CONTROL OF CELL ADHESION AND FUNCTIONS

USING SELF-ORGANIZED HONEY COMB-PATTERNED

POLYMER FILMS

Masaru Tanaka

Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1Katahira, Aoba-ku, Sendai, Japan

Akinori Tsuruma, Sadaaki Yamamoto

Sadaaki Yamamoto Creative Research Initiative “Sousei” (CRIS), Hokkaido University, N20W10 Kita-ku, Sapporo, Japan

Masatsugu Shimomura

Institute of Multidisciplinary Research for Advanced Materials, and World Premier International Research Center

Tohoku University, 2-1-1Katahira, Aoba-ku, Sendai, Japan

Keywords: Self-organization, Honeycomb, Scaffold, Tissue engineering, Cell adhesion, Stem cell.

Abstract: The design of nano- and microstructures based on self-organization is a key area of research in the search

for new biomaterials and biodevices, and such structures have a variety of potential applications in tissue

engineering scaffolds and medical implants. 3D scaffolds of appropriate pore size and porosities and with

interconnected pores are required to facilitate cell adhesion, proliferation, differentiation, and eventual tissue

regeneration in a natural manner. We have reported the honeycomb-patterned polymer film with highly

regular pores that is formed by self-organization. The honeycomb films exerted a strong influence on cell

morphology, proliferation, cytoskeleton, focal adhesion, and ECM production profiles. Our studies

demonstrated that the neural stem / progenitor cells morphology, proliferation, and differentiation are

controlled by the pore size of the honeycomb film. It is expected that the honeycomb films will have great

potentials as biomaterials for tissue regeneration in a growth factor free proliferation process of stem cells.

1 INTRODUCTION

It is well established that surface topography

influences implant integration. Many in vitro studies

have extended these observations to cells in culture,

demonstrating that scaffold architecture and surface

chemistry considerably influence cell behavior.

Therefore, cell adhesion, proliferation, and

differentiation can be regulated by controlling

surface topography.

We have reported a honeycomb-patterned

polymer film (honeycomb film) with regular pores,

which is formed by self-organization. The

honeycomb films strongly affected cell morphology,

proliferation, cytoskeleton, focal adhesion, and

extracellular matrix (ECM) production profiles

(Tanaka, 2006., Tanaka, 2007., Yamamoto, 2007.,

Mcmillan, 2007., Tanaka, 2008., Arai, 2008.,

Tsuruma, 2008.). These studies were performed on

cells cultured in the absence of growth factors.

In neural tissue engineering, the preparation of

neural stem/progenitor cells (NSCs) is required for

the treatment of diseases of the nervous system

(Steinman, 2003). NSCs are self-renewing,

immature, undifferentiated, and multipotent cells.

They can differentiate into cells constituting the

central neural system, such as neurons, astrocytes,

and oligodendrocytes (Roberti, 2000., Wurmser,

2004., Wang, 2006.). The use of NSCs is a potential

therapy for diseases of the nervous system. In order

to increase the feasibility of this technique, viable

method, the preparation, culture, and seeding of

NSCs are steps that have to be carefully controlled.

The main problem associated with the use of stem

cells is that when these cells are extracted from an

390

Tanaka M., Tsuruma A., Yamamoto S. and Shimomura M. (2009).

CONTROL OF CELL ADHESION AND FUNCTIONS USING SELF-ORGANIZED HONEY COMB-PATTERNED POLYMER FILMS.

In Proceedings of the International Conference on Biomedical Electronics and Devices, pages 390-393

DOI: 10.5220/0001545403900393

 SciTePress

individual, they start differentiating (specializing

into a specific cell type); thus, they lose their stem

cell characteristics. Thus, the reintroduction of these

cells into patients is problematic. In order to

overcome this problem, a culture environment

wherein the stem cells can be maintained in the

undifferentiated state is required. The proliferation

of self-renewing NSCs is required for cell therapy.

We studied the effects of the pore size of the

honeycomb films on the proliferation and

differentiation of NSCs.

2 MATERIALS AND METHODS

Honeycomb films were fabricated using

biodegradable polymers poly(ε-caprolactone) (PCL)

and a copolymer of dodecylacrylamide and ω-

carboxyhexylacrylamide. The honeycomb film was

prepared on a glass substrate by employing a

previously described method (Sato, 2002., Tanaka,

2004., Tanaka, 2007). The flat film was prepared by

a spin coater in dry condition. NSCs were derived

from the cerebral cortex of embryonic 14 day mice.

The NSCs were seeded on the films at a density of 2

× 10

cells/cm

. NSCs were cultured in serum

medium (Opti-MEM, 10 % Fetal Bovine Serum, 55

μM 2-mercaptoethanol) for 24 hr. After that, NSCs

were cultured in serum-free medium. The

morphologies of neurons were examined by a

scanning electron microscope (SEM) and a confocal

laser scanning microscope. NSCs were

immunostained for Nestin and BrdU. Cell number

was estimated by measuring of DNA concentration

from the extracted samples.

3 RESULTS AND DISCUSSION

Scanning electron microscopy (SEM) revealed a

highly regular hexagonal arrangement of pores

(honeycomb pattern), and a well-interconnected,

uniform pore structure (Fig. 1a–d). NSCs cultured

on flat films differentiated into round neurons with

neurites that extended randomly on the film. The

morphology of cells on the honeycomb films

depended on the pore size of the films. On films

with a pore size of 1.5 μm, the cell bodies were flat.

Their neurites extended randomly and jumped over

the pore of the film. On films with a pore size of 10

μm, the cell bodies were round and the cells adhered

to the rims of the films. Their neurites extended

along the rims forming a simple network. The

honeycomb film provided positive cues to support

neurite extension. The cells on films with pore sizes

of 5 and 8 μm exhibited a morphology similar to that

of cells on films with a pore size of 10 μm.

