Conjugation of hyaluronic acid

with ascorbic acid and

evaluation of

its in vitro activity on MC3T3-E1

Dongjeong Park

Department of Medical Science

The Graduate School, Yonsei University

Conjugation of hyaluronic acid

with ascorbic acid

and evaluation of its

in vitro activity on MC3T3-E1

Directed by Professor Jong-Chul Park

The Master's Thesis

submitted to the Department of Medical Science,

the Graduate School of Yonsei University

in partial fulfillment of the requirements for the degree of

Master of Medical Science

Dong jeong Park

December 2008

This certifies that the Master's Thesis

of Dongjeong Park is approved.

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

Thesis Supervisor: Jong-Chul PARK

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

Jin Woo LEE

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

Dong Hee LEE

The Graduate School

Yonsei University

December 2008

Acknowledgements 지난 시간을 돌이켜보면 항상 아쉬움이 남지만, 저에게 있어 석사 2년

은 그 어느 때 보다 아쉬움과 부족함이 많은 2년 이였던 거 같습니다.

우선, 대학원이란 곳에서 공부할 수 있는 기회를 주신 지도교수이신 박

종철 교수님께 감사 드립니다. 실험이 잘 되지 않을 때에도 항상 격려하

여 주시고 실험에 전념할 수 있게 배려해 주신 교수님의 지도하에 미약

하나마 이렇게 성과를 낼 수 있었습니다. 바쁘신 와중에도 심사위원으로

서 많은 지도말씀 해주신 이진우 교수님께도 감사의 인사 드립니다. 그

리고 이 논문의 아이디어를 제공해 주신, 멀리 수원에서도 많은 가르침

을 주신 이동희 박사님께도 감사의 말씀 드립니다. BMP 실험에서부터 이

동희 박사님이랑은 실험적으로 계속 인연이 이어지는 것 같습니다.

실험실 막내로 들어왔던 제가 어느덧 졸업을 앞두고 있습니다. 실험실에

서 부족하고 실험에 대해 아무것도 모르는 나를 인내로써 잘 이끌어준

내겐 사수나 다름없는 미희언니에게도 감사드려요. 그리고 우리 실험실

의 군기반장이자 왕 고참이신 현숙 언니 결혼을 축하 드립니다. 행복하

세요~ 실험실의 조력자로 항상 든든한 버팀목이 되어 준 연이 언니와 지

금은 결혼하셔서 행복한 신혼을 즐기고 계실 혜련 언니에게도 감사 인사

드립니다. 입학 동기이자 우리 실험실의 청일점인 수창오빠도 졸업 축하

드립니다. 똑 부러지면서도 왠지 친근한 혜리 언니, 새롭게 실험 배우시

느라 고생이 많으신 대형선생님, 마지막 신관 파트너이자 BMP실험 열심

히 하고 있는 슬로바키아 댁 바보라, 늦둥이이자 실험실의 귀염둥이 들

인 재경이와 병주, 연구비와 방비 관리까지 고생이 많으셨던 금주언니까

지 지난 2년 동안 참 많은 일들이 있었고, 좋은 추억 많이 가져갑니다.

또한, 제 첫 논문인 AEPSE 손보시느라 많이 고생하셨을 한동욱박사님 정

말로 감사 드립니다. 박사님 덕분에 논문에 대해 많이 배웠습니다. 부산

대에서 훌륭한 업적 많이 만드세요! 결혼도 축하드립니다!

이렇게 막상 감사 인사 드리려니 생각나는 분들이 너무나도 많습니다.

무엇보다도 학위과정 무사히 마칠 수 있도록 승환이 보시면서 내색한 번

많고 힘들게 도와주신 어머님 아버님. 두 분의 도움이 없었으면 아마 이

렇게 논문을 쓰지 못했으리라 생각합니다. 2년 동안 부족한 며느리 챙겨

주시느라 정말 고생 많으셨습니다. 그리고 곁에서 누나 도시락 싸주면서

잘 챙겨준 하나뿐인 동생 동민이와 틈틈이 영양제 가져다 주시며 힘을

주셨던 학업과 업무에 바쁘신 하나뿐인 도련님에게도 감사합니다. 항상

저에게 힘이 되어 주시는 정신적 지주이신, 멀리 있지만 마음만은 느껴

지는, 더 뵙고 싶은 부모님께 이 논문을 바칩니다. 그리고 마지막으로

항상 옆에서 힘이 되어 주는 나의 조력자이자 영원한 파트너 효철 오빠

에게 이 자리를 빌어 고맙다는 인사 다시 전합니다.

2008년 12월 박동정 드림

i

TABLE OF CONTENTS Abstract ··············································································································1

І. INTRODUCTION ··························································································3

1. Hyaluronic acid ························································································3

2. Modification of hyaluronic acid ······························································4

3. Role of ascorbic acid in bone ··································································4

4. Objective of this study ············································································ 6

II. MATERIALS AND METHODS ··································································7

1. Materials ··································································································7

2. Conjugation of hyaluronic acid with sodium ascorbate ··························7

3. Conjugational analysis ·············································································7

4. Stability of ascorbic acid ·········································································8

5. Cell culture ······························································································ 8

6. Cytotoxicity & Proliferation test ······························································8

7. Assay of alkaline phosphatase activity ·····················································8

8. Type Ι collagen expression ·······································································8

9. Bone mineralization test ···········································································9

10. Statistical analysis ··················································································10

III. RESULTS ···································································································11

1. Conjugation scheme ···············································································11

2. Cytotoxicity ···························································································13

3. Proliferation test······················································································15

ii

4. ALP activity ························································································17

5. Type Ι collagen expression ··································································19

A. Western blotting ···········································································19

B. Sirius red staining ········································································21

C. ELISA results ···············································································26

6. Bone mineralization test ·····································································28

A. Alizarin red staining ·····································································28

B. Von Kossa staining ·······································································31

7. Browning test·······················································································33

8. GPC analysis ·······················································································35

IV. DISCUSSION ·························································································37

V. CONCLUSION ························································································40

REFERENCES······························································································41

ABSTRACT (IN KOREAN) ········································································46

iii

LIST OF FIGURES

Figure 1. Chemical modification point of HA. HA can be variously modified for

application. Hydroxyl group of glucosamine can be modified by

acetlyation, ether, aldehyde, epoxy reaction, carboxyl group of glucoronic

acid by ester cross linking. ····················································5

