U.S. patent application number 14/388013 was filed with the patent office on 2015-07-02 for hyaluronic acid-calcium phosphate composite for growth factor support and method for producing same.
This patent application is currently assigned to BIOALPHA INC.. The applicant listed for this patent is Su Hyun Jung, Jung Ju Kim, Hyun Seung Ryu, Jun Hyuk Seo. Invention is credited to Su Hyun Jung, Jung Ju Kim, Hyun Seung Ryu, Jun Hyuk Seo.
Application Number | 20150182666 14/388013 |
Document ID | / |
Family ID | 49260574 |
Filed Date | 2015-07-02 |
United States Patent
Application |
20150182666 |
Kind Code |
A1 |
Kim; Jung Ju ; et
al. |
July 2, 2015 |
HYALURONIC ACID-CALCIUM PHOSPHATE COMPOSITE FOR GROWTH FACTOR
SUPPORT AND METHOD FOR PRODUCING SAME
Abstract
The present invention relates to a carrier for growth factor
related to regeneration of bone tissues that is capable of
arbitrarily controlling the delivery rate of growth factors related
to bone regeneration and thus especially applicable to a bone void
filler in the fields of the dental or orthopedic applications. The
carrier for controlling the delivery rate of the growth factor in
the present invention is composed of a hyaluronic acid hydrogel
having a distribution of interconnected pores and a calcium
phosphate microsphere being distributed in the pores of the
hyaluronic acid hydrogel. The calcium phosphate microsphere having
a porosity suitable for delivery of the growth factor is positioned
into the pores of the cross-linked hyaluronic acid hydrogel to
complete the carrier.
Inventors: |
Kim; Jung Ju; (Yongin-si,
KR) ; Jung; Su Hyun; (Jeongeup-si, KR) ; Ryu;
Hyun Seung; (Gyeonggi-do, KR) ; Seo; Jun Hyuk;
(Gyeonggi-do, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kim; Jung Ju
Jung; Su Hyun
Ryu; Hyun Seung
Seo; Jun Hyuk |
Yongin-si
Jeongeup-si
Gyeonggi-do
Gyeonggi-do |
|
KR
KR
KR
KR |
|
|
Assignee: |
BIOALPHA INC.
SEOUL
KR
|
Family ID: |
49260574 |
Appl. No.: |
14/388013 |
Filed: |
March 30, 2012 |
PCT Filed: |
March 30, 2012 |
PCT NO: |
PCT/KR2012/002397 |
371 Date: |
March 10, 2015 |
Current U.S.
Class: |
424/422 ;
514/770 |
Current CPC
Class: |
A61L 27/20 20130101;
C08L 5/08 20130101; A61L 27/12 20130101; A61L 2430/02 20130101;
C08K 2201/005 20130101; A61L 2300/414 20130101; A61L 27/46
20130101; A61L 27/46 20130101; A61L 27/58 20130101; C08K 7/18
20130101; A61L 27/56 20130101; C01B 25/327 20130101; A61L 27/52
20130101; C01P 2004/32 20130101; C08K 3/32 20130101; C08K 2003/325
20130101; C08L 5/08 20130101; C01P 2004/61 20130101; A61L 27/54
20130101 |
International
Class: |
A61L 27/46 20060101
A61L027/46; A61L 27/12 20060101 A61L027/12; A61L 27/58 20060101
A61L027/58; A61L 27/56 20060101 A61L027/56; A61L 27/54 20060101
A61L027/54; A61L 27/20 20060101 A61L027/20; A61L 27/52 20060101
A61L027/52 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 29, 2012 |
KR |
10-2012-0032121 |
Claims
1. A hyaluronic acid/calcium phosphate composite for loading a
growth factor, comprising: a hyaluronic acid hydrogel having a
distribution of interconnected pores and being cross-linked in the
presence of a cross-linking agent; and a calcium phosphate
microsphere being distributed in the pores of the hyaluronic acid
hydrogel and having a size of 45 .mu.m to 75 .mu.m.
2. The hyaluronic acid/calcium phosphate composite for loading a
growth factor as claimed in claim 1, wherein the hyaluronic acid
has a molecular weight in the range of 1,000,000 to 5,000,000.
3. The hyaluronic acid/calcium phosphate composite for loading a
growth factor as claimed in claim 1, wherein the hyaluronic acid
has a degree of cross-linking of 40% or less.
