Wound healing activity of

bioactive compound and

application of composite scaffold

Hye – Lee Kim

Department of Medical Science

The Graduate School, Yonsei University

Wound healing activity of

bioactive compound and

application of composite scaffold

Directed by Professor Jong – Chul Park

The Doctoral Dissertation submitted to

the Department of Medicine Science,

the Graduate School of Yonsei University

in partial fulfillment of the requirements for the degree

of Doctor of Philosophy

Hye – Lee Kim

December 2012

This certifies that the Doctoral

Dissertation of Hye – Lee Kim is

approved.

------------------------------------------------

Thesis Supervisor : Jong–Chul Park

------------------------------------------------ Thesis Committee Member#1 : Jeong Koo Kim

------------------------------------------------

Thesis Committee Member#2 : Dong Kyun Rah

------------------------------------------------ Thesis Committee Member#3 : Kwang–Hoon Lee

-----------------------------------------------

Thesis Committee Member#4 : Kyung Sik Kim

The Graduate School

Yonsei University

December 2012

ACKNOWLEDGEMENTS

새로운 분야에 대한 도전으로 모든 일들이 두려움으로 다가왔었지만,

그 두려움이 설렘이 되면서 어느새 박사과정도 마무리되고 있습니다. 지금

까지 응원해주신 모든 분들께 감사드립니다.

처음 많이 낯설어하던 저에게 박사과정의 앞길을 밝혀주시고 학문과

인생에 대해 많은 가르침을 주신 박종철 교수님께 깊이 감사드립니다. 바

쁘신 중에도 미흡한 제 논문이 발전할 수 있도록 항상 관심 가져주시고 훌

륭하신 조언을 아낌없이 해주신 성형외과 나동균 교수님과 피부과 이광훈

교수님, 인제대학교 김정구 교수님, 외과 김경식 교수님께 진심으로 감사드

립니다. 그리고 동물실험에 대해 아낌없는 조언을 해주신 건국대학교 도선

희 교수님께도 감사드립니다.

박사과정 동안 함께해주신 박봉주 박사님과 한동욱 박사님, 김학희 박

사님, 김정성 박사님, 한인호 박사님, 백현숙 언니, 임혜련 언니, 이미희 박

사님께 앞으로 바라시는 바 모두 이루시길 기원하며 감사드립니다. 그리고

우연이, 이대형, 진수창, 서혁진, 강재경, 김민성, Barbora Vagaska, 박동정,

권병주, 이정현, 조혜진에게도 감사드리며, 앞날에 행운이 가득하길 빕니다.

세종대에서의 인연으로 지속적인 관심과 도움을 주신 권언혜 언니에게도

감사합니다.

언제나 아낌없는 가르침과 많은 사랑을 주시는 세종대학교 이원준 교

수님께 감사드립니다. 항상 격려해주시는 세종대학교 김대준 교수님께도

감사드립니다. 졸업 후에도 끊임없이 관심을 가져주시고 격려해주신 나노

신소재공학과 모든 교수님께도 감사드립니다.

새로운 가족이 되어 저를 응원해주시는 시부모님께도 진심으로 감사의

인사를 드립니다. 이제는 나의 인생의 동반자가 된 한별에게도 감사합니다.

마지막으로 지금까지 긴 시간 동안 응원해주시고 후원해주신 사랑하는 부

모님과 동생에게 진심으로 고마움을 전하며, 이 논문을 바칩니다.

2013년 01월

김혜리 드림

TABLE OF CONTENTS

ABSTRATE ·································································· 1

I. INTRODUCTION ····················································· 4

1. Wound healing processes ··········································· 4

2. Skin substitute ························································ 6

3. Biodegradable polymer ············································· 9

4. Natural bioactive compound ······································· 9

5. Objectives of this study ············································· 10

6. References ···························································· 11

II. FABRICATION OF POLY (LACTIC-CO-GLYCOLIC ACID)

(PLGA) SCAFFOLDS ················································ 13

1. Introduction ························································· 13

2. Materials and methods ············································ 15

A. Materials ··························································· 15

B. Electrospinning setup ············································ 16

C. Measurement of surface morphology on fibrous PLGA

scaffolds ··························································· 18

D. Measurement of pore size on fibrous PLGA scaffolds ····· 18

E. Measurement of cell infiltration ································ 20

F. Sterilization methods ············································ 21

G. Sterility test ······················································· 23

H. Statistical analysis ················································ 23

3. Results ································································ 25

A. Characterization of electrospun PLGA scaffolds ············ 25

B. Influence of dry ice on pore size ································ 29

C. Influence of the pore sizes on the cell infiltration ············ 29

D. Sterilization effects on electrospun PLGA scaffolds ········· 32

4. Discussion ····························································· 37

5. Conclusion ···························································· 39

6. References ···························································· 40

III. EVALUATION OF ELECTROSPUN (1,3),(1,6)-β-D-GLUCANS

(BGs)/PLGA SCAFFOLDS ·········································· 43

1. Introduction ·························································· 43

2. Materials and methods ············································· 44

A. Evaluation of wound healing effects of BGs using the skin-

derived cells ······················································· 44

B. Fabrication of electrospun BGs/PLGA scaffolds ············· 45

C. Characterization of electrospun BGs/PLGA scaffolds ······ 45

D. Viability and proliferation of HDFs on electrospun BGs/PLGA

scaffolds ···························································· 46

E. Animals and surgical manipulation ···························· 47

F. Determination of wound area ··································· 50

G. Immunohistochemical analysis of wound area ··············· 50

H. Statistical analysis ················································ 51

3. Results ································································· 52

A. Evaluation of wound healing effects of BGs on HDFs and

NHEKs ····························································· 52

B. Characterization of electrospun BGs/PLGA scaffolds ······· 55

C. BGs release pattern from BGs/PLGA scaffolds ·············· 57

D. Viability and proliferation of HDFs on electrospun BGs/PLGA

scaffold ····························································· 60

E. Wound healing and histological examination ················· 63

F. Immunohistochemical analysis of wound healing ············ 67

4. Discussion ····························································· 69

5. Conclusion ···························································· 72

6. References ···························································· 73

IV. EVALUATION OF ELECTROSPUN EPICALLOCATECHIN

-3-O-GALLATE (EGCG) /PLGA SCAFFOLDS ················ 76

1. Introduction ·························································· 76

2. Materials and methods ············································· 77

A. Viability of the skin-derived cells on EGCG ·················· 77

B. Electrospun EGCG/PLGA scaffolds ··························· 77

C. Characterization of electrospun EGCG/PLGA scaffolds ···· 77

D. Viability of HDFs on electrospun EGCG/PLGA scaffolds ·· 77

E. Animals and surgical manipulation ····························· 78

F. Determination and immunohistochemical analysis of wound

area ·································································· 78

G. Statistical analysis ················································· 78

3. Results ································································· 80

A. Viability of the skin-derived cells on EGCG ·················· 80

B. Characterization of electrospun EGCG/PLGA scaffolds ··· 82

C. Viability and proliferation of HDFs on electrospun EGCG/

PLGA scaffolds ··················································· 85

D. Wound healing and histological examination ················· 87

E. Immunohistochemical analysis of wound healing ············ 87

4. Discussion ····························································· 92

5. Conclusion ····························································· 93

6. References ····························································· 94

ABSTRACT(IN KOREAN) ················································ 96

LIST OF FIGURES

Fig. 2-1. The schematic of electrospinning system is used in this

study (A). The surfaces are shown in the collector

deposited ice crystals and polymer fibers (B). ··········· 17

Fig. 2-2. Images of surface morphologies for the measurement of

pore areas (magnification of 1000). Surface morphology

of the scaffolds was observed by a SEM (A), and

controlled of the contrast of each grayscale image (B).

The controlled image was assigned a threshold value (C).

············································································ 19

Fig. 2-3. The electrolysis vessel used in this study. (A), Schematic

of electrolysis vessel; (B), Experimental electrolysis

vessel for treatment with direct current. ···················· 22

Fig. 2-4. Digital photograph of the electrospun PLGA inoculated

with Escherichia coli (A - C) and Staphylococcus aureus

(D - F) after incubation on standard agar at 36 oC for 7

days. (A, D), Untreated electrospun PLGA; (C, E),

electrospun PLGA treated with 6 V for 10 sec; and (D, F),

electrospun PLGA treated with 6 V for 50 sec (n = 10,

scale bar: 30 mm). ················································· 24

Fig. 2-5. Fibers of the electrospun PLGA were detected with a

SEM. Fibers of the electrospun PLGA were deposited

with various parameters such as the PLA/PGA ratio of

PLGA (A), the kind of solvent (B) and the PLGA

concentration of polymer solution (C). ······················ 26

Fig. 2-6. Fiber morphology of the electrospun PLGA scaffolds

was measured by a SEM. The electrospun fibers were

deposited using HFIP (A) and THF/DMF (B). ··········· 27

Fig. 2-7. SEM images of cross-section of the scaffolds. The

scaffolds were fabricated with HFIP (A) and THF/DMF

(B). ····································································· 28

Fig. 2-8. Images of surface morphology (magnification of 1000)

were assigned a threshold value (A), and the pore areas

of the electrospun scaffolds were calculated with the

images (B). The scaffolds were fabricated using HFIP

without dry ice (a) and with dry ice (b). The scaffolds

were electrospun using THF/DMF without dry ice (c)

and with dry ice (d) (*p < 0.05 versus the pore size

fabricated without dry ice, as analyzed by a t-test).

············································································ 30

Fig. 2-9. Infiltrated cells into the scaffolds were fixed and stained

with Pi (red). Cells (red) and scaffolds (white) were

observed with a digital fluorescence microscope (A)

(scale bar: 500 µm). The calculated the cells distances

from the surfaces using image J (B). ························· 31

Fig. 2-10. SEM images of Escherichia coli (A, B) and

Staphylococcus aureus (C, D) in the electrospun PLGA.

(A, C), untreated; (B, D), treated with 6 V for 60 sec.

··········································································· 34

Fig. 2-11. Shape and surface morphology of the electrospun PLGA.

The images were observed by (A) a digital photograph

(scale bar: 1mm) and (B) a SEM. ···························· 35

Fig. 2-12. The number average and the weight average of molecular

weight (Mw and Mn) of the electrospun PLGA (n = 5).

············································································ 36

Fig. 3-1. Surgical procedure of the full-thickness wound model in

nude mice (scale bar: 12 mm). ································· 48

Fig. 3-2. Growth rate effects of BGs on HDFs (A) and NHEKs (B)

(*p < 0.05 versus the control group (0 mg/mL), as

analyzed by a t-test). ·············································· 53

Fig. 3-3. Migration effects of BGs on HDFs (A) and NHEKs (B)

(scale bar: 100 µm). ··············································· 54

Fig. 3-4. SEM images of the PLGA scaffolds and the BGs/PLGA

scaffolds. The collector surface images were detected by

a SEM after electrospinning of BGs without PLGA.

············································································ 56

Fig. 3-5. Cumulative release of BGs from the electrospun

scaffolds containing different amounts of BGs into PBS

at 37 oC during 16 days. The BGs concentration in PBS

at each time point was calculated with a BGSTAR kit.

············································································ 58

Fig. 3-6. SEM images of (A, C) the PLGA scaffolds and (B, D)

the 50BGs/PLGA scaffolds incubated for 7 days at 37 oC.

············································································ 59

Fig. 3-7. Influence of HDFs proliferation on the scaffolds

containing different amounts of BGs. After incubation

for 1, 3, 5 and 7 days, the cell viability was detected by

an ATP assay. (*p < 0.05 versus the PLGA scaffolds at

the same time, as analyzed by a t-test with n = 5)

············································································ 61

Fig. 3-8. SEM images of HDFs incubated for 5 days at 37 oC on

(A, C) the PLGA scaffold and (B, D) the 50BGs/PLGA

scaffold. ······························································· 62

Fig. 3-9. Appearance of the wounds in mice at 0, 1, 2 and 4 weeks

after treatment with the PLGA scaffolds and the

50BGs/PLGA scaffolds. (scale bar : 6 mm) ·············· 64

Fig. 3-10. Histological morphology with hematoxylin and eosin of

wound sites treated with the PLGA scaffolds and the

50BGs/PLGA scaffolds. (S: scaffold, Orange line:

Normal epidermal, Yellow line: Re-epithelialization,

scale bar: 2 mm) ··················································· 65

Fig. 3-11. Interaction with the surrounding cells and the elect-

rospun scaffolds. The wound sites were treated with he-

matoxylin and eosin. (Scale bar: 200 µm) ·················· 66

Fig. 3-12. Immunohistochemical observation of wounds treated

with the PLGA scaffolds and the 50BGs/PLGA scaffolds,

and stained with anti Ki-67 and anti CD 31. (A),

Microscopic observation of immunehistochemical results

(S: scaffold, scale bar: 200 µm); (B), quantitative

analyses of cells showing immunopositivity for Ki-67 or

CD 31 (*p < 0.05, as analyzed by one-way followed by

the Tukey HSD test). ·············································· 69

Fig. 4-1. Cell viability effects of EGCG on (A) HDFs and (B)

NHEKs (*p < 0.05 versus the control group (0 µM), as

analyzed by t-test). ················································· 81

Fig. 4-2. SEM images of the PLGA scaffolds and the

EGCG/PLGA scaffolds. ········································ 83

Fig. 4-3. Cumulative release of EGCG from the electrospun

scaffolds containing different amounts of EGCG into

PBS at 37 oC. ························································ 84

Fig. 4-4. SEM images of HDFs incubated for 5 days at 37 oC on

the PLGA scaffold (A), the 1EGCG/PLGA scaffold (B)

and the 5EGCG/PLGA scaffold (C). ························ 86

Fig. 4-5. Appearance of the wounds in mice at 0, 1, 2 and 4 weeks

after treatment with the PLGA scaffolds and the 1EGCG/

PLGA scaffolds. (scale bar: 6 mm) ·························· 88

Fig. 4-6. Histological morphology with hematoxylin and eosin of

wound sites treated with the PLGA scaffolds and the

1EGCG/PLGA scaffolds. (S: scaffold, Orange line:

Normal epidermal, Yellow line: Re-epithelialization,

scale bar: 2 mm) ··················································· 89

Fig. 4-7. Interaction with the surrounding cells and the electrospun

scaffolds. The wound sites were treated with

hematoxylin and eosin. (Scale bar: 200 µm) ·············· 90

Fig. 4-8. Immunohistochemical observation of wounds treated

with PLGA scaffolds and 1EGCG/PLGA scaffolds, and

stained with anti Ki-67 and anti CD 31. (A), Microscopic

observation of immunehistochemical results (S: scaffold,

scale bar: 200 µm); (B), quantitative analyses of cells

showing im munopositivity for Ki-67 or CD 31 (*p <

0.05, as analyzed by one-way followed by the Tukey

HSD test). ····························································· 91

LIST OF TABLES

Table 1-1. Kinds of commercially created skin substitute. ·········· 8

Table 2-1. Sterilization effects of DC on Escherichia coli (E. coli)

and Staphylococcus aureus (S. aureus) in the electrospun

PLGA scaffolds. (n = 10) ········································ 33

Table 3-1. The experimental numbers of nude mice for the animal

study. ··································································· 50

Table 4-1. The experimental numbers of nude mice for the animal

study. ··································································· 79

- 1 -

<ABSTRACT>

Wound healing activity of bioactive compound and

application of composite scaffold

Hye – Lee Kim

Department of Medical Science

The Graduate School, Yonsei University

(Directed by Professor Jong – Chul Park)

Wound sites in skin must be immediately repaired because the skin

isolates and protects from the outside. Wound sites become healing with three

processes such as inflammation, proliferation and tissue remodeling. Treatment

in wound sites is aiming to accelerate the wound healing, to protect the wound

sites and to reduce the pain. A skin substitute, one of the treatment methods in

wound sites, is used in complete section that requires epidermis and dermis. A

skin substitute used commercially is made of the extracellular matrix (ECM),

the living cells, the cytokines and the growth factors. However, these materials

have some problems to induct the over expression of matrix metalloproteinase

in wound sites and to require the cell culture times and the less validity date. In

this study, we investigated the biodegradable scaffolds as a skin substitute. The

scaffolds made of poly (lactic-co-glycolic acid) (PLGA) what is one of the most

used biodegradable polymers was fabricated using electrospinning.

