preparation and characterization of demineralized bone ... · preparation and characterization of...

9
Polymer (Korea), Vol. 28, No. 5, pp 382-390 (2004)// , \28Ì \5x, 2004, pp 382-390 41#Ý# í>rÖ~#e)ZÁ#Ù#-u©Ñc#eÁ#åÙ# =r®#==Õ#í##u#$®# "=./#ñV/#ÙºÕ . /#½µ .. /#í .. /#ññ¦ .. /#µ ... / %# Àý@# ÄÐ5</# @èí# <@# UU¸</# # -0¬# ظôì0¬@/# DÁ@# ÄÐZظ5<# # +5337#4# 53¼# QX/#5337#<#5¼# ,# Preparation and Characterization of Demineralized Bone Particle-loaded PLGA Scaffold for Tissue Engineered Bone Ji Wook Jang, Bong Lee, Chang Whan Han*, Mun Suk Kim**, Sun Hang Cho**, Hai Bang Lee**, and Gilson Khang*** ,% Department of Polymer Engineering, Pukyong National University, San 100, Nam Ku, Busan 608-739, Korea *Department of Orthopedic Surgery, College of Medicine, The Catholic University of Korea, 520-2, Daeheung 2 Dong, Jung Ku, Daejeon 301-723, Korea **Nanobiomaterials Laboratory, Korea Research Institute of Chemical Technology, P. O. Box 107, Yuseong, Daejeon 305-600, Korea ***Department of Polymer¯Nano Science and Technology, Chonbuk National University, 664-14, Dukjin Dong 1Ga, Dukjin Ku, Jeonju 561-756, Korea e-mail : [email protected] (Received January 20, 2004;accepted September 2, 2004) 5¨¹ôA©q Ü0ì Q ظ 8dÄ\ (Ä (demineralized bone particle, DBP) (Uq(q (BMP) ¨` Èô (XD=Dpx, (BMSC S ) ļ `$. 8 0¬\Ô DBP¼ ¨` 4ì ½´ (PLA) ½´-@ì¼´ 5Q©ô (PLGA) $5q ô¼ é$ ÐäE/ <ÔÜÕ| \ °@, X$5aU O DÐ<ìD8ý ´é, ùqðU @$. BMSCsÔ (Ä p¼ ´é , °(x, Ä\ Wright-Giemsa, Alizarin red, von Kossa O ALP <| ¸@$. DBP@ ¨`\ ô DBP@ ¨`X ô BMSCs¼ LÅ ´-ð Dðä | ýÅ, ´ $ (Uq U¼ T, 4$. ô $5Ô 90.2% ´´ $5¬p 69.1 µm ´´$. BMSCsÔ Wright-Giemsa, Alizarin red, von Kossa O ALP <ð< °(x, Ä@ @åÈ |°, |$ XÉ ð< DBP@ ¨`\ ô\ ¼Ø¨i A- ¸ X È DBP@ ¨` X ô\Ô ¼Ø¨i A- ¸ û@$. ð A| DBP¼ ¨` ô\ DBP BMSCs@ (Uq QÔ Ô¸| Ñé$ ì\$. ,-897,.9¹One of the significant natural bioactive materials is demineralized bone particle (DBP) whose has a powerful inducer of new bone growth. In this study, we developed the DBP loaded poly-lactide (PLA) and poly(L- lactide-co-glycolide) (PLGA) scaffolds for the possibility of the application of the tissue engineered bone. PLA/DBP and PLGA/DBP scaffolds were prepared by solvent casting/salt leaching method and were characterized by porosimeter, scanning electron microscopy. BMSCs were stimulated by osteogenic medium and characterized by histological stained Wright-Giemsa, Alizarin red, von Kossa, and alkaline phosphate activity (ALP). DBP impregnated scaffolds with BMSCs were implanted into the back of athymic nude mouse to observe the effect of DBP on the osteoinduction compared with control scaffolds. It can be observed that the porosity was above 90.2% and the pore size was above 69.1 µm. BMSCs could be differentiated into osteoprogenitor cells as result of wright- giemsa, alizarin red, von Kossa and ALP staining. In in vivo study, we could observed calcification region in PLA/DBP and PLGA/DBP groups, but calcification did not occur almost in control scaffolds. From these results, it seems that DBP as well as BMSCs play an important role for bone induction in PLA/DBP and PLGA/DBP scaffolds. Keywords¹scaffold, DBP, BMSCs, osteogenesis, tissue engineered bone.

