U.S. patent application number 14/239663 was filed with the patent office on 2014-07-31 for composition for regeneration of cartilage.
This patent application is currently assigned to National University Corporation Hokkaido University. The applicant listed for this patent is Norimasa Iwasaki, Akio Minami, Nobuo Ohzawa, Atsushi Sukegawa. Invention is credited to Norimasa Iwasaki, Akio Minami, Nobuo Ohzawa, Atsushi Sukegawa.
Application Number | 20140213524 14/239663 |
Document ID | / |
Family ID | 47746586 |
Filed Date | 2014-07-31 |
United States Patent
Application |
20140213524 |
Kind Code |
A1 |
Iwasaki; Norimasa ; et
al. |
July 31, 2014 |
COMPOSITION FOR REGENERATION OF CARTILAGE
Abstract
A novel composition for regenerating a cartilage has been
demanded, which can achieve a good effect of regenerating a hyaline
cartilage that is a nearly normal cartilage without requiring the
use of any transplanted cell. The present invention provides a
composition for regenerating a cartilage, wherein (a) a monovalent
metal salt of low endotoxin alginic acid and (b) SDF-1 are used in
combination.
Inventors: |
Iwasaki; Norimasa; (Kita-ku,
JP) ; Sukegawa; Atsushi; (Kita-ku, JP) ;
Minami; Akio; (Kita-ku, JP) ; Ohzawa; Nobuo;
(Shinjuku-ku, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Iwasaki; Norimasa
Sukegawa; Atsushi
Minami; Akio
Ohzawa; Nobuo |
Kita-ku
Kita-ku
Kita-ku
Shinjuku-ku |
|
JP
JP
JP
JP |
|
|
Assignee: |
National University Corporation
Hokkaido University
Mochida Pharmaceutical Co., Ltd.
|
Family ID: |
47746586 |
Appl. No.: |
14/239663 |
Filed: |
August 22, 2012 |
PCT Filed: |
August 22, 2012 |
PCT NO: |
PCT/JP2012/071749 |
371 Date: |
February 19, 2014 |
Current U.S.
Class: |
514/17.1 |
Current CPC
Class: |
A61L 27/54 20130101;
A61L 2430/06 20130101; A61L 2300/252 20130101; C08L 5/04 20130101;
A61K 31/734 20130101; A61L 27/20 20130101; A61L 27/20 20130101;
A61L 2300/414 20130101; A61K 38/195 20130101 |
Class at
Publication: |
514/17.1 |
International
Class: |
A61K 38/19 20060101
A61K038/19; A61K 31/734 20060101 A61K031/734 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 23, 2011 |
JP |
2011-181662 |
Claims
1. A composition for cartilage regeneration wherein (a) a
monovalent metal salt of a low endotoxin alginic acid and (b) SDF-1
are used in combination.
2. The composition according to claim 1, wherein the composition
does not contain a cell growth factor.
3. The composition according to claim 1, wherein the SDF-1 is
contained in the composition at a concentration of 0.2 .mu.g/ml or
higher and less than 100 .mu.g/ml.
4. The composition according to claim 1, wherein the composition is
applied to a cartilage injury lesion, and is cured by applying a
cross-linking agent to the surface of the composition.
5. The composition according to claim 4, wherein the cross-linking
agent is at least one metal ion compound selected from the group
consisting of Ca.sup.2+, Mg.sup.2+, Ba.sup.2+ and Sr.sup.2+.
6. The composition according to claim 1, wherein the monovalent
metal salt of alginic acid has a weight-average molecular weight of
500,000 or more in gel filtration chromatography.
7. The composition according to claim 1, wherein viscosity of the
composition is 400 mPas to 20,000 mPas.
8. The composition according to any claim 1, wherein the
composition does not contain cells for cartilage regeneration.
9. The composition according to claim 1, wherein the composition
does not contain a scaffold material for cells except (a) the
monovalent metal salt of low endotoxin alginic acid.
10. The composition according to claim 1, wherein the monovalent
metal salt of alginic acid is potassium alginate or sodium
alginate.
11. The composition according to claim 1, wherein the monovalent
metal salt of low endotoxin alginic acid has an endotoxin content
of 100 EU/g or less.
12. The composition according to claim 1, wherein the composition
for cartilage regeneration is used for treating a cartilage injury
lesion or for treating a cartilage disease.
Description
TECHNICAL FIELD
[0001] The present invention relates to a composition for cartilage
regeneration and the like.
BACKGROUND ART
[0002] Articular cartilage is hyaline cartridge that is composed of
a small number of cells, collagenous extracellular matrix, abundant
proteoglycans and water. Since vascular and neural networks are
present in the bone and the bone has self-repairing ability, even
when a bone is fractured, the fractured site is often adequately
repaired. Articular cartilage, however, lacks vascular and neural
networks. Accordingly, it has very little self-repairing ability
and thus a cartilage defect lesion is not adequately repaired
particularly when a large defect lesion is formed in the cartilage.
Even when the lesion is repaired, the lesion is remodeled with
fibrous cartilage that has different mechanical property from that
of hyaline cartilage. Therefore, a cartilage defect lesion leads to
pain in the joint and loss of joint function, which often result in
development of osteoarthritis. In addition, an early stage of
osteoarthritis, where abrasion of the surface of articular
cartilage starts due to aging or excessive joint usage, may lead to
cartilage defect over a large region as a result of progression of
the symptoms.
[0003] Thus, since articular cartilage is inadequate in
self-repairing ability, surgical procedures for treating cartilage
lesion, for example, autologous osteochondral implantation
(mosaicplasty), a procedure of perforating with a pick
(microfracture), drilling, a procedure of shaving subchondral bone
with a burr (abrasion procedure) and resection of injured cartilage
(debridement) are required. Among these procedures, the
microfracture procedure, drilling and the abrasion procedure are
called marrow stimulation techniques, which stimulate bleeding from
bone marrow to induce marrow-derived cartilage precursor cells in
expectation of them to differentiate into cartilage. These
techniques, however, have limitation for a widespread cartilage
defect and cartilage regenerated by these procedures is fibrous
cartilage that has different mechanical property from that of
hyaline cartilage.
[0004] In 1984, Peterson et al. and Grande et al. tested autologous
chondrocyte implantation (ACI) technique in the non-full thickness
of rabbit articular cartilage. ACI is a technique involving
harvesting and culturing tissue from patient's own normal
cartilage, implanting the cultured cells suspended in a medium in
the affected area and covering the cartilage defect lesion with a
piece of periosteum to prevent leakage of the cells. ACI technique
was first clinically applied in 1994 and now has been used for more
than 15 years. Until now, several clinically successful examples
have been reported. Recent clinical examinations, however, also
report that ACI technique did not show significantly superior
results over other surgeries with respect to repair of an articular
cartilage defect.
[0005] There are two major reasons for such poor results from the
ACI technique. One reason is the technical difficulties in
anchoring cells and a scaffold to the joint defect lesion and
covering them with a periosteum patch. In ACI technique, the joint
needs to be widely exposed by arthrotomy to suture the periosteum
patch to cover the cell suspension. Moreover, several complicated
issues related to the periosteum patch including thickening, defect
and intra-articular adhesion of periosteum have also been reported.
The other reason is the limitation to the use of chondrocytes.
Chondrocytes rapidly lose their differentiation phenotypes in
monolayer culture and transform into fibroblasts. Another problem
is that harvest of cartilage from a non-weight-bearing site of the
affected joint required by the ACI technique leaves the problem of
the donor site that has been harvested of chondrocytes to remain
problematic.
