Advance Online Publication

ODEP Vol. 1, No. 1

Original Article

Development of Multilayered Dental Pulp Cell Sheets
and Consideration for Calcification Ability
KOYANAGI Katofumi, MAEDA Munehiro, SEKIYA Miki,
NISHIDA Taro and IGARASHI Masaru
Department of Endodontics, The Nippon Dental University, School of Life Dentistry at Tokyo

Abstract
Purpose: The purpose of this study was to analyze the calcification characteristics of three-dimensional（3D）culture of multi-layered dental pulp cell（DPC）sheets that have potential application in pulp regeneration and evaluation of dental materials and pharmaceuticals.
Methods: DPC were isolated from third molars extracted from three healthy patients aged 18 30 years
old. DPC sheets were constructed using a temperature-responsive culture dish（UpCell）and a gelatin stamp（Cell Stamp for UpCell）. Histological examination of three-layer and six-layer DPC sheets was performed
using hematoxylin and eosin staining（HE）. DPC sheets on mineral trioxide aggregates（MTA）
were observed
using a scanning electron microscope（SEM）to examine the microstructure. The LOX-1 probe was used to
examine the effects of hypoxia. Calcification ability was analyzed by real-time PCR and Alizarin Red S staining（ARS）. In this study, two-dimensional（2D）culture（monolayer）served as a control.
Results: Thick multilayered cell sheets were confirmed under the macroscopic view and with HE. The
SEM images showed that the DPC sheets covered the MTA. Higher osteo/odontogenic differentiation wasseen in the multi-layered cell sheets than in the monolayer cell sheets. The expression levels of alkaline phosphatase（）, type Ⅰ collagen（）, osteocalcin（）, and dentin sialoprotein（）genes werehigher in the DPC sheets. With ARS, characteristic nodules of mineralization were observed in the cellsheets.
Conclusion: DPC sheets possessed higher mineralizing ability than the monolayer sheets. Cell sheets may
be useful for comparative examination of pharmaceuticals, materials, and pulp regeneration in a 3D environment.
Key words: dental pulp cell（DPC）, osteo/odontogenic differentiation, three-dimensional culture, cell sheets

Corresponding author: Dr. KOYANAGI, Department of Endodontics, The Nippon Dental University, School of Life Dentistry atTokyo, 1 9 20, Fujimi, Chiyoda-ku, Tokyo 102 8159, Japan
TEL: ＋81 3 3261 5698, FAX: ＋81 3 5216 3718, E-mail: k-koyanagi̲d2117006@tky.ndu.ac.jp
Received for Publication: January 7, 2021/Accepted for Publication: January 15, 2021
DOI: 10.11471/odep. 2021 003

at Tokyo with informed consent. DPC was cultured
according to the method described by Huang et al14）.

Introduction

Pulp tissues were minced and digested in a solution
In recent years, three-dimensional（3D）culture has

containing 3 mg/m collagenase type Ⅰ（Sigma, St.

been performed using various methods. It has been

Louis, MO, USA）and 4 mg/m dispase（Gibco, Invitro-

reported that 3D culture provides results that closely

gen, Carlsbad, CA, USA）at 37℃ for 60 minutes. Cell

mimic natural tissues as compared to those of two-di-suspensions were obtained using a 70μm strainer andmensional（2D）culture for accurate modeling of living

then seeded in 60 mm culture dishes at 37℃ under 5％organisms1） and analysis of medicines2,3）. In dentistry,

CO2 and cells with passage numbers of 3 6 were used

3D cultures can be used to assess biocompatibility ofin all experiments. The growth medium used was asandfollows; minimum essential medium α（MEM-α, Gibco）,

periodontal ligament , using scaffolds . However,

10％ fetal bovine serum（FBS, Hyclone, Logan, UT,

hypoxia, promotion of calcification in the spheroid struc-USA）, 100 units/m penicillin G, 100μg/m streptomy-

dental materials

4,5）

and for regeneration of pulp
7）

6）

8）

9）

ture , and a decrease in viability due to a decrease incin（Wako Pure Chemical Industries, Ltd., Osaka,

oxygen and nutrient supply can occur due to the scal-

Japan）, 2 mmol/ L-glutamine（Wako）, and 100μmol/

10）ing-up of the 3D structure . Therefore, there may be

L-ascorbicacid-2-phosphate（Sigma）. The osteo/odonto-

underestimation or overestimation of the results whengenic differentiation medium（OM）used was as follows:

evaluating drugs and materials. In view of these facts,

10 nmol/ dexamethasone（Sigma）, 10 mmol/ β-glycer-

we wondered if it would be possible to perform 3D cul-

ophosphate（Sigma）, 50μg/m L-ascorbic acid phos-

ture that maintains a specified thickness that does notphate（Sigma）, and 10 nmol/ 1,25 dihydroxyvitamin D3

cause hypoxia without using a scaffold and that focuses（Cayman Chemical, Ann Arbor, MI, USA）.

