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Int Chin J Dent 2006; 6: 115-122.

Microarray analysis of osteogenesis gene of human dental follicle
cells stimulated with dexamethasone in vitro
Yongkuan Zhang, DDS, PhD,a Haiyan Sun, DDS, PhD,a Zhu Lin, DDS, PhD, ZuoLin Jin,
DDS, PhD, Yingchun Bi, DDS, PhD, and Xueying Han, DDS a, Equally contribute to the manuscript.
Department of Orthodontics, College of Stomatology, The Fourth Military Medical University, Xi’an,
P. R. China
Purpose: To examine osteoblastic genes expression of freshly isolated human dental follicle cells duringosteogenic differentiation in vitro using cDNA microarrays.
Materials and Methods: The dental follicle cells isolated from dental follicle were cultured in DMEM media
with 10-8 mol/L Dex for 4 weeks, the control without Dex. Total RNA was extracted from the cultured follicle
cells with Trizol. An analysis of osteoblastic genes expression of dental follicle cells treated with Dex and thecontrol using cDNA microarrays. The image file was inverted and spots were digitized using ScanAlyze
software.
Results: There were differences in the expression of 28 genes by human dental follicle cells cultured in thepresence of Dex. Among the 28 genes, the expression of 20 genes up-regulated and eight genes down-regulated
in Dex-treated cells than control.
Conclusion: Our work demonstrates differential osteoblastic genetic expression of dental follicle cells with a
Dex-based protocol by microarray for the first time. Some of these selective changes in gene activity might thus
reflect the fundamental events that underlie osteoblastic differentiation of human dental follicle cells in vitro.
(Int Chin J Dent 2006; 6: 115-122.)
Key Words: cDNA microarray, dental follicle cell, dexamethasone, differentiation, osteoblast.

Introduction
The dental follicle or dental sac is a loose ectomesenchymally derived connective tissue surrounding theenamel organ and the dental papilla of the developing tooth germ prior to eruption.1 Cells within the dental
follicle region, a loose connective tissue surrounding the developing tooth, harbours progenitor cells for theperiodontium. The periodontium as the supporting tissue of the tooth is composed of the periodontal ligament(PDL), alveolar bone and the mineralized bone-like cementum covering the tooth root surfaces. Substantial
evidence exists indicating that follicle cells are progenitors of periodontal cells including cementoblasts, PDL
fibroblasts, and alveolar osteoblasts.1,2 When triggered appropriately, dental follicle cells are considered to beable to differentiate toward a cementoblast/osteoblast phenotype. The differentiation capacity of these follicle
cells was proven by in vivo tests with SCID mice. Here, cells formed a cementumlike matrix as opposed tobovine PDL fibroblasts or bovine alveolar osteoblasts.3,4 Zhao et al. reported that BMP-2 promotes
differentiation of immortalized murine dental follicle cells towards an osteoblast or cementoblast phenotype.5
Recently, precursor cells (PCs) were isolated from dental follicle of human third molar teeth anddemonstrated that cultured PCs are unique undifferentiated lineage committed cells residing in the periodontium
prior or during tooth eruption. PCs differentially express osteocalcin (OCN) and bone sialoprotein (BS) after
transplantation in immunocompromised mice but without any sign of cementum or bone formation.6 Gene
expressions of osteocalcin (OCN), bone morphogenic protein (BMP)-2 and nestin were increased during theboth differentiation approaches.7
Bone formation is a complex process involving proliferation of osteoprogenitors and/or pre-osteoblasts andexpansion of the osteoblast population followed by osteoblast differentiation resulting in matrix maturation andmineralization. This sequence of events requires the up-regulation and inhibition of several genes that encode
phenotypic proteins, e.g. collagen type I, alkaline phosphatase (ALP), and osteocalcin.8 Transcription factors

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such as c-fos, c-jun, Cbfa1 are also important for osteoblast growth and differentiation.9,10 The expression ofthese key genes is further modulated by several factors including growth factors, peptide and steroid hormones.
Although several target genes of which relate to bone formation on human dental follicle cells are known, adetailed analysis of target genes involved in the osteoblastic differentiation of human dental follicle cells is stilllacking. In this study, the osteogenic gene expression changes in human dental follicle cells treated withdexamethasone were investigated by functional microarray technique.