[0006] Meanwhile, there has been an attempt to utilize natural
polymers such as collagen, chitosan, agarose and alginic acid in
regenerative medicine for articular cartilage. It is, however,
difficult to acquire sufficient cartilage regeneration effect with
polymer alone, and polymer is usually used in combination with
cultured chondrocytes and mesenchymal stem cells. For example,
Patent Document 1 discloses that mesenchymal stem cells embedded in
a composition containing a monovalent metal salt of a low endotoxin
alginic acid can be applied to a cartilage injury lesion to gain
favorable hyaline cartilage regeneration that is almost comparable
to normal cartilage. In addition, an attempt to use growth factors
and cytokines has also been made in cartilage regeneration
medicine. TGF-.beta. and bFGF are known as typical factors for
differentiating/proliferating chondrocytes.
[0007] SDF-1 (Stromal cell-derived factor 1) is one type of
chemokines SDF-1 is expressed in ischemic tissues caused by cardiac
infarction, brain infarction, and skin lesion and the like to allow
the vascular precursor cells to migrate toward the ischemic site
for angiogenesis. Non-patent Document 1 describes that SDF-1
expression was confirmed in the bone implantation site and that
SDF-1 played a role in guiding mesenchymal stem cells to the local
site upon bone healing. Patent Document 2 discloses a
sustained-release composition comprising SDF-1 and a hydrogel of
modified gelatin. This composition is utilized for the treatment of
ischemic diseases and the like because it allows sustained release
of SDF-1. On the other hand, whether or not SDF-1 is expressed at
the cartilage injury lesion and whether or not it exerts a
beneficial effect upon administration to the cartilage injury
lesion have previously been unknown. There is also a report saying
that SDF-1 is present at a higher concentration in a pathological
tissue of osteoarthritis or rheumatoid arthritis than in a normal
tissue, and that SDF-1 of a higher concentration (200 ng/ml or
higher, which is less than 100 ng/ml in a normal tissue) can result
in necrosis of chondrocytes (Non-patent Document 2).
[0008] Patent Document 3 discloses a cell-free graft comprising an
open porous structuring matrix and a serum. This cell-free graft is
described of its potential to be used for cartilage regeneration
since the serum in the graft can stimulate migration of mesenchymal
precursor cells to the defect lesion, but whether or not it
actually exerts a cartilage regeneration effect in vivo remains
unrevealed. Generally, in cartilage regeneration medicine, it is
difficult to predict the in vivo effect of implantation therapy
only by in vitro cartilage regeneration tests or cell migration
tests. Specifically, a graft used in cartilage regeneration
medicine needs to provide performances, for example, to remain at
the injury lesion for weeks, to successfully graft with the
surrounding tissue as regenerated cartilage and the like. It is
difficult to confirm such performances other than in vivo.
[0009] Patent Document 4 describes that a cell-free scaffold
containing SDF-1 or TGF-.beta. is arranged in a manner to allow
fluid communication with cells, thereby rending the cells to
migrate toward the scaffold. The scaffold actually used in Patent
Document 4 is collagen sponge covered with cross-linked calcium
alginate. Gelatin microspheres containing SDF-1 and/or TGF-.beta.
are embedded in the cross-linked calcium alginate layer of the
scaffold. Patent Document 4 reports that when this scaffold was
brought into contact with bone-marrow-derived mesenchymal stem
cells (MSC), human adipose-derived stem cells (ASC) and synovial
stem cells (SYN), with the collagen sponge side facing down, the
cells in some cases had migrated to the collagen sponge of the
scaffold. It also describes that although cartilage formation was
confirmed with the scaffold containing TGF-.beta., the cell
migration thereof was moderate, whereas the scaffold containing
both TGF-.beta. and SDF-1 resulted in good cell migration and
cartilage formation were confirmed. Patent Document 4, however,
does not show that the cells migrate to the cross-linked calcium
alginate layer of the scaffold. Patent Document 4 also concludes
that although MSC and ASC migrated to the scaffold containing only
SDF-1 and not TGF-.beta., it was inadequate to induce cartilage
formation with SDF-1 alone.
PRIOR ART DOCUMENTS
Patent Documents
[0010] [Patent Document 1] WO2008/102855 (pamphlet) [0011] [Patent
Document 2] WO2009/060608 (pamphlet) [0012] [Patent Document 3]
WO2007/003324 (pamphlet) [0013] [Patent Document 4] WO2010/048418
(pamphlet)
Non-Patent Documents
[0013] [0014] [Non-patent Document 1] Toshiyuki Kitaori et al., The
Journal of Clinical Orthopaedic Association, Vol. 45, No. 1, pp.
72-75 (2010) [0015] [Non-patent Document 2] Lei Wei et al., The
Journal of Rheumatology, Vol. 33, pp. 1818-1826 (2006)
DISCLOSURE OF INVENTION
[0016] Under the above-described circumstances, there has been a
need in the cartilage regeneration medicine for a composition for
cartilage regeneration, which can reduce the burden on patients
such as cell harvest, arthrotomy and the like, which can easily be
applied to a cartilage injury lesion, and which can exert a
cartilage regeneration effect in vivo. In particular, a novel
composition for cartilage regeneration has been expected that can
produce a good hyaline cartilage regeneration effect that resembles
that of normal cartilage without using graft cells.
[0017] In order to solve the above-described problems, the present
inventors have gone through intensive studies and found that good
cartilage regeneration can be induced at a cartilage injury lesion
without using graft cells by applying to the cartilage injury
lesion a composition for cartilage regeneration wherein (a) a
monovalent metal salt of a low endotoxin alginic acid and (b) SDF-1
are used in combination, thereby accomplishing the present
invention.
[0018] Thus, the present invention provides a composition for
cartilage regeneration as follows.
[1-1] A composition for cartilage regeneration wherein (a) a
monovalent metal salt of a low endotoxin alginic acid and (b) SDF-1
are used in combination. [1-2] The composition according to [1-1]
above, wherein the composition does not contain a cell growth
factor. [1-3] The composition according to either one of [1-1] and
[1-2], wherein the SDF-1 is contained in the composition at a
concentration of 0.2 .mu.g/ml or higher and less than 100 .mu.g/ml.
[1-4] The composition according to any one of [1-1] to [1-3] above,
wherein the composition is applied to a cartilage injury lesion,
and is cured by applying a cross-linking agent to the surface of
the composition. [1-5] The composition according to [1-4] above,
wherein the cross-linking agent is at least one metal ion compound
selected from the group consisting of Ca.sup.2+, Mg.sup.2+,
Ba.sup.2+ and Sr.sup.2+. [1-6] The composition according to any one
of [1-1] to [1-5] above, wherein the monovalent metal salt of
alginic acid has a weight-average molecular weight of 500,000 or
more in gel filtration chromatography. [1-7] The composition
according to any one of [1-1] to [1-6] above, wherein viscosity of
the composition is 400 mPas to 20,000 mPas. [1-8] The composition
according to any one of [1-1] to [1-7] above, wherein the
composition does not contain cells for cartilage regeneration.
[1-9] The composition according to any one of [1-1] to [1-8] above,
wherein the composition does not contain a scaffold material for
cells except (a) the monovalent metal salt of low endotoxin alginic
acid. [1-10] The composition according to any one of [1-1] to [1-9]
above, wherein the monovalent metal salt of alginic acid is
potassium alginate or sodium alginate. [1-11] The composition
according to any one of [1-1] to [1-10] above, wherein the
monovalent metal salt of low endotoxin alginic acid has an
endotoxin content of 100 EU/g or less. [1-12] The composition
according to any one of [1-1] to [1-11] above, wherein the
composition for cartilage regeneration is used for treating a
cartilage injury lesion or for treating a cartilage disease.
[0019] In addition, the present invention also provides a method of
regenerating cartilage as follows.
