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

Promoter: Prof. Dr. Shinji Takeoka

Referees: Prof. Dr. Yasuo Ikeda

Prof. Dr. Nobuhito Goda

Prof. Dr. Thorsten Lang

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

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

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

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

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

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.

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.

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

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.

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.

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

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.

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].

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].

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

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].

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

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

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

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

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

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

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

Chapter 1

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].

Chapter 1

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].

Chapter 1

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).

Chapter 1

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].

Chapter 1

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..

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

Chapter 1

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

Chapter 1

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

Chapter 1

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].

Chapter 1

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].

Chapter 1

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

Chapter 1

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].

Chapter 1

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)

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].

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)

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].

Chapter 1

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

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].

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.

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.

Chapter 1

40

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Chapter 1

41

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Chapter 1

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Chapter 2

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

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.

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.

Chapter 2

46

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

Chapter 2

47

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)

Chapter 2

48

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].

Chapter 2

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.

Chapter 2

50

Chapter 3

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

Chapter 3

52

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].

Chapter 3

53

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.

Chapter 3

54

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

Chapter 3

55

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.

Chapter 3

56

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)

Chapter 3

57

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.)

Chapter 3

58

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.

Chapter 3

59

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.

Chapter 3

60

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.

Chapter 3

61

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

Chapter 3

62

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)

Chapter 3

63

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.

Chapter 3

64

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)

Chapter 3

65

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).

Chapter 3

66

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

(%

)

Chapter 3

67

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.

Chapter 3

68

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)

Chapter 3

69

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.

Chapter 3

70

Chapter 4

71

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

Chapter 4

72

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

Chapter 4

73

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..

Chapter 4

74

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

Chapter 4

75

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

Chapter 4

76

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.

Chapter 4

77

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.

Chapter 4

78

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.

Chapter 4

79

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].

Chapter 4

80

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).

Chapter 4

81

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).

Chapter 4

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.

Chapter 4

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.

Chapter 4

84

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.

Chapter 4

85

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.

Chapter 4

86

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.

Chapter 4

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].

Chapter 4

88

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).

Chapter 4

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.

Chapter 4

90

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.

Chapter 4

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.

Chapter 4

92

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.

Chapter 5

93

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

Chapter 5

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

Chapter 5

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;

Chapter 5

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].

Chapter 5

97

Table 5.1. Barrier devices marketed under development.

Chapter 5

- 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.

Chapter 5

- 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

Chapter 5

- 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,

Chapter 5

- 101 -

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

Chapter 5

- 102 -

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.

Chapter 5

- 103 -

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.

Chapter 5

- 104 -

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.

Chapter 5

- 105 -

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.

Chapter 5

- 106 -

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

Chapter 5

- 107 -

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.

Chapter 5

- 108 -

[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.

Chapter 5

- 109 -

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

Chapter 5

- 110 -

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.

Chapter 5

- 111 -

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.

Chapter 5

- 112 -

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.

Chapter 5

- 113 -

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.

Chapter 5

- 114 -

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.

Chapter 5

- 115 -

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

Chapter 5

- 116 -

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.

Chapter 5

- 117 -

[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.

Chapter 5

- 118 -

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.

Chapter 5

- 119 -

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.

Chapter 5

- 120 -

References

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2. T. Fujie et al., Adv. Funct. Mater., 2009, 19, 2560.

3. E. Ersoy et al., J. Surg. Res., 2008, 147, 148.

4. T. Fujie et al., Adv. Mater., 2007, 19, 3549.

5. Y. Yeo et al., Eur. J. Pharm. Biopharm. 2008, 68, 57.

6. M. A. Erb et al., Albes, Eur J Cardiothorac Surg. 2009, 36, 703.

7. Y. Okamura et al., Adv. Mater. 2009, 21, 4388.

8. E. Ersoy et al., J. Gastrointest. Surg. 2009, 13, 282.

9. K. Kramer et al, Arch. Surg. 2002, 137, 278.

10. A. Rickenbacher et al., Expert. Opin. Biol. Ther. 2009, 9, 897.

11. Y. Fukuhira et al., J Biomed Mater Res B Appl Biomater 2008, 86B, 353.

12. C. Weis et al., J Biomed Mater Res B Appl Biomaterials 2004, 70, 191.

13. YH. Lee et al., J Biomed Mater Res. 2003, 64, 88.

14. N. Okuyama et al., J Surg Res. 1998, 78, 118.

15. C. Wei et al., Biomaterials 2009, 30, 5534.

16. S. Avital et al., Dis. Col. Rec. 2005, 48, 153.

17. C. Schug-Paβ et al., Surg. Endosc. 2008, 22. 2433.

18. W. C. Welch et al., J. Neurosurg. (Spine 4) 2002, 97, 413.

19. Z. Tang et al., Nature Mater., 2003, 2, 413.

20. R. Vendamme et al., Nature Mater. 2006, 5, 494.

21. J. Matsui et al., J. Am. Chem. Soc. 2004, 126, 3708.

22. J. Zhang et al., Biomaterials 2005, 26, 3353.

23. M. A. Priolo et al., ACS Appl. Mater. Interface 2010, 2, 312.

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24. C. Jiang et al., Adv. Mater. 2004, 16, 157.

25. S. Markutsya et al., Adv. Funct. Mater. 2005, 15, 771.

26. J. D. Cashman et al., J Surg. Res. 2010, 1.

27. G. S. diZerega et al., Hum Reprod. Update 2001, 7, 547.

28. D. Niwa et al., J. Biomater. Appl., 2011, in press.

29. Y. Hong et al., Biomaterials 2005, 26, 6305.

30. P.B. van Wachem et al., Biomaterials 1985, 6, 403.

31. C. Z. Wei et al., Biomaterials 2009, 30, 5534.

32. C. A. Falabella et al., Dis. Surg. 2009, 26. 476.

33. X. Z. Shu et al., Biomaterials 2003, 24, 3825.

34. F. Boradldi et al., Tissue & Cell 2003, 35 37.

35. T. Fujie et al., Surgery 2010, 148. 48.

36. G. E. Folk et al., Biol. Rhythm Res. 2010, 41, 1.

37. R.T. Carbon et al., Pharma Medica, 2005, 23, 134.

38. E. Bhunchet et al., Hepatology 1998, 27, 481.

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Chapter 5

- 122 -

Chapter 6

123

Chapter 6

Conclusions and Future prospects

1. Conclusions

2. Future prospects

References

Chapter 6

124

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

Chapter 6

125

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

Chapter 6

126

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).

Chapter 6

127

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.

Chapter 6

128

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.

Chapter 6

129

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

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.

Chapter 6

131

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.

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)

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