4. The hyaluronic acid/calcium phosphate composite for loading a
growth factor as claimed in claim 1, wherein the cross-linking
agent of the hyaluronic acid is selected from the group consisting
of polyoxyethylene bis(glycidyl ether), 1,2,3,4-diepoxybutane,
1,2,7,8-diepoxyoctane, diethylene glycol diglycidyl ether, and
1,4-butanediol diglycidyl ether.
5. The hyaluronic acid/calcium phosphate composite for loading a
growth factor as claimed in claim 1, wherein calcium phosphate
constituting the calcium phosphate microsphere is selected from the
group consisting of monocalcium phosphate
(Ca(H.sub.2PO.sub.4).sub.2), dicalcium phosphate (CaHPO.sub.4),
calcium dihydrogen phosphate (Ca(H.sub.2PO.sub.4).sub.2),
tricalcium phosphate (Ca.sub.3(PO.sub.4).sub.2), and octacalcium
phosphate (Ca.sub.8H.sub.2(PO.sub.4).sub.6.5H.sub.2O).
6. The hyaluronic acid/calcium phosphate composite for loading a
growth factor as claimed in claim 1, wherein the growth factor is
at least one selected from the group consisting of epidermal growth
factor (EGF), heparin-binding EGF-like growth factor (HB-EGF),
fibroblast growth factor (FGF), vascular endothelial growth factor
(VEGF), and bone morphogenetic protein (BMP), including BMP-2,
BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10,
BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, or
BMP-18.
7. A method for preparing a hyaluronic acid/calcium phosphate
composite for loading a growth factor, comprising: sintering
calcium phosphate powder at 1,050.degree. C. to 1,250.degree. C. to
obtain a spherical calcium phosphate microsphere having a size of
45 .mu.m to 75 .mu.m; and mixing the calcium phosphate microsphere
with a hyaluronic acid hydrogel cross-linked in the presence of a
cross-linking agent to obtain a hyaluronic acid/calcium phosphate
composite for loading a growth factor.
8. The method as claimed in claim 7, wherein the hyaluronic acid
has a molecular weight in the range of 1,000,000 to 5,000,000.
9. The method as claimed in claim 7, wherein the hyaluronic acid
has a degree of cross-linking of 40% or less.
10. The method as claimed in claim 7, wherein the cross-linking
agent of the hyaluronic acid is selected from the group consisting
of polyoxyethylene bis(glycidyl ether), 1,2,3,4-diepoxybutane,
1,2,7,8-diepoxyoctane, diethylene glycol diglycidyl ether, and
1,4-butanediol diglycidyl ether.
11. The method as claimed in claim 7, wherein calcium phosphate
constituting the calcium phosphate microsphere is selected from the
group consisting of monocalcium phosphate
(Ca(H.sub.2PO.sub.4).sub.2), dicalcium phosphate (CaHPO.sub.4),
calcium dihydrogen phosphate (Ca(H.sub.2PO.sub.4).sub.2),
tricalcium phosphate (Ca.sub.3(PO.sub.4).sub.2), and octacalcium
phosphate (Ca.sub.8H.sub.2(PO.sub.4).sub.6.5H.sub.2O).
12. The method as claimed in claim 7, wherein the growth factor is
at least one selected from the group consisting of epidermal growth
factor (EGF), heparin-binding EGF-like growth factor (HB-EGF),
fibroblast growth factor (FGF), vascular endothelial growth factor
(VEGF), and bone morphogenetic protein (BMP), including BMP-2,
BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10,
BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, or BMP-18.
Description
TECHNICAL FIELD
[0001] The present invention relates to a hyaluronic acid/calcium
phosphate composite for loading growth factors and a preparation
method thereof and, more particularly to, a hyaluronic acid/calcium
phosphate composite and a preparation method thereof, where the
hyaluronic acid/calcium phosphate composite is capable of
arbitrarily controlling the delivery rate of a growth factor
related to bone regeneration and used as a carrier for growth
factor in regeneration of bone tissue applicable to the bone void
filler for dental or orthopedic applications.
BACKGROUND ART
[0002] A number of patent and non-patent documents have proposed
the results of many studies on the carrier to deliver growth
factors.
[0003] For example, Korean Patent Application No. 10-2008-0100315
discloses a preparation method for porous calcium phosphate
granules and a manufacturing method for a functional bone graft
material using the same.
