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Title

Changes in the homeostatic mechanism of dental pulp

with age: expression of the core-binding factor

alpha-1, dentin sialoprotein, vascular endothelial

growth factor, and heat shock protein 27 messenger

RNAs

Author(s)

Alternative

Matsuzaka, K; Muramatsu, T; Katakura, A; Ishihara,

K; Hashimoto, S; Yoshinari, M; Endo, T; Tazaki, M;

Shintani, M; Sato, Y; Inoue, T

Journal Journal of Endodontics, 34(7): 818-821

URL http://hdl.handle.net/10130/561

Right

Abstract

Aim: Dental pulp has various characteristics in the pulp chamber, but only a

few biological evaluations about the effect of age on dental pulp tissue have

been reported. The purpose of this study was to compare dental pulp from

young and from adult rats to characterize the homeostatic mechanism.

Methodology: Dental pulp cells (DPCs) were obtained from the first molar of

rats, weighing 150 g each for the young group and 350 g each for the adult

group. Expression of Cbfa-1, VEGF or HSP27 mRNAs by cultured pulp cells

was determined using a quantitative real time PCR system after 3, 7 or 14

days.

Results: The expression of Cbfa-1 mRNA in the young group was higher than

in the adult group. Expression of VEGF and HSP27 mRNAs in the adult

group was higher than in the young group.

Conclusion

The self defense system in young DPCs is undertaken by calcification, but in

adult DPCs it is carried out by the expression of self defense proteins and the

regeneration of vessels.

Key words: aging; dental pulp; homeostasis; cbfa-1; VEGF; dentin

sialoprotein; HSP27; mPCR

Introduction

Dental pulp has various characteristics in the pulp chamber, including

fibrosis, atrophy, cellularity loss, dyscalcification and the degeneration of

odontoblasts (1, 2). Further, reduction of the chamber size is a typical

phenomenon in teeth from older individuals (3). These phenomena affect the

dental pulp and root properties, and suggest that aged pulp retains the

ability to create dentine but at a diminished rate (4). Further, vital

pulpotomy is the method that determines the characteristics of

dyscalcification. However, the success ratio of vital pulpotomy is lower in

adult pulp than in young pulp. This phenomenon depends on the function of

the homeostatic mechanism. Many kinds of cells can be observed in pulp tissue, in

which we can confirm odontoblast, fibroblast, endothelial cells, leukocyte, nerve fiber,

etc. It is known that mesenchymal cells in pulp have a potential for mineralization from

the formation of denticle and secondary or thirdly dentin induced by a variety of stimuli.

This phenomenon is known the protective response of pulp against the stimuli.

Vascularization plays an important role concerning mineralization and hard tissue

formation excluding dystrophic calcification. So, not only expression of self defense

protein but also mineralization and vasculariztion are inextricable for the homeostatic

mechanism in the pulp. There are many mechanisms regulating the

homeostasis of dental pulp. Many kinds of bone related protein concern for

the mineralization in dental pulp. Core binding factor alfa-1 (Cbfa-1) is

known as one of the required transcription factors that are produced when

pre-osteoblasts differentiate into osteoblasts (5). Odontoblasts also express

Cbfa-1 while differentiating from preodontoblasts (6, 7). In addition to this,

dentin sialo-protein (DSP) is specific protein of dental pulp for dentin

formation. Vascular endothelial growth factor (VEGF) is known as an

angiogenic growth factor that elicits cellular responses to hypoxia (8, 9).

Induction of VEGF has also been investigated in rat astrocytes and in the

heart under hypoxic conditions (10, 11). The development of endothelial

cell-based microvascularization and microcirculation is undoubtedly crucial

for the preservation of structure and for regeneration (12). Furthermore,

heat shock proteins (HSP) are expressed for self-defense against injury.

HSP-27 is involved in the cell death of dental pulp cells during stress due to

modification and activation (13).

To clarify the differences of characteristics between young dental

pulp cells (DPCs) and adult DPCs is beneficial for the prognostic of vital

pulpotomy. So, the purpose of this study was to evaluate the young and adult

DPCs to characterize their cell proliferation and their expression of Cbfa-1,

DSP, VEGF or HSP27 mRNAs .

