Invest Clin 65(2): 179 - 191, 2024 https://doi.org/10.54817/IC.v65n2a05

Corresponding author. Wen-jing Li. Department of Oral Medicine. The Second Hospital of Hebei Medical Universi-

ty. Shijiazhuang City, Hebei province, China. Tel: +86-0311-66002729. E-mail: 27801072@hebmu.edu.cn

Insulin-like growth factor-1 promotes the

proliferation and odontogenic differentiation

of human dental pulp cells in vitro

and in vivo.

Yan Wang, Nan Du, Cong-na Liu and Wen-jing Li

Department of Oral Medicine, The Second Hospital of Hebei Medical University,

Shijiazhuang, China.

Keywords: dental tissue regeneration; odontoblasts; osteogenic differentiation.

Abstract. Human dental pulp cells (hDPCs) have emerged as a potential

alternative for the regeneration of dental tissues. Insulin-like growth factor-1

(IGF-1) is involved in the proliferation and osteogenic differentiation of hDPCs

in vitro. However, the effect of IGF-1 on the proliferation and odontogenic dif-

ferentiation of hDPCs in vivo remains unknown. This study collected hDPCs

from healthy premolars and third molars by collagenase type I and dispase.

Immunocytochemical staining showed positive vimentin staining and negative

cytokeratin staining in hDPCs. Treatment with IGF-1 (50, 75, and 100 ng/mL)

significantly increased the proliferation ability of hDPCs in a concentration-de-

pendent manner. In vivo experiments, hDPCs were seeded into an acellular der-

mal matrix and transplanted subcutaneously into nude mice. After two and four

weeks of transplantation, the hematoxylin and eosin staining revealed more

cells and extracellular matrix in implants from the IGF-1 treatment group, and

Alizarin Red staining revealed more mineralized tissue compared to the control

group. Transmission electron microscopy (TEM) analysis of hDPCs showed an

abundance of mitochondria, rough endoplasmic reticulum, and Golgi complex-

es. In conclusion, IGF-1 promotes the proliferation of hDPCs in vitro and odon-

togenic differentiation of hDPCs in vivo, indicating that modifying IGF-1 signal-

ing may provide potential strategies for the regeneration of dental tissues.

180 Wang et al.

Investigación Clínica 65(2): 2024

El factor de crecimiento similar a la insulina-1 promueve

la proliferación y diferenciación odontogénica de células

de pulpa dental humana in vitro e in vivo.

Invest Clin 2024; 65 (2): 179 – 191

Palabras clave: regeneración del tejido dental; odontoblastos; diferenciación

osteogénica.

Resumen. Las células de la pulpa dental humana (hDPCs) han emergido

como una alternativa prometedora para la regeneración de tejidos dentales. El

factor de crecimiento insulínico tipo 1 (IGF-1) juega un rol crucial en la pro-

liferación y diferenciación osteogénica de las hDPCs en condiciones in vitro.

No obstante, el impacto del IGF-1 sobre la proliferación y diferenciación odon-

togénica de las hDPCs en un contexto in vivo aún no ha sido completamente

elucidado. En el presente estudio, se extrajeron hDPCs de premolares y terceros

molares sanos mediante el uso de colagenasa tipo I y dispasa. La tinción inmu-

nocitoquímica de las hDPCs reveló una reactividad positiva para vimentina y ne-

gativa para citoqueratina. El tratamiento con IGF-1 en concentraciones de 50,

75 y 100 ng/mL incrementó de manera significativa y dependiente de la dosis la

capacidad proliferativa de las hDPCs. En experimentos in vivo, las hDPCs fueron

implantadas en una matriz dérmica acelular y posteriormente trasplantadas de

manera subcutánea en ratones desnudos. Tras 2 y 4 semanas de trasplante, la

coloración con hematoxilina y eosina evidenció un aumento en la cantidad de

células y matriz extracelular en los implantes tratados con IGF-1, mientras que

la coloración con Rojo de Alizarina indicó una mayor formación de tejido mine-

ralizado en comparación con el grupo control. El análisis mediante microscopía

electrónica de transmisión (TEM) de las hDPCs mostró una abundante presen-

cia de mitocondrias, retículo endoplásmico rugoso y complejos de Golgi. En

conclusión, nuestros hallazgos sugieren que el IGF-1 favorece la proliferación

de las hDPCs in vitro así como su diferenciación odontogénica in vivo, lo cual

señala que la modificación de la señalización del IGF-1 podría ofrecer estrate-

gias potenciales para la regeneración de tejidos dentales.

Received: 24-10-2023 Accepted: 02-03-2024

INTRODUCTION

Dental pulp, situated at the innermost

tissue within teeth, primarily comprises

loose connective tissue and plays an essen-

tial role in the repair and regeneration of

dental tissues. Traumatic incidents or the

impact of infectious agents and other patho-

genic stimuli can readily give rise to pulpi-

tis

. Human dental pulp cells (hDPCs) are

present within dental pulp tissue, constitut-

ing a mixture of fibroblasts, inflammatory,

immune cells, odontoblasts, and undifferen-

tiated mesenchymal cells

2,3

. Among these

components, the undifferentiated mesen-

chymal cells possess a multipotent cellular

phenotype with the potential to differenti-

ate into various cell lineages, such as osteo-

blasts, adipocytes, neural progenitors, and

chondrocytes

4-6

. When dental pulp tissue is

Growth changes of human dental pulp cells 181

Vol. 65(2): 179 - 191, 2024

exposed to external stimuli, dental pulp cells

can differentiate into odontoblasts under

the regulation of various factors, which is

a significant factor in the regeneration and

repair capacity of dental tissue damage

7,8

Therefore, exploring the mechanisms of hD-

PCs proliferation and odontoblastic differen-

tiation is essential.