[2-1] A method for regenerating cartilage comprising the step of
applying to a cartilage injury lesion a composition wherein (a) a
monovalent metal salt of low endotoxin alginic acid and (b) SDF-1
are used in combination. [2-2] The method according to [2-1] above,
wherein the composition does not contain a cell growth factor.
[2-3] The method according to either one of [2-1] and [2-2],
wherein the concentration of SDF-1 in the composition is 0.2
.mu.g/ml or higher and less than 100 .mu.g/ml. [2-4] The method
according to any one of [2-1] to [2-3] above, wherein the
composition is applied to the cartilage injury lesion, and is cured
by applying a cross-linking agent to the surface of the
composition. [2-5] The method according to [2-4] above, wherein the
cross-linking agent is at least one metal ion compound selected
from the group consisting of Ca.sup.2+, Mg.sup.2+, Ba.sup.2+ and
Sr.sup.2+. [2-6] The method according to any one of [2-1] to [2-5]
above, wherein the monovalent metal salt of alginic acid has a
weight-average molecular weight of 500,000 in gel filtration
chromatography. [2-7] The method according to any one of [2-1] to
[2-6] above, wherein viscosity of the composition is 400 mPas to
20,000 mPas. [2-8] The method according to any one of [2-1] to
[2-7] above, wherein the composition does not contain cells for
cartilage regeneration. [2-9] The method according to any one of
[2-1] to [2-8] above, wherein the composition does not contain a
cell scaffold material for cells except (a) the monovalent metal
salt of low endotoxin alginic acid. [2-10] The method according to
any one of [2-1] to [2-9] above, wherein the monovalent metal salt
of alginic acid is potassium alginate or sodium alginate. [2-11]
The method according to any one of [2-1] to [2-10] above, wherein
the monovalent metal salt of low endotoxin alginic acid has an
endotoxin content of 100 EU/g or less. [2-12] The method according
to any one of [2-1] to [2-11] above, wherein the method is used for
treating a cartilage injury lesion or for treating a cartilage
disease.
[0020] The present invention provides a composition for cartilage
regeneration in cartilage regeneration medicine, which can reduce
the burden on patients such as cell harvest, arthrotomy and the
like, which can easily be applied to a cartilage injury lesion, and
which can exert cartilage regeneration effect in vivo. A
composition for cartilage regeneration according to a preferred
embodiment of the present invention is capable of inducing hyaline
cartilage regeneration that resembles that of normal cartilage
without using graft cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 shows expression of SDF-1 protein in rabbit
osteochondral defect models in Example 1. (A-D) are SDF-1
immunohistostaining images at cartilage injury lesions at
predetermined time points following the procedure of creating
full-thickness osteochondral defect. The black arrow indicates a
cell that is expressing SDF-1 protein. (E-H) are SDF-1
immunohistostaining images at intact joint surfaces of a sham
surgery group at predetermined time points. The scale bar is 50
.mu.m. (I) is a low magnification image of a cartilage injury
lesion following a week after the procedure. The scale bar is 1 mm.
(J) shows Western blots. "Sham" represents the sham surgery group
and "Defect" represents the full-thickness osteochondral defect
created group.
[0022] FIG. 2 shows images showing macroscopic findings at Week 4
(A-D) and macroscopic findings at Week 16 (E-H) in rabbit
osteochondral defect models in Example 2. "Defect" represents an
untreated group, "Vehicle" represents an alginic acid group,
"SDF-1" represents an alginic acid+SDF-1 administration group, and
"AMD3100" represents an alginic acid+AMD3100 administration group.
(I) shows macroscopic scores at Week 4 and (J) shows macroscopic
scores at Week 16 (n=10). *p<0.05, **p<0.01,
.dagger.p<0.001
[0023] FIG. 3 shows images of histological sections of the rabbit
osteochondral defect models of Example 2 at Week 4. "Defect"
represents the untreated group, "Vehicle" represents the alginic
acid group, "SDF-1" represents the alginic acid+SDF-1
administration group, and "AMD3100" represents the alginic
acid+AMD3100 administration group. (A-D) are results from
safranin-O staining, (E-H) are results from staining with an
anti-type I collagen antibody, and (I-L) are results from staining
with an anti-type II collagen antibody. The scale bar is 500 .mu.m.
(M) shows histological scores at Week 4 (n=5). *p<0.01,
**p<0.001
[0024] FIG. 4 shows images of histological sections of the rabbit
osteochondral defect model of Example 2 at Week 16. "Defect"
represents the untreated group, "vehicle" represents the alginic
acid group, "SDF-1" represents the alginic acid+SDF-1
administration group, and "AMD3100" represents the alginic
acid+AMD3100 administration group. (A-D) are results from
safranin-O staining, (E-H) are results from staining with an
anti-type I collagen antibody, and (I-L) are results from staining
with an anti-type II collagen antibody. The scale bar is 500 .mu.m.
(M) shows histological scores at Week 16 (n=5). *p<0.01,
**p<0.001
[0025] FIG. 5 shows images showing macroscopic findings at Week 4
(A-D) and macroscopic findings at Week 12 (E-H) with respect to
"(2-5) Assessment of SDF-1 dosage" in Example 2.
[0026] FIG. 6 shows histological sections at Week 12 with respect
to "(2-5) Assessment of SDF-1 dosage" in Example 2. (A-D) are
results from safranin-O staining, (E-H) are results from staining
with an anti-type I collagen antibody, and (I-L) are results from
staining with an anti-type II collagen antibody.
[0027] FIG. 7 shows images of H-E staining of implanted alginic
acid gel for assessing in vivo cell homing in Example 3. (A-D) show
sample sections following a week after the surgery. (A) is a low
magnification image with a scale bar of 1 mm. (B-D) shows high
magnification images with a scale bar of 100 .mu.m. (B) shows an
alginic acid group, (C) shows an alginic acid+SDF-1 administration
group and (D) shows an alginic acid+AMD3100 administration group.
(E) shows the cell counts within the fields (n=5). *p<0.01,
**p<0.001
[0028] FIG. 8 shows the results from the in vitro assessment of the
effect of SDF-1 on the behavior of BMSC in Example 4. (A) shows the
results from the cell migration test using CytoSelect.TM. in media
with or without SDF-1. The vertical axis represents fluorescence
intensity that reflects the cell counts (n=16). (B) shows the
results from the cell proliferation test. The vertical axis
represents absorbance that reflects the cell counts (n=5).
BEST MODES FOR CARRYING OUT THE INVENTION
[0029] Hereinafter, the present invention will be described in
detail. The following embodiments, however, are examples for
illustrating the present invention, and the present invention may
be carried out in various embodiments without departing from the
scope of the invention.
[0030] 1. Cartilage Regeneration
[0031] "Cartilage" is found in joints, thoracic walls,
intervertebral discs, meniscus, and in tubular structures such as
larynx, respiratory tract and ears, and can be classified into
three types, namely, hyaline cartilage, elastic cartilage and
fibrous cartilage. For example, articular cartilage is hyaline
cartilage which is composed of chondrocytes, collagenous
extracellular matrix, proteoglycans and water and which is free of
vascular distribution. Hyaline cartilage is characteristic in that
it is abundant in type II collagen that is stained with an
anti-type II collagen antibody, and it is stained red upon
safranin-O staining that stains proteoglycans.
[0032] "Cartilage injury" refers to states where the cartilage has
been injured due to aging, traumatic injury or other various
factors, including states with deteriorated cartilage functions,
for example, a state where viscoelasticity unique to cartilage
(meaning that the cartilage is slowly compressed with a load and
slowly returns to its original state once released from the load)
is decreased for some reason so that the ability of the cartilage
to support a load while retaining the mobility thereof is
interfered. The cartilage injuries can also be seen in diseases
such as osteoarthritis and rheumatoid arthritis. The present
invention relates to a composition for cartilage regeneration which
can be applied to such cartilage injury lesion. Cartilage defect
refers to a site that lacks a cartilage layer, including a cavity
in the cartilage tissues and the surrounding tissues that form said
cavity. Cartilage defect is one aspect of cartilage injury, and a
composition of the present invention can favorably be used for
treating a cartilage defect lesion.