on cell sheet engineering. Cell sheet engineering refers

2 ．Laminating procedure for the DPC sheets

to the use of a temperature-responsive culture dish

DPC sheets were laminated by a modified procedure（UpCell）to collect cell sheets that retain extracellularof the method described by Haraguchi et al15） and thematrix without using enzymes such as trypsin fromnumber of seeded cells was determined with referencemonolayer culture, and to construct 3D structures by

to the manufacturerʼs protocol. DPC was seeded at a11）

layering these cell sheets . It is possible to collect cell

cell density of 1.5×105/well into a 24-well UpCell（Cell-

sheets that retain the binding and extracellular matrix

Seed, Tokyo, Japan）and incubated for 5 days in GM,

and stack these cell sheets to construct a 3D struc-then the 7.5％ gelatin stamp（CellStamp for UpCell;

ture11）. In recent years, cell sheet technology has been

CellSeed, Tokyo, Japan）was placed in the well and left

12,13）. However, there are few

at 25℃ for 30 minutes. The cell sheets were trans-studies on the effects of calcification and hypoxia in cellferred to a gelatin gel, which was then transferred tosheets that retain the specified thickness by multi-lay-

the next well. This process was repeated to create mul-ering of dental pulp cell（DPC）sheets using UpCell.

tiple layers. After the stacking was complete, the gela-

Therefore, we used a gelatin stamp（CellStamp fortin gel was heated at 37℃ for 30 min to dissolve theclinically tested in humans

UpCell）to stack cell sheets and analyze the character-gelatin and was washed three times in the medium

istics of DPC sheets for application in pulp regeneration（Fig. 1）.

and evaluation of dental materials and pharmaceuticals.

3 ．Obser vation by hematoxylin and eosin（HE）
staining

Materials and Methods

The monolayer, three-layer, and six-layer cell sheetsin the GM were cultured on a polytetrafluoroethylene

1 ．Cell isolation and culture

membrane for 3 days and fixed with 10％ neutral for-In this study, DPC was collected from three third

malin. Then, the cell sheets were embedded with opti-

molars of three healthy patients（18 30 years old）who

mal cutting temperature（OCT）frozen compound andhad undergone orthodontic treatment, following a pro-a 7μm section was cut from the OCT blocks andtocol approved by the Institutional Review Board of

stained with HE. HE was observed using an optical

The Nippon Dental University, School of Life Dentistry

microscope.

Advance Online Publication①Preparation of gelatin stamp②For UpCell③Placement on UpCell

・7.5％ gelatin stamp

ODEP Vol. 1, No. 1

In case of
6 layers

Repeat3 times④Incubate at 23 ℃ for 30 min⑤24 well plate
Dissolution ofgelatin（37℃，30min）⑥In culture
medium3 washes

Used forexperiments

Fig．1 Procedure for laminating cell sheets①Gelatin gel（7.5％）was prepared by molding and placed on a 24 well UpCell plate. ② ④：Cells were attached tothe bottom of the gelatin stamp at 23℃ for 30 min, and the process was repeated three times to make three layer
sheets. Six layer sheets were prepared by layering a three layer sheet on top of another three layer sheet after dissolving the gelatin. ⑤Gelatin was dissolved by heating at 37℃ for 30 min in the 24 well plate. ⑥Cell sheets were
washed 3 times with GM.

4 ．Obser vation by scanning electron microscopy（SEM）mean size of calcification（n＝3）.
6 ．Analysis of hypoxia

The monolayer, three-layer, and six-layer cell sheets

Monolayer, three-layer, and six-layer cell sheets werein the GM were cultured on MTA（ProRoot MTA; Den-

cultured in the OM for 3 days, and the hypoxia probe

sply Tulsa Dental Specialties, Johnson City, TN, USA）

solution LOX-1（Medical & Biological Laboratories,

with a diameter of 6 mm for 3 days. The samples were

Aichi, Japan）was used in the medium at a final concen-

fixed with 2.5％ glutaraldehyde solution for 30 min andtration of 2μ/ . The cells were observed under a fluo-

post-fixed with 1％ osmium tetroxide for 2 h at room

rescence microscope（Carl Zeiss LSM 700, Oberkochen,temperature. The samples were then dehydrated withGermany）.

50, 70, 80, 90, 95％, and 99.5％ ethanol for 10 min, followed by replacement with t-butanol and freeze-drying.
The samples were observed using SEM.

7 ．Real-time polymerase chain reaction（PCR）
analysis
Monolayer, three-layer, and six-layer sheets were cul-

5 ．Alizarin Red S staining

tured in the GM and collected at 3, 7, and 14 days.