Cell-based tissue engineering has be-

come an irreplaceable method for regen-

erating dental tissues

. Its fundamental

mechanism involves inducing stem cells

to differentiate into odontoblast-like or

osteoblast-like cells, ultimately achieving

the regeneration of the dental pulp-dentin

complex

10,11

. Furthermore, hDPCs exhibit

some advantages, characterized by no ethi-

cal controversy, low immunogenicity, and

easy obtainment from impacted or orth-

odontic extraction

12,13

. Therefore, hDPCs

have emerged as a potential alternative for

future use in regeneration therapy. In addi-

tion to stem cells and biomimetic materials,

suitable growth factors are imperative in tis-

sue regeneration by promoting cell prolifera-

tion, differentiation, and locomotion

14,15

Insulin-like growth factor-1 (IGF-1),

a member of the insulin-like peptide fam-

ily, exerts essential roles in the bone forma-

tion and periodontal regeneration of teeth

14,16,17

. Local controlled delivery of IGF-I from

dextran-co-gelatin hydrogel microspheres

enhances new bone formation and alveolar

bone reconstruction in the periodontal de-

fects

. Moreover, IGF-1 is also involved in

the odontoblastic differentiation of hDPCs or

human dental pulp stem cells (hDPSCs). In

vitro experiments showed 100 ng/mL IGF-1

promotes proliferation and increases the os-

teogenic differentiation-related expression

of DPSCs via mammalian target of rapamy-

cin (mTOR) signaling pathway

, as well as

the enhancement of proliferation and osteo/

odontogenic differentiation was found in the

human periodontal ligament stem cells via

activating MAPK pathways

. Similarly, oth-

er in vitro research indicates that combined

use of 100 ng/ml VEGF and 100 ng/mL IGF-

1 also improve the proliferation, migration,

osteogenesis, and angiogenesis of hDPSCs

via activation of the phosphoinositide 3-ki-

nase (PI3K)/Akt signaling pathway

This study aimed to investigate the ef-

fect of IGF-1 on the proliferation and odon-

togenic differentiation of hDPCs in vitro and

in vivo. These findings may offer valuable in-

sights into the potential utility of hDPCs in

treating dental pulp diseases.

MATERIALS AND METHODS

Isolation of human DPCs

Healthy premolars for orthodontic

needs or third molars were collected from

patients without dental caries or periodon-

tal tissue diseases at the dental clinic of The

Second Hospital of Hebei Medical University.

All study protocols were approved by the Eth-

ics Committee of The Second Hospital of He-

bei Medical University, and written informed

consent was obtained from all patients. The

hDPCs were obtained as described previous-

ly. To completely collect the dental pulp, the

tooth surface attachments were scraped off,

and the teeth were split in a sterile environ-

ment. After washing three times with phos-

phate buffer solution (PBS), dental pulp tis-

sues were cut into pieces of 1 mm×1 mm

in size, then digested in a solution of 3mg/

mL collagenase type I (Sigma-Aldrich) and

4mg/mL dispase (Sigma-Aldrich) at 37°C on

a shaker for one hour. Following filtration

and centrifuging, cell suspensions of dental

pulp were cultured in Dulbecco’s modified

Eagle’s medium (DMEM, Gibco, Grand Is-

land, NY, USA) supplemented with 20% fetal

bovine serum and 1% penicillin-streptomycin

(Gibco, Grand Island, NY, USA) at 37°C in a

5% CO

incubator. Observe whether dental

pulp cells crawl out of the tissue blocks and

cell morphology and growth. Once cells ad-

here to the culture surface, the medium was

refreshed every three days. When cell fusion

reached over 80%, the adherent cells were

digested with 0.25% trypsin (Sigma-Aldrich),

and passage culture was performed at a 1:3

182 Wang et al.

Investigación Clínica 65(2): 2024

ratio. The hDPCs from the mixed population

at passages 3 to 6 were used for subsequent

experiments.

Immunohistochemical staining

Passage 4 hDPCs were digested with

0.25% trypsin and prepared as cell suspen-

sion, then seeded into 6-well cell culture

plates at a concentration of 1×10

cells per

well. Immunohistochemical staining for vi-

mentin and cytokeratin was carried out on

the day following cell adherence. Briefly, cell

slides were fixed with 4% formaldehyde for

10 minutes, then treated with 0.5% Triton

X-100 (diluted in PBS) for 10 minutes at

room temperature. Following PBS washing,

cells were blocked with 10% FBS for 20 min-

utes and subsequently incubated overnight

at 4°C with primary antibodies anti-vimentin

and anti-cytokeratin (1:200, Abcam). After

washing three times with PBS, cells were in-

cubated with biotin-conjugated secondary

antibodies (1:1000, Abcam) at room tem-

perature for 20 minutes. DAB staining was

performed, and counterstaining with hema-

toxylin was carried out to visualize the cell

nuclei. After mounting coverslips with neu-

tral balsam, photography was conducted un-

der an optical microscope (Olympus, Tokyo,

Japan).