Materials and Methods
Materials
Expression profiling was performed with the use of the GEArray DNA Q Series Microarrays: Human
Osteogenesis Gene Array containing 96 genes correlated with bone formation (Superarray Bioscience Corp.,
Bethesda, MD, USA). This particular array was selected because it contains genes involved in genes ofosteoblastic differentiation, some of which are makers of bone formation, including Growth factor andassociated molecule, Matrix and its associated protein, Cell adhesion molecule.
Cell culture
Human dental follicles from third mandibular molars were surgically removed from patients who needorthodontic treatment and informed content, the age of the patients varied between 12 and 13 years. Dental
follicles below the crown were separated from the tissue. The surfaces of the follicle tissues were cleaned andminced by using a sterilized scalpel. Also dental papilla tissue was discarded. Then tissues were digested andcultured as previously described.6 Minced and digested tissues of dental follicle explants were seeded into 60
mm plates in DMEM media (Gibco, Los Angeles, CA, USA) at 37˚C in 5% CO2 in a humidified atmosphere.
After single cells had attached on the plastic surface, non-adherent cells were removed by change of medium.
Plastic adherent cells were continuously propagated until confluence. The dental follicle cells isolated fromdental follicle were cultured at an initial density of approximately 5,000 cells/cm2 in DMEM media (Gibco) until
first passage.
Cell exposure to Dex
For dexamethasone analysis, cells were cultured as described previously.7 Dex was dissolved in DMEM
media with 10% FBS at a concentration of 10-8 mol/L, the control without Dex, Long-term cultures were madewith follicle cells at passage 5 for 4 weeks with media change twice a week, but without cell passaging. After 4
weeks, the culture medium was removed from the cells, which were then washed twice with PBS and then for
RNA extraction.
RNA extraction
Total RNA was extracted from the cultured follicle cells with Trizol. The pellet of total RNA was washedbriefly with 75% ethanol, resuspended in 20 µL DEPC-treated water. The total RNA was then treated with
DNase I (Gibco) for removal of any DNA contamination. The quantity and purity of the RNA were measured
by means of a spectrophotometer.
Expression profiling and data analysis
A GEArray Q series human osteogenesis gene array kit (SuperArray Inc., www.superarray.com, Bethesda,
MD, USA) was obtained. RNA (1.5 mg) from Dex-treated and the control were used as a template to generate
Biotin-16-dUTP labeled cDNA probes, according to the manufacturer’s instructions. The cDNA probes were116

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Int Chin J Dent 2006; 6: 115-122.

denatured and hybridized at 60˚C with the osteogenesis SuperArray membrane. The membrane was thenwashed, exposed using a chemiluminescent substrate, and analyzed by scanning the X-ray film and importing
the image into Adobe Photoshop as a TIFF file. The image file was inverted and spots were digitized using
ScanAlyze software, and normalized by subtracting the background as the average intensity value of three spots
containing plasmid DNA (PUC18). The average of two GAPDH spots were used as positive controls and set asbaseline values to which the signal intensity of other spots was compared. Using this normalized data, the signal
intensity from the membranes was compared using the GEArray Analyzer program. This analysis was repeatedthree times.
The detection parameters were arbitrarily set to report greater than two-fold changes in expression levels
either up or down. Differences in gene expression greater than two-fold are presented as the ratios of gene
expression at DEX treated cells compared with the control cells. To assess the reliability and repeatability of themethods, we performed the expression profiling experiments twice, each time point in duplicate and each set of
RNA labeled and hybridized twice. Comparison of the individual replicate filters showed that there were nodifferences between them within the threshold limits set as described above (data not shown). For the final
analysis, the replicate images were therefore merged to produce a composite image.

Results
Using the GEArray Analyzer software settings, Global expression analysis of genes associated with boneformation regulated by Dex. The representative array image of overall expression of the 96 genes in Dex-treatedcells (Fig. 1) and control (Fig. 2) was shown. Each gene was deposited as a duplicate for statistic analysis andquality control. We obtained evidence that there were differences in the expression of 28 genes by human dental
follicle cells cultured in the presence of Dex. Among the 28 genes, the expression of 20 genes was identified as
Dex-promoted genes which showed an up-regulation at least 2-fold in Dex-treated cells than control. And theexpression of eight genes was identified as Dex-suppressed genes which showed a down-regulation of more than
50% in Dex-treated cells than contro1. Among those, the expressions of five genes were inhibited more than
5-fold (Table 1). In Table 1, these 28 differentially expressed genes have been assigned to two groups based on
the functional activity of the corresponding protein; growth factor and associated molecule, matrix and its
associated protein. Cell adhesion molecules were not different between two groups.

