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Construction of Heterofunctional Nanosheets
Page 1
Construction of Heterofunctional Nanosheets
with Different Cytocompatibilities in Two Sides
and
Application as an Anti-Adhesion Barrier
表裏で異なる細胞親和性を持つナノシートの調製と
癒着防止剤としての応用
February 2011
Daisuke NIWA
丹羽 大輔

Page 2
Promoter: Prof. Dr. Shinji Takeoka
Referees: Prof. Dr. Yasuo Ikeda
Prof. Dr. Nobuhito Goda
Prof. Dr. Thorsten Lang

Page 3
Preface
Nanotechnology and nanomaterials are currently being applied to a variety of products in the
electronics and optronics industries, and are contributing to impressive technical innovation. Remarkable
developments have also been made in the field of nanobiotechnology, a fusion of nanotechnology and
biotechnology, including thousands of diagnostic techniques, drug delivery systems (DDS), and
innovative diagnostic or medical treatment techniques proposed in the tissue engineering field with the
cooperation of medical and biological engineering. Following the work of Dr. Langer who implanted a
biodegradable polylactide scaffold in the shape of a human ear into a mouse for tissue regeneration, Dr.
Okano developed “cell sheet engineering” in which a cell sheet was prepared on cell culture dish covered
with thermo-responsive polymer and implanted into the damaged area of cornea or cardiac muscle to
regenerate tissue. These technical developments will be the essence of individualized medicine in the near
future with implantation of custom tissues or organs reconstructed from iPS cells. Recently, we have
developed an ultra-thin film (nanosheet) that is biodegradable, biocompatible, and very thin (nm
thickness). We have accumulated data that suggest this technology would be useful as a wound dressing
material after surgery due to its anti-adhesion properties. Characteristics of this nanosheet include a high
aspect ratio derived from its nm thickness, high flexibility and physical adhesion. There is also merit to
the fact that lesions can be covered with a very small amount of the nanosheet. Since bulk preparation and
sterilization are possible in a short time, unlike cell sheets, development as a wound dressing material in
emergency is promising, and would be available to anyone at a low price and for a wide range of
applications.
This thesis consists of six chapters. The first chapter deals with the fundamentals of self-assembly,
self-organization and supramolecules. Examples of techniques or their molecular design for chemistry or
engineering were also introduced in this chapter. In chapter 2, the fundamental characteristics of a
poly-L-Lactic acid (PLLA) nanosheet is described in reference to cell adhesion property. In chapter 3, the
author focused on the thrombin coated PLLA nanosheet and evaluated its hemostatic characteristics and
anti-adhesion property. In chapter 4, based on the findings of anti-cell adhesion properties from chapter 2,
the author prepared a heterofunctional nanosheet in which one side had anti-cell-adhesion property and
the other side had cell adhesion property that would lead to a material that could promote healing with the
application of a collagen spin-coating method. In chapter 5, the author established the use of a PLLA
nanosheet as an anti-adhesion material in combination with TachoComb that is famous as a hemostat.
Finally, the conclusions and future prospects of this thesis were described in Chapter 6.
The author supports the use of functionalization of nanosheets in both materials and methods to
advance biomedical application of this technology. These materials should be an innovative alternative for
conventional wound dressing in the next generation of postoperative materials.
Daisuke Niwa

Page 4

Page 5
i
CONTENT
Preface
Chapter 1: Principal, Design and Construction of Molecular Assembly
and Molecular Organized Materials
1. Introduction
2
2. Fundamental aspects of self-Assembly and self-organization and supramolecule
2
2.1. Principle of self assembly and self-organization and supramolecule
2
2.2. Secondary bonding for self assembly
10
3. Ultra thin film based on molecular organizations
25
3.1. Nanomaterials based on nanotechnology
25
3.2. Ultra-thin film based on self-assembly
30
3.3. Evaluation tool for ultra-thin film
34
3.4. General uses for the ultra-thin film
35
4. Polymer nanohseets utilized in the biomedical field
36
4.1. Biomedical application of ultra-thin film
36
4.2. Functionalisation of ultra-thin film
39
References
40
Chapter 2: Cell adhesive property on the nanosheet
1. Introduction
44
2. Construction of poly-L-lactic acid (PLLA) nanosheet
45
2.1. Preparation of PLLA nanosheet
45
2.2. Evaluation of PLLA nanosheet
46
3. Cell adhesive property on the PLLA nanosheet
47
4. Summary
48
References
49
Chapter 3: Construction of Thrombin loaded nanosheet and evaluation
of its hemostat ability and anti-adhesion ability
1. Introduction
51

Page 6
ii
2. Biology and management of wounds
51
2.1. Wound biology
51
2.2. Steps of wound healing
52
2.3. Classification of wound dressing products
54
3. Hemostatic materials
55
3.1. Basic hemostasis
55
3.2. Humoral hemostasis
56
3.3. General hemostatic materials
58
3.4. TachoSil and TachoComb
60
4. Preparation of Thrombin loaded PLLA nanosheet (Thr-PLLA)
60
5. Analysis of Thr-PLLA
61
5.1. Preparation of FITC-Thr-PLLA
61
5.2. Evaluation of Thr-PLLA nanosheet by Infrared (IR) spectrum
62
5.3. Quantification of adsorbed thrombin
63
5.4. Blood coagulation assay
63
6. Evaluation of Thr-PLLA using an animal model
64
6.1. Animal viability
64
6.2. Anti-adhesion property of Thr-PLLA
65
7. Summary
67
References
68
Chapter 4: Construction of heterofuncitonal nanosheets for the cell
adhesive and non-adhesive material
1. Introduction
70
2. Construction of heterofunctional nanosheets
71
2.1. Polylactic acid nanosheet bearing collagen layer (Col-Spin-PLLA)
71
2.2. Col-Spin-PLLA obtained by “Supporting film Method” and “Sacrificing film
method”
72
3. Structural characterisation of heterofunctional nanosheets
73
3.1. Surface and thickness characterization by AFM
73
3.2. IR measurement
75
3.3. Wettability of each sample
75
3.4. Observation of surface morphology of each sample from AFM measurement 77
4. Cell adhesion property of heterofunctional nanosheet
78
4.1. Cell Attachment test
79

Page 7
iii
4.2. Analysis of cell spreading ration with time
82
4.3. Immunofluorescence of the cells on actin filaments
84
4.4. Heterofunctionality of the Col-Spin-PLLA nanosheet
86
5. Summary
88
References
89
Chapter 5: Nanosheet as post-surgical adhesion barrier
1. Introduction
92
2. Anti-adhesion barrier
93
3. Construction of PLLA nanosheet
96
3.1 Manipulation of free-standing nanosheet
96
3.2 Application of nanosheet for the biomedical usage
96
4. The application of the nanosheet as an anti-adhesion barrier
97
4.1 In vitro model of nanosheet (cell adhesion and blood attachment assay) 101
4.2 In vivo experiment of nanosheet in the liver defect model for combined
application with hemostatic materials
107
5. Summery
117
References
118
Chapter 6: Conclusions and Future prospects
1. Conclusions
121
2. Future Prospects
122
References
128

Page 8

Page 9
Chapter 1
1
Chapter 1
Principal, Design and Construction of Molecular Assembly and
Molecular Organized Materials
1. Introduction
2. Fundamental Aspects of Self-Assembly and Self-Organization
3. Ultra thin film based on molecular organizations
4. Polymer nanohseets utilized in the biomedical field
References

Page 10
Chapter 1
2
1. Introduction
Biomolecules such as cells, viruses, organelles, biomembranes and enzymes,
which exist in nature, are composed of smaller molecules via self organization.
Biomacromolecules, such as nucleic acids, proteins, lipids and polysaccharides, are also
made from self-assembling small molecules via weak interaction. In general, chemical
entities in which several small molecules self-assemble and self-organize through
molecular interaction are called supramolecules. Lehn proposed this new concept in
1989 and was awarded a Nobel Prize in chemistry. Starting with an investigation of the
basis of design, supramolecular chemistry that creates new structure and function has
progressed in its relevance to organic chemistry, inorganic chemistry, analytical
chemistry, physical chemistry, biochemistry, cellular biology, structural biology and
polymer chemistry. In this chapter, I describe the fundamentals of supramolecular
chemistry based on self-assembly through secondary interactions represented by
noncovalent bonds such as electrostatic interaction, hydrogen bonding, coordination
bonding, hydrophobic interaction and van der Waals interaction. In section 2, the
fundamental characteristics of these secondary interactions are described in more detail,
using current research projects as examples. In section 3, self-organizing materials are
described, with a particular focus on thin film. In section 4, an example of a recent
practical biomedical application was explained.
2. Fundamental Aspects of Self-Assembly and Self-Organization
2.1. Principals of self assembly and self-organization
Self-assembly is a common principle in nature and technological innovation.

Page 11
Chapter 1
3
Whitesides et al. summarized several reasons why self-assembly is quite interesting [1].
1) Humans are attracted by the appearance of order from disorder.
2) Living cells self-assemble, and understanding life will therefore require
understanding self-assembly. The cell also offers countless examples of functional
self-assembly that stimulate the design of non-living systems.
3) Self-assembly is one of the few practical strategies for making ensembles of
nanostructures. It will therefore be an essential part of nanotechnology.
4) Manufacturing and robotics will benefit from applications of self-assembly.
5) Self-assembly is common to many dynamic, multicomponent systems, from smart
materials and self-healing structures to netted sensors and computer networks.
6) The focus on spontaneous development of patterns bridges the study of systems
with many interacting components.
It thereby connects reductionism to complexity and emergence.
There are two commonly recognized types of self-assembly: static (S) and
dynamic (D). Whitesides et al. also discriminate two additional self-assembly types:
templated self-assembly and biological self-assembly (B). These four types of
self-assembly are summarized in Table 1.1.

Page 12
Chapter 1
4
Crystal structure and most folded globular proteins are formed by static
self-assembly. Static self-assembly is the focus of most research due to its stability once
formed.
System
Type
Applications / important
Atomic, ionic, and molecular crystals
Phase-separated and ionic layered polymers
Self-assembled monolayers (SAMs)
Lipid bilayers and black lipid films
Liquid crystals
Colloidal crystals
Bubble rafts
Macro- and mesoscopic structures (MESA)
Fluidic self-assembly
“Light matter”
Oscillating and reaction-diffusion reactions
Bacterial colonies
Swarms (ants) and schools (fish)
Weather patterns
Solar systems
Galaxies
S
S
S, T
S
S
S
S
S or D, T
S, T
D, T
D
D, B
D, B
D
D
D
Material, optoelectronics
Microfabrication, sensors, nanoelectronics
Biomembranes, emulsions
Displays
Band gap materials, molecular sieves
Models of crack propagation
Electronic circuits
Microfabrication
Biological oscillations
New models for computation/optimization
Figure 1.1. Examples of static self-assembly. (A) Crystal structure of a ribosome [2].
(B) Self-assembled peptide-amphiphile nanofibers [3]. (C) An array of millimeter-sized
polymeric plates assembled at a water/perfuorodecalin interface by capillary interactions.
(D) Thin film of nematic liquid crystal on an isotropic substrate. (E) Micrometer-sized
metallic polyhedrafolded from planer substrates [4]. (F) A three-dimensional aggregate
of micrometer plates assembled by capillary forces [5].
Table 1.1. Examples of self-assembly (S, static, D, dynamic, T, Templated, B, biological

Page 13
Chapter 1
5
Although most researchers have an interest in static self-assembly, life is dynamic.
Living cells contain many reacting chemicals and environmental sensors, and allow heat
and particular chemicals to pass through. At the molecular level, static self-assembly
describes the formation of a lipid bilayer or base-pairing and protein folding. The
behavior of critical structures in the cell – including actin filaments, histones and
chromatin, and protein aggregates in signaling pathways – involve dynamic
self-assembly.
Examples and possible applications of dynamic self-assembly include:
(1) Crystallization at all scales.
(2) Robotics and Manufacturing. Self-assembly offers a new approach to the
Figure 1.2. Examples of dynamic self-assembly. (A) Can optical micrograph of a cell
with fluorescently laveled cytoskeleton and nucleus; microtubules (~24 nm in
diameter) are colored red [6]. (B) Reaction-diffusion waves in a Belousov-Zabatinski
reaction in a 3.5-inch Petri dish. (C) A simple aggregate of three millimeter-sized,
rotating, magnetized disks interacting with one another via vortex-vortex interactions [7].
(D) A school of fish. (E) Concentric rings formed by charged metabolic beads 1 mm in
diameter rolling in circular paths on a dielectric support. (F) Convention cells formed
above a micropatterned metallic support. The distance between the centers of the cells is
~2 mm.

Page 14
Chapter 1
6
assembly of parts with nano- and micrometer dimensions.
(3) Nanoscience and Technology. Bottom-up and top-down are two approaches for
constructing a nanosystem.
(4) Microelectronics. Self-assembly offers a possible route to three dimensional
microsystems.
(5) Netted system.
Figure 1.3. Application of self-assembly. (A) A 2 by 2 cross array made by sequential
assembly of n-type InP nanowires with orthogonal flows [8]. (B) Diffraction grating
formed on the surface of a poly(dimethylsiloxane) sphere ~ 1 mm in diameter. The
sphere was compressed between two glass slides, and its free surface was exposed to
oxygen plasma. Upon release of compression, the oxidized surface of the polymer
buckled with uniform wavelength of ~ 20 μm [9]. (C) Three-dimensional electronic
circuits self-assembled from millimeter-sized polyhedra with electronic components
(LEDs) enmbossed on their faces. (D) An artificial, ferromagnetic opal prepared by
temnplated self-assembly of polymeric microbeads [10]. The opal properties of the
aggregate can be adjusted by modifying external magnetic field.

Page 15
Chapter 1
7
Furthermore, as mentioned above, self-assembly with more complex and superior
function is referred to as a supramolecule. Supramolecules develop highly complex
systems through noncovalent intermolecular interaction between small molecules and
components [11].
Supramolecules are based on the selective binding between natural products or
even synthesized macrocyclic compounds such as crown ether and alkali metal. The
concept of molecular recognition began as a new branch of chemistry, and through
expansion into different fields including general molecular interactions and molecular
processes, it evolved into the field of supramolecular chemistry. The concept of
supramolecular chemistry can vary, but in general it deals with sophisticated and
complicated organization arising from the association of two or more types of chemicals
through van der Waals interaction. Supramolecules are characterized by spatial
placement and ultrastructure and even by their intermolecular binding. Supramolecules
have a defined character that is structural, conformational, thermodynamic,
kinetically-controlled or dynamic. Discriminating interaction patterns include
coordination of metals, static interaction, hydrogen bonding, van der Waals interaction,
Figure 1.4. From molecular, to supramolecular and to Constitutional Dynamic
Chemistry under Preorganization and Self-organization by design and with selection

Page 16
Chapter 1
8
Synthesize
Covalent binding
Receptor
Substrates
Interaction
Molecular
bindign
Supramolecule
Moleular
recognition
Changing
Mobility
Molecular device
Supramolecular device
Functional
component
Self-assembly
Self-organization
Chemistry
Molecule
Supramolecule
A
B
C
D
E
donor-acceptor interaction, etc. and these lead to different strength, directionality and
dependency of diverse distances and angles. Strength can range from weak or moderate
hydrogen bonds to strong metal interactions. Whereas the former interactions lead to
stability between enzymes and their substrates, the latter substantiate the strength of
diverse interactions such as antigen-antibody interactions. However, intermolecular
interactions are generally weaker than covalent binding and supramolecular interactions
are thermodynamically unstable, highly kinetically reactive and dynamically soft. So,
supramolecular chemistry is related to soft binding, and therefore represents “soft
chemistry”.
Molecular recognition, transformation, and translocation are fundamental
functions. Assembly of multi-molecules and phases like vesicles, interface and liquid
crystals make it possible to further develop supramolecules into supramolecular devices.

Page 17
Chapter 1
9
Numerous molecular receptors capable of selectively binding specific substrates via
non-covalent interactions have been developed. They perform molecular recognition
functions that rely on the molecular information stored in the interacting species.
Suitably functionalized receptors may affect supramolecular catalysis and selective
transport processes.
A wide variety of phenomena are regarded as spontaneous structures in various
scientific disciplines, and the definitions applied differ. The following selection is taken
from the relevant literature [13]:
● Chemistry: Self-organization = well-defined structures result spontaneously from
the components of a system by noncovalent forces (self-assembly); for example, liquid
crystals, micelles, or oscillating reactions.
● Biology: Self-organization = spontaneous accumulation of complex structures under
adequate environment conditions solely on the basis of the respective molecular
property, namely, without the effect of external factors; for example, protein folding,
Figure 1.5. Supramolecular science as the science of informed matter at the interfaces
of chemistry with biology and physics [12].

Page 18
Chapter 1
10
formation of liquid double layers, or morphogenesis.
● Physics: Self-organization = spontaneous formation of new three-dimensional and
temporal structures in complex systems that results from the cooperative effect of partial
systems; for example, ferromagnetism, superconductivity, or convection cells.
2.2. Secondary bonding for self assembly
J-Marie Lehn defines supramolecular chemistry as “chemistry beyond the
molecule”, whose goal is to gain control over the intermolecular non-covalent bond. As
described before, it incorporates coordination of metals, static interactions, hydrogen
bonding, van der Waals interactions, donor-acceptor interaction, etc. The interactions
that are needed for self-organization or assembly of suparmolecular polymers are
described below.
(1) Coordination of metals
The merit of coordination of metals, or hydrogen bonding, is reversibility of
self-organization. One example is the dendrimer and dendrimer connection via
Ruthenium19. This type of self-assembly uses a ruthenium (II) metal centre to form
stable complexes between the discrete terpyridine receptor units attached to different
cascade macromolecules [14].