Flow cytometry analysis

The purity of isolated hDPCs was as-

sessed using flow cytometry analysis with

specific antibodies for CD44 and CD45

The cells were trypsinized and fixed in 4%

paraformaldehyde in PBS. Subsequently,

hDPCs were permeabilized with 0.1% Triton

X-100 (Sigma-Aldrich) in PBS and then in-

cubated with conjugated antibodies against

CD44 (1:100, Biotech, Minneapolis, USA)

and anti-CD45 (1:100, Biotech, Minneapo-

lis, USA). After washing three times with

PBS, flow cytometric analysis was conducted

using a FACS (Becton, Dickinson, San Jose,

CA) with Quest CELL software (Becton,

Dickinson).

Cell proliferation assay

The proliferation ability of hDPCs was

assessed using the MTT (3-(4, 5-dimethylthi-

azol-2-yl)-2, 5-diphenyl nyltetrazolium bro-

mide, Sigma) assays. Passage 4 hDPCs were

prepared as a cell suspension with 0.25%

trypsin and seeded into 96-well cell culture

plates at a concentration of 2×10

cells per

well. After 24 hours of cultivation, cells were

randomly divided into four groups. In the ex-

perimental groups, DMEM culture medium

containing different concentrations of IGF-

1 (50 ng/mL, 75 ng/mL, 100 ng/mL, Pep-

rotech, USA) was added, while in the con-

trol group, DMEM without IGF-1 was added.

Each group had five replicates. The cells

were then cultured at 37°C for 1, 3, 5, and 7

days, respectively. Subsequently, 20μl of MTT

(2 mg/mL in PBS) was added to each well.

After 4 hours of incubation, the supernatant

was removed, and 150μL DMSO were added.

The absorbance values at A450 for each well

were determined using a microplate reader

(Bio-Rad).

Odontoblastic differentiation

in vivo

The acellular dermal matrix (ADM, Bei-

jing Qingyuan Weiye Company, China) un-

derwent aseptic cutting into pieces measur-

ing 5mm×5mm×1mm. These pieces were

subjected to three washings with PBS and a

2-hour soak in 10% FBS within a sterile en-

vironment. The 2x10

hDPCs were prepared

as a cell suspension. After removing the FBS,

cells were gently seeded onto the scaffold

using a pipette, ensuring even distribution

within the scaffold interstices through gen-

tle shaking. The composite was co-cultured

at 37°C for 24 hours. The culture medium

was replaced with 100 ng/mL IGF-1 culture

medium supplemented with mineralization

induction solution, consisting of 10% FBS,

DMEM, 10 mmol/L β-glycerophosphate, 100

mg/mL vitamin C, and 10 nmol/L dexa-

methasone. The culture medium in the con-

trol group was replaced with a mineraliza-

tion induction solution without IGF-1. The

Growth changes of human dental pulp cells 183

Vol. 65(2): 179 - 191, 2024

induction culture was maintained for three

days. For the in vivo phase, eight 4-week-old

immunodeficient mice were selected. Un-

der sterile conditions, incisions were made

in the skin along their dorsal midlines. The

hDPCs-scaffold complexes were transplanted

subcutaneously into the left dorsal region,

while control scaffolds were transplanted as

controls into the right dorsal region. The

odontoblastic differentiation in the hDPCs-

scaffold complexes within the mice was ob-

served at two and four weeks post-transplan-

tation.

Histological staining

Hematoxylin and Eosin (HE) staining

was used for the histological observation,

and Alizarin red staining was performed to

identify calcium-containing osteocytes or

odontoblasts. The mice were euthanized at

two and four weeks post-transplantation.

The scaffold and surrounding tissues were

immediately harvested and fixed in 4% para-

formaldehyde. Subsequently, 4-μm-thick

sections were prepared using a freezing

microtome. The sections were stained with

hematoxylin for 3 minutes for HE staining,

followed by differentiation and a 20-second

rinse in alkaline water. Next, sections were

stained with eosin for 20 seconds. For the

alizarin red staining, sections were fixed in

95% anhydrous ethanol for 10 minutes. Af-

ter rinsing in double-distilled water thrice,

sections were stained in 0.1% alizarin red-

Tris-HCl at 37°C for 30 minutes. All sections

were observed and counted under a light mi-

croscope (Olympus, Tokyo, Japan).

Transmission electron microscopy (TEM)

TEM was used to assess the morpholo-

gies of hDPCs-scaffold complexes. After

two and four weeks post-transplantation,

the scaffold was immediately harvested and

washed three times with PBS, then fixed in

4% (v/v) glutaraldehyde for 2 hours at room

temperature. The complexes were dehy-

drated with varying ethanol concentrations

and embedded with epoxy resin. Subse-

quently, 4-μm-thick sections were prepared

and stained with the toluidine blue staining.

Ultimately, all the samples were air-dried

overnight and viewed with a Hitachi Model

H-7500 TEM (Hitachi, Japan).

