Clinical Research
Microarray Evaluation of Age-related Changes in Human
Dental Pulp
Michelangelo Tranasi, DDS,* Maria Teresa Sberna, MD, DDS,† Vincenzo Zizzari, DDS,*
Giuseppe D’Apolito, DDS,* Filiberto Mastrangelo, MD, DDS, PhD,* Luisa Salini, DDS,‡
Liborio Stuppia, MD, DDS,§ and Stefano Tetè, MD, DDS*
Abstract
Introduction: The dental pulp undergoes age-related
changes that could be ascribed to physiological, defensive,
or pathological irritant-induced changes. These changes
are regulated by pulp cell activity and by a variety of extra-
cellular matrix (ECM) macromolecules, playing important
roles in growth regulation, tissue differentiation and orga-
nization, formation of calcified tissue, and defense mecha-
nisms and reactions to inflammatory stimuli. The aim of
this research was to better understand the genetic changes
that underlie the histological modification of the dental
pulp in aging. Methods: The gene expression profile of
the human dental pulp in young and older subjects was
compared by RNA microarray analysis that allowed to
simultaneously analyze the expression levels of thousands
of genes. Data were statistically analyzed by Significance
Analysis of Microarrays (SAM) Ingenuity Pathway Analysis
(IPA) software. Semiquantitative and real-time reverse-
transcriptase polymerase chain reaction analyses were per-
formed to confirm the results. Results: Microarray analysis
revealed several differentially expressed genes that were
categorized in growth factors, transcription regulators,
apoptosis regulators, and genes of the ECM. The compar-
ison analysis showed a high expression level of the biolog-
ical functions of cell and tissue differentiation,
development, and proliferation and of the immune,
lymphatic, and hematologic system in young dental pulp,
whereas the pathway of apoptosis was highly expressed
in older dental pulp. Conclusions: Expression profile anal-
yses of human dental pulp represent a sensible and useful
tool for the study of mechanisms involved in differentia-
tion, growth and aging of human dental pulp in physiolog-
ical and pathological conditions. (J Endod 2009;35:
1211–1217)
From the *Department of Oral Science University ‘‘G. d’An-
nunzio,’’ Chieti, Italy; †Department of Oral Science, University
‘‘Ateneo Vita-Salute San Raffaele,’’ Milan, Italy; ‡Base Sanitary
District (D.S.B), Francavilla al Mare (Ch), Italy; and §Center of
Excellence of Aging (CESI), Department of Clinical Sciences
and Bio-imaging, Department of Biomedical Sciences, University
‘‘G. d’Annunzio,’’ Chieti, Italy.
Address requests for reprints to Prof. Stefano Tete,
University of Chieti, Via dei Vestini 66013 Chieti, Italy, E-mail
address: tete@unich.it.
0099-2399/$0 - see front matter
Copyright ª 2009 American Association of Endodontists.
doi:10.1016/j.joen.2009.05.026
JOE — Volume 35, Number 9, September 2009
Key Words
Age-related changes, dental pulp, extracellular matrix, gene expression, microarray
T he dental pulp is a loose connective tissue characterized by its specific anatomic
location and organization being surrounded by dentin, which is a hard and inelastic
tissue. Dental pulp is composed of cells (fibroblasts, odontoblasts, and undifferentiated
mesenchymal cells) in contact with a complex chain of macromolecules secreted extra-
cellularly that form the extracellular matrix (ECM) (1). The extracellular components
are largely responsible for the physiological properties of the tissue.
The ECM plays an important role in growth regulation and tissue differentiation
and organization: important processes like defense mechanisms, inflammatory reac-
tions, and the formation of calcified tissue occur extracellularly. The main macromol-
ecules in dental pulp ECM are collagenous proteins (type I, III, and V collagen) (2),
bone sialoprotein, osteopontin, dentin matrix protein-1, osteocalcin, and osteonectin
(3, 4). Odontoblasts also secrete tooth-specific proteins, such as dentin sialoprotein
(DSP), dentin phosphoprotein, and dentin glycoprotein (DGP), which are encoded
by a single gene called dentin sialophosphoprotein (DSPP) (5, 6). DSPP is an ECM
protein of primary importance in tooth formation, and it plays an essential role in phys-
iological tooth development (7–9).
The dental pulp, like other body tissues, undergoes age-related changes that are
difficult to differentiate from physiological, defensive, and pathological irritant-induced
changes (10). One of the most obvious features of aging is a reduction in size of the pulp
chamber caused by the continual secretion of dentinal matrix (physiological secondary
dentinogenesis) by odontoblasts (11). This process tends to narrow the originally wide-
open root apex; with aging, the apical deposition of secondary dentin and cementum
increases, and circulation and innervation are compromised. As the pulp aging process
continues, there is a great decrease in the number of cells (odontoblasts, fibroblasts,
and mesenchymal cells) and a corresponding apparent increase in the number of
collagenous fibers; the number of blood capillaries, lymphatics, and nerves decrease,
