependent on cell size, as also reported by other investigators [26] . Moreover, the higher proliferation activity of DFCs may be an advantage for tissue regeneration applications due to efficient ex vivo expansion and cryopreservation in large quantities. On the ultrastructural comparison, less heterochromatin was observed in the nucleus of DFCs than that in PDLCs. There were more abundant organelles and bundles of microfilaments in cytoplasm of PDLCs, while DFCs demonstrated rich in free ribosomes. These differences indicated that DFCs were more immature and pluripotent than PDLCs [27–29] . A large number of lysosomes in DFCs, including primary and secondary lysosomes, might be related to a more active process of autophagy, a lysosome-dependent degradation pathway, which is crucial for maintaining cellular homeostasis as well as remodeling during normal development and plays a critical role in stem cell renewal, proliferation and aging  [30] . Besides, more rough endoplasmic reticula and mitochondria in PDLCs indicated the stronger ability for protein synthesis and secretion [31] .

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Research Article  Tian, Bai, Guo et al.

Control Thickness of ND (µm)

PDLCs/TDM

H&E

DFCs/TDM

150 100 50 0 PDLCs

Masson

DFCs

Figure 6. In vivo dentin-like tissue formation from dental follicle cells and periodontal ligament cells. After induced by TDM under the dorsal surface of immunocompromised mice for 8 weeks, DFCs could regenerate complete dentin-like tissues, including dentin, predentin and calcospherites (red triangles). However, there was no layering in the dentin generated by PDLCs. There was no significant differences in amount of newly formed dentin matrix of the two groups. de: Dentin; DFC: Dental follicle cell; H&E: Hematoxylin and eosin; ND: Neo-dentin; PDLC: Periodontal ligament cell; TDM: Treated dentin matrix. For color images please see online www.futuremedicine.com/doi/full/10.2217/RME.15.21

The analysis of stem cell properties revealed differences in clone-forming ability, stem cell surface epitopes and multipotential differentiation potential of DFCs and PDLCs. DFCs represented stronger clone-forming ability comparing to PDLCs. Both these two cell populations demonstrated the capacity to undergo adipogenic and osteogenic differentiation. DFCs were more effective to form adipocytes following in vitro induction, while PDLCs showed stronger osteogenic differentiation potential, which may be attributed to their potential role in benefiting alveolar bone remodeling. Additionally, DFCs exhibited higher expression levels of STRO-1 and CD146, which are putative surface markers for mesenchymal stem cells [32,33] . These findings demonstrate the existence of stem/progenitor cells in these two heterogeneous cell populations, which also contain transit amplifying cells, progenitor cells of more lineage restricted characteristics and differentiated cells. Meanwhile, these data suggest that DFCs have a higher proportion of cells that possess stem cell properties than PDLCs, implying the relatively stronger regenerative potential, which might be attributed to the fact that they are derived from tissue at an earlier stage of tooth development. Furthermore, 35 differentially expressed protein spots in DFCs and PDLCs were identified by MALDI-TOF-MS. These proteins are mainly located in cytoplasm, effecting protein folding, cell growth, metabolic enzymes, etc. For example, CNN2 is an actin-binding protein implicated in cytoskeletal organization, which is physiologically responsive to

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mechanical stimuli [34] . The higher expression of CNN2 in PDLCs may function in regulating cellular interactions with the mechanical environment of periodontium. Additionally, previous studied have demonstrated CNN2’s function as a suppressor of cell proliferation [35] , suggesting that CNN2 is a possible contributor to the finding that PDLCs were less capable of proliferation than DFCs. CSTB, another protein increased in PLDCs, has recently been found to be a modulator of bone metabolism, which can inhibit bone resorption [36] , and was confirmed to be host defense-related protein present in gingival crevicular fluid of periodontal healthy people [37] , indicating the vital function of PDLCs in maintaining the homeostasis of periodontal tissue. TBCA is a tubulin folding cofactor, which is essential for cell cytoskeleton function [38] . The up-regulated TBCA in PDLCs may be associated with the unique function of PDLCs in the maintenance of the periodontium as well [39] . Cell differentiation is strongly influenced by physical, chemical and cellular signals provided by the local microenvironment. During tooth development, DFCs penetrate into the Hertwig’s epithelial root sheath, and then attach to the surface of the dentin, initiating the development of periodontium [13] . Therefore, dentin is an important microenvironment factor for the differentiation of DFCs. In this study, a bioactive material, TDM [16,17] , mimicking the structure of natural dentin, was used as the scaffold to provide the microenvironment inducing odontogenic differentiation of DFCs and PDLCs.