Materials and Methods

Cell culture

All animal studies were conducted in compliance with the guidelines for the

treatment of experimental animals at the Tokyo Dental College. Pulp cells

were obtained from 150 g (5-week-old) Wistar male rats as young DPCs, and

from 350 g (15-week-old) Wistar rats as adult DPCs. Animals were

sacrificed with an overdose of thiopental (Ravonal®; Tanabe, Japan). Both

the upper and lower first molars were extracted from each animal, and were

washed for 5 min in ? -minimal essential medium (? -MEM; GIBCO, Carlsbad,

CA, USA) containing 500 µg/ml gentamycin and 0.3 µg/ml fungizone. The

periodontal ligament was then removed mechanically (using a knife

observed through a loupe) and the outer surface of the root was then

immersed in a solution of sodium hypochlorite for avoiding the

contamination. Following washing with PBS, each tooth was broken using a

dental chisel and a mallet. Each pulp was then carefully collected with a

dental explorer and a dental scalar using a microscope into a 35 mm culture

dish. Cells were cultured in ?-MEM containing 10% fetal bovine serum (FBS,

inactivated at 56°C for 35 minutes, GIBCO) and antibiotic (40 µg/ml

gentamycin, SIGMA, St Louis, MO, USA) in a humidified atmosphere of 95%

air and 5% CO2 at a temperature of 37°C. The culture medium was changed

every 2 days. Fourteen days after primary culture, the cells were detached

using trypsin / EDTA (0.1% trypsin, 0.02% EDTA, pH 7.2) and were

subcultured. Cells from the 4th subculture were then used in the following

experiments.

Quantitative RT-PCR

Approximately 5×104 DPCs were seeded in 6-well dishes and were cultured.

The culture medium was changed every 2 days. Total RNA was extracted

from each sample using the acid guanidium thiocyanate / phenol-chloroform

method as follows. The culture medium of each dish was removed and cells

were then rinsed twice using PBS. The cells were homogenized in 1 ml

TRIsol® Reagent (Invitrogen, Carlsbad, CA, USA) after 3, 7 or 14 days of

incubation. Each solution was transferred to a 1.5 ml tube containing

chloroform and was mixed. The tubes were centrifuged at 14,000 rpm at 4°C

for 20 minutes, after which the supernatants were placed in 1.5 ml tubes

containing 250 µl 100% isopropanol (a half amount of the TRIsol® Reagent)

at -80 °C for one hour. After centrifugation at 13200 rpm for 20 minutes at 4

°C, the supernatants were discarded, and the remaining total RNA pellets

were washed with 70% cold ethanol. The total RNA pellets were dissolved in

50 µl RNAase-free (DEPC-treated) water. Total RNAs were reverse

transcribed and amplified in 20 µl volumes using a Reverse Transcription Kit

(Quanti Tect®, Qiagen, MD, USA) containing RNA PCR Buffer, 2 U/µl

RNAase inhibitor, 0.25 U/µM reverse transcriptase, 0.125 µM oligo

dt-adaptor primer, 5 mM MgCl2 and RNAase-free water. RT-PCR products

were analyzed by quantitative real-time RT-PCR in TaqMan® Gene

Expression Assays (Applied Biosystems; Foster City, CA, USA) for the target

genes: Cbfa-1 (Rn 01512296-m1, 116 bp), DSP (Rn02132391_s1, 87bp), VEGF

(Rn 00582935-m1, 75 bp) and HSP 27 (Rn00583001_g1, 136 bp). The

TaqMan® Endogenous Control (Applied Biosystems) for the target gene

ß-actin (4352340E) was used as an endogenous control. All PCR reactions

were performed using a real time PCR 7500 fast system. Gene expression

quantitation using TaqMan® Gene Expression Assays was performed as the

second step in a two -step RT-PCR. Assays were done in 20 µL singleplex

reactions containing TaqMan® Fast Universal PCR Master Mix, TaqMan®

Gene Expression Assays, distilled water and cDNA, according to the

manufacturer's instructions (Applied Biosystems). Reaction conditions

consisted of a primary denaturation at 95°C for 20 sec, then cycling for 40

cycles of 95°C for 3 sec and 62°C for 30 sec. PCR data were supplemented by

the value of house keeping gene with and were ß-actin reported compared to

the corresponding three days of incubation in the young DPCs.

Statistical analysis

Three experimental runs were conducted, and data were analyzed using

one-way analysis of variance (ANOVA) and a multiple comparison test

(Scheffe’s test) (p<0.05).