Figure 2. Expected reaction scheme of HA-AA conjugation 2 steps were processed

for conjugation. Scheme 1 shows carboxyl group activation by NHS,

scheme 2 shows AA substitution process. Reactions were performed in

phosphate buffered saline solution at pH 7.5. ········································12

Figure 3. Cytotoxicity of HA-AA conjugate. Cell cytotoxicity on MC3T3-E1 cells

was investigated by MTT assay performed 24 hours after seeding. The

data reported for conjugation made by HA: AA ratio 1:1 (w/v), and

concentration up to 2.0mg/ml. There was no significant difference

between all tested groups (by Bonferroni test). ·····································14

Figure 4. Cellular proliferation assay of HA-AA conjugate. HA, AA, HA-AA

conjugate proliferation test on MC3T3-E1 cells up to 7 days. HA and

HA-AA conjugate treated concentration 0.5 and 1.0mg/ml, AA treated at

25, 50uM. (n=4, *: p < 0.05 by student’s t- test) ···································16

Figure 5. ALP assay. Dose dependent effect of HA-AA conjugate on alkaline

phosphatase up to 14 days (up to 2mg/ml). HA-AA conjugate showed

ALP activity up to 2.0mg/ml dose dependently. (n=3, *: p < 0.05 by

student’s t- test) ······················································································18

Figure 6-1. Type Ι collagen expression by western blotting (at 7 day). 0.5mg/ml,

1.0mg/ml conjugate induced type 1 collagen. AA induced collagen

expression dose dependently. However, HA suppressed collagen

expression. ······························································································20

Figure 6-2. Type Ι collagen expression by sirius red staining (without osteogenic

media). (A~E) showed stained collagen fiber at 7 day, (F~J) at 21 day.

iv

(A, F) - negative control, (B, G) - 50ug/ml ascorbic acid, (C, H) -

50ug/ml ascorbic acid, 0.5mg/ml hyaluronic acid mixture, (D, I) -

0.5mg/ml hyaluronic acid, (E, J) - 0.5mg/ml HA-AA conjugate

·················································································································22

Figure 6-3. Quantitative analysis by Sirius red staining (without osteogenic media).

For quantitative analysis, collagen bound sirius red was extracted using

0.01N NaOH. After 7, 14, 21, 28 day. (n=4, *: p < 0.05 by student’s t-

test) ·········································································································23

Figure 6-4. Type Ι collagen expression by sirius red staining (with osteogenic

media). (A~E) showed stained collagen fiber at 7 day, (F~J) at 21 day.

(A, F) - negative control, (B, G) - 50ug/ml ascorbic acid, (C, H)-

50ug/ml acorbic acid, 0.5mg/ml hyaluronic acid mixture, (D, I) -

0.5mg/ml hyaluronic acid, (E, J)- 0.5mg/ml HA-AA conjugate

·················································································································24

Figure 6-5. Quantitative analysis by sirius red staining (with osteogenic media). For

quantitative analysis, collagen bound sirius red was extracted using

0.01N NaOH. After 7, 14, 21, 28 day. (n=4, *: p < 0.05 by t- test) ······25

Figure 6-6. Type Ι collagen expression by ELISA. Quantitative analysis of

expressed type Ι collagen in 100ul cell culture conditioned media at 21

day with osteogenic media (n=2). With standard curve calibrated. ·······27

Figure 7.1 Effect of HA-AA conjugate on mineralization: Alizarin red staining. 7,

21 and 28 day results are shown. (A) - negative control, (B) - 50ug/ml

ascorbic acid, (C) - 50ug/ml ascorbic acid, 0.5mg/ml hyaluronic acid

mixture, (D) - 0.5mg/ml hyaluronic acid, (E) - 1.0mg/ml hyaluronic acid,

(F) - 0.5mg/ml HA-AA conjugate, (G) - 1.0mg/ml HA-AA conjugate

·················································································································29

Figure 7-2. Quantitative analysis of alizarin red staining. Data were quantified by

Alizarin Red S (ARS) assay via extraction with etylpyridinium chloride

(CPC). The amount of released dye was quantified by microplate reader

v

at 570 nm. Mineralization was significantly decreased in HA-AA

conjugate treatment compared to the control. *Versus cultures in

osteogenic medium alone (*: p<0.05 by t-test). The result is

representative of four different experiments. ·········································30

Figure 7.3 Effect of HA-AA conjugate on mineralization: von Kossa staining. The

black spots are reduced calcium. 7, 21 and 28 day results were showed.

(A) - negative control, (B) - 50ug/ml ascorbic acid, (C) - 50ug/ml

ascorbic acid, 0.5mg/ml hyaluronic acid mixture, (D) - 0.5mg/ml

hyaluronic acid, (E) - 1.0mg/ml hyaluronic acid, (F) - 0.5mg/ml HA-AA

conjugate, (G) - 1.0mg/ml HA-AA conjugate ········································32

Figure 8. Browning test. The change of color was observed for 21 days. Data show

sample before acceleration and at 3, 7 day (A), 21 day (B) ··················34

Figure 9. GPC results of HA-AA conjugate. HA, AA and HA-AA conjugate was

examined by Gel Permeation Chromatography analysis. With infrared

ray detector, molecular weight of HA, AA and HA-AA were calculated.