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Comparison of human DFCs and PDLCs for dentin tissue regeneration 

COL-1

PDLCs/TDM

BGN

DMP-1

DFCs/TDM

Blank

Mitochondria

Control

DCN

PDLCs/TDM

DSP

DFCs/TDM

Research Article

Figure 7. Immunohistochemical examination of dentin-like tissues formed by dental follicle cells and periodontal ligament cells in vivo. Both the tissues generated by DFCs and PDLCs were positive for COL-I, DSP, DMP-1, BGN and human specific mitochondria antibody staining. However, as for DCN, DFCs-generated tissues were positive, while the PDLCs-generated tissues were negative. Negative control of PBS substituted for the primary antibody showed no staining (blank). DFC: Dental follicle cell; PDLC: Periodontal ligament cell; TDM: Treated dentin matrix.

This study found that the dentinogenesis-associated genes expressed differently in DFCs and PDLCs with or without the microenvironment of TDM. For instance, DSPP is the dentin-specific marker and believed to actively promote and control the mineralization of collagen fibers and crystal growth within predentin. The human and mouse genetic studies showed the association of DSPP gene mutations or ablations with mineralization defects in the dentin [40,41] . Additionally, DSP, cleaved from DSPP, is thought to be a functional marker for the secretory activity of odontoblasts [42] . As DSPP is critical for dentin mineralization, the higher expression of DSPP in DFCs may indicate its better dentinogenesis ability than PDLCs. BGN and DCN are two of predominant proteoglycans present in predentin and dentin, mainly secreted by odontoblasts. They are known to interact with type I collagen fibrils recruited to form dentin matrix and play a significant role in the regulation of collagen fibrillogenesis and mineralization [43–45] . It has been reported that the addition of BGN increased apatite formation in vitro, suggesting the potential role of

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BGN as a nucleator of mineralization [46] . More significantly, the gene knockout experiments found that BGN may have a positive effect on the coalescence of calcospherites during dentin mineralization [47] . In our study, after induction of TDM, the expression of BGN in PDLCs was up-regulated and higher than that in DFCs, implying their different capability to modulate the coalescence of calcospherites during the odontogenesis. DCN is thought to function as a negative regulator for dentin mineralization, as DCN can particularly binds to the gap region in the collagen fibril, which is believed to block initiation of mineralization  [48] . In vitro experiments have shown that decorin expression appears to be inversely correlated to matrix mineralization [45,49] . More importantly, Haruyama et al. showed that the excess DCN was responsible for inhibiting conversion of predentin to dentin at the mineralization front [47] . Our study showed that expression of DCN in PDLCs was lower than that in DFCs after induction of TDM, suggesting the weaker ability of PDLCs for the negative control of matrix mineralization. In addition, recent data provide evidence for the important ability