Upload: others

Post on 19-Mar-2020

8 views

Category:

Documents


0 download

TRANSCRIPT

Page 1: Preparation and Characterization of Demineralized Bone ... · Preparation and Characterization of Demineralized Bone Particle-loaded PLGA Scaffold for Tissue Engineered Bone Ji Wook

Demo

���� Polymer (Korea), Vol. 28, No. 5, pp 382-390 (2004)� �

���� ���, �28� �5�, 2004�, pp 382-390�

������

����������������������������

�������������������

������������������

�������

�������

�������

������

������������������������������ �

����������������������������������������

� ������� ��� �������������� ��� ������

Preparation and Characterization of Demineralized Bone Particle-loaded PLGA Scaffold for Tissue Engineered Bone

Ji Wook Jang, Bong Lee, Chang Whan Han*, Mun Suk Kim**, Sun Hang Cho**, Hai Bang Lee**, and Gilson Khang***,�

Department of Polymer Engineering, Pukyong National University, San 100, Nam Ku, Busan 608-739, Korea *Department of Orthopedic Surgery, College of Medicine, The Catholic University of Korea,

520-2, Daeheung 2 Dong, Jung Ku, Daejeon 301-723, Korea **Nanobiomaterials Laboratory, Korea Research Institute of Chemical Technology,

P. O. Box 107, Yuseong, Daejeon 305-600, Korea ***Department of Polymer�Nano Science and Technology, Chonbuk National University,

664-14, Dukjin Dong 1Ga, Dukjin Ku, Jeonju 561-756, Korea †e-mail : [email protected]

(Received January 20, 2004;accepted September 2, 2004)

�������� ���� � ��� ������ �� (demineralized bone particle, DBP)� ������ (BMP) ��� �� �������� (BMSCS)� ��� ���. � ����� DBP� �� ��

���� (PLA)� ����-������ ���� (PLGA) ��� ���� �� ���/���� � �����, ������ � ������� ��� ���� ���. BMSCs� ��� ��� ���

����� ���� Wright-Giemsa, Alizarin red, von Kossa � ALP �� � �����. DBP� �� ���� DBP� ��� �� ���� BMSCs� ��� � ��� ������ ��� ��� ��� ��� ��� ��� ���. ��� ���� ���� 90.2% ����� �� ����� 69.1 µm �����. BMSCs� Wright-Giemsa, Alizarin red, von Kossa � ALP ���� ����� ��� ���

�, ���� �� �� DBP� �� ����� ���� �� � ���� DBP� �

�� �� ������ ���� ���� ����. ��� � DBP� �� ����� DBP� BMSCs� ���� ��� �� � ����� ����. ���������One of the significant natural bioactive materials is demineralized bone particle (DBP) whose has a powerful inducer of new bone growth. In this study, we developed the DBP loaded poly-lactide (PLA) and poly(L-lactide-co-glycolide) (PLGA) scaffolds for the possibility of the application of the tissue engineered bone. PLA/DBP and PLGA/DBP scaffolds were prepared by solvent casting/salt leaching method and were characterized by porosimeter, scanning electron microscopy. BMSCs were stimulated by osteogenic medium and characterized by histological stained Wright-Giemsa, Alizarin red, von Kossa, and alkaline phosphate activity (ALP). DBP impregnated scaffolds with BMSCs were implanted into the back of athymic nude mouse to observe the effect of DBP on the osteoinduction compared with control scaffolds. It can be observed that the porosity was above 90.2% and the pore size was above 69.1 µm. BMSCs could be differentiated into osteoprogenitor cells as result of wright-giemsa, alizarin red, von Kossa and ALP staining. In in vivo study, we could observed calcification region in PLA/DBP and PLGA/DBP groups, but calcification did not occur almost in control scaffolds. From these results, it seems that DBP as well as BMSCs play an important role for bone induction in PLA/DBP and PLGA/DBP scaffolds. Keywords�scaffold, DBP, BMSCs, osteogenesis, tissue engineered bone.