Fig. 1. The representative array image of in Dex-treated cells.

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Fig. 2. The representative array image of in control cells.

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Table 1.

Int Chin J Dent 2006; 6: 115-122.

The 28 genes differentially expressed by human dental follicle cells in response to Dex.

GenBank

Description

NM_000478
NM_000711
NM_006128
NM_001200
NM_001201
NM_001202
NM_021073
NM_001718
NM_001719
NM_001720
NM_002006
NM_000660

Growth factor and associated molecule alkaline phosphatase
ALP
Bone gamma-carboxyglutamate (gla) protein (Osteocalcin)
Osteocalcin
Bone morphogenetic protein 1
BMP1
Bone morphogenetic protein 2
BMP2
Bone morphogenetic protein 3 (Osteogenic)
BMP3
Bone morphogenetic protein 4
BMP4
Bone morphogenetic protein 5
BMP5
Bone morphogenetic protein 6
BMP6
Bone morphogenetic protein 7 (Osteogenic protein 1)
BMP7
Bone morphogenetic protein receptor type IA
ALK-3
Fibroblast growth factor 2 (Basic)bFGF2
Transforming growth factor, beta 1 1
TGFβ1(Camurati-Engelmann disease)
Colony stimulating factor 3 (Granulocyte)
G-CSF
Fibroblast growth factor 1 (Acidic)
FGF1
MAD, Mothers against decapentaplegic homolog 1 (Drosophila)
Smad1
MAD, Mothers against decapentaplegic homolog 6 (Drosophila)
Smad6
MAD, Mothers against decapentaplegic homolog 7 (Drosophila)
Smad7
Transforming growth factor, beta 2
TGFβ2
Fibroblast growth factor 3
FGF3(Murine mammary tumor virus integration site (v-int-2) oncogene homolog)
Fibroblast growth factor receptor 1
FGFR1(fms-related tyrosine kinase 2, Pfeiffer syndrome) Matrix and its associated proteins
Collagen, type I, alpha 1
COL1A1
Collagen, type X, alpha 1 (Schmid metaphyseal chondrodysplasia) COL10A1
Collagen, type XV, alpha 1
COL15A1
Collagen, type XVI, alpha 1
COL16A1
Collagen, type XIX, alpha 1
COL19A1
Collagen, type III, alpha 1
COL3A1(Ehlers-Danlos syndrome type IV, autosomal dominant)Collagen, type IV, alpha 5 (Alport syndrome)
COL4A5Collagen, type VII, alpha 1
COL7A1(Epidermolysis bullosa, dystrophic, dominant and recessive)

NM_000759
NM_000800
NM_005900
NM_005585
NM_005904
NM_003238
NM_005247
NM_000604
NM_000088
NM_000493
NM_001855
NM_001856
NM_001858
NM_000090
NM_033380
NM_000094

Gene Name

Ratio
2.252E+05.857E+02.303E+03.336E+02.910E+03.474E+04.818E+0
3.109E+0#DIV/0!
2.353E+02.682E+0
3.097E+01.744E-1
4.720E-1
2.650E+02.326E-1
1.057E-1
4.016E-1
2.205E-1
1.662E-1
1.074E+1
2.980E+02.928E+0
2.007E+04.378E+02.646E-2
2.114E+02.204E+0

Discussion
Morsczeck and colleagues documented isolation of dental follicle cells from two individual human dental
follicles by their ability to adhere on a plastic surface. Both isolated cell lines were able to differentiate into amembrane-like structure with mineralized foci.6 We isolated dental follicle cells from human dental follicle
before the root began its formation using Morsczeck’s methods and applied osteogenic differentiation protocols
with dexamethasone. The process of osteogenic differentiation can be divided into several steps, consisting ofcell proliferation, extracellular matrix synthesis, maturation and mineralization. The process is complex, theregulators and participators consist of growth factor and associated molecule, matrix and its associated protein,and cell adhesion molecule. The TGF-β superfamily of growth factors includes BMPs, the TGF-βs, activins,
inhibins, myostatin, and others.11,12
BMPs are potent osteoblast differentiation factors in vitro. Various BMPs, including BMP-2, BMP-4, andBMP-7, induce the differentiation of multipotential mesenchymal cells (e.g., C3H10T1/2 cells) into both
osteochondrogenic lineage cells and osteoblast precursor cells.13-16 The expression patterns of several of BMPs,
particularly BMP-2, BMP-4, and BMP-7, suggest a role in the generation of facial bone.17,18