- 2 -

Electrospinning easily fabricates the multi-fiber structure with the polymers.

This structure has the advantages to provide the large surface area for cell

attachment and the fluid passages. The fabricated scaffolds were investigated to

composite with natural bioactive compounds instead of the cytokines and the

growth factors what accelerate the wound healing. The natural bioactive

compounds instead of them were used (1,3)-(1,6)-β-D-glucans (BGs) and

epigallocatechin-3-O-gallate (EGCG). BGs, a natural product of glucose

polymers, have immune stimulatory activity that is especially effective in

wound healing. EGCG is the major polyphenolic compound in green tea, and

has been investigated to regulate the secretion of cytokines and the activation of

skin cells during wound healing.

First of all, the porous PLGA scaffolds were investigated by

electrospinning using different process parameters. The electrospun scaffolds

showed the uniformity structures when the scaffolds were fabricated with

PLGA (PLA:PGA = 75:25) in HFIP or THF/DMF at 20% (w/v). These process

parameters had been used for this study because the scaffolds as a skin

substitute were demanded the uniformity shapes. The scaffolds processed with

dry ice had a loose connection between the fibers and showed large pore sizes.

The infiltration of the adult human fibroblasts was directly improved with the

increase of the pore sizes of the scaffolds. Biodegradable scaffolds were

sometimes deformed by heat, high temperature, pressure and moisture during

sterilization. In this study, the electrospun scaffolds were treated with direct

current (DC) for sterilization. During sterilization with DC treatment, the fiber

structures were maintained and the microorganism sterilized with a short time.

Therefore, DC treatment has the possibility of applying in the sterilization

method for biodegradable scaffolds.

The scaffolds composed of PLGA and BGs (BGs/PLGA scaffold) or

EGCG (EGCG/PLGA scaffold) was investigated using electrospinning. The

growth rate of human dermal fibroblasts (HDFs) increased in 50BGs/PLGA

- 3 -

scaffolds (PLGA scaffolds included 50 wt% BGs) because BGs improved the

HDFs proliferation. HDFs showed the cytotoxicity in PLGA scaffolds included

5 wt% EGCG (5EGCG/PLGA scaffolds) because EGCG induced caspase-3

activity to enhance that caused apoptosis. Therefore, 50BGs/PLGA scaffolds

and 1EGCG/PLGA scaffolds (PLGA scaffolds included 1 wt% EGCG) were

examined the wound healing effect in the animal study. According to H&E

staining results, the re-epithelialization in wound sites and the cell infiltration in

scaffolds were significantly accelerated the sites treated with the scaffolds

included BGs or EGCG at 2 weeks after scaffold implantation. At 2 weeks, the

proliferating cells (Ki-67 staining) in basal layers and the angiogenesis (CD 31

staining) in new dermis were also improved with the scaffolds included BGs or

EGCG.

In conclusion, BGs/PLGA scaffolds and EGCG/PLGA scaffolds should

be applied as a skin substitute for accelerated wound healing. Before application

to a skin substitute, the EGCG concentration in a skin substitute should be

regulated because the EGCG has cytotoxicity at high concentration.

----------------------------------------------------------------------------------------------------------

Key words: wound healing, skin substitute, biodegradable scaffold, Poly (lactic-

co-glycolic acid), (1,3),(1,6)-β-D-glucan, epigallocatechin-3-o-gallate, electrospinning,

direct-current treatment

- 4 -

Wound healing activity of bioactive compound and

application of composite scaffold

Hye – Lee Kim

Department of Medical Science

The Graduate School, Yonsei University

(Directed by Professor Jong – Chul Park)

I. INTRODUCTION

1. Wound healing processes

The skin, the largest organ in the body, is composed of the epidermis

and the dermis with a complex nerve and blood supply. The skin provides vital

barrier function and protects the outside world. Trauma, disease, burn or

surgery cause the wound sites in skin, and any wound in it must rapidly and

efficiently heal. Wound healing is a dynamic, interactive process involving

soluble mediators, blood cells, keratinocytes, fibroblasts and extracellular

matrix. Wound healing process divides three phases such as inflammation,

proliferation and tissue remodeling.1 First of all, a clot immediately formed for

hemostasis, and a clot also provides a provisional extracellular matrix for cell

migration in wound sites. A clot consists of the platelet aggregation, the blood

coagulation and fibrinogen. Several mediators for wound healing are also

present in a clot, and the mediators attract and activate the cells such as

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inflammatory cells, fibroblasts and keratinocytes. Inflammatory cells migrate in

a clot and begin the wound repair. During the inflammation, foreign particles

and bacteria are extruded or phagocytosed by neutrophils and macrophage for

cleaning the wound sites. Growth factors and inflammatory cytokines are

expressed and secreted for the migration and proliferation of cells involved in

the next phase. In the proliferation phase, keratinocytes grow and divide for

re-epithelialization. Macrophages and fibroblasts induce the collagen deposition

and granulation tissue formation, and compose the dermis layer. Fibroblasts

stimulated by transforming growth factor have differentiated into

myofibroblasts, and myofibroblasts induce wound contraction by connection to

the ECM. Epithelial cells proliferate and crawl for formation new blood vessels

(angiogenesis). Finally, disorganized collagen is remodeled and rearranged in

the dermis layer. Over-expressed extracellular matrix degrades by matrix

metalloproteinase that secreted macrophages and fibroblasts, and the cells that

are no longer needed are removed by apoptosis. According to these processes,

wound healing is the active participation of blood cells, tissue types, cytokines

and growth factors. Recently, researchers have investigated that wound healing

is accelerated and efficient by cells, cytokines and growth factors.1-5

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2. Skin substitute

The wound sites commonly are treated with ointment and coverage with

wound dressing and grafts. These treatments are proposed to protect the wound

site, to accelerate the wound healing, to reduce the pain and to decrease the

scarring in wound sites. Wound dressing is one of the immediate treatment

methods, and they removed from wound sites after wound healing. Grafts

degrade during repair in wound sites. Grafts usually applied the damaged

full-thickness wound sites that are complete sections of epidermis and dermis.

The grafts used to wound sites improve the wound healing by the excellent

coverage and the reduction of wound contraction. Allograft and heterograft

skins are tissues that are removed from person or animals. The grafts required to

transplant within a short time after harvest and the ultimate rejection following

withdrawal of immune-suppression therapies. Therefore, a skin substitute has

being investigated through tissue engineering and regenerative medicine. A skin

substitute is commercially fabricated with the protein components of

extracellular matrix (ECM) such as collagen, fibrin, laminin and elastin or the

combination with the protein components and the living cells, and the

commercially used the a skin substitute shows in Table 1-1.6

A skin substitute is widely applied to the scaffold that has

three-dimension (3D) structure. The 3D scaffold encourages ingrowth of

fibroblast and epithelial cells and improves granulation tissue deposition when

implanted in wound sites. Some scaffolds combined with the living cells such as

keratinocytes and fibroblasts. Keratinocytes and fibroblasts cultured in the

scaffolds have the advantages of the rapid proliferation in wound sites, the

producing collagen and the secreting growth factors to aid wound healing in

wound sites. According to Table 1-1, the cytokines and the growth factors have

been applied to mix with the scaffolds for improvement of wound healing.

However, the skin substitute created commercially has the problems that cause

the failure of wound healing. ECM, a main composition of scaffolds, induce the

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over expression of matrix metalloproteinase (MMP) in wound sites. The need

for cell culture and proliferation time requires upwards of two or three weeks.

Cell age and other factors may affect viability and persistence of donor cells.

Also, the ECM proteins and the processing of a skin substitute fabrication are

too expensive, and a skin substitute has a short shelf life.4,6-8

Therefore, the biodegradable polymers for the scaffolds used to a skin

substitute are required to develop and to research. The additives to instead of

the cytokine for improvement of wound healing were also desired to research.

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Table 1-1. Kinds of commercially created a skin substitute.6

Structure Composition

Dermal

substitutes

• Collagen coated nylon mesh

+ Neonatal allogenic fibroblasts

• 3D scaffold (Collagen, GAGs, Growth factor)

+ Allogenic neonatal fibroblasts

• Bovine type I collagen + GAGs

Composite

substitutes

• Human allogenic neonatal keratinocytes

+ Human allogenic neonatal foreskin fibroblasts

+ Bovine type I collagen + ECM proteins

+ Cytokines

• Human allogenic neonatal keratinocytes

+ Human allogenic neonatal foreskin fibroblasts

+ Bovine collagen sponge

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3. Biodegradable polymer

A skin substitute is demanded to degrade during wound healing.

Biodegradable polymers break down and lose their initial integrity inside body,

and degrade inside body. Therefore, biodegradable polymers have potential

materials for artificial compounds. Biodegradable scaffolds made of

biodegradable polymers are not necessary to be removed surgically once they

are no longer needed. Using the properties, biodegradable polymers have been

used for the solid dispersions, the suture, the drug delivery system and the

biodegradable scaffolds for the injured organs. Poly (lactic-co-glycolic acid)

(PLGA) is one of the most widely used biodegradable polymers. Because the

PLGA properties such as the biodegradable rate, the mechanical strengths, the

molecular weights and the glass transition temperature are easily controlled.

These PLGA properties are regulated by the chemical composition of poly

(lactic acid) (PLA) and poly (glycolic acid) (PGA) because PLGA degrade by

hydrolytic attack of their ester bonds, resulting in the formation of PLA and

PGA.9,10

PLGA also has excellent biocompatibility. Therefore, PLGA was

applied to the skin substitute compound in this study.

4. Natural bioactive compound

Beta-glucans are natural products of glucose polymers, and have been

found to promote immune stimulatory activity.11

Particularly in wound sites, it

was researched that β-glucan enhanced wound healing by increasing infiltration

of macrophages, stimulating tissue granulation, collagen deposition, and

re-epithelialization.12-14

The combination with β-glucans and collagen matrix

has been investigated to reduce the scarring and to decrease the pain.15

Kwon et

al. demonstrated that Sparassis crispa, a medicinal mushroom that has more

than 40% of β-glucans, significantly improved the wound healing in patients

with diabetes by oral administration.16

Epigallocatechin-3-O-gallate (EGCG) is the major polyphenolic comp-

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ound in green tea, and has excellent antioxidant activities.17

Moreover, EGCG

has other advantageous effects such as antimicrobial, anti-inflammatory, and

immunomodulatory activities.18

These advantageous effects including anti-

oxidant effects can be applied to regulate the wound healing process. EGCG has

been demonstrated effectively protect against UV-induced damage to skin.

Collagen production in keloid fibroblasts were suppressed by EGCG

treatment.19

These studies suggest that EGCG can be applied to a skin substitute

that can promote the activity of skin cells.

According to these reports, it can be suggested that β-glucans can be

applied to the addition instead to the cytokine and the growth factors for the

acceleration of wound healing.

5. Objectives of this study

In the present study, we investigated to fabricate PLGA scaffolds using

electrospinning and to combine with β-glucans or EGCG for the improvement

of wound healing. The behaviors of human dermal fibroblasts were researched

in the fabricated PLGA scaffolds with β-glucans or EGCG. Furthermore, the

wound healing effects of the scaffolds were evaluated in an animal study.

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6. References

1. Singer AJ, Clark RAF. Cutaneous wound healing. New Eng J Med

1999;341:738-46.

2. Prathiba V, Gupta PD. Cutaneous wound healing: significance of

proteoglycans in scar formation. Curr Sci 2000;78:1-5.

3. Bowcock AM, Krueger JG. Getting under the skin: the immunogenetics of

psoriasis. Immunology 2005;5:699-711.

4. Martin P. Wound healing-aiming for perfect skin regeneration. Science

1997;276:75-81.

5. MacKay D, Miller AL. Nutritional support for wound healing. Altern Med

Rev 2003;8:359-77.

6. Anthony DM, Mark WJF. Tissue engineering of replacement skin: the

crossroads of biomaterials, wound healing, embryonic development, stem

cells and regeneration. J R Soc Interface 2007;4:413-37.

7. Kim CH, Park H-S, Son YS. The status and prospect of bioartificial skin.

Polymer 2002;13:48-55.

8. Hench LL, Jones JR, Fenn MB. New materials and technologies for

healthcare. 1st ed. Covent Garden (London): Imperial Collage Press; 2012

9. Miller RA, Brady JM, Cutright DE. Degradation rates of oral oesorbable

implants (polylactates and polyglycolates): rate modification with changes

in PLA/PGA copolymer ratios. J Biomed Mater Res 1977;11:711-9.

