Chapter 23

Cell Biological Assays for Measuring Odontogenic

Activities of CCN Proteins
Koichiro Muromachi, Hiroshi Sugiya, and Nobuyuki Tani-Ishii

Abstract
In postnatal dentin formation, odontoblast differentiation occurs in the pulp tissue regenerative process under
pathological condition. Odontoblasts and newly differentiated odontoblast-like cells beneath the caries lesion
form tertiary dentin and are highly odontogenic. To observe the activity of dentinogenesis occur within the
hard tissue, a combination of immunohistological analysis and immunodetection of dentinogenesis specific
molecules, such as dentin sialophosphoprotein (DSPP) and/or its cleaved products dentin sialoprotein (DSP)
and dentin phosphoprotein (DPP), is a reliable approach. Besides, recent studies have revealed that the expres-
sion of CCN family member 2 (CCN2), a member of the CCN family protein, is confirmed in accordance
with tooth development and reparative dentin formation. Therefore, CCN2 could serve as a marker for den-
tinogenesis. Here, we describe a method for visualizing the CCN2 signal as an odontogenic activity in forma-
lin-fixed paraffin-embedded (FFPE) sections of demineralized human teeth and human dental pulp cells.

Key words Immunohistochemistry, Western blot, Human teeth, Dentinpulp complex, Odontoblasts,
Dentinogenesis

1 Introduction

Odontoblasts are one of the most unique cells in orofacial tissues due
to its morphological and functional features. These cells are aligned
along the predentin and all of them extend its cellular processes in an
outward direction [1]. Previous immunohistochemical studies have
revealed that odontoblast is divided into several stages by their meta-
bolic status [2, 3]. Once primary dentin formation is settled, dentin-
secreting activity of odontoblast is reduced in accordance with aging
[3]. In this resting phase, odontoblasts deposit secondary dentin
faintly onto originally formed dentin. The rate of secondary dentin
secretion is limited from one-tenth to one-fiftieth than that of pri-
mary dentinogenesis [4]. However, even after the tooth eruption,
tertiary dentinogenesis recurs upon harmful external stimuli such as
dental caries, trauma, and cavity preparation during restorative proce-
dures in dentistry as a protective response of pulp tissue [4].

Masaharu Takigawa (ed.), CCN Proteins: Methods and Protocols, Methods in Molecular Biology, vol. 1489,
DOI 10.1007/978-1-4939-6430-7_23, Springer Science+Business Media New York 2017

251
252 Koichiro Muromachi et al.

Concerning the status of odontoblast activity and differentiation,

it is thought that dentin sialophosphoprotein (DSPP) and/or its
processed products dentin sialoprotein (DSP) and dentin phospho-
protein (DPP) are valuable markers for characterizing the phase of
odontoblast maturation, especially dentin secretion [2]. DSPP,
DSP, and DPP are the most prominent component of non-
collagenous protein in dentin [5]. The two DSPP derived products
play different roles; DSP involves in the initiation of dentin miner-
alization, and DPP facilitates the dentin maturation [6].
In developing tooth germs, the epithelialmesenchymal inter-
action is necessary for tooth growth [7]. This interplay between
each type of cells leads the CCN family member 2 (CCN2) expres-
sion [8]. In addition, experimentally neutralized CCN2 function
provides the impairment of tooth growth, as a result of suppression
of ameloblasts and odontoblasts differentiation [8]. Meanwhile,
the expression of CCN2 is increased in odontoblast-like cells
beneath the reparative dentin in carious teeth [9]. Thus, we
hypothesized that the expression patterns of CCN2 and DSPP in
pathological conditioned dental pulp will be instrumental in under-
standing the odontogenic activity in mature teeth [10]. To this
end, we applied a methodology of immunohistochemistry and
western blot. This document provides the full methods we have
employed as a demonstration of CCN2 expression in dentinpulp
complex to measure the odontogenic activity.