Interestingly, on honeycomb films with a pore size

of 3 μm, several large spheroids were observed. The

neurites gathered to form large bundles, which

radiated out from the periphery of the spheroids.

NSCs on honeycomb films with a pore size of 3 μm

formed spheroids of diameter 30~90 μm (Fig. 2);

such spheroid formation was not observed for NSCs

either on other honeycomb films (with pore sizes of

1.5, 5, 8, and 10 μm) or on flat films.

Top

layer

Bottom

layer

Pillar

１

0 μm

Tilted

μm

Closs

Top

　Pillar

Bottom

10 μ m10

Top

(a)

(b)

(c)

(d)

Top

layer

Bottom

layer

Pillar

１

0 μm

Tilted

μm

Closs

Top

　Pillar

Bottom

10 μ m10

Top

(a)

(b)

(c)

(d)

Top

layer

Bottom

layer

Pillar

１

0 μm

Tilted

μm

Closs

Top

　Pillar

Bottom

10 μ m10

Top

(a)

(b)

(c)

(d)

Top

layer

Bottom

layer

Pillar

１

0 μm

Tilted

μm

Closs

Top

　Pillar

Bottom

10 μ m10

Top

(a)

(b)

(c)

(d)

Figure 1: SEM images of the honeycomb film in different

views. (a) Surface, (b) tilted, and (c) cross-section. (d)

Schematic representation of the 3D structure of the

honeycomb film.

Culture time (days)

Diamet er (μm)

100

120

35 710

3 days

7 days

10 days

Culture time (days)

Diamet er (μm)

100

120

35 710

3 days

7 days

10 days

Figure 2: Spheroids diameter on the honeycomb film (pore

size:3 µm). Fluorescent images indicate spheroids stained

for Nestin. Bar: 50 µm.

In order to characterize the cells on the flat and

honeycomb films, the cells were immunostained for

nestin and microtubule-associated protein 2 (MAP2)

and were labeled with bromodeoxyuridine (BrdU).

Nestin and MAP2 are selective markers for NSCs

and neurons, respectively. Nestin expression

decreases when NSCs differentiate and mature into

neurons. BrdU is selectively incorporated into the

nuclei of proliferating cells, and thus indicates cell

CONTROL OF CELL ADHESION AND FUNCTIONS USING SELF-ORGANIZED HONEY COMB-PATTERNED

POLYMER FILMS

391

growth. Immunostaining for nestin and labeling with

BrdU revealed that the spheroids were aggregates of

self-renewed NSCs. The diameter of the spheroids

increased with the culture time (Fig. 2).

Figure 3: The removability and phenotype of the spheroids

on the honeycomb film (pore size: 3 μm).

Figure 4: Comparison of our method for proliferation of

NSCs using honeycomb films with the conventional

neurosphere method.

These results implied that honeycomb films with

pore size less than the cell size promoted the

proliferation of NSCs, but prevented their

differentiation. We found that the number of total

neural cells increased after 3 days owing to the

maintenance of the undifferentiated state and to the

proliferation of NSCs.

In order to determine the removability of the

spheroids from honeycomb films and to ascertain the

phenotypes of the cells, cells obtained from the

spheroids were cultured in a standard culture dish

(Fig. 3). All cells of the spheroids adhered to the

culture dish. The cells extended neurites after 2 d

and were positive for MAP 2, suggesting their

differentiation into neurons. This result implied that

the cells in the spheroids could differentiate into

neurons and confirmed the finding of the

immunostaining experiment that the spheroids were

aggregates of self-renewed NSCs.

The conventional neurosphere culture method is

widely used for the proliferation of NSCs (Cattaneo,

1990., Louis, 2004). This technique involves the use

of serum-free culture medium supplemented with

growth factors (fibroblast growth factor-2: FGF-2

and/or epidermal growth factor: EGF) (Fig. 4). The

NSCs obtained by this method are expected to be

supplied to lost and dysfunctional nervous systems

in order to regenerate neural tissue. In this

technique, NSCs are cultured without the attachment

to a surface (floating culture) because the NSCs

immediately differentiate into neurons when they are

attached to the substrate surface. Improvements in

the neurosphere culture technique, which include the

use of U-bottomed wells, have recently been

reported (Mori, 2006). However, our technique

required neither growth factors nor the floating

culture system wherein the cells are not attached to a

surface.

In our technique, some NSCs were observed to

be encapsulated by the 3 μm pores immediately after

cell seeding. At this adhesion arraignment, the NSC

contacts to the pore at around the cell body. Such

circular adhesion may result in a small adhesion

area; thus, the NSCs are in an environment similar to

that in the neurosphere method, that is, they are

suspended to prevent contact with surfaces. Thus,

cell encapsulation, in contrast to cell adhesion that is

observed on flat surface, is probably the reason for

the control of NSC proliferation by surface

topography; such encapsulation is characteristic to

the honeycomb film with a pore size of 3 μm.

4 CONCLUSIONS

Our study revealed that the morphology,

proliferation, and differentiation of NSCs are

controlled by the pore size of the honeycomb film.

This is a novel approach to NSC culture in

regenerative medicine, wherein the proliferation and

differentiation of the NSCs are controlled by the

surface topography of scaffolds. Honeycomb films

are potentially useful biomaterials for neural tissue

regeneration, which can help in the proliferation of

NSCs in the absence of growth factors.

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ACKNOWLEDGEMENTS

This work is supported by Grants-in-Aid and

CREST from Japan Science and Technology

Corporation (JST), and Special Coordination Funds

for Promoting Science and Technology of Ministry

of Education, Culture, Sports, Science and

Technology, Japan.

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