Clinical Research

Variation in the Expression of Insulin-like Growth Factor-1

in Human Pulp Tissue According to the Root-development
Stage
Javier Caviedes-Bucheli, DDS, MSc, Patricia Angel-Londoo, DDS, Alberto Daz-Perez, DDS,
Mara Patricia Orozco, DDS, Jos Luis lvarez, DDS, Nelson Lombana, MSc,
Eduardo Daz, MSc, and Hugo Roberto Muoz, DDS, MSc
Abstract
Insulin-like growth factor-1 (IGF-1) plays an important
role in cell proliferation and differentiation. It actively
participates during odontogenesis and mineralization
of dental hard tissues. The purpose of this study is to
use a radioimmunoassay (RIA) to determine whether
there are differences in the expression of IGF-1 in
human pulp from teeth having incomplete or complete
root development. Twenty-six pulp samples were obtained from freshly extracted human third molars, half
of them from teeth with incomplete root development.
All of the samples were processed and labeled using an
IGF-1 RIA kit. Results showed IGF-1 presence in all
human pulp samples with a statistically significant
higher expression in pulps having complete root development. The present findings are consistent with the
hypothesis that IGF-1 contributes toward forming and
mineralizing dental tissues in teeth with incomplete
root development as well as in pulp-repairing process in
teeth with complete root development. (J Endod 2007;
33:12931295)

Key Words
Human pulp tissue, insulin-like growth factor-1, root
development stage

From the School of Dentistry, Pontificia Universidad Javeriana, Bogot, Colombia.

Address requests for reprints to Dr Javier Caviedes-Bucheli,
School of Dentistry, Pontificia Universidad Javeriana, Cra 7 No.
40-62 Building 26, Bogot, Colombia. E-mail address:
javiercaviedes@cable.net.co
0099-2399/$0 - see front matter
Copyright 2007 by the American Association of
Endodontists.
doi:10.1016/j.joen.2007.07.026

rowth factors are highly specific proteins that regulate cell proliferation and differentiation, playing a major role in several physiological processes during embryogenesis and regulating many functions in organs and tissues during postnatal growth
and development, including dental pulp (1, 2). Insulin-like growth factor-1 (IGF-1) is
an anabolic peptide that mediates the biological effects of growth hormone (3, 4) and
constitutes an essential factor during cell cycle, stimulating cell proliferation (1, 5, 6).
Several growth factors, including IGF-1, act as inductors for odontogenesis, which
is regulated by cell-extracellular matrix communications and epithelial-mesenchymal
interactions (7, 8). Different levels of IGF-1 immunoreactivity have been detected in
ameloblasts and odontoblasts of rat incisors according to developmental stage, suggesting that this growth factor participates in the formation and mineralization of dental
tissues (9). Exogenous IGF-1 added to mouse pulp cultures has stimulated mineralization, mitotic activity, and cell differentiation, promoting ameloblast and odontoblast
development and consequently leading to enamel and dentin formation (10). Polymerase chain reaction studies have shown that these functions are mediated by induction of
specific genes (2).
It has been shown that, in serum-free cultures, IGF-1 induces proliferation and
differentiation of dog dental pulp cells into odontoblast-like cells (11). Furthermore,
there is an increase in proliferative activity of human pulp cells in the presence of IGF-1
in primary cultures (6). This evidence supports the hypothesis that this growth factor
plays a significant role in the pulp-repairing process. During dentinogenesis, IGF-1
could get embedded in the dentin matrix structure and could be released into dental
pulp after an injury to the pulpodentin complex, stimulating repair processes (12, 13).
A previous study (14) revealed statistically significant differences in the expression
of the IGF-1 receptor (IGF-1R) when comparing pulps from teeth having complete or
incomplete root development, being its expression higher in the latter. Results from that
study are consistent with the hypothesis that IGF-1 contributes toward forming and
mineralizing dental tissues as well as in pulp-repairing processes. However, to support
this hypothesis, it is still relevant to determine if there are also differences in the level of
free (unbound) IGF-1 in pulps from teeth having complete or incomplete root development. Therefore, the purpose of this study was to use a radioimmunoassay (RIA) to
determine the difference in the expression of IGF-1 in pulp tissue of teeth having
incomplete or complete root development.

Materials and methods

Human pulp was obtained from 26 freshly extracted noncarious and nonrestored
third molars from different patients who gave their written informed consent to donate
their pulp tissue. Half of the samples were from teeth having incomplete root development and the other half from teeth with complete root development. Determining root
development stage was performed both visually and radiographically. All of the teeth
included in the incomplete root-development group had an apical orifice diameter
larger than 3 mm mesiodistal or buccolingual. The teeth included in the complete
root-development group had an apical orifice diameter no larger than 0.5 mm. Teeth
with an apical orifice diameter between 0.5 and 3 mm were discarded.