Results

Quantitative RT-PCR

Cbfa-1 mRNA expression in young DPCs increased from 3 to 14 days of

culture, but in adult DPCs the expression of Cbfa-1 mRNA is very low.

Further, the expression of Cbfa-1 mRNA in young DPCs was higher than in

adult DPCs. There were significant differences between young and the adult

DPCs at all time periods (Fig. 1). DSP mRNA expression in young DPCs also

increased from 3 to 14 days of culture, but in adult DPCs the expression of

DSP mRNA is slightly increase. DSP mRNA expression in young group at 14

days of incubation was significantly higher than that in adult group (Fig. 2).

VEGF mRNA expression in both the young and the adult DPCs tended to

increase day by day. The rate of increased VEGF mRNA expression in adult

DPCs was higher than in young DPCs. There were significant differences

between the young and the adult DPCs at all time periods (Fig. 3). HSP27

mRNA expression in the young group did not vary with culture time, but the

expression of HSP27 mRNA in adult DPCs was slightly increased from 3 to 7

days of culture. The HSP27 mRNA expression in adult DPCs at 7 and 14

days of culuter was significantly higher than in young DPCs (Fig. 4).

Discussion

It is well known that the volume of the pulp chamber decreases with age, and

that this phenomenon occurs as a result of homeostatic mechanisms (14).

Only a few biological evaluations about aged dental pulp tissues have been

reported (15-17). Further, a reduction of osteocalcin mRNA expression may

also be associated with the loss of viability in dental pulp (18). Although

Muramatsu et al. evaluated the aged dental pulp tissue from old people, We

used pulp cells from the 350 g rats (15-week-old) as not senescence DPCs but

adult DPCs in this study. In this study, we analyzed the mRNA expression of

several important markers to further evaluate the characteristics of adult

dental pulp.

Cbfa-1 is a transcription factor that belongs to the runt domain gene

family (20-22). Cbfa-1 is a factor that not only regulates osteoblast

maturation, but also chondroblast maturation (20, 22). Furthermore, Cbfa-1

regulates not only the expression of bone and cartilage related genes, but

also is involved in dystrophic calcification. Further, DSP is the specific

protein for dentin related protein secreted from DPCs and odontoblast.

Dentin phosphoprotein (DPP) and dentin sialoprotein (DSP) are two types of

dentin non-collagenous matrix proteins. These proteins were originally

thought to be dentin-specific until Qin et al. (23) To examine the DSP

expression is one of indicators for DPCs’ differentiation. So, a dentin bridge is

formed under the necrotic tissue after vital pulpectomy with Ca2OH. Further,

the expression of Cbfa-1 in dental pulp tissue is well known (24). In this

study, a little expression of Cbfa-1 and DSP mRNAs was seen in adult DPCs,

but young DPCs expressed high levels of Cbfa-1 and DSP mRNAs. This

phenomenon demonstrates that young DPCs may readily produce dentin

bridges or ectopic calcification in the pulp chamber.

Angiogenesis plays an important role in maintaining homeostasis.

Booth et al. demonstrated that VEGF is involved in angiogenic processes in

healthy as well as in diseased periodontal tissues (25). Further, there are

some reports about the relationship of VEGF in dental pulp (26, 27). Many

studies have investigated angiogenesis in the DPCs, and have gradually

clarified its function. However, age-related differences in angiogenesis are

less well understood. VEGF is a crucial regulator of vascular development

during embryogenesis (vasculogenesis), as well as in blood-vessel formation

(angiogenesis) in adult tissues (28). Yoshino et al. reported that mechanical

stress stimulates the production of VEGF (29). In this study, the rate of

increase in VEGF mRNA expression in the adult group was higher than in

the young group. Further, it is known that the dental pulp canal becomes

stenosed with age which could explain why VEGF mRNA expression of adult

DPCs is higher than that of young cells.

Heat shock protein (HSP) 27 is a member of the small HSP family

that plays a part in regulating cell growth, cell differentiation, wound

healing, apoptosis and cell protection against stress (30). Further, HSP serve

as molecular chaperones in maintaining homeostasis and are expressed

physiologically in response to various types of stress. Furthermore, HSP has

remarkable potencies to induce self-defense mechanisms necessary to

maintain the homeostatic mechanism in aged tissues. HSP 27 expression can

be associated with cellular vulnerability or tolerance to stress and can even

be used as an index of cellular stress (31, 32). In this study, the rate of

increase in HSP 27 mRNA expression in adult DPCs was higher than in

young DPCs, which means that the self defense ability in adult DPCs is high.