Page 2: Preparation and Characterization of Demineralized Bone ... · Preparation and Characterization of Demineralized Bone Particle-loaded PLGA Scaffold for Tissue Engineered Bone Ji Wook

Polymer (Korea), Vol. 28, No. 5, 2004� Demineralized Bone Panticle-loaded Porous Scaffold � �����

���, �28� �5�, 2004� ������ �� �� ��� ��� � ����

�����

���� ��� ���� ��� ���� �� ��

� ��� ����� ��� �� ��� �� ��

���� �� ��� �� ��������� ��.1

������� �� ���, ����, ���� ���

���� ����, ��� ���� �� ��� �

���� ����� ����� ��� � � �

� ��� ��� �� � �� ���� �� ��

��. ������� ���� ������ � �

�� ��� ���� ����� ��� ���� �

��� ��� �� ���� � �� ��� �

� ��� �� ���. ��� ��� ��� �� �

�� � ��, ���� ��� ���� ���� �

��� ��� �� ���� ���� ��� ���

����, ������ ��� ��� ���� ���

��.2,3

�� α-������ �� �������� (PGA),

������ (PLA), � ��� ����� ��(L-lactide-

co-glycolide) (PLGA)� �� FDA� �� �� ���

���� ����� ��� ���, ���� ���

�� ����� �� ���� ��.4-6 PLA� PLGA�

�� �����, ����, ��� ��� � �,

�� PLGA� ���� ��� �� ���� �

� � ��.7-9 ���� ����� ������ �

� (demineralized bone particle, DBP)� ��� ����

�� � ���� �� ��. Urist �� DBP� �

� ��� ���� ������� ��� �� ��

�� �����.10,11 DBP� ��� ��� ���� ��

�� ����� � ���� ��� � � � �

�� ��� ����� ��� � ��� �� �

�.12-18 �� ������ �� � ��� �� ���

���� �� � ��, ��� �� � �� ��

�� ��, ��� ��� � �� ��� � �

� �� ���� �� � ��� ���� ��

��� �� � ���� ��.19 ���� ���

��� �� �� ���� �� ��� �� ��.

�� ���� ����� ��� ���� �� �

�� � � �� � �, ��� ��� ��, �

�, �� �� � ���� �� � �� �� ��

�� ����� ��� ��, ��, ��� �� ��

�� �� �� � ��� �� �� �� �

�� ��.20-22

������ ���� � �� ���� ���

�� � ��� � ��� ��� ���, �

����� �� �� �� ��� ��� ��

� �� ��� ����� ��. ����� �� �

��� �� �� � �� ��� �����,

� ���� ��� ���� �� �� � �� �

�, ��� ��� ��� ���� ���� ��

������ ���.23 �� � ���� ����

��� ��� PLGA ��,24 ���� ������

��� PLGA ��,25 ������� ��� PLGA �

�,26,27 � ��� D3� �� PLGA ��,28,29 ���

��� �� �� �30 ��� ��� ����

��� ����� ��� �� �� �����. �

� �� ���� DBP� ��� DBP � DBP� �

� PLGA ��� �� ��� ��� ��� ���

� � �����.31

��� ��� ��� � � ����� PLA �

PLGA ��� ������ ��� �� �� �

���, ��� ��� �� ��� DBP� ��� �

� ��� �� ��� ���. �� �������

� (bone-marrow derived mesenchymal stem cells, BMSCs)

� ���� ��� � ���� ���. DBP� PLGA

� �� ���/� ��� ��� ��� ����

�����32 ���� � � ������� (scan-

ning electron microscopy, SEM) � ��� � ���

�����. BMSCs� �� ��� ��� ����

�, ��� ��� ��� ����� �����.

���� ��� Wright-Giemsa, Alizalin red, von Kossa

�� � ���� �, in vivo ��� ����� �

�� ���� �� ��� �� ��� ���

� �� �����.

�������

�������� PLA� (Boehringer Ingelheim Chem. Co.

Ltd., Germany)�� �����, PLGA (lactide/glicolide

��, 75/25, Resomer® RG 756, Boehringer Ingelheim Chem.

Co. Ltd., Germany)� ������ 90000 g/mole� �

�����. ����� (Orient Chem. Co., Korea)� �

� ����� ������ � ���� ���� ��

� ��� �� ��� ���� �����. DBP

� (Osteotech, Inc., Shrewsbury, NJ, USA) ��� ���

��, ������� (MC, Tedia Co. Inc., USA) ��

�� ����� ���� HPLC � �����.

PLA� PLGA� �� ���� Figure 1� �����

DBP� �� Figure 2� �����.