[0033] More particularly, a composition of the present invention is
a composition for cartilage regeneration wherein (a) a monovalent
metal salt of low endotoxin alginic acid and (b) SDF-1 are used in
combination. By using the composition of the present invention,
better cartilage can be regenerated as compared to the case where a
monovalent metal salt of alginic acid is used alone. Since the
composition of the present invention has good adhesive property to
a cartilage injury lesion and can be applied with a syringe or the
like, it can easily be applied to a cartilage injury lesion. It can
be administered under an arthroscope when extensive arthrotomy can
be avoided. A composition of a preferable embodiment of the present
invention can induce good hyaline cartilage regeneration that
resembles normal cartilage without using graft cells.
[0034] According to the present invention, "cartilage regeneration"
or "cartilage tissue regeneration" means to regenerate the function
of cartilage at a cartilage injury lesion with functional disorder
or dysfunction. According to the present invention, regeneration of
the function does not have to be complete regeneration of the
function as long as the function is recovered compared to the state
of the cartilage injury lesion before application of the
composition of the present invention. Provided that the state of
normal cartilage before receiving injury is 100% and that the state
of a cartilage injury lesion prior to application of the
composition of the present invention is 0%, the state is preferably
recovered to 30% or higher, more preferably 50% or higher, more
preferably 80% or more and particularly preferably almost to the
state before the injury. A rate of occurrence of cartilage other
than hyaline cartilage, such as fibrous cartilage, is preferably
low during the cartilage regeneration. Moreover, the phrase
"treatment of a cartilage injury lesion" or "treatment of a
cartilage defect lesion" means that the cartilage at the cartilage
injury lesion or the cartilage defect lesion found in aging,
traumatic injury, osteoarthritis, intervertebral disc damage,
meniscus damage, osteochondral dissecans or the like is regenerated
so as to alleviate or heal these conditions. Furthermore, the
phrase "treatment of a cartilage disease" means that cartilage at a
cartilage injury lesion or a cartilage defect lesion found in the
cartilage disease such as osteoarthritis, rheumatoid arthritis or
neurogenic arthropathy is regenerated so as to alleviate or heal
these conditions. One embodiment of a composition for cartilage
regeneration of the present invention is a composition for
regenerating hyaline cartilage. Hyaline cartilage regeneration has
a purpose of regenerating cartilage in which the proportion of the
hyaline cartilage is higher than that of the fibrous cartilage, and
intends to regenerate cartilage tissues that are abundant in type
II collagen and proteoglycans.
[0035] In addition, the phrase "application to a cartilage injury
lesion" means to bring the composition for cartilage regeneration
to make contact with a cartilage injury lesion, where a composition
of the present invention is preferably injected into a cartilage
defect lesion so as to fill in the defect lesion. Alternatively,
one or more relatively small holes may be formed into a cartilage
injury lesion, preferably a cartilage defect lesion, so as to
inject the composition of the present invention to fill in the
holes. Application to a cartilage injury lesion is preferably
injection that sufficiently fills the cavity volume of the affected
area. The affected area is preferably subjected to a necessary
pretreatment, and if necessary, washed, prior to the application of
the composition of the present invention. The phrase "to wash an
affected area" means to remove blood components, other unwanted
tissues and the like from a site that is to be applied with the
composition of the present invention, for example, with
physiological saline or the like. At the end of washing, the
affected area is preferably dried, for example, by wiping off the
remaining unwanted fluid components before applying the composition
of the present invention.
[0036] 2. Monovalent Metal Salt of Alginic Acid
[0037] A "monovalent metal salt of alginic acid" contained in a
composition for cartilage regeneration of the present invention is
a water-soluble salt that is formed through ion exchange between a
hydrogen atom of carboxylic acid at position 6 of alginic acid and
a monovalent metal ion such as Na.sup.+ or K.sup.+. Specific
examples of monovalent metal salts of alginic acid include sodium
alginate and potassium alginate, while sodium alginate that can be
obtained as a commercially available product is particularly
preferable. A solution of a monovalent metal salt of alginic acid
forms a gel when mixed with a cross-linking agent.
[0038] An "alginic acid" used in the present invention is a
biodegradable polymeric polysaccharide, which is a polymer
resulting from linear polymerization of two types of uronic acids
called D-mannuronic acid (M) and L-guluronic acid (G). More
specifically, alginic acid is a block copolymer which has a
homopolymer fraction of D-mannuronic acid (MM fraction), a
homopolymer fraction of L-guluronic acid (GG fraction) and a
fraction having randomly arranged D-mannuronic acids and
L-guluronic acids (MG fraction), arbitrarily linked together. A
composite ratio of D-mannuronic acid to L-guluronic acid (M/G
ratio) of alginic acid varies primarily according to the type of a
biological origin thereof such as seaweed, and is affected by the
habitat and seasons of said biological origin. The M/G ratio widely
ranges from about 0.4 that is rich in G to about 5 that is rich in
M.
[0039] Since a monovalent metal salt of alginic acid is a polymeric
polysaccharide, it is difficult to accurately determine the
molecular weight thereof but in general its weight-average
molecular weight is in a range of 10,000 to 10,000,000, and
preferably 50,000 to 3,000,000. Since a lower molecular weight
results in a poor cartilage regeneration effect, particularly in a
poor hyaline cartilage regeneration effect, at a cartilage injury
lesion, a monovalent metal salt of alginic acid used in the present
invention preferably has a weight-average molecular weight of
500,000 or more.
[0040] In general, calculation of a molecular weight of a polymeric
polysaccharide by gel filtration chromatography may result in 10 to
20% measurement error. For example, a value of 400,000 may vary
within a range of 320,000 to 480,000, 500,000 may vary within a
range of 400,000 to 600,000, and 1,000,000 may vary within a range
of 800,000 to 12,000,000. Therefore, a preferable range of a
weight-average molecular weight of a monovalent metal salt of
alginic acid that is particularly favorable regarding an effect on
cartilage is, at least 500,000 or more, more preferably 650,000 or
more, and still more preferably 800,000 or more. Since an
excessively high molecular weight is difficult to be produced and
causes problems such as excessively high viscosity upon making an
aqueous solution thereof and decreased solubility, the
weight-average molecular weight is preferably 5,000,000 or less and
more preferably 3,000,000 or less.
[0041] In general, since a polymeric substance derived from a
natural origin do not have a single molecular weight but rather
consists of an aggregate of molecules of various molecular weights,
they are measured in a molecular weight distribution that has a
certain range. A typical measurement method is gel filtration
chromatography. Typical examples of information of a molecular
weight distribution acquired by gel filtration chromatography
include weight-average molecular weight (Mw), number-average
molecular weight (Mn) and variance ratio (Mw/Mn).
[0042] A weight-average molecular weight places emphasis on the
contribution of a polymer having a larger molecular weight to an
average molecular weight, and is represented by the following
formula:
Mw=.SIGMA.(WiMi)/W=.SIGMA.(HiMi)/.SIGMA.(Hi)
[0043] A number-average molecular weight is calculated by dividing
the total weight of the polymers by the total number of the
polymers:
Mn=W/.SIGMA.Ni=.SIGMA.(MiNi)/.SIGMA.Ni=.SIGMA.(Hi)/.SIGMA.(Hi/Mi)
[0044] Here, W represents the total weight of the polymers, Wi
represents the weight of the "i"th polymer, Mi represents the
molecular weight at the "i"th elusion time, Ni represents the
number of molecular weights Mi, and Hi represents the height at the
"i"th elusion time.