BMP-2 isexpressed during facial skeletogenesis, in the developing tooth germ, and in the facial ectomesenchyme andMeckel’s cartilage.19 BMP-2 and BMP-4 also play important roles in mandibular induction20 and in other oral

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and facial structures.21 We performed microarray analysis on human dental follicle cells in which BMP1-BMP8
were included to obtain more insight into the mechanisms that govern Dex-induced differentiation of dental
follicle cells. In the present study, we observed BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7
transcripts up-regulated, and only BMP-8 not in dental follicle cells treated with Dex for 4 weeks. Morsczeck
and colleague7 also reported up-regulation of expression of BMP-2, the differentiation-related gene within
osteoblast differentiation, followed by commitment and establishment of the bone phenotype in human dental
follicle cells of Dex treatment. Zhao et al. reported that BMP-2, in a dose- and time-dependent manner, induced
cementoblast/osteoblast differentiation of follicle cells in CD-1 mice, as reflected by enhanced core binding
factor a1 (Cbfa1), bone sialoprotein (BSP), and osteocalcin (OCN) mRNA expression and enhanced mineralformation.5

It suggests the BMPs play an important role in the differentiation of dental follicle cells toosteoblast-like cells. It was reported that BMP-3 is a negative determinant of bone density.22 But in our
experiment, BMP-3 was also up-regulated.
Smad1 is expressed mainly in the inner enamel organ epithelium and the adjacent dental mesenchyme,suggesting that Smad1 mediated BMP signaling is critical for epithelial-mesenchymal interaction during toothmorphogenesis.23

Smad6 and Smad7 are inhibitors of TGF-β, activin and BMP signaling.

One of themechanisms proposed to explain the inhibitory effects of Smad6 and Smad7 is that each of these Smads can bindto diverse TGF-β family receptors and interfere with phosphorylation of R-Smads.

For instance, Smad7

associates stably with the TGF-β receptor complex and inhibits TGF-β- mediated phosphorylation of Smad2 andSmad3. Alternatively, another mechanism which may help to explain the selective inhibition of BMP signaling
by Smad6 suggests that, at low concentration, Smad6 can compete with Smad4 for binding to activated Smad1
and block the BMP signaling pathway.24

In our experiments, a significant increase in BMPs, Bonemorphogenetic protein receptor and Smad1, moreover decreased in Smad 6, Smad 7 of Dex-treated dental
follicle cells than controls. This suggests that the Smads play an important role in differentiation of humandental follicle cells to osteoblasts, and Dex induced osteoblastic differentiation involved in BMP and Smadpathways.
While in vitro studies on TGF-β2 and TGF-β3 expression are poorly documented, the rapid up-regulation of
TGF-β1 mRNA has previously been shown to occur in bonelike cells as a result of fluid flow-induced shear
stress25 and also during mechanical stretching of osteoblasts.26 Recombinant TGF-β1 alone could not replace
the components and its inclusion in cultures resulted only in stimulation of matrix secretion by existing cells of
the dental papillae and no differentiation of odontoblasts. However, when recombinant TGF-β1 was combinedwith either an inactive preparation of dentine extracellular-matrix components, heparin or fibronectin,
odontoblast differentiation was induced.27 Dex not only increased the gene expression of TGF-β1 in our
experiments, but also enhanced the expression of the Fibronectin in the works of Morsczeck and colleague. It
suggests that TGF-β1 plays an important role in the process of differentiation of dental follicle cells toosteoblast-like cells.

Signals for TGF-β1 and TGF-β3 transcripts in the mouse increase significantly asodontoblasts differentiated, whereas TGF-β2 transcripts were high in preodontoblasts and diminished withpolarization during odontoblast cytodifferentiation.28 Other studies also show only weak detection of TGF-β2
transcripts in mouse and human odontoblasts.29 Using microarray technique, we found that Dex up-regulated
the TGF-β1 and down-regulated TGF-β2, but not the TGF-β3 on the human dental follicle cells at first time.
ALP is not a bone matrix protein, yet it is well known that it is closely related to bone matrix mineralization.