Statistical analysis

Data were shown as mean ± SD, and

statistical analysis was performed using

SPSS 17.0 software (IBM SPSS, Armonk, NY,

USA). Group comparisons were performed

using one-way ANOVA followed by a least sig-

nificant difference (LSD) post-hoc compari-

sons. P<0.05 was considered to indicate a

statistically significant difference.

RESULTS

Identification of human dental pulp cells

(hDPCs)

The inverted microscopy showed that

hDPCs displayed clustered growth after sev-

en days of adherent culture, primarily with a

spindle-shaped morphology, while a smaller

fraction of polygonal shapes (Fig. 1A). After

passaging, the hDPCs adopted a vortex-like

growth pattern, characterized by a more

uniform, short spindle-shaped morphology

with extending cytoplasmic processes (Fig.

1B). Immunocytochemical analysis was per-

formed using passage 4 hDPCs, and showed

positive staining for vimentin (Fig. 1C) and

negative staining for cytokeratin (Fig. 1D),

indicating their mesenchymal origin. Addi-

tionally, the flow cytometry analysis showed

that the purity of hDPCs reached more than

95% (Fig. 1E). These morphological features

were aligned with the typical biological char-

acteristics of hDPCs.

IGF-1 promoted proliferation of hDPCs

in vitro

MMT assay was performed to assess

the effect of different IGF-1 concentrations

(50, 75, and 100ng/mL) on the prolifera-

tion ability of hDPCs. As shown in Fig. 2,

there was no significant difference among

groups (p>0.05), and 100ng/mL IGF-1 sig-

184 Wang et al.

Investigación Clínica 65(2): 2024

nificantly increased the proliferation of hD-

PCs three days after treatment compared

to the control group (p<0.01). At day five

and day seven, treatment with IGF-1 signifi-

cantly increased the proliferation of hDPCs

compared with the control group (p<0.05

and p<0.01), 100 ng/mL IGF-1 displayed

a more substantial enhancement compared

to 50 ng/mL and 75 ng/mL (p<0.05 and p

<0.01). These results indicated that IGF-1

promoted the proliferation of hDPCs in a

concentration-dependent manner.

Fig. 1. Identification of hDPCs. A: Morphology after seven days of primary culture. B: Morphology of hDPCs

in passage 3. C: Immunocytochemical staining for vimentin. D: Immunocytochemical staining for

cytokeratin. E: Flow cytometric analysis for CD44 and CD45.

Fig. 2. IGF-1 promoted the proliferation of hDPCs in vitro. MMT assay showed treatment with 100 ng/mL

IGF-1 significantly increased the proliferation of hDPCs from day 3, as well as 50 ng/mL and 75 ng/

mL IGF-1 in day 5 and day 7 cultures. *p<0.01 and **p<0.01 vs control (DMEM without IGF-1).

Growth changes of human dental pulp cells 185

Vol. 65(2): 179 - 191, 2024

IGF-1 promoted odontogenic

differentiation of hDPCs in vivo

In order to investigate the effect of

IGF-1 on osteogenic differentiation in vivo,

hDPCs were seeded into an ADM scaffold

for the induction of odontogenic differen-

tiation, and the hDPCs-scaffold complexes

were transplanted subcutaneously into im-

munodeficient mice for two or four weeks.

HE staining conducted at 2 and 4 weeks

post-transplantation revealed more cells and

extracellular matrix in implants from IGF-1

treatment group compared to the control

group (Fig. 3). Alizarin red staining dem-

onstrated that mineralized nodules in the

IGF-1 group exhibited time-dependent en-

hancement, including the differentiation of

odontoblast-like cells (Fig. 4). TEM analysis

of hDPCs at 2 and 4 weeks post-transplanta-

tion showed an abundance of mitochondria,

rough endoplasmic reticulum and Golgi

complexes (Fig. 5). These findings indicated

that IGF-1 progressively promoted the odon-

togenic differentiation of hDPCs in vivo.

DISCUSSION

Cell-based tissue engineering has be-

come an irreplaceable method for regen-

erating dental tissues

, and the potential

value of hDPCs has been widely accepted in

bone tissue engineering due to their high

self-renewal capacity and stemness

22,23

. The

process of dentinal regeneration involves

the proliferation and differentiation of hD-

PCs into odontoblasts, dental pulp healing,

and reparative dentin formation

. Increas-

ing studies have implicated IGF-1 in the

maintenance of proliferation and differen-

tiation of various stem cells, such as embry-

onic stem cells, bone marrow mesenchymal

stem cells (BMSC), periodontal ligament

stem cells, and hDPSCs

. However, the ef-

fect of IGF-1 on the proliferation and differ-

entiation of hDPCs in vitro and in vivo war-

rants further investigation. In this study, we

found that exogenous IGF-1 promoted the

proliferation of hDPCs in a concentration-

dependent manner.

Fig. 3. HE staining revealed more cells and extracellular matrix in implants from the IGF-1 group compared

to the control group (without IGF-1) at 2 and 4 weeks post-transplantation.

186 Wang et al.

Investigación Clínica 65(2): 2024

Fig. 4. IGF-1 promoted odontogenic differentiation of hDPCs in vivo. Alizarin red staining was performed to

identify calcium-containing odontoblast, showing more mineralized tissues (arrows) in IGF-1 group

compared to the control group (without IGF-1) at 2 and 4 weeks post-transplantation.