10. Wei G, Pettway GJ, McCauley LK, Ma PX. The release profiles and

bioactivity of parathyroid hormone from poly (lactic-co-glycolic acid)

microspheres. Biomaterials 2004;25:345-52.

11. Ruiz HJ, Biosynthesis of beta-glucans in fungi. Antonie Van Leeuwenhoek

1991;60:72-81.

12. Tokunaka K, Ohno N, Adachi Y, Tanaka S, Tamura H, Yadomae T.

Immunopharmacological and immunotoxicological activities of a

water-soluble (1→3)-beta-D-glucan, CSBG from Candida spp. Int J

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Immunopharmacol 2000;22:383-94.

13. Rose GD, Vervicka V, Yan J, Siz Y, Vetvickova J. Therapeutic

intervention with complement and beta-glucan in cancer.

Immunopharmacology 1999;42:61-74.

14. Kougias P, Wei D, Rice PJ, Ensley HE, Kalbfleisch J, Williams DL et al.

Normal human fibroblasts express pattern recongnition receptors for fungal

(1/3),(1/6)-glucan. Mycol Res 2001;69:3933-8.

15. Delatte SJ, Evans J, Hebra A, Adamson W, Othersen HB, Tagge EP.

Effectiveness of beta glucan collagen for treatment of partial thickness

burns in children. J Pediatr Surg 2001;36:113-8.

16. Kwon A-H, Qiu Z, Hashimoto M, Yamamoto K, Kimura T. Effects of

medicinal mushroom (Sparassis crispa) on wound healing in

streptozotocin-induced diabetic rats. Am J Surg 2009;197:503-9.

17. Elbling L, Weiss L-M, Teufelhofer O, Uhl M, Knasmueller S, Rolf S-H et

al. Green tea extract and epigallocatechin-3-gallate, the major tea catechin,

exert oxidant but lack antioxidant activities. FASEB J 2005;19:807-9.

18. Wolfram S. Effects of green tea and EGCG on cardiovascular and

metabolic health. J Am Coll Nutr 2007;26:373S–88S.

19. Hsu S, Yu F, Lewis J, Singh B, Borke JL, Athar M et al. Induction of p57

is required for cell survival when exposed to green tea polyphenols.

Antican Res 2011;22:4115–20.

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II. FABRICATION OF POLY (LACTIC-CO-GLYCOLIC ACID) (PLGA)

SCAFFOLDS

1. Introduction

A skin substitute is demanded to mimic the structures of ECM because

the skin dermal structure is comprised of ECM.1,2

The structure of natural ECM

is 3D network composed of multi-fibers. The scaffolds which have 3D network

are made by several methods such as solvent casting method3, gas

forming/salt-leaching method4 and electrospinning method.

5,6 Especially,

electrospinning method easily fabricated the multi-fiber structure with the

synthetic and the natural polymers. Electrospinning technology enables to

generate micro/nano scale features with polymers. These structures provide

large surface-to-volume ratios and offer a large area for cells attachment and

cells growth.6-8

Thus, scaffolds fabricated electrospinning were suggested to

apply to a skin substitute. Recently, research has been developed on high

porosity scaffolds using electrospinning by mixing with other materials9 and

control of the process parameters such as solvents10

and voltage11

. These

methods have to use their process parameters, and have a limitation of materials.

Nam J. et al. previously investigated a simple method to increase the porosity of

mesh by ice crystals. According to the research, polymer were manufactured as

a high porous structure with ice crystals at high humidity.12,13

Biodegradable scaffolds sometimes deform after sterilization, due to

instability of composing polymer at high temperature, pressure and moisture.

To avoid the deforming, biodegradable scaffolds are sterilized by ethylene

oxide (EO) because EO sterilization method is processed at low temperature

under low humidity.14

However, EO treatment can potentially leave residues on

the surface and within the scaffolds. Especially, the high porosity scaffolds can

be easily remained EO residues in harmful quantities. Sterilization methods

using irradiation do not need chemical reagents, heat and moisture, but cause

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sectioning of polymer chemical bonds.14,15

Therefore, the sterilization without

deforming and sectioning of chemical bonds should be investigated. In the

previous study, we researched the electrical treatment including direct-current

could inactivate microorganism in solution. The experimental results were

suggested that the electrical treatment leads to sterilization of microorganism in

solution by breakdown or disruption of their membranes. The bacteria were

sterilized after electrical treatment at a short time (>3 msec) with low electric

currents (>300 mA) in electric vessels. Moreover, the electrical treatment

method does not require chemical reagents and heat.16-18

The present study was investigated the ability of the electrospinning

using various process parameters such as the solvent, the ratio of PLA and PGA,

the PLGA concentration in solution and the processing temperature. Cells

infiltration effects into the scaffolds were also researched. The sterilization

effects on the fabricated PLGA scaffolds were investigated.

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2. Materials and methods

A. Materials

PLGA polymer was purchased from Lakeshore Biomaterials

(Birmingham, AL, USA). The molecular weights (Mw) were 120 kDa of the

75:25 (75% PLA and 25% PGA) and 65 kDa of the 50:50 PLGA (50% PLA

and 50% PGA). PLGA was dissolved in a 1:4 mixture of dimethylformamide

(DMF, Duksan Pure Chemicals Co. Ltd., Ansan city, Gyeonggi do, Korea) and

tetrahydrofuran (THF, Duksan Pure Chemicals Co. Ltd.). 1,1,1,3,3,3,-

hexafluoro-2-propanol (HFIP, Wako pure chemical industries, Ltd., Chuo-ku,

Osaka, Japan) and acetone (Duksan Pure Chemicals Co. Ltd.) were also used

for solvent. The electrospinning system was purchased from NanoNC (Seoul,

Korea). Adult human dermal fibroblasts (HDFs) were obtained from Cambrex

BioScience Walkersville Inc. (Walkersville, MD, USA), and maintained in

Dulbecco’s modified Eagle’s medium (DMEM) containing 4.0 mM

L-glutamine, 1.5 g/L sodium bicarbonate 4.5 g/L glucose, 1.0 mM sodium

pyruvate, 10% fetal bovine serum and 1% antibiotic antimycotic solution. All

supplements for HDFs were obtained from Welgene Inc. (Daegu, Korea). HDFs

were incubated in a humidified atmosphere containing 5% CO2 at 37 oC.

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B. Electrospinning setup

The electrospinning system is shown in Fig. 2-1 (A). The polymer

solution was then loaded into a syringe with a 21 gauge metal needle tip. The

metal needle tip was connected to 20 kV of a high-voltage source, and a metal

collection drum was connected to ground. The distance between the needle tip

and the collector drum was measured at 10 cm, and the polymer solution was

ejected at 2 mL/h. For low temperature process, dry ice (Fine dryice, Seoul,

Korea) was loaded in the metal collector drum. Dry ice causes the deposition of

ice crystals on the collector surfaces and maintained at low temperature on the

collector surfaces. The collector deposited ice crystals and polymer fibers are

shown in Fig. 2-1 (B). The condition without dry ice was processed at room

temperature. Both environmental humidities were maintained at 50% using a

boiling water bath. All scaffolds were dried by a deep freezer.

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Fig. 2-1. The schematic of electrospinning system is used in this study (A). The sur-

faces are shown in the collector deposited ice crystals and polymer fibers

(B).

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C. Measurement of surface morphology on fibrous PLGA scaffolds

The shapes of fibrous PLGA scaffolds were photographed by a digital

camera (Sony Inc, Minato-ku, Tokyo, Japan). A scanning electron microscopy

(SEM, Minato-ku, Tokyo, Japan) was used to observe the surface morphology

of the fibrous PLGA scaffolds and bacteria. Before the observed the SEM, the

scaffolds observed the surface morphology were coated with Pt for 120 sec. The

inoculated samples were fixed using 2.5% glutaraldehyde solution. After

fixation, samples were dehydrated using graded ethanol, and dried. The dried

samples were coated with gold before observing by a SEM. The number

averages and the weight averages of molecular weight (Mn and Mw) of each

sample (n = 5) were determined by gel permeation chromatography (Viscotek

Corp., Houson, TX, USA) with chloroform.

D. Measurement of pore size on fibrous PLGA scaffolds

Surface morphology images were used for a measurement of the

scaffolds porosity. For the calculation of the pore sizes of the scaffolds, SEM

images (a magnification of 1,000) were estimated by Image J software

(National Institutes of Health, Bethesda, MD, USA), as shown in Fig. 2-2. The

original images improved the contrast by expanding the grayscale range of the

histogram (B). After controlling the contrast, their binary images were created

using a threshold of Image J software (C). Finally, the pore sizes of the

scaffolds were selected using a wand tool, and then the selected areas were

measured.

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Fig. 2-2. Images of surface morphologies for the measurement of pore areas

(magnification of 1000). Surface morphology of the scaffold was observed

by a SEM (A), and controlled of the contrast of each grayscale image (B).

The controlled image was assigned a threshold value (C).

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E. Measurement of cell infiltration

Cell infiltration into the scaffolds was investigated with HDFs and an

inverted fluorescent microscope (Olympus, Hachioji-shi, Tokyo, Japan). The

scaffolds were punched at Ф12 mm, and treated with plasma. The prepared

scaffolds were placed on well-plates, and seeded HDFs with 5 x 105 cells. After

the incubation for 1 day, the scaffolds were fixed with 70% ethanol diluted with

distilled water (DW), and stained with propidium iodide (PI, Sigma-Aldrich

Corp., St. Louis, MO, AR, USA) for observation of cell infiltration. To

characterize the HDFs infiltration, the cross-sectional areas of the fixed

scaffolds were observed with a blue filter on an inverted fluorescent microscope.

The depth of infiltrated cells was calculated by Image J using fluorescent

images.

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F. Sterilization methods

The electrolysis vessel used for treatment with DC is shown in Fig. 2-3.

The electrolysis vessel was made of two platinum electrodes (25 mm x 25 mm)

which were 10 mm apart. Each platinum electrode was connected to a

computer-based timing control through a parallel port interface which was used

to control the power transistor. The electrolysis vessel was filled with 3 ml of

saline solution, and then the inoculated sample was soaked in the solution. The

inoculated sample in the electrolysis vessel was treated with DC at 4 V or 6 V

for 10 sec to 60 sec.

The traditional sterilization methods for comparing with the DC

treatment methods were used EO gas, gamma ray and electron beam. One group

of samples was sterilized by EO. The fibrous PLGA scaffolds were exposed to

EO at 37 oC for 4 hours in 3M Steri-Vac 5XL sterilizer (3M Health Care Ltd.,

St. Paul, MN, USA), and vented over 12 hours at room temperature in the same

space. The other groups of samples were irradiated with gamma or e-beam.

Gamma irradiation was carried out in the air with a 60

Co source at a 1 kGy/h

dose rate to 25 kGy final dose at room temperature (Greenpia Tech. Inc.

Yujoo-Kun, Gyeonggi do, Korea). The e-beam irradiation of fibrous PLGA

scaffolds were carried out by a tray and exposed with a surface dose of 15 - 25

kGy at room temperature in air, using electron acceleration (EB Tech Co., Ltd.,

Daejeon, Korea). Electron energy was maintained at 1 eV and with the tray

speed of 1 cm/min.

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Fig. 2-3. The electrolysis vessel used in this study. (A), Schematic of electrolysis

vessel; (B), Experimental electrolysis vessel for treatment with direct

current.

- 23 -

G. Sterility test

Escherichia coli (E. coli, ATCC 8739) and Staphylococcus aureus (S.

aureus, ATCC 6358p) were used for the sterilization test because these bacteria

cause significant public health problems worldwide.19,20

The bacteria suspension

for inoculation into the samples were prepared with saline solution (0.9% NaCl

in DW). The density of the suspensions was 1 x 106 colony forming units/ml.

Each electrospun PLGA was inoculated by the immersion in the each

suspension for 10 min with shaking. The inoculated samples were then treated

with DC or traditional sterilization methods. After treatment, the samples were

incubated in standard agar media (Becton Dickinson, Franklin Lakes, NJ, USA)

at 36 oC for 14 days. The sterilization efficiency of DC treatment was

determined by the number of colonies, and the methods are shown in Fig. 2-4.

The samples inoculated with E. coli or S. aureus are shown in Fig. 2-4 after

incubation. Non-sterilized samples inoculated with E. coli or S. aureus were

used as controls (Fig. 2-4. (A) and (D)). Appearance of colonies on the agar

media after 14 days indicated contamination and inefficient sterilization (Fig.

2-4. (B) and (E)); while a clear, uncontaminated media indicated efficient

sterilization (Fig. 2-4. (C) and (F)), producing a sterile product. Five days after

putting the inoculated samples on the agar media pictures were taken using a

digital camera (Sony Inc.). All the treatment conditions were independently

repeated 10 times.

H. Statistical analysis

All quantitative data are reported as means ± SDs. Statistical analyses

were performed using Student’s t-test. A p-value < 0.05 was considered

statistically significant.

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Fig. 2-4. Digital photograph of the electrospun PLGA inoculated with Escherichia

coli (A - C) and Staphylococcus aureus (D - F) after incubation on standard

agar at 36 oC for 7 days. (A, D), Untreated electrospun PLGA; (C, E),

Electrospun PLGA treated with 6 V for 10 sec; and (D, F), Electrospun

PLGA treated with 6 V for 50 sec (n = 10, scale bar: 30 mm).

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3. Results

A. Characterization of electrospun PLGA scaffolds

Electrospun PLGA scaffolds were affected by the various parameters

such as the ratio of PLA and PGA, the solution and the polymer concentration,

as shown in Fig. 2-5. These parameters influenced the fiber shapes, the fiber

thickness and the surface morphology of the PLGA scaffolds. The ratio of PLA

and PGA and the PLGA concentration in solution influenced the formation of

bead. The solvents have an effect on the dispersion of the fiber thickness. A

skin substitute is demanded to the uniformity shapes because of the uniformity

quality. Therefore, the electrospun PLGA (PLA:PGA = 75:25) scaffolds solved

at 20% (v/v) in HFIP or THF/DMF had been used for this study.