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During the late stage, osteocalcin (OC) production is observed. OC is thought to be associated with matrix
calcification and is regarded as a late maturation marker. OPN is an extracellular matrix protein and a cytokine
that has been reported to act in a number of physiological and pathological events including bone
remodeling.30-33 The results of our experiments revealed that ALP, OCN of the human dental follicle cells
up-regulated after being treated with Dex for 4 weeks, but OPN not. OCN transcripts increased by about 5-foldat day 28 of differentiation in Dex-treated cells, compared to untreated cells. This is consistent with previous
reports.7 Dexamethasone affects the differentiation of bone marrow stromal cells to osteoblastic cells. It
stimulates ALP activity,34-36 osteonectin34 and osteocalcin28,34 the type I collagen synthesis.34 These results wereconsistent with our experiments.
Ninety percent of the organic bone matrix is collagen, mostly type I. Our study showed that Dex increased
the mRNA levels for type I, type X, type XV, type XVI, type XIX, type IV and type VII respectively, anddecreased the mRNA level of Collagen III for the analysis of the microarray data. A similar up-regulation of
COL X mRNA has been reported with the differentiation of embryonic stem cells into chondrocytes,
hypertrophic chondrocytes, and osteoblasts.37
Collagen III and bone sialoprotein are useful markers in that they aid in differentiating among cementum,
dentin, and bone (absent or low collagen III, modest bone sialoprotein expression).38,39 Both insulin-like growth
factors and receptors (IGF-RI and IGF-RII) have also been implicated as regulators of bone turnover,40 and
IFG-I did not affect the proliferation or early osteogenic differentiation of human marrow stromal cells. The
beneficial effects of IGF-I on the skeleton are not mediated primarily via an effect on osteoprogenitor fraction
and are thus consistent with the hypothesis that the effects of IGF-I are differentiation dependent and restricted
largely to the more mature cells of the osteoblast lineage.41 In this study, we demonstrated by microarray thatDex increased the expression of IGF-IR, but decreased the expression of IFG-II, but not IGF-I in human dental
follicle cells. The lack of effect of Dex on the expression of the expression IGF-I is consistent with the results ofearlier studies using human marrow stromal cells.42 This suggests that IGF-I does not play a major role indirecting dental follicle cells toward the osteogenic pathway of differentiation.
Cam et al.43 and Russo et al.44 elucidated the expression of FGF-2 in the cells differentiating from the dental
papilla and dental follicle in the region of the crown during early tooth development. They showed that FGF-2
expression is specific to the epithelium and ectomesenchyme associated with the developing crown and showed
progression with time. The results of our experiment revealed that Dex up-regulated the expression of FGF-2,
but down-regulated the expression of FGF-1, FGF-3 and FGFR-1. It suggested that FGF-1, FGF-2, FGF-3 and
FGFR-1 were involved the differentiation of human dental follicle cells.
Our results revealed that the expression of G-CSF was decreased by Dex, which suggested G-CSF was
involved in the osteoblastic differentiation progress of human dental follicle cells. Osteoblast activity, as
measured by histomorphometry and osteocalcin expression, is strongly down-regulated during G-CSF treatment.
However, the G-CSF receptor is not expressed on osteoblasts; accordingly, G-CSF had no direct effect on
osteoblast function.45
In conclusion, using a microarray approach, we have identified a number of genes that are associated with theprocess of human dental follicle cells differentiation at first time. In our own studies, we observed 20 transcripts
up-regulated and eight transcripts down-regulated in human dental follicle cells treated with Dex for 4 weeks.
Our results presented here demonstrate conclusively that human dental follicle cells display osteoblastic feature

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after treated with Dex for 4 weeks, and the progress involved a number of osteoblastic genes. It might be highlyinteresting to examine what role these gene products play in differentiation and how they work together.

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Correspondence to:Dr. ZuoLin Jin
Department of Orthodontics, College of Stomatology, The Fourth Military Medical University
145 Changle Xi Road, Xi’an, 710032, P. R. China
Fax: +86-29-84776138 E-mail: zuolinj@fmmu.edu.cn
Received September 23, 2006. Accepted November 21, 2006.
Copyright ©2006 by the International Chinese Journal of Dentistry.

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