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Expression of IGF-1 in Human Pulp Tissue

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The extracted teeth were washed with 5.25% sodium hypochlorite
after extraction to eliminate remains of periodontal ligament that could
contaminate the pulp sample. The teeth were then sectioned by using a
Zekrya bur (Dentsply Maillefer, Tulsa, OK) in a high-speed handpiece
irrigated with saline solution. The pulp tissue was obtained by using a
sterile endodontic excavator, fixed in 4% paraformaldehyde, weighed,
and kept frozen at !70C until use.
Pulp samples were ultrasonically disaggregated (Ultrasonic Processor S-2028-130; ISC BioExpress, Kaysville, UT) for their homogenization; 250 !L of acetic acid was added and double boiled for 10
minutes. Disaggregated tissue was spun at 2200 rpm for 20 minutes
(GS-6KR Centrifuge; Beckman, Fullerton, CA) and supernatants were
transferred to another tube.
One hundred microliters of each samples supernatant were submitted to competition binding assays using a RIA kit for IGF-1 following
manufacturers instructions (Ref. 035-10; Phoenix Peptide Pharmaceuticals, Belmont, CA). Assays were performed in duplicate. After 2 hours
of incubation, the suspensions were spun at 5000 rpm for 1 hour
(Beckman) to precipitate the bound fractions. The supernatants were
aspirated, and pellet radioactivity was read on a Gamma Counter
(Gamma Assay LS 5500, Beckman). Analysis of the binding data assessed the amount of IGF-1 present in every sample.

Statistical analysis
Values are presented as IGF-1 amount in nanograms per gram of
pulp weight. Mean and maximum/minimum values are presented for
each group; t test was performed for establishing statistically significant
differences (p " 0.05) between the groups of pulps from teeth having
incomplete and complete root development.

Results
IGF-1 was found to be expressed in all human pulp samples. The
mean expression for the group of pulps from teeth having incomplete
root development was 11.18 ng/g pulp tissue, with a standard deviation
of 24.71. The maximum and minimum values were 2.84 and 77.58 ng/g
pulp tissue, respectively (Table 1).
Expression for the group of pulps from teeth having complete root
development was 1950.73 ng/g pulp tissue, with a standard deviation of
1313.91. The maximum and minimum values were 35.64 and 4886.50
ng/g pulp tissue, respectively (Table 2); a t test showed statistically
significant differences between both groups (p " 0.0002).

TABLE 1. IGF-1 Expression in Human Pulp Samples from Teeth Having

Incomplete Root Development
Sample

Pulp Weight (g)

IGF-1 (ng/g)*

1
2
3
4
5
6
7
8
9
10
11
12
13
Mean
Standard deviation

0.128
0.109
0.108
0.106
0.076
0.178
0.037
0.069
0.073
0.255
0.590
0.170
0.180
0.160
0.142

9.82
6.78
2.84
9.96
20.34
26.80
77.58
3.20
9.73
9.75
6.85
69.90
8.25
20.14
24.71

*Values are given in nanograms of IGF-1 per gram of pulp tissue.

1294

Caviedes-Bucheli et al.

TABLE 2. IGF-1 Expression in Human Pulp Samples from Teeth Having

Complete Root Development
Sample

Pulp Weight (g)

IGF-1 (ng/g)*

1
2
3
4
5
6
7
8
9
10
11
12
13
Mean
Standard deviation

0.003
0.001
0.007
0.003
0.008
0.002
0.008
0.003
0.006
0.013
0.001
0.008
0.003
0.005
0.003

1408.16
4886.50
35.64
1667.33
981.62
3163.25
949.25
2800.16
1380.83
661.69
2179.50
664.81
2665.66
1803.42
1313.91

*Values are given in nanograms of IGF-1 per g of pulp tissue.