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Research Article  Tian, Bai, Guo et al. of BCN and DCN to interact with signaling molecules, such as BMP/TGF-β and Wnt/β-catenin pathways, which could also potentially modulate proliferation, migration and development of the osteoblast phenotype [43,50] . Since all of these above information indicate that the normal expression of DSPP, BGN and DCN plays an important role in the highly orchestrated process of dentin formation, it is plausible to postulate that different expression of these proteins in DFCs and PDLCs may result some differences in dentinogenesis. To evaluate the odontogenic characteristics of these two cell types in vivo, DFCs and PDLCs were seeded onto TDM respectively and then implanted into the dorsum of immunodeficient mice for 8 weeks. The results showed that under the induction of TDM in vivo, both DFCs and PDLCs could regenerate dentin-like tissues which were positive stained for identified markers for dentin and odontoblasts: DMP-1 and DSP [42,51,52] , although PDLCs are known for their important role in promoting periodontal regeneration [1] . Histological examination revealed a complete dentin regeneration from DFCs, including dentin, predentin and calcospherites, which was consistent with our previous result [17] . However, in the dentin formed by PDLCs, the boundary between dentin and perdentin was barely visible that seemed only a single layer. Further, immunostaining showed that these two types of newly generated tissues were positive for BGN. As for DCN, the dentin regenerated by DFCs exhibited a positive staining, but in PDLCs-generated dentin, no positive staining for DCN was detected, indicating that the lack of DCN may relate to the mechanisms underlying the formation of single layer structure of the regenerated dentin. Thus, the above results suggest that PDLCs could contribute to regenerate dentin-like tissues in the inductive microenvironment of TDM, and DFCs have a better potential for dentin regeneration than PDLCs, possibly due to the different expressions of dentinogenesis-associated genes and the different proportions of stem/progenitor cells in these two heterogeneous cell populations. Conclusion The present study showed that PDLCs could contribute to regenerate dentin-like tissues in the inductive microenvironment of TDM scaffold. However, DFCs exhibited more remarkable dentinogenic capability than PDLCs did. Besides, DFCs presented a higher proliferation activity and different multipotential differentiation potential compared with PDLCs. The proteomic analysis revealed that PDLCs expressed more proteins related to maintaining the homeostasis

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of periodontal tissue. These findings extend our understanding on tooth development, contribute to the improvement of current dental tissue engineering protocols via cell-based approaches and imply that a pure population of stem cells is not necessary. Future perspective Dental follicle and periodontal ligament, which are from periodontium of different developmental stages, have attracted wide attention in the field of tissue engineering as easily accessible postnatal tissue source of stem cells. The potential application of PDLCs for periodontal tissue regeneration has been extensively documented. The results of our study suggest that PDLCs retained the potential to perform dentinogenesis but were inferior to DFCs as a source of multipotent stem cells, expanding the application scope of PDLCs in dental tissue regeneration. Nonetheless, there remain many important issues that must be addressed, including the concrete mechanisms of cellular differentiation and dentin regeneration. For example, TGF-β or Wnt/β-catenin signaling pathway [53,54] , which could be modulated by BGN and DCN [43,50] , might be involved in this odontogenesis. Financial & competing interests disclosure This study was supported by National Basic Research Program of China (2010CB944800), National HighTechnology Research and Development Program of China (2011AA030107), Nature Science Foundation of China (11172190, 81271095, 81271119, 81200792 and 81300848), International Cooperation Program of China (2013DFG32770 and 2011DFA51970), Doctoral Foundation of Ministry of Education of China (20110181120067, 20110181110089 and 20120181120013), Key Technology R&D Program of Sichuan Province (2012SZ0013, 12ZC0493, 13ZC0971, 2013GZX0158, 2013SZ0015 and 13ZC0979) and Basic Research Program of Sichuan Province (2011JY0125, 12JC0212, 2012JY0077 and 2013JY0019). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

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Comparison of human DFCs and PDLCs for dentin tissue regeneration 

Research Article

Executive summary Isolation & culture of dental cells • The primary cells were grown from dental follicle of wisdom tooth germ and periodontal ligament of premolars respectively. After subculturing, dental follicle cells (DFCs) and periodontal ligament cells (PDLCs) showed typical morphology of mesenchymal cells with spindle shape. • Both cell phenotypes were negative for the epithelial cell marker, CK-14, and positive for vimentin, a mesenchymal cell marker.

Ultrastructural comparison • Homogeneous electron-dense granules, an identifying marker of DFCs, were observed in DFCs by transmission electron microscopy evaluation. • A large number of lysosomes were observed in the cytoplasm of DFCs. • There are more mitochondria and rough endoplasmic reticula in the cytoplasm of PDLCs.