The monolayer, three-layer, and six-layer cell sheets

Total RNA was extracted using TRIzol reagent

were cultured in the OM for 21 days, washed with

（Thermo Fisher Scientific, Waltham, MA, USA）andphosphate-buffer solution, and fixed with 10％ neutral

Maxwell RSC simplyRNA Cells Kit（Promega Corpora-formaldehyde at room temperature for 15 min. Aftertion, Madison, WI, USA）
according to the manufacturerʼ

washing twice with dH2O, 300 m /well of 40 mmol/s instructions. Complementary DNA synthesis was per-

ARS（pH 4.1）was added, incubation at room tempera-

formed from total RNA（1μg）using SuperScript VILO

ture was performed for 20 min, then the wells were

Master Mix（Life Technologies, Foster City, CA, USA）.

washed with 1 m dH2O 4 times for 5 min each. The

Real-time PCR was performed with a THUNDERBIR

samples were observed using an optical microscope andProbe qPCR Mix（TOYOBO, Osaka, Japan）using amacroscopic views. Three random points on the well

StepOnePlus real-time PCR system（Applied Biosys-

were measured by ImageJ software to determine thetems Waltham, Massachusetts, USA）under the follow-

Table 1
Genes

Sequences of human primers used for real time PCR
Upper primers

Lower primers

5 TCCCTGATGTTATGCATGAGC 35 CGAGAGTGAACCATGCCA 3

5 CGCCTGGGTCTCTTCACT 3
5 TTCTGTACGCAGGTGATTGG 3
5 TGACACATTTGATCTTGCTAGGA 3

5 CTCACACTCCTCGCCCTAT 3
5 GACATGTTCAGCTTTGTGGAC 3
5 TTTGGGCAGTAGCATGGG 3monolayer

three－layer

six－layer

A

B

C

D

E

F

G

H

I

macroscopic
views

Hemotoxylin
and eosin
staining

scanning
electronmicroscope

Fig．2 Histological and SEM findings of cell sheets
Macroscopic views of the monolayer（A）, three layer（B）, and six layer（C）cell sheets. Hemotoxylin and eosin staining of the monolayer（D）, three layer（E）, and six layer（F）cell sheets.
Scanning electron microscope view of the monolayer（G）, three layer（H）, and six layer（I）cell
sheets.
Scale bar＝50 μm

ing conditions: 95℃ for 3 min, followed by 40 cycles at
95℃ for 15 s. The primers for gene amplification were

Resultssubjected to 40 cycles at 60℃（45s）for ALP, COL1A1,
and OCN and at 62℃（45s）for DSPP. β（NM̲001101.2）was used as an endogenous control. The
primer sequences used are shown in Table 1.
8 ．Statistical Analysis

1 ．Histological analysis
The macroscopic view revealed membranous cellsheets with a thickness of three and six layers. The
thickness of the monolayer, three-layer, and six-layer

Statistical significance was analyzed using one-way

cell sheets, as seen with HE staining, was approxi-

analysis of variance followed by Tukeyʼs test. Datamately 7μm, 25μm, and 45μm, respectively. For SEM,

were expressed as mean±standard deviation, and p＜

when the cell suspension was seeded on the sample, the0.05 was considered statistically significant.

cells were scattered and attached to the MTA, but on
the cell sheets, an image covering the sample wasobserved（Fig. 2）.

Advance Online Publication

ODEP Vol. 1, No. 1

2 ．Analysis of hypoxia

ization were larger in the six-layer cell sheets（1,573.4±

The monolayer cell sheets showed a few LOX-1 posi-

669.1μm2）than in the three-layer sheets（895.4±269.7

tive cells. More hypoxic cells were observed in the six-

μm2）
（Fig. 4）.
4 ．Real-time PCR

layer cell sheets than in the monolayer and three-layer
cell sheets（Fig. 3）.

The expression of

3 ．Alizarin Red S staining

was upregulated in themonolayer, three-layer, and six-layer cell sheets at day 7

Several nodules of mineralization were observed inand downregulated at day 14, but tended to be signifi-

the three-layer and six-layer cell sheets but not in thecantly higher in the three-layer and six-layer cell sheetsmonolayer sheets. In addition, the nodules of mineral-than in the monolayer sheets（p＜0.05）. The expressionof

was upregulated in the monolayer, three-layer, and six-layer cell sheets on day 7 and was significantly higher in the six-layer cell sheets than in themonolayer sheets. The expression of

was upregu-

lated in the monolayer, three-layer, and six-layer cellsheets at day 14 and was significantly higher in themonolayer

three－layer

six－layer

Fig．3 Analysis of hypoxia
24 well Viewed from above hypoxia was detectedwith the LOX 1 probe in the monolayer, three layer,and six layer cell sheets.
Scale bar＝100 μm
macroscopic
view

three-layer（p＜0.05）and six-layer（p＜0.05）cell sheets
than in the monolayer sheets. The expression of
was upregulated in the monolayer, three-layer, and sixlayer cell sheets at day 14 and was significantly higher
in the six-layer cell sheets than in the monolayer sheets（p＜0.05）（Fig. 5）.