and their connective tissue sheaths become part of the remaining fibrous pulp. Other
age-related changes include the increase of reticular fibers, lipid infiltration, and calci-
fication (12).
The vitality of dentin-pulp complex during tissue homeostasis, after injury and in
aging, is dependent on pulp cell activity and on the signaling processes that regulate the
behavior of these cells. Not only do the cells of the pulp maintain tissue homeostasis
after tooth development, but they also underpin the defense reactions taking place in
response to injury, such as caries, and the reparative events leading to tissue regener-
ation. Growth factors are the key group of molecules responsible for signaling a variety
of cellular processes during pulp development (13). The roles of growth factors in cell
signaling during tooth development and regeneration have only recently been identified.
They play a central role in signaling various aspects of tissue development and regen-
eration and may regulate genes controlling cell proliferation, cell differentiation, or the
secretory products of the cells (14).
Growth factors are responsible for signaling many of the key events in tooth
morphogenesis and differentiation; the recapitulation of these processes after dental
injury allows tissue regeneration. Apoptosis is another key phenomenon in tooth life;
SAM Evaluation of Age-related Changes in Human Dental Pulp 1211
Clinical Research
it is a genetically regulated form of cell death that is implicated in bio-
logical processes ranging from embryonic development to aging and
plays an essential role in tooth development and aging (15, 16).
The aim of this research was to evaluate gene expression profile of
young and older human dental pulp in physiological conditions in order
to better understand the molecular mechanism playing a role in tooth
development and aging. In this research, changes in the gene expres-
sion profile were determined by comparing pulp samples using the mi-
croarray technique. This allows us to simultaneously analyze the
expression levels of thousands of genes in a single experiment and to
compare the most important function of dental pulp in aging.
Materials and Methods
Pulp Samples
Dental pulp samples were obtained from human third molar teeth,
extracted for orthodontic or prosthetic reasons, from patients under-
going treatment in the clinics of the Department of Oral Science at
the University ‘‘G. d’Annunzio’’ of Chieti after informed patient consent.
The pulp of 10 third molars of young subjects (during apex maturation:
age 18-20) and 10 third molar pulps of older subjects (age 57-60) were
obtained. Only teeth with no clinical or radiographic evidence of caries,
periodontal disease, or restoration were considered for this study.
Immediately after extraction, teeth were sliced for the removal of
the pulps. The external surfaces were cleaned with chlorhexidine 0.2%,
and all external soft tissue remnants were mechanically removed. A 2-to
3-mm deep groove was cut around the teeth using a diamond cutter,
avoiding exposure of the pulp. Then, the teeth were fractured through
the cutting line, and pulps were removed from the coronary pulp cham-
bers. The specimens were divided into two pools of five third molars
from young subjects (named respectively Young-1 and Young-2
groups) and two pools of five third molars from older subject (named
respectively Old-1 and Old-2 groups).
RNA Isolation
Pulps were placed into RNA later (Ambion, Austin, TX) immedi-
ately after removal. Total RNA was isolated by using the SV Total RNA
Isolation System (Promega, Madison, WI). RNA concentration and
purity were determined by measuring absorbencies at 260 and 280
nm, and a 260:280 ratio of 1.7 was considered acceptable for analysis.
RNA was amplified using the ‘‘Amino Allyl MessageAmp II amplified RNA
(aRNA) Amplification kit’’ (Ambion), which is able to produce aRNA
containing 5-(3-aminoallyl)-UTP modified nucleotides and specifically
bind fluorescent dyes Cyanin3 (Cy3) and Cyanin5 (Cy5).
Microarray Expression Profiling
The obtained aRNA (10-20 mg) was labeled with Cy3 or Cy5
(Amersham; Pharmacia Biotech, Buckinghamshire, UK) and hybridized
on the arrays. In order to correct differences in dye efficiencies and
minimize such a source of bias, we performed dye-swap experiments
using the assumption that both channels should be equally bright.
Four different experiments were performed using a high-density array
containing about 42,658 sequences (21,329 transcripts present in
replicates) in the form of oligonucleotidic sequences of about 70 bases
each, specific for different sequences of the human transcriptome
(Micro Cribi, Padova, Italy).
Fluorescent signals were captured by a ScanArray 5000 Packard
laser scanning (Packard BioChip Technologies, Billerica, MA) and
analyzed using the ‘‘ScanArray Express’’ software. For normalization,
a single scaling factor was applied to each specimen so that the average
Cy3:Cy5 ratio was 1.0 for meaningful comparison. Obtained data were