Profile of HA (A), AA (B) and HA-AA conjugate (C) ··························36

vi

Abbreviations

HA: hyaluronic acid

AA: ascorbic acid

HA-AA conjugate: hyaluronic acid- ascorbic acid conjugate

NHS: n- hydroxysuccinimide

FBS: fetal bovine serum

α-MEM: minimum essential medium – alpha modification

MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

β –GP: β-glycerophosphate

ALP: alkaline phosphatase

ELISA: enzyme-linked immunosorbent assay

ARS: alizarin red staining

CPC: cetylpyridinium chloride

GPC: gel permeation chromatography

IR: infrared ray

NMR: nuclear magnetic resonance

1

Abstract

Conjugation of hyaluronic acid with ascorbic acid and evaluation of

its in vitro activity on MC3T3-E1

Dongjeong Park

Department of Medical Science

The Graduate School, Yonsei University

(Directed by Professor Jong-Chul Park)

Hyaluronic acid (Hyalronan, HA), β-1,4-linked D-glucuronic acid and β-1,3 N-

acetyl-D-glucosamine polysaccharide, is a nonsulfated glycosaminoglycan (GAG)

conserved in extracellular matrix (ECM). Due to its biocompatibility, biodegradable

properties, HA is widely applied for tissue engineering. However, HA also has

defects for tissue engineering such as mechanical properties, difficulty of handling.

Thus, it is variously modified by chemical reactions to produce HA derivative. HA

plays an important role in tissue morphogenesis, proliferation and cell

differentiation. Ascorbic acid (AA) has an effect on collagen synthesis and bone

mineralization. Ascorbate levels also have a significant effect on osteoblast

proliferation and alkaline phosphatase (ALP) expression. However, AA is weak to

heat and light, thus it is easily degradable. Therefore, we conjugated HA with AA in

order to make it more stable and effective. In this study, we prepared HA-AA

conjugate and evaluated activity of products in pre-osteoblast.

To produce more effective conjugation, we synthesized HA derivative, HA-N-

hydroxysuccinimide, an activated ester of the glucuronic acid moiety. This HA-

2

active ester intermediate is a precursor for drug-polymer conjugates. The degree of

substitution was calculated by NMR analysis. The modified HA was dialyzed and

lyophilized. The yield of conjugation is calculated by Gel Permeation

Chromatography (GPC). After the process, HA was conjugated with AA once again

as previously mentioned. In this study, the resultant HA-AA conjugate was tested on

MC3T3-E1, murine pre-osteoblast cells. We examined cellular viability

(cytotoxicity), proliferation and type Ι collagen expression. The expression of Type І

collagen was examined by sirius red staining, ELISA assay kit and western blot.

Alkaline phosphatase assay was also performed. For confirmation on bone

mineralization, alizarin red staining and von Kossa staining was performed.

In conclusion, the in vitro data demonstrate that HA-AA conjugate has an

important role in bone formation, as it can increase proliferation and osteogenic

differentiation of MC3T3-E1 cells. These observations further support its use in in

vivo system for tissue engineering applications.

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

Key words: hyaluronic acid, sodium ascorbate, conjugation, MC3T3-E1, type І

collagen

3

Conjugation of hyaluronic acid with ascorbic acid and evaluation of

its in vitro activity on MC3T3-E1

Dongjeong Park

Department of Medical Science

The Graduate School, Yonsei University

(Directed by Professor Jong-Chul Park )

I. INTRODUCTION

1. Hyaluronic acid (Hyaluronan, HA)

Hyaluronic acid is one of the widely used materials in tissue engineering, which is

linear highly polymerized compound consisting of β-(1→4)-linked D-glucuronic

and β-(1→3)-N-acethyl-D-glucosamine. HA is universally found in ECM,

especially in connective tissues1,2. Because HA has various molecular weight from

1000 to 10,000,000 dalton, shows unique physicochemical properties and

distinctive biological functions3. The effects of HA are not only non-biological

functions such as, hydration of tissue4, structure support1 but also biological activity

such as, development of embryonic stem cells5 and cancer cell development6,7,8,

anti-bacterial effect9. HA also interacts with other molecules and participates in

regulating cell behaviors (cell migration, differentiation9,10 and adhesion11). Also

because of its universality and easy hydrolysis by hyaluronidase, HA is known to

have good bio-adaptability.

In tissue engineering, HA has many important application in drug delivery and

surgery. For example, HA is used as an adjuvant for ophthalmic drug delivery12, and

it was reported to enhance the absorption of drugs and proteins via mucosa tissues 13,14. Locally applied HA does not result in any phagocytosis or immunological

4

reactions15.

But, even though HA has such advantages, HA has difficulties in application due

to its physical chemical properties. To overcome this problem, modified HA was

widely used.

2. Modification of hyaluronic acid

Chemical modification of HA allows enhancement of physical and chemical

properties while retaining its natural biocompatibility, biodegradability, and lack of

immunogenicity16. Functional modification of carboxylic groups on HA, polymer-

drug conjugate for controlled drug release, uses of HA-derived biochemical

probes17 have been recently studied (Figure 1).

In this study, ascorbic acid was conjugated with hyaluronic acid for controlled

ascorbic acid release.

5

Figure 1. Chemical modification points of HA. HA can be variously modified for

application. Hydroxyl group of glucosamine can be modified by acetylation, ether,

aldehyde, epoxy reaction, carboxyl group of glucoronic acid by ester cross linking.