SEM images of the electrospun PLGA scaffolds using dry ice are shown

in Figs. 2-6 and 2-7. Using HFIP, the average fiber diameter of the scaffolds

without dry ice was 0.95 ± 0.32 μm. With dry ice, the scaffolds fabricated using

HFIP showed 0.94 ± 0.26 μm as the average fiber diameter. The average fiber

diameters of the meshes using the THF/DMF with and without dry ice were and

0.95 ± 0.57 μm and 0.93 ± 0.40 μm (Fig. 2-6), respectively. Their cross-section

images were different between them (Fig. 2-7). At both temperatures, the fibers

manufactured with HFIP showed a similar connection with the surrounding

fibers, and their connections were compact. The fibers deposited with

THF/DMF at room temperature also had a compact connection with the

surrounding fibers, and consequently showed a large number of fibers. However,

the fibers fabricated with THF/DMF at low temperature showed loose

connections, and the scaffolds manufactured with ice crystals showed large void

spaces.

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Fig. 2-5. Fibers of the electrospun PLGA were detected with a SEM. Fibers of the

electrospun PLGA were deposited with various parameters such as the

PLA/PGA ratio of PLGA (A), the kinds of solvent (B) and the PLGA

concentration of polymer solution (C).

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Fig. 2-6. Fiber morphology of the electrospun PLGA scaffolds was measured by a

SEM. The electrospun fibers were deposited using HFIP (A) and THF/DMF

(B).

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Fig. 2-7. SEM images of cross-section of the scaffolds. The scaffolds were fabricated

with HFIP (A) and THF/DMF (B).

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B. Influence of dry ice on pore size

The pore sizes of the scaffolds were calculated by the Image J software

(Fig. 2-8). The pore sizes average of the scaffolds fabricated using HFIP were

5.67 ± 3.91 μm without dry ice and 25.42 ± 13.94 μm with dry ice. Using

THF/DMF, the scaffolds without and with dry ice showed 11.84 ± 9.99 μm and

126.1 ± 86.08 μm. For both solvent systems, the pore sizes of the scaffolds

using dry ice are significantly higher than those of the scaffolds fabricated

without dry ice. This suggests that the electrospinning process using dry ice

caused a decrease in the loose connections of the fibers and their loose

connections induced the pore size to expand. Also, the average of pore sizes

gradually increased from 25.42 ± 13.94 μm to 126.1 ± 86.08 μm with

THF/DMF.

C. Influence of the pore sizes on the cell infiltration

For the influence of the pore sizes on the cell infiltration, the scaffolds

using THF/DMF were applied to the cell infiltration because the scaffolds have

the significantly different connection with the surrounding fibers. After HDFs

seeded on the scaffolds were incubated for 1 day, the location of HDFs on the

scaffolds are shown in Fig. 2-9. HDFs infiltrated 14.83 ± 10.78 μm of the

scaffolds fabricated without dry ice and 141.48 ± 60.20 μm of the scaffolds

fabricated with dry ice. As a result, the scaffolds fabricated with dry ice

improved the infiltration of the cells, compared with the scaffolds fabricated

without dry ice.

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Fig. 2-8. Images of surface morphologies (magnification of 1000) were assigned a

threshold value (A), and the pore areas of the electrospun scaffolds were

calculated with the images (B). The scaffolds were fabricated using HFIP

without dry ice (a) and with dry ice (b). The scaffolds were electrospun

using THF/DMF without dry ice (c) and with dry ice (d) (*p < 0.05 versus

the pore size fabricated without dry ice, as analyzed by a t-test).

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Fig. 2-9. Infiltrated cells into the scaffolds were fixed and stained with Pi (red). Cells

(red) and scaffolds (white) were observed with a digital fluorescence

microscope (A) (scale bar: 500 µm). The calculated the cells distances from

the surfaces using image J (B).

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D. Sterilization effects on electrospun PLGA scaffolds

The different of the DC treatment times and the treatment voltages were

tested, as summarized in Table 2-1. At 4 V, the complete sterility was achieved

over 30 sec for E. coli and 40 sec for S. aureus. At 6 V, E. coli was sterilized

over 20 sec and S. aureus was sterilized over 30 sec. These results were

concluded that sterility could be using DC treatment at 4 V with longer

treatment times. Both bacteria were completely sterilized within 40 sec

independently of process voltage without any electrolysis. According this

results, subsequent studies compared DC treatment of electrospun PLGA at 6 V

and 50 sec. The morphologies of E. coli and S. aureus in samples are shown in

Fig. 2-10. Both of bacteria were distributed inside and outside of samples. The

inoculated bacteria in the samples showed normal morphology before treatment

with DC (Fig. 2-10 (A) and (C)). After DC treatment, all inoculated bacteria in

samples were reduced in size and exhibited transformed morphologies (Fig.

2-10 (B) and (D)).

Images of the scaffolds and fiber shapes of the electrospun PLGA are

shown in Fig. 2-11. The photography was observed by a digital camera and the

surface morphology was observed by a SEM. The results of the Mn and Mw of

the electrospun PLGA are illustrated in Fig. 2-12. After DC treatment and

irradiation using gamma or e-beam, the shapes and the fibers of electrospun

PLGA were preserved. However, the electrospun PLGA were stick to each

other after sterilization using EO (Fig. 2-11). The Mn and Mw of the electrospun

PLGA after treatment with DC or EO were preserved. Their Mn and Mw

decreased after irradiated with gamma and e-beam (Fig. 2-12).

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Table 2-1. Sterilization effects of DC on Escherichia coli (E. coli) and Staphylococcus

aureus (S. aureus) in the electrospun PLGA scaffolds. (n = 10)

Treatment

Voltage

Treatment Time

(in seconds) E. coli S. aureus

4V 10 X X

20 X X

30 O X

40 O O

50 O O

6V 10 X X

20 O X

30 O O

40 O O

50 O O

X, inefficient sterilization after treatment with DC

O, efficient sterilization after treatment with DC

- 34 -

Fig. 2-10. SEM images of Escherichia coli (A, B) and Staphylococcus aureus (C, D)

in the electrospun PLGA. (A, C), Untreated; (B, D), Treated with 6 V for 60

sec.

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Fig. 2-11. Shape and surface morphology of the electrospun PLGA. The images were

observed by (A) a digital photograph (scale bar: 1 mm) and (B) a SEM.

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Fig. 2-12. The number average and the weight average of molecular weight (Mw and

Mn) of the electrospun PLGA (n = 5).

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4. Discussion

The porous structures provide the passage for the fluid exchange and the

cell infiltration into the scaffolds. Increase of the pore sizes improves their

passage. The pore sizes of the electrospun scaffolds were increased by the

process using dry ice. Dry ice induces the collector drum to deposit of the ice

crystals. The ice crystals were deposited with polymer fiber during

electrospinning. After drying the scaffolds, the sites of ice crystals were pores in

scaffolds. Therefore, the pore sizes of scaffolds increased using dry ice.12

However, the scaffolds using HFIP at both temperatures showed a similar

connection with the surrounding fibers, and their connections were compact.

HFIP and THF have different vapor pressures, such as 269 hPa and 357 hPa at

30 oC. The vapor pressures induce the residual solvent in the scaffolds after

electrospinning. The residual solvent in scaffolds causes to connect the

surrounding fibers.5 In this study, the electrospinning was processed at low

temperature for the formation of ice crystals. The low temperature has disturbed

the solvent volatilization, especially HFIP. When electrospinning used the

solvent had a low vapor pressure, such as HFIP, the ice crystals did not have an

effect on the connection. The ice crystals should improve the pore sizes of the

electrospun scaffolds using a high vapor pressure solvent without the

morphology transformation of the fibers, according to the Figs. 2-6 and 2-8. The

increase the pore sizes by ice crystals also enhances the cell infiltration (Fig.

2-9). Therefore, the pore sizes and the cell infiltration of the electrospun

scaffolds should be easily improved by the dry ice.

Sterilization is an essential process for biodegradable polymers to be

used as biomaterials or tissue engineered scaffolds. The characteristics of

biodegradable scaffolds can change due to decomposition of constituent

polymers due to high temperature, pressure or moisture during sterilization. In

this study, DC treatment was applied to sterilization methods for biodegradable

scaffolds. In the previous studies, the treatment with electric current caused

- 38 -

breaks and ruptures in bacteria scaffolds.18-20

The cellular contents were

released into the surrounding surface from the parts of broken scaffolds. As a

result, the bacteria treated with DC reduced their sizes and were sterilized.

Sterilization method using EO requests several days for removed the resided EO.

Irradiation methods also need to several days for sterilization depending on the

size and quantity of the medical devises.15

Schlapp M. et al consisted that

deformation of the microparticles occurs as a consequence of the high pressure

at low sterilization temperature (32 oC), which is below the glass transition

temperatures (40 oC - 60

oC).

21 Thus, EO gas under applied conditions (32

oC, 4

bar for 6 h) is incompatible for the biodegradable polymers. The scaffolds

sterilized EO were transforming during imaging at magnification higher than

1,000 using a SEM. Because EO treatment reduced the physical properties of

the scaffolds, including the yield stress and break stress.22

The molecular weight

of PLGA decreased after the irradiation with gamma or e-beam. Many

researchers have reported that gamma or e-beam irradiation caused polymer

degradation through chain scission. In particular, backbone chain scission

results from exposure to gamma or e-beam radiation, and is the reason for the

rapid decrease in the molecular weights. The decrease in the molecular weights

of polymers is also related to significantly less crystalline structure. It has been

demonstrated that the crystal structure can delay degradation because the

crystalline regions in the polymer have resistant chains that are more oriented

and closely packed compared to amorphous regions.14,23

These results suggested

that the treatment method with DC was considered the sterilization methods of

biodegradable polymers without deforming and change the chemical bonding.

- 39 -

5. Conclusion

The electrospinning using ice crystals should improve the porosity of the

structure by disturbing the contact with the fibers. However, the polymer should

be solved with a solvent that has a high vapor pressure. Cell infiltration was

accelerated on the scaffolds that had large pore sizes due to the ice crystals.

Therefore, the electrospinning method using ice crystals with low volatile

solvents can improve the pore sizes of the scaffolds, and induce the acceleration

of the cells infiltration.

All the above results show that the treatment method with DC did not

deform the electrospun PLGA and change molecular weight of PLGA. This

method also requires within 40 sec of treatment time and process than

traditional sterilization methods without any harmful effect. These results

suggested that the DC treatment method provides proper sterilization method

for the biodegradable polymer used in tissue engineering without any damage.

- 40 -

6. References

1. Rovert MN, Athanassions S. Tissue engineering: form biology to

biological substitutes. Tissue Eng 1995;1:193-4.

2. Joshua SB, Kerr HM, Howard NES, Gillian ME. Wound healing dressings

and drug delivery systems: a review. J Pharm Sci 2008;97:2892-923.

3. Miller DC, Thapa A, Haberstroh KM, Webster TJ. Endothelial and

vascular smooth muscle cell function on poly (lactic-co-glycolic acid) with

nano structured surface features. Biomaterials 2004;25:53-61.

4. Yoon JJ, Park TG. Degradation behaviors of biodegradable macroporous

scaffolds prepared by gas foaming of effervescent salts. J Biomed Mater

Res B: Appl Biomat 2001;55:401-8.

5. Pham QP, Sharma U, Mikos AG. Electrospinning of polymeric nanofibers

for tissue engineering applications: a review. Tissue Eng 2006;12:

1197-211.

6. Duan B, Yuan X, Zhang Y, Li X, Zhang Y, Yao K. A nanofiberous

composite membrane of PLGA chitosan/PVA prepared by electrospinning.

Eur Polymer J 2006;42:2013-22.

7. Rho KS, Jeong L, Lee G, Seo B-M, Park YJ, Hong S-D et al.

Electrospinning of collagen nanofibers: effects on the behavior of normal

human keratinocytes and early-stage wound healing. Biomaterials

2006;27:1452-61.

8. Chen J-P, Chang G-Y, Chen J-K. Electrospun collagen/chitosan

nanofibrous membrane as wound dressing. Colloids Surf A: Physicochem

Eng Aspects 2008;313:183-8.

9. Mutsuga M, Narita Y, Yamawaki A, Satake M, Kaneko H, Suenmatsu Y,

et al. A new strategy for prevention of anastomotic stricture using

tacrolimus-eluting biodegradable nanofiber. J Thorac Cardiovasc Surg

2009;137:703-9.

10. Schneider OD, Loher S, Brunner TJ, Uebersax L, Simonet M, Grass RN,

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et al. Cotton wool-like nanocomposite biomaterials prepared by

electrospinning: in vitro bioactivity and osteogenic differentiation of

human mesenchymal stem cells. J Biomed Mater Res Part B: Appl

Biomater 2008;84B:350-62.

11. Fu L, Liu Y, Hu P, Xiao K, Yu G, Zhu D. Ga2O3 nanofibons: synthesis,

characterization, and electronic properties. Chem Mater 2003;15:4287-91.

12. Nam J, Huang Y, Agarwal S, Lannutti J. Ultraporous 3D polymer meshes

by low-temperature electrospinning: using of ice crystals as a removable

void template. Polym Eng Sci 2007;47:2020-6.

13. Greiner A, Wendorff JH. Electrospinning: a fascinating method for the

preparation of ultrathin fibers. Angew Chem Int Ed 2007;46:5670-703.

14. Holy CE, Cheng C, Davies JE, Shoichet MS. Optimizing the sterilization

of PLGA scaffolds for use in tissue engineering. Biomaterials

2001;22:25-31.

15. McDonnell GE. Antisepsis, disinfection, and sterilization: types, action,

and resistance. 1st ed. Washington (DC): AMS Publisher; 2007.

16. Park JC, Lee MS, Han DW, Lee DH, Park BJ, Lee I-S et al. Inactivation of

bacteria in seawater by low-amperage electric current. Appl Environ

Microbiol 2003;70:2405-8.

17. Lee MH, Han D-W, Woo YI, Uzawa M, Park J-C. Inactivation of Listeria

monocytogenes in brine and saline by alternating high-voltage pulsed

current. J Microbiol Biotechnol 2008;18:1274-7.

18. Jin SC, Yoo H, Woo YI, Lee MH, Vagaska B, Kim JS et al. Selective

sterilization of Vibro parahaemolyticus from a bacterial mixture by

low-amperage electric current. J Microbiol Biotechnol 2009;19:537-41.

19. Madigan MT, Martinko JM, Parker J. Biology of microorganisms. 1st ed.

London (UK): Benjamin Cummings Publisher; 1997.

20. The Korean Pharmacopoeia - Part 9: Sterility Test. Tests for sterility test,

2008. 9.