optical microscope view

A

B

C

D

E

F

G

H

I

monolayer

three－layer

six－layer

Fig．4 Macroscopic and optical views
Macroscopic view of Alizarin Red S staining of the monolayer（A）, three layer（D）, and six layer（G）cell sheets in the 24 well plate. Optical microscope view ofthe monolayer（B, C）, three layer（E, F）, and six layer（H, I）cell sheets. Nodulesof mineralization（indicated with black arrows）can be observed in relation to thethree layer and six layer cell sheets.
Scale bar＝500μm

＊

2.5

2.5
＊

ALP/β－actin

COL1A1/β－actin

＊

2.0
1.5
1.0
0.5
0

3 Days

7 Days

2.0
1.5
1.0
0.5
0

14 Days＊18

14 Days
＊
＊

6

14

＊

12
10
8
6
4
2
3 Days

7 Days

14 Days

Monolayer

DSPP/β－actin

OCN/β－actin

7 Days7

16

0

3 Days

5
4
3
2
1
0

3 Daysthree layers

7 Days

14 Days

six layers

Fig．5 Real time PCR analysis of mineralization related marker genes
Real time polymerase chain reaction was performed for, and（n＝3）. The results of two independent experiments performed in triplicate are expressedas the mean（±SD）（＊：p＜0.05）.

Phase contrast imageof DPC
Scale bar=100μm

Fig．S1

Fig．S2 Macroscopic and optical views
Macroscopic view of ARS of the monolayer（A）was cultured in the GM
for 21 days. Optical microscope view of the monolayer（B, C）. Calcification
was not observed（Scale bar=500μm）.

Advance Online Publication

ODEP Vol. 1, No. 1

tor-1α（HIF-1α）22）. The effect of hypoxia on cells may

Discussion

cause activation of angiogenesis23）by inducing vascular

In the present study, it was possible to fabricate cellHIF-1α, which may be advantageous for pulp regenera-

sheets to form in membranous by multilayering, astion by transplantation. The above results suggest thatshown by Kikuchi et al16）. In addition, some studies oncell sheet technology may be further developed by

MTA have reported the necessity of direct contact

changing the application depending on the thickness of

between cells and MTA for their differentiation into

the cell sheets.

endothelial growth factor（VEGF）expression related to17）odontoblast-like cells . Based on these studies, if it isNote that there were some limitations to this study.

possible to cover the MTA as shown by SEM, it may

We were not able to produce stable cell sheets. There-

be possible to analyze the interaction with cells that

fore, it is necessary to consider a more appropriate pro-occurs in the surface layer of the material in more

tocol.

detail and to study the material properties in an envi-

In the future, we plan to conduct a more detailed. Considering the miner-

study to investigate the material science of multilay-

alization-associated marker gene, after the initialered cell sheets with DPC for regeneration of dental

expression of calcification and in

pulp.

ronment similar to that, which is an

important factor for mineral deposition18）, downregulation was observed after 14 days in the monolayer

Conclusion

sheets, while the levels were high in the three-layer andsix-layer sheets, which may have been due to the

DPC sheets possessed higher mineralizing ability

occurrence of heterogeneous calcified areas within thethan monolayer sheets. Cell sheets may be useful for

sheets.

comparative examination of pharmaceuticals, materials,, a major collagen in dentin and bonematrix, also showed a similar trend. The expression ofand pulp regeneration in a 3D environment.

, a non-collagenous protein and a marker of osteo/odontogenic differentiation, and, a representative

marker of odontogenic differentiation, was also higher

The authors have no conflicts of interest directly relevantto the content of this article.

in the multilayered sheets. The expression trends of themarkers were consistent with those seen in previous

Supplemental Date

19）

studies . The above-described upregulation of the mineralization-related marker and increased nodular calcification seen with ARS were observed, especially in the

DPC image（Fig. S1）and ARS staining control（Fig.
S2）were added as Supplemental Date.

six-layer sheets. This may have been due to the thickness of the cells（as seen with HE）, causing the expansion of hypoxic areas and the upregulation of mineralization-associated marker expression20） induced by
localized hyper-calcified areas and hypoxic areas in
ARS. However, when considered in terms of materialogical characterization, since many dental materials
are often compared with cells for their odontogenic
induction21）, a comparative study of calcification-related
factors using six-layer sheets may result in an overestimation of calcification. Therefore, we hypothesized thata 3D culture with about three layers would not beaffected by scaffolding or hypoxia and would allow us toperform comparative material studies in a 3D environment similar to that in vivo. In addition, there is a correlation between LOX-1 and hypoxia-inducible fac-

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