6

3. Role of ascorbic acid in Bone

Ascorbic acid, also known as vitamin C, plays important role in connective tissue

in synthesis of collagen and in differentiation of undifferentiated osteoblasts. In case

of osteogenesis mediated by BMP, AA regulated osteogenesis18, which was proved

by increase of alkaline phospatase activity and type І collagen expression19.

Ascorbic acid is important for collagen synthesis in connective tissue due to its role

as a cofactor for proline hydroxylase and lysine hydroxylase20,21, which are involved

in hydroxylation of collagen. At the post translational modification process, AA

plays a critical role in formation of triple helix structure of collagen. In addition,

osteoblast differentiation mediated by 1,25(OH)2 vitamin D3, retinoic acid, and

bone morphogenetic proteins was also affected by the concentration of ascorbic

acid22,23,24,25. Thus, ascorbic acid appears to be essential for normal bone

formation26,27.

4. Objective of this study

In this study, we made HA-AA conjugate for ascorbic acid delivery system. This

study focused on released AA from the conjugate with HA and, its ability to

enhance new bone formation effect in vitro. Also earlier study reported that ascorbic

acid plays a role as potential hyaluronidase inhibitors1 that means AA could help to

prolong the effect of HA. Our previous research observed that AA was more stable

in a conjugated state in conditions of high temperature and humidity. Based on these

results, conjugated HA-AA derivative would have more stability and activity for

bone repair.

7

II. MATERIALS AND METHODS

1. Materials

Hyluronic acid sodium salt (Mw 12kD, from Streptococcus equi spp.) was

purchased from Fluka Bio-Chemika (Buchs, Switzerland). N-hydroxysuccinimide, a

conjugate intermediate28, sodium ascorbate, dialysis tubing cellulose membrane

were obtained from Sigma-aldrich (St Louis, MO). In this study we used ascorbic

acid derivative, sodium ascorbate because it is buffered and less acidic. So it has

more bioavailability.

2. Conjugation of hyaluronic acid with sodium ascorbate

At first step, HA was dissolved in PBS solution, pH value was adjusted at 3.5, 7.5,

10.5. NHS was mixed in HA solution in ratio 1:1% (w/v) at room temperature. The

reaction was performed under nitrogen gas fuzzing system to prevent the influence

of oxygen. Product was dialyzed for overnight to removed unreacted NHS and

lyophilized. Then, the same procedure was reported repeated with sodium ascorbate

to produce HA-AA conjugate. HA: AA ratio was modified 1:1 or 1:2% (w/v).

3. Conjugational analysis (GPC analysis)

Gel permeation chromatography (GPC) analyses were performed on a Waters GPC

system using PL aqua gel - OH 60 (7.8 x 300 mm) column with Waters 515 pump

system. Analyses were performed at 35℃ with mobile phase of Buffer pH 7

solution at a flow rate of 1.0 mL/min and monitored using Waters 2410 Differential

Refractometer + Waters 490E Programmable Multi wavelength Detector

(measurement wavelength : 254nm). The injection volume was 100μL, and result

data was dealt with EMPOWER. Standard curve was calculated by polysaccharide.

The substitution of carboxyl group was determined by 1H-NMR 400 MHz FT-NMR

spectrometer (Varian Inova AS 400, Varian, USA) 29.

8

4. Stability of ascorbic acid

The stability of conjugated AA was monitored by browning test. Oxidized AA

turned brown in water solution30. We studied free AA and conjugated AA under

severe conditions, at 50℃ for 21 days, monitored and photographed.

5. Cell culture

Murine pre-osteoblast, MC3T3-E1 cells, obtained from ATCC (subclone 4, CRL-

2593) cultured in α-MEM media with 10% fetal bovine serum and 1% penicillin-

streptomycin purchased from Welgene (Daegu, Korea). Cells were cultured in at 37℃

in a 5% CO2 atmosphere incubator.

6. Cytotoxicity & Proliferation test

For cytotoxicity test, 1X105cells were seeded on 24 well plate and added HA-AA

conjugate. After 2 days, increase in cell number was measured by 3-[4,5-

dimethylthiazol-2]-2,5-dipheniltetrazolium bromide (MTT) viability assay.

Proliferation assay was studied 1X104 cells/well in 12-well plate for 7 days. Media

was changed 2-3times a week.

7. Assay of alkaline phosphatase activity

Cell were plated at 3X103 cells/ well in 48 well plate and cultivated in the presence

of HA-AA conjugate. ALP activity was determined using 4mg/ml phosphate

substrate and 1.5mM alkaline buffer solution which were mixed and treated with

cells. Optical density was measured directly by ELISA at 405nm31. Cell number (by

MTT assay), p-nitrophenol concentration was calibrated to determine ALP level.

8. Type 1 collagen expression

Our target gene, type І collagen expression was studied by western blotting, sirius

red staining using The Sircol™ Collagen Assay kit (biocolor, Northern Ireland)32

and with enzyme-linked immunosorbent assay (ELISA) kit (USCN Life Science

Technology Company, Wuhan, China) as described by the manufacturers. In briefly,

9

sirius red staining was performed like this33,34,34,35. Cells were seeded at 5X104 cells/

well in 24 well plate for 21 days. Cells were incubated with or without osteogenic

media (consisting of basal media, 10nM dexamethasone, 10mM β-

glycerophosphate31). Before staining, cells were fixed by 10% formalin solution for

12 hours at 4℃. After the cells were washed twice with distilled water, they were

incubated with sirius red stain for an hour at room temperature. After the staining,

cells were washed with 0.1N HCl twice and dried. Stained collagen fibers were

photographed. For quantification, cells stained with sirius red (n =4) were destained

with 0.01N NaOH for 30 min , then the extracted stain was transferred to a 96-well

plate, and the absorbance at 570 nm was measured using a microplate reader, as

previously described36.