Fig 5. TEM analysis. A-B: The subcellular structure of hDPCs in scaffold 2 weeks (A) and 4 weeks (B) post-

transplantation in IGF-1 group (magnification × 6K). C-D: The subcellular structure of hDPCs in

scaffold 2 weeks (C) and 4 weeks (D) post-transplantation in IGF-1 group (magnification × 15K).

Mitochondria and rough endoplasmic reticulum were highlighted using arrows.

Growth changes of human dental pulp cells 187

Vol. 65(2): 179 - 191, 2024

Additionally, IGF-1 progressively pro-

moted odontogenic differentiation of hD-

PCs in vivo by subcutaneous transplanta-

tion into nude mice.

As a unique type of dental stem cell,

hDPCs are derived from the pulp in deep

carious teeth and display stronger prolifera-

tion and osteogenic differentiation capabil-

ity due to distinctive environmental stimulus

26,27

. This distinctive trait provides valuable

insights into the cellular mechanisms under-

lying tooth development and regenerative

repair, offering the potential for dental pulp

regeneration. The proliferation ability of col-

ony-forming cultures of hDPCs and BMSCs

was assessed by bromodeoxyuridine, show-

ing that the number of proliferating cells in

hDPC cultures was significantly higher than

that in BMSC cultures. This result proves

the more substantial proliferation capability

of hDPCs compared to BMSCs

. Moreover,

under specific induction conditions, hDPCs

can be induced to differentiate into various

cell types, including odontoblast-like and

osteoblast-like cells. Almushayt et al.

ex-

perimentally verified the capacity of hDPCs

to differentiate into odontoblast-like cells in

vitro. This study also found the odontoblast

differentiation potential of hDPCs in vivo

by subcutaneous transplantation into nude

mice. Zhang et al.

isolated dental pulp

cells from healthy third molars through en-

zymatic digestion, confirming their identity

as hDPSCs. Subsequent induction assays re-

vealed their multilineage differentiation po-

tential, including differentiation potential

toward adipogenic, osteogenic, fibrogenic,

chondrogenic, and neurogenic lineages.

Yang et al.

demonstrated the capacity of

dental pulp stem cells to differentiate into

chondrocytes and myocytes under specific

induction conditions.

Growth factors are critical in tooth tis-

sue regeneration, as they have essential roles

in regulating cell functions, such as platelet-

derived growth factor, fibroblast growth fac-

tor, and epidermal growth factor

32,33

. IGF-1,

one of the ubiquitous peptide hormones, has

been identified to facilitate various cellular

processes, including cell proliferation, differ-

entiation, migration, apoptosis, and survival

. Previous studies have reported that IGF-

1 can affect hDPCs proliferation, promote

osteogenesis, and help reconstruct tooth-

supporting tissues

34-36

. A report by Onishi

et al.

reveals multiple biological effects of

IGF-1 on hDPCs. IGF-1 can stimulate hDPCs

proliferation, increase mucin and extracel-

lular matrix protein synthesis, enhance DNA

synthesis, and increase alkaline phosphatase

(ALPase) activity when hDPCs were cultured

in a serum-free medium supplemented with

IGF-1. Increasing investigations have illus-

trated the capacity of IGF-1 to induce the

differentiation of ameloblasts and odonto-

blasts, facilitating dentin regeneration and

the development of structures resembling

enamel-dentin complexes

34,35

. IGF-1 also in-

fluences the proliferation and differentiation

of other odontogenic cell types, specifically

periodontal ligament stem cells and stem

cells from apical papilla

19,38

. In this study, we

also found that exogenous IGF-1 promoted

the proliferation of hDPCs in vivo in a con-

centration-dependent manner and progres-

sively promoted odontogenic differentiation

of hDPCs in vivo.

IGF-1 receptor (IGF-1R) is a cell-surface

receptor tyrosine kinase with its specific li-

gands IGF-1

. It activates downstream sig-

naling pathways, namely the phosphoinosit-

ide 3-kinase (PI3K)/AKT and the RAS/

mitogen-activated protein kinase (MAPK)

pathways. These pathways are widely in-

volved in stem cell growth, proliferation, and

differentiation

40,41

. Previous studies have re-

ported that the activation of IGF-1R signal-

ing contributes to maintaining the self-re-

newal and differentiation of hDPCs

. It has

been demonstrated that treatment with IGF-

1 can up-regulate the expression of phospho-

ERK and phospho-p38 of hDPCs, suggesting

MAPK signaling pathway during differentia-

tion of hDPSCs and human periodontal liga-

ment stem cells

. The interactions between

IGF-1R and the p38 MAPK signaling pathway

188 Wang et al.

Investigación Clínica 65(2): 2024

control the quiescence and activation of

hDPSCs. In addition, the downstream effec-

tors between the MAPK signaling pathway

and the PI3K/Akt signaling pathway partly

overlap, resulting in the interaction effect of

two signaling pathways. Some studies show

that IGF-1 can promote the differentiation

of adipose-derived stem cells and endothe-

lial cells by the PI3K/AKT signaling pathway

. Meanwhile, the IGF-1-induced activation

of the AKT signaling pathway also involved

the proliferation, migration, osteogenesis,

and angiogenesis of hDPSCs

. However, the

underlying molecular mechanism of prolif-

eration and odontogenic differentiation for

hDPCs needs further research.