[0045] Since it is considered that a cartilage regeneration effect
(particularly, a hyaline cartilage regeneration effect) at a
cartilage injury lesion largely owes to molecular species with a
larger molecular weight, a weight-average molecular weight can be
employed as an indicator of the molecular weight.
[0046] A molecular weight measurement of a naturally-occurring
polymeric substance is known to make a difference in the resulting
value according to the measurement method (examples for hyaluronic
acids: Chikako YOMOTA et. al. Bull. Natl. Health Sci., Vol. 117, pp
135-139(1999), and Chikako YOMOTA et. al. Bull. Natl. Inst. Health
Sci., Vol. 121, pp 30-33(2003)). Molecular weight measurements of
alginate are described in a literature, including a method of
calculating a molecular weight from intrinsic viscosity and a
method of calculating a molecular weight by SEC-MALLS (Size
Exclusion Chromatography with Multiple Angle Laser Light Scattering
Detection) (ASTM F2064-00 (2006), published by ASTM International).
This literature suggests, in order to measure a molecular weight by
size exclusion chromatography (i.e., gel filtration
chromatography), use of a multi-angle laser light scattering
detector (MALLS) in combination with a calibration curve using
pullulan as a standard substance (i.e., measurement by SEC-MALLS).
There is also an example of using the molecular weight determined
by SEC-MALLS as a standard value of alginate in a catalogue (FMC
Biopolymer, PRONOVA.TM. sodium alginates catalogue).
[0047] Unless otherwise stated, a molecular weight of alginate
specified herein is a weight-average molecular weight calculated by
gel filtration chromatography.
[0048] Typical conditions for gel filtration chromatography include
use of a calibration curve using pullulan as a standard substance.
The molecular weight of pullulan as a standard substance is
preferably at least 1,600,000, 788,000, 404,000, 212,000 or
112,000. In addition, an eluent (200 mM sodium nitrate solution),
column conditions and the like can be specified. As the column
conditions, at least one column with an exclusion limit molecular
weight of 10,000,000 or more is preferably used with a
polymethacrylate resin filler. A typical column is TSK gel
GMPW.times.1 (diameter 7.8 mm.times.300 mm) (Tosoh
Corporation).
[0049] Although a monovalent metal salt of alginic acid that is
initially extracted from a brown alga has a large molecular weight
and high viscosity, the molecular weight becomes smaller and the
viscosity becomes lower during the processes of heat drying,
lyophilization, purification and the like. Accordingly, appropriate
temperature management at each step in production allows production
of monovalent metal salts of alginic acid having different
molecular weights. Monovalent metal salts of alginic acid having
larger molecular weights can be obtained by managing the
temperature at each step in production to be lower whereas
monovalent metal salts of alginic acid with smaller molecular
weights can be obtained with higher temperature. Additionally,
techniques such as appropriate selection of brown algae as the raw
material or fractionation based on molecular weights during the
production process can also allow production of monovalent metal
salts of alginic acid having different molecular weights.
Furthermore, after the molecular weight or the viscosity of the
monovalent metal salt of alginic acid produced by a certain
technique is measured, it can be mixed with a monovalent metal salt
of alginic acid from other lot having different molecular weight or
viscosity so as to obtain a monovalent metal salt of alginic acid
having a molecular weight of interest.
[0050] Although alginic acid used in the present invention may
either be naturally derived or synthesized, it is preferably
naturally derived. Examples of naturally-occurring alginic acids
include those extracted from brown algae. Although brown algae
containing alginic acid grow in the coastal regions around the
world, seaweeds that can actually be used as a raw material for
alginic acid are limited with typical examples being Lessonia from
South America, Macrocystis from North America, Laminaria and
Ascophyllum from Europe, Durvillea from Australia and the like.
Examples of brown algae that can serve as raw materials of alginic
acids include Lessoni species, Macrocystis species, Laminaria
species, Ascophyllum species, Durvillea species, Eisenia species
and Ecklonia species.
[0051] Preferably, the content of the monovalent metal salt of
alginic acid in the composition for cartilage regeneration of the
present invention is about 0.5 to 10% w/v.
[0052] The monovalent metal salt of alginic acid in the composition
for cartilage regeneration of the present invention is an active
element that exerts an effect by providing environment suitable for
cells that contribute to cartilage regeneration, for example,
host-derived mesenchymal stem cells that have migrated to the
affected area, to differentiate into chondrocytes, and the
chondrocytes to regenerate cartilage.
[0053] 3. Low Endotoxin Treatment
[0054] The monovalent metal salt of alginic acid contained in the
composition for cartilage regeneration of the present invention is
a monovalent metal salt of low endotoxin alginic acid. Low
endotoxin means that the level of endotoxin is lowered to an extent
that does not raise substantial inflammation or fever.
Specifically, the alginic acid has been subjected to a low
endotoxin treatment. By using a low endotoxin alginic acid in a
composition of the present invention, a composition with high
bioaffinity can be obtained, which causes less degeneration and low
inflammation response in the surrounding cartilage.
[0055] The low endotoxin treatment can be performed according to a
known method or a method pursuant thereto. For example, the
treatment can be carried out according to the method of Suga et al.
involving purification of sodium hyaluronate (see, for example,
Japanese Unexamined Patent Application Publication No. Heisei
9-324001), the method of Yoshida et al., involving purification of
.beta.1,3-glucan (see, for example, Japanese Unexamined Patent
Application Publication No. Heisei 8-269102), the method of William
et al. involving purification of a biopolymer salt such as alginate
or gellan gum (see, for example, Japanese Unexamined Patent
Application Publication (Translation of PCT Publication) No.
2002-530440), the method of James et al. involving purification of
a polysaccharide (see, for example, International Publication No.
93/13136), the method of Lewis et al. (see, for example,
specification of U.S. Pat. No. 5,589,591), the method of
Hermanfranck et al. involving purification of alginate (see, for
example, Appl Microbiol Biotechnol (1994) 40:638-643) or methods
pursuant thereto. The low endotoxin treatment of the present
invention is not limited thereto, and can be carried out by a known
method such as washing, filtration with a filter (such as an
endotoxin-removing filter or an electrically-charged filter),
ultrafiltration, purification with a column (such as an endotoxin
adsorption affinity column, a gel filtration column or an
ion-exchange resin column), adsorption to a hydrophobic substance,
a resin or activated charcoal, a treatment with an organic solvent
(extraction with an organic solvent, deposition/precipitation
through addition of an organic solvent, or the like), a surfactant
treatment (see, for example, Japanese Unexamined Patent Application
Publication No. 2005-036036), or an appropriate combination
thereof. The steps in these treatments may appropriately be
combined with a known method such as centrifugation. Preferably,
the treatment is suitably selected according to the type of the
alginic acid.
[0056] An endotoxin level can be confirmed and measured according
to a known method such as a method using a limulus agent (LAL), or
a method using Endospecy (registered trademark) ES-24S set
(Seikagaku Corporation). Although a method for treating endotoxin
of an alginic acid contained in the composition of the present
invention is not particularly limited, the resulting endotoxin
content of a monovalent metal salt of alginic acid is preferably
500 endotoxin unit (EU)/g or less, more preferably 100 EU/g or
less, still more preferably 50 EU/g or less, and particularly 30
EU/g or less upon endotoxin measurement using a limulus agent
(LAL). Sodium alginate that has been subjected to a low endotoxin
treatment is available, for example, as a commercially available
product such as Sea Matrix (sterilized) (Kimika Corporation-Mochida
International Ltd.) and PRONOVA.TM. UP LVG (FMC).
[0057] 4. SDF-1
[0058] A composition for cartilage regeneration of the present
invention is characterized in that SDF-1 is used in combination
with the monovalent metal salt of low endotoxin alginic acid.