Page 19
Chapter 1
11
Another example of a supramolecule, constructed by Hunter et al., was made
from self-assembled porphyrin polymers. In this system, an ethyl hexyl group was
introduced at the meso-position to improve solubility in an organic solvent [14]. In this
model, cobalt porphyrins with a pyridyl group coordinated with each other on the order
of μM (generally, porphyrins coordinate with each other on the order of mM).
Furthermore, the cobalt is trivalently oxygenated in air with a high ligand affinity that
reduces the kinetic speed of exhange, but bivalent cobalt porphyrin dominates in this
model and cannot eliminate the trivalent cobalt porphyrin [15].
Figure 1.6. Bis-dendric complex.
Figure 1.7. Self-assembly of a cobalt porphyrin polymer via coordination of two
covalently attached pyridine ligands, one on each face of the porphyrin.
Rh

Page 20
Chapter 1
12
(2) Electrostatic interaction (ion-ion interaction)
Electrostatic interaction is described in Figure 1.1. It is formed by interactions
between a positively charged molecule and a negatively charged molecule. Coulomb‟s
force (electrostatic interaction) works between two charged atoms (molecules). The
attracting force between these two charged molecules is proportional to the product of
both charges and inversely proportional to the distance between the molecules and the
dielectric constant. However, a reduction in force can occur if the nuclei are too close
Therefore, the Coulombs interaction energy U is reflected by the equation described
below. (Q1Q2, each charge; ε, dielectric constant; r, distance between atoms; a, b,
constant numbers)
U = Q1Q2/4πεr + be
-r/a
From this equation, the force is attractive when there are different charges, and is
maximal under vacuum or in an apolar solvent and relatively weaker in a polar solvent.
In a neutral solvent, the carboxyl group and amide group of the glutamic acid or lysine
residue in the protein get dissociated and protonated to exist as an ion that has a
negative or positive charge and becomes one of the driving forces forming the steric
structure of protein. When the environment surrounding the carboxyl or amide group is
hydrophobic, there is sometimes no ionization. In this case, ion dipole interaction or
dipole-dipole interaction sometimes play a major role.
Shunguang Zhang focused on fabricating several self-assembling peptides and
proteins for a variety of studies and biomaterials that were representative as ionic self
–complementary peptides [16].

Page 21
Chapter 1
13
(3) Hydrophobic interaction
This interaction is important in the area of surfactants, such as membranes,
micelles, and vesicles. Hydrophobic molecules are rejected from water molecules
resulting in hydrophobic molecule clusters (Figure 1.9.). There are no forces on the
hydrophobic molecules, only a passive force of water. Hydrophobic interactions proceed
via the donation of a large entropy term and the hydrophobic interaction tends to be
stable following thermal elevation. This characteristic is unique from other interactions
that tend to become unstable as temperature increases. Hydrophobic interactions play an
important role since much molecular recognition, including biological molecules,
occurs in water.
Figure 1.8. Fabrication of various peptide materials. (a) the ionic self-complementary
peptide has 16 amino acids that form kind of β-sheet structure.
Figure 1.9. Hydrophobic interaction
Hydrophobic molecule
Water molecule

Page 22
Chapter 1
14
(4) van der Waals interaction
The force between molecules is called molecular interaction (van der Waals
interaction). When a general gas becomes cold, it aggregates and changes to a solid.
This phase change is caused by molecular interaction. The observations that real gas
behaviour is different from the behaviour of an ideal gas, that gas fluid has viscosity,
and that there is a change in temperature associated with a change in gas expansion due
to pressure changes (Joule-Thomsen effect) provides evidence of interaction between
molecules. For example, a variety of compensation formulas have been presented over
time to describe the gas state equation. A representative compensation formula is the
van der Waals equation.
(P+a/V
2
)(V-b)=RT
(V, gas volume of 1 mol; P, the pressure; T, absolute temperature; R, gas constant; a,b,
the constant depending on the type of gas)
In this case, the formula represents an ideal gas when both a and b are 0. The value a/v
2
,
the intrinsic pressure, reflects the action caused by forces other than pressure P. The
constant b is assumed to be the compensation for molecular size. This equation is
correlated with a more proper Virial expansion:
PV=RT{1+(NA/V)B(T)+…}
(NA, Avogadro number; B(T), second virial coefficient relating to the amount of
molecular interaction) J.E. Lennard-Jones presumed intramolecular potential as –μ/r
m
+
ν/r
n
(r, intramolecular distance; μ, ν, m, n, constants) and analysed B(T). The first term
of this potential represents the attraction force and the second term represents the
repulsive force, analysed in detail in the case of m=6 and n=12. From the ratio of the
result and the actual measurement, μ and ν were estimated. The term of the attractive

Page 23
Chapter 1
15
force corresponds to intrinsic pressure and is caused by van der Waals force. Most
important is the dispersion force that came from polarizability of gas. For apolar
molecules, dipolar interactions, inductive effects, and quadrupolar interactions other
than polarizability are also important, but they are mostly small and localized on free
rotation when the gas molecules are rotating freely. The repulsive force is generated by
exchange repulsion and Coulomb repulsive force when the molecules are too close. The
intermolecular force in the gas phase was mainly referred; however, this would change
if the molecules were too close to rotate independently. The orientation is dominated by
dipolar interactions, especially in apolar molecules. Without dipole moments,
quadrupolar interactions are superior. Of course the dispersion force is also important as
an aggregative force, but its role in influencing the relative molecular orientation is
small because its anisotropy is smaller than the dipole moment or quadrupolar
interaction. When the molecules are close together, charge transfer interaction and
hydrogen bonding are added as new forces. The former one is an intermolecular
interaction proposed by R. S. Mulliken to explain the binding strength between
electron-releasing and electron-accepting molecules and is generally thought to exist in
homogeneous molecules and has been confirmed in aromatic molecules with
unsaturated bonds or radical crystals. Hydrogen bonding plays an important role in
water molecules, alcohol molecules, carboxylic molecules, proteins and other
biologically important molecules.
(5) π-π interaction
Electron clouds are delocalized in benzene both above and below the planar
molecule. There is an overlap of the molecular orbital of each electron cloud during the
stacked state when aromatic rings, including porphyrin, superimpose each molecular

Page 24
Chapter 1
16
plane. π-electron clouds of both molecules have increased energy stability with an
increase in delocalization. This stability arises because of π-π interactions.
Molecules with π-bonds, especially relatively large π-conjugated systems, tend to
form molecular complex with atoms or molecules with high electron affinity or with
low ionization potential (ionization energy). This molecular complex is a π-complex.
There are many π-complexes that could be considered crystals, however, there are also
many that are not crystals even though they may form crystals in solution. The
representative example for a crystallized π-complex is a quinon-quinon complex
(quinhydrone). A π-complex has a characteristic absorbance band in the visible range,
and is strongly stained. These appearances of an absorbance band or binding force
between the components are explained by charge-transfer theory. Considering
molecules with small ionization potential and high electron donor potential as D, and
molecules with high electron affinity and high electron acceptor potential as A, the
ground electric state of complex DA is represented as a quantum mechanical resonance
of the nonbonded state (D…A) where D and A are bound only by dispersion force and
charge-transfer state (D
+
D
-
) with electron transfer from D to A. It is hypothesized that
the above actions contribute to stability of the compound. Charge transfer interaction is
more prominent when the ionization potential of the electron donor is low and the
electron affinity of the electron acceptor is large, but molecules with a large π electron
conjugated system could work as both donors and acceptors. Some π-complexes may
act as intermediaries in chemical reactions, and many π-complex crystals have
interesting properties as an organic semiconductor.
(6) C-H…π interaction
It is clear that in crystals, hydrogen atoms that are weakly depolarized to carbon

Page 25
Chapter 1
17
atoms interact with π electrons in aromatic rings of [C-H…π]. A typical example is the
interaction between benzene and chloroform. As seen in Figure 1.10., a weak hydrogen
atom in chloroform is oriented perpendicular to the π electron clouds in benzene. C-H
δ+
π interaction is seen not only between organic molecules but also in host guest
complex crystals.
(7) Hydrogen bonding
Hydrogen bonding is a form of chemical bonding. A hydrogen atom has only one
electron around the nucleus (proton) and when this electron participates in bonds with
atoms with high electronegativity, such as N, O, of F, it is sequestered by the other atom
to form a bare nucleus with a positive charge. When there is a strong negative atom near
a hydrogen atom, a weak attraction bond is formed. A hydrogen bond is approximately
1/10
th
the strength of a typical covalent bond, however this force is enough to influence
the orientation of molecules in solid or liquid. Melting heat (6.01 kJ/mol) is higher than
sublimation heat (45.05 kJ/mol) due to hydrogen bonding rather than van der Waals
interactions. Furthermore, a high dielectric constant and finite value of entropy at 0 K
are also caused by hydrogen bonding. Moreover, the high rate of dimer formation of
organic acids like formic acid, acetic acid or benzoic acid is also explained by hydrogen
bonding.
Figure 1.10. C-H
δ+π interaction

Page 26
Chapter 1
18
a
Hydrogen bonding between molecules
(Related to many molecules)
Ice
b Formic acid
Hydrogen bonding between molecules
(Related to two molecules)
Cis
Trans
c o-chlorophenol
Intermolecular hydrogen bonding
d (FHF)-
Intermolecular hydrogen bonding
e (HF)n in liquid
Polymerization
Hydrogen bonding also plays a key role in biochemistry, including the formation of
carbohydrates, protein and DNA. An equivalent condition can often be obtained in
aggregated states by hydrogen bonding with exchange of a covalent bond and hydrogen
bond, therefore ferroelectricity can sometimes be detected. Hydrogen bonding is studied
in detail not only by thermal measurement but also by IR spectroscopy, Raman
spectroscopy and UV spectroscopy. In the case of strong hydrogen bonding, a new
absorbance is measured in the UV range that is not seen in the component molecules,
and it is interpreted as a charge-transfer absorption band. Under hydrogen bond
formation, a hydrogen atom from an hydroxyl group (O-H) or amino group (N-H), often
affect carbonyl oxygen (C=O) or imino nitrogen (C=N), and the strength of hydrogen
bonding is reflected in the distance of H…Y. Basic strength of hydrogen bonding is
listed in Table 1.2..
Figure 1.11. Example of hydrogen bonding

Page 27
Chapter 1
19
OH・・・O
0.272
O-O
0.281
N-O
OH・・・N
0.279
O-N
0.295
N-N
OH・・・S
0.331
O-S
0.27
O-N
OH・・・F
0.272
O-F
0.291
N-N
OH・・・Cl
0.312
O-Cl
0.301
N-O
OH・・・Br
0.328
O-Br
NH・・・O
0.289
N-O
NH・・・N
0.298
N-N
NH・・・S
0.342
N-S
NH・・・F
0.292
N-F
NH・・・Cl
0.323
N-Cl
NH・・・Br
0.337
N-Br
FH・・・F
0.244
F-F
Adenine(A)-Thymine(T)
Guanine(G)-Cytocine(C)
bond length /nm
bond length /nm
N
N
NH
N
N
N
HN
O
O
H
H
H
A
T
N
N
O
N
N
N
N
O
N
H
H
H
H
H
N
C
G
Figure 1.12. Central structure of hydrogen bonding
For example, in the case of hydrogen bonding in a peptide [N-H・・・O=C] in an
α-helix formation, the distance of N…O is 2.87Е in which N-H is 1.00Е and H…O is
1.87Е and H is in alignment with N and O. The main characteristic of hydrogen
bonding is that it has the same orientation as a covalent bond even though it is weak.
In other words, the binding strength is strongest and most stable when aligned. When
the repulsive force of van der Waals interaction is working between H and O that form
hydrogen bonds, both atoms repulse each other and the H atom is out of alignment but
the force of hydrogen bonding overcomes the repulsive force and forms a bond. Though
the distance between X and Y is shorter than the sum of van der Waals radius in general
(the sum of van der Waals radius between a hydrogen atom and oxygen atom is
1.2+1.4=2.6Е. Hydrogen bonding is 1.8~2.0Е and this is shorter than the sum of van
der Waals), diversity is seen in hydrogen bonding of type [X―H・・・Y] and there have
been some examples suggesting that the sum of van der Waals is larger than X…Y. This
binding strength is relatively weak and forms a central structure as decribed in Figure
Table 1.2. The binding length of hydrogen bonding

Page 28
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20
Figure 1.13. Tetramer structure
of hydrogen bonding.
1
2
3
4
Figure 1.14. Triple hydrogen bonded complexes
1.11, where the two hydrogen receptors Y1 and Y2 share the same hydrogen.
Examples of molecular structure are described below.
First, the simplest example of triple hydrogen
bonding is shown. As described before, formic acid
forms a homo dimer. There is an example of a double
bonded tetramer (Figure 1.13.). This tetramer has a
total of eight hydrogen bonds.
Next, examples of simple triple hydrogen bonds are shown. This hydrogen
bonded complex between guanine and cytosine (1) is central to the structure of nucleic
acids. Other similar examples are also shown (Figure 1.14.-2,3,4) [17].
Using this concept of triple hydrogen bonding, attempts were made to construct a
diversity of complexes and estimate their equilibrium constants (Figure 15) [18].

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21
Figure 1.15. More complex structure through hydrogen bonding
Kimizuka et al. constructed an amphiphilic supramolecular structure by employing a
hydrogen-bond-mediated aromatic sheet as the strongly interacting (solvophobic) unit
and alkyl chains as the solvophilic unit (Figure 1.16.). Transmission and scanning
microscopy show the bundles of 100Е (Figure 1.17.) [19].

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22
Figure 1.16. Schematic illustration of conceivable supramolecular structure
Figure 1.17. Electron microscope examination of melamine/diimide complexes.
(a) Transmission electron micrograph of melamine-1/diimide complex. The
sample was poststained with uranyl acetate. (b) Scanning electron micrograph of
melamine-2/diimide complex. The sample was coated with Pt (ca. 10Å).

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23
Figure 1.18. Schematic illustration of phase transition characteristics of aqueous
2(C12OC3)-Mela/CA-phC10N
+
. Reversibly dissociation was observed with a
dilute dispersion (5mM).
Kimizuka et al. also reported the first example of water soluble supramolecules directed
by complementary hydrogen bonds. This assembly is thermally reversible with repeated
forming and breaking of the assembly [20].
There are some examples of self-assembly of porphyrin derivatives. In the case
described below, Drain et al. built a closed tetrameric square with two sets of
complementary porphyrin building blocks (Figure 1.19.) [21]. There are many
examples of assemblies composed of porphyrin derivatives and its complementary units.
A famous example is the use of porphyrin derivative assembly for making
photo-switched conductors. Linear porphyrin arrays self-assembled by either hydrogen
bonding or metal ion coordination into liquid bilayer membranes were shown in Figure
1.20. [22].

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24
Figure 1.19. Closed cyclic tetramers composed of uracyl porphyrin and
diaminopyridyl porphyrin.
Figure 1.20. Photocurrent device composed of self-assembled porphyrin via
hydrogen-bonded system..

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Chapter 1
25
Figure 1.21. The porhyrin assembling structure obtained by controlling
molecular structure.
Previously, we synthesized a 5, 15 meso-substituted methyl uracil derivative
bearing 6-methyl uracil units directly at the meso positions [23]. In this case, we
regulated the atropisomers of porphrin as a means of determining direction. This is quite
an interesting example because there are two forms of assembly, one that leads to dimer
formation and another that leads to formation of a polymer through hydrogen bonding.
In this model, it was shown that we could build different complexes or assemblies by
controlling the molecular structure.
3. Ultra thin film based on molecular organization
3.1. Nanomaterials based on nanotechnology
The representative example of a nanomaterial based on nanotechnology
should be nanoparticles, liposomes or micelles, which are types of particles or
nanomembranes. The nanomembrane or nanofilm will be discussed in the next section,
and in this section we focus on particles.
These nanoparticles are used for drug delivery system (DDS). Nanoparticles
can be made from a variety of organic and inorganic materials including non-degradable
and biodegradable polymers, lipids (liposomes, nanoemulsions, and solid-liquid

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26
Figure 1.22. Various type of drug carrier for DDS.
nanoparticles) self-assembling amphiphilic molecules, dendrimers, metals, and
inorganic semiconductor nanocrystals (quantum dots) [24]. They are colloidal
assemblies.
[Liposomes]
A lipid bilayer is a membrane-like structure that, according to the biomembrane
model, functions as the membrane of cells. The polarized lipid has an amphiphilic
character with both hydrophilicity and hydrophobicity. The hydrophilic group is
oriented towards the water through the formation of hydrogen bonds or electrostatic
interactions. The hydrophobic group is aggregated to avoid water. Amphiphilic
molecules form a lipid bilayer as this is the stable structure in water. In other words, the
hydrophilic groups line up with each other and once the lipid bilayer is fused together
the bare hydrophobic groups are completely separate from water. Therefore a globular
compartment is formed; this molecular polymer is called a liposome or vesicle, and is
filled with water.
Presently, advances in pharmaceutical liposomes are quite noteworthy. For

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27
Figure 1.23. Evlolution of liposome.
example, one issue with liposomes is their fast elimination from blood and capture of
the liposome by cells of the peticulo-endothelial system, primarily in the liver. A
number of trials to overcome these limitations on the use of liposomes have been
initiated. One of these projects is the development of a long-circulating liposome. In
general, to improve the circulation time in the blood, biocompatible polymers such as
PEG are introduced onto the surface of the liposome that act as a protective layer from
opsonins [25].
The evolution of the liposome is shown in Figure 1.23. A is the early traditional
phospholipid „plain‟ liposome with a water insoluble drug. The letter (a) is the water
soluble drug or plasmid entrapped in the liposome and (b) is the liposome membrane to
which (a) is incorporated. B is the immunoliposome in which the antibody is covalently
coupled with liposome that is targeted by antigen. In case (c), the antibody is bound
through covalent binding and in case (d) through hydrophobic interaction. C is
constructed for long circulation using PEG (e) or some other protective polymer which
shields the liposome surface from interaction with opsonizing proteins (f). D is a long
circulation model that contains an antibody (h) and protective polymers (g) in the distal
end of the liposome. New generation liposomes contain diagnostic labels that are
incorporated or attached to the lipid membrane (k), or positively charged lipids (l) that
form complexes with negatively charged DNA, stimuli-sensitive lipids (n), attachment

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28
stimuli sensitive polymers (o), cell penetrating peptides (p) or viral components (q)
other than those described in D (i) and (j). In addition to use as a drug, liposomes can be
loaded with magnetic particles (r) for magnetic targeting and/or with colloidal gold or
silver particles (s) for electron microscopy. Obata et al. developed a new generation
liposome that was composed of cationic 1,5-ditetradecyl-N-lysyl-L-glutamate or
1,5-ditetradecyl-N-alginyl-L-glutamate. Compared to Lipofectoamine2000, there was
no reduction in transfection efficiency in the presence of fetal bovine serum and
remarkably low cytotoxity was observed [26].
[Micelles]
Hydrophobic or hydrophilic structures of amphiphilic molecules assemble
together and are distributed in laminae called micelles. When amphiphilic substances
form micelles, the dispersal system is stable. For example, anionic surfactants such as
soap make oil soluble in water by orienting its hydrophobic moiety to the oil side, and
its carboxylate anion group to the water side. On the other hand, the anionic negative
charge of the carboxylate anion group attracts the polarized water to form an electric
double layer by water anionic charge and polarized water charge. In this phenomenon,
the emulsion is stable by existence of a repulsive charge at the surface of the oil droplet.
Polymer micelles are becoming useful as a therapeutic application for cancer
due to their small size (10-100nm), in vivo stability, ability to solubilize water-insoluble
anti-cancer drugs, and prolonged blood circulation times [27].