statistically analyzed using the SAM system (Significance Analysis of
1212 Tranasi et al.
Microarrays). Expression level of genes was obtained from the log2
of the ratio between older pulp (numerator) and young pulp (denom-
inator) fluorescent signal intensity. The ratios were calculated by two
independent analyses of each couple of samples (Old-1/Young-1 and
Old-2/Young-2); then, the average of ratios obtained was used for
fold change analysis. In each experiment, a 1.5-fold change in the signal
of each spot was considered as the evidence of a different expression of
the specific transcript.
Ingenuity pathway analysis (IPA) software (Ingenuity Systems,
Redwood City, CA) was used to understand and investigate gene inter-
actions, molecular pathways, and main gene functions involved during
dental pulp aging. Gene accession numbers were imported into the IPA
software, and significant genes were selected using cutoffs of p < 0.05
and fold change >1.5. Using the software, a comparison analysis was
performed to compare biochemical, biologic, and molecular functions
in the two stages. The identified genes were also mapped to genetic
networks available in the Ingenuity database and then ranked by score.
IPA was performed to gather even more information on gene changes
and to link them to biological functions in the model system of aging.
Semiquantitative Reverse Transcriptase Polymerase
Chain Reaction
Semiquantitative reverse transcriptase polymerase chain reaction
(RT-PCR) analysis was performed using the pooled RNA samples previ-
ously described for microarray analysis. Differential expression analysis
was performed for the genes transforming growth factor alpha (TGFA),
transforming growth factor beta 1 (TGFB1), fibroblast growth factor 1
(FGF1), caspase 1, apoptosis-related cysteine peptidase (CASP1), dentin
sialophosphoprotein (DSPP), alkaline phosphatase (ALPL), osteonectin
(SPARC), and connective tissue growth factor (CTGF). First-strand comple-
mentary DNA (cDNA) was synthesized from 1.5 mg of total RNA using the
RT-PCR system RETROscript (Applied Biosystem, Milan, Italy) with random
hexamers. The resultant cDNA (2 mL) was amplified in a 100-mL reaction
volume containing PCR reaction buffer, 1.5 mmol/L MgCl2, 0.2 mmol/L
each deoxy-deoxyribonucleotide triphosphate (dNTP), 1 mmol/L oligonu-
cleotide primers (MWG Biotech, Ebersberg, Germany), and 2.5 U Ampli-
Taq Gold DNA polymerase (Applied Biosystems). The housekeeping gene
glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was used as the
control. The identity of the products was confirmed by cycle sequencing
the amplified cDNA. Amplification products were resolved by 1.5% agarose
gel electrophoresis and visualized by ethidium bromide staining. Primers
for semiquantitative RT-PCR are listed in Table 1.
Quantitative Real-Time PCR
Quantitative real-time PCR analysis was performed using RNAs ob-
tained from pulps from young and older dental pulps. The expression
levels of the genes TGFA, TGFB1, FGF1, caspase 1, CASP1, DSPP, ALPL,
SPARC, and CTGF were analyzed. The validation analysis was performed
by using the HT 7900 sequence detection system (Applied Biosystems,
Foster City, CA). TaqMan probes (TaqMan Gene Expression Assays,
Applied Biosystems) were used for signal analysis. Housekeeping GADPH
was used as the internal control for relative gene expression quantization.
Results
Microarray Analysis
The gene expression data were analyzed through different analysis
techniques, including ScanArray Express software, the SAM system, and
IPA. All the methods were consistent and showed that over 1,300 genes
were significantly expressed. It was possible to identify the top biological
functions and to compare their expression in the human dental pulp at
two different development stages.
JOE — Volume 35, Number 9, September 2009
 Clinical Research
TABLE 1. GenBank Accession Codes and Primers Used for Genes Analyzed with Semiquantitative RT-PCR
Selected Genes Gene Bank Primers
Transforming growth factor, NM_003236 Forward: AGG TCT CTA ATG ACC TTA Reverse: TGG TCT GAA
alpha (TGFA) GAG CCC AGA
Transforming growth factor, NM_000660 Forward: CAT CCA TGA CAT GAA CCG ACC CTT Reverse: ACA GAA
beta 1 (TGFB1) GTT GGC ATG GTA GCC CTT
Fibroblast growth factor 1 (FGF1) NM_000800 Forward: ATG GCC GAA GGG GAG ATC ACA ACC Reverse: TTA GTC
AGA AGA TAC CGG GAG GGG
Caspase1, apoptosis-related cysteine NM_033292 Forward: TGG TCT TGT GAC TTG GAG GA Reverse: TGG CTT CTT ATT
peptidase (CASP1) GGC ACG AT
Dentin sialophosphoprotein (DSPP) NM_014208 Forward: AAT GGG ACT AAG GAA GCT G Reverse: GAG GAT AAA GGA
CAA CAT GG
Alkaline phosphatase (ALPL) NM_000478 Forward: ATC TTT GGT CTG GCC CCC ATG Reverse: ATG CAG GCT
GCA TAC GCC AT
Osteonectin (SPARC) NM_003118 Forward: ACA TGG GTG GAC ACG G Reverse: CCA ACA GCC TAA
TGT GAA
Connective tissue growth NM_001901 Forward: GAA GAC ACT TAC GGC CCA GA Reverse: AAA CTT GAT
factor (CTGF) GGG CTT GGA GA
GAPDH (Housekeeping) NM_002046 Forward: TGA TGA CAT CAA GAA GGT GGT GAA G Reverse: TCC TTG
GAG GCC ATG TGG GCC AT