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21. Friess W, Schlapp M. Sterilizationof gentamicin containing collagen

/PLGA microparticle composites. Eur J Parm Biopharm 2006;63:176-87.

22. Athanasiou KA, Niederauer GG, Agrawal CM. Sterilization, toxicity,

biocompatibility and clinical applications of polylactic acid/polyglycolic

acid copolymers. Biomaterials 1996;17:93-102.

23. Loo JSC, Ooi CP, Tan MLF, Boey FYC. Isothermal annealing of

poly(lactide-co-glycolide) (PLGA) and its effect on radiation degradation.

Polym Int 2005;54:636-43.

- 43 -

III. EVALUATION OF ELECTROSPUN (1,3)-(1,6)-β-D-GLUCANS (BGs)

/PLGA SCAFFOLDS

1. Introduction

Treatment of a wound site is thought to improve wound healing by

stimulating the host to produce various cytokines.1 β-glucans are natural

products of glucose polymers, and have been found to promote immune

stimulatory activity.2 β-glucans improve wound healing in wound sites because

of the acceleration of the macrophage infiltration and the stimulation of tissue

granulation by the deposited collagen increase.3-6

In previous studies, the

wound healing effects of water-soluble (1,3)-(1,6)-β-D-glucans (BGs) have

been demonstrated on the skin-derived cells such as HDFs and adipose

tissue-derived mesenchymal stem cells (ADSCs). BGs accelerated the

proliferation and the migration of them. In addition, the contraction of the

collagen gels formed by fibroblasts was accelerated after BGs treatment. Thus,

BGs should be infiltrated into HDFs and ADSCs in order to activate them in

wound sites.7,8

Tsubaki K. et al. who produced BGs for this study have been

researched that BGs strongly induced the production of various cytokines,

especially interferon γ (IFN- γ), tumor necrosis factor-α (TNF- α) and interlukin

6 (IL-6).9-11

These previous results suggest that BGs may accelerate wound

healing, and that a skin substitute containing BGs could improve wound

healing.

In the present study, we purposed to fabricate scaffolds with BGs and

PLGA using electrospinning, and investigated the behavior of fibroblasts in

these scaffolds. Furthermore, the wound healing effects of the scaffolds were

evaluated in an animal study.

- 44 -

2. Materials and methods

A. Evaluation of wound healing effects of BGs using the skin-

derived cells

Water-soluble BGs powder was obtained from Adeka Corp. (From

Aureoubasidium pullulans). HDFs and adult normal human epidermal

keratinocytes (NHEKs) were used in this study. NHEKs were obtained from

Lonza group. Ltd. (Basel, Switzerland). HDFs were maintained in DMEM with

all supplements. NHEKs were maintained in KGM-Gold kit (keratinocytes

growth medium, Lonza group Ltd.). After reaching confluence, the cells were

washed with PBS, and detached using 0.025% trypsin in 0.04mM

ethylenediaminetetraacetic acid (EDTA, Welgene Inc.).

These cells were seeded in culture well (24-well culture plate, Falcon,

NJ, SUA) with 1 mL growth medium containing 2 x 104 cells. After 1 day

incubation, the cells were treated with BGs in medium without growth factors.

The proliferating cells were quantified using MTT assay which measures the

mitochondrial dehydrogenase activity of living cells. The yellow tetrazolium

salt-3-(4,5-dimethylthiazol-2-yi)-2,5-diphenyltetrazolium bromide (MTT,

Sigma) was reduced by metabolically activity cells to form insoluble purple

formazan crystals. The cell viability and the cell proliferation were estimated by

detected the produced formazan salts. The produced formazan salts were

dissolved with dimethyl sulphoxide (DMSO, Duksan Pure Chemicals Co. Ltd.),

and the solution was transferred to a 96-well plate (Falcon, Atlanta, CA, USA).

The absorbance was measured using the ELISA reader at a wavelength of 570

nm. Migration assay for wound closure experiment was used with the silicon

culture insert (Ibidi, Munich, Germany). HDFs and NHEKs were seeded in the

silicon culture insert at a culture dish, and attached for 1 day in incubation. The

silicon culture inserts were removed from culture dishes after 1 day, and treated

with BGs. The cells migration was observed by microscope.

- 45 -

B. Fabrication of electrospun BGs/PLGA scaffolds

The solvent for polymer solution used was HFIP. The PLGA

concentration was 20% (w/v) of the solvent volume, and the BGs

concentrations were 25 wt% and 50 wt% of the total PLGA weight. Only BGs

were dispersed in solvent, and the concentration of BGs was 50% (w/v) of the

solvent. All solutions were used to manufacture the scaffolds at 15 kV by an

electrospinning system. The solution flow rate was maintained at 2 mL/h using

a syringe infusion pump. After electrospinning, the scaffolds were dried for 2

days at room temperature. The scaffolds were sterilized with 25 kGy gamma

irradiation at room temperature.

C. Characterization of electrospun BGs/PLGA scaffolds

The surface morphology of the scaffolds was observed by a SEM. The

scaffolds were Pt sputter-coated for 120 sec before a SEM analysis. For release

kinetics of BGs from the scaffolds, the scaffolds were immersed in 2 mL PBS

which was boiled at 36 oC in incubation. During the immersion, the scaffolds in

PBS were protected from light for 16 days in a shaking incubator. The weight of

the scaffolds was determined at 2 mg/mL before their immersion in PBS. The

BGs concentration in PBS at each time point was calculated with a BGSTAR

kit (Wako Pure Chemical Industries Ltd.). The samples were conjugated with

the detected reagent, and then reacted in 37 oC for 30 min. The reacted samples

were added the color-developing reagents in numerical order, and detected by

ELISER reader.

- 46 -

D. Viability and proliferation of HDFs on electrospun BGs/PLGA

scaffolds

Scaffolds were cut out into circles 12 mm in diameter, and put onto

24-well culture plates. HDFs were placed in scaffolds at a density of 5 x 104

cells per scaffold for a proliferation assay. HDFs on scaffolds were incubated in

humidified atmosphere containing 5% CO2 at 36 oC for 1, 3, 5, and 7 days. The

proliferation assays were performed with an ATP bioluminescence assay kit

(Roche Molecular Biochemicals, Manheim, Germany). At each time point, the

scaffolds were immersed in 0.1% Triton-X in 100 mM Tris and 4 mM EDTA

(pH 7.81) during 30 min for the cell lysis. ATP from the cells lysis was

collected by a centrifuge at 1000 g for 60 sec, and then each supernatant was

transferred to each fresh tube. The luciferase reagent was added to the

supernatant, and the ATP concentration was quantified using a microplate

luminometer (Berthold Technologies GmbH & Co., Germany). To calculate the

cell numbers using ATP concentrations, the ATP standard curve was plotted

using serial dilutions of the cells and the ATP concentrations. The morphology

of the cells incubated for 5 days in scaffolds was observed by a SEM. The cells

in the scaffolds were fixed in PBS included 2% glutaraldehyde (Merck KGaA,

Darmstadt, Germany), 2% paraformaldehyde (Merck KGaA) and 0.5% CaCl2

for 6 h. Then, the scaffolds were treated with 1% OsO4 (Polysciences, Inc.,

Warrington, PA, USA) in 0.1 M PBS for 2 h, and washed twice with 0.1M PBS.

The fixed cells in the scaffolds were dehydrated with absolute alcohol (Merck

KGaA). The dehydrated cells were treated with isoamylactete (Merck KGaA)

and dried. Finally, the cells in the scaffolds were coated with gold by an ion

coater (Eiko Engineering Co. Ltd., Kawachinagano City, Osaka, Japan) and

observed with a SEM (Hitachi Ltd.).

- 47 -

E. Animals and surgical manipulations

Male BALB/c nude mice (7-week old) were used for the animal study.

This animal experiment was performed in accordance with the Guidelines for

the Care and Use of Laboratory Animals, and the protocol was approved by the

Animal Care and Use Committee of Yonsei University College of Medicine.

A single full-thickness skin wound (diameter of 12 mm) was created on

the upper back of each mouse. The animal models were described in Fig. 3-1.

There were the study groups (n = 6): PLGA scaffolds, 50BGs/PLGA scaffolds

(PLGA scaffolds including 50 wt% BGs). The study groups were shown in

Table 3-1. Each scaffold was placed into the wound site, and then the scaffolds

were attached to the surrounding tissue by suturing. The sutured wound sites

were covered with Tegaderm® (3M Co.). At 1, 2 and 4 weeks after the

scaffolds were transplanted, the mice were euthanized using zoletile (120 mg/kg;

Boehringer Ingelheim Agrovet, Hellerup, Denmark) and rompun (40 mg/kg;

Bayer, Pittsburgh, PA, USA). The transplanted scaffolds and the surrounding

tissues were isolated, and fixed with 10% formaldehyde for 2 days. After

fixation, they were embedded in paraffin for the histological and the

immunohistochemical evaluation.

- 48 -

Fig. 3-1. Surgical procedure of the full-thickness wound model in nude mice (scale

bar: 12 mm).

- 49 -

Table 3-1. The experimental numbers of nude mice for the animal study.

PLGA scaffolds: Fabrication with PLGA

50BGs/PLGA scaffolds: Fabrication with PLGA and 50 wt% BGs

Treatment times Experimental scaffolds Number of

nude mouse

1 week PLGA scaffolds

50BGs/PLGA scaffolds

6

6

2 weeks PLGA scaffolds

50BGs/PLGA scaffolds

6

6

4 weeks PLGA scaffolds

50BGs/PLGA scaffolds

6

6

- 50 -

F. Determination of wound sites

The wound sites were measured with a digital camera (Canon Inc.,

Ohta-ku, Tokyo, Japan), and hematoxylin and eosin staining (H&E,

Dakocytomation Inc., Carpinteria, CA, USA). The wound sites were

photographed at 1, 2 and 4 weeks with a digital camera, and isolated at each day

for H&E staining. The isolated tissues were fixed using 10% neutral buffered

formaldehyde. The fixated tissues were formed into paraffin-embedded tissue

blocks. The blocks were sectioned at a thickness of 4 μm, and then underwent

deparaffinization and rehydration. The H&E stained sections were prepared for

the examination of epithelial regeneration and interaction with original tissues.

The sections were scanned with a digital virtual microscope (Olympus) and

measured with OlyVIA 6711 (Olympus). Viewing programs were also used

with OlyVIA 6711.

G. .Immunohistochemical analysis of wound area

The isolated tissues for staining Ki-67 and CD 31 were fixed with 10%

neutral buffered formaldehyde. The fixed tissues were formed into

paraffin-embedded tissue blocks. The blocks were sectioned at a thickness of 4

μm, and then underwent deparaffinization and rehydration. The rehydrated

sections were placed in target retrieval solution (0.01 M citrate buffer) and

heated in a microwave oven at two times, followed by washing with PBS. The

sections were the incubated with rat monoclonal anti-mouse Ki-67

(Dakocytomation Inc.) and mouse monoclonal antibody CD 31 (Novacastra,

Newcastle, UK). The sections treated with anti Ki-67 were incubated for 30 min

at room temperature, and then rinsed with PBS. The rinsed sections were

exposed to polyclonal rabbit anti-rat Ig/HRP (Dakocytomation Inc.) for 30 min

at room temperature, and then treated with α-rabbit Envision-HRP solution

(Dakocytomation Inc.) for 30 min. The sections treated with anti CD 31 were

stored at 4 oC overnight, and then rinsed with PBS. Prior to the exposition of

- 51 -

anti CD 31, the sections were blocked at non specific binding sites with 1%

bovine serum albumin in Tris-buffered saline for 10 min. Both antibody binding

sites were visualized by incubation with Envision-HRP-DAB (Dako-

cytomation Inc.). The sections were counterstained for 3 min with hematoxylin

(Dakocytomation Inc.), and then dehydrated with sequential ethanol for sealing

and microscopic observation. The sections that expressed anti Ki-67 and anti

CD 31 were observed under 200 magnifications using a microscope (Olympus).

The number of Ki-67 positive cells at the basement layers of the epidermis was

counted in both wound margin areas. The numbers of Ki-67 positive cells were

calculated the percentage of the total numbers of the cells at the basement layers

of the epidermis. The positive stained capillaries of CD 31 were measured in

three different areas at both ends of the operated sites, the middle of the

operated sites.

H. Statistical analysis

Cells proliferation assays were conducted in three independent cultures

for each experiment. Quantitative data are expressed as means ± SDs. Statistical

comparisons were carried out using the Student’s t-test (one-way). A p-value <

0.05 was considered statistically significant.

Animal studies were conducted with 6 mice per group. The results are

reported as mean ± SDs and were compared to those of PLGA scaffolds.

Statistical analyses were performed using ANOVA, followed by Tukey’s HSD

test and Fisher’s PLSD test using SPSS software (12.0 Ko for windows). A

p-value < 0.05 was considered statistically significant.

- 52 -

3. Results

A. Evaluation of wound healing effects of BGs on HDFs and

NHEKs

The growth rate effects of BGs are illustrated in Fig. 3-2. The

proliferation of HDFs and NHEKs for 5 days was enhanced with the BGs dose

dependently. HDFs and NHEKs proliferation was significantly enhanced on the

treated 0.5 mg/mL and 1 mg/mL BGs after 5 days. At 5 days after treatment

with 1 mg/mL BGs, the growth rate of HDFs and NHEKs increased by 4.38

times and 3.70 times, especially. Cell movement is combined effect of cell

division and cell migration. The migration speeds of HDFs and NHEKs are

illustrated in Fig. 3-3. Both cells were improved the migration speed after

treatment with 1mg/mL BGs. These results can be beneficial for acceleration of

wound healing by proliferation and cell migration.

- 53 -

Fig. 3-2. Growth rate effects of BGs on HDFs (A) and NHEKs (B). (*p < 0.05 versus

the control group (0 mg/mL), as analyzed by a t-test).

- 54 -

Fig. 3-3. Migration effects of BGs on HDFs (A) and NHEKs (B) (scale bar: 100 µm).