Discussion
IGF-1 belongs to a polypeptide family related to insulin, which
includes 2 ligands (IGF-1 and IGF-2), 2 cell-surface receptors (IGF-1R
and IGF-2R), at least 6 high-affinity binding proteins (IGFBP-1 to IGFBP6), and multiple IGFBP proteases (2, 5, 15). IGF-1 is a highly mitogenic
and differentiation factor that exerts endocrine, paracrine, and autocrine functions to control pre- and postnatal growth and development.
It is a potent stimulator of cell proliferation, differentiation, and maintenance of specialized functions in several tissues (1).
IGF receptors, the binding proteins, and the proteases regulate the
activity of the ligands in several tissues. IGFBPs function as transport and
storage peptides for IGF in the intravascular and extracellular environment, lengthening its half-life and potentiating or inhibiting its activity by
regulating its interactions with receptors (1, 2, 15, 16). On the other
hand, proteases cleave IGFBPs and, therefore, modulate ligand accessibility (1).
It has been shown that IGF-1 regulates cell growth and differentiation in developing tissues. It also modulates some functions in mature
cells in which IGF-1 is important for maintaining homeostasis and regulating metabolism necessary for tissue survival (1, 4, 5, 9). This could
explain IGF-1 presence in both groups of the present study.
All IGF-1 biological actions are mediated through interaction with
its specific cell-surface receptor (IGF-1R), which transduce the signal
into the cell via mitogen-activated protein-kinase or phosphatidyl-inositol 3-kinase (5, 17). Previous studies have confirmed the presence of
IGF-1R in the dental pulp of teeth with incomplete or complete root
development (14, 15).
The odontogenesis process is regulated by epithelial-mesenchymal and cell-extracellular matrix interactions (7, 8, 18) that lead to
dentinogenesis and amelogenesis (8, 19). Odontoblast differentiation is
controlled by the expression of some growth factors as well as several
transcription factors (7, 13, 20).
Immunohistochemistry and hybridization studies have shown that
IGF-1 acts in a paracrine/autocrine manner during odontogenesis, promoting tissue mineralization (21, 22). Reverse-transcription polymerase chain reaction studies in mouse pulp cultures have also shown that
IGF-1 enhances mineralization of enamel and dentin by inducing expression of specific genes (2). Another study corroborated the role of
IGF-1 in the pulp-repairing process by adding this growth factor in pulp
exposures in rat molars, showing better healing when compared with
the control teeth (23).
Results from the present study showed a statistically significant
lower IGF-1 expression in pulps from teeth with incomplete root develJOE Volume 33, Number 11, November 2007

Clinical Research
opment. It is important to understand that the laboratory test used (RIA)
only measured IGF-1 in tissue. A previous radioreceptor study (14)
showed a statistically significant higher expression of IGF-1 receptor in
the same type of pulps. With these results in mind, it could be hypothesized that locally produced IGF-1 in pulps from teeth having incomplete root development is rapidly bounded to receptors in pulp cells,
making it undetectable by RIA. Further kinetic studies are needed to
ascertain this.
On the other hand, there was a higher IGF-1 expression in pulps
from teeth having complete root development; the previous RRA study
(14) showed a lower expression of the IGF-1 receptor by pulp cells.
IGF-1 presence in these pulps sustains the theory that IGF-1 is necessary
for tissue homeostasis, and it could be useful in the event of an injury to
the pulpodentin complex.
In a systematic study in human permanent teeth, the presence
and distribution of the IGF-1 family (ligand, receptors, and IGFBPs)
were analyzed by immunohistochemistry. Expression of IGF-1 and
IGFBP-1, 3, 5, and 6 was found in dental pulp, especially close to
odontoblasts and pulp stones. IGF-1R was found to be expressed in
odontoblasts, which suggests that this growth factor is involved in
the biology of odontoblast and in the formation of pulp stones. There
was a weak to moderate response for IGF-1 in dentin; however, it is
important to take into account that during the laboratory sample
processes (cutting teeth, decalcification, and immunohistochemistry per se) some of the growth factor embedded in dentin matrix
could get denaturalized or eliminated (15).
All the evidence analyzed suggests the therapeutic potential of
IGF-1 in pulp tissue, both in reparation and regeneration of the pulpodentin complex when insulted by external irritants because this growth
factor is not only present in systemic circulation, but it also could be
synthesized in a variety of tissues, including dental pulp. It is known that
in systemic circulation IGF-1 is bounded to specific proteins, particularly in developing tissues (24). This fact may account for the lower
levels of free IGF-1 in developing pulp.
When IGF-1 is applied in combination with other growth factors,
such as platelet-derived growth factor, the neoformation of tissue is
increased, thus repairing or regenerating the previously lost tissue
(25). It would be interesting to consider possible IGF-1 therapies in
combination with the vasoactive intestinal peptide for the maintenance
of health and vitality of pulpodentin complex because of the anti-inflammatory and cytoprotective characteristics of this neuropeptide (26).
IGF-1 levels found in the present study suggest that this growth factor
plays an important role in homeostasis and reparative process of the mature
pulp, where it can facilitate the formation of reactionary or reparative dentin
in response to irritants, such as caries, abrasion, or dental trauma. Moreover, it also participates in tooth development stimulating the process of
odontogenesis and mineralization of dental structures. Further research
about the interactions of this growth factor in the pulpodentin complex
could lead to developing future biological pulp therapies.

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