2 Materials

2.1 Immuno- 1. 4 % paraformaldehyde phosphate buffer solution.

histochemistry 2. Decalcifying solution B (EDTA method, Wako) (see Note 1).
2.1.1 Tissue Preparation 3. Sample Pack.
Reagents 4. Series of graded ethanols (70, 80, 90, 95, and 100 % in DW).
5. Xylene.
6. Tissue-Tek MEGA-CASSETTE (Sakura Finetek) or
equivalent.
7. Automatic embedding equipment Sakura Rotary (Sakura
Finetek, RH12-DM) or equivalent.
8. Tissue-Tek TEC Embedding Center (Sakura Finetek) or
equivalent.

2.1.2 Tissue Sectioning 1. Sliding microtome (Leica, SM2000R) or equivalent.

Equipments,
2. Disposable microtome blade (Feather, A35).
Deparaffinization
and Rehydration Reagents 3. Water bath.
4. Superfrost Micro Slide Glass (MAS-coated).
5. Tissue-Tek Slide Warmer (Sakura Finetek) or equivalent.
 Measuring CCN2 Signals In Human Dental Pulp 253

6. Small paintbrush.
7. Xylene.
8. Series of graded ethanols (100, 95, 90, 80, and 70 % in DW).

2.1.3 Blocking Reagents 1. Endogenous Peroxidase Blocking Reagent: Add 30 % of

for Endogenous Peroxidase Hydrogen peroxide to Methanol just before use.
and Nonspecific Antibody 2. Blocking buffer: Dissolve 4 g of Block Ace (DS Pharma
Interaction Biomedical) powder in a 100 ml of Millipore water. Add 0.5 ml
of Tween 20 and make up to a volume of 1 l with Millipore
water (see Note 2).

2.1.4 Primary and 1. Anti-human CCN2/CTGF rabbit polyclonal antibody

Secondary Antibodies (Peprotech) (see Note 3).
2. Anti-human DSPP mouse monoclonal antibody (LFMb-21)
(Santa Cruz Biotechnology) or equivalent (see Note 4).
3. Dako pen (Dako).
4. Dako REAL EnVision Detection Reagent Peroxidase
Rabbit/Mouse (Dako).

2.1.5 Detection 1. Dako EnVision kit (Dako) containing the substrate buffer
and Counterstaining and the DAB + Chromogen Solution.
Reagents 2. Mayers hematoxylin solution.
3. Series of graded ethanols (70, 80, 90, 95, and 100 % in DW).
4. Xylene.
5. Entellan (Merck) or equivalent.
6. Coverslip (24 50).

2.2 Western Blot 1. -MEM supplemented with 10 % FBS and antibiotics (20 U/ml
penicillin G, 100 g/ml kanamycin, 250 ng/ml fungizone).
2.2.1 Cell Culture
2. Trypsin.

2.2.2 Cell Preparation 1. Recombinant human BMP-1 (R&D systems) (see Note 5).
2. Recombinant human MMP-3 (Acris Antibodies) or equivalent
(see Note 5).
3. Protease inhibitor cocktail solution: Dissolve one tab of
Complete Mini EDTA-free (Roche) in a 500 l of CelLytic
M Cell Lysis Reagent (Sigma-Aldrich).
4. Cell lysis buffer: Mix the Protease inhibitor cocktail solution
and the CelLytic M Cell Lysis Reagent in a 1 : 20 ratio. Add
100 M PMSF (Cell Signaling Technology), 0.2 mM EGTA,
and 2 mM EDTA.
5. Cell scraper.
254 Koichiro Muromachi et al.

6. Sample buffer: We routinely use the Red Loading Buffer pack

(New England BioLabs) containing 3 SDS sample buffer and
30 DTT. Mix the 3 SDS sample buffer and the 30 DTT in
a 9:1 ratio before using.