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29
Figure 1.24. Schematic illustration of the core-shell structure
There are some merits of using micelles. First of all, the solubility of water-insoluble
drugs at the core of a micelle is dramatically improved because of encapsulation.
Second, the half-lives are prolonged because of PEG protection from opsonization.
Third, their small size (10-100 nm) is well-suited for injection and further deposition to
the tumor due to an enhanced permeability and retention (EPR) effect stemming from
the leakiness of tumor vasculature [28].
[Polymer based nanoparticle]
It is known that cytotoxity and side effects are reduced using nanoparticles
whereas therapeutic efficacy is improved, so, nanoparticles are receiving attention as
aniticancer drugs. Nanoparticles are known to escape from the vasculature through the
leaky endotherial tissue that surrounds a solid tumor. Another important advantage of
using nanoparticles is their ease of preparation. Kumar et al. prepared
poly(lactic-co-glycolide) (PLGA) nanoparticles as a DNA carrier [29].

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30
Figure 1.25. Schematic representation of PLGA nanosphere preparation process.
3.2. Ultra-thin film based on self-assembly
In section 3.1., we described nanomaterials based on self-assembly rather than
nanofilms or nanomembranes. In this section, we describe an ultra-thin film based on
self-assembly. The most famous ultrathin film was fabricated using the
Langmuir-Blodgett (LB) technique and the Layer-by-Layer technique.
[Langmuir-Blodgett film]
Organization of surfactants at the molecular level has been investigated for more
than a century [30]. Techniques for handling monolayers of surfactants were developed
by Agnes Pockels in 1891 [31], and Irving Langmuir and Katherine Blodgett
demonstrated in the 1930s that compressed monolayers of surfactants could be
transferred, layer-by-layer, onto solid substrates to form ultrathin stable films, which are
now referred to as Langmuir-Blodgett (LB) films [32]. LB films are routinely and
reproducibly formed by moving a clean plate through a monolayer, supported on an

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31
aqueous solution [33]. Hydrophobic substrates preferentially attract the tails of
surfactants and the monolayer is transferred during immersion. Conversely, polar
substrates favor the surfactant headgroups and monolayer transfer preferentially occurs
during the withdrawal process. Repeated withdrawal and dipping of a hydrophilic
substrate through the monolayer leads to the buildup of a
substrate-head-tail-tail-head-head y-type multilayer LB film.
[Layer-by-layer deposition film]
Layer-by-layer (LbL) film is usually fabricated through electrostatic interaction
between polycations and polyanions, such as polyelectrolytes. Recently, there have been
reports of the fabrication of a freely suspended membrane using conventional LbL
assembly [34-40] combined with sacrificial or pH sensitive substrates [41,42]. However,
it takes a long time to fabricate these LbL membranes, so Tsukruk et al. developed a
new method of fabrication by using a spin-coater [34,43]. This fabrication method
combined a spin-coating technique with the conventional LbL technique, thereby
making LbL assembly more simple, time-efficient, and cost effective. Spin-coating
techniques have usually been used for gas sensing and light emission. Free-standing
LbL multilayers with thickness below 100 nm have been fabricated with a spin-coater
by Kotov [44]. This spin-coating film made conventional LbL film more robust, tough
and mechanically strong.
Kunitake et al. constructed an LbL membrane composed of organic/inorganic
interpenetrating networks (IPN) in the form of a free-standing nanomembrane (around
35-nm thick) with unprecedented macroscopic size and characteristics [45].

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32
Figure 1.26. Preparative procedure for self-supporting hybrid IPN nanofilm.
Figure 1.27. (a) Microscopic characterization of free-standing hybrid nanofilm.
SEM top-view image of a hybrid nanomembrane. (b) Micrographs of a large
free-standing nanofilm in the air supported by a wire loop. The nanophilm is
transparent and can reflect the light.
a)
b)

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Chapter 1
33
Figure 1.28. a) Schematic of layer-by-layer assembly of polyethyleneimine and clay,
with (b) a cross-sectional illustration of the resulting brick wall nanostructure, and (c)
a structureal representation of an MMT platelet form ref. and PEI
It is common during LbL film construction that one or more of the deposition
ingredients are charged nanoparticles [46]. Quantum dots, clay, silica nanoparticles, and
carbon nanotubes have been deposited in LbL assemblies to import, respectively,
optoelectronic behaviour, strength, anti-reflectivity, and electrical conductivity. In
addition to exhibiting the strength of steel, assemblies containing clay have oxygen
barrier properties that rival ceramic or thin metallic film. Tsukruk et al. evaluated the
growth of assemblies made with cationic polyethylenimine (PEI) and anionic
montmorillonite clay.
Fujie et al. fabricated an LbL nanofilm (nanosheet) that was composed of chitosan
as a source of polycations and alginate acid as a source of polyanions using the
techniques described above. The thickness of this nanosheet is approximately 30.2 ±
4.3 nm and can be altered by the number of layers. This nanosheet is flexible, has a high
aspect ratio, and is strongly adhesive [47].

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Chapter 1
34
Figure 1.29. The LbL nanosheet made from chitosn and alginate acid via electrostatic
interaction.
3.3. Evaluation of ultra-thin film
Characterization of materials at increasingly small dimensions is a critical part of
many manufacturing industries, including semiconductors, optoelectronics, and
automotive and aerospace components. The uses of proven analytical tools to
characterize products of nano- and micro-technology are reported here [48].
1. TEM (Transmission Electron Microscopy)
2. XPS (X-ray Photoelectron Spectroscopy)
3. XRD (X-Ray Diffraction)

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Chapter 1
35
4. AES (Auger Electron Spectroscopy)
5. TOF-SIMS (Time of Fright Secondary Ion Mass Spectroscopy)
6. Contact angle
7. AFM (Atomic Force Microscopy)
8. Ellipsometry
9. IRAS (Infrared Reflection Absorption Spectroscopy)
10. UV-visible spectroscopy
11. Zeta potential measurement
12. Fluorescent microscopy
3.4. General uses for ultra-thin film
LbL assemblies can exhibit a wide array of properties, including electrical
conductivity [49-51], sensing [52-54], superhydrophobicity [55-57], antimicrobial
[58-60], drug delivery [61,62] and controlled molecule release [63].

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36
Figure 1.30. Schematic representation of a polyelectrolyte coated gel bead (100 μm)
loaded with LbL microcapsules in size.
4. Polymer nanohseets utilized in the biomedical field
4.1. Biomedical application of ultra-thin film
The benefits of ultra-thin films include their ability to coat complicated and
difficult geometries, adherence to many surface chemistries, and ease of fabrication.
Hammond et al. fabricated an LbL film composed of linear polyethylenimine and
sulfonated polystyrene and analysed the release of a model protein. This LbL film can
be controlled for any geometry and size scale. The protein rate and timescale of release
from the film are tunable by choosing the appropriate anion and considering the
properties of the degradable polymer [62].
Hennink et al. constructed a microcapsule in which a dextran sulfate/
poly-L-alginine) LbL coated a calcium core that was filled with FITC-dextran (as a
model drug and to allow visualization by fluorescence microscopy) (Figure 1.30.) [61].
The authors suggest that the materials described in this study may find applications for
single shot vaccinations delivering microparticles that contain antigen.
Fujie et al. used LbL nanothickness film as plaster in a post-surgical model for
lung tissue-defect repair [64]. They constructed a chitosan/alginate acid LbL described
in 3.2. and applied it to the lung tissue defect. The authors suggest that it would be

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Chapter 1
37
Figure 1.31. Tissue defect repaired by a polysaccharide nanosheet using a
water-soluble supporting membrane. a) A bilayerd LbL film peeled with PVA
supporting membrane(75nm). b) Free-standing LbL film in water. c) LbL film
supported by a PVA membrane sealing the visceral pleural defect of a beagle dog.
Figure 1.32. Murine cecal puncture model treated with PVAc-TC-nanosheets: (a)
schematic presentation and (b) macroscopic images after treatment with the
PVAc-TC-nanosheet where (c) illumination of TC under black light suggested the
location of the PVAc-TC-nanosheet.
possible to repair the tissue defect with a 75 nm nanosheet and no conventional surgical
intervention. The details of this application are described in Chapter 5.
These authors also demonstrated the sealing effect of the nanosheet on a murine cecal
puncture model. They fabricated a Layer-by-Layer nanosheet composed of chitosan and
alginate acid covered by polyvinyl acetyl cellulose containing tetracycline [65].

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Chapter 1
38
On the other hand, a free-standing biodegradable poly-L-lactic acid (PLLA)
nanosheet made by a spin-coater was used for a stomach incision model in a similar
fashion. Okamura et al. demonstrated a potential biomedical application of the
nanosheet as a novel wound dressing that lacks adhesive reagents and is able to repair a
gastric incision without suturing [66]. The ultra-thin PLLA nanosheet was found to have
an excellent sealing efficacy for gastric incision as a novel wound dressing that does not
require adhesive agents. Furthermore, the sealing operation completely repaired the
incision without scars and tissue adhesion. This approach would be ideal as an
alternative to conventional suture/ligation procedures not only because the procedure is
a less invasive surgical technique but also because of a reduction in operation time. This
result suggested that the PLLA nanosheet could function as a bio-interface for balancing
conflicting biophenomena involved in tissue repair and resistance to tissue adhesion.
Specifically, when the surface of the PLLA nanosheet adhered directly to the stomach, it
was exposed to blood and tissue fluid that contain various growth factors. On the other
hand, it would be intrinsically difficult to adhere various cells to the outer surface of the
PLLA nanosheet.
Conventional suture
and ligation
Sealing with the
PLLA nanosheet
Figure 1.33. Macroscopic and microscopic observations of the stomach seven days after
treatments. Sealing with the PLLA nanosheet.

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Chapter 1
39
Figure 1.34. immunochemical and histochemical staining of troponin T (A-E) and
ALP (A‟-E‟) of C2C12 on rhBMP-2-loaded films for increasing BMP-2 initial
concentration: A) 0, B) 0.5, C) 1, D) 10, and E) 50 μg mL
-1
(scale bar:150μm)
4.2. Functionalisation of ultra-thin film
Piccart et al. developed a rhBMP-2 loaded LbL film made from poly-L-lysine and
hyaluronan to alter myoblasts to osteoblasts [67]. They provided evidence that very thin
films (1μm in thickness) can be used as a tunable reservoir for rhBMP-2 delivery to
cells. BMP-2 was trapped in the film and remaining bioactive for more than 10 days.

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40
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43
Chapter 2
Cell adhesion property on the nanosheet
1. Introduction
2. Construction of poly-L-lactic acid (PLLA) nanosheet
3. Cell adhesive property on the PLLA nanosheet
4. Summary
References

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Chapter 2
44
1. Introduction
It is well-known that ten years ago, when Prof. Langer from MIT and Prof.
Vacanti from Harvard Medical school presented prospects for tissue engineering, the
very idea that living flesh could be “constructed” following engineering principles and
combining nonliving materials with cells sounded fantastical to many [1]. These authors
placed a scaffold of copolymer made from Polyglycol acid and poly lactide (PGA/PLA)
in the shape of a human ear under the stretched skin of a nude mouse, seeded it with ear
cartilage cells, and generated tissue in the shape of a human ear Figure 2.1. [2].
Thus, Poly-L-lactic acid (PLLA) is such a famous material for its biodegradability
and biocompatibility. In this chapter, we describe the nanosheet made from PLLA that
was also used in the mouse ear study. PLLA is suitable for applications in wound
dressing or tissue regeneration. PLLA is known for its non-toxicity, ease of fabrication
and biodegradability. Nonetheless, surface modification of PLLA is still required to
provide cytocompatibility [3,4].
Figrue 2.1. Tissue-engineered cartilage in the shape of a human ear. The
scaffolding was seeded with cells and implanted on the dorsum of a nude
(hairless) mouse.

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Chapter 2
45
Dropping of
1.0wt%PLLA
Silicon wafer
Dipping
10wt%PVA
solution
Peeling
off
2. Construction of poly-L-lactic acid (PLLA) nanosheets
2.1. Preparation of PLLA nanosheets
Freestanding PLLA nanosheets
Poly-L-lactic acid (PLLA) and methylene chloride were purchased from
Polyscience, Inc. (Warrington, PA). A PLLA nanosheet was constructed as described
below (Figure 2.2.). A PLLA nanosheet was prepared on a silicon substrate using a
spin-coater, Opticoat MS-A150 (MIKASA Corp., Tokyo, Japan), at 4,000 rpm for 20 s
and dried on a hotplate at 80
o
C. The thickness of the resulting nanosheet was
approximately 60 nm. Then, a 10 % wt polyvinyl alcohol (PVA) solution was dropped
onto the PLLA nanosheet and dried on a hotplate at 80
o
C for preparation of a supporting
film. The PLLA nanosheet complexed with the PVA supporting film was then peeled
from the silicon substrate.
PLLA nanosheet attached to a dish
These nanosheets are suitable for decal transfer to other substrates via a polyvinyl
alcohol (PVA) cast membrane as a supporting film (Figure 2.3.).The nanosheet with the
supporting film was attached to a dish. Water was then added to the dish to allow the
PVA to dissolve overnight. The dish was washed twice with water to completely remove
the PVA and air-dried for at least 3 hrs.
Figure 2.2. Preparative scheme of PLLA nanosheets.

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2.2. Evaluation of PLLA nanosheet
AFM measurement
Atomic force microscopy (AFM) was used to observe the surface morphology of
the nanosheets. The thickness of the nanosheet sample prepared on the silicon wafer
was also determined. The thickness of this nanosheet was approximately 60 nm.
Assessment of the surface profile
The thickness of each nanosheet was also determined using a surface profiler
α-step (KLA-Tencor Corp., San Jose, CA). From assessment of the surface profile, the
thickness of this nanosheet was also 59.5±9.5 nm.
Figure 2.3. Transference of a PLLA nanosheet to a PS dish.
Figure 2.4. AFM images of nanosheets prepared on silicon wafers: A; top view
(left; silicon wafer, right; nanosheet, scale bar: 10 μm), B; cross-sectional images

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100μm
100μm
100μm
PLLA
3. Cell adhesive property on the PLLA nanosheet
To evaluate the cell behavior on the PLLA nanosheet, we observed the cells on the
PLLA nansoheet 24 hours after the murine cell line were seeded. Murine fibroblast cell
line NIH3T3 was purchased from DS Pharma (Osaka, Japan). NIH3T3 cells were
seeded on a nanosheet sample and cultured under a DMEM-containing 10% fetal bovine
serum and 1% penicillin streptomycin solution (Wako Chemical Co.) in a humidified
atmosphere of 5% CO2 at 37
o
C. After reaching confluence, the cells were dissociated
with a 0.05 w/v% trypsin-0.53 mmol/l EDTA/4Na solution supplemented with phenol
red (Wako Chemical Co.). The nanosheet samples of PLLA with the PVA supporting
film were attached to a polystylene cell culture dish (IWAKI Corp, Japan). PVA was
dissolved in distilled water and then dried. The dish was filled with medium that
contained 3.67 x 10
4
cells / cm
2
of NIH3T3 cells and cultured for 24 hours, followed by
observation with an optical microscope (Olympus, Co., Tokyo, Japan). When murine
fibroblast NIH3T3 cells were seeded on the PLLA nanosheet, there was no cell
attachment observed on the nanosheet as expected (Figure 2.5.).
Figure 2.5. Behaviour of Murine cell line NIH3T3 on the PLLA nanosheet (Left;
PLLA nanosheet, right; Polystylene cell culture dish)

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4. Summary
From these observations on cell adhesiveness it was clear that adsorption or
attachment onto the organs comes from physical adsorption, not from biological
adhesion. This physical adsorption is likely because of its nm thickness and flexibility.
This phenomenon is closely related to thickness and the scratching test [5].

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49
References
1. A. Khademhosseini et al., Biotechnology, 2009, 64.
2. C. A. Vacanti, MRS Bulletin, 2001, 798.
3. Y. Hong et al., Biomaterials, 2005, 26, 6305.
4. P. B. van Wachem et al., Biomaterials, 1985, 6, 403.
5. Y. Okamura et al., Adv. Mater. 2009, 21, 4388.