�������������������������� ���

Page 3: Preparation and Characterization of Demineralized Bone ... · Preparation and Characterization of Demineralized Bone Particle-loaded PLGA Scaffold for Tissue Engineered Bone Ji Wook

���� � � Jang et al. Polymer (Korea), Vol. 28, No. 5, 2004�

���� � ������� ���, �28� �5�, 2004��

PLA, PLA/DBP (50%), PLGA, � PLGA/DBP (50%) ��

� ���� �� ���/� ��� ��� ���

��. PLA � PLGA� MC� �� ��� ��

� � DBP� ��� ���� �� ���� �

�� ����� ���� �� (180�250 µm, 250

�355 µm) ����� �� ��� ����� (Table

1). ��� PLA, PLA/DBP, PLGA, � PLGA/DBP� ��

��� �� 10 mm � �� 5 mm ��� ��� �

�� �� � ��� (MH-50Y, CAP 50 tons, Japan)� ��

� ���� 60 kgf/cm2� � � 24�� �� �

���. ���� ��� �� ��� 3� ����� 48

�� �� �� �, 8 mTorr, -55 � ��� 48��

�� �� ����. ����� MC� ���� ��

� 1�� �� 25 � ������ ��� �,

�� ��� ��� ��� �����. ��� ��

���� Figure 3� �����.

����������� ��� � �� ���

��� ��� ���� �� �� ��, �����, �

� ���� � ���� ���� �� �� ���

(Micromeritics Co., Model AutoPore II 9220, USA)� �

����. ��� PLA, PLGA ���� ��, ����

��, ��� � ���� 0.1 g, 6.7 ~ 7.3 mL,

3.4 KPa � 414 MPa ���. ��� P� �� ���

r� �� ��� Washburn �33

pr ⋅−= θγ cos2 (2)

� ��� ����. �� γ� ������ � 485

(a)

O CH C O CH C

CH3 CH3

� �

��

O CH2 C O CH2 C

� �

��

O CH C O CH C

CH3 CH3

� �

��

O CH2 C O CH2 C

� �

��

(b)�

�������� Chemical structures of (a) PLA and (b) PLGA.

(a)�

(b)

������� SEM microphotographys of (a) DBP and (b) DBP powder.

������� � Schematic representation of the solvent casting/salt leaching method to fabricate PLA/DBP and PLGA/DBP scaffolds.

� ��������������������� ���� ��������������������

�� ���������� � !��������"�!��# ���!��� ����� �$�!��

polym er D B P

con ten t (% )

size o f N aC l (µm )

vo lum e of P L G A to N aC l

(w /w % )

porosity (% )

m edian pore d iam eter

(µm ) P L A 0 180�250 90 94 .1 92 .1 P L A 0 250�355 90 92 .5 109 .3 P L A 50 180�250 90 92 .9 69 .1 P L A 50 250�355 90 90 .2 80 .4

P L G A 0 180�250 90 97 .3 120 .8 P L G A 0 250�355 90 96 .5 133 .7 P L G A 50 180�250 90 96 .6 96 .5 P L G A 50 250�355 90 94 .8 111 .9

���������������� �������������������������������������������������

Page 4: Preparation and Characterization of Demineralized Bone ... · Preparation and Characterization of Demineralized Bone Particle-loaded PLGA Scaffold for Tissue Engineered Bone Ji Wook

Polymer (Korea), Vol. 28, No. 5, 2004� Demineralized Bone Panticle-loaded Porous Scaffold � �����

���, �28� �5�, 2004� ������ �� �� ��� ��� � ����

dyne/cm� ���� � θ� ��� PLA� PLGA �

�� �� �� �� ��� (Model 100-0, Rame-

Hart Inc., USA)� ��� ��� �� 160°���.34,35

��� ε � ��� �

ρ

ε1

i

i

+=

V

V (3)

� ��� ����. �� Vi� �� �� ���� ρ� �� ����.

������������������ ��� �� ��

����� ������ 14 ���� �� 3000 U

�� ��� ���� � ���� ��� 20�

30 mL� ���� ���� � ��� � 2� ��

���. � ��� 50% �� (Percoll, Sigma, Chem. Co.,

USA) ������� ��� ���� �� � �

�� ��� ���. �� ������ 500 g�� 25

�� ����� ��� , �� , �� � ��

� ���� � ���� �� ��� ��

� ��� ��� � �� ��� 1000 rpm��

10�� �������. ��� ���� ��� 103

�104 ��/cm2� ��� �� ����� �����.