2.2.3 SDS-PAGE 1. 7.5 % Mini-PROTEAN TGX Precast Protein Gels

and Immunoblot (Bio-Rad).
2. Precision Plus Protein Dual Color Standard (Bio-Rad).
3. Running buffer: Dissolve one pack of Tris-Glycine-SDS pow-
der (Takara) in a 500 ml of Millipore water.
4. Transfer buffer: Mix 100 ml of 10 Tris Glycine buffer (Bio-
Rad) to 700 ml of Millipore water and 200 ml of Methanol.
5. Supported Nitrocellulose Membranes 0.2 m (Bio-Rad) or
equivalent.
6. TBS buffer: Mix 5 ml of 10 TBS Buffer pH 7.5 (Rockland
Immunochemicals) and 45 ml of Millipore water.
7. Blocking buffer: Dissolve 4 g of Block Ace powder in a 100 ml
of Millipore water (see Note 2).
8. Anti-human CCN2/CTGF and anti-human DSPP antibodies:
the same as in Subheading 2.1.4.
9. Anti--actin antibody (e.g., anti--actin rabbit monoclonal
antibody, Cell Signaling Technology).
10. PE/PET pouch (Yamamoto Manufacturing).
11. Wash buffer: Add 0.5 ml of Tween 20 to 100 ml of blocking
buffer. Make up to a volume of 1 l with Millipore water.
12. Anti-rabbit IgG HRP-linked antibody (Cell Signaling
Technology) or equivalent.
13. Anti-mouse IgG HRP-linked antibody (Cell Signaling
Technology) or equivalent.
14. ECL Prime Western Blotting Detection Reagent (GE).
15. Hyperfilm ECL (GE).
16. LAS 3000 (Fuji Film) or equivalent.

3 Methods

All steps are performed at room temperature unless noted other-

wise. The procedure to obtain the tooth samples must be approved
by the ethics committee.
 Measuring CCN2 Signals In Human Dental Pulp 255

3.1 Immuno- 1. After extraction, cut the intact and carious teeth horizontally at the
histochemical cervical region in order to obtain a secure fixation of dental pulp.
Staining of CCN2 in 2. Immerse the samples immediately with 4 % paraformaldehyde
Odontoblasts Under for 1 day at 4C (see Note 6).
Pathological Condition 3. After fixation, rinse the samples with distilled water.
3.1.1 Preparation 4. Put the samples into the Sample Packs.
of Teeth
5. Hang the Sample Packs and demineralize the samples with decal-
cifying solution B for 4 weeks at 4 C with stirring (see Note 7).
6. Process the demineralized tissues through a series of graded
ethanols (70, 80, 90, 95, and 100 %) for dehydration, immerse
in xylene, and infiltrate in paraffin using the automatic embed-
ding equipment Sakura Rotary.
7. Embed the samples in paraffin using Tissue-Tek TEC
Embedding Center.

3.1.2 Sectioning, 1. Cut sections of 4-m thickness from paraffin blocks on a

Deparaffinization, microtome with a disposable blade suited for hard tissue block.
and Rehydration 2. Pick up the cut sections from the microtome using wetted
of Samples paintbrush.
3. Float the thin sections on water bath at 40 C to stretch them.
4. Mount the stretched sections on slide glasses using paintbrush.
5. Wipe off the excess water on the slide glasses.
6. Dry the tissue mounted slides using a paraffin stretching plate
(Slide Warmer) at 37 C for 1 day.
7. To melt the paraffin, take the slides on paraffin stretching plate
at 37 C for 30 min or in paraffin oven at 60 C for 10 min.
8. Deparaffinize the slides two times with xylene for 3 min.
9. Rehydrate the slides by 3 min processes through a series of
graded ethanols (100, 95, 90, 80, and 70 %).

3.1.3 Inactivation 1. Incubate the sections with Endogenous Peroxidase Blocking

of Endogenous Peroxidases Reagent for 30 min.
and Blocking Nonspecific 2. To block nonspecific antibody interactions, rinse the sections
Interactions with blocking buffer for 3 min, at least three times.

3.1.4 Primary and 1. Wipe off the excess buffer on the slide glasses.
Secondary Antibodies 2. Encircle the sections with a Dako pen.
3. Add 40 l/slide of primary antibody diluted in blocking buffer
(see Note 8).
4. Put the slides into the moist chamber and incubate overnight
at 4 C.
5. After incubation, remove the primary antibody solution care-
fully using blotting paper (see Note 9).
256 Koichiro Muromachi et al.