[0059] SDF-1 according to the present invention is capable of
exerting an improved cartilage regeneration effect by being added
to an alginic acid, as compared to the effect of the alginic acid
alone. In the composition for cartilage regeneration of the present
invention, SDF-1 is an active element that exerts an effect of
promoting migration of host-derived cells (e.g., cells that
contribute to cartilage regeneration, such as mesenchymal stem
cells) to the affected area.
[0060] SDF-1 (Stromal cell-derived factor 1) is a protein that is
also called CXCL-12 or PBSF, and belongs to the CXC chemokine
family having four conserved cysteine residues.
[0061] SDF-1 used in the present invention may be derived from
human or a non-human mammal such as bovine, monkey, cat, mouse,
rat, guinea pig, hamster, pig, dog, rabbit, sheep or horse.
Preferably, SDF-1 is derived from human.
[0062] Isoforms of human SDF-1 have been confirmed to exist, such
as SDF-1.alpha. (Genbank registration number NP.sub.--954637),
SDF-1.beta. (Genbank registration number NP 000600), SDF-1.gamma.
(Genbank registration number NP 001029058) and SDF-16 (Genbank
registration number NP.sub.--001171605). SDF-1.alpha. is a peptide
of 89 a.a. The sequence of 1-88 a.a. of SDF-1.alpha. is conserved
among the isoforms, where .beta., .gamma. and .delta. have addition
of +5 a.a., +31 a.a. and +52 a.a. at the C-terminal side,
respectively. Moreover, 1-21 a.a. of SDF-1.alpha. at the N-terminal
side is cleaved upon becoming a mature peptide. Thus, 22-88 a.a. of
SDF-1.alpha. is considered as the minimum active unit, and any
peptide can be used as SDF-1 as long as it has this unit.
Preferably, SDF-1 used in the present invention is human mature
SDF-1.alpha. or human mature SDF-1.beta..
[0063] SDF-1 used in the present invention may have one or more
amino acids in its amino acid sequence substituted, inserted,
deleted and/or added, and/or may have a sugar chain substituted,
deleted and/or added, as long as it has an activity as a chemokine
Amino acid mutation is accepted as long as SDF-1 retains at least
four cysteine residues (Cys30, Cys32, Cys55 and Cys71 in the case
of human SDF-1.alpha.) and as long as it has identity in a range of
90% or more to the original natural amino acid sequence.
Alternatively, it may be provided in a form of a
physiologically-accepted salt. While salts with
physiologically-accepted bases (e.g., alkali metals, etc.) or acids
(organic and inorganic acids) can be used as
"physiologically-accepted salts", physiologically-accepted acid
addition salts are particularly favorable. Examples of such salts
include salts with inorganic acids (for example, hydrochloric acid,
phosphoric acid, hydrobromic acid and sulfuric acid) and salts with
organic acids (for example, acetic acid, formic acid, propionic
acid, fumaric acid, maleic acid, succinic acid, tartaric acid,
citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic
acid and benzenesulfonic acid).
[0064] SDF-1 used in the present invention may readily be prepared
according to a known technique or otherwise available as a
commercially available product. In the case where SDF-1 is to be
prepared, for example, it may be prepared as a recombinant SDF-1 by
a genetic engineering procedure or may be prepared by a peptide
synthesis procedure. In the case where SDF-1 is to be obtained as a
commercially available product, for example, Recombinant
Human/Rhesus Macaque/Feline CXCL12/SDF-1 alpha (#350-NS-050,
R&D Systems Inc.), Recombinant Human/Rhesus Macaque/Feline
CXCL12/SDF-1 beta (#351-FS-050, R&D Systems Inc.), Human
SDF-1.alpha. (#130-096-137, Miltenyi Biotec Inc.) or the like can
be used.
[0065] When SDF-1 is to be contained in a composition for cartilage
regeneration of the present invention, it is added such that the
SDF-1 content in the composition for cartilage regeneration of the
present invention is preferably 0.2 .mu.g/ml or more and less than
100 .mu.g/ml and more preferably 1 .mu.g/ml or more and less than
60 .mu.g/ml.
[0066] 5. Preparation of Composition for Cartilage Regeneration
[0067] A composition for cartilage regeneration of the present
invention is characterized in that (a) a monovalent metal salt of
low endotoxin alginic acid and (b) SDF-1 are used in combination.
Hereinafter, (a) a monovalent metal salt of low endotoxin alginic
acid may be referred to as "component (a)" and (b) SDF-1 as
"component (b)".
[0068] According to the present invention, to "used in combination"
refers to concomitant use, and means that it is acceptable as long
as both components (a) and (b) are contained when the composition
of the present invention is applied to a cartilage injury lesion
and the form during the distribution process including sale does
not particularly matter. For example, it may be provided either (1)
in a form of a compounding agent that has both components (a) and
(b) mixed and formulated together, or (2) in a form of two
separately formulated formulations, i.e., components (a) and (b),
provided in combination as a kit or provided separately, so as to
be mixed and used upon application. In other words, SDF-1 as
component (b) may be provided in a form of a compounding agent that
is formulated by blending component (b) with component (a) in
advance, or as a kit obtained by combining and packaging two types
of separately formulated formulations, components (a) and (b).
[0069] Each or a mixture of components (a) and (b) may be provided
in a solution state by using a solvent or provided as a solid
material such as a lyophilization product. In this regard, even if
the components are provided as solid materials, the composition of
the present invention is made into a solution state with a solvent
to have a fluidity upon administration.
[0070] The solvent is not particularly limited as long as it is
biologically applicable, and examples include injectable water,
purified water, distilled water, ion-exchanged water (or deionized
water), Milli-Q water, physiological saline and phosphate buffered
saline (PBS). Preferably, the solvent is injectable water,
distilled water, physiological saline or the like.
[0071] In a composition for cartilage regeneration according to a
preferable embodiment of the present invention, the content of the
monovalent metal salt of alginic acid is about 0.5 to 10% w/v, and
the content of SDF-1 is 0.2 .mu.g/ml or more and less than 100
.mu.g/ml in the composition.
[0072] For example, in order to provide a composition of 2% w/v
alginic acid10 .mu.g/ml SDF-1, an aqueous solution formulation
already containing defined amounts of alginic acid and SDF-1 can be
produced and provided as a compounding agent, or 1 mL of a 4% w/v
aqueous alginic acid solution and 1 mL of 20 .mu.g/ml aqueous SDF-1
solution can be included in a kit as separate formulations such
that the whole amounts of them are mixed upon use to give a
composition containing a final concentration of 2% w/v alginic
acid10 .mu.g/ml SDF-1.
[0073] 6. Viscosity of Composition for Cartilage Regeneration
[0074] Although the viscosity of a composition for cartilage
regeneration of the present invention is not particularly limited
as long as the effect of the present invention can be obtained, it
is preferably 400 mPas to 20,000 mPas. For example, it can be
prepared to have an appropriate viscosity by using the
above-described solvent or the like. Viscosity within such range
gives good adhesive property to a cartilage injury lesion, and also
allows injection into an articular cavity or a cartilage injury
lesion with a syringe or the like. Moreover, when the composition
for cartilage regeneration of the present invention has a viscosity
of about 2,000 mPas or more, the adhesive property to a cartilage
injury lesion is further improved. In particular, for example, in a
case where a cartilage injury lesion of a human femoral joint
surface is to be manipulated under an arthroscope, if the viscosity
is about 5,000 mPas or more, the composition of the present
invention can be injected into the cartilage defect lesion even if
the open side of the cartilage defect lesion is facing down so that
the composition of the present invention can be brought into
contact with and attached to the cartilage injury surface for at
least 1 minute without anchoring. If necessary, the surface of the
composition can be anchored during the attachment. The adhesive
property to the cartilage injury lesion can further be improved by
increasing the viscosity. For example, when the viscosity is 10,000
mPas, the composition can be attached to the affected area without
anchoring for a longer period of time as compared to the case where
viscosity is 5,000 mPas. Therefore, when the opening of the
cartilage defect lesion or the hole formed in the cartilage injury
lesion or the cartilage defect lesion is inclined or facing down,
the composition of the present invention is preferably attached to
the injury lesion for at least 5 seconds, preferably 10 seconds or
longer, more preferably 30 seconds or longer and particularly
preferably 1 minute or longer without using anchoring means. By
adjusting the viscosity, the composition of the present invention
can ensure time for performing anchoring means on the surface of
the composition. Here, "attached to the injury lesion" means that
the composition of the present invention stays on the injury lesion
without falling from the injury lesion. Accordingly, the
composition of the present invention is advantageous in that the
treatment can be conducted by a simple injection method even when
the position of the site is difficult for the practitioner to
treat, for example, the affected area is facing down, upon
treatment, by adjusting the viscosity thereof.