In conclusion, we found that exogenous

IGF-1 promoted the proliferation of hDPCs in

a concentration-dependent manner in vitro

and progressively promoted odontogenic dif-

ferentiation of hDPCs in vivo. This suggests

that modifying IGF-1 signaling may offer po-

tential strategies for hDPCs-based tissue en-

gineering to regenerate dental tissues.

Declarations Ethics approval and consent

to participate

This study was conducted following

the Helsinki Declaration. This study was

conducted with approval from the Ethics

Committee of the Second Hospital of Hebei

Medical University. Written informed con-

sent was obtained from all participants. This

study was conducted under the principles of

ethical animal research outlined in the Basel

Declaration and the ethical guidelines by the

International Council for Laboratory Animal

Science (ICLAS). This study was conducted

following NC3Rs ARRIVE guidelines.

Competing interest

The authors had no separate personal,

financial, commercial, or academic conflicts

of interest.

Funding

Not applicable.

Authors ORCID’number

• Yan Wang (YW):

0009-0004-4323-0268

• Nan Du (ND):

0009-0009-3942-3872

• Cong-na Liu (CNL):

0009-0002-2022-0329

• Wen-jing Li (WJL):

0009-0007-4067-5845

Author contributions

Conception and design: Wang W; ad-

ministrative support: YW and ND; provision

of study materials or patients: ND and CNL;

collection and assembly of data: CNL and

WJL; data analysis and interpretation: YW

and WJL; manuscript writing: all authors

and final approval of manuscript: all authors.

REFERENCES

1. Liu S, Wang S, Dong Y. Evaluation of a

bioceramic as a pulp capping agent in vi

tro and in vivo. J Endod. 2015;41(5):652-

657. https://doi.org/10.1016/j.joen.2014.

12.009.

2. Cooper PR, Takahashi Y, Graham LW,

Simon S, Imazato S, Smith AJ. Inflam

mation-regeneration interplay in the den-

tine-pulp complex. J Dent 2010;38(9):687-

697. https://doi.org/10.1016/j.jdent.2010.

05.016.

3. Wang YL, Hu YJ, Zhang FH. Effects of GP

NMB on proliferation and odontoblastic

differentiation of human dental pulp cells.

Int J Clin Exp Pathol 2015;8(6):6498-

6504.

4. Miura M, Gronthos S, Zhao M, Lu B,

Fisher LW, Robey PG, Shi S. SHED:

stem cells from human exfoliated deci

duous teeth. Proc Natl Acad Sci USA.

2003;100(10):5807-5812. https://doi.org/

10.1073/pnas.0937635100.

5. Jo YY, Lee HJ, Kook SY, Choung HW,

Park JY, Chung JH, Choung YH, Kim

ES, Yang HC, Choung PH. Isolation and

Growth changes of human dental pulp cells 189

Vol. 65(2): 179 - 191, 2024

characterization of postnatal stem cells

from human dental tissues. Tissue Eng.

2007;13(4):767-773. https://doi.org/10.

1089/ten.2006.0192.

6. Paino F, Ricci G, De Rosa A, D’Aquino

R, Laino L, Pirozzi G, Tirino V, Papac

cio G. Ecto-mesenchymal stem cells from

dental pulp are committed to differentia

te into active melanocytes. Eur Cell Ma-

ter. 2010;20:295-305. https://doi.org/10.

22203/ecm.v020a24.

7. Kim BC, Bae H, Kwon IK, Lee EJ, Park

JH, Khademhosseini A, Hwang YS. Os

teoblastic/cementoblastic and neural di-

fferentiation of dental stem cells and their

applications to tissue engineering and

regenerative medicine. Tissue Eng Part

B Rev. 2012;18(3):235-244. https://doi.

org/10.1089/ten.TEB.2011.0642.

8. Smith JG, Smith AJ, Shelton RM, Coo

per PR. Dental pulp cell behavior in

biomimetic environments. J Dent Res.

2015;94(11):1552-1559. https://doi.org/

10.1177/0022034515599767.

9. Chen FM, Zhang J, Zhang M, An Y,

Chen F, Wu ZF. A review on endogenous

regenerative technology in periodon

tal regenerative medicine. Biomaterials.

2010;31(31):7892-7927. https://doi.org/

10.1016/j.biomaterials.2010.07.019.

10. Liang C, Liao L, Tian W. Stem cell-based

dental pulp regeneration: insights from

signaling pathways. Stem Cell Rev Rep.

2021;17(4):1251-1263. https://doi.org/

10.1007/s12015-020-10117-3.

11. Suzuki T, Lee CH, Chen M, Zhao W, Fu SY,

Qi JJ, Chotkowski G, Eisig SB, Wong A,

Mao JJ. Induced migration of dental pulp

stem cells for in vivo pulp regeneration. J

Dent Res. 2011;90(8):1013-1018. https://

doi.org/10.1177/0022034511408426.

12. Huang GT, Gronthos S, Shi S. Mesenchy

mal stem cells derived from dental tissues

vs. those from other sources: their biology

and role in regenerative medicine. J Dent

Res. 2009;88(9):792-806. https://doi.org/

10.1177/0022034509340867.