In conclusion, the self defense system in young DPCs is achieved by

calcification, but in adult dental pulp cells it is carried out by the expression

of self defense proteins and the regeneration of vessels.

Acknowledgements

The authors would like to thank Ms. Saori Takano, Dr. Daisuke Sato and Dr

Hideki Soumiya for their technical assistance.

This research was in part supported by an Oral Health Science

Center Grant HRC7 from the Tokyo Dental College and by a “High-Tech

Research Center” Project for Private Universities: matching fund subsidy

from MEXT (Ministry of Education, Culture, Sports, Science and Technology)

of Japan, 2006-2010, and 2007-2010 (No. 19592414).

References

1. Bernic S, Nedelman C. Effect of aging on the human pulp. J Endod 1975;

1: 88-94.

2. Ketterl W. Age-induced changes in the teeth and their attachment

apparatus. Int Dental J 1983; 33: 262-71.

3. Oi T, Saka H, Ide Y. Three dimensional observation of pulp cavities in the

maxillary first premolar tooth using micro-CT. Int Endodontic J 2004; 37:

46-51.

4. Woods MA, Robinson QC, Harris EF. Age –progressive changes in pulp

widths and root lengths during adulthood: a study of American blacks and

whites. Gerodontology 1990; 9: 41-50.

5. Komori T, Yagi H, Nomura S, et al. Targeted disruption of cbfa-1 results in

a complete lack of bone formation owing to maturational arrest of osteoblasts.

Cell 1997; 89: 755-64.

6. Priam F, Ronco V, Locker M, et al. New cellular models for tracking the

odontoblast phenotype. Arch Oral Biol 2005; 50: 271-7.

7. Huang GT, Sonoyama W, Chen J, Park SH. In vitro characterization of

human dental pulp cells: various isolation methods and culturing

environments.

Cell Tissue Res 2006; 324: 225-36.

8. Ferrara N. The role of vascular endothelial growth factor in pathological

angiogenesis. Breast Cancer Res Treat 1995; 36: 127–37.

9. Carmeliet P, Collen D. Vascular development and disorders: molecular

analysis and pathogenic insights. Kidney Int 1998; 53: 1519–49.

10. Ijichi A, Sakuma S, Tofilon PJ. Hypoxia-induced vascular endothelial

growth factor expression in normal rat astrocyte cultures. Glia 1995; 14:

87–93.

11. Ladoux A, Frelin C. Hypoxia is a strong inducer of vascular endothelial

growth factor mRNA expression in the heart. Biochem Biophys Res Commun

1993; 195: 1005–10.

12. Schmid J, Wallkamm B, Hammerle CH, Gogolewski S, Lang NP. The

significance of angiogenesis in guided bone regeneration. A case report of a

rabbit experiment. Clin Oral Implants Res 1997; 8: 244-8.

13. Kitamura C, Nishihara T, Ueno Y, et al. Effects of sequential exposure to

lipopolysaccharide and heat stress on dental pulp cells. J Cell Biochem 2006;

99: 797-806.

14. Gómez PA, Cabrini RL. Anatomic variations of the root canal of the rat

according to age. Acta Odontol Latinoam 2004; 17: 39-42.

15. Muramatsu T, Hamano H, Ogami K, Ohta K, Inoue T, Shimono M.

Reduction of connexin 43 expression in aged human dental pulp. Int Endod J

2004; 37: 814-8.

16. Uitto MA, Ranta R. Protocollagen proline hydroxylase activity in human

dental pulp at various stages of development. Proc Finnish Dental Soc 1973;

69: 254-7.

17. Van Amerongen JP, Lemmens IG, TOnino GJ. The concentration,

extractability and characterization of collagen in human dental pulp. Arch

Oral Biol 1983; 28: 339-45.

18. Muramatsu T, Hamano H, Ogami K, Ohta K, Inoue T, Shimono M.

Reduction of osteocalcin expression in aged human dental pulp. Int Endod J

2005; 38: 817-21.

19. Matsuzaka K, Tsuruoka M, Kokubu E, et al. Age-related differences in

expression of vascular endothelial growth factor by periodontal ligament

cells in vitro. Bull Tokyo Dental College 2007; 48: 143-6.