Immunophenotypic characterization • DFCs and PDLCs shared a similar immunophenotype, which was positive for mesenchymal cell markers (CD105, CD90, CD44 and CD29), and negative for hematopoietic lineage markers (CD34 and CD45) and endothelium antigen (CD31). • DFCs showed higher expression levels of mesenchymal stem cell markers CD146 and STRO-1 than PDLCs.

Cell growth & colony forming • The proliferation activity and colony-forming efficiency of DFCs were higher than that of PDLCs.

Adipogenic & osteogenic differentiation • Both these two cell populations demonstrated the capacity to undergo adipogenic and osteogenic differentiation. DFCs were more effective to form adipocytes following in vitro induction, while PDLCs showed stronger osteogenic differentiation potential.

Comparative proteomic analysis • The proteomic analysis identified 32 differentially expressed proteins in DFCs and PDLCs, which affect protein folding, cell growth, metabolic enzymes, etc. • PDLCs expressed more proteins related to maintaining the homeostasis of periodontal tissue.

Regenerative capacity of DFCs & PDLCs induced by treated dentin matrix • The different expressions of DSPP, biglycan and decorin were detected in DFCs and PDLCs with or without the microenvironment of treated dentin matrix (TDM). • PDLCs could regenerate dentin-like tissues induced by TDM under the dorsal surface of mice for 8 weeks. However, the structure of dentin tissues generated by DFCs were more complete than that of PDLCs.

Conclusion • PDLCs could contribute to regenerate dentin-like tissues in the inductive microenvironment of TDM scaffold, although DFCs presented more remarkable dentinogenic capability than PDLCs did. • DFCs presented higher proliferation activity and colony-forming efficiency compared with PDLCs. • PDLCs showed stronger osteogenic differentiation potential after in vitro induction. • This study expands our understanding on the odontogenic potential of PDLCs and provides evidence for the seed cell selection in tooth regeneration.

References

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Sonoyama W, Liu Y, Yamaza T et al. Characterization of the apical papilla and its residing stem cells from human immature permanent teeth: a pilot study. J. Endod. 34(2), 166–171 (2008).

5

Miura M, Gronthos S, Zhao M et al. SHED: stem cells from human exfoliated deciduous teeth. Proc. Natl Acad. Sci. USA 100(10), 5807–5812 (2003).

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Morsczeck C, Gotz W, Schierholz J et al. Isolation of precursor cells (PCs) from human dental follicle of wisdom teeth. Matrix Biol. 24(2), 155–165 (2005).

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Luan X, Ito Y, Dangaria S, Diekwisch TG. Dental follicle progenitor cell heterogeneity in the developing mouse periodontium. Stem Cells Dev. 15(4), 595–608 (2006).

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Vollner F, Ernst W, Driemel O, Morsczeck C. A two-step strategy for neuronal differentiation in vitro of human dental follicle cells. Differentiation 77(5), 433–441 (2009).

Papers of special note have been highlighted as: • of interest; •• of considerable interest 1

Seo BM, Miura M, Gronthos S et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 364(9429), 149–155 (2004).  

•

Describes for the first time that periodontal ligament contains stem cells that have the potential to generate cementum/periodontal ligament like tissue in vivo.

2

Yao S, Pan F, Prpic V, Wise GE. Differentiation of stem cells in the dental follicle. J. Dent. Res. 87(8), 767–771 (2008).

•

Describes the existence of a possibly puripotent stem cell population in the dental follicle.

3

Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc. Natl Acad. Sci. USA 97(25), 13625–13630 (2000).

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Guo W, Gong K, Shi H et al. Dental follicle cells and treated dentin matrix scaffold for tissue engineering the tooth root. Biomaterials 33(5), 1291–1302 (2012).

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Describes a feasible strategy for tooth roots regeneration using dental follicle cells combined with treated dentin matrix scaffold in the alveolar fossa.

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