- 55 -

B. Characterization of electrospun BGs/PLGA scaffolds

Influence of BGs concentration in scaffolds on their surface morphology

is shown in Fig. 3-4. The fibers made of PLGA had 1.96 ± 0.09 μm of an

average fiber diameter. The average fiber diameter of the scaffolds with 25 wt%

BGs (25BGs/PLGA scaffolds) was 1.42 ± 0.34 μm. In the scaffolds with 50 wt%

BGs (50BGs/PLGA scaffolds), their fiber diameter ranged from 1.25 to 0.52 μm,

with an average fiber diameter of 0.89 ± 0.37 μm. The fiber diameters were

reduced with the increase of BGs concentration on the scaffolds. While the

solution containing a mixture of PLGA and BGs was electrospinning, several

beads were deposited on the collector. The bead number on the scaffolds

increased with the increase of the BGs concentration in the polymer solutions.

The BGs in solvent without PLGA were deposited as several sizes of the

particles on the collector during electrospinning because BGs were insoluble in

the solvent.

- 56 -

Fig. 3-4. SEM images of the PLGA scaffolds and the BGs/PLGA scaffolds. The

collector surface images were detected by a SEM after electrospinning of

BGs without PLGA.

- 57 -

C. BGs release pattern from BGs/PLGA scaffolds

Figure 3-5 illustrates the cumulative release of BGs at a 16 day period

from the scaffolds. After incubation for 4 days in PBS, the cumulative amount

of BGs released from the 25BGs/PLGA scaffolds was 0.41 ± 0.5 mg, which

was 51.86 ± 6.52% of the total BGs concentration on the scaffolds. The BGs on

the 25BGs/PLGA scaffolds were released in a logarithmic manner for up to 6

days, after which the release rate decreased with time. In the 50BGs/PLGA

scaffolds, 0.81 ± 0.09 mg of BGs had been released in 4 days, which was 61.12

± 7.12 % of the total BGs concentration on the scaffolds. The amount of BGs

released from the 50BGs/PLGA scaffolds decreased after 4 days. As shown in

Fig. 3-5, the release profiles of BGs from the 50BGs/PLGA scaffolds were an

initial burst release within 4 days upon contact with PBS. Figure 3-6 shows the

surface morphology of the scaffolds incubated for 7 days. Some beads on the

50BGs/PLGA scaffolds specifically had holes in their surfaces.

- 58 -

Fig. 3-5. Cumulative release of BGs from the electrospun scaffolds containing

different amounts of BGs into PBS at 37 oC during 16 days. The BGs

concentration in PBS at each time point was calculated with a BGSTAR kit.

- 59 -

Fig. 3-6. SEM images of (A, C) the PLGA scaffolds and (B, D) the 50BGs/PLGA

scaffolds incubated for 7 days at 37 oC.

- 60 -

D. Viability and proliferation of HDFs on electrospun BGs/PLGA

scaffolds

The viability and growth rate of HDFs on scaffolds were measured using

an ATP assay and a SEM (Figs. 3-7 and 3-8). The same amounts of cells were

seeded onto all scaffolds. For the 5 days after cell seeding, there were no

distinct differences in the growth rate associated with the BGs concentrations in

the scaffolds. The cell density significantly (*p < 0.05) increased in the

50BGs/PLGA scaffolds after 7 days. The 50BGs/PLGA scaffolds were

considered a significant effect on the growth rate of HDFs after 7 days. The

morphology of HDFs on scaffolds incubated for 5 days at 36 oC is shown in Fig.

3-8. On the both scaffolds, HDFs exhibited the polygonal shape that is typical

of the normal cell morphology. The growth rate of the HDFs was improved in

the 50BGs/PLGA scaffolds, and the morphology of the HDFs was the normal

shapes changes to the morphology of the HDFs in both scaffolds showed the

normal shapes.

- 61 -

Fig. 3-7. Influence of HDFs proliferation on the scaffolds containing different

amounts of BGs. After incubation for 1, 3, 5 and 7 days, the cell viability

was detected by an ATP assay. (*p < 0.05 versus the PLGA scaffolds at the

same time, as analyzed by a t-test with n = 5).

- 62 -

Fig. 3-8. SEM images of HDFs incubated for 5 days at 37 oC on (A, C) the PLGA

scaffold and (B, D) the 50BGs/PLGA scaffold.

- 63 -

E. Wound healing and histological examination

The full-thickness skin wounds were made on the back of each mouse

for a wound-healing test. Figures 3-9, 3-10 and 3-11 show the appearance of the

wounds after the operations. At 2 weeks after treatment, the connective tissues

had slightly repaired with some local infection. Microscopic findings did not

reveal any significant differences between the PLGA and the 50BGs/PLGA

scaffolds with respect to wound contraction at 2 weeks (Fig. 3-9). However, the

rate of re-epithelialization at 2 weeks is 29.23 ± 2.34% of PLGA scaffolds and

77.26 ± 10.08 % of 50BGs/PLGA scaffolds (Fig. 3-10). Also, the 50BGs/PLGA

scaffolds significantly enhanced the infiltration of the surrounding cells (Figs.

3-10 and 3-11). PLGA scaffolds can be clearly distinguished from the tissue 2

weeks after the operations. In contrast, the boundary line between the

50BGs/PLGA scaffold and the tissue was barely distinguishable in the wound

site, and cells were abundantly evident inside the 50BGs/PLGA scaffolds. At 2

weeks, the ECM was sufficiently expressed the surrounding of the

50BGs/PLGA compared with PLGA scaffolds.

- 64 -

Fig. 3-9. Appearance of the wounds in mice at 0, 1, 2 and 4 weeks after treatment

with the PLGA scaffolds and the 50BGs/PLGA scaffolds. (scale bar: 6 mm)

- 65 -

Fig. 3-10. Histological morphology with hematoxylin and eosin of wound sites treated

with the PLGA scaffolds and the 50BGs/PLGA scaffolds. (S: Scaffold,

Orange line: Normal epidermal, Yellow line: Re-epithelialization, scale bar:

2 mm)

- 66 -

Fig. 3-11. Interaction with the surrounding cells and the electrospun scaffolds. The

wound sites were treated with hematoxylin and eosin. (Scale bar: 200 µm)

- 67 -

F. Immunohistochemical analysis of wound healing

Figure 3-12 shows the representative immune localization of anti Ki-67

and anti CD 31 after 2 weeks in a wound treated with PLGA or 50BGs/PLGA

scaffolds. Ki-67 positive cells were counted at the basal layers of the epidermis

(Fig. 3-12). The positive cells that were stained Ki-67 were 77.2 ± 5.6 % in the

50BGs/PLGA scaffolds and 51.3 ± 7.0 % in the PLGA scaffolds. The strongest

positive staining for Ki-67 was evident after 2 weeks in the wound treated with

the 50BGs/PLGA scaffolds. Moreover, the wounds treated with the

50BGs/PLGA scaffolds showed significantly higher positive staining for CD 31

(34 ± 8.6 capillaries) than the PLGA scaffolds (22.7 ± 8.6 capillaries). After 4

weeks in the wound treatment, the wound sites treated with the 50BGs/PLGA

scaffolds were completely closing. Therefore, the Ki-67 positive cells in the

basal layer decreased compared with them which were treated after 2 weeks.

- 68 -

Fig. 3-12. Immunohistochemical observation of wounds treated with the PLGA

scaffolds and the 50BGs/PLGA scaffolds, and stained with anti Ki-67 and

anti CD 31. (A) Microscopic observation of immunohistochemical results

(S: scaffold, scale bar: 200 µm); (B) Quantitative analyses of cells showing

immunopositivity for Ki 67 or CD31 (*p < 0.05, as analyzed by one-way

followed by the Tukey HSD test).

- 69 -

4. Discussion

HDFs are the main cell type involved in the regulation of ECM protein

production in wound sites, and the formation of ECM promotes the granulation

tissue. Improvement of the HDF activity induces the acceleration of ECM

production, and the HDF activity is reflected by their growth rate.12-14

Keratinocytes is in epidermal layer and formed the barrier against

environmental damage such as pathogens, UV radiation and water loss.

Keratinocytes in wound sites grow from the survive cells and the hair follicle,

and then migrate for filling the gap created by the wound.15,16

The growth rate

and the migration of HDFs and NHEKs were improved by BGs in media (Figs.

3-2 and 3-3). The researchers have demonstrated that BGs stimulated fibroblast

NF-κB nuclear binding activity and IL-6 gene expression in a time-dependent

manner.17

NF- κB plays an important role in wound healing, inflammation and

apoptosis because the expression of the pro-inflammation cytokines such as

TNF-α, interleukin 1β (IL-1β) and bacterial lipopolysaccharides (LPS) are

controlled.18,19

According to the researches, these results suggest that BGs

directly enhance the wound healing in skin. Therefore, BGs has the potentiality

as the addition in the skin substitute for the accelerated wound healing.

The skin substitute was manufactured with the scaffolds composted with

PLGA and BGs using electrospinning (Fig. 3-4). The beads in scaffolds can be

expected BGs particles because the beads showed the holes after incubation for

7 days (Fig. 3-6). BGs particles suggested the disruption of the polymer chain

entanglement. The polymer chain entanglement causes the fiber formation, and

the high concentration of the entanglement induces the increase of the fiber

thickness.20

It suggested that BGs dispersed in the solution and then disrupted

the entanglements. Therefore, the increase of BGs concentration induced

increase of the bead numbers and the decrease of the fiber thickness. However,

BGs/PLGA scaffolds were able to be manufactured using electrospinning, and

BGs can be supplied in the wound site using BGs/PLGA scaffolds. Researchers

- 70 -

demonstrated that the attachment area and the growth rate of HDFs have been

reduced dramatically by the decrease in fiber diameters.12,21

According to the

results, the 50BGs/PLGA scaffolds cannot support sufficient areas for the

attachment and growth of HDFs as compared with the PLGA scaffolds.

However, HDFs showed normal shapes on both scaffolds (Fig. 3-8). The

growth rate of HDFs was improved in the 50BGs/PLGA scaffolds after

incubating for 7 days (Fig. 3-7). In previous studies, BGs dose dependently

enhanced the proliferation of HDFs on well plates for 5 days.6,22

According to

the release profile (Fig. 3-5), the BGs continually released from the scaffolds

during 16 days. The released BGs from the scaffolds can accelerate the HDF

activity by the improvement of the attachment area and the growth rate of HDFs

on the scaffolds. Therefore, the 50BGs/PLGA scaffolds could accelerate wound

healing by improving the HDF activity.

Clinically, it is a significant factor to wound repair as fast as possible.

For the acceleration of wound healing, the skin substitute required the

interaction with the surrounding cells and the excellent infiltration of them.23

Their interaction should directly influence cellular activity such as the adhesion,

proliferation, and migration of cells.13,22

BGs have been demonstrated to

stimulate the immune system and to enhance macrophage infiltration into injury

sites.9 In the present study, 50BGs/PLGA scaffolds significantly improved the

interaction with the surrounding cells and the infiltration of them (Figs. 3-10

and 3-11). These suggest that the BGs in the wound site can improve the wound

healing by the induction of the interaction with the surrounding cells and the

stimulation of inflammatory. It is demonstrated that phagocytes, natural killer

cells, dendritic cells and langerhans cells of skin has specific BGs receptor sites

that are termed dectin-1 and toll like receptor 2 and 6.22

The receptors initiate

the immune activity, and accelerate the pro-inflammatory factors such as

interferon-γ, TNF-α, IL-6 and IL-1β. Also, BGs have been demonstrated to

increase the expression of pro-inflammatory factors such as IL-6 and

- 71 -

interferon-γ in splenocytes.23,24

These factors are secreted by macrophages and

T cells to stimulate an immune response and promote proliferation, migration,

and activation.25

As a result, proliferated cells (the positive cells stained Ki-67)

were obtained at high levels in the wound site treated with 50BGs/PLGA

scaffolds as compared with those treated with PLGA scaffolds (Fig. 3-12). The

stronger staining of Ki-67 was considered that the keratinocytes were

proliferated on the basement layer. Improvement of keratinocytes proliferation

can enhance the re-epithelialization, because keratinocyte is a main cell of the

epidermal layer.26

Thus, 50BGs/PLGA scaffolds were regarded to improve the

re-epithelialization by the increase of keratinocyte proliferation on the basement

layer. IL-6 had been reported to have multiple functions in angiogenesis and

vascular remodeling. Fan et al. had concluded that IL-6 significantly improved

the proliferation of endothelial cells.27

Therefore, 50BGs/PLGA scaffolds

enhanced angiogenesis because of the promotion of IL-6 expression by BGs.

- 72 -

5. Conclusion

BGs have been demonstrated to enhance the cellular responses,

proliferation, and migration of HDFs. In this study, we investigated the possible

roles of the electrospun scaffolds including BGs for the acceleration of skin

wound healing. The proliferation of HDFs improved as the ratio of BGs

increased in the electrospun scaffolds. At 2 weeks after scaffolds application,

the 50BGs/PLGA scaffolds had enhanced the interaction of the wound with

surrounding cells, proliferation, and angiogenesis as compared with the PLGA

scaffolds. Therefore, the BGs/PLGA scaffolds may be applied as a skin

substitute for accelerated wound healing.

- 73 -

6. References

1. Peter K, Angela M. Wounds-from physiology to wound dressing. Dtsch

Arztebl Int 2008;105:239-48.

2. Ruiz HJ. Biosynthesis of beta-glucans in fungi. Antonie Van Leeuwenhoek

1991;60:72-81.

3. Portera CA, Love EJ, Memore L, Zhang L, Muller A, Browder W et al.

Effect of macrophage stimulation on collagen biosynthesis in the healing

wound. Am Surg 1997;63:125-30.

4. Delatte SJ, Evans J, Hebra A, Adamson W, Othersen HB, Tagge EP.

Effectiveness of beta glucan collagen for treatment of partial thickness

burns in children. J Pediatr Surg 2001;36:113-8.

5. Rose GD, Vervicka V, Yan J, Siz Y, Vetvickova J. Therapeutic

intervention with complement and beta-glucan in cancer.

Immunopharmacology 1999;42:61-74

6. Kougias P, Wei D, Rice PJ, Ensley HE, Kalbfleisch J, Williams DL et al.

Normal human fibroblasts express pattern recongnition receptors for fungal

(1, 3), (1, 6)-glucan. Mycol Res 2001;69:3933-8.

7. Woo YI, Park BJ, Kim H-L, Lee MH, Kim J, Yang YI et al. The biological

activities of (1, 3), (1, 6)-β-D-glucans and porous electrospun PLGA

membranes containing b-glucan in human dermal fibroblasts and adipose

tissue-derived stem cells. Biomed Mater 2010;5:044109.