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51
Chapter 3
Construction of Thrombin loaded nanosheet and evaluation of
its hemostat ability and anti-adhesion ability
1. Introduction
2. Biology and management of wounds
3. Hemostatic materials
4. Preparation of thrombin loaded PLLA nanosheet (Thr-PLLA)
5. Analysis of Thr-PLLA
6. Evaluation of Thr-PLLA using an animal model
7. Summary
References

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1. Introduction
As described in Chapter 1, a PLLA nanosheet that has been constructed in our
laboratory and which is biodegradable and biocompatible is known to be useful as an
anti-adhesion barrier from in vivo experimentation. In this research, we tried to fabricate
a wound dressing material coated with thrombin that had hemostatic ability using
hydrophobic interaction between thrombin and a PLLA nanosheet. In this chapter, we
constructed a thrombin-loaded nanosheet and evaluated its material properties and
performed an evaluation mainly on its hemostatic ability in vitro and in vivo
experiments.
2. Biology and management of wounds
2.1. Wound biology
The overlapping segments of the repair process are conceptually defined as
inflammation, proliferation, and remodeling. During the inflammatory phase,
hemostasis occurs, and an acute inflammatory infiltrate ensues. The proliferation phase
is characterized by fibroplasias, granulation, contraction, and epithelialization. The final
phase is remodeling, which is commonly described as scar maturation [1].

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a)
b)
c)
e)
d)
2.2. Stage of wound healing.
Wound healing proceeds through a series of interdependent and overlapping
stages in which a variety of cellular and matrix components act together to reestablish
the integrity of damaged tissue and replacement of lost tissue. There are five stages for
wound healing, classified as: hemostasis, inflammation, migration, proliferation, and
remodeling.
Figure 3.1. The temporal relationship of repair stages and cellular infiltrates into the
wound. Overlap occurs between the stages, and the beginning and endpoints are
approximate.
Figure 3.2. The 5 stages of wound healing; a) hemostat, b) inflammation, c) migration,
d) proliferation, e) remodeling process.

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Hemostatic phase
Immediately after tissue injury, hemostasis is stimulated by platelet degranulation
and exposure of tissue thromboplastic agents (Figure 3.2. a). After tissue injury, the
lacerated vessels immediately constrict, and thromboplastic tissue products,
predominantly from the subendothelium, are exposed. Platelets aggregate and form the
initial hemostatic plug, and coagulation and complement cascades are initiated. The
intrinsic and extrinsic coagulation pathways lead to activation of prothorombin to
thrombin, which converts fibrinogen to fibrin, which is subsequently polymerized into a
stable clot. When the thrombus is formed, homeostasis in the wound is achieved.
Inflammation phase
Within 24 h, a neutrophil efflux into the wound occurs. The neutrophils scavenge
debris and bacteria and secrete cytokines for monocyte and lymphocyte attraction and
activation. Keratinocytes begin migration when a provisional matrix is present.
Migration phase
At two to three days after injury, the macrophage becomes the predominant
inflammatory cell type in clean, noninfected wounds. These cells then regulate the
repair process by secretion of myriad growth factors, including types that induce
fibroblast and endothelial cell migration and proliferation. Bone marrow-derived
mesenchymal progenitor cells are present in the wound.
Proliferation phase
Local fibroblasts and elicited progenitor cells are activated and present at the
wound by three to five days after injury. These cells secrete matrix components and
growth factors that continue to stimulate healing. Keratinocyte migration (epiboly)
begins over the new matrix. Migration starts from the wound edges as well as from

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epidermal cell nests at sweat glands and hair follicles in the center of the wound.
Remodeling phase
Scar formation is the outcome of healing in postnatal skin. Scar is composed of
densely packed, disorganized collagen fiber bundles. Remodeling occurs up to one to
two years after injury and consists of further collagen cross-linking and regression of
capillaries, which account for the softening of scar and its color change from red to
white.
2.3. Classification of wound dressing products
Wound dressings are generally classified as: 1. passive products; 2. interactive
products; and 3. bioactive products, based on their nature of action [2]. Traditional
wound dressings like gauze and tulle dressings that account for the largest market
segment are passive products. Interactive products are comprised of polymeric films and
forms, which are mostly transparent, permeable to water vapor and oxygen but
impermeable to bacteria. These films are recommended for low exuding wounds.
Bioactive dressings deliver substrates active in wound healing, either by delivery of
bioactive compounds or with dressing constructed of material with intrinsic bioactivity.
These materials include proteoglycans, collagen, noncollagenous proteins, alginates or
chitosan.

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3. Hemostatic materinals
3.1. Basic hemostasis
Generally, activation of the hemostatic process is proportional to the extent of
vascular damage and limited to the site of injury [3]. These features of the coagulant
response require that the coagulation mechanism functions in an amplified and localized
manner. The response to vascular injury begins when blood is exposed to subendothelial
structures and rapid activation of the hemostatic process is initiated. A sequence of
events occurs in the initial formation of platelet thrombin, which includes platelet
adhesion to expose subendothelium and release of a platelet plug by the formation of
Table 3.1. A comparative chart of some commercial dressing material
(Interactive and bioactive products)

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thrombin. Thrombin induces more platelets to aggregate and forms fibrin that stabilizes
the platelet plug. This cascade arrangement of the blood coagulation reaction is clearly
one mechanism by which the initiating stimulus is amplified to obtain a sufficient
coagulant response.
There are two major defense mechanisms against bleeding: fibrin generation
(humoral hemostasis) and platelet aggregation (cellular hemostasis). Fibrin and
activated platelets constitute the major components of hemostatic plugs. These
mechanisms operate in tandem, although fibrin generation may be relatively more
important for hemostasis in veins and venules, and platelet aggregation may be
relatively more important for hemostasis in arteries, arterioles, and capillaries. In
addition, the vascular component was found to play a major role (vascular hemostasis).
3.2. Humoral hemostasis
Fibrin generation is a series of coordinated and calcium-dependent
proenzyme-to-serine protease conversions likely localized on the surface of activated
cells in vivo. It culminates with the conversion of prothrombin to thrombin by the
coagulant complex known as prothrombinase. The apparent end point of blood
coagulation is the conversion of the soluble plasma protein, fibrinogen, into insoluble
fibrin. This is only one of several necessary reactions that are catalyzed by thrombin.
The traditional concept of the coagulation system consists of a strict cascade that is
divided into the extrinsic and intrinsic system (Figure 3.3.)

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In contrast, the current model of blood coagulation differs from the original “cascade”
scheme, in which an intrinsic pathway begins with contact activation of factor XII
(FXII), leading to factor IX activation, followed by factor X activation. The primary
ambiguity of the old model was the importance of FXII, because clinical bleeding is
absent in persons affected by hereditary FXII deficiency. In contrast, patients with FXI
deficiency have a severe bleeding disorder when provoked, indicating the importance of
FXI in normal hemostasis [4]. The current model of coagulation helps reconcile these
observations by describing hemostasis as a process involving three overlapping phases
(Figure 3.4.)
Figure 3.3. The “classical” coagulation cascade is described as a pathway of proteolytic
enzymatic reactions, resulting in the formation of fibrin via the cleavage of fibrinogen, a
process mediated by the serine protease thrombin. PL, phospholipids.

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3.3. General hemostatic materials
Numerous topical hemostatic agents are currently available on the market. A
recent review summarized different groups of hemostats and the most commonly used
products. An overview is summarized in Table 3.2 [5].
Figure 3.4. The cell-based model of coagulation according to Hoffman and Mocre. Fibrin
formation occurs on different cell surfaces in three phases (from left to right). During the
initiation (left), only small amounts of thrombin are formed, which coactivates platelets as
an amplification step (middle). Several factors are cleaved and activated to form the
“intrinsic tenase”. The cell-based model of coagulation according to (IXa/VIIIa) on the
surface of activated platelets (right). This finally leads to the formation of large amounts of
thrombin and subsequent fibrinogen cleavage to form the clot.

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Collagen-based hemostats attract and activate platelets leading to thrombous
formation, whereby collagen seems to be an anchoring material for clotting factors and
platelets. Therefore, some collagen-based products are combined with other substances,
mainly thrombin, to support the clotting cascade.
Gelatin-based hemostats are available in sponge, powder or paste form. The
water-insoluble material has hemostatic properties and is usually resorbed within days.
The mechanism is not fully understood, but it is thought to crosslink with adjacent
tissue, thus inducing a physical reaction. Certain products are also combined with
procoagulant factors.
Cellulose-containing hemostats have a low pH and therefore react with blood
components leading to the formation of blood clots. In addition they have bacteriostatic
properties.
Polysaccharides are positively charged and therefore attract blood cells, which
Table 3.2. Different groups of local haemostats with the most commonly used
products.

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have a negative charge. They are also thought to release vasoactive substances and
activate platelets. Inorganic and polymeric products absorb fluid that leads to swelling
of the material and therefore occludes the small blood vessels of the wound but may
also attract and activate platelets.
Several companies produce fibrin sealants with slightly different compounds.
Tisseel (Baxter Healthcare), Quixil (Ethicon), Vivostat (Vivolution) and
TachoComb/TachoSil (Nycomed) are members of this group, however only the latter
contains a collagen fleece.
3.4. TachoSil and TachoComb
TachoSil, a collagen-bound fibrin sealant, underwent several improvements over
the years. The initial version was called TachoComb and was introduced to the market
in the early 1990s after it was found to be effective in the management of diffuse
parenchymatous bleeding in animal studies. It consisted of a sponge-like equine
collagen patch coated with a mixture of human fibrinogen [5,6], bovine thrombin and
bovine aprotinin. This product was followed by TachoComb H with the same
components except for bovine thrombin, which was replaced by human thrombin.
4. Preparation of thrombin loaded PLLA nanosheet (Thr-PLLA)
Data from in vivo experimentation indicated that a biodegradable and
biocompatible PLLA nanosheet fabricated in this laboratory could be quite useful as an
anti-adhesion barrier. In this research, we aimed to fabricate the wound dressing
material as a functional hybrid of hemostatic activity and an anti-adhesion barrier by
preparing a thrombin-coated PLLA nanosheet (Thr-PLLA) using hydrophobic

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interaction. In this experiment, we focused on the evaluation of fundamental properties
of Thr-PLLA using in vitro and in vivo experimentation.
4..1. Fabrication of Thr-PLLA nanosheet
The preparation scheme of Thr-PLLA was described above (Scheme 3.1.). 1.0
wt% polyvinyl alcohol (PVA) solution and 1.0 wt% PLLA solution was spin-coated on a
SiO2 substrate to produce the film by a spin-coating method. After 1 U/mL thrombin
solution was cast and remained at rest at 4
o
C for 4 hours, Thr-PLLA was washed three
times with with PB.
5. Analysis of Thr-PLLA
5.1. Preparation of FITC-Thr-PLLA
A FITC-labelled Thr-PLLA nanosheet was prepared using
Fluorescent-5-isothiocyanate (FITC, final conc. 0.01 mM) and observed with
fluorescent microscopy (exposure time; 2.5 s) (Figure 3.5.). Comparison of Thr-PLLA
with FITC cast PLLA nanosheet as a control suggested homogeneous coating of
thrombin.
Thrombin
Deposition of thrombin
(4oC, 4 hrs)
Suspending in water
Thrombin-PLLA nanosheet
Thrombin
Deposition of thrombin
(4oC, 4 hrs)
Suspending in water
Thrombin-PLLA nanosheet
Scheme 3.1. Preparation of Thrombin nanosheet.
SiO2 substrate
PVA
PLLA
PVA solution
Spin-coating
(4000 rpm,20 s)
PLLA solution
Spin-coating
(4000 rpm,20 s)
SiO2 substrate
SiO2 substrate
PVA
PLLA
PVA solution
Spin-coating
(4000 rpm,20 s)
PLLA solution
Spin-coating
(4000 rpm,20 s)

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300 μm
(b)
300 μm
300 μm
300 μm
(b)
(a)
300 μm
(a)
300 μm
300 μm
60
70
80
90
100
500
1000
1500
2000
2500
3000
3500
4000
100
90
80
70
60
4000
3500
3000
2500
2000
1500
1000
500
%
T
Wavenumber (cm-1)
PLLA
Thr-PLLA
5.2. Evalution of Thr-PLLA nanosheet by Infrared (IR) spectrum
To evaluate Thr-PLLA nanosheet qualitatively, we used IR spectrum to detect the
amide vibration bonding at 1640 cm
-1
(Figure 3.6.).
We prepared a 1 U/mL thrombin-coated nanosheet on the PLLA nanosheet and detected
amide vibration of thrombin using a CaF substrate and comparing to PLLA as a control.
However, no differences could be detected and the amide vibration of thrombin could
not be detected. This was likely due to a minimum detectable quantity of thrombin or
possibly because there was no thrombin. However, since we could see the fluorescent
image of thrombin on the PLLA nanosheet, the former speculation is likely correct.
Figure 3.5. (a) FITC-PLLA nanosheet, (b) FITC-thrombin nanosheet
Figure 3.6. IR spectrum of PLLA and Thr-PLLA nanosheet.

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5.3. Quantification of adsorbed thrombin
To more precisely quantify the amount of thrombin, the quantification of adsorbed
thrombin was performed by quartz crystal microbalance (QCM) measurement after the
thrombin solution was cast (0.5, 1.0, 2.5 U/mL) and and allowed to rest for 4 hrs at 4
o
C
(Figure 3.7., n=3).
Whereas almost no thrombin was adsorbed on the PLLA nanosheet with 0.5 U/mL
thrombin solution, approximately 10 ng thrombin was adsorbed with more than 1.0
U/mL and it was suggested that the loading mass was controllable depending on the
thrombin units.
5.4. Blood coagulation assay
The glass substrates covered by the PLLA nanosheet and the Thr-PLLA nanosheet were
set on a 12-well dish and dropped with 200 μl of rat whole blood (30 minutes after
administration of heparin intravenously; 2ml of 10-fold dilution of somunopentyl 64.8
mg/mL with saline). After 15 min incubation, these substrates were washed with 3mL
PBS and the supernatants were centrifuged. The images of whole blood that remained
0
50
100
150
200
250
300
0.5
1.0
2.5
Concentration (U/ml)
L
o
a
d
in
g
ma
ss (n
g
/cm
2 )
Figure 3.7. Loading mass depending on the concentration of thrombin on
the PLLA nanosheet (n=3)

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after washing depended on thrombin concentration (0, 1.0, 2.5, 5.0 U/mL) and are
shown in Figure 3.8. After precipitates were dissolved with MilliQ water on the 1.0
U/mL sample only, absorbance at 412 nm was measured using a microplate reader. The
absorbance was slightly lower on the Thr-PLLA nanosheet compared to the PLLA
nanosheet (Figur 3.9.).
6. Animal experiment for evaluation of Thr-PLLA
6.1. Animal viability
To check the effects of Thr-PLLA, we performed an animal experiment using a
0
0.2
0.4
0.6
0.8
1
1.2
Thr
PLLA
A
b
s
a
t 4
1
2
n
m
(-)
Figure 3.8. Coagulation ability of Thr-PLLA nanosheet depending on thrombin amount
0.5 U/mL
1.0 U/mL
2.5 U/mL
5.0 U/mL
Figure 3.9. Absorbance of blood washed water for PLLA and Thr-PLLA nanosheet (1.0
U/mL concentration of thrombin) covered glass plates (n=3).

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liver defect model approximately 1.5 cm in diameter, covered by a Thr-PLLA and
PLLA nanosheet. The rat was administered with 200 U of heparin and monitored for 30
minutes. We checked survival rate after suturing (Figure 3.10.). The untreated and
PLLA nanosheet group died, whereas 100% viability was confirmed in the Thr-PLLA
group and this result indicated that the Thr-PLLA nanosheet provided some hemostatic
activity.
6.2. Anti-adhesion property of Thr-PLLA
As mentioned in Section 5.2, the Thr-PLLA nanosheet held some promise for the
extension of animal viability. At the same time we evaluated the PLLA side of the
Thr-nanosheet as an anti-adhesion barrier. To evaluate anti-adhesive property, we
observed the macroscopic appearance of the liver at five days postoperative. We
experienced difficulty putting the nanosheet on the liver and this process was
accompanied by heavy hemorrhaging. The nanosheet did not seem to prevent the heavy
hemorrhage from oozing such that the nanosheet seemed to float on the surface of the
blood. Heavy postoperative adhesion was confirmed at five days postoperative and
documented in Figure 3.11.
Figure 3.10. Viability of the liver wounded model rat with thrombin-sheet and PLLA
nanosheet (n=3)
0
20
40
60
80
100
Thr
PLLA
Untreated
V
ia
b
ility
(%
)

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a)
b)
It was assumed that since severe adhesion was confirmed on an untreated mouse,
there was no evidence of discriminating differences between two groups. Histological
sectional images are shown in Figure 3.12.
A dissected liver sample was fixed with 10 % formaldehyde for more than four
hours with shaking. A 10%, 15%, and 20% PBS solution of sucrose was prepared and
the liver was immersed in a stepwise fashion for more than four hours at each
concentration. The organs were quickly chilled in O.C.T. compounds (Sakura Finetek
Japan Co., Ltd., Tokyo) using liquid nitrogen. Frozen tissue was cut into a 15 μm slice
and dried on an O/N prepared slide and incubated in 20% PBS solution of formalin for
20 min at room temperature for refixation. After washing with distilled water, samples
were immersed in Mayer’s Hematoxilin solution (MUTO PURE CHEMICALS CO.,
LTD., Tokyo) and incubated for 15 min. After washing with MilliQ water, samples were
immersed in an 80% ethanol solution of Eosin Y Solution (MUTO PURE CHEMICALS
CO., LTD., Tokyo) and washed with MilliQ water, 70%, 80%, 90%, and 100% ethanol
solution in a stepwise fashion. The samples were immersed through xylene solution
several times, and encapsulated glycerol was placed between the cover glass and
prepared slide.
Figure 3.11. Severe adhesion at postoperative 5 days on Thr-PLLA. Adhesion between a)
liver and omentum, b) liver left lobe and right lobe.

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a)
b)
7. Summary
All the data indicated that the Thr-PLLA was not suitable as a hemostatic material.
This was mainly because the nanosheet was washed away by heavy bleeding and the
loaded thrombin was too low to be effective as a hemostatic material. To produce a
nanosheet for use as hemostatic material, a higher physical intensity must be attached to
the nanosheet.
Figure 3.12. Tissue adhesion at postoperative 5 days on Thr-PLLA between liver right
lobe and left lobe. a) low magnification, b) high maginification. (scale bar; 400 μm)

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References
1. HP. Lorenz et al., Wounds : Biology, Pathology, and Management. CHPTER 10.
191-208.
2. W. Paul et al., Trends Biomater. Artif. Organs, 2004, 18. 18.
3. A. Jorres et al, Management of Acute Kidney Problems, 2010, 181.
4. M. Hoffman et al, Thromb. Haemost, 2001, 85, 958.
5. A. Rickenbacher et al, Expert Opin. Biol. Ther., 2009, 9, 898
6. H. Seyednejad et al, Br. J. Surg., 2008, 95, 1197.