��� ������ Dulbecco's modified eagle medium

(DMEM, Gibco BRL, USA)� 10% �� (FBS, Gibco

BRL)� ��� (10 U/mL, ���� G ��� 10 µg/mL

���� B, Gibco BRL)� �����. ��� ���

3�� ��� ��� ���� � 9�� ��� �

�����.

�������������������� ������

��� ������ ��� ��� �� (DMEM�10%

FBS�1% ����10 mM β-����������10-8

M �����10-8 M ��� D3)� �����. 4� �

Wright-Giemsa, Alizarin red, von Kossa �� �� �

����� ��� �����. ��� ��� 4�� �

� ��� β-���������, ����, ���

D3� ��� ������ ��� ��� � �

��� (Table 2). � I� ���� (DMEM)� β-��

�������� ��� ���, � II� �����

β-���������� ��� D3� ��� �, � III

� ����� β-���������� ����

��� ��� ��� � IV� ����� β-���

������, ��� D3, � ���� ��� ��

�. ��� ������ ��� ALP ��� ALP ��

��� �����.

��������������������� In vivo ����

����� ��� ���� �� ��� �� �

�� �����. �� 4�� �� �� �� 2�3

cm �� ���� PLA, PLA/DBP, PLGA � PLGA/DBP

���� �� BMSCs� 1�105�/cell ���� PLA/

BMSCs, PLA/DBP/BMSCs, PLGA/BMSCs, � PLGA/DBP/

BMSCs� ����, 4�� �� �� ���� 10% �

��� �����. ��� ���� ��� �� �

�� �� 3 µm� �� � ����� ���� ���

� ��� �� �� H&E ��� von Kossa ��

�����.

������������

����������������������������

�� PLA, PLA/DBP (50%), PLGA, � PLGA/DBP (50%)

��� ���� �� ���/� ��� ��� �

����. �� ���/� ��� � ��� ��� �

��� SEM ��� Figure 4� �����. ��� �

� ��� ��� � ��� �� ��� �� �

�� �, ��, ���, �� ��� �� � �

��� �� � � �����. ��� �� � �

� ���� ��� ���� �, �� ��� ��

���� ��� ��� ����, �� � ��� �

�� � � ���. �� �� (180�250 µm, 250�355

µm)� �� ������ �� �� ��� ��� �

���. �� �� ��� � ��� �� ����

����� ���� �� � ��� ���. ���

� ��, ��� �� ��� ��� �� ��� ��

� ���� ���� �� ��� �� �� � �

����. DBP� �� ���� DBP� ��� �

� ���� ���� ��� � � ��� ��

�� �� � ��� ��� DBP� ���� �

� �� ���� ����.

����������� Table 1�� ����� �� �

�� �����. ���� ��� �� ��� 180�250

� ���� � �$��#�!�����!� ���%���� � ����&������!�����

����%�#��

g ro u p s co n d itio n o f m ed iu m G ro u p I D M E M�F B S�an tib o d y�β-g ly ce ro p h o sp h a te G ro u p II D M E M�F B S�an tib o d y�β-g ly ce ro p h o sp h a te�v itam in D 3 G ro u p III D M E M�F B S�an tib o d y�D ex am etaso n e�β-g ly ce ro p h o sp h a te

G ro u p IV D M E M �F B S�a n tib o d y�d e x a m e ta so n e�β-g ly c e ro p h o sp h a te �v itam in D 3

Page 5: Preparation and Characterization of Demineralized Bone ... · Preparation and Characterization of Demineralized Bone Particle-loaded PLGA Scaffold for Tissue Engineered Bone Ji Wook

���� � � Jang et al. Polymer (Korea), Vol. 28, No. 5, 2004�

���� � ������� ���, �28� �5�, 2004��

µm �� 250�355 µm� ����� � � ����

�, ���� �� ���� 90%��� �� ����

�����. �� ��� �������� �����

����, DBP� ��� �� � �� �� �

�����. Figure 5� �� ��� ������, DBP

� ��� 50%�� ��� ���� � ���� �

� �� � ��� �� ��� ���� ���

��� � � ����.

������������������ ��������

� ������ ��� ���� �, 4� �� �

�� � ��� ��� ��, ���� �������

�� �� �� � ���. 4� �� ��� ��

� �� ��� ��� � � ���.