USCN mouse type 1 collagen ELISA kit was designed for cell culture supernatauts,

serum, plasma. We used cell culture conditioned media with HA-AA conjugate for

21 days. The microtiter plate provided has been pre-coated with a biotin-conjugated

immobilized antibody specific to mouse type 1 collagen. Standard and samples

were added to the appropriate microtiter plate. Following a wash to remove any

unbound antibody-enzyme reagent, a substrate solution was added to the wells and

color developed in proportion to the amount of collagen Type Ⅰ bound in the

initial step. The color development was stopped and the intensity of the color was

measured spectrophotometically at a wevelengh of 450nm. The concentration of

collagen in the samples was then determined by comparing the optical density of the

samples to the standard curve.

9. Bone mineralization test

To detect calcium deposition, two bone mineralization assays were

preformed37,38,39.

MC3T3-E1 cells were incubated in a 6-well plate for a month. After incubation

culture medium was aspirated and the cells were washed with PBS three times.

Before staining, cells were fixed with methanol: 10% (w/v) formalin: distilled water

=1:1:1 solution during overnight. For alizarin red staining, with 40mM alizarin red

10

staining solution (pH 6.8) treated for 15 minutes, followed by rinsing with distilled

water five times. In fresh PBS, the stain was photographed. For quantification, cells

were destained with 10% (w/v) cethylpyridinium chloride (CPC) for 15 minutes,

and the extracted staining solution was transferred into a 96-well plate. Absorbance

was measured at 570nm40,41,42,43,44.

At the same time, von Kossa staining method was performed to detect phosphate

deposits in bone nodules45,46,47. 5% Silver nitrate treated plates were exposed to UV

lamp for an hour after which the plates were rinsed with distilled water. Fresh 5%

sodium carbonate (in 25% formalin) was added up to 5 min for development, the

plates were then rinsed with water three times, and 5% sodium thiosulphate was

added for fixation. The plates were washed with water and dried for image analysis.

10. Statistical analysis

Results are expressed as means standard error (SE). Statistical analysis was carried

out by analysis of variance (ANOVA) and specific comparisons between groups

were analyzed by Student t- test or the Bonferroni (SPSS 12.0 for Windows

software, Chicago, IL, USA)

11

II. RESULTS

1. Conjugation of hyaluronic acid with ascorbic acid

HA-AA conjugation was processed by two steps (Figure 2). At first, n-

hydroxysuccinimide (NHS) was utilized to activate carboxyl group of hyaluronic

acid. Then, unreacted NHS was removed by dialysis membrane and the product was

lyophilized.

Then, with dialysis membrane NHS which is not reacted was removed and

lyophilized. Second, with NHS AA was substituted via ester bond caused by

dehydration. Dialyzed and lyophilized HA-AA conjugate resultant was produced.

Whole substitution reaction was performed under nitrogen gas fuzzing system to

prevent influence of oxygen.

12

Figure 2. Expected reaction scheme of HA-AA conjugation. 2 steps were processed

for conjugation. Scheme 1 shows carboxyl group activation by NHS, scheme 2

shows AA substitution process. Reactions were performed in phosphate buffered

saline solution at pH 7.5.

13

2. Cytotoxicity of HA-AA conjugate

Before application to MC3T3-E1 cells, cellular toxicity was investigated. There

was no toxicity under concentration 2.0mg/ml. According to HA: AA ratio, toxicity

showed different. Cytotoxicity of HA: AA = 1: 2 (w/v) product showed more

toxicity than concentration 1.5mg/ml. But, three different pH conditions (pH 3.5,

7.5, 10.5) did not affect toxicity at the same HA: AA conjugation ratio. We tested in

vitro system on HA-AA conjugate made at pH 7.5 and HA-AA ratio 1:1 (Figure 3).

14

Figure 3. Cytotoxicity of HA-AA conjugate. Cellular cytotoxicity on MC3T3-E1

cells was investigated by MTT assay performed 24 hours after seeding. The data

reported for conjugation made by HA: AA ratio 1:1 (w/v), and concentration up to

2.0mg/ml. There was no significant difference between all tested groups (by

Bonferroni test).

15

3. Cellular proliferation assay of HA-AA conjugate

We found that HA-AA conjugate inhibited cellular proliferation until 7 days

(Figure 4). Both HA and AA were also shown to have inhibitory effect in

comparison with the untreated groups (n=4, t-test, significantly differences at *:

p<0.05). This result coincided with previous study on HA to MC3T3-E1.

16

Figure 4. Cellular proliferation assay of HA-AA conjugate. HA, AA, HA-AA

conjugate proliferation test on MC3T3-E1 cells up to 7 days. HA and HA-AA

conjugate treated concentration 0.5 and 1.0mg/ml, AA treated at 25, 50uM (n=4, * :

p < 0.05 by student’s t- test).

0

20

40

60

80

100

120

140

160

1 day 3 day 5 day 7 day

prol

ifera

tion

(% o

f con

trol

)

Day

control 0.5mg/ml conjugate1.0mg/ml conjugate25uM AA50uM AA0.5mg/ml HA1.0mg/ml HA

*

*

*

*

*

**

*

**

*

*: p< 0.05

*

*

**

*

17

4. Activity of alkaline phosphatase

Alkaline phosphatase activity on MC3T3-E1 cells was significantly increased after

culture with HA-AA conjugate. Up to 2.0mg/ml the expression was increased, on

the other hand, more than 2.0mg/ml the expression was rather than decreased. This

result was similar with previous study about HA induced ALP activity. HA and AA

also induced ALP level but, the increase was less than HA-AA conjugate (Figure 5).