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Chapter 4
Construction of heterofunctional nanosheets for the cell
adhesive and non-adhesive materials
1. Introduction
2. Construction of heterofunctional nanosheet
3. Structural Characterisation of Heterofunctional nanosheets
4. Cell adhesion property of heterofunctional nanosheet
5. Summary
References

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1. Introduction
Ultra-thin films are widely paid attention because of their unique mechanical
properties such as huge aspect ratio or high flexibility, following high adhesiveness and
mechanical stability in both water and air [1,2,3]. They include a variety of materials
such as cross-linked Langmuir-Blodgett membrane [4], layer-by-layer (LbL) membrane
composed of polycation and polyanion [5], hybrid type of organic and inorganic
compounds [6] and organic/inorganic interpenetrating networks [2]. The fabrication
method using a spin-coater is also developed and the construction of LbL membrane
becomes more simply, time-efficiently, and cost-effectively [7,8] We have developed
biocompatible and biodegradable PLLA several tens of nanometer thickness film
(PLLA nanosheet) as well as the LbL film (LbL nanosheet) composed of
chitosan/sodium alginate using spin-coating techniques [9,10] for application in wound
dressing or tissue regeneration. When the murine fibroblast NIH3T3 cells were seeded
on the PLLA nanosheet, there was no cell attachment seen on the nanosheet. In general,
though PLLA is famous for its nontoxity, processibility and biodegrability, the surface
modification is still required to provide cytocompatibility [11,12]. This observation
explains the absence of tissue adhesion in vivo. On the other hand, collagen is a
fundamental biomacromolecule that has many domains recognized by integrin on the
surface of cell membrane. Collagen is a kind of extracellular matrix (ECM) such as
laminine, fibronectin, vitronectin, elastin [13] and widely used for fabricating ECM-like
scaffold [5,14] or coating material for cell adhesion and proliferation [15,16]. Here, we
propose a biomaterial where one side is made up of a PLLA nanosheet, which behaves
as an adhesion barrier, and the other side has immobilized collagen, which is predicted
to enhance wound healing. Typically, several methods such as covalent bonding, LbL

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assembly and graft-coating are used for constructing collagen coated materials [11]. In
this research, two immobilization methods were tested; (i) collagen cast on the surface
of a PLLA nanosheet (Col-Cast-PLLA) and (ii) collagen spin-coated on the nanosheet
(Col-Spin-PLLA). We anticipated both kinds of nanosheet would improve the cell
adhesion properties. However, it was difficult to generate a homogeneous nanosheet for
Col-Cast-PLLA and it took considerably longer to prepare because of the dry-in-air
procedure. By contrast, Col-Spin-PLLA gave a homogeneous collagen surface with a
short preparation time. Then, we compare the morphology, wettability and cell behavior
on the surface of three types of nanosheet; PLLA, Col-Cast-PLLA and Col-Spin-PLLA.
2. Construction of heterofunctional nanosheet
2.1. Polylactic acid nanosheet bearing collagen layer (Col-Spin-PLLA)
[Materials and Methods]
A collagen solution; Atelo Cell 1AC-30 (native collagen bovine dermis) at pH 3.0,
was purchased from KOKEN Corp. (Tokyo, Japan) and diluted 6 folds (f.c. 0.5 mg/ml)
with 70% ethanol. A PLLA nanosheet was spin-coated with the solution for 20 sec at
4,000 rpm. After casting a PVA supporting film on the resulting Col-Spin-PLLA
nanosheet, the complex film was peeled from the substrate. The PLLA nanosheet was
prepared in the totally same fashion as described in page 45. For the comparison with
the protocol constructing Col-Spin-PLLA, the PLLA nanosheet preparative scheme was
shown again in Figure 4.1..

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2.2. Col-Spin-PLLA obtained by “Supporting film Method” and “Sacrificing film
method”
The nanosheets were prepared with a spin-coater as described in 2.1. The
thickness of the nanosheet was adjusted by the rotating speed and/or the concentration
of the PLLA solution. It was initially difficult to coat the hydrophilic collagen
homogeneously on the hydrophobic PLLA nanosheet because the collagen aqueous
solution is repelled. Then, we diluted the collagen solution (Atelo Cell 1AC-30 Native
collagen Bovine dermis, KOKEN, Tokyo) to 0.5 mg/ml with 70% ethanol in order to
increase the affinity of the collagen solution with the PLLA surface. These nanosheets
have a capability of decal transferring to the other substrates via a polyvinyl alcohol
(PVA) cast membrane as a supporting film as described in Chapter 2.
Figure 4.1. Preparative scheme of nanosheets. a) PLLA nanosheet and b)
Col-Spin-PLLA nanosheet

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3. Structural Characterisation of Heterofunctional nanosheets
3.1. Surface and thickness characterization by AFM
[Materials and Characterizaiton]
The thickness of each nanosheet was determined using a surface profiler α-step
(KLA-Tencor Corp., San Jose, CA). Atomic force microscopy (AFM) was used to
observe the surface morphology of the nanosheets. The thicknesses of the nanosheet
samples prepared on the silicon wafer were also determined.
Using the ethanol solution, we coated collagen homogeneously onto the PLLA
surface and prepared three kinds of nanosheet samples; PLLA, Col-Cast-PLLA (where
collagen was cast on the PLLA) and Col-Spin-PLLA (where collagen was spin-coated
on the PLLA). We used atomic force microscopy (AFM) and ellipsometry to analyze
the surface morphology and thickness of each nanosheet. The AFM results are shown in
Figure 4.2.a. Both the PLLA and Col-Spin-PLLA samples formed a smooth surface,
whereas Col-Cast-PLLA showed some roughness. This roughness is likely to be caused
by deposition of collagen from the 70% ethanol solution due to the water enriched
condition in the late stage of the dry-in-air process. The ellipsometric data also
supported the proposition that a homogeneous collagen layer was generated by
Col-Spin-PLLA. The thickness of the PLLA nanosheet was 59.5±9.5 nm, whereas
Col-Spin-PLLA and Col-Cast-PLLA had a thickness of 67.1±5.2 nm and 180.2 ±62.5
nm, respectively (Figure 4.2.b). The difference in thickness between PLLA and
Col-Spin-PLLA suggested a 5-10 nm thick collagen layer was formed on the PLLA
nanosheet. The spin-coater is thought to instantaneously generate an ultra-thin
homogeneous layer of hydrophilic collagen on the hydrophobic PLLA surface.
Unevenness in the collagen layer is derived from differences in the distribution of

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collagen density during the dry-air-process on Col-Cast-PLLA. Surprisingly, there was
no collagen fibril on both Col-Cast-PLLA and Col-Spin-PLLA, which was generally
seen on the type-I-collagen substrates. This result suggests that the collagen is denatured
in both Col-Cast-PLLA and Col-Spin-PLLA (Figure 4.2.a). Taken together, the AFM
and ellipsometry results indicate that the preparation of a homogeneous nanosheet
covered with an ultra-thin collagen layer is feasible using a spin-coating rather than
casting methodology.
Figure 4.2. a) AFM images of nanosheets prepared on silicon wafers: A; top view (left;
silicon wafer, right; nanosheet), B; cross-sectional images. b) Thickness of nanosheets
prepared on silicon wafers measured by ellipsometry. Mean values ±SD of three
measurements are shown.

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3.2. IR measurement
[Materials and Characterization]
The PLLA, Col-PLLA and Col-Spin-PLLA nanosheets were measured using
FT-IR (FT/IR-410 Fourier Transform Infrared Spectrometer, JASCO, JAPAN). The
nanosheets were scooped onto a NaCl plate and air dried for 1 day. Infrared
transmittance was performed with 32 times resolution in the range of 500 - 4000 cm
-1
.
Though there was no amide vibration on PLLA nanosheet, The amide vibration mode of
collagen was detected at around 1640 cm
-1
derived from the amide I peak of collagen in
IR spectrum (Figure 4.3.) on Col-Cast-PLLA and Col-Spin-PLLA.
3.3. Wettability of each sample
Water wettability of the various surfaces were analyzed with Drop Master
DM-301 (Kyowa-Interface Science Co. Ltd) using the sessile drop technique. Static
water contact angle was analyzed on the PS, Col-Cast-PS, PLLA and Col-Spin-PLLA.
Measurements were carried out at room temperature in air with deionized water as the
probe liquid. Liquid droplets with a volume of 2 μl were deposited onto the sample
Figure 4.3. IR spectrum of PLLA, Col-Cast-PLLA and Col-spin-PLLA nanosheet.

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surface through a 22 gauge dispensing needle. The contact angle was then measured 5
seconds later. Each contact angle reported here is an average of at least three
independent measurements. The Col-Spin-PLLA (53.1±1.06°) showed the highest
hydrophilicity, whereas Col-Cast-PLLA (72.3 ± 1.39°) showed almost the same
hydrophobicity as PLLA (71.3±0.22°) (Figure 4.4.). As a reference, PS showed a
higher hydrophilicity (36.5±1.46°) than Col-Spin-PLLA. The high hydrophilicity of
Col-Spin-PLLA compared with PLLA indicates the coating effect of collagen on the
PLLA surface is completely different from that of Col-Cast-PLLA.
For a reference, we spin-coated Albumin solution (0.5 wt%; 3.0 mg/ml D2W was
diluted to 0.5 mg/ml with 70% EtOH) on the PLLA nanosheet. The contact angle of the
surface was 72.8±0.17°. It was suggested that the raise of hydrophobic interaction by
spin-coating method was characteristic mainly on the fibrous protein like collagen not
on the globular protein like albumin. It was speculated that the reason of blood
coagulation was not induced so much might be the interaction between globular protein
and PLLA nanosheet was quite week.
Figure 4.4. The contact angles of water on various substrates. Data are expressed as
means ±S.E. mean of values obtained from three different fields.

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3.4. Observation of surface morphology of each sample from AFM measurement
[Materials and Characterization]
Atomic force microscopy (AFM) was used to observe the surface morphology of
the nanosheets at 0, 1.0 and 4.5 hrs-incubation in 10% FBS-containing medium at 37
o
C
that is the same condition as the cell culturing. Noteworthy was the appearance of many
collagen fibrils on the Col-Cast-PLLA after 1.0-hr incubation, which was not apparent
for the PLLA and Col-Spin-PLLA preparations. Moreover, it was also clarified that the
fibrils grew during incubation for 4.5-hrs (Figure 4.5). The unevenness of
Col-Cast-PLLA is due to the formation of collagen fibrils as mentioned above. However,
there was no fibril formation on Col-Spin-PLLA even after 4.5 hrs incubation, although
the amide vibration mode of collagen was detected at around 1640 cm
-1
derived from
the amide I peak of collagen in IR spectrum (Figure 4.3.), suggesting the presence of
procollagen as a precursor of collagen fibrils. Liang et al. reported that collagen
morphology was induced by dewetting and self-assembly [19]. The collagen layer was
less rough after fast drying than after slow drying. The spin-coating method is
considered to be a fast drying process, which leads to the formation of smaller and more
filamentous collagen fibrils. Furthermore, we believe that the hydrophobic interaction
between collagen and the PLLA nanosheet is too strong to allow formation of collagen
fibrils. Alternatively, the amount of collagen on the Col-Spin-PLLA is too small to
initiate the development of collagen fibrils as evident from the AFM measurements. The
hydrophilic RGD moiety of the non-fibril collagen is present on the molecular surface
of Col-Spin-PLLA (Figure 4.5.). Indeed, Col-Spin-PLLA is more hydrophilic than
Col-Cast-PLLA [20].

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4. Cell adhesion property of heterofunctional nanosheet
[Materials and Methods]
Murine fibroblast cell line NIH3T3 was purchased from DS Pharma (Osaka,
Japan). NIH3T3 cells were seeded on a nanosheet sample and cultured under a
DMEM-containing 10% fetal bovine serum and 1% penicillin streptomycin solution
(Wako Chemical Co.) in a humidified atmosphere of 5% CO2 at 37
o
C. After reaching
confluence, the cells were dissociated with a 0.05 w/v% trypsin-0.53 mmol/l EDTA/4Na
solution supplemented with phenol red (Wako Chemical Co.). The nanosheet samples of
PLLA, Col-Spin-PLLA and Col-Cast-PLLA with the PVA supporting film were
attached to a 24-well PS dish (IWAKI Corp, Japan). PVA was dissolved in distilled
water and then dried. The dish was filled with medium that contained the same number
of NIH3T3 cells and cultured for an appropriate time, followed by observation with an
optical microscope (Olympus, Co., Tokyo, Japan).
Figure 4.5. Changes in collagen morphology with time. Samples were incubated at
37
o
C in medium containing 10% FBS (Left; low magnification, Right; high
magnification, scale bar: 5 μm).

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4.1.
Cell attachment test and cell spreading test
[Materials and Methods]
The attachment assay was carried out using the 24-well dish prepared with PLLA,
Col-Cast-PLLA, Col-Spin-PLLA or untreated 24-well dish (PS) as a control. We
initially seeded 1.5 x 10
5
cells in 500 μl of medium and incubated the cells for 60 min.
The cells were gently washed twice in PBS and then fresh medium (450 μl) was added,
which was supplemented with MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-phenyl-
2
H
tetrazolium bromide, 5mg/ml, 60 μl; Sigma-Aldrich, St Louis, MO). After the cells were
incubated for 5 hrs in this medium, the supernatant was removed, and then 450 μl of
DMSO was added to solubilize the formazan precipitates. The 24-well dish was shaken
for 10 min and the absorbance at 550 nm measured with a Benchmark Plus microplate
spectrophotometer (BIO-RAD Corp., Hercules, CA).
We also studied the cell behavior on the nanosheet samples using a NIH3T3 cell
line. The appearance of NIH3T3 cells on each sample was evaluated by using
Fluo3-AM (Figure 4.7. a). The cell spreading assay was performed on a 24-well-dish
prepared with PLLA, Col-Spin-PLLA or Col-Cast-PLLA. Initially, 1.5 x 10
5
NIH3T3
cells were plated on the 24-well dish and incubated for 60 min at 37
o
C. The cells were
washed and then stained with 6 μM Fluo3-AM (Molecular Probes, Dojin Chemical,
Tokyo) in a fresh medium for 35 min at 37
o
C and then for 15 min at room temperature.
Cell spreading was estimated as described previously [17]. After washing the cells, the
dish was mounted on a fluorescence microscope BIOREVO BZ-9000 (KEYENCE,
Tokyo, Japan). Cell spreading area on the substrate or nanosheet was then quantified
using Image J software (JAVA).

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82
[Results and Discussion]
The cells on both PLLA and Col-Cast-PLLA did not spread, but instead
maintained a rounded morphology (tethering). The majority of cells were confirmed to
be strongly attached to Col-Spin-PLLA, where they adopted an elongated shape. The
commercially available PS culture dish gave almost the same number of attached cells
in comparison with Col-Spin-PLLA. Quantification of the data using Image J software
(Figure 4.7.b) indicated that the order of the contact area of cells on the nanosheet
sample was Col-Spin-PLLA≧PS>Col-Cast-PLLA=PLLA. This result was confirmed
using the MTT assay (compare Figure 4.6. to Figure 4.7.a). Specifically, the
Col-Spin-PLLA nanosheet showed the greatest amount of cell attachment, with
Col-Cast-PLLA having less than half that of Col-Spin-PLLA, and PLLA the lowest. As
a reference, the commercial PS surface showed a slightly lower level of cell attachment
than that of Col-Spin-PLLA.
Figure 4.6. The amount of cell attachment at 1-hour incubation after washing the various
substrates with PBS. NIH3T3 cells were detached from the culture dish and resuspended
in fresh medium containing 10% FBS. After 1 hour incubation, the substrates were
washed with PBS and cells were incubated for 5 hours in fresh medium containing MTT
(3-(4,5-dimethyl-2-thiazonyl)-2,5-diphenyl-
2
H tetrazolium bromide. 5 mg/ml). Then the
supernatants were decanted, and the formazan precipitates were solubilized by the
addition of 450 μl of 100% DMSO and placed on a plate shaker for 10 min. Absorbance
at 550 nm was determined on a microplate reader. Living cell number was proportional
to the absorbance of MTT at 550 nm.

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83
Figure 4.7. a) Image of fluo3-AM stained NIH3T3 cells deposited on the various
samples (scale bar: 50 μm). b) Cell spreading area on the various samples.

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4.2.
Analysis of cell spreading ratio with time
[Materials and Methods]
The attachment assay was carried out using the 24-well dish prepared with PLLA,
Col-Cast-PLLA, Col-Spin-PLLA or untreated 24-well dish (PS) as described above. 1.5
x 10
5
NIH3T3 cells in 500 μl of DMEM medium were re-plated and incubated under a
humidified atmosphere containing 5% CO2. Cells were viewed under a phase-contrast
microscope, and random fields were photographed after 30, 90, 180 and 270 min at a
total magnification of 200 x. We defined cells that had failed to spread as phase-bright
and rounded [18]. By contrast, spread cells were not phase-bright, exhibited extensive
membranous protrusions, and lacked a rounded morphology [18]. The numbers of
spread and rounded cells were counted in three fields in triplicate. The spreading cell
ratio was calculated and presented graphically. Furthermore, we studied the early stage
of cell attachment at 30, 90, 180, 270 min after seeding in order to analyze the rate of
cell adhesion (Figure 4.8.). Cells were photographed at the appropriate time, and the
numbers of spread and rounded cells were counted.
[Results and Discussion]
After 30 min, almost all cells showed a rounded shape on Col-Cast-PLLA,
whereas as many as 25% of cells had begun to spread on the PS and Col-Spin-PLLA.
After 90 min, >90% of cells still showed rounded morphology on Col-Cast-PLLA,
whereas > 60~70% of cells had begun to spread on Col-Spin-PLLA and PS. By 180 min,
most of the cells had spread and formed membranous protrusions on Col-Spin-PLLA
and PS, whereas approximately half of the cells still showed rounded morphology on
Col-Cast-PLLA.