�������������������� ������

��� ����� ����� ���� �� ���

��� ���� 4� � Wright-Giemsa, Alizarin red, �

von Kossa �� �����. �� ��� Figure 6� �

����, von Kossa �� ���� ������ ��

�� ���� �� � ���. �� ��� ���

� ��� ���� � ���� �� 4�� �

� ��� ��� ��� �� ��� 4� � ALP

��� ALP �� � ��� ��� Figure 7� Table 3�

�����. ALP ���� � I, III, II, � IV� � �

��� ��� �� �, � II� III ��� ��

� ���� �� ��� D3� �� ��� ��

���� ����, β-���������, ����, �

�� D3� �� ���� ��� �� ��� ��

���. �� ALP �� ��� ��� � I�� 13.7

� � � � � � � � � � �

� � � � � � � � � � � � � � �

� � � � � �

�������' SEM micrographs of DBP/PLGA scaffolds by means of the solvent casting/salt leaching. (A) PLA (180�250 µm), (B) PLA (250�355µm), (C) PLA/DBP (180�250 µm), (D) PLA/DBP (250�355 µm), (E) PLGA (180�250 µm), (F) PLGA (250�355 µm), (G) PLGA/DBP (180�250 µm), and (H) PLGA/DBP (250�355 µm).

Page 6: Preparation and Characterization of Demineralized Bone ... · Preparation and Characterization of Demineralized Bone Particle-loaded PLGA Scaffold for Tissue Engineered Bone Ji Wook

Polymer (Korea), Vol. 28, No. 5, 2004� Demineralized Bone Panticle-loaded Porous Scaffold � �����

���, �28� �5�, 2004� ������ �� �� ��� ��� � ����

�4.63, � II�� 30.3�19.53, � III�� 18.88�7.47,

� IV�� 40.24�24.2� ����. � III�� ��

� II�� �� ALP �� �� �, � IV�� �

� �� ALP �� ���.

������� �������������� PLA� PLGA�

DBP� ��� ���� ���� �� �����

���� ���� ��� ��� ����� ���. � �� ������ ��� ���� ���� 4� �

��� �� 10% ��� ��� � ��� � 3

µm� ��� ����� ����� H&E, von Kossa

�� ���. ���� �� ���� ��

H&E �� ���. Figure 8 �� ���� PLA�

PLGA� �� � ��, ����� ��� ��� �

� �� � ��. PLA/DBP� PLGA/DBP ����

���� DBP � ���� ��� � �� � �

��� ��� � � ���� �� �� � �

�. �� DBP ��� ������ ���� ����

� �� �� � ��, �� DBP� �� ��� �

��� � �� ���� ����. DBP� ����

� ��� ��� ���� �� ����

�� � �� von Kossa �� �����. von Kossa

���� PLA� PLGA ������ ���� ��

�� ����. ��� PLA/DBP� PLGA/DBP ���

��� ��� ���� �����. ���

� PLA� PLGA� BMSCs� ���� ������ �

��� ���� �����, PLA/DBP� PLGA/DBP�

Incr

emen

tal l

ntru

sion

(m

L/g)

� ���� � ���

� � Pore size Diameter (µm)

(a)

���

���

���

���

���

���

Incr

emen

tal l

ntru

sion

(m

L/g)

� ���� � ��

� � Pore size Diameter (µm)�

(b)

���

���

���

���

���

���

���

�������( Pore size distribution of (a) PLA/DBP and (b) PLGA/ DBP scaffolds by means of the solvent casting/salt leaching.

(a)

(b)

(c)

�������) Photographs of histochemical staining for (a) Wright-Giemsa, (b) Alizarin reds, and (c) von Kossa.

Page 7: Preparation and Characterization of Demineralized Bone ... · Preparation and Characterization of Demineralized Bone Particle-loaded PLGA Scaffold for Tissue Engineered Bone Ji Wook

���� � � Jang et al. Polymer (Korea), Vol. 28, No. 5, 2004�

���� � ������� ���, �28� �5�, 2004��

BMSCs� ���� ������ ���� ��

� ���. ��� ��� PLA� PLGA� � ��

� ��� ���� ��� �� ��� �� � �,

�� ��� ��� �� ���� BMSCs� ��� �

��� � �� ���� ����. �� ��� �

���� BMP� ��� �� DBP� �� ����

�� ��� ��� �� �� � ���� �� �

��� ����� ��� ���� �����, ��

BMSCs� ��� ������ BMSCs� ���� ��

�� �� �� �� � ��� ����� ��

�� ���� �� ����. BMSCs� ��� ���

��� ����� � � � ��� �����,

���� � ���� ����� ����.