[0004] However, this technology involves loading growth factors in
a simple manner of adsorption, but without using a material to bind
the bone morphogenetic proteins to the support, so the bone
morphogenetic proteins are released in a short period of time (fast
release) with the difficulty in controlling the release rate, more
likely causing an adverse effect that the tissue grows excessively
faster than usual in the course of tissue regeneration [Yeh, T. T.,
S. S Wu et al., Osteoarthritis Cartilage 15(12): 1357-1366, 2007].
In addition, the technology adopts adsorption and freeze-drying of
proteins, thus more possibly ends up transforming the structure of
the bone morphogenetic proteins and has the difficulty in
sterilizing the bone graft material on which the bone morphogenetic
proteins are adsorbed.
[0005] The carrier for growth factor is a pharmaceutical
preparation injected into the human body and hence subjected to
proven sterilization methods, such as gamma radiation
sterilization, E-beam sterilization, or high-pressure steam
sterilization, ethylene oxide (E.O.) gas sterilization, in the
manufacturing process of medical equipment. These sterilization
methods involve irradiation of heat or radioactive rays to have an
effect on the structure of the proteins. Therefore, the bone
morphogenetic proteins after the sterilization process not only
fail to achieve their intended effects sufficiently but also become
recognized as transformed proteins in the human body, with high
possibility of causing an adverse effect [Chen, J. B. et. Al., J
Biomed Mater Res A, 80(2): 435-443, 2007].
[0006] On the other hand, International Patent Application No.
PCT/EP2008/005340 discloses the technology related to a composite
bone repair material including a porous block type ceramic scaffold
and a stabilizing polymer arranged in the support.
[0007] The ceramic scaffold is immersed in an aqueous solution of
polyethylene glycol thio (PEG-thiol)containing bioactive
substances, such as parathyroid hormone (PTH), bone morphogenetic
protein (BMP), enamel matrix derivative (EMD), etc., and a polymer
mixture of polyarm polyethylene glycol acrylate (polyarm
PEG-acrylate) to form a bone repair material that contains
bioactive substances. But, the polymers used in this method, that
is, the aqueous solution of PEG-thiol and polyarm PEG-acrylate are
free from the portion to bind the bioactive substances to the
support, so it is impossible to achieve a controlled release of the
growth factors as specified above. The results of the PTH release
test proposed in this document show that the release of the growth
factors is completed in 5.8 days. When such a release profile is
applied to the bone morphogenetic proteins (BMPs) that are growth
factors for bone generation and organization, the bone
morphogenetic proteins (BMPs) can be released excessively fast
(fast release) to cause an adverse effect on the bone generation or
bone regeneration, such as generating bone in the regions other
than the bone tissue or failing to achieve a fast regeneration of
bone in the damaged bone tissue.
[0008] Further, International Patent Application No.
PCT/IB2009/005235 discloses a bone morphogenetic composition
prepared by mixing a bone morphogenetic growth factor/amphipathic
anionic polysaccharide composite and at least one at least divalent
cationic soluble salt and processed into an open implant in the
freeze-dried form. This document suggests the use of hyaluronic
acid to promote the effect of the growth factors, but no approach
to the method for controlling the delivery rate of the growth
factors.
[0009] In addition, Korean Patent Application No 10-2008-0038777
specifies a hyaluronic acid bone void filler composite and a
preparation method thereof, which the hyaluronic acid bone void
filler composite is prepared by adding a calcium phosphate
derivative to a matrix including hyaluronic acid to induce bone
regeneration by the osteoconductive action of the hyaluronic
acid.
[0010] However, the technology disclosed in the document relates to
the bone void filler used to fill in the bony voids but does not
suggest any technology regarding the carrier for growth factor
promoting bone generation. Moreover, the calcium phosphate compound
used as a bone regeneration inducer composed of hydroxyapatite and
.beta.-TCP is prepared by the chemical precipitation reaction, and
it is thus impossible to control the porosity of the bone void
filler. Further, the porosity of the bone void filler cannot be
controlled when the calcium phosphate compound is used as a carrier
for growth factor. As the pores are not interconnected with one
another, neither the growth factor can be loaded in the bone void
filler nor the release rate of the growth factor can be under
control. Furthermore, there possibly occurs a fast release of the
growth factors. As a result, the bone void filler is not suitable
as a carrier for growth factor.