SAM analysis shortlisted 560 genes upregulated in older pulp and
758 genes in young dental pulp (showing a 4:1 ratio) involved in pulp
development and homeostasis. We focused on the most significant gene
family involved in dental pulp development, which can be categorized
into the following: growth factors, transcription regulators, apoptosis
regulators, and genes of the ECM.
In our study, SAM analysis showed the upregulation in young
healthy dental pulp (negative fold change) of several genes linked to
growth factors like the bone morphogenetic protein 15, the growth
differentiation factor family, and the vascular endothelial growth factor
A. The FGF family, the CTGF, and vascular endothelial growth factor B,
instead, resulted upregulated in older dental pulp (positive fold
change) (Table 2). As for genes belonging to transcriptional reculators
(Table 3), we found that in young dental pulp there was a high expres-
sion of Jun Oncogene (JUN) and Jun B Proto-Oncogene (JUNB),
whose products are members of the transcription factors activating
protein-1.
Table 4 shows the expression of genes involved in the activation
and regulation of apoptotic processes in human dental pulp; it is
possible to observe that in older dental pulp several proapoptotic
genes, such as apoptosis-inducing factor mitochondrion-associated
1 (AIFM1) and programmed cell death family (PDCD5 and PDCD7)
resulted upregulated, whereas the X-linked inhibitor of apoptosis
(BIRC4), an important apoptosis inhibitor, appears downregulated.
Finally, the expression of the encoding of important genes for
collagenous and noncollagenous ECM protein was reported (Table 5).
Collagen type 1 (COL1A1, COL1A2) resulted upregulated in young
dental pulp, whereas the expression of collagen type 3 (COL3A1) and
collagen type IX (COL9A2, COL9A3) was higher in older dental pulp.
Regarding noncollagenous ECM protein, the data show a very high
expression of DSPP and AMBN in young dental pulp.
Then, the top biological functions generated by IPA for the signif-
icant genes were compared in the samples from young dental pulp and
from old dental pulp (Fig. 1); the p value represents the statistical signif-
icance of the specific biological function: -log (p value) increases with
statistical significance (-log (p value) = 1.30 when p value = 0.05).
Canonical pathway comparison analyses identified top biological func-
tions that were most significant to our data set and showed their expres-
sion in young and older pulp. The presence of biological process is
reported in relation to tissue vitality and aging. In particular, the expres-
sion levels of genes linked to ‘‘cellular growth and proliferation’’; ‘‘gene
expression’’; ‘‘tissue development’’; ‘‘immune, lymphatic, and hemato-
logical system development’’; and ‘‘cell death’’ have been evaluated.
Worthy of note are the significant lower expression of all these functions
and the higher expression of genes involved in cell death regulation in
older dental pulp compared with young dental pulp.
Semiquantitative and Real-Time RT-PCR Analyses
To validate the microarray data, semiquantitative and real-time RT-
PCR analyses were performed on selected genes. Data confirmed that
the eight genes analyzed by semiquantitative RT-PCR (Fig. 2) were
differentially expressed as predicted by microarray data. Furthermore,