18

Figure 5. ALP assay. Dose dependent effect of alkaline phosphatase on HA-AA

conjugate up to 14 days (up to 2mg/ml). HA-AA conjugate induced ALP activity up

to 2.0mg/ml dose dependently (n=3, * : p < 0.05 by student’s t- test).

0

500

1000

1500

2000

2500

3000

1 3 5 7 10 14

ALP

act

ivity

(% o

f co

ntro

l )

day

0.0mg/ml1.0mg/ml2.0mg/ml3.0mg/ml4.0mg/ml

*

*

*

**

* *

* *

**

*

*

19

5. Type І collagen expression

5-1. Type І collagen expression by western blotting

Expression of type І collagen, our target protein, was increased in presence of HA-

AA conjugate. Figure 6-1 shows western blotting result of conjugate at 7 days.

Although expression of collagen was examined for a month, due to difficulties to

detect collagen protein by western blotting, the data show only results at 7 day. AA

also induced collagen expression, but conjugate showed more stronger effect.

20

Figure 6-1. Type 1 collagen expression by western blotting (at 7 day). 0.5mg/ml,

1.0mg/ml conjugate induced type 1 collagen. AA induced collagen expression is

dose dependently. However, HA suppressed collagen expression.

21

5-2. Type І collagen expression by Sirius red staining

Sirius red staining was performed with (Figure 6-2,3) or without (Figure 6-4,5)

osteogenic media. Osteogenic media was used with 10nM dexamethasone, 10mM β

-glycerophosphate. These results are shown without osteogenic media. As time

increases, the expression of collagen also increased especially in 50ug/ml AA,

0.5mg/ml conjugate treated groups.

For quantitative analysis, collagen bound sirius red was extracted using 0.01N

NaOH. After 7 days, 50ug/ml AA and 0.5mg/ml conjugate treated cells significantly

formed collagen fibers. On the other hand, HA showed rather on inhibition effect.

At 28 day, conjugate was most effective to synthesize of collagen fibers (Figure 6-

3,5).

22

Figure 6-2. Type І collagen expression by Sirius red staining (without osteogenic

media). (A~E) showed stained collagen fiber at 7 day, (F~J) at 21 day. (A, F) -

negative control, (B, G) - 50ug/ml ascorbic acid, (C, H) - 50ug/ml acorbic acid,

0.5mg/ml hyaluronic acid mixture, (D, I) - 0.5mg/ml hyaluronic acid, (E, J) -

0.5mg/ml HA-AA conjugate

23

Figure 6-3. Quantitative analysis by Sirius red staining (without osteogenic media).

For quantitative analysis, collagen bound sirius red was extracted using 0.01N

NaOH. After 7, 14, 21, 28 day (n=4, * : p < 0.05 by student’s t- test).

0

50

100

150

200

250

300

350

400

7 14 21 28

Qua

ntita

tive

anal

ysis

(% o

f con

trol

)

day

control

0.5mg/ml HAHA,AA mixture50ug/ml AA0.5mg/ml conjugate

*

*

*

**

*

*

*

**

24

These results show sirius red staining with osteogenic media.

Figure 6-4. Type І collagen expression by Sirius red staining (with osteogenic

media). (A~E) showed stained collagen fiber at 7 day, (F~J) at 21 day. (A, F) -

negative control, (B, G) - 50ug/ml ascorbic acid, (C, H)- 50ug/ml ascorbic acid,

0.5mg/ml hyaluronic acid mixture, (D, I) - 0.5mg/ml hyaluronic acid, (E, J)-

0.5mg/ml HA-AA conjugate

25

Figure 6-5. Quantitative analysis by sirius red staining (with osteogenic media). For

quantitative analysis, collagen bound sirius red was extracted using 0.01N NaOH.

After 7, 14, 21, 28 day (n=4, * : p < 0.05 by t- test).

0

50

100

150

200

250

7 14 21 28

Qua

ntita

tive

anal

ysis

(% o

f con

trol

)

day

control

0.5mg/ml HA

HA, AA mixture

50ug/ml AA

0.5mg/ml conjugate

**

* **

*

* * *

*

26

5-3. Type І collagen expression by ELISA

Collagen expression was also studied by ELISA. USCN ELISA kit was designed

to detect collagen protein presence in cell culture supplement. After 21 days,

collagen expression was observed on HA and HA-AA conjugate. 1.0mg/ml HA-AA

conjugate showed about 2.7ng/ml collagen concentration (Figure 6-6).

27

Figure 6-6. Type І collagen expression by ELISA assay. Quantitative analysis of

expressed type І collagen in 100ul cell culture conditioned media at 21 day with

osteogenic media (n=2). With standard curve calibrated.

28

6. Bone mineralization assay

6.1 Alizarin red staining

The last step of bone differentiation, mineralization assay was studied for 28 days.

In alizarin red staining, only 1.0mg/ml HA- AA conjugate treated cells showed

mineralization (Figure 7-1). Using CPC, absorbance was measured at 570nm

(Figure 7-2).

29

Figure 7-1 Effcet of HA-AA conjugate on the mineralization: Alizarin red staining.

7, 21 and 28 day results were showed. (A) - negative control, (B) - 50ug/ml ascorbic

acid, (C) - 50ug/ml ascorbic acid, 0.5mg/ml hyaluronic acid mixture, (D) -

0.5mg/ml hyaluronic acid, (E) - 1.0mg/ml hyaluronic acid, (F) - 0.5mg/ml HA-AA

conjugate, (G) - 1.0mg/ml HA-AA conjugate

30

Figure 7-2. Quantitative analysis of alizarin red staining. Data was quantified by

Alizarin Red S (ARS) assay via extraction with etylpyridinium chloride (CPC). The

amount of released dye was quantified by microplate reader at 570 nm.