[0011] In other words, as the most important thing is that the
carrier for growth factor for regeneration of bone tissues can be
injected into the human body, the carrier for growth factor is
required to be sterilized with little toxicity. In addition, the
carrier for growth factor has to be biodegradable and capable of
controlling the delivery rate of the growth factors arbitrarily as
suitable to the size or degree of the bone voids and reducing the
adverse effect possibly caused in the case of the fast release of
the growth factors. Most of all, an efficient regeneration of the
bone tissues takes place when the growth factors are released
suitably according to the regeneration rate of the damaged tissue
and only on a confined region of the damaged tissue. However, the
above-specified conventional technologies do not suggest any
solution to the above-mentioned problems with the carrier for
growth factor for regeneration of bone tissues and there is still a
need for the carrier for growth factor for regeneration of bone
tissues to solve the problems.
DISCLOSURE OF INVENTION
[0012] In order to solve the problems with the prior art, it is an
object of the present invention to provide a carrier for growth
factor for regeneration of bone tissue and a preparation method
thereof, where the carrier for growth factor for regeneration of
bone tissue is capable of loading the growth factor for
regeneration of bone tissue while making it possible to control the
release rate of the growth factor that helps bone regeneration,
especially in the fields of dentistry and orthopedics, for example,
related to the periodontal bone loss, implant peripheral bone,
osteoporosis, surgery, traumatic bone damage, and so forth.
[0013] In order to achieve the object of the present invention,
there is provided a hyaluronic acid/calcium phosphate composite for
loading a growth factor that includes: a hyaluronic acid hydrogel
having a distribution of interconnected pores and being
cross-linked in the presence of a cross-linking agent; and a
calcium phosphate microsphere being distributed in the pores of the
hyaluronic acid hydrogel and having a size of 45 .mu.m to 75
.mu.m.
[0014] In accordance with the present invention, there is also
provided a method for preparing a hyaluronic acid/calcium phosphate
composite for loading a growth factor that includes: sintering
calcium phosphate powder at 1,050.degree. C. to 1,250.degree. C. to
obtain a spherical calcium phosphate microsphere having a size of
45 .mu.m to 75 .mu.m; and mixing the calcium phosphate microsphere
with a hyaluronic acid hydrogel cross-linked in the presence of a
cross-linking agent to obtain a hyaluronic acid/calcium phosphate
composite for loading a growth factor.
EFFECTS OF THE INVENTION
[0015] According to the present invention, it is advantageous in
that the hyaluronic acid/calcium phosphate composite is prepared
using calcium and phosphate naturally present in the bone tissue
and hyaluronic acid naturally present in the extracellular matrix
and thus suitable to deliver growth factors related to bone
regeneration and effectively useful in promoting the bone
regeneration.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a mimetic diagram showing the net charge of the
growth factor according to the iso-electric point of the growth
factor and the pH value of the environment.
[0017] FIG. 2 is an SEM (Scanning Electron Microscopic) image of a
.beta.-TCP microsphere according to the sintering temperature of
the .beta.-TCP microsphere.
[0018] FIG. 3 is a mimetic diagram showing a carrier capable of
controlling the delivery rate of the growth factor using a
hyaluronic acid hydrogel and a calcium phosphate microsphere
according to the present invention.
[0019] FIG. 4 shows the results of the release of growth factors
related to the bone regeneration using the delivery system capable
of controlling the delivery rate of the growth factor according to
the present invention.
[0020] FIG. 5 is an SEM image of the hyaluronic acid hydrogel
showing that the pore size is different depending on the degree of
cross-linking (A: 20% cross-linking degree, B: 10% cross-linking
degree).
BEST MODES FOR CARRYING OUT THE INVENTION
[0021] Hereinafter, the present invention will be described in
further detail.
[0022] The present invention provides a carrier capable of
controlling the delivery rate of the growth factor. The carrier is
composed of a hyaluronic acid hydrogel having a distribution of
interconnected pores and a calcium phosphate microsphere being
distributed in the pores of the hyaluronic acid hydrogel. The
calcium phosphate microsphere of a different porosity is positioned
in the pores having a different degree of cross-linking to complete
the carrier.
[0023] The first principle of the present invention to control the
delivery rate of the growth factor is electrical attraction between
the growth factor and the carrier. The bone morphogenetic growth
factor involved in the bone regeneration has an iso-electric point
of approximately 8 to 10 and takes electrically positive charges at
about pH 7 that is the in-vivo environment (Refer to FIG. 1) [T.