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Figure 4.8. a) Spreading cell ratio on the various substrates. NIH3T3 cells were
detached from their culture dishes and re-plated in fresh medium containing 10% FBS.
The cells were then allowed to adhere and spread at 37
o
C for 30 min, 90 min, 180 min or
270 min, at which times they were photographed with the use of a phase-contrast
microscope and the numbers of spread and rounded cells were counted. The results are
representative of three separate experiments (scale bar: 100 μm). b) The spreading cell
ratio with time was calculated and is shown graphically.

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Indeed, even after 270 min approximately 40% of cells on Col-Cast-PLLA had
failed to spread. For PLLA, the cells neither attached nor spread throughout the
observation period and had aggregated with each other by 270 min. In the early stage of
cell attachment, the number of spreading cells showed the following order;
Col-Spin-PLLA≧PS>>Col-Cast-PLLA>PLLA. From the above data, the amount of
spreading cells is closely related to the collagen morphology. Specifically, the number
of spreading cells was considerably greater for Col-Spin-PLLA in comparison with that
for Col-Cast-PLLA. Thus, the spin-coating method generates a better cell adhesive
surface compared with PLLA or Col-Cast-PLLA.
4.3.
Immunofluorescence of the cells on actin filaments
[Materials and Methods]
The NIH3T3 cell line was incubated in DMEM containing 10% fetal bovine
serum and 1% trypsin-streptomycin for 4.5 hrs after the cells were seeded on the PS,
PLLA, Col-Cast-PLLA or Col-Spin-PLLA. The medium was removed from the dish
and the cells were washed with PBS. Cells were then fixed with 4% p-formaldehyde
(Wako Chemical Co.) for 1 or 2 hrs at room temperature. After quenching in
p-formaldehyde with 50 mM NH4Cl for 20 min, the dish was washed with PBS three
times for 10 min each. The cells were incubated in a 1% BSA PBS solution containing
1/200 Alexa Fluor
®
594 phalloidin for 45 min. After three washings with PBS, the cells
were stained with 1/1000 of the DAPI PBS solution (1 mg DAPI was dissolved in a 1
ml methanol solution 2 μl was put into the 2 ml of PBS solution) and the morphology of
the actin filaments was observed.

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87
[Results and Discussion]
In order to obtain information concerning focal adhesion, we observed
fluorescent-labeled actin filaments inside the cells to evaluate cell adhesiveness with
each surface (Figure 4.9.). There was no detectable fluorescence intensity in the case of
PLLA due to the lack of cell adhesion after the extensive washing step in the
immunostaining procedure. The elongation of actin filaments was barely observed for
the Col-Cast-PLLA sample. However, quite good elongation was observed on
Col-Spin-PLLA, indicating a reasonable biological affinity with the cells. Intriguingly,
PS as a control seemed to show poorer actin filament elongation than Col-Spin-PLLA.
Taken together, our results show that cell adhesive properties tend to be closely related
to collagen morphology. Indeed, our results are in good agreement with previous reports,
which showed that the cells expand on the films of native collagen with a lower density
of large fibrils [21,22]. These earlier papers suggest that collagen fibrils control
excessive proliferation of cells [21-25]. The differences in cell behavior between
Col-Cast-PLLA and Col-Spin-PLLA were reproduced in our findings described here.
Daniel et al. reported that the thermal or proteolytic denaturation of collagen I unwinded
the triple-helical structure to expose RGD-motifs [26,27,28,29]. RGD-motifs trigger the
binding of α5β1- and αv-integrins, which signals the initiation of cellular processes such
as adhesion, spreading, motility and differentiation. Our results from collagen
morphology and contact angle measurements show that non-fibril Col-Spin-PLLA is
more hydrophilic than fibril Col-Cast-PLLA. Our contact angle result of
Col-Spin-PLLA showed almost the same value as RGD-immobilized PLLA film [30].
Hence, the RGD moiety might be exposed in Col-Spin-PLLA. Therefore, the
Col-Spin-PLLA nanosheet appears to mimic the situation during wound repair [26].

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4.4.
Heterofunctionality of the Col-Spin-PLLA nanosheet
We used a PVA sacrificial film method for evaluating the heterofunctionality of
the Col-Spin-PLLA nanosheet. Initially, we spin-coated 2.0 wt% PVA at 4,000 rpm for
20 s and dried on a hotplate at 80
o
C and the Col-Spin-PLLA nanosheet was prepared on
a PVA sacrificed film. Then, the PVA layer was dissolved in distilled water and the
floating Col-Spin-PLLA membrane adhered to the Ecoli petri dish and dried in air for 3
hrs. To evaluate the heterofunctionality of the nanosheet, the edge of the nanosheet was
folded. Confluent NIH3T3 cells in 10-cm PS dish were detached and 25% of the
confluent cells were seeded on the nanosheet and cultured for 16 hrs. Finally, we
prepared a heterofunctional free-standing PLLA nanosheet with a spin-coated collagen
Figure 4.9. Actin filaments of the NIH3T3 cell line were stained using phalloidin 4.5
hours after seeding (x 40 image). The cell nucleus was stained with DAPI (scale bar: 50
μm).

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89
side and an uncoated side. The sacrificial film was used for the convenient collection
and manipulation of the nanosheets [3]. Next, we attached cells to the nanosheet with
the collagen side on top and the uncoated PLLA side exposed by folding the edge of the
nanosheet. There was no cell attachment on the folded area, while cells were confluent
on the collagen side after 16 hrs incubation (Figure 4.10.). The extensive formation of
actin filaments was confirmed inside the cells on the collagen side of the nanosheet.
Therefore, we were able to prepare a heterofunctional nanosheet that controls cell
adhesive properties. Moreover, cell attachment was confined specifically to one side
of the nanosheet.
Figure 4.10. Heterofunctionality of the Col-spin-PLLA nanosheet. The PLLA side (a),
and Col side (b) of the Col-Spin-PLLA, Upper side of (c); PLLA side of the folded
Col-spin-PLLA nanosheet, down side of (c); Col side of Col-spin-PLLA nanosheet on
the E-coli culture dish.

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5. Summary
In conclusion, we have established a technology, using a modified spin-coating
process, to homogeneously coat collagen onto a non-cell adhesive PLLA nanosheet,
thereby furnishing the surface with cell adhesive properties. The development of a
heterofunctional nanosheet will be extremely valuable for the generation of novel
biomaterials, such as post-surgical wound dressings. Specifically, these nanosheets have
cell adhesive properties on one side, for attachment to the wound, and a non-adhesive
surface on the other side, which acts as an adhesion barrier. Additional in vivo
experiments are currently being conducted to further evaluate these heterofunctional
nanosheets.

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91
References
1. Z. Tang et al., Nat. Mater., 2003, 2, 413.
2. R. Vendamme et al., Nature Mater., 2006, 5, 494.
3. T. Fujie et al., Adv. Mater., 2007, 19, 3549.
4. J. Matsui et al., J. Am. Chem. Soc., 2004, 126, 3708.
5. J. Zhang et al., Biomaterials, 2005, 26, 3353.
6. MA. Priolo et al., ACS Appl. Mater. Interface, 2010, 2, 312.
7. C. Jiang et al., Adv. Mater., 2004, 16, 157.
8. S. Markutsya et al., Adv. Funct. Mater., 2005, 15, 771.
9. T. Fujie et al., Adv. Funct. Mater., 2009, 19, 256.
10. Y. Okamura et al., Adv. Mater., 2009, 21, 4388.
11. Y. Hong et al., Biomaterials, 2005, 26, 6305.
12. PB van Wachem et al., Biomaterials, 1985, 6, 403-408.
13. H. Fernandes et al., J. Mater. Chem., 2009, 19, 5474.
14. T. Fujie et al., Soft Matter, 2010, 6, 4672.
15. M. Morra et al., J. Biomed. Mater. Res. A., 2006, 78A, 449.
16. X. Li et al., Appl. Surface Sci., 2008, 255, 459.
17. M. Jin et al., Invest. Ophthalmol. Vis. Sci., 2000, 41, 4324.
18. K. Inagaki et al., Oncogene, 2000, 19, 75-84.
19. ZH. Liang et al., Mater. Lett., 2009, 63, 927.
20. HE. Yang et al., Macromol. Res., 2007, 15, 256.
21. JT. Elliott et al., Matrix Biol., 2005, 24, 489.
22. JT. Elliott et al., Langmuir, 2003, 19, 1506.
23. GE. Davis et al., G. E., Biochem. Biophys. Res. Commun., 1992, 182, 1025.

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24. BK. Pilcher et al., Arch. Dermatol. Res., 1998, 290, S37.
25. S. Rhee et al., Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 5425.
26. AV. Taubenberger, Biomaterials, 2010, 31, 2827.
27. J. Heino et al., J., Bioessays, 2007, 29, 1001.
28. M. Yamamoto et al., Exp. Cell Res., 1995, 219, 249.
29. I. Aukhil et al., Periodontology 2000, 2000, 22, 44.
30. HJ. Jung et al., Macromol. Res., 2005, 13, 446.

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Chapter 5
Nanosheet as post-surgical adhesion barrier
1. Introduction
2. Anti-adhesion barrier
3. Construction of PLLA nanosheet
4. The application of the nanosheet as anti-adhesion barrier
5. Summary
References

Page 102
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94
1. Introduction
Postoperative adhesion formation is the most frequent complication of surgery,
although often not recognized as much [1]. With the incidence of 55%-100% in all
abdominal operations, adhesions are responsible for an increased risk of small bowl
obstruction, chronic abdominal pain, and infertility. In the case of surgery that is
accompanied by heavy hemorrhage, fibrin glue is generally used. However, this will
cause pleural adhesion. In a case of high-risk patients with respiratory failure, such a
severe pleural adhesion might further deteriorate pulmonary dysfunctions, a serious
complication for the compromised patients [2]. Hence, a novel tissue sealant that does
not cause tissue adhesion is required.
Biodegradable physical barriers have been successfully used to prevent
adhesion formation by mechanically limiting tissue apposition during the critical period
of mesothelial repair and healing. By minimizing the development of a fibrin matrix
between serosal tissue surfaces, such membranes may prevent adhesion formation [3].
In this chapter, we evaluated the efficacy of nanosheet as a postoperative
adhesion barrier in the liver defect model that was used in combination with
TachoComb which is quite famous as hemostatic material and accordingly followed by
strong tissue adhesion. At first, to understand the fundamental of adhesion, we described
about the anti-adhesion barrier. TachoComb is conventionally used in the operation
accompanied by heavy hemorrhage as described in Chapter 3. However, TachoComb
composed of fibrin and collagen that would be a scaffold of fibroblast will cause severe
postoperative adhesion. In general, it seems like difficult to use hemostat materials in
combination with anti-adhesion barrier. In one reason, it is caused by low physical
adhesiveness because of its thickness. The merit of nanosheet is

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95
nanometer-size-thickness literally and attach to any substrates, skins, organs, whatever
via physical adhesion as described until now [4].
Secondary, we described about the findings of nanosheet up to the present time.
We also introduced nanosheet in Chapter 1, but the more detail was described here.
Nanosheet made from chitosan and alginate acid was used for the lung defect model,
nanosheet made from poly-L-lactic acid was used for the stomach incision model. We
have never take notice on the anti-adhesion property, but have piled up findings on both
nanosheets as tendency of anti-adhesion barrier. Nanosheet has possibility to use
anti-adhesion barrier when using it with TachoComb too.
Thirdly, we described nanosheet property as an anti-adhesion barrier from in vitro
and in vivo experiment. In detail, we checked the anti-cell adhesion and anti-blood
adhesion property from in vitro experiment, and evaluated the adhesion score, observed
histological analysis and morphology from the scanning electron microscopy.
2. Anti-adhesion barrier
In injured state, the apparent imbalance between deposition of fibrin and its
destruction appeared and the balance shifted to the formation of fibrinous strands
eventually infiltrated by cells to create nascent adhesions [5].
Ideally, a barrier device should be easy to use via laparoscopic and open
procedures, provide unrestricted coverage of the affected peritoneum, and remain
effective throughout healing [6]. Barrier devices have been tested or commercialized in
various forms including polymer solutions, solid membranes, pre-formed or in situ
cross-linkable hydrogels. Among many devices, 5 materials were approved by Food and
Drug Administration of the United States (FDA) and are currently in market;

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96
regenerated cellulose (Interceed
®
), and expanded polytetrafluoroethylne (Preclude
®
),
both of which are largely used in gynecological surgery. Hyaluronic acid-carboxymethyl
cellulose (Seprafilm
®
) which is used for general and gynecological surgery in the U.S.
and Europe; Polylactide membrane (Surgiwrap
®
) and most recently, 4% icodextrin
solution (Adept
®
). Representative of barrier devices that have come as far as eliciting
corporate interest are summarized in Table 5.1. [5].

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97
Table 5.1. Barrier devices marketed under development.

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- 98 -
3. Construction of PLLA nanosheet
3.1. Manipulation of free-standing nanosheet
Polylactic acid (PLLA) nanosheet was prepared as described before. PLLA is
prepared in both sacrificing membrane and supporting membrane methods. In this
experiment, we used sacrificing membrane using with wire loop both in vitro and in
vivo to remove the influence of poly vinyl alcohol as possible as we could.
3.2. Application of nanosheet for the biomedical usage.
We described about the general wound dressing materials in Chapter 3. In
general, we used nanosheet as wound dressing materials. For example, LbL nanosheet
made from chitosan and alginate acid was tested in the lung defect model [2]. A
nanosheet of 75 nm thickness, ciritical load of 9.1 x 10
4
Nm
-1
, and an elastic modulus of
9.6 GPa is used for the minimally invasive repair of a visceral pleural defect in beagle
dogs without any pleural adhesion caused by wound repair.
Figure 5.1. LbL flm secured the repaired defect when pressurized by over 50 cm H2O
pressure at 3 h after repair. The region indicated by arrows shows the nanosheet-sealed
area.

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- 99 -
As described in Chpater 3, the usefulness of PLLA nanosheet has been indicated
in the same fashion from previous expreriment [7]. In this thesis, PLLA nanosheet was
mainly used as well as Chapter 2, 3 and 4.
4. The application of the nanosheet as an anti-adhesion barrier
In consideration with all the biomedical findings, we thought about the usage of
nanosheet as anti-adhesion barrier especially in liver defect model that is accompanied
with heavy hemorrhage. As described in Chapter 3, there have been no useful materials
that have both hemostat and anti-adhesion barrier functions. In this experiment, we
established a model in which we use TachoComb that was quite useful for the hemostat
and nanosheet that was likely to be useful for the postoperative adhesion barrier.
Moreover, since the collagen itself is known for its cell adhesion property that we
showed in Chapter 4, TachoComb is not likely to be a perfect anti-adhesion barrier.
Hence this method is totally meaningful if we prevent postoperative adhesion derived
from TachoComb with combination use with nanosheet.
Figure 5.2. Representative histological findings at different time points after repair
(H&E staining).
3 h
3 d
7 d

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- 100 -
Postoperative adhesion occurs after nearly all abdominal operations. They often
lead to serious complicated symptoms such as bowl obstruction, chronic abdominal pain,
pelvic pain, and infertility [1,8,9]. In injured state, balance between fibrin formation and
its destruction is lost to form the fibrinous strands and also forms nascent adhesion
under infiltration of fibroblast cells or inflammatory cells [5]. Hemostatic agent such as
fibrin glue was required for the surgery accompanied by heavy bleeding, however, it
often induce severe postoperative adhesion. In general, hemostatic agent such as
TachoComb or TachoSil has been used, which were composed of a sponge-like equine
collagen patch coated with a mixture of human fibrinogen that was explained in
Chapter 3. While it was proved to be effective for hemostat material, and indicated
minor postoperative adhesions, that cannot be shared by the outcome of the present
and there was no evidence of a considerable reduction of postoperative adhesions [6,
10]. On the other hand, many polymers such as poly(lactide) (PLA), polyvinyl alcohol)
(PVA), chitosan-based hydrogel and polyethylene glycol have been used for the
adhesion barrier [11-15]. The materials for adhesion barrier have to be anti-adhesive
without restraint of wound healing, efficient even in the presence of blood or other body
fluids, anti-inflammatory, biodegradable and idiotproof [9]. Seprafilm is commercially
available that is composed of hyaluronate sodium and carboxymethylated cellulose
(CMC). Polylactide resorbable films have been widely developed and evaluated these
anti-adhesion property in orthopedic, neurosurgical way [11,16-18]. However, some
experiment from them showed there was no benefit from using these films because of
these unsufficient bioadhesivity. In fact, it has to be sutured to the tissues. Furthermore,
there have been no reports on the effective modification on PLA for improving its
bioadhesivility. This unsufficient property is for one reason, derived from the thickness,

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because the polylactic acid film was known to be less adhesive to substrate when the
film was thicker. The thickness should be thinner for the improvement of bioadhesive
property.
On the other hand, as described in Chapter 4, the ultra-thin film has been an
object of interest for a long time for its unique mechanical property like huge aspect
ratio or high flexibility and high adhesiveness [4,19,20]. Starting from
Langmuir-Blodgett [21] or layer-by-layer (LbL) membrane composed of polycation and
polyanion [22], hybrid type of organic and inorganic compounds [23] and
organic/inorganic interpenetrating networks [20] to spin-coating LbL membrane [24,25],
many ultra-thin films were developed. We have developed LbL film (LbL nanosheet)
composed of chitosan/sodium alginate using spin-coating techniques for lung defect
model and it was suggested that novel therapeutic tools for overlapping tissue wounds
will be possible [2]. We showed the PLLA ultra-thin film also will be applicable for
gastric incision model [7]. These results showed that PLLA nanosheet could function as
a bio-interface for balancing conflicting phenomena involved in tissue repair and
resistance to tissue adhesion. These nanohseets had several tens of nanometer thickness
and likely have the extremely good bioadhesive property, and in fact, showed quite
brilliant property for application in wound dressing or tissue regeneration. However, we
never applied these nanosheets to liver defect model that is accompanied by the heavy
hemorrhage. In the case of liver defect model, the main reason of postoperative
adhesion seemed to be the blood components and cell infiltration from liver and
damaged peritoneum during operation (Figure 5.3.). It was easy to be imagined that
nanosheet should be short of mechanical intensity for the heavy hemorrhage and
required supporting membrane to prevent rinsing away from the surface of the wound

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peritoneum
liver
Blood outside the lesion site
Blood from liver ablation
liver
Peritoneum
with gushing blood.
In this report, we examined whether the PLLA nanosheet is functionalized as an
adhesion barrier in combination use with TachoComb that is quite useful as hemostat
materials and promote post-operative adhesion. We describe the application of a
free-standing PLLA nanosheet with an extremely high aspect ratio of greater than 10
6
and evaluate its property as an adhesion barrier in a liver defect model as it was used
with TachoComb that functions as hemostatic material and supporting membrane. We
found the potential of nanosheet as biointerfacial physical barrier by combined with
hemostat materials even when applying it for the organs such as liver that is
accompanied by heavy hemorrhage.
Figure 5.3. Rat liver ablation model and bleeding site.