Mineralization was significantly decreased in HA-AA conjugate treatment

compared to the control. *Versus cultures in osteogenic medium alone (*: p<0.05 by

t-test). The result is representative of four different experiments.

0

100

200

300

400

500

600

700

800

900

7 14 21 28

Qua

ntita

tive

anal

ysis

of A

RS

(% o

f con

trol

)

Day

control

HA (0.5mg/ml)HA (1.0mg/ml)AA (50ug/ml)HA, AA mixtureconjugate (0.5mg/ml)conjugate (1.0mg/ml)

*

*

*

*

31

6.2 Von Kossa staining

As experimental period increased, calcium deposition was significantly increased.

The black spots are reduced calcium, these were seen by naked eyes. HA-AA

conjugate induced new bone generation until last step of bone differentiation

process. In HA-AA conjugate treated groups, calcium deposition was observed

especially 1.0mg/ml treated (Figure 7-3).

32

Figure 7-3 Effect of HA-AA conjugate on mineralization: von Kossa staining. The

black spots are reduced calcium. 7, 21 and 28 day results were showed. (A) -

negative control, (B) - 50ug/ml ascorbic acid, (C) - 50ug/ml acorbic acid, 0.5mg/ml

hyaluronic acid mixture, (D) - 0.5mg/ml hyaluronic acid, (E) - 1.0mg/ml hyaluronic

acid, (F) - 0.5mg/ml HA-AA conjugate, (G) - 1.0mg/ml HA-AA conjugate

33

7. Browning test

To prove whether ascorbic acid was stable or not in conjugated state browning test

was performed (Figure 8). Ascorbic acid is known as very weak to heat and light.

Oxidized AA turned brown in water solution. At 50℃, a condition of high

temperature and humidity state accelerator, the change of color was observed for 21

days. This result indicated that ascorbic acid was more stable in conjugated state.

And it is also shown that volume of distilled water was decreased in high

temperature due to evaporation, but water with HA-AA conjugation did not show

the decrease. This result is thought to be due to high water retention capacity of HA.

34

Figure 8. Browning test. The change of color was observed until 21 days, Data were

shown at before acceleration, 3, 7 day - (A) and 21 day- (B).

21 DayB

Before 7 Day3 Day

free conjugated conjugatedconjugated freefree

A

conjugated free

35

8. GPC analysis

Molecular weight of HA , AA and HA-AA conjugate were determined by GPC

Results (Figure 9). We used 12000kD HA, the peak was 16290kD Mw. HA has

tendency to show higher molecular weight in water soluble solvent shows higher

than actual in GPC analysis. In profile of HA-AA conjugate (C), two peaks were

shown. One of HA (retention time at 7.4 min) the other is AA (retention time at 10

min). With these results, we knew the presence of HA and AA in HA-AA conjugate,

but whether HA and AA was correctly conjugated or not was not proved. Also,

further study by UV detection would be needed to prove conjugation.

36

Figure 9. GPC results of HA-AA conjugate. HA, AA and HA-AA conjugate was

examined by Gel Permeation Chromatography analysis. With infrared ray detector,

molecular weight of HA, AA and HA-AA were calculated. Profile of HA (A), AA

(B) and HA-AA conjugate (C)

37

V. DISCUSSION

HA was widely used for scaffold or drug delivery system in tissue engineering.

Due to its bio-adaptability, bio-degradable and non immunogenic properties, it is

known as good biomaterial for application. Our study was designed for HA not only

as scaffold but also AA delivery system. We made HA-AA conjugate via ester bond

that would be fragile to esterase cleavage in cell presence culture media or body

serum. Released AA could induce synthesis of collagen to generate new bone.

Our conjugation scheme used in this study was 2 had way steps (Figure 2). NHS

has been found to modify side-groups on proteins to make them reactive with other

side groups and to mediate the ester bond formation between the hydroxyl and

carboxyl groups of HA. Therefore, it has been used in modification of HA.

In the present study, our HA-AA conjugate did not show cellular cytotoxicity

(Figure 3). Results of cytotoxicity were similar with cytotoxicity of HA in a

previous study48. Resultant conjugate inhibited cellular proliferation (Figure 4) same

as hyaluronic acid and ascorbic acid used separately. It was reported that depending

on the tissue or origin, exogenous HA induced or inhibited proliferation49.

At first, the activity of conjugate was examined by ALP expression test (Figure 5).

Up to 2.0mg/ml the expression was increased, on the other hand, at concentrations

higher than 2.0ml/ml, the expression has rather decreased. This result was similar

with the previous study on HA induced ALP activity. HA and AA induced also ALP

activity but, the increase was lower than in case of HA-AA conjugate (data did not

shown).

Type 1 collagen expression also increased in presence of HA-AA conjugate

(Figure 6). Ascorbic acid is required for the formation of collagen matrix. AA

influences hydroxylation of proline and lysine necessary for molecular stabilization

of collagen. It is well known tool to detect protein expression by western blotting.

However, it was not easy to detect type І collagen expression by western blotting

(Figure 6-1) due to its own triple helix structure. Therefore, sirius red staining and

ELISA method was also performed.

38

Sirius red staining is presented as a method for collagen determination, enabling

quantitative morphometric measurements to be performed. HA-AA conjugate

showed as collagen expression like 50ug/ml ascorbic acid (Figure 6-2,3). The

concentration of 50ug/ml was used as a conditioned media for bone differentiation.

This concentration is known to be effective concentration for in vitro differentiation.