Boix et al., Journal of Inorganic Biochemistry 99 (2005), Atsushi
Iwakura et al., J. thorac Cardiovasc Surg. 126: (2003), Jeroen J.
J. P. et al., Tissue Engineering, Volume 13, Number 4, (2007)].
[0024] Based on the understandings of this point, the present
invention employs hyaluronic acid and calcium phosphate as
constituent substances of a carrier for delivering the growth
factor related to bone regeneration, where the hyaluronic acid and
the calcium phosphate take electrically negative charges at the
in-vivo pH value. Hyaluronic acid contains functional groups such
as hydroxyl groups (--OH) or carboxyl groups (--COOH) that carry
electrically negative charges, and its surface takes electrically
negative charges by the phosphate group (--PO.sub.4) of calcium
phosphate. The growth factor related to bone regeneration is loaded
on the functional groups of the carrier having electrically
negative charges through the electrical bonding, and the release
rate of the growth factor from the carrier can be controlled
according to the structure of the hyaluronic acid hydrogel and the
calcium phosphate microsphere.
[0025] Further, the second principle of the present invention to
control the delivery rate of the growth factor is related to the
porosity of the carrier for the growth factor. The carrier for
delivering the growth factor is composed of a calcium phosphate
microsphere having a distribution of interconnected pores and a
hyaluronic acid hydrogel having a desired porosity. When the
calcium phosphate microsphere has a high porosity, the growth
factor is positioned deep and wide in the central portion of the
microsphere and the pores of the microsphere and released slowly.
Further, with an increase in the degree of cross-linking of the
hyaluronic acid hydrogel, the hydrogel has the increased porosity,
higher strength and lower degradation rate. This leads to a
decrease in the exposure rate of the calcium phosphate microsphere
and thus makes the growth factor bound to the carrier released
slowly.
[0026] In the present invention, the term "degree of cross-linking"
refers to the permanent structure that individual molecules or
monomer chains of the hyaluronic acid polymer are interconnected.
Moreover, for the purpose to achieve the object of the present
invention, the degree of cross-linking is defined as the percentage
(%) weight ratio of the cross-linking agent with respect to the
unit of the hyaluronic acid monomer in the cross-linked portion of
the hyaluronic acid-based composition. This can be measured as the
weight ratio of the hyaluronic acid monomer to the cross-linking
agent (i.e., hyaluronic acid monomer: cross-linking agent).
[0027] The hyaluronic acid as used in the present invention is not
specifically limited, but in general a natural substance,
preferably derived from vertebrates or microorganisms. Typically,
the hyaluronic acid has a molecular weight of 600,000 to 7,000,000.
But, the molecular weight of the hyaluronic acid used in the
present invention is preferably in the range of 1,000,000 to
5,000,000 in consideration of viscosity, degradability, and so
forth.
[0028] The hyaluronic acid can be extracted from the tissues or
bio-synthesized. In this regard, many documents are known (Korean
Patent Nos. 1993-0001320 (Feb. 25, 1993) and 1987-0001815 (Oct. 13,
1987)). For example, the hyaluronic acid may be extracted from the
tissues, such as of chicken crest, synovial fluid of joints, humane
umbilical cord tissue, bovine bronchial tubes, etc. or obtained
from the culture of microorganisms, such as non-hemolytic
streptococcus.
[0029] The hyaluronic acid hydrogel constituting the carrier for
growth factor according to the present invention is cross-linked.
The porosity and pore size of the hydrogel can be controlled by
adjusting the degree of cross-linking, and the retention ratio and
the affinity of the growth factor to the hydrogel can be controlled
by changing the structure of the hydrogel. Further, the degradation
rate of the hydrogel can be under control to regulate the release
rate of the growth factor through mass erosion. The hyaluronic acid
hydrogel can be cross-linked in the presence of polyoxyethylene
bis(glycidyl ether), 1,2,3,4-diepoxybutane, 1,2,7,8-diepoxyoctane,
diethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl
ether, etc., preferably polyoxyethylene bis(glycidyl ether) or
1,4-butanediol diglycidyl ether.
[0030] The degree of cross-linking of the hyaluronic acid hydrogel
as desirable to serve as a carrier for growth factor in the present
invention is 40% or less. This is to adjust the degradation rate of
the hyaluronic acid hydrogel to the initial period of bone
regeneration. Generally, the initial period of bone regeneration is
known to be about 2 months. For adjusting the rate of bone
regeneration to this range, it is desirable for the scaffold to
degrade about 2 months after installation. If the scaffold remains
without degradation 3 months or more after installation, it is
known to interfere with bone regeneration or bone formation. Hence,
there is required a carrier having an appropriate degradation
rate.