13. Pierdomenico L, Bonsi L, Calvitti M, Ron

delli D, Arpinati M, Chirumbolo G, Bec-

chetti E, Marchionni C, Alviano F, Fossati

V, Staffolani N, Franchina M, Grossi A,

Bagnara GP. Multipotent mesenchymal

stem cells with immunosuppressive ac

tivity can be easily isolated from dental

pulp. Transplantation. 2005;80(6):836-

842. https://doi.org/10.1097/01.tp.0000

173794.72151.88.

14. Chen FM, Zhang M, Wu ZF. Toward delivery

of multiple growth factors in tissue engi

neering. Biomaterials. 2010;31(24):6279-

6308. https://doi.org/10.1016/j.biomate

rials.2010.04.053.

15. Kim SG, Zhou J, Solomon C, Zheng Y, Su

zuki T, Chen M, Song S, Jiang N, Cho S,

Mao JJ. Effects of growth factors on den

tal stem/progenitor cells. Dent Clin Nor-

th Am. 2012;56(3):563-575. https://doi.

org/10.1016/j.cden.2012.05.001.

16. Fang Y, Wang LP, Du FL, Liu WJ, Ren GL.

Effects of insulin-like growth factor I on

alveolar bone remodeling in diabetic rats.

J Periodontal Res. 2013;48(2):144-150.

https://doi.org/10.1111/j.1600-0765.

2012.01512.x.

17. Chen FM, Zhao YM, Wu H, Deng ZH,

Wang QT, Zhou W, Liu Q, Dong GY, Li K,

Wu ZF, Jin Y. Enhancement of periodontal

tissue regeneration by locally controlled

delivery of insulin-like growth factor-I from

dextran-co-gelatin microspheres. J Control

Release. 2006;114(2):209-222. https://

doi.org/10.1016/j.jconrel.2006.05.014.

18. Feng X, Huang D, Lu X, Feng G, Xing J,

Lu J, Xu K, Xia W, Meng Y, Tao T, Li L,

Gu Z. Insulin-like growth factor 1 can pro

mote proliferation and osteogenic diffe-

rentiation of human dental pulp stem cells

via mTOR pathway. Dev Growth Differ.

2014;56(9):615-624. https://doi.org/10.1

111/dgd.12179.

19. Yu Y, Mu J, Fan Z, Lei G, Yan M, Wang S,

Tang C, Wang Z, Yu J, Zhang G. Insulin-

like growth factor 1 enhances the proli

feration and osteogenic differentiation of

human periodontal ligament stem cells via

ERK and JNK MAPK pathways. Histochem

Cell Biol. 2012;137(4):513-525. https://

doi.org/10.1007/s00418-011-0908-x.

20. Lu W, Xu W, Li J, Chen Y, Pan Y, Wu B.

Effects of vascular endothelial growth

190 Wang et al.

Investigación Clínica 65(2): 2024

factor and insulin growth factor1 on

proliferation, migration, osteogenesis

and vascularization of human carious

dental pulp stem cells. Mol Med Rep.

2019;20(4):3924-3932. https://doi.org/

10.3892/mmr.2019.10606.

21. Lins-Candeiro CL, Paranhos LR, de Oli

veira Neto NF, Ribeiro RAO, de-Souza-

Costa CA, Guedes FR, da Silva WHT,

Turrioni AP, Santos Filho PCF. Viability

and oxidative stress of dental pulp cells

after indirect application of chemome

chanical agents: An in vitro study. Int En-

dod J. 2023; 57(3):315-327. doi: 10.1111/

iej.14013.

22. Schreier C, Rothmiller S, Scherer MA,

Rummel C, Steinritz D, Thiermann H,

Schmidt A. Mobilization of human me

senchymal stem cells through different

cytokines and growth factors after their

immobilization by sulfur mustard. Toxi

col Lett. 2018;293:105-111. https://doi.

org/10.1016/j.toxlet.2018.02.011.

23. Martin-Del-Campo M, Rosales-Ibañez

R, Alvarado K, Sampedro JG, Garcia-

Sepulveda CA, Deb S, San Román J,

Rojo L. Strontium folate loaded biohy

brid scaffolds seeded with dental pulp

stem cells induce in vivo bone regenera

tion in critical sized defects. Biomater

Sci. 2016;4(11):1596-1604. https://doi.

org/10.1039/c6bm00459h.

24. Woo SM, Lim HS, Jeong KY, Kim SM,

Kim WJ, Jung JY. Vitamin D promotes

odontogenic differentiation of human

dental pulp cells via ERK activation. Mol

Cells. 2015;38(7):604-609. https://doi.

org/10.14348/molcells.2015.2318.

25. Teng CF, Jeng LB, Shyu WC. Role of

insulin-like growth factor 1 receptor

signaling in stem cell stemness and

therapeutic efficacy. Cell Transplant.

2018;27(9):1313-1319. https://doi.org/

10.1177/0963689718779777.

26. Werle SB, Lindemann D, Steffens D, De

marco FF, de Araujo FB, Pranke P, Casa-

grande L. Carious deciduous teeth are a po-

tential source for dental pulp stem cells. Clin

Oral Investig. 2016;20(1):75-81. https://doi.

org/10.1007/s00784-015-1477-5.