TABLE 2. List of Growth Factors in Young and Older Dental Pulp

Growth factors

Name Description Gene bank Fold change Type

BMP15 Bone morphogenetic protein 15 NM_005448 –1.655 Growth factor
CTGF Connective tissue growth factor NM_001901 1.759 Growth factor
FGF1 Fibroblast growth factor 1 (acidic) NM_000800 1.632 Growth factor
FGF5 Fibroblast growth factor 5 NM_004464 2.578 Growth factor
GDF10 Growth differentiation factor 10 NM_004962 –1.581 Growth factor
PDGFA Platelet-derived growth factor alpha NM_002607 –2.051 Growth factor
polypeptide
TGFA Transforming growth factor, alpha NM_003236 –1.573 Growth factor
TGFB1 Transforming growth factor, beta 1 NM_000660 1.668 Growth factor
VEGFA Vascular endothelial growth factor A AF022375 –1.590 Growth factor
VEGFB Vascular endothelial growth factor B NM_003377 2.677 Growth factor
Positive values of fold change indicate genes upregulated in older dental pulp, and a negative value indicates genes expressed in young dental pulp.

JOE — Volume 35, Number 9, September 2009 SAM Evaluation of Age-related Changes in Human Dental Pulp 1213
Clinical Research
TABLE 3. Expression of Transcription Regulators in Young and Older Dental Pulp
Transcription regulators

Name Description Gene Bank Fold change Type

JUN jun oncogene NM_002228 –1.547 Transcription regulator
JUNB jun B proto-oncogene NM_002229 –2.262 Transcription regulator
FOS v-fos FBJ murine osteosarcoma viral NM_005252 –4.216 Transcription regulator
oncogene homolog
PAX6 paired box 6 NM_001604 –1.505 Transcription regulator
NOTCH1 Notch homolog 1, translocation- NM_017617 –1.701 Transcription regulator
associated (Drosophila)
NOTCH2 Notch homolog 2 (Drosophila) NM_024408 –2.39 Transcription regulator
NOTCH3 Notch homolog 3 (Drosophila) NM_000435 –1.95 Transcription regulator
Oncogenes and genes of the Notch signaling pathway result expressed in young dental pulp.