[0031] The other constituent component of the carrier of the
present invention is calcium phosphate. In the present invention,
calcium phosphate in the form of porous microsphere is used in
order to control the delivery rate of the growth factors. The
porosity of the calcium phosphate microsphere can be adjusted
arbitrarily in the manufacturing process to control the affinity of
the growth factor loaded on the microsphere. The present invention
uses this principle to control the delivery rate of the growth
factor. The porosity of the calcium phosphate microsphere can be
controlled in the process to control the sintering temperature,
which is preferably in the range of 1,050.degree. C. to
1,250.degree. C.
[0032] It is also necessary in the present invention to control the
diameter of the calcium phosphate microsphere in order for the
calcium phosphate microsphere to effectively function in the
delivery of the growth factor. In particular, the minimum diameter
of the calcium phosphate microsphere is 45 .mu.m so as to prevent
the phagocytosis of macrophages in the initial immune reaction so
that the calcium phosphate microsphere can be used in the delivery
of growth factor without being degraded in a short period of time.
Further, the maximum diameter of the calcium phosphate microsphere
is 75 .mu.m in consideration of the pore size of the humane
trabecular bone, which varies depending on the region of the human
body but generally ranges from 100 .mu.m to 300 .mu.m.
[0033] The calcium phosphate microsphere may be prepared with
monocalcium phosphate (Ca(H.sub.2PO.sub.4).sub.2), dicalcium
phosphate (CaHPO.sub.4), calcium dihydrogen phosphate
(Ca(H.sub.2PO.sub.4).sub.2), tricalcium phosphate(TCP)
(Ca.sub.3(PO.sub.4).sub.2), octacalcium phosphate
(Ca.sub.8H.sub.2(PO.sub.4).sub.6.5H.sub.2O), etc. The calcium
phosphate microsphere prepared in the present invention is a
carrier for growth factor as implanted in vivo and preferably uses
.beta.-TCP that is advantageously biodegradable, absorbed by the
osteoclasts and useful in the bone regeneration when absorbed as a
constituent component for bone.
[0034] In general, the growth factor related to bone regeneration
may include transforming growth factor family (TGF family),
epidermal growth factor (EGF), fibroblast growth factor (FGF),
vascular endothelial growth factor (VEGF), and so forth. Among
these, the growth factor related to bone regeneration may be the
bone morphogenetic proteins (BMPs) that are an important factor for
the bone regeneration in the transforming growth factor family (TGF
family). Specific examples of such BMPs may include BMP-2, BMP-3,
BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11,
BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, BMP-18, etc.
[0035] FIG. 1 is a mimetic diagram showing the net charge of the
growth factor according to the iso-electric point of the growth
factor and the pH value of the environment. As shown in FIG. 1, the
growth factor related in the bone regeneration at the iso-electric
point of 8 to 10 takes electrically positive charges, so it can be
loaded on the functional groups of the carrier having electrically
negative charges, such as the hydroxyl group (--OH) and the
carboxyl group (--COOH) of hyaluronic acid and the phosphate group
(--PO.sub.4) of calcium phosphate, through electrical bonding. The
release rate of the growth factor in the carrier can be controlled
depending on the structures of the hyaluronic acid hydrogel and the
calcium phosphate microsphere.
[0036] The hyaluronic acid hydrogel that is the constituent
component of the carrier to control the delivery rate of the growth
factor in the present invention has a distribution of
interconnecting pores. For example, hyaluronic acid having a high
molecular weight of at least 1,000,000 Da is dissolved in distilled
water at pH 7.0 to a content of 1 wt. % to 20 wt. % and then
sufficiently reacted with 1 wt.% to 40 wt. % of a cross-linking
agent, such as polyoxyethylene bis(glycidyl ether),
1,2,3,4-diepoxybutane, 1,2,7,8-diepoxyoctane, diethylene glycol
diglycidyl ether, or 1,4-butanediol diglycidyl ether, to obtain a
hyaluronic acid hydrogel having a degree of cross-linking in the
range of 1% to 40%.