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4.1. In vitro model of nanosheet ( cell adhesion and blood attachment assay)
4.1.1. The preparation of PLLA nanosheet
[Materials and Characterization]
The freestanding PLLA nanosheet was prepared by the following method (Figure
5.4.). It was totally the same as Figure 2.2.. At first, 10 mg mL
-1
was dissolved in
distilled water and PVA solution was spin-coated on SiO2 substrate and dried on a
hotplate at 80
o
C. Then a PLLA was dissolved in methylene chloride at a concentration
of 10 mg mL
-1
. A PLLA solution was dropped on the SiO2 substrate and spin-coated at
4,000 rpm for 20s, followed by drying at 80
o
C for 5 min. The resulting substrate was
immersed to distilled water and PVA was dissolved in water to obtain free-standing
PLLA nanosheets. In this case, a PVA film was used as a sacrificing membrane. The
thickness was analyzed by atomic force microscopy (AFM, KEYENCE, Tokyo, Japan)
and the thickness of the resulting nanosheet was around 60 nm. The macroscopic
morphology of the PLLA nanosheet was photographed with a digital camera RICOH
GX200 (RICHO Co., Ltd., Tokyo, Japan). The thickness of the nanosheet was adjusted
by the rotating speed and/or the concentration of the PLLA solution. We used 60
nm-thickness nanosheet because of its high adhesiveness. Its critical load is
significantly decreased over 100 nm-thickness and leads to low flexibility. Because of
its flexibility, the nanosheet was afforded to physically attach to any substrates, skins,
organs or whatever for using as interface as described before.

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Dropping of
1.0wt%PVA
Silicon wafer
Dipping
1.0wt%PLLA
solution
Suspending
in water
Adhesions form when sarosal surfaces are traumatized, resulting in an
inflammatory reaction with release of cytokines, growth factors, and angiogenic factors
from activated macrofages, neutrophils, and other inflammatory cells. The presence of
fibrin-rich exudates, coupled with a decrease in peritoneal fibrolytic capacity leads to
the development of a fibrin matrix, which provides a scaffold for the adherence and
proliferation of fibroblasts, mesothelial cells, and endothelial cells. As tissue remodeling
proceeds, cell differentiation and growth, extracellular matrix deposition, and
angiogenesis occur, resulting in the formation of dense, vascularized fibrous bands
[26,27]. Anti-adhesion barrier required the anti-blood attachment property and anti-cell
adhesion property and physical barrier property such as prevention of protein
permeability or cell infiltration.
4.1.2. In vitro Anti-PLLA blood attachment assay
[Materials and Methods, Results]
First of all, to elucidate the adequacy of PLLA nanosheet as the postoperative
Figure 5.4. Preparative scheme of PLLA nanosheet.

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TachoComb
TachoComb + PLLA
adhesion barrier, we conducted the anti-blood adhesion test on each sample. We
evaluated blood attachment property of PLLA nanosheet. We lapped 1cm x 1cm of
TachoComb with PLLA nanosheet that was made by sacrificing layer method. 100 μl of
blood each was dropped on TachoComb and PLLA covered TachoComb and incubated
at 37
o
C for 30 min and each sample was rinsed vigorously with 2ml of saline. While
the blood remained after washing vigorously with saline, the blood on TachoComb +
PLLA was almost washed away. This result showed the blood attachment was reduced
by PLLA nanosheet (Figure 5.5.).
[Results and Discussion]
It is known that the postoperative adhesion is caused by not only fibrin formation
by bleeding from wounded surface but also bleeding by incision of the peritoneum.
Even with an optimal surgical technique intending to minimize mesothelial injury,
peritoneal trauma is inevitable [9]. PLLA nanosheet is promising for the anti-blood
attachment materials from outside of TachoComb. Furthermore, we observed the
NIH3T3 cells on PLLA nanosheet in the former paper [28] and in Chapter 2. PLLA
nanosheet could not afford any cells to attach on the PLLA nanosheet while the cells
reach to almost confluent on the PS cell culture dish. It is commonly shared that PLLA
Figure 5.5. Anti-blood attachment assay on samples, a) TachoComb, b) TachoComb +
PLLA.

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is poor for its cytocompatibility, though PLLA is famous for its nontoxity, processibility
and biodegrability [29,30]. PLLA nanosheet is making good use of its character.
Cytocompatibility is one of the main reasons of post-operative adhesion because of cell
attachment is usually followed by cell migration and proliferation to form firm
undissectable adhesion. There was a former paper that concluded adhesions prevention
was mainly depend on the inhibition of fibroblast [31]. There was also a former paper
on cross-linked hyaluronic acid and in that report, lubricating surface of HA film
discourage significant cell attachment [32-34]. The roughness of PLLA nanosheet was
known to be very small and smooth, so it was also the main reason of
anti-cell-attachment property. Any cell attachment to an adhesion barrier material could
possibly encourage the barrier to be used as an additional junction point between injured
sites [33]. That is to say, PLLA nanosheet is expected to be effective for preventing
TachoComb from being scaffold for the cells from other organs such as liver and
omentum. This quite high anti-blood attachment property and anti-cell adhesiveness
would be promising for the anti-biointerfacial barrier separating from other site of
defect lesion.
4.1.3 In vitro transmembrane assay for evaluation of blood and protein
permeability
[Materials and Methods, Results]
We used a Transwell Membrane
®
(TM) with a pore size of 3 μm (Corning Co.,
Inc., Corning, NY). We removed the mesh and put on PLLA, TachoComb and PLLA +
TachoComb instead. The patching order of TachoComb and PLLA was followed to that
was used in animal experiment. The sample with hole by removal of mesh was also

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Saline
TachoComb
Nanosheet
a)
b)
TachoComb
TachoComb + PLLA
3500
3000
2500
2000
1500
1000
500
0
-500
-1000
Prote
in
con
c
entratio
n /
μ
g mL
-1
Time / min
0
50
100
150
200
prepared for control. Novo-Heparin 5,000 units / 5mL for Injection was purchased from
MOCHIDA PHARMACEUTICAL CO., LTD. (Tokyo, Japan). Blood was collected
from 12 wk old Male Slc:SD rat, and 100 μl of blood each was dropped on the samples.
The macroscopic blood permeability and protein assay was performed for evaluation of
the blood and protein permeation. Albumin, from Bovine Serum, Cohn Fraction V, pH
7.0 was purchased from Wako Chemical Co. (Tokyo, Japan) and used for protein assay.
Figure 5.6. Whole blood and protein permeability test using a transmembrane assay
a) schematic presentation of the transwell assay and b) the amount of permeated
protein after assay with time.

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[Results and Discussion]
We evaluated the barrier effect of PLLA, TachoComb and TachoComb+PLLA
against blood. The transmembrane coated with each sample was set in an in vivo
Transwell Membrane
(TM) kit [35]. To eliminate extra influence of mesh, we
removed the mesh (Figure 5.6.a.). In general, the time required for blood coagulation or
clotting is few minutes that are followed by fibrinolysis [36]. We evaluated the protein
or blood permeability in the early stage until 150 min, in which time was expected to be
enough for uncoagulated blood or fibrinogen to form an adhesion with the other organs
through clot. In TachoComb+PLLA, there was almost no blood or protein penetrated
while the proteins were confirmed to permeate through the TachoComb (Figure 5.6.b.).
In usual surgical operation followed by mild degree of hemorrhage like capillary
bleeding, 3~7 cm H2O of sealing pressure is required and 20-30 hPa is required for the
venous bleeding in liver and kidney [37]. It was known that the 60 nm thickness PLLA
nanosheet can stand up to at least 7 kPa from bulging experiment. This data showed that
the nanosheet can stand enough for the liver defect model. However, from the
macroscopic image, one of three of PLLA nanosheets was broken and red blood cells
dropped through the sheet to well, because of its low intensity owing to no supporting
membrane (Data not shown). In Tachocomb, the protein amount was considerably high
including proteins inside of the blood and from TachoComb. On the other hand, the
permeated protein in TachoComb+PLLA was considerably reduced, because of the
combination of PLLA’s barrier function and hemostat function and in addition, intensity
enhanced function of TachoComb as supporting substrate.

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4.2. In vivo experiment of the nanosheet in the liver defect model
4.2.1 Animal experiment
[Materials and Methods]
Based on these in vitro experiments above, we performed the animal experiment
to clarify the usages of PLLA nanosheet as wound dressing and anti-adhesion material.
All animal experiments were approved by the Animal Research Committee of Waseda
University. Male Slc:SD rat were studied. The rats were deeply anesthetized with
diethyl ether and 2 ml of 10 folds dilution of Somunopentyl with saline was
administered intramuscularly. Then their hair was shaved around the anterior abdominal
wall. After laparotomy, ablation of liver was performed. Immediately after ablation,
gushing blood was wiped off and each sample was pasted. A PLLA+TachoComb or
TachoComb (without PLLA nanohseet) was placed onto a liver ablation lesion (1.5 cm
diameter). PLLA nanosheet was prepared at the size of 4 cm x 4 cm. We used
sacrificing layer method for easy handling making good use of high flexibility, to
transfer it on the TachoComb that has large roughness. On the TachoComb + PLLA,
TachoComb was placed onto the ablation lesion and then PLLA nanosheet cut with
surgical scissor was covered on the TachoComb after scooped it with 3 cm x 3 cm
wireloop. It was easy to cover PLLA nanosheet on TachoComb, because of its flexibility
and high adhesiveness.An ablation of approximately 1.5 cm diameter was made in left
lobe of liver using wire loop and surgical knife. Immediately after ablation, gushing
blood was wiped off and stuck each samples. 4~6 animals each for TachoComb,
TachoComb+PLLA. For TachoComb and TachoComb+PLLA, their wounded surfaces
were held for a few minutes as prescribed in the manual. We also prepared untreated
sample with just laparotomy as a control. After sutured peritoneum, the surviving rats

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a)
b)
were monitored for 5 days. The livers were removed from the rats 5 days after surgical
intervention, fixed with 10% formaldehyde, and stained with hematoxylin-eosin (H&E)
or observed with SEM.
[Results and Discussion]
We used 8~9-week-old rats at the weight of around 250-300 g (Table 5.2.). The
weight of ablation lesion of liver was around 250 mg (Table 5.3.). Quite many blood
vessels ran inside of the liver such as central vein, sinusoids, portal veins and hepatic
artery [38], it sometimes seemed like floating sheet inside of the pool of blood. Since
PLLA nanosheet that has a possibility to be washed away by the gushing blood,
TachoComb is also likely to be quite useful for supporting substrate of nanosheet.
Figure 5.7. Protocol of nanosheet pasting on the rat liver.
Figure 5.8. a) Fluorescent labeled nanosheet on the rat liver, b) in the black light,
Pyrene-labeled-nanosheet on the left hand, EuTTA-labeled nanosheet on the rat
liver respectively.

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0
No adhesions
1
Firm adhesion with easily dissectable plane
2
Adhesion with dissectable plane causing tissue trauma
3
Fibrous adhesion with non-dissectable tissue planes
Score
Description
Untreated
Sample
TachoComb
TachoComb + PLLA
n
Weight / g
6
7
6
297.8 ± 21.4
305.9 ± 28.5
299.3 ± 36.9
Untreated
Sample
TachoComb
TachoComb + PLLA
n
Ablation weight / mg
6
7
6
267.7 ± 11.5
269.2 ± 36.8
275.1 ± 41.2
4.2.2. Adhesion score at postoperative 5 days
[Materials and Methods]
5 days after surgery, tissue adhesion prevention efficacy to adjacent tissues was
evaluated according to the following table (Table 5.4.).
The data are presented as mean values ±SD. Statistical analyses were performed
using Stat View 4.02J software package (Abacus Concepts, Berkeley, CA). Any other
statistical evaluations were compared using an unpaired t-test with *p<0.05 and **p <
0.01 set as the level of statistical significant.
[Results and Discussion]
From the evaluation of adhesion score at postoperative 5 days, we confirmed the
high scores in the TachoComb. This seemed to be caused by oozing blood through
Table 5.2. Weight of 8~9 week old rats using experiment.
Table 5.3. Ablation weight of livers.
Table 5.4. Criteria of adhesion score.

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Untreated
Sample
PLLA
TachoComb
TachoComb + PLLA
n
Score
5
4
6
6
2.8 ± 0.45
1.5 ± 0.58
2.3 ± 0.52
1.0 ± 0.63
a)
b)
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
Ad
h
e
sio
n
Sco
re
/
-
Untreated
TachoComb
TachoComb + PLLA
**
Untreated
TachoComb
TachoComb + PLLA
TachoComb and adhered blood during operating procedure. Actually we tried to
confirm its barrier function using bare PLLA nanosheet. As we expected during
operative procedure, PLLA might not have banked up the gushing blood and be washed
away. Because quite many blood vessels ran inside of the liver such as central vein,
sinusoids, portal veins and hepatic artery [38], it sometimes seemed like floating sheet
inside of the pool of blood.
Figure 5.9. a) The appearance of liver ablation lesion of each sample at postoperative
5 days. b) Comparison of adhesion score of each sample.
Table 5.5. Adhesion score at postoperative 5 days.

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As we checked the adhesion score of PLLA only, postoperative severe adhesion was
seen in two animals out of four (data not shown). Since PLLA nanosheet that has a
possibility to be washed away by the gushing blood, TachoComb is also likely to be
quite useful for supporting substrate of nanosheet. In fact, we confirmed reduction of
adhesion score in TachoComb+PLLA. This result means PLLA nanosheet might act as
an interfacial barrier between another organs and TachoComb or blood oozing from
inside of TachoComb or attached blood during operative procedure. The efficacy was
shown, using the combination of PLLA nanosheet as an anti-adhesion barrier and
TachoComb as hemostat material with a reinforced function as supporting substrate for
nanosheet.
4.2.3. Assessment of liver function
[Materials and Methods, Results]
For evaluating liver functions, Alanine transaminase (ALT) measurement was
done 5 days after surgery. Blood was collected from their heart into the 500 ml of
Capiject tube (TERUMO Medical Corp., NJ, USA). Blood samples were centrifuged at
3,500 G for 90 sec. for collecting only serum or plasma. ALT measurement was done
under the instruction of manual. The serum enzymes ALT is normally sensitive to
comprises in liver function and rise dramatically (10-100 fold) in hepatic injury and
disease [39]. ALT value at 5 days after operation was normal that was between 0~35
IU/L on all of four samples (Table 5.6.). From the ALT measurements, all of the liver
function were normal in not only TachoComb but also PLLA nanosheet indicating no
influence for the body to function normally and applicable as a biomaterial inside of the
body.

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Untreated
Sample
TachoComb
TachoComb + PLLA
n
ALT value
3
5
7
18.0 ± 6.44
18.1 ± 4.70
14.7 ± 3.87
4.2.4. Histological sectional analysis
[Materials and Methods, Results]
To support the visual evaluation of adhesion score, we analyzed the histological
section of each samples. At first, the removed liver sample was fixed with 10 %
formaldehyde for more than 4 hours with shaking. 10%, 15%, 20% PBS solution of
sucrose was prepared and liver was immersed in a stepwise fashion for more than 4
hours each. The organs were quickly chilled in the O.C.T. compounds (Sakura Finetek
Japan Co., Ltd., Tokyo) using liquid nitrogen. Frozen tissue were cut with 15 μm slice
and dried on prepared slide O/N and incubated in 20% PBS solution of formalin for 20
min at r.t. for refixation. After washing with distilled water, samples were immersed in
Mayer’s Hematoxilin solution (Wako Chemical Co., Tokyo) and incubated for 15 min.
After washing with miliQ, samples were immersed in 80% ethanol solution of 0.5 %
Eosin Y Ethanol Solution (Wako Chemical Co., Tokyo) and washed with miliQ, 70%,
80%, 90%, 100% ethanol solution in a stepwise fashion. The samples were immersed
through xylene solution several times, and encapsulate glycerol between cover glass and
prepared slide.
Table 5.6. Alanine transaminase (ALT value) of liver covered with each sample.