20. de Crombrugghe B, Lefebvre V, Nakashima K. Regulatory mechanisms in

the pathways of cartilage and bone formation. Curr Opin Cell Biol 2001; 13:

721-8.

21. Drissi H, Luc Q, Shakoori R, et al. Transcriptional autoregulation of the

bone related CBFA1/RUNX2 gene. J Cell Physiol 2000; 184: 341-50.

22. Enomoto H, Enomoto-Iwamoto M, Iwamoto M, et al. Cbfa1 is a positive

regulatory factor in chondrocyte maturation. J Biol Chem 2000; 275:

8695-702.

23. Qin C, Brunn JC, Cadena E, Ridall A, Tsujigiwa H, Nagatsuka H, Nagai N, Butler

WT. The expression of dentin sialophosphoprotein gene in bone. J Dent Res. 2002; 81:

392-4.

24. Yildirim S, Yapar M, Sermet U, Sener K, Kubar A. The role of dental pulp

cells in resorption of deciduous teeth. Oral Surg Oral Med Oral Pathol Oral

Radiol Endod 2007; (in press).

25. Booth V, Young S, Cruchley A, Taichman NS, Paleolog E. Vascular

endothelial growth factor in human periodontal disease. J Periodontal Res

1998; 33: 491-9.

26. Grando Mattuella L, Westphalen Bento L, de Figueiredo JA, Nor JE, de

Araujo FB, Fossati AC. Vascular endothelial growth factor and its

relationship with the dental pulp. J Endod. 2007; 33: 524-30.

27. Botero TM, Shelburne CE, Holland GR, Hanks CT, Nor JE. TLR4

mediates LPS-induced VEGF expression in odontoblasts. J Endod. 2006; 32:

951-5.

28. Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L. VEGF receptor

signaling - in control of vascular function. Nat Rev Mol Cell Biol 2006; 7:

359-71.

29. Yoshino H, Morita I, Murota SI, Ishikawa I. Mechanical stress induces

production of angiogenic regulators in cultured human gingival and

periodontal ligament fibroblasts. J Periodontal Res 2003; 38: 405-10.

30. Leonardi R, Villari L, Caltabiano M, Travali S. Heat shock protein 27

expression in the epithelium of periapical lesions. J Endod 2001; 27: 89-92.

31. Schlesinger MJ. Heat shock proteins. J Biol Chem 1990; 265: 12111–4.

32. Amemiya K, Kaneko Y, Muramatsu T, Shimono M, Inoue T. Pulp cell

responses during hypoxia and reoxygenation in vitro. Eur J Oral Science

2003; 111: 332-8.

Legends of figures

Fig. 1Cbfa-1 mRNA expression

Cbfa-1 mRNA expression in the young group increased from 3 to 14 days of

culture. Cbfa-1 mRNA expression in adult group did not change at all. The

data is shown as the fold increase of corresponding three days of incubation in the

young DPCs.

Fig. 2 DSP mRNA expression

DSP mRNA expression in both groups increased from 3 to 14 days of culture.

However, DSP mRNA expression in adult group increased only slightly. So

there is a significant difference between young and adult groups at 14 days.

The data is shown as the fold increse of corresponding three days of

incubation in young DPCs.

Fig. 3 VEGF mRNA expression

VEGF mRNA expression in both the young and the adult DPCs increased

day by day. The rate of increase of VEGF mRNA expression in adult DPCs

was higher than in the young group. The data is shown as the fold inrease of

corresponding three days of incubation in the young DPCs.

Fig. 4 HSP27 mRNA expression

HSP27 mRNA expression in young DPCs was not different at various culture

times, but HSP27 mRNA expression in adult DPCs was slightly increased

from 3 to 7 days of culture. The data is shown as the fold increase of corresponding

three days of incubation in the young DPCs.

1

Fig. 1 Cbfa-1 mRNA expression

0

2

4

6

8

10

3 7 14

youngadult

days

* : p<0.01

*

*

*Fol

d In

crea

se

2

Fig. 2 DSP mRNA expression

3 7 14 days

* : p<0.01

youngadult

*

02468

101214

Fol

d In

crea

se

3

Fig. 3 VEGF mRNA expression

0

2

4

6

8

10

3 7 14

young

days

* : p<0.01

*

**

adult

Fol

d In

crea

se

4

Fig. 4 HSP27 mRNA expression

00.20.40.60.81

1.21.4

young

3 7 14 days

* : p<0.01

* *

adult

Fol

d In

crea

se