6. Wash the slides with blocking buffer for 3 min, at least three
times.
7. Wipe off the excess buffer on the slide glasses.
8. Add one to two drops of the Dako REAL EnVision
Detection Reagent Peroxidase Rabbit/Mouse.
9. Put the slides into the moist chamber and incubate for 1 h.

3.1.5 Detection 1. After incubation, remove the secondary antibody solution

and Counterstaining carefully using blotting paper.
2. Wash the slides with blocking buffer for 3 min, at least three
times.
3. Wipe off the excess buffer on the slide glasses.
4. Mix the substrate buffer and the DAB + Chromogen Solution
just before use.
5. Add 40 l/slide of the DAB mixture solution.
6. Incubate for 15 min and visualize the antigenantibody reac-
tion under a microscope.
7. Wash the slides with distilled water for 5 min to stop the
reaction.
8. Immerse the slides into Mayers Hematoxylin for 2 min.
9. Wash the slides with distilled water for 5 min.
10. Dehydrate the slides by 10 s processes through a series of
graded ethanols (70, 80, 90, 95, and 100 %) (see Note 10).
11. To permeate, process the sections two times with xylene for
10 s.
12. Add 12 drops of the Entellan onto the sections and put a
coverslip on the mounting solution.
13. Dry the slides for 1 h on slide tray.
14. Observe the slides using the microscope.

3.2 Measuring CCN2 1. After extraction, cut the intact teeth horizontally at the cervical
Expression in Human region.
Dental Pulp Cell Lysate 2. Strip off the pulp tissue from pulp chamber using tweezer
by Western Blot under aseptic conditions.
3.2.1 Cell Culture 3. Mince the dental pulp using scalpel with one drop of the
-MEM supplemented with 10 % FBS.
4. Place the minced tissue on a tissue culture dish and cover with
a sterilized glass coverslip.
5. Culture the explants in -MEM supplemented with 10 % FBS and
antibiotics at 37 C in a humidified atmosphere of 5 % CO2 in air.
6. Once cell growth from the explants had reached confluence,
detach the cells with 0.025 % trypsin in PBS.
 Measuring CCN2 Signals In Human Dental Pulp 257

7. Add same volume of -MEM and harvest the floating cells.

8. After centrifugation, subculture the pelleted cells in culture dishes.

3.2.2 Cell Preparation 1. Seed human dental pulp cells (passages 35) in 100-mm tissue
culture dishes at 2 105 cells/ml in -MEM containing 10 % FBS.
2. For the experiments, starve the cells in medium containing 1 %
FBS for 24 h before stimulation.
3. After serum starvation, stimulate the cells with rhBMP-1 or
rhMMP-3 at appropriate concentrations.
4. After stimulation, aspirate the medium and wash the cells twice
with PBS.
5. Add 250 l/dish of the cell lysis buffer and collect the cell
lysates using cell scraper (see Note 11).
6. Centrifuge for 1 min at 10,000 g and transfer the supernatant
to new 1.5 ml sample tubes.
7. Mix the supernatant and the sample buffer in a ratio of 2:1.
8. Then, boil the samples at 95 C for 5 min.
9. Storage the heated samples at 80 C prior to SDS-PAGE.
10. Adjust the protein concentration of samples using the method
of Bradford [11].