[0037] The hyaluronic acid hydrogel with a different degree of
cross-linking has a different porosity, which results in a
different degradation rate of the hyaluronic acid hydrogel and
hence a different release rate of the growth factors bonded to the
hyaluronic acid hydrogel. As can be seen from Table 1, which
presents the degradation rate of the hyaluronic acid hydrogel
depending on the degree of cross-linking, the degradation rate of
the hyaluronic acid hydrogel is remarkably dependent upon the
degree of cross-linking
TABLE-US-00001 TABLE 1 Degradability of hyaluronic acid hydrogel
controlled in degree of cross-linking in the presence of 100 units
of enzyme for breaking down hyaluronic acid hydrogel Degree of
cross-linking (%) 1 20 40 Degradability (%) 0 hour 0 0 0 3 hours
21.61 13.56 8.31 6 hours 42.53 25.40 12.28 24 hours 69.82 39.43
13.05 36 hours 84.1 47.52 15.89 48 hours 100 52.61 17.23
[0038] The other constituent component of the carrier for
controlling the delivery rate of the growth factor in the present
invention, the calcium phosphate microsphere is, for example,
.beta.-TCP that has high in-vivo affinity and is biodegradable and
absorbable in vivo. Firstly, .beta.-TCP powder is synthesized and
spray-dried into spherical particles, which are then sintered at
1,050.degree. C. to 1,250.degree. C. to prepare porous .beta.-TCP
microspheres. The sintered .beta.-TCP microsphere is a spherical
particle having an isotropic structure and its porosity is
dependent upon the sintering temperature. In other words, as can be
seen from Table 2, the increase in the sintering temperature leads
to a gradual reduction in the porosity of the .beta.-TCP
microsphere and hence the decreased absorption rate of the
.beta.-TCP microsphere.
[0039] The surface pore structure of the .beta.-TCP microsphere
depending on the sintering temperature is shown in FIG. 2.
According to FIG. 2, as the sintering temperature increases, the
inter-cohesion of the surface particles becomes stronger to cause
an abrupt reduction in the size and distribution of the pores
connected to the exterior. Therefore, the amount of the loadable
growth factor such as BMP-2 and the affinity to the growth factor
are decreased with an increase in the sintering temperature. It is
thus possible to prepare a carrier capable of arbitrarily
controlling the release rate of the growth factor using the
above-specified feature of the .beta.-TCP microsphere.
EXAMPLES
[0040] Hereinafter, the present invention will be described in
further detail with reference to the examples, which are not
intended to limit the scope of the present invention.
Examples 1 to 9
[0041] 1. Preparation of .beta.-TCP Microsphere
[0042] 10 g of amorphous .beta.-TCP powder slurry dissolved in 67
ml of distilled water is spry-dried at 150.degree. C. to obtain
approximately spherical .beta.-TCP powder having an average
particle size of 2 The B-TCP powder put in an alumina crucible to a
thickness of 10 mm is subjected to a first sintering in an electric
furnace at 600 .degree. C. for one hour and then a second sintering
at 1,050.degree. C. to 1,250.degree. C. to produce spherical
.beta.-TCP microspheres, which are then size-screened through a
sieve. Among the size-sorted B-TCP microspheres, .beta.-TCP
microspheres having a size of 45 .mu.m to 75 .mu.m are chosen,
washed with an ultrasonic washer for 30 minutes and then dried out
at 70.degree. C. for one hours to obtain the final B-TCP
microspheres. The surface pore structure of the B-TCP microspheres
thus obtained depending on the sintering temperature is illustrated
in FIG. 2.
[0043] FIG. 2 is an SEM (Scanning Electron Microscopic) image
showing the surface of the .beta.-TCP microsphere controlled in
porosity in order to regulate the delivery rate of the growth
factors. Referring to the surface pore structure shown in FIG. 2,
as the sintering temperature rises, the inter-cohesion of the
surface particles becomes stronger to cause an abrupt reduction in
the size and distribution of the pores connected to the
exterior.
[0044] 2. Preparation of .beta.-TCP Microsphere
[0045] 1 g of hyaluronic acid powder having a molecular weight of
1,000 kDa is dissolved in 100 ml of distilled water under agitation
at 1,000 rpm for one hour. 1 to 40 wt % of 1,4-butanediol
diglycidyl ether as a cross-linking agent is added to the solution,
which is then adjusted to a pH 9.0. The mixture is subjected to
mixing with agitator for one hour and then kept at 25.degree. C.
for 10 hours to obtain a cross-linked hyaluronic acid hydrogel.