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H
F
T
H
F
T
O
[Results and Disucussion]
Histological sectional analysis from H&E staining supported the evaluation of
appearance and adhesion score (Figure 5.9.). It was taken for granted that the blood
drained from the affected area was the main reason of post-operative adhesion. On the
untreated sample, intestine was adhered to the liver on the obverse side through the
blood drained from affected area, and stomach was also adhered on the other side
through the blood running around. Severe symptom like bowl obstruction might be
occurred when this condition would be left untreated (Data not shown). On the
TachoComb sample that we couldn’t peel the left lobe off from the right lobe or
omentum because of heavy adhesion via fibroblast and the gigantic hematoma
confirmed between two livers. It was known that the temporary fibrin matrix persists
and gradually becomes more organized as collagen-secreting fibroblasts and leads to
adhesion formation under no fibrinolysis condition [40]. On the other hand,
PLLA+TachoComb had no adhesion and it was clear that the gushing blood never go
out from inside of lapping PLLA nanosheet. These results showed that the PLLA
nanosheet surely separated fibrin matrix and fibroblast from other organs or tissues in
Figure 5.10. Histological analysis of liver ablation lesion covered with each sample
at postoperative 5 days.
TachoComb
TachoComb+PLLA

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nanosheet
Fibroblast
Red blood cell
TachoComb
TachoComb + PLLA
TachoComb+PLLA, though the nanosheet could not afford to be seen because of
nanometer-thickness.
4.2.5. SEM (Scanning electron microscopy) measurement
[Materials and Methods, Results]
At first, the removed liver sample was fixed with 10 % formaldehyde for more than 4
hours with shaking. Then the samples were immersed in 70%, 80%, 90%, 100%
dehydrated ethanol in a stepwise fashion. After immersed in tertiary butyl alcohol /
ethanol = 1 / 1 solution, in totally tertiary butyl alcohol. Then the samples were
freeze-dried O/N, and were coated with an Au layer using an ion-sputtering coater
(HITACHI E-1045; 18 mA, 40 s., Hitachi Co. Tokyo, Japan). SEM observation of liver
sample was undertaken using HITACHI S-4300 instrument (Hitachi Co., Tokyo, Japan).
Figure 5.11. SEM image of liver ablation lesion covered with each sample at
postoperative 5 days.

Page 125
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[Results and Discussion]
SEM analysis was performed to check the existence of nanosheet and we
analyzed the morphology of wounded surface at postoperative 5 days (Figure 5.11.).
We observed TachoComb and TachoComb+PLLA on the liver. While we could not see
any cells and red blood cells on the surface of TachoComb+PLLA, we could see many
cells and blood cells on the TachoComb. The non existence of whole red blood cells and
fibroblast-like-cells on the surface of TachoComb+PLLA as opposed to TachoComb
showed that PLLA nanosheet has a property preventing cell infiltration and blood
leakage. These phenomena fit to in vitro experiment on cell attachment, whole blood
attachment and protein permeation experiment. Surprisingly, the nanosheet on the
TachoComb was likely to adhere tightly to and reflect the shape of cells or red blood
cells or TachoComb itself under the sheet. This result supported high flexibility and
adhesiveness of nanosheet. Furthermore, we could see many blood cells under the
breaking sheet by artificially, and that means PLLA nanosheet plays a key role as a
barrier and prevents the oozing blood from running out from inside of the TachoComb.
4.2.6. The degradation of PLLA
It is required that the degradation speed of PLLA nanosheet is slower than
TachoCombs’ for the nanosheet to be used with TachoComb, because cell lines tend to
attach easily with fibrin or collagen of TachoComb. Actually we evaluated the
morphology of nanosheet and TachoComb at postoperative 14 days. We could not see
any difference between the TachoComb only and TachoComb + PLLA on an adhesion
score.

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Untreated
TachoComb
TachoComb + PLLA
Hole by degradation
border
From the SEM analysis of the appearance of nanosheet, the tiny holes could be seen all
over the picture. From figure, many red white cells and fibrlblast-like cells appeared
from underneath the nanosheet. It was speculated that the adhesion occurred when the
tiny holes connected together as time passed and the clacks and gaps appeared, and the
TachoComb and cell lines appeared to bind the cell line from omentums or the other
organs.
Figure 5.12. The appearance of the liver at postoperative 14 days.
Figure 5.13. The appearance of nanosheet at postoperative 14 days.

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5. Summary
Consistent with our in vitro findings, the PLLA nanosheet drastically reduced the
penetration of blood or protein. Because there has no hemostat ability and no intensity
to prevent the gushing blood in PLLA nanosheet, Tachocomb is quite useful for not only
hemostat but also the supporting substrate. Furthermore, since cells could not afford to
adhere and blood was difficult to attach onto the PLLA nanosheet, it should be useful
for anti-adhesion barrier from other tissues or organs. In addition, because neither most
of the blood leakages translocate through the PLLA nanosheet nor fibroblast did
infiltrate through the PLLA nanosheet, the PLLA nanosheet acted as an efficient barrier.
However, the adhesion tended to exacerbate at postoperative 14 days and there seemed
no differentiation between TachoComb and TachoComb+PLLA (Data not shown). It
was supposed to be by the degradation of PLLA nanosheet. For immaculately proper
anti-adhesion materials, the delay of degradation speed might be needed at least rather
than TachoComb degradation. An entire absence of collagen or fibrin glue remnants was
observed after 3 month in coronary artery surgery in pig model [6]. The development of
biocompatible materials that is more difficult to be biodegradable than PLLA nanosheet
is imperative and now ongoing. The merit of nano-meter-thickness is its minimal
amount of materials and should be the most harmless for the animal bodies. This usage
of nanosheet should be quite promising for the next generational anti-adhesion barrier.

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Chapter 6
123
Chapter 6
Conclusions and Future prospects
1. Conclusions
2. Future prospects
References

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1. Conclusions
1. No cell adhesion was observed on the PLLA nanosheet.
2. It became clear that adsorption or attachment onto the organs comes from
physical adsorption, not from biological adhesion.
3. The Thr-PLLA nanosheet was washed away with heavy bleeding.
4. The thrombin loaded onto the Thr-PLLA was too low to be effective as a
hemostatic material.
5. We have established a technology, using a modified spin-coating process, to
homogeneously coat collagen onto a non-cell adhesive PLLA nanosheet, thereby
furnishing the surface with cell adhesive properties.
6. A Col-Spin-PLLA nanosheet has cell adhesive properties on one side, for
attachment to the wound, and a non-adhesive surface on the other side, which
acts as an adhesion barrier.
7. A high anti-blood attachment property and anti-cell adhesiveness were
confirmed on the PLLA nanosheet.
8. The permeated protein in TachoComb+PLLA was considerably reduced
compared to TachoComb, because of PLLA’s barrier function.
9. From animal experimentation of each sample, it was suggested that the PLLA
nanosheet remarkably reduced adhesion score.
10. From ALT measurement of livers at five days postoperative, it was shown that
the nanosheet was not harmful to the body.
11. SEM observation showed the PLLA nanosheet had a property preventing cell
filtration and blood leakage.
12. Severe adhesion was observed at 14 days postoperative and the degradation of

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the nanosheet was indicated.
2. Future prospects
(1) Hybrid model of hemostat and nanosheet
As described in Chapter 5, the PLLA nanosheet was indicated to function as an
interfacial physical barrier between TachoComb and the blood or other body fluids from
the outside of the wounded surface in the early stage of hemostasis. However, it was
clearly shown from SEM images that degradation of the nansoheet occurs. A PLLA
nanosheet is known to be degraded within one week under pancreatic conditions [1]. It
is imminent to assess whether the degradation speed is slower than the TachoComb
itself. TachoComb is supposed to be degraded in at least three months [2]. We have to
search for biodegradable or biocompatible materials that will retain barrier function for
at least three months. We have examined the biomaterials that are used for sutures,
stents, contact lenses or breast implants. However, they were often difficult for
maintaining nanosheet structure or rather promoted the wound adhesion; some
examples included polymethylmethacrylate (PMMA), polyvinylacetyl cellulose (PVAc),
polydimethylsiloxane (PDMS), and polyurethane. The information that we obtained to
present was that the degradation speed of the biodegradable polymer could be controlled
by molecular weight or nanosheet thickness. For instance, PMMA or polycaprolactone
(PCL) could not exist as a nanosheet at a small molecular weight, but could function as
needed at a high molecular weight. The more stable the sheet, the slower the
degradation speed. Future work involves assessment of a PCL nanosheet and
polydioxanone (PDO) nanosheet at some range of thickness. The molecular structures

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a)
b)
c)
* O CH
O C *
n
CH3
O
* C
(CH2)5 O
*
n
O
* O
(CH2)2 O CH2 C
O
*
n
were described in Figure 6.1. [3].
Once a nanosheet is developed with a slower degradation speed, it will be promising
for the construction of a functional hybrid of hemostat and nanosheet. I believe that
the final goal of this experiment is to fabricate the biomaterials that will be useful
for hemostatic activity and anti-adhesion barrier at the same time. Here I want to
describe one example that I have been developing.
A limitation exists with tissue engineering and wound healing on a 2D ultra
thin membrane, whereas the molecular cell biological niche space is highlighted
[4,5]. Furthermore, limitation of a loaded protein or low molecular weight
compound likely also exists. Therefore, when we think about the fabrication of
wound healing materials from the perspective of regenerative medicine, a 2D matrix
is not suitable. Therefore, we fabricated a dimensional extension nanosheet that was
a hybrid of a 2D and 3D matrix casting the fibrin gel on the collagen spin-coated
nanosheet. Then we focused on the gel-loaded nanosheet that was almost the same
composition as the fibrin gel known as TachoComb and developed the research.
Fibrin gel is widely used as a hemostat during operation and this material is known
for its usefulness in hemostasis ability. However, as described in Chapter 3 and 5,
inflammation can occur due to organ scraping and adhesion. The dimensionally
expanded nanosheet has adhesion barrier ability and can act as a scaffold for wound
cure and overcome the handling difficulty of the gel itself.
Figure 6.1. Molecular structures of biodegradable polymers. a) poly(lactic
acid) (PLA), b) poly (e-caprolactone) (PCL), c) polydioxanone (PDO).

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Glass
PLLA nanosheet
Gel sheet
A)
B)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
PLLA nanosheet
Glass
Gel sheet
A
B
S
/ -
C)
Here we described the effectiveness of this dimensionally expanded gel sheet.
Moreover, we tried to prepare a free-standing gel sheet that contained the cells
Figure 6.1. A) Lee-White experiment on the samples, B) Absorbance of
washed blood at 412 nm, C) SEM image of blood on the gel sheet.
Figure 6.2. A) 5 days after putting gel sheet on the wounded surface of the
liver. B) Histological sectional image of wounded surface at postoperative
5days.

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inside of the gel. After cells were cultured for three days, we checked the cells inside
of the gel growing in the matrix (Figure 6.3.).
The actin filament was stained by 594-alexa fluoro phalloidin and the nucleus was
stained with DAPI. This is likely the first example of this type of cell culture.
Recently, there was a report that cell differentiation could be controlled by the
rigidity of gel [6][7]. Another merit was the ability to contain a large amount of
protein. Gel rigidity is controlled by the amount of fibrinogen and degradation speed
depends on the amount of aprotinin. Because the gel is always under a wet
environment, for example, a liposome could be loaded (whereas it could not be
loaded on a nanosheet) and could lead to further medical development such as
anticancer drugs. In the near future, a dimensional expansion nanosheet that has
both adhesion barrier and hemostatic ability and also controls cell differentiation
will be possible and might lead to development of scaffolds for tissue engineering
such as bone regeneration that is followed by heavy bleeding after an operation.
(2) Drug containing nanosheet
We noticed that the PLLA nanosheets could contain some substances such as
antibiotic drugs, fluorescent dyes or hydroxyapatite using a controlled solvent and
concentration, even though they are not hydrophobic at all. We provided some
examples (Figure 6.4.). As we showed in Chapter 5, we could prepare a nanosheet
Figure 6.3. Gel sheet containing NIH3T3 murine fibroblast cell line.

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containing fluorescent dye. This result is shown again, and the blue is a nanosheet
containing pyrene and the red is a nanosheet containing EuTTA. Using this
technology, nanosheets could be labeled even inside the body. Moreover, we could
contain the metronidazole that is useful for trichomonad, periodontal disease and
Helicobacter pylori bacteria. We can see the homogeneous dispersion of
nanoparticles or crystals of metronidazole inside the nanosheet. This mechanism is a
sustained-release system following nanosheet degradation.
(3) Porous nanosheet
We could construct a nanosheet that has many pores inside of the nanosheet.
We controlled the pore numbers or sizes by adjusting the ratio of PEG and PLLA in
the solution. Thus, we also could control the permeation of proteins or particles and
it will lead to application as masks for virus or for artificial dialysis.
Figure 6.4. A. Flurescent dye containg nanosheet. a) Under the fluorescent
light, b) Under the black light. SEM image of metronidazole containing
nanosheet a) PLLA nanosheet, b) metronidazle containing nanosheet
a)
b)
a)
b)
A
B

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Chapter 6
130
120
100
80
60
40
20
0
Pe
rme
a
tio
n
p
e
rce
n
ta
g
e
/ %
0
10
20
30
40
time / hour
PLLA / PEG = 1 / 0
PLLA / PEG = 1 / 0.25
PLLA / PEG = 1 / 0.5
PLLA / PEG = 1 / 1
PLLA / PEG = 1 / 2
PLLA/PEG
= 1/0
PLLA/PEG
= 1/0.25
PLLA/PEG
= 1/0.5
PLLA/PEG
= 1/1
PLLA/PEG
= 1/2
A
B
Figure 6.5. A. Surface morphology of porous nanosheet. B. Albumin
permeation of each nanoseheet.

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References
1. Y. Okamura et al., Adv. Mater. 2009, 21, 4388.
2. M. A. Erb et al., Albes, Eur J Cardiothorac Surg. 2009, 36, 703.
3. SJ. Holland et al., J. Control. release, 1986, 4, 155.
4. M. Schindler et al., Cell Biochem. Biophys. 2007, 45, 215.
5. R. Langer. Adv. Mater. 2009, 21, 3235.
6. A. J.Engler, et al., Cell. 2006. 126. 677.
7. W. P. Daley et al., J.Cell. Sci. 2007. 121. 255.

Page 140
Academic achievement
(List of Publication)
(1) D. Niwa, T. Fujie, T. Lang, N. Goda, S. Takeoka, “Heterofunctional nanosheet
controlling cell adhesion properties by collagen coating”, J. Biomater. Appl., (in
press).
(2) T. Fujie, S. Furutate, D. Niwa, S. Takeoka, “A nano-fibrous assembly of
collagen-hyaluronic acid for controlling cell-adhesive properties”, Soft Matter, 6,
4672-4676 (2010).
(International Symposium)
(1) D. Niwa, T. Fujie, T. Lang, N. Goda, S. Takeoka. “Analysis of the interaction
between cells and the different types of nanosheets”, American Chemical Society
239th National Meeting & Exposition, San Francisco, 2010. 4.
(2) D. Niwa, N. Goda, T. Lang, S. Takeoka. “The analysisi of „nano-adhesive plaster‟ in
the molecular biological way”, The 3rd Global COE International Symposium on
„Practical Chemical Wisdom‟, Tokyo, 2009. 1.
(3) D. Niwa, N. Goda, T. Lang, S. Takeoka.“The analysis of the interaction between
Cells and Hetrofunctional Nanosheets”, The 4th Global COE International
Symposium on „Practical Chemical Wisdom‟, Tokyo, 2010. 1.
(4) D. Niwa, M. Koide, T. Fujie, Y. Okamura, N. Goda, S. Takeoka. “Application of
nanosheet as an anti-adhesion barrier in liver defect model.”, The 2nd NIMS
(MANA)-Waseda Interventional Symposium, Tsukuba, 2010. 12.
(Domestic Symposium)
(1) 丹羽大輔, 藤枝俊宣, 合田亘人, 武岡真司. 「物性の異なる高分子超薄膜の構築と細胞
との相互作用解析」日本化学会 第 3 回関東支部大会(2009 年 9 月, 東京)
(2) 古舘祥, 藤枝俊宣, 丹羽大輔, 武岡真司. 「細胞接着性表面を有する自己支持性コラーゲ
ン/ヒアルロン酸ナノシートの構築」日本化学会 第 3 回関東支部大会(2009 年 9 月, 東
京)
(3) 古舘祥, 藤枝俊宣, 丹羽大輔, 武岡真司. 「細胞接着性コラーゲン/ヒアルロン酸ナノシ
ートの構築」 第 31 回日本バイオマテリアル学会大会 (2009 年 11 月, 京都)

Page 141
Acknowledgements
The presented thesis is the collection of the author’s studies, which have been carried out
under the direction of Prof. Dr. Shinji Takeoka at the Department of Life Science and Medical
bioscience, Waseda University during 2008-2011. The author expresses the greatest
acknowledgement to Prof. Dr. Shinji Takeoka for his valuable suggestions, helpful discussions, and
continuous encouragement throughout this work. The author also expresses his sincere gratitude to
Prof. Dr. Yasuo Ikeda, Prof. Dr. Nobuhito Goda and Prof. Dr. Thorsten Lang for the efforts as
members of the judging committee for the doctorial thesis.
The author would like to express special thanks to the members of University of Bonn. In
particular, grateful acknowledgement is made to Prof. Dr. Thorsten Lang, Mr. David Walrafan and
Mr. Luis Spitta for their valuable suggestions.
The author gratefully acknowledges to Prof. Dr Nobuhito Goda for his helpful and valuable
suggestions, remarks and discussions, and also thanks to Assoc. Prof. Dr. Nanami Matsuda, Dr. Mai
Kanai, Miss, Miss. Tsutsumi Kanako and other members for their encouragement.
The author expresses acknowledgement to his mentor from bachelor to doctorial course, Dr.
Yosuke Okamura and Dr.Toshinori Fujie for the discussions with full passion on his research. The
author also @expresses his acknowledgement to Dr. Keitaro Sou, Dr. Satoshi Arai, Dr. Yosuke Obata,
Mr. Atsushi Murata and the other members of Takeoka Lab.
The author expresses the remarkable thanks to the member of Team Nanosheets, Dr. Toshinori
Fujie, Mr. Zhang Hong, Mr. Akihiro Saito, Mr. Hiroki Hanyuda, Mr. Masatsugu Koide and Miss.
Yuko Kawamoto.
All members in Laboratory of Biomolecuar Assembly and Laboratory of Biochemistry offered
kind assistance for which the author would like to thank deeply. The author acknowledges to Global
COE Programme, MEXT.
Finally, the author expresses his deepest gratitude to his family, Mr. Masami Niwa and Mrs.
Mineko Niwa for their affectionate contributions.
January, 2011
Daisuke Niwa