3.2.3 SDS-PAGE 1. Set the 7.5 % Mini-PROTEAN TGX Precast Gels into the
and Immunoblot electrode assembly.
2. Fill the buffer chamber with the running buffer.
3. Load an equal concentration of samples to gels.
4. Run the gels with the running condition at 200 V.
5. After electrophoresis, cut the excess part of gels using wide and
flat-ended spatula.
6. Remove the gels gently from the gel cassettes and overlay the
0.2 m nitrocellulose membrane onto the gel (see Note 12).
7. Set the gel and the nitrocellulose membrane to transfer
assembly.
8. Fill the transfer module with the transfer buffer.
9. Transfer the proteins with a condition at 12.5 V for
overnight.
10. After transfer, trim the nitrocellulose membranes to avoid the
nonspecific reaction of primary antibody.
11. Incubate the membranes with the TBS buffer at 60 rpm shak-
ing for 10 min.
12. Incubate the membranes with blocking buffer at 60 rpm shak-
ing for 50 min.
258 Koichiro Muromachi et al.

13. Dilute the primary antibodies in wash buffer (excluding Tween

20) at appropriate ratio (see Note 13).
14. Place the membrane into heat-sealed PE/PET pouch and add
primary antibody solution (see Note 14).
15. Incubate at 200 rpm shaking for 2 h.
16. Dilute the secondary antibodies in wash buffer (excluding
Tween 20) at appropriate ratio (see Note 15).
17. Pick out the membranes from the pouch and wash with the
wash buffer for 3 min at least three times.
18. Incubate the membranes with secondary antibodies at 60 rpm
shaking for 90 min.
19. Wash the membranes with the wash buffer for 3 min at least
three times.
20. After the final wash, detect the immunoreactivity using an ECL
Prime Western Blotting Detection Reagent.

4 Notes

1. We recommend the Decalcifying solution B (EDTA method,

Wako) for demineralizing the tooth samples with good success
[9, 10], although we have also tried other acidic decalcifying
reagents (e.g., formic acid).
2. We routinely use the Block Ace (DS Pharma Biomedical) for
blocking buffer to prevent the nonspecific antibody interac-
tion. This method is easy and displays good results [9, 10] than
skim milk. Other commercially available blocking reagents can
also be used.
3. We recommend the anti-human CCN2/CTGF rabbit poly-
clonal antibody (Peprotech) with excellent results [9, 10],
although we have also tried other anti-human CCN2/CTGF
mouse monoclonal antibody (Data not shown). We think that
it is worth trying other commercially available antibodies.
4. We have also tried to observe the immunoreaction for dentin
matrix protein-1 (DMP-1) in human teeth, but the specific
signals were shown in overall the dentin and pulp tissue (Data
not shown). Hence, we thought that DMP-1 is not suitable as
an odontoblast specific marker.
5. To induce the expression of CCN2, we routinely stimulate the
dental pulp cells with rhMMP-3 or rhBMP-1 [10]. The expres-
sion of both metalloproteases under pathological conditioned
dental pulp is well investigated [9].
6. We recommend that the volume of paraformaldehyde should
be at least ten times greater than the size of samples.
 Measuring CCN2 Signals In Human Dental Pulp 259

7. We recommend that the volume of decalcifying solution B

should be at least 50 times greater than the size of samples.
When the surface of samples is softened, cut off the redundant
area of samples using a scalpel for secure permeation of the
decalcifying solution.
8. We routinely dilute the primary antibodies (anti-CCN2/
CTGF and anti-DSPP) at 1:100 ratio.
9. Do not touch the tissue section. Carefully blot the antibody
solution only.
10. Shrinkage of specimens and separation of dentin and odonto-
blast layer may occur owing to long time exposure to
dehydration.
11. Place the sample tubes on ice to avoid the sample
degradation.
12. To prevent the gel drying, we perform this procedure in the
stainless steel tray filled with transfer buffer. When overlaying
the nitrocellulose membrane on the gel, carefully remove the
bubbles between the membrane and the gel.
13. We routinely dilute the primary antibodies at following ratio;
anti-CCN2/CTGF and anti-DSPP (diluted 1:1000), anti--
actin (diluted 1:2000).
14. Remove bubbles carefully before seal the pouch.
15. We routinely dilute the secondary antibodies at 1:2000 ratio.

Acknowledgments

We would like to thank Dr. Naoto Kamio for helpful suggestions

with western blot. This work was supported by JSPS KAKENHI
Grant-in-Aid for Young Scientists (B) #26861609.

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