8. Woo YI, Son HJ, Lim HR, Lee MH, Baek HS, Tsubaki K et al. Effects of

(1, 3), (1, 6)-β-D-glucans behavior in human dermal fibroblast cells under

serum starvation. Key Eng Mater 2007;4001:342-3.

9. Tada R, Yoshikawa M, Kuge T, Tanioka A, Ishibashi K-I, Adachi Y et al.

Granulocyto macrophage colony-stimulating factors is required for

cytokine induction by a highly 6 branched 1,3-β-D-glucan from

Aureobasidium Pullulans in mouse-derived splenocytes. Immnopharm

Immunotox 2011;33:302-8.

- 74 -

10. Tada R, Yoshikawa M, Kuge T, Tanioka A, Ishibashi K-I, Adache Y et al. A

highly branched 1,3-β-D-glucan extracted from Aureobasidium pullulans

induces cytokine production in DBA/2 mouse-derived splenocytes. Int

Immunopharmacology 2009;9:1431-6.

11. Tsubaki K, Tanioka A, Hatashima K, Shoji Y, Iwasawa H, Yamazaki M.

Immuno-modulation activities of new β-glucan from cereal and

fermentation food. Bromacil: Pharmacol Food Component 2008:147-69.

12. Tettamanti G, Grimaldi A, Rinaldi L, Arnaboldi F, Congiu T et al. The

multifunctional role of fibroblasts during wound healing in Hirudo

medicinalis (Annelida, Hirudinea). Biol Cell 2004;96:443-55.

13. Leong MF, Rasheed MZ, Lim TC, Chian KS. In vitro cell infiltration and

in vivo cell infiltration and vascularization in a fibrous, highly porous poly

(D,L-lactide) scaffold fabricated by cryogenic electrospinning technique. J

Biomed Mater Res 2009;91A:231-40.

14. Darby IA, Hewitson TD. Fibroblast differentiation in wound healing and

fibrosis. Int Rev Cytol 2007;257:143-79.

15. Werner S, krieg T, Smola H. Keratinocyte-fibroblast interactions in wound

healing. J Invest Dermatol 2007;127:998-1008.

16. Deters A, Dauer A, Schnetz E, Fartash M, Hensel A. high molecular

compounds (polysaccharides and proanthocyanidins) from Hammelis

virginiana bark: influence on human skin keratinocyte proliferation and

differentiation and influence on irritated skin. Phytochemisty

2001;58:949-58.

17. Ougias P, Wei D, Rice PJ, Ensley HE, Kalbfleisch J, Williams DL et al.

Normal human fibroblasts express pattern recognition receptors for fungal

(13)-beta-D-gluancs. Infect Immun 2001;69:3933-8.

18. Stoltz RA, Abraham NG, Schwartzman ML. The role of NF-κB in the

angiogenic respose of coronary microvessel endothelial cells. Proc Natl

Acad Sci 1996;93:2832-7.

- 75 -

19. Werner S, Krieg T, Smola H. Keratinocyte-fibroblast interaction in wound

healing. J Invest Dermatol 2007;127:998-1008.

20. Fujihara K, Teo W-E, Lim T-C, Ma Z, Ramakishna S. An introduction to

electrospinning and nanofibers. 1st ed. Singapore: World Sci Publisher;

2005.

21. Lowery JL, Datta N, Rutledge GC. Effect of fiber diameter, pore size and

seeding method on growth of human dermal fibroblasts in electrospun poly

(e-caprolactone) fibrous mats. Biomaterials 2010;31:491-504.

22. Sheterian A, Borboa A, Sawada R, Costantini T, Potenza B, Coimbra R et

al. Realtimeanalysis of the kinetics of angiogenesis and vascular

permeability in an animal model of wound healing. Burns 2009;35:811-7.

23. Gregory SS, Annette W. Interactions between extracellular matrix and

growth factors in wound healing. Wound Rep Reg 2009;17:153-62.

24. Chan GCF, Chan WK, Sze MY. The effects of β-glucan on human immune

and cancer cells. J Hematol Oncol 2009;2:25.

25. Barrientos S, Stojadinovic O, Golinko MS, Brem H, Carnic MT. Growth

factors and cytokines in wound healing. Wound Rep Reg 2008;16:585-601.

26. Rho KS, Jeong L, Lee G, Seo B-M, Park YJ, Hong S-D et al.

Electrospinning of collagen nanofibers: effects on the behavior of normal

human keratinocytes and early-stage wound healing. Biomaterials

2006;27:1452-61.

27. Fan Y, Ye J, Shen F, Zhu Y, Yeghiazarians Y, Shu W et al. Interleukin-6

stimulates circulating blood-derived endothelial progenitor cell

angiogenesis in vitro. J Cereb Blood Flow Metab 2008;28:90-8.

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IV. EVALUATION OF ELECTROSPUN EPIGALLOCATECHIN-3-O-

GALLATE (EGCG)/PLGA SCAFFOLDS

1. Introduction

In the normal physiology of wound healing, the levels of reactive

oxygen species and oxidative stress have influences on the wound healing

process. Wound sites exposed to the high levels of oxidative stress become

repaired.1 To inhibit wound site injury by oxidative stress, antioxidants have

been applied to balance oxidative stress on the wound sites.2-4

The green tea

extracts have antioxidant activities, antimicrobial effects and anti-inflammatory

effects. It has been studied that catechin epicatechin gallate (ECG), found in

green tea extract, improves the quality of wound healing in a full-thickness

wound healing model in rats. ECG enhances angiogenesis, nitric oxide synthase

activation, and cyclooxygenase-2 activation in the earliest phase of wound

healing. After this phase, ECG promotes the inhibition of these compounds.

These effects of ECG accelerate the compacts the epidermal layer.5 Especially,

epigallocatechin-3-O-gallate (EGCG), the major polyphenolic compound in

green tea, has much stronger antioxidant activities than vitamin C that is one of

the most utilized antioxidants.6 EGCG is regulated of mitochondrial integrity

and antioxidative enzyme activity of HDFs, resulting the increase of the HDFs

viability.7 The researches have focused on the wound healing effects of collagen

sponge immersed in EGCG with saline. Those studies showed that collagen

sponge immersed in EGCG solution can improve wound healing in diabetic

mice by accelerating re-epithelialization and angiogenesis.8 The studies suggest

being a potentially effective a skin substitute to the scaffolds including EGCG.

In the present study, we fabricated scaffolds with EGCG and PLGA

using the electrospinning method, and investigated the behavior of fibroblasts in

these scaffolds. Furthermore, the wound healing effects of the scaffolds were

evaluated in an animal study.

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2. Materials and methods

A. Viability of the skin-derived cells on EGCG

EGCG was purchased from TeavigoTM (DSM Nutritional Products Ltd.,

Basel, Switzerland), and solved in DMSO. HDFs and NHEKs were inoculated

in 24-weel culture plate with 1 mL medium. After 1 day incubation, the cells

were treated with EGCG in medium without growth factors. Cell viability was

quantified using MTT assay and measured using ELISA reader at a wavelength

of 570 nm.

B. Electrospun EGCG/PLGA scaffolds

PLGA and EGCG dissolved in DMF/THF. The PLGA concentration

was 20% (w/v) of the solvent volume, and the ratio between DMF and THF was

1:4. The EGCG concentrations were 1 wt% and 5 wt% of the total PLGA

weight. All scaffolds dried for 2 days were sterilized with 25 kGy gamma

irradiation at room temperature.

C. Characterization of electrospun EGCG/PLGA scaffolds

The surface morphology of the electrospun scaffolds was determined by

a SEM. For the analysis of the released EGCG patterns, the pre-warmed PBS

(37 oC in an incubation) was used. The calculated scaffolds were placed in the

pre-warmed PBS, and incubated at 36 oC in a shaking incubator. The scaffolds

in the PBS were protected from light until each time point. The EGCG

concentration in the PBS analyzed with ELISER reader at 275 nm.

D. Viability of HDFs on electrospun EGCG/PLGA scaffolds

HDFs were maintained in DMEM with all supplements. The HDFs were

incubated in humidified atmosphere containing 5% CO2 at 37 oC. The HDFs

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were placed in the scaffolds at a density of 5 x 104 cells per scaffold. HDFs on

the scaffolds were incubated for 5 days. The cell morphology was fixed and

determined using a SEM.

E. Animals and surgical manipulation

A single full-thickness skin wound (diameter of 12 mm) was created on

the upper back of each mouse (Male BALB/c nude mice, 7-week old). There

were the study groups (n = 6): PLGA scaffolds, 1EGCG/PLGA scaffolds

(scaffolds including 1 wt% EGCG). The study groups were shown in Table 4-1.

At 1, 2 and 4 weeks after implanted the scaffolds, the transplanted scaffolds and

the surrounding tissue were isolated, and then fixed. The fixed samples were

embedded in paraffin.

F. Determination and immunohistochemical analysis of wound area

The wound areas were observed by a digital camera and H&E staining.

The sections were scanning with a digital virtual microscope. Others isolated

samples were stained with Ki-67 and CD 31.

G. Statistical analysis

Cells proliferation assays were conducted in three independent cultures

for each experiment. Quantitative data are expressed as means ± SDs. Statistical

comparisons were carried out using the Student’s t-test (one-way). A p-value <

0.05 was considered statistically significant.

- 79 -

Table 4-1. The experimental numbers of nude mice for the animal study.

PLGA scaffolds: Fabrication with PLGA

1EGCG/PLGA scaffolds: Fabrication with PLGA and 1 wt% EGCG

Treatment times Experimental scaffolds Number of

nude mouse

1 week PLGA scaffolds

1EGCG/PLGA scaffolds

6

6

2 weeks PLGA scaffolds

1EGCG/PLGA scaffolds

6

6

4 weeks PLGA scaffolds

1EGCG/PLGA scaffolds

6

6

- 80 -

3. Results

A. Viability of the skin-derived cells on EGCG

In treated with EGCG, the viability of HDFs and NHEKs was reduced

with the increase of EGCG concentration (Fig. 4-1). EGCG has been

demonstrated to involve an apoptotic mechanism by the enhancement of

caspase-3 activity and the caspases activety, consequently show cytotoxicity at

the high concentration. The proliferation assay and the migration assay of HDFs

and NHEKs treated with EGCG did not experiment because EGCG has

cytotoxicity to HDFs and NHEKs according to viability assay.

- 81 -

Fig. 4-1. Cell viability effects of EGCG on (A) HDFs and (B) NHEKs. (*p < 0.05

versus the control group (0 µM), as analyzed by t-test).

- 82 -

B. Characterization of electrospun EGCG/PLGA scaffolds

Figure 4-2 shows the surface images of electrospun scaffolds observed

by a SEM. The fibers of the PLGA scaffold had 1.44 ± 0.14 μm of an average

fiber diameter, as measured from SEM images. The average fiber diameter of

the electrospun scaffolds with 1% EGCG (1EGCG/PLGA scaffold) and 5%

EGCG (5EGCG/PLGA scaffold) were 1.54 ± 0.36 μm and 1.07 ± 0.26 μm,

respectively. With the increase of EGCG concentration in the PLGA scaffolds,

the average fiber diameter decreased.

The release kinetics of EGCG from the scaffolds were investigated

using PBS. Figure 4-3 illustrates the cumulative release of EGCG and the

remaining EGCG concentration on the scaffolds. The EGCG on the scaffolds

were released in a logarithmic manner for up to 12 days. At 12 days after

immersion in PBS, the number of released EGCG from the 5EGCG/PLGA

scaffolds was about 5 times higher than that released from the 1EGCG/PLGA

scaffolds. The released EGCG percentage in the total EGCG on the scaffolds

was 16.5% for the 1EGCG/PLGA scaffolds and 16.2% for the 5EGCG/PLGA

scaffolds. These results suggest that EGCG is regularly released from scaffolds

without a burst phase.

- 83 -

Fig. 4-2. SEM images of the PLGA scaffolds and the EGCG/PLGA scaffolds.

- 84 -

Fig. 4-3. Cumulative release of EGCG from the electrospun scaffolds containing

different amounts of EGCG into PBS at 37 oC.

- 85 -

C. Viability and proliferation of HDFs on electrospun EGCG/

PLGA scaffolds

To examine the cytocompatibility of the PLGA scaffolds containing

EGCG, HDFs were incubated on them for 5 days. Figure 4-4 shows the HDFs

morphology on the PLGA scaffolds with and without EGCG. The HDFs

maintained normal cell morphology of the standard spindle form on the PLGA

scaffolds and the 1EGCG/PLGA scaffolds. HDFs lost their spindle shape and

exhibited a round shape on the 5EGCG/PLGA scaffolds. Their shapes

suggested that the 5EGCG/PLGA scaffolds supplied the EGCG that produce the

cytotoxicity. The PLGA scaffolds and the 1EGCG/PLGA scaffolds were used

for an animal study.

- 86 -

Fig. 4-4. SEM images of HDFs incubated for 5 days at 37 oC on the PLGA scaffold

(A), the 1EGCG/PLGA scaffold (B) and the 5EGCG/PLGA scaffolds (C).

- 87 -

D. Wound healing and histological examination

The appearance of the wound sites and the H&E stained sections is

illustrated on Figs. 4-5, 4-6 and 4-7. According to the photographic results (Fig.

4-5), there was no significant difference in the wound reduction effects between

the wounds treated with scaffolds with and without EGCG on any day. In

contrast, the histological observations showed significant results. According to

the H&E stained sections (Fig. 4-6), the rate of re-epithelialization at 2 weeks is

29.23 ± 2.34% of the PLGA scaffolds and 35.56 ± 1.46% of the 1EGCG/PLGA

scaffolds. However, the surrounding cells infiltrated well into the 1EGCG/

PLGA scaffolds compared to the PLGA scaffolds (Fig. 4-7). The ECM of the

surrounding of the 1EGCG/PLGA scaffolds also stronger expression than that

of the surrounding of the PLGA scaffolds. The inflammatory cells and the

epidermal cells were abundant within the 1EGCG/PLGA scaffolds, where the

epidermal cells migrated to the tops of the scaffolds. The inflammatory cells

and epidermal cells surrounded the PLGA scaffolds.