a statistical analysis was performed to correlate fold change with differ-
ential expression detected by PCR (Table 6).
Discussion
Age-related changes of the dental pulp have been extensively
studied. As a tooth ages, vascular, lymphatic, and nerve supplies
decline and fibroblasts decrease in size and number. In fact, a reduc-
tion of 15.6% in crown odontoblasts, a reduction of 40.6% in root
odontoblasts, and a decreased secretory activity have been observed,
suggesting that the reparative capacity of the pulp is compromised
with aging (17). Furthermore, age-related changes include increases
in cross-linkages and the number of collagen fibers, lipid infiltration,
and calcifications (18). Despite many studies having investigated the
mechanisms underlying these age-related changes, we still have
much to learn of the biological control mechanisms responsible for
cellular activity and survival throughout life.
In this study, we performed a RNA microarray comparison anal-
ysis, focusing on changes that occur in the gene expression profile of
the human dental pulp during its development and aging. We investi-
gated dental pulp tissue at two different developmental stages: young
dental pulp during apex maturation and old dental pulp.
Microarray is a recent and useful tool that provides an indication
of the changing gene expression between two samples that, supported
by the appropriate software statistical analysis, provide significant data
(19). Over the past 2 decades, genetic dissection of the tooth develop-
ment process has identified signaling molecules and factors that
partner with them allowing precise development of the tooth at the
appropriate developmental stage. The availability of the complete
genomic of humans as well as their representative transcriptomes
has provided a platform for large-scale interrogation of genes in
specific organs (20). In this research, IPA identified the most signifi-
cant biological functions in human dental pulp at two different develop-
ment stages and showed the different expression levels of the genetic
pathways involved in aging.
A significant reduction of the vitality in older dental pulp results
from the ‘‘gene expression,’’ ‘‘cellular and tissue development,’’
‘‘cellular growth and proliferation’’ charts. These data are confirmed
by the higher expression of growth factors like bone morphogenetic
protein, TGFA, growth factor differentiation family, platelet-derived
growth factor a, vascular endothelial growth factor a, and FGF family
in young dental pulp. The roles of growth factors in cell signaling during
tooth development and regeneration have only recently been identified
(21, 22): high expression of growth factors in young dental pulp may
indicate that they regulate the genes controlling cell proliferation and
cell differentiation and that they are responsible for signaling many of
the key events in tooth morphogenesis and differentiation (23).
On the other hand, our research shows the up-regulation of
growth factors such as CTGF, FGFs, and TGFB1 in old dental pulp. These
proteins, secreted extracellularly, perform many cellular functions,
including the control of cell growth, cell proliferation, cell differentia-
tion and apoptosis. TGFB and FGFs family appear to be important mole-
cules mediating this signaling of odontoblast differentiation (24).

TABLE 4. List of Genes Involved in the Activation and Regulation of Apoptotic Processes
Apoptosis regulators

Name Description Gene bank Fold change Type

AIFM1 apoptosis-inducing factor, mitochondrion- NM_004208 1.706 Enzyme
associated, 1
MOAP1 modulator of apoptosis 1 AK024029 2.472 Other
PDCD5 programmed cell death 5 NM_004708 2.045 Other
PDCD7 programmed cell death 7 BC016992 2.926 Other
PDCD6IP programmed cell death 6 interacting protein AB037796 1.795 Other
CAPN11 calpain 11 NM_007058 2.396 Peptidase
CASP1 caspase 1, apoptosis-related cysteine peptidase NM_033292 3.104 Peptidase
CASP3 caspase 3, apoptosis-related cysteine peptidase NM_004346 3.176 Peptidase
CASP6 caspase 6, apoptosis-related cysteine peptidase NM_001226 2.761 Peptidase
CASP7 caspase 7, apoptosis-related cysteine peptidase NM_033339 2.1 Peptidase
AATF apoptosis antagonizing transcription factor NM_012138 1.841 Transcription
LYST lysosomal trafficking regulator NM_000081 3.885 Transporter
SYVN1 synovial apoptosis inhibitor 1 AB058713 –1.574 Transporter
BIRC4 X-linked inhibitor of apoptosis NM_001167 –1.494 Other
Positive values indicate genes upregulated in older dental pulp, and negative values indicate genes downregulated in older dental pulp.