�������

� ����� ����� ���� PLA� PLGA�

��� �� ��� DBP� ��� ��� ���

� ��� ��� ��� ����� ���.

� ��� ��� ���� ��� �� ���/� �

�� ��� ����� ��� ���� ����

90% ��, 100 µm ��� ����� ���.

� ��� ��������� ��� ��� ���

��� �����. ������ ��� ���� ��

Wright-Giemsa, Alizarin red, � von Kossa �� ��

� ������ ���� ���� ����. �

� �� �� ��������� ��� ��� ��

� ����� �����, β-���������, ��

� D3, � ����� ���� ��� ����, �

����� ��� D3� ��� �� ��� �

���.

� ��� DBP� BMSCs� ��� ��� ��� �

� � �� ���. H&E ��� ���� 4� �

��� ��� �� �� � �� �, DBP� �

� ������ DBP ��� ����� ���� ��

� ���� � � �� ������ ���� ���

����� �����. �� von Kossa ���� DBP

��� � ��� ��� ���� �� �

���. �� DBP� �� BMP �� ���� ��

�� ��� �� � ��� ���� �����

���� ������, �� ���� BMSCs� ���

� ��� ���� ����.

��� � PLA � PLGA� DBP� 50% ��� �

��� ��� ���� � DBP� �� ����

�� ��� ��� ��� ����� � H&E�

von Kossa �� �� �����. �� DBP� �

�� ���� ��� �� ��� �� ���� �

�������* Photographs of histochemical staining for H&E (After 4 weeks): (A) PLA (�100), (B) PLA/DBP (�100), (C) PLGA (�100), and (D) PLGA/DBP (�100).

� ��������������"��������%�#��

g ro u p s 2 w eek s (n�1 2 ) G ro u p I 1 3 .7�4 .6 3 G ro u p II 3 0 .3�1 9 .5 3 G ro u p III 1 8 .8 8�7 .4 7 G ro u p IV 4 0 .2 4�2 4 .2 ρ - V a lu e 0 .6 8

�������� + Photographs of histochemical staining for ALP (A) group I medium, (B) group II medium, (C) group III medium, and (D) group IV medium.

Page 8: Preparation and Characterization of Demineralized Bone ... · Preparation and Characterization of Demineralized Bone Particle-loaded PLGA Scaffold for Tissue Engineered Bone Ji Wook

Polymer (Korea), Vol. 28, No. 5, 2004� Demineralized Bone Panticle-loaded Porous Scaffold � ����

���, �28� �5�, 2004� ������ �� �� ��� ��� � ���

� �����. �� DBP� ��� �� ���� �

�, DBP ��� ��� ��, ��� �� ����

����, BMSCs�� ���� ���� � �� ��

� ��� �� � �� �� ��.

��� ��� ��� ��� (01-PJ11-PG9-01NT00-

0011, 0405-BO01-0204-0006)� �� � � �� ��

�� ������.

�� ���� 1. G. Khang, S. J. Lee, and H. B. Lee, “Polymer-cell Interaction”,

in Tissue Engineering; Concepts and Application, 2nd Eds., J. J. Yoo, and I. Lee, Editors, Korea Med. Pub. Co., Seoul, p

297 (2002). 2. E. Wintermantel, J. Mayer, J. Blum, K. L. Eckert, P. Luscher,

and M. Mathey, Biomaterials, 17, 83 (1996). 3. S. P. Baldwin and W. M. Saltzman, Adv. Drug Deliv. Rev., 33,

71 (1998). 4. C. M. Agrawal, G. G. Niederauer, D. M. Micallef, and K. A.

Athanasiou, “The use of PLA-PGA Polymers in Orthopedics”, in Encyclopedic Handbook of Biomaterials and Bioengi-neering, 2nd Eds., Marcel Dekker, New York, p 1055 (1995).

5. J. O. Hollinger and J. P. Schmitz, J. Oral Maxillofac. Surg., 45, 594 (1987).

6. O. Bostman, J. Bone Joint Surg., 73, 148 (1991). 7. K. A. Athanasiou, A. R. Singhal, C. M. Agrawal, and B. D.

Boyan, Clin Orthop., 315, 272 (1995). 8. L. G. Cima, J. P. Vacanti, C. Vacanti, D. Ingber, D. Mooney,

� � � � � � �

� � � � � � �

� � � � �

������� , Photographs of histochemical staining for von Kossa (After 4 weeks): (A) PLA, (B) PLA/DBP, (C) PLA/BMSCs, (D) PLA/DBP/BMSCs, (E) PLGA, (F) PLGA/DBP, (G) PLGA/ BMSCs, and (H) PLGA/DBP/BMSCs.

Page 9: Preparation and Characterization of Demineralized Bone ... · Preparation and Characterization of Demineralized Bone Particle-loaded PLGA Scaffold for Tissue Engineered Bone Ji Wook

�� � � Jang et al. Polymer (Korea), Vol. 28, No. 5, 2004�

�� � ������� ���, �28� �5�, 2004��

and R. Langer, J. Biomech. Eng., 113, 143 (1991). 9. A. G. Mikos, G. Sarakinos, S. M. Leite, J. P. Vacanti, and R.

Langer, Biomaterials, 14, 323 (1993). 10. M. R. Urist, Science, 150, 893 (1965). 11. C. B. Huggins and M. R. Urist, Science, 167, 896 (1971). 12. J. Wang and M. J. Glimcher, Calcif. Tissue Int., 65, 156

(1999). 13. J. Wang and M. J. Glimcher, Calcif. Tissue Int., 65, 486

(1999). 14. J. Wang, R. Yang, L. C. Gerstenfeld, and M. J. Glimcher,

Calcif. Tissue Int., 67, 314 (2000). 15. H. D. Adkinsson, J. Strauss-Schoerenberger, M. Gillis, R.

Wilkins, M. Jackson, and K. A. Hruska, J. Orthop. Res., 18, 503 (2000).

16. S. Mizuno and J. Glowacki, Exp. Cell. Res., 227, 89 (1996). 17. C. Lasa, J. Hollinger, W. Drohan, and M. MacPhee, Plast.

Resconst. Surg., 96, 1409 (1995). 18. S. Mizuno and J. Glowacki, Biomaterials, 17, 1819 (1996). 19. K. Whang, C. H. Thomas, and K. E. Healy, Polymer, 36, 837

(1995). 20. M. B. Habal, Clin. Orthop., 188, 239 (1984). 21. J. M. Lane and H. S. Sandhu, Orthop. Clin. North Am., 18,

213 (1997). 22. H. E. Jergesen, J. Chua, R. T. Kao, and L. B. Kaban, Clin.

Orthop., 268, 253 (1991). 23. N. C. Rath and A. H. Reddi, Nature, 278, 855 (1979).

24. G. Khang, P. Shin, I. Kim, B. Lee, S. J. Lee, Y. M. Lee, H. B.

Lee, and I. Lee, Macromol. Res., 10, 158 (2002).

25. M. K. Choi, G. Khang, I. Lee, J. M. Rhee, and H. B. Lee,

Polymer(Korea), 25, 318 (2001).

26. E. K. Jeon, J. Y. Shim, H. J. Whang, G. Khang, I. Jo, I. Lee, J.

M. Rhee, and H. B. Lee, Biomater. Res., 5, 23 (2001).

27. E. K. Jeon, G. Khang, I. Lee, J. M. Rhee, and H. B. Lee,

Polymer(Korea), 25, 893 (2001).

28. H. S. Kim, I. Lee, J. M. Lee, C. W. Han, J. H. Sung, M. Y.

Park, G. Khang, and H. B. Lee, J. Korea Soc. Endocrinology,

17, 206 (2002).

29. S. J. Lee, D. H. Lee, G. Khang, Y. M. Lee, and H. B. Lee,

Macromol. Chem. Symp., 15, 201 (2002).

30. J. W. Jang, B. Lee, C. W. Han, I. Lee, H. B. Lee, and G.

Khang, Polymer(Korea), 27, 226 (2003).

31. G. Khang, C. S. Park, J. M. Rhee, S. J. Lee, Y. M. Lee, I. Lee,

M. K. Choi, and H. B. Lee, Korea Polym. J., 9, 267 (2001).

32. L. E. Freed, J. C. Marquis, A. Nohria, J. Emmanual, A. G.

Mikos, and R.Langer, J. Biomed. Mater. Res., 11, 27 (1993).

33. H. L. Ritter and L. C. Drake, Ind. Eng. Chem., 17, 782 (1945).

34. D. M. Smith, D. W. Hua, and W. L. Earl, MRS Bull., 19, 44

(1994).

35. A. G. Mikos, G. Sarakinos, S. M. Leite, J. P. Vacanti, and R.

Langer, Biomaterials, 14, 323 (1993).