HA did not increase type І collagen expression and showed no synergetic effect

with ascorbic acid. Expression of collagen was significantly induced by 50ug/ml

AA and 0.5mg/ml HA-AA conjugate, in the presence or absence of osteogenic

media (Figure 6-4,5). In case of osteogenic media untreated cells, expression of type

a collagen was more significant.

Secreted collagen concentration was also measured (Figure 6-6). Using ELISA,

collagen in cell cultured 100ul conditioned media was quantified. HA treated cells

showed 0.07ng/ml (0.5mg/ml HA treated), 0.13ng/ml (1.0 HA treated) of collagen.

In case of 1.0mg/ml HA-AA conjugate 2.27ng/ml collagen was secreted (Figure 6-

6).

AA also induced calcium deposition and bone mineralization of bone. Alizarin red

staining and von Kossa staining are well known methods for mineralization assay.

In alizarin red staining, 3 weeks after 1.0mg/ml conjugate treatment, mineral

deposition was stained. But, other experimental groups did not shown mineral

deposition (Figure 7-1, 2). As a case of von Kossa staining, at 4 week reduced

calcium was detected (Figure 7-3). But AA alone did not induce bone mineral

deposition. These results indicate our conjugate is more effective to induce than

bone calcium deposition more than ascorbic acid alone.

We confirmed HA-AA conjugate prolonged activity by browning test (Figure 8).

AA has its activity only in a reduced form. Factors such as heat, air, metal make AA

oxidized form, and cause loss of activity. According to these results, oxidation of

AA was protected by conjugated state. Conjugated AA was more stable to heat and

water solution. We tested ALP assay again after incubated severe condition. HA, AA

mixture and HA-AA conjugate were incubated 50℃ for a month, and then treated

into culture media. At 14th day, the activity of conjugate was decreased much less

39

than HA and AA mixture (data did not shown).

However our conjugation protocol has also limitations. First, whether HA and AA

was correctly conjugated or not was not proved. Through GPC analysis, we

confirmed molecular weight of HA, AA HA-AA conjugate using RI detector

(Figure 9). But conjugate state could be examined by UV detector. Conjugated AA

would be detected in area of macro molecular range of retention time. Also, using

such as Multi-angle light scattering macromolecular (MALS), conjugational yield

could be calculated. The substitution of carboxyl group of hyaluronic acid was

tested by 1H- and solid NMR. But data could not be analyzed.

Based on these results, further analysis would be needed, and other modifications

could be made to HA-AA conjugate. Also because our HA-AA conjugate model

was designed to be degradable by esterase present in body serum, release profile of

AA, with esterase should be investigated.

40

V. CONCLUSION

In conclusion, the HA-AA conjugate had increased activity on MC3T3-E1 cells. It

can be suggested that our conjugate induced ALP activity, expression of type І

collagen and bone mineralization. Due to high molecular weight of HA, NMR and

GPC results could not be successfully explained. Thus, correct analysis should be

performed in further studies. Although it was not proved that our conjugate

conjugated correctly, it showed cellular activity and stability in in vitro assay. In this

study, HA-AA conjugate showed highest effectivity at concentration 0.5mg/ml and

1.0mg/ml which is similar to the effective concentration of hyaluronic acid.

This conjugation system could be adapted also for use in other drug delivery

system. Based on this study, our HA-AA conjugate derivative could be used for

further applications.

41

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Abstract (In Korean)

히아루론산-아스코르브산의 접합과 미분화 골아세포에서의

활성 효과

<지도교수 박 종 철 >

연세대학교 대학원 의과학과

박 동 정

골 이식재로 최근 많이 사용되는 히아루론산은 조직의 수화작용 및 구

조 유지, 세포의 발달 및 운동성에 있어 중요한 역할을 하며, 생체적합

성이 뛰어나고 hyaluronidase에 의하여 생분해 되어 생체 친화적이라는

이점을 가지고 있다. 아스코르브산은 골 세포의 분화 및 증식 작용에 있

어 필수적인 성장요소이나 빛과 열에 약하다는 단점을 가지고 있다.

본 연구에서는 골이식재를 위한 히아루론산에 아스코르브산을 접합하여

히아루론산 지지체에 본래의 기능에 아스코르브산의 활성을 더하여 조골

세포의 좀 더 높은 활성을 가진 골 이식재를 만들고자 하며, in vitro

상에서 이 복합체가 각각의 순 물질보다 활성 및 물질의 안정성이 더 뛰

어남을 보이고자 한다.

히아루론산과 아스코르브산의 접합을 위해선 N- hydroxysuccinimide

(NHS)를 사용하여 두 단계의 반응으로 좀 더 접합이 잘 일어나도록 하였

으며, 접합한 복합체는 투석 과정을 통하여 정제하였다. 접합 과정에서

47

pH 및 히아루론산, 아스코르브산의 비율을 달리하여 높은 수율의 접합이

일어나는 조건을 찾도록 하였고, GPC 분석으로 확인하였다. 생성된 복합

체는 미 분화 조골세포인 MC3T3-E1을 이용하여 생체 적합성 시험(세포

생존도 및 증식시험)과 활성시험(Type І collagen 과 Alkaline

phosphatase의 발현 정도 확인), 뼈의 미네랄 침착시험을 알리자린 레드

염색과 본 코사 염색으로 칼슘의 침착을 확인하였다.

이를 바탕으로 접합된 히아루론산과 아스코르브산 유도체는 골 이식을

위한 높은 활성도를 보였으며 이를 근거로 향후 연구를 전망해 보고자

한다.

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핵심되는 말: 히아루론산, 아스코르브산, 접합, 미분화 골아세포, 제 І

형 콜라겐