1214 Tranasi et al. JOE — Volume 35, Number 9, September 2009

 Clinical Research
TABLE 5. List of Genes Involved in the ECM Formation and Mineralization
Genes encoding for collagenous and non-ollagenous ECM proteins

Name Description Gene bank Fold change Type

COL10A1 Collagen, type X, alpha 1(Schmid NM_000493 2.645 Other
metaphyseal chondrodysplasia)
COL1A1 Collagen, type I, alpha 1 NM_000088 –1.885 Other
COL1A2 Collagen, type I, alpha 2 NM_000089 –1.719 Other
COL2A1 Collagen, type II, alpha 1 NM_001844 –1.529 Other
COL3A1 Collagen, type III, alpha 1 AK021531 1.744 Other
COL5A2 Collagen, type V, alpha 2 NM_000393 –1.573 Other
COL5A3 Collagen, type V, alpha 3 NM_015719 1.868 Other
COL8A1 Collagen, type VIII, alpha 1 NM_020351 1.981 Other
COL9A2 Collagen, type IX, alpha 2 NM_001852 2.722 Other
COL9A3 Collagen, type IX, alpha 3 NM_001853 2.606 Other
AMBN Ameloblastin (enamel matrix protein) NM_016519 –5.900 Other
DMP1 Dentin matrix acidic phosphoprotein NM_004407 2.286 Other
ALPL Alkaline phosphatase NM_000478 2.123 Phosphatase
IBSP Bone sialoprotein, bone sialoprotein II NM_004967 3.198 Other
DSPP Dentin sialophosphoprotein NM_014208 –8.992 Other
OGN Osteoglycin NM_033014 1.541 Other
SPARC Osteonectin NM_003118 –1.268 Other
MGP Matrix Gla protein NM_000900 2.262 Other

Positive values indicate genes upregulated in older dental pulp, and negative values indicate genes expressed in young dental pulp.

Secretion of these growth factors by the inner dental epithelium, their
sequestration within the dental basement membrane and their presen-
tation to the undifferentiated dental papilla cells, provide the control of
odontoblasts differentiation in the tooth germ (25). However, in the
mature tooth, similar processes occur to allow recruitment of dental
pulp stem cells and their differentiation toward odontoblast cells for
dentin bridge formation (23, 26).
CTGF is an ECM-associated signaling molecule having mitogenic
and chemotactic activities that stimulate synthesis of extracellular matrix
components, promoting both the growth and the differentiation of most
mesenchymal cells (27). In literature, it has been documented that
CTGF significantly enhances the expression of collagens, especially of
collagen type III. In our opinion, and according to existing findings,
TFGB1, FGF1, and CTGF upregulation in older pulp may play a role in

Figure 1. The top biological functions generated by the IPA comparison analysis; the p value represents the statistical significance of the specific biological
function: -log (p value) increases with statistical significance.

JOE — Volume 35, Number 9, September 2009 SAM Evaluation of Age-related Changes in Human Dental Pulp 1215
Clinical Research
Figure 2. Eight genes selected for semiquantitative RT-PCR analysis. Expression levels are normalized to GAPDH housekeeping gene levels.
the reactivation of odontoblast activity during reparative dentinogenesis
processes (28, 29).
The expression in young dental pulp of several transcription
factors like JUN and JUNB and the presence of the NOTCH pathway
suggest a role for these genes in the control of odontoblast differentia-
tion. In fact, JUN and JUNB products are members of the transcription
factors activating protein-1, which probably controls most of the ECM
components (30), whereas NOTCH is reported to be involved in cell
fate specification and proliferation during odontogenesis (31).
Moreover, IPA comparison analysis shows other interesting func-
tions, differently expressed during pulp aging, that are related to the
immune, lymphatic, and hematologic system development. The low
expression in older dental pulp of these functions could be related to
physiological dental pulp changes. During aging, the deposition of
secondary dentin increases, and the blood, lymphatic, and nerve supply
become compromised; blood vessels undergo arteriosclerotic changes,
the lymphatic system shows age-related degenerative changes, and the
pulpal nerve shows progressive mineralization (12). Mitsiadis asserts
that the dental pulp volume decreases gradually upon aging because
of the continuous production of dentin matrix by odontoblasts and
that this age-related pulp chamber reduction is associated with the elim-
ination of a certain number of odontoblasts by apoptosis (32). In agree-
ment with this statement, our research shows a rise of apoptosis
regulators in older dental pulp and the upregulation of proapoptotic
genes like AIFM1, MOAP1, PDCD5, and PDCD7, confirming a possible
correlation between apoptosis and the reduction of dental pulp volume.
In this research, the expression of the most significant genes of the
ECM was also analyzed. Type I and III collagen constitute the major
molecular proteins of the dental tissues. COL1A1, COL1A2, COL2A1,
and COL5A2 resulted highly expressed in young dental pulp. Previous
studies report a high expression of collagen type I in young dental
pulp, suggesting that this protein may be involved in odontoblastic
differentiation or that it is a component of predentin (10). However,
COL3A1, COL5A3, COL8A1, COL9A2, COL9A3, and COL10A1 resulted
upregulated in older dental pulp. Collagen type III is a fibrillar collagen
TABLE 6. Results of Q-RT-PCR in Selected Genes
Selected genes
Transforming growth factor alpha (TGFA)
Transforming growth factor beta 1 (TGFB1)
Fibroblast growth factor 1 (FGF1)
Caspase 1, apoptosis-related cysteine peptidase (CASP1)
Dentin sialophosphoprotein (DSPP)
Alkaline phosphatase (ALPL)
Osteonectin (SPARC)
Connective tissue growth factor (CTGF)
1216 Tranasi et al.
that is found in extensible connective tissues such as skin, lung, and the
vascular system, frequently in association with type I. The low expres-
sion reported for COL3A1 in young dental pulp may suggest that type
III collagen synthesis is not highly represented during the odontoblast
differentiation process and so not strictly related to the odontogenic
process (33). Then, the upregulation of collagen type IX in older dental
pulp could have a role in the polymerization and aggregation of previ-
ously existing smaller units of collagen (34). In fact, type X collagen has
been previously observed to be strongly distributed in the developing
human enamel organ and ameloblasts (35), and it is considered one
of the major candidate molecules of the enamel matrix involved in its
mineralization. Anyhow, our data suggest that its expression still
remains elevated during tooth aging and its secretion may also take
place during physiological reparative events.
It is well known that odontoblasts first synthesize and secrete
collagen type I into the extracellular matrix as a foundation for subse-
quent mineralization. Acidic noncollagenous proteins, including DSP
and dentin phosphoprotein, are then secreted to initiate the mineraliza-
tion processes. The expression in young dental pulp of genes involved in
dentin matrix mineralization, like DSPP and AMBN, encoding some of
the principal proteins of the dentin ECM of the tooth, is significantly
higher than in adult dental pulp. These proteins are synthesized and
secreted by odontoblasts and dental pulp cells to form components
of dentin ECM. AMBN encodes for ameloblastin, a protein of enamel
also found in odontoblasts (7–9), whereas the product of DSPP gene
is a phosphorylated parent protein that is cleaved post-translationally
into three dentin components: dentin sialoprotein, dentin glycoprotein,
and dentin phosphoprotein (3). Conditions related to mutations of
these genes result in pathological defects of matrix mineralization
like dentinogenesis imperfecta type II and type III and dentin dysplasia
type II (3). In our opinion, this study confirms that the vitality of the pulp
dentinal complex decrease with aging as shown by the low expression of
genes encoding for transcription regulators and the high expression of
genes involved in apoptotic processes. However, the expression in older
dental pulp of growth factors like CTGF, FGF1, FGF5, and TGFB1 and of
Fold change ± SD Fold change
Gene bank by Q-RT-PCR by microarray
NM_003236 –4.2  2.5 –1.573
NM_000660 3.1  1.3 1.668
NM_000800 2.9  1.3 1.632
NM_033292 3.1  0.3 3.104
NM_014208 –7.2  1.5 –8.992
NM_000478 1.8  0.6 2.123
NM_003118 –2.5  1.1 –1.268
NM_001901 –4.4  2.6 1.759
JOE — Volume 35, Number 9, September 2009