27. Gnanasegaran N, Govindasamy V, Abu Ka

sim NH. Differentiation of stem cells derived

from carious teeth into dopaminergic-like

cells. Int Endod J. 2016;49(10):937-949.

https://doi.org/10.1111/iej.12545. Epub

2015 Oct 3.

28. Gronthos S, Mankani M, Brahim J, Ro

bey PG, Shi S. Postnatal human den-

tal pulp stem cells (DPSCs) in vitro

and in vivo. Proc Natl Acad Sci U S A.

2000;97(25):13625-13630. https://doi.

org/10.1073/pnas.240309797.

29. Almushayt A, Narayanan K, Zaki AE,

George A. Dentin matrix protein 1 in

duces cytodifferentiation of dental pulp

stem cells into odontoblasts. Gene Ther.

2006;13(7):611-620. https://doi.org/10.1

038/sj.gt.3302687.

30. Zhang W, Walboomers XF, Shi S, Fan M,

Jansen JA. Multilineage differentiation

potential of stem cells derived from human

dental pulp after cryopreservation. Tissue

Eng. 2006;12(10):2813-2823. https://doi.

org/10.1089/ten.2006.12.2813.

31. Yang X, Zhang W, van den Dolder J, Wal

boomers XF, Bian Z, Fan M, Jansen JA.

Multilineage potential of STRO-1+ rat

dental pulp cells in vitro. J Tissue Eng Re

gen Med. 2007;1(2):128-135. https://doi.

org/10.1002/term.13.

32. Moeller M, Pink C, Endlich N, Endlich K,

Grabe HJ, Völzke H, Dörr M, Nauck M,

Lerch MM, Köhling R, Holtfreter B, Ko

cher T, Fuellen G. Mortality is associated

with inflammation, anemia, specific disea

ses and treatments, and molecular markers.

PLoS One. 2017;12(4):e0175909. https://

doi.org/10.1371/journal.pone.0175909.

33. Alarçin E, Lee TY, Karuthedom S, Moham

madi M, Brennan MA, Lee DH, Marrella A,

Zhang J, Syla D, Zhang YS, Khademhos

seini A, Jang HL. Injectable shear-thinning

hydrogels for delivering osteogenic and an

giogenic cells and growth factors. Bioma-

ter Sci. 2018;6(6):1604-1615. https://doi.

org/10.1039/c8bm00293b.

34. Shinohara Y, Tsuchiya S, Hatae K, Honda

MJ. Effect of vitronectin bound to insulin-

like growth factor-I and insulin-like growth

factor binding protein-3 on porcine ena

Growth changes of human dental pulp cells 191

Vol. 65(2): 179 - 191, 2024

mel organ-derived epithelial cells. Int J

Dent. 2012;2012:386282. https://doi.org/

10.1155/2012/386282.

35. Catón J, Bringas P Jr, Zeichner-David M.

Establishment and characterization of an

immortomouse-derived odontoblast-like

cell line to evaluate the effect of insulin-

like growth factors on odontoblast differen

tiation. J Cell Biochem. 2007;100(2):450-

463. https://doi.org/10.1002/jcb.21053.

36. Schminke B, Vom Orde F, Gruber

R, Schliephake H, Bürgers R, Mios

ge N. The pathology of bone tissue du-

ring peri-implantitis. J Dent Res. 2015;

94(2):354-361. https://doi.org/10.1177/

0022034514559128.

37. Onishi T, Kinoshita S, Shintani S, So

bue S, Ooshima T. Stimulation of proli-

feration and differentiation of dog dental

pulp cells in serum-free culture medium

by insulin-like growth factor. Arch Oral

Biol. 1999;44(4):361-371. https://doi.

org/10.1016/s0003-9969(99)00007-2.

38. Wang S, Mu J, Fan Z, Yu Y, Yan M, Lei G,

Tang C, Wang Z, Zheng Y, Yu J, Zhang G.

Insulin-like growth factor 1 can promote

the osteogenic differentiation and osteo

genesis of stem cells from apical papilla.

Stem Cell Res. 2012;8(3):346-356. https://

doi.org/10.1016/j.scr.2011.12.005.

39. Chen C, Xu Y, Song Y. IGF-1 gene-modified

muscle-derived stem cells are resistant to

oxidative stress via enhanced activation of

IGF-1R/PI3K/AKT signaling and secretion

of VEGF. Mol Cell Biochem. 2014;386(1-

2):167-175. https://doi.org/10.1007/s110

10-013-1855-8.

40. Liu C, Zhang Z, Tang H, Jiang Z, You

L, Liao Y. Crosstalk between IGF-1R and

other tumor promoting pathways. Curr

Pharm Des. 2014;20(17):2912-1921.

https://doi.org/10.2174/1381612811319

9990596.

41. Girnita L, Worrall C, Takahashi S, Se

regard S, Girnita A. Something old, so-

mething new and something borrowed:

emerging paradigm of insulin-like growth

factor type 1 receptor (IGF-1R) sig

naling regulation. Cell Mol Life Sci.

2014;71(13):2403-2427. https://doi.org/

10.1007/s00018-013-1514-y.

42. Wan Y, Zhuo N, Li Y, Zhao W, Jiang D.

Autophagy promotes osteogenic diffe

rentiation of human bone marrow mes-

enchymal stem cell derived from osteo-

porotic vertebrae. Biochem Biophys Res

Commun. 2017;488(1):46-52. https://doi.

org/10.1016/j.bbrc.2017.05.004.