[0075] Meanwhile, injection with a syringe or the like is easier
when the viscosity is about 20,000 mPas or less. Injection with a
syringe or the like is possible even when the viscosity is, for
example, about 20,000 mPas, but when injection is difficult due to
high viscosity, the composition of the present invention can be
applied to the cartilage injury surface by using other means. In
terms of easy manipulation with a syringe, the viscosity of the
composition of the present invention is preferably 20,000 mPas or
less, and more preferably 15,000 mPas or less. Thus, when the
opening of the cartilage defect lesion or the hole formed in the
cartilage injury lesion or the cartilage defect lesion is inclined
or facing down, the viscosity of the composition of the present
invention that is suitable for application to the cartilage injury
lesion is preferably about 2,000 mPas or more in terms of adhesive
property and 20,000 mPas or less in terms of easy handling of the
composition. The viscosity is preferably 3,000 mPas to 15,000 mPas,
more preferably 4,000 mPas to 10,000 mPas, and particularly
preferably 5,000 mPas to 6,000 mPas.
[0076] The viscosity of the composition for cartilage regeneration
can be adjusted by controlling, for example, the concentration of
the alginic acid, the molecular weight of the alginic acid, the M/G
ratio of the alginic acid or the like.
[0077] The viscosity of a solution of a monovalent metal salt of
alginic acid becomes higher when the concentration of the alginic
acid in the solution is high, and becomes lower when the
concentration of the alginic acid in the solution is low. The
viscosity becomes higher when the molecular weight of the alginic
acid is large, and becomes lower when the molecular weight is
small. For example, in order to obtain a viscosity of 400 mPas to
20000 mPas with an alginic acid having a molecular weight of about
1,700,000 Da (Sea Matrix), an aqueous alginic acid solution of
about 1% w/v to 3% w/v may be used. When an alginic acid with a
smaller molecular weight is used, the concentration of the alginic
acid needs to be increased than the above case. The viscosity of
the aqueous alginic acid solution can be measured according to a
known method by using, for example, rotational viscometer
(cone-plate type) (TVE-20LT, TOKI SANGYO CO., LTD., JAPAN) or the
like.
[0078] Since the viscosity of a solution of a monovalent metal salt
of alginic acid is affected by the M/G ratio, an alginic acid
having a preferable M/G ratio can appropriately be selected, for
example, according to the viscosity of the solution or the like.
The M/G ratio of the alginic acid used in the present invention is
about 0.4 to 4.0, preferably about 0.8 to 3.0, and more preferably
about 1.0 to 1.6.
[0079] As previously described, since the M/G ratio is determined
primarily by the type of the seaweed, the type of a brown alga used
as the raw material has an effect on the viscosity of the solution
of the monovalent metal salt of alginic acid. The alginic acid used
in the present invention is preferably derived from a brown alga of
Lessonia species, Macrocystis species, Laminaria species,
Ascophyllum species or Durvillea species, more preferably derived
from a brown alga of Lessonia species, and particularly preferably
derived from Lessonia nigrescens.
[0080] 7. Gelation of Surface of Composition
[0081] Some embodiments of the composition for cartilage
regeneration of the present invention do not contain a
cross-linking agent. When a composition for cartilage regeneration
does not contain a cross-linking agent, the composition for
cartilage regeneration containing a solution of a monovalent metal
salt of alginic acid may be applied to a cartilage injury lesion
and then a cross-linking agent may be applied to the surface of the
composition. By gelating the surface of the composition to cure the
surface, the composition can effectively be prevented from leaking
from the cartilage injury lesion.
[0082] Such a cross-linking agent is not particularly limited as
long as it can cross-link and anchor the surface of the solution of
a monovalent metal salt of alginic acid, and examples include
bivalent or higher metal ion compounds of Ca.sup.2+, Mg.sup.2+,
Ba.sup.2+ and Sr.sup.2+, and cross-linking reagents that have two
to four amino groups within their molecules. More specifically,
examples of bivalent or higher metal ion compounds include
CaCl.sub.2, MgCl.sub.2, CaSO.sub.4, BaCl.sub.2 and SrCl.sub.2
(preferably, CaCl.sub.2, CaSO.sub.4, BaCl.sub.2, etc.), while
examples of cross-linking reagents having two to four amino groups
within their molecules comprise diaminoalkanes optionally having a
lysyl group on a nitrogen atom
(--COCH(NH.sub.2)--(CH.sub.2).sub.4--NH.sub.2), that is,
diaminoalkane and derivatives thereof that form lysyl amino groups
by substituting an amino group with a lysyl group, specific
examples being diaminoethane, diaminopropane and
N-(lysyl)-diaminoethane. In terms of easy access, gel strength and
the like, the cross-linking agent is preferably a bivalent or
higher metal ion compound of Ca.sup.2+, Mg.sup.2+, Ba.sup.2+ or
Sr.sup.2+ (e.g., CaCl.sub.2, MgCl.sub.2, CaSO.sub.4, BaCl.sub.2,
SrCl.sub.2, etc.), more preferably CaCl.sub.2, CaSO.sub.4,
BaCl.sub.2 or the like, and particularly preferably a CaCl.sub.2
solution.
[0083] A method for applying a bivalent or higher metal ion to the
surface of the composition is not particularly limited, and an
example includes a method in which a solution of a bivalent or
higher metal ion is applied to the composition surface with a
syringe, a spray or the like. The timing of applying the
cross-linking agent to the surface of the composition of the
present invention may be subsequent to or simultaneously with the
application of the composition of the present invention to the
defect lesion.
[0084] Preferably, a suitable amount of the cross-linking agent is
suitably adjusted according to the size of the defect lesion to
which the composition of the present invention has been applied.
The cross-linking agent gradually penetrates inside from the
surface of the composition, by which the cross-linking takes place.
In order to prevent major impact by the cross-linking agent on the
contact region between the composition of the present invention and
the injury surface, the amount of the cross-linking agent applied
is adjusted not to be excessive. The amount of the bivalent or
higher metal ion applied is not particularly limited as long as it
allows the surface of the composition containing the monovalent
metal salt of alginic acid to be cured. When, for example, a 100 mM
CaCl.sub.2 solution is to be added, however, the amount added is
preferably about 0.3 to 0.6 ml for a defect with a diameter of 5 mm
and a depth of about 2 mm, and the dosage may be determined in
proportion to the surface area of the affected area. For example,
in the case of a defect with widths (10 mm.times.20 mm) and a depth
of about 5 mm, the amount is preferably about 1 to 12 ml, and more
preferably about 2 to 10 ml. The amount can appropriately be
increased or decreased while observing the state of the injury
lesion. The application to the surface of the composition
containing a monovalent metal salt of alginic acid may take place
as long as, for example, several seconds to 10-odd seconds.