genes for collagenous proteins, like COL10A1, COL3A1, COL5A3,
COL8A1, COL9A2, and COL9A3, confirm that reparative processes are
continuous during the entire tooth life. We still have much to learn of
the biological control mechanisms responsible for cellular activity
and survival, even if the expression profile analysis during pulp devel-
opment can represent a sensible and useful tool for the study of the
mechanisms involved in the differentiation, growth, and evolution of
human dental pulp in normal and pathological conditions, offering
exciting opportunities for novel treatments and tissue engineering
approaches for tissue restoration.
References
1. Abrahão IJ, Martins MD, Katayama E, et al. Collagen analysis in human tooth germ
papillae. Braz Dent J 2006;17:208–12.
2. Linde A. The extracellular matrix of the dental pulp and dentin. J Dent Res 1985;64
Spec No:523–9.
3. Butler WT, Ritchie H. The nature and functional significance of dentin extracellular
matrix proteins. Int J Dev Biol 1995;39:169–79.
4. D’Souza RN, Cavender A, Sunavala G, et al. Gene expression patterns of murine
dentin matrix protein 1 (Dmp 1) and dentin sialophosphoprotein (DSPP) suggest
distinct developmental functions in vivo. J Bone Miner Res 1997;12:2040–9.
5. Feng J, Luan X, Wallace J, et al. Genomic organization, chromosomal mapping, and
promoter analysis of the mouse dentin sialophosphoprotein (Dspp) gene, which
codes for both dentin sialoprotein and dentin phosphoprotein. J Biol Chem
1998;273:9457–64.
6. MacDougall M, Simmons D, Luan X, et al. Dentin phosphoprotein and dentin sia-
loprotein are cleavage products expressed from a single transcript coded by
a gene on human chromosome 4. J Biol Chem 1997;272:835–42.
7. Begue-Kirn C, Krebsbach PH, Bartlett JD, et al. Dentin sialoprotein, dentin phospho-
protein, enamelin and ameloblastin: tooth-specific molecules that are distinctively
expressed during murine dental differentiation. Eur J Oral Sci 1998;106:963–70.
8. Yamakoshi Y, Hu JC, Iwata T, et al. Dentin sialophosphoprotein is processed by
MMP-2 and MMP-20 in vitro and in vivo. J Biol Chem 2006;281:38235–43.
9. Begue-Kirn C, Ruch JV, Ridall AL, et al. Comparative analysis of mouse DSP and DPP
expression in odontoblasts, preameloblasts, and experimentally induced odonto-
blast-like cells. Eur J Oral Sci 1998;106(Suppl 1):254–9.
10. Quigley MB. Functional and geriatric changes of the human pulp. Oral Surg Oral
Med Oral Pathol 1971;32:795–806.
11. Morse DR, Esposito JV, Schoor RS. A radiographic study of aging changes of the
dental pulp and dentin in normal teeth. Quintessence Int 1993;24:329–33.
12. Morse DR. Age-related changes of the dental pulp complex and their relationship to
systemic aging. Oral Surg Oral Med Oral Pathol 1991;72:721–45.
13. Sloan AJ, Rutherford RB, Smith AJ. Stimulation of the rat dentine-pulp complex by
BMP7 in vitro. Arch Oral Biol 2000;45:173–7.
14. Sloan AJ, Smith AJ. Stimulation of the dentine-pulp complex of rat incisor teeth by
transforming growth factor beta isoforms 1-3 in vitro. Arch Oral Biol 1999;44:
149–56.
JOE — Volume 35, Number 9, September 2009
Clinical Research
15. Renehan AG, Booth C, Potten CS. What is apoptosis, and why is it important? BMJ
2001;322:1536–8.
16. Opferman JT, Korsmeyer SJ. Apoptosis in the development and maintenance of the
immune system. Nat Immunol 2003;4:410–5.
17. Murray PE, Stanley HR, Matthews JB, et al. Age-related odontometric changes of
human teeth. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;93:474–82.
18. Saad AY. Regressive changes of the dental pulp complex in retained primary molars
with congenitally missing successor teeth. J Clin Pediatr Dent 1997;22:63–7.
19. Pääkkönen V, Vuoristo J, Salo T, et al. Effects of TGF-beta 1 on interleukin profile of
human dental pulp and odontoblasts. Cytokine 2007;40:44–51.
20. Buchaille R, Couble ML, Magloire H, et al. A substractive PCRbased cDNA library
from human odontoblast cells: identification of novel genes expressed in tooth
forming cells. Matrix Biol 2000;19:421–30.
21. Tziafas D, Smith AJ, Lesot H. Designing new treatment strategies in vital pulp therapy.
J Dent 2000;28:77–92.
22. Smith AJ, Lesot H. Induction and regulation of crown dentinogenesis. Embryonic
events as a template for dental tissue repair? Crit Rev Oral Biol Med 2001;12:425–37.
23. Smith AJ. Vitality of the dentin-pulp complex in health and disease: growth factors as
key mediators. J Dent Educ 2003;67:678–89.
24. Ruch JV, Lesot H, Begue-Kirn C. Odontoblast differentiation. Int J Dev Biol 1995;39:
51–68.
25. Bègue-Kirn C, Smith AJ, Ruch JV, et al. Effects of dentin proteins, transforming
growth factor beta 1 (TGF beta 1) and bone morphogenetic protein 2 (BMP2)
on the differentiation of odontoblast in vitro. Int J Dev Biol 1992;36:491–503.
26. Bègue-Kirn C, Smith AJ, Loriot M, et al. Comparative analysis of TGF betas, BMPs,
IGF1, msxs, fibronectin, osteonectin and bone sialoprotein gene expression during
normal and in vitro-induced odontoblast differentiation. Int J Dev Biol 1994;38:
405–20.
27. Wang JJ, Ye F, Cheng LJ, et al. Osteogenic differentiation of mesenchymal stem cells
promoted by overexpression of connective tissue growth factor. J Zhejiang Univ Sci
B 2009;10:355–67.
28. Mekin M, Joffre-Romeas A, Farges J-C, et al. Effects of TGFb1 on dental pulp cells in
cultured human tooth slices. J Dent Res 2000;79:1689–96.
29. Mitsiadis TA, Rahiotis C. Parallels between Tooth development and repair:
conserved molecular mechanism following carious and dental injury. J Dent Res
2004;83:896–902.
30. Yates S, Rayner TE. Transcription factor activation in response to cutaneous injury:
role of AP-1 in reepithelialization. Wound Repair Regen 2002;10:5–15.
31. About I, Mitsiadis TA. Molecular aspects of tooth pathogenesis and repair: in vivo
and in vitro models. Adv Dent Res 2001;15:59–62.
32. Thimios A, Mitsiadis TA, De Bari C, et al. Apoptosis in developmental and repair-
related human tooth remodeling: a view from the inside. Exp Cell Res 2008;314:
869–77.
33. Andujar MB, Couble P, Couble ML, et al. Differential expression of type I and type III
collagen genes during tooth development. Development 1991;111:691–8.
34. Pihlajamaa T, Vuoristo MM, Annunen S, et al. Human COL9A1 and COL9A2 genes.
Two genes of 90 and 15 kb code for similar polypeptides of the same collagen mole-
cule. Matrix Biol 1998;17:237–41.
35. Felszeghy S, Hollo K, Modis L, et al. Type X collagen in human enamel development:
a possible role in mineralization. Acta Odontol Scand 2000;58:171–6.
SAM Evaluation of Age-related Changes in Human Dental Pulp 1217