E. Immunohistochemical analysis of wound healing

Sections exposed to anti Ki-67 and anti CD 31 were used to determine

the number of proliferated and the endothelia cells. Figure 4-8 shows the

sections exposed to anti Ki-67 and anti CD 31, and the results of the

quantitative analysis of the positive cells. In wounds treated with the

1EGCG/PLGA scaffolds, the positive cells stained of Ki-67 and CD 31

significantly improved. The percentage of Ki-67 and the capillaries in wound

sites treated with the 1EGCG/PLGA scaffolds increase by 1.6 times and 1.2

times. These results suggested that re-epithelialization in wounds treated with

the 1EGCG/PLGA may improve the proliferation of basal epidermal cells, and

provide fluid from new capillaries.

- 88 -

Fig. 4-5. Appearance of the wounds in mice at 0, 1, 2 and 4 weeks after treatment

with the PLGA scaffolds and the 1EGCG/PLGA scaffolds. (scale bar: 6

mm)

- 89 -

Fig. 4-6. Histological morphology with hematoxylin and eosin of wound sites treated

with the PLGA scaffolds and the 1EGCG/PLGA scaffolds. (S: Scaffold,

Orange line: Normal epidermal, Yellow line: Re-epithelialization, scale bar:

2 mm)

- 90 -

Fig. 4-7. Interaction with the surrounding cells and the electrospun scaffolds. The

wound sites were treated with hematoxylin and eosin. (Scale bar: 200 µm)

- 91 -

Fig. 4-8. Immunohistochemical observation of wounds treated with the PLGA

scaffolds and the 1EGCG/PLGA scaffolds, and stained with anti Ki-67 and

anti CD 31. (A), Microscopic observation of immunohistochemical results

(S: scaffold, scale bar = 200 µm); (B), Quantitative analyses of cells

showing immunopositivity for Ki 67 or CD 31 (*p < 0.05, as analyzed by

one-way followed by the Tukey HSD test)

- 92 -

4. Discussion

The scaffolds composted with PLGA and EGCG for the skin substitute

was manufactured using electrospinning method, as shown in Fig. 4-1. The

fiber diameters decrease with the increase of EGCG concentration in the

electrospun scaffolds. Polymer fibers are formed by polymer chain

entanglement during electrospinning. The polymer chain entanglement is

influenced by the molecular weights of the polymers. The reduction molecular

weight suppresses the entanglement and reduces the average fiber diameter.8

The molecular weight of EGCG is 458.4 Da, and the molecular weight is lower

than other polymers’. The entanglement of PLGA chains can be disrupted by

EGCG during electrospinning; thus their fiber diameters decrease with an

increase of EGCG concentration (Fig. 4-2). However, the skin substitute can be

fabricated with PLGA and EGCG using electrospinning.

EGCG is expected to be release from the scaffolds during wound

healing when the EGCG/PLGA scaffolds are used for a skin substitute. EGCG

should be applied in the early phase of wound healing because neutrophils, mast

cells, macrophages, and lymphocytes are activated and proliferate in the wound

sites within 2 days after the injury.9 According to the release kinetics, EGCG is

released from the scaffolds over the span of 12 days (Fig. 4-3). As a result, the

EGCG/PLGA scaffolds can be released in the early phase of wound healing to

influence the process. The released EGCG from the scaffolds was also in direct

proportion to the EGCG concentration on the scaffolds. Therefore, the exposed

EGCG concentration in wound sites can easily be regulated by the EGCG

concentration in scaffolds. However, this suggests that the 5EGCG/PLGA

scaffolds may have cytotoxicity (Fig. 4-4) because HDFs on the 5EGCG/PLGA

scaffolds were transformed into uncommon shapes. Caspase-3 activity and

caspases in cells were enhanced by a high concentration of EGCG, and then

cells become an apoptosis.10,11

These studies suggest that the 5EGCG/PLGA

scaffolds may supply the EGCG concentration that entails the cytotoxicity.

- 93 -

The evidence indicates that wound healing has multiple mechanisms,

and these responses consist of migration of immune cells to wound sites,

proliferation of skin cells and angiogenesis.4,9,12

The migration of immune cells

to wound sites and the proliferation of skin cells induce the reduced wound size

which is important to alleviate suffering, protect from infection and avoid of

dehydration. Newly formed capillaries in wound sites, one of the responses of

the wound healing mechanism, provides nutria supplements and the required

cytokines to the cells that are located in the wound sites.13

According Figs. 4-8,

wound sites treated with the 1EGCG/PLGA scaffolds significantly improved

with the percentage of Ki-67 and CD31 positive cells. Ki-67 is expressed in

proliferating cells and is widely used as a proliferation marker. The expression

of CD 31 in wound sites is suggested to cause the formation of new capillaries

because CD 31 is a maker of the endothelial cells. Thus, wound sites treated

with the 1EGCG/PLGA scaffolds experience accelerated proliferation of

epithelial cells, and formation of new capillaries, compared to the wound sites

treated with the PLGA scaffolds. The 1EGCG/PLGA scaffolds can enhance

re-epithelialization through the acceleration of proliferation of epithelial cells.

Therefore, the 1EGCG/PLGA scaffolds can be used as a skin substitute for the

improvement of wound healing.

5. Conclusion

In conclusion, the wound healing effects of the fabricated scaffolds were

investigated using a full-thickness wounds created in nude mice. The

5EGCG/PLGA scaffolds induced morphologic transformation of the HDFs. The

1EGCG/PLGA scaffolds enhanced the infiltration of the surrounding cells, and

significantly increased the immunoreactive of proliferation cells and

angiogenesis. Therefore, the scaffolds containing low EGCG concentration can

be applied to a skin substitute to influence wound healing with EGCG.

- 94 -

6. Reference

1. Fitzmauricea SD, Sivamania RK, Isseroffa RR. Antioxidant therapies for

wound healing: a clinical guide to currently commercially available

products. Skin Pharmacol Physiol 2011;24:113-26.

2. Davis RH, Leitner MG, Russo JM, Byrne ME. Wound healing, oral and

topical activity of aloe vera. J Am Podiatr Med Assoc 1989;79:559-62.

3. Maheshwari RK, Singh AK, Gaddipati J, Srimal RC. Multiple biological

activities of curcumin: a short review. Life Sci 2006;78:2081-7.

4. Sen CK, Khanna S, Gordillo G, Bagchi D, Bagchi M, Roy S. Oxygen,

oxidants, and antioxidants in wound healing. Ann NY Academy Sci

2002;957:239-49.

5. Kapoor M, Howard R, Hall I, Appleton I. Effects of epicatechin gellate on

wound healing and scar formation in a full thickness incisional wound

healing model in rat. Am J Pathol 2004;165:299-307.

6. Elbling L, Weiss L-M, Teufelhofer O, Uhl M, Knasmueller S, Rolf S-H et

al. Green tea extract and epigallocatechin-3-gallate, the major tea catechin,

exert oxidant but lack antioxidant activities. FASEB J 2005;19:807-9.

7. Meng Q, Velalar CN, Ruan R. Effects of epigallocatechin-3-gallate on

mitochondrial integrity and antioxidative enzyme activity in the aging

process of human fibroblast. Free Radic Biol Med 2008;44:1032-41.

8. Kim HH, Kawazoe T, Han D-W, Matsumara K, Suzuki S, Tsutsumi S et al.

Enhanced wound healing by an epigallocatechin gallate-incorporated

collagen sponge in diabetic mice. Wound Rep Reg 2008;16:714–20.

9. Fujihara K, Teo W-E, Lim T-C, Ma Z, Ramakishna S. An introduction to

electrospinning and nanofibers. 1st ed. Singapore: World Sci Publisher;

2005.

10. Diegelmann FR, Evans MC. Wound healing: an overview of acute, fibrotic

and delayer healing. Front Biosci 2004;9:283-9.

11. Islam S, Islam N, Kermode T, Johnstone B, Mukhtar H, Moskowitz RW et

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al. Involvement of caspase-3 in epigallocatechin-3-gallate-mediated

apoptosis of human chondrosarcoma cells. Biochem Biophys Res Commun

2000;270:793-7.

12. Babich H, Krupka ME, Nissim HA, Zuckerbraun HL. Differential in vitro

cytotoxicity of epicatechin gallate (ECG) to cancer and normal cells from

the human oral cavity. Toxicol in Vitro 2005;19:231-42.

13. Priya SG, Jungvid H, Kumar A. Skin tissue engineering for tissue repair

and regeneration. Tissue Eng Part B Rev 2008;14:105-18.

14. Numata Y, Terui T, Okuyama R, Hirasawa N, Sugiura Y, Miyoshi I et al.

The accelerating effect of histamine on the cutaneous wound healing

process through the action of basic fibroblast growth factor. J Invest

Dermatol 2006;126:1403-9.

- 96 -

< ABSTRACT(IN KOREAN)>

생리활성물질의 창상치유 작용과

복합 지지체로써의 적용

<지도교수 박종철>

연세대학교 대학원 의과학과

김 혜 리

피부손상은 다양한 세포와 여러 성장인자들에 의해 염증기와

증식기, 재형성기를 통해 치유된다. 심한 화상이나 외과수술로 인해

피부손상이 진피까지 광범위하고 형성되면 3차원 구조의 피부 대치

물을 사용한다. 현재 상용화된 피부 대치물은 천연고분자를 이용하

여 지지체를 만들거나, 제조한 지지체에 피부유래세포 또는 성장인

자를 내포시켜 제조한다. 그러나 천연고분자는 기질금속단백질 분해

효소의 과발현을 유도하며, 세포를 배양하기 위해 제조시간은 긴데

반해 짧은 유통기한을 갖는다는 문제점이 있다. 그러므로 본 연구에

서는 생분해성 합성고분자 중 Poly (lactic-co-glycolic acid) (PLGA)를

이용하여 3차원 구조의 피부 대치물을 만들고자 하였다. PLGA는 생

분해속도를 쉽게 조절할 수 있으며, 생체적합성이 우수하여 주로 사

용되는 고분자이다. 또한 고분자를 섬유형태로 제조하여 적층하는

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방법인 전기방사법을 이용하여 다공성의 지지체를 제조하고자 하였

다. 또한 지지체에 천연 생리활성물질을 혼합하여 창상치유를 촉진

하고자 하였다. 천연 생리활성물질로는 창상치유 효과가 있다가 보

고되고 있는 물질 중 (1,3)-(1,6)-β-D-glucans (BGs)과 Epigallocatechin-

3-O-gallate (EGCG)를 사용하였다. BGs는 글루코스 폴리머로 창상부

위의 면역반응을 활성화시키고, 녹차추출물인 EGCG는 창상부위의

피부세포와 cytokine의 분비를 조절하여 창상치유를 촉진시킨다고

보고되고 있다.

첫 번째로 전기방사법의 공정조건에 따른 PLGA 지지체의 변화

를 확인하였다. 제조된 인공피부의 균일한 물성을 위하여 전기방사법

으로 제조한 PLGA 섬유의 획일성을 확인하였다. PLA와 PGA의 비율

이 75:25인 PLGA를 tetrahydrofuran (THF)와 N,N dimethylmethanamide

(DMF)이 혼합된 용매 (THF/DMF)나 1,1,1,3,3,3,-hexafluoro-2-propanol

(HFIP)에 녹였을 때 균일한 섬유가 형성되는 것을 확인하였다. 또한

드라이 아이스를 이용하여 공정온도를 낮추어 주면 PLGA 섬유와 얼

음 알갱이가 같이 적층되어 pore의 크기를 증가시켰다. Pore 사이즈가

증가함에 따라 human dermal fibroblasts (HDFs)의 침투도 향상되었으므

로 드라이 아이스에 의한 pore 크기의 증가가 주변 세포의 침투를 용

이하게 하여 창상부위의 치유를 촉진시킬 수 있을 것으로 판단된다.

생분해성 폴리머는 고열과 고온, 압력, 고습에 약하여 멸균하는 동안

형태의 변형이 유발된다. 본 연구에서 전기방사법으로 제조한 PLGA

지지체를 direct current (DC) treatment를 이용하여 단시간에 지지체의 모

양 및 분자량의 변화 없이 멸균할 수 있었다. 그러므로 전기방사한

지지체와 유사하게 가능 파이버로 만들어진 지지체의 멸균으로 DC

treatment의 활용이 가능할 것으로 판단된다.

제조한 PLGA 지지체에 생리활성물질인 BGs (BGs/PLGA)와

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EGCG (EGCG/PLGA)를 혼합하고자 하였다. BGs이 HDFs의 성장을 촉

진시키므로 BGs/PLGA 지지체에서도 HDFs의 성장이 촉진되었다. 반

면 세포독성을 보이는 EGCG는 고농도의 EGCG를 포함하고 있는

EGCG/PLGA 지지체에서 HDFs의 독성이 확인되었다. 그러므로, 총

PLGA 무게의 50 wt% BGs를 혼합한 50BGs/PLGA 지지체와 총 PLGA

무게의 1 wt% EGCG를 혼합한 1EGCG/PLGA 지지체를 이용하여 동물

실험을 진행하였다. H&E 염색 결과를 토대로 50BGs/PLGA 지지체와

1EGCG/PLGA 지지체로 인공피부로 사용하였을 때, PLGA 지지체보다

창상부위의 재상피화가 촉진되었으며, 지지체로의 세포 침투도 활성

화된 것을 확인할 수 있었다. 또한 50BGs/PLGA 지지체와 1EGCG

/PLGA 지지체로 창상부위에 처리하였을 때, the basal layer에서 증식세

포의 양 (Ki-67 staining)과 형성된 진피층에서 신생혈관 (CD 31)이 확

연하게 증가하였음을 확인할 수 있었다.

본 연구를 통하여 전기방사법을 이용하여 세포의 침투가 용이

한 지지체를 만들 수 있었으며, DC treatment를 이용하여 변형 없이 멸

균을 할 수 있는 것을 확인할 수 있었다. 또한 BGs 또는 EGCG를

PLGA와 혼합하여 지지체를 제조함으로써 지지체의 재생과 창상부위

의 신생혈관 형성 및 재상피화를 촉진하여 창상치유 효과를 촉진시킬

수 있을 것으로 판단된다. 따라서 BGs 또는 EGCG를 포함하고 있는

지지체는 창상치유를 촉진시킬 수 있는 피부 대치물로 활용이 가능할

것으로 판단된다.

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핵심되는 말: 창상치유, 피부 대치물, 생분해성 지지체, poly (lactic-co-glycolic

acid), (1,3)-(1,6) 베타글루칸, 에피갈로카테킨-3-O-갈레이트, 전기방사, direct-

current treatment