[0085] Furthermore, the composition of the present invention may
contain a cross-linking agent that promotes gelation due to the
difference in time or temperature or change in the environment such
as contact with the calcium ion in vivo so that the composition of
the present invention can keep a solution state before the
administration and gelates by itself after the administration in
vivo. Examples of such cross-linking agents include calcium
gluconate, CaSO.sub.4 and calcium alginate.
[0086] Here, when the cross-linking agent contains calcium, a
higher calcium concentration is known to develop gelation faster
and form harder gel. Since, however, calcium has cytotoxicity, when
the concentration is too high, it may adversely affect the
cartilage regeneration effect of the composition for cartilage
regeneration of the present invention. Accordingly, when, for
example, a CaCl.sub.2 solution is used in order to cure the surface
of a composition containing a monovalent metal salt of alginic
acid, the concentration is preferably 25 mM to 200 mM, and more
preferably 50 to 100 mM.
[0087] Since the surface of the composition of the present
invention is gelated with a cross-linking agent or the whole
composition is gelated by mixing the composition with a
cross-linking agent in advance upon applying the composition to the
cartilage injury lesion, the composition of the present invention
is cured at the affected area and can be located at the applied
cartilage injury lesion in a closely attached state. Thus, the
component (b) can allow migration and accumulation of the
host-derived cells to the affected area. In addition, since the
composition of the present invention is closely attached to the
cartilage injury lesion, the cartilage regeneration effect, in
particular, the hyaline cartilage regeneration effect, of the
composition of the present invention is more strongly produced.
[0088] 8. Application of Composition for Cartilage Regeneration to
Cartilage Injury Lesion
[0089] A composition for cartilage regeneration of the present
invention can be applied to a cartilage injury lesion of human or
an organism other than human, for example, a non-human mammal such
as bovine, monkey, avian, cat, mouse, rat, guinea pig, hamster,
pig, dog, rabbit, sheep or horse to promote regeneration of the
cartilage.
[0090] The composition for cartilage regeneration of the present
invention is preferably, in a liquid state with fluidity, namely, a
solution state. According to the present invention, the phrase
"having fluidity" means to have a property of changing its form
into indefinite shapes. For example, the composition preferably has
fluidity so that it can be included in a syringe or the like and
injected into an affected area. The composition of the present
invention in a solution state can also readily be applied to a
cartilage injury lesion with a syringe, a gel pipette, a
specialized syringe or the like. In addition, it allows the
composition to adapt to a injury or defect lesion of any shape, and
allows the composition to fill or make contact with the whole
defect lesion.
[0091] In some embodiments, the composition of the present
invention can cover the entire injury lesion, has good adhesiveness
to the cartilage defect lesion and can easily make contact with
cells or tissues of the injury lesion of an organism. Accordingly,
component (b) can easily promote migration of the host-derived
cells to the affected area. In some embodiments, the composition of
the present invention applied to the injury lesion fuses to the
tissues of the organism such that it is indistinguishable at the
applied site in about four weeks or so following the application,
with high compatibility to the organism.
[0092] When the viscosity of the composition for cartilage
regeneration of the present invention is too high such that it is
difficult to apply it with a syringe to fill in a cartilage defect
lesion, a pressurized or an electrically-powered syringe may be
used. Instead of using a syringe or the like, the composition may
also be applied to the cartilage injury lesion, for example, with a
spatula, a stick or the like. When the composition is injected with
a syringe, for example, a 16G to 18G needle is preferably used. The
composition of the present invention is used such that it is
directly injected into a cartilage defect lesion preferably under
an arthroscope or an endoscope with a syringe, a gel pipette, a
specialized filling instrument or the like. Alternatively, an
affected area may be exposed by a known surgical procedure, for
example, arthrotomy by medial parapatellar approach before directly
injecting the composition into the cartilage defect lesion with a
syringe, a gel pipette, a specialized filling instrument or the
like but since the composition of the present invention can be
applied to the cartilage injury lesion by a simple procedure with a
syringe or the like, there is no need of widespread arthrotomy.
[0093] An amount of the composition for cartilage regeneration of
the present invention to be applied is not particularly limited and
may be determined according to the size of the cartilage defect
lesion or the size of the hole formed in the injury lesion where
the composition is to be applied. When the composition is directly
injected into the cartilage defect lesion, the amount is, for
example, 0.05 to 10 ml, and more preferably 0.1 to 2 ml.
Preferably, the composition is applied to the cartilage injury
lesion by injection so as to sufficiently fill in the cavity volume
of the affected area.
[0094] A composition for cartilage regeneration of the present
invention may also contain, if necessary, other pharmaceutically
active ingredients and components ordinarily used in
pharmaceuticals, such as commonly used stabilizers, emulsifiers,
osmotic pressure adjusters, buffers, isotonic agents,
preservatives, pain relievers or colorants.
[0095] In one embodiment of the present invention, the composition
of the present invention does not contain any component that exerts
pharmacological action to the cartilage, other than a monovalent
metal salt of low endotoxin alginic acid and SDF-1. Such a
composition of the present invention can exert a sufficient
cartilage regeneration effect.
[0096] Furthermore, in some embodiments, the composition for
cartilage regeneration of the present invention does not contain
cells, especially cells for cartilage regeneration. Examples of
cells for cartilage regeneration include, for example, stem cells
and stromal cells, where their source is not particularly limited
but examples thereof include bone marrow, adipose cells and
umbilical cord blood. Particular examples include mesenchymal stem
cells and bone marrow mesenchymal stromal cells. Additional
examples include cells such as cartilage precursor cells,
chondrocytes, synovial cells, hematopoietic stem cells and ES
cells. The phrase "to contain cells for cartilage regeneration"
refers to addition of cells that are prepared by a process in which
cells of interest are collected and concentrated from bone marrow
or the like or a process where the cells are cultured to increase
the amount thereof. Specifically, the phrase means that cells for
cartilage regeneration is contained, for example, for
1.times.10.sup.5 cells/ml or more.
[0097] The composition for cartilage regeneration of the present
invention may also contain a factor for promoting cell growth.
Examples of such factors include BMP, FGF, VEGF, HGF, TGF-.beta.,
IGF-1, PDGF, CDMP, CSF, EPO, IL and IF. These factors may be
produced by a recombinant method or purified from a protein
composition.
[0098] In one embodiment of the present invention, the composition
of the present invention does not contain these growth factors.
Even when the composition does not contain such growth factors,
regeneration of the cartilage is sufficiently favorable.
[0099] In some embodiments of the present invention, the
composition of the present invention does not contain any scaffold
material for cells except (a) a monovalent metal salt of low
endotoxin alginic acid. A "scaffold material for cells" refers to a
material that serves as a scaffold for migrated cells that
contribute to cartilage regeneration, for example, host-derived
mesenchymal stem cells, to differentiate and/or to proliferate.
Examples of such scaffold materials include those described in page
5, line 29 to page 6, line 16 of WO2010/048418 (except a monovalent
metal salt of low endotoxin alginic acid or a gel obtained by
curing said monovalent metal salt).
[0100] Furthermore, the present invention also provides use of (a)
a monovalent metal salt of low endotoxin alginic acid and (b) SDF-1
for producing a composition for cartilage regeneration.
[0101] In addition, the present invention provides a combination of
(a) a monovalent metal salt of low endotoxin alginic acid and (b)
SDF-1 used for cartilage regeneration.
[0102] 9. Therapeutic Method
[0103] The present invention further provides a method of cartilage
regeneration by using the above-described composition for cartilage
regeneration of the present invention. Specifically, the present
invention provides a cartilage regeneration method comprising the
step of applying a composition wherein (a) a monovalent metal