[0046] The hyaluronic acid hydrogel thus obtained is freeze-dried
and then ground to powder having a particle size of 500 .mu.m or
less, After passing through a sieve, 5 g of the hyaluronic acid
hydrogel powder is mixed with 5 g of the .beta.-TCP microsphere
prepared above under agitation to form 10 g of a carrier for growth
factor. FIG. 3 is a mimetic diagram showing the carrier for growth
factor prepared from hyaluronic acid hydrogel and calcium phosphate
microspheres according to the examples of the present
invention.
[0047] The porosity and water absorption of the carrier for growth
factor thus prepared are presented in Table 2.
TABLE-US-00002 TABLE 2 Physical properties of carrier for growth
factor depending on sintering temperature of .beta.-TCP microsphere
and degree of cross-linking of hyaluronic acid hydrogel Sintering
Degree of Water temperature cross-linking Porosity absorption
Example (.degree. C.) (%) (%) (wt %) 1 1,050 1 68.54 75 2 20 66.23
73.1 3 40 64.37 71.8 4 1,150 1 61.96 62.5 5 20 60.24 56.4 6 40
58.32 50 7 1,250 1 56.21 45.9 8 20 53.58 43.4 9 40 50.9 39.2
[0048] Referring to Table 2, the carrier for growth factor varies
in the porosity and the water absorption depending on the sintering
temperature and the degree of cross-linking Further, the porosity
and the water absorption have an effect on the release and delivery
rates of the growth factors.
[0049] Bone Morphogenetic Growth Factor (BMP-2) Loading And Release
Test
[0050] 100 mg of the carrier for growth factor composed of the
hyaluronic acid hydrogel and the .beta.-TCP microsphere as prepared
in Example 1, 3, 6, or 9 is filled in a syringe, and 100 pl of a
solution of the bone morphogenetic growth factor, BMP-2 is then
sucked into the syringe. The syringe is put into connection with
another syringe via a straight-line connector and then subjected to
a mixing process for about five times to load the growth factor on
the carrier.
[0051] 100 mg of the carrier loading the bone morphogenetic growth
factor, BMP-2, is added to 1 ml of a phosphate buffer solution. The
buffer solution containing BMP-2 released for 1, 4, 6, or 14 days
is collected as a sample. The sample thus obtained is measured in
regards to the amount of BMP-2 with a BMP-2 ELISA kit using the
antigen-antibody reaction. The measurement results are presented in
FIG. 4.
[0052] Referring to FIG. 4, the release and delivery rates of the
growth factor are changed depending on the sintering temperature
and the degree of cross-linking of the carrier for growth factor.
In 14 days, almost all of the bone morphogenetic growth factor
loaded on the carrier of Example 9 is released, while no more than
40% of the growth factor loaded on the carrier of Example 6 is
released. Further, less than about 10% of the bone morphogenetic
growth factor loaded on the carrier of Example 1 or 3 is released
in 14 days.
[0053] The results show that BMP-2 is loaded on the carrier without
deformation or damage and that the release and delivery rates of
the growth factor are dependent upon the sintering temperature and
the degree of cross-linking of the carrier for growth factor. This
implicitly demonstrates that the release and delivery rates of the
growth factor can be arbitrarily controlled.
[0054] On the other hand, FIG. 5 is an SEM image of the hyaluronic
acid hydrogel showing that the pore size is dependent upon the
degree of cross-linking (A: 20% cross-linking degree, B: 10%
cross-linking degree), where the hyaluronic acid hydrogel has the
porosity and the pore size varied depending on the degree of
cross-linking As can be seen from FIG. 5, the pore size is about
180 .mu.m when the degree of cross-linking is 20% in (A) and about
240 .mu.m when the degree of cross-linking is 10% in (B).
INDUSTRIAL AVAILABILITY
[0055] The hyaluronic acid/calcium phosphate composite for loading
growth factors according to the present invention is capable of
controlling the delivery rate of the growth factor related to bone
regeneration and thus can be usefully applied as a carrier for
growth factor related to the regeneration of bone tissue applicable
to the bone void fillers for dental or orthopedic applications.
[0056] The present invention has been described with reference to
the particular illustrative examples, which are susceptible to many
changes and modifications without departing from the scope and
spirit of the present. All such changes and modifications are
deemed to be covered by the claims of the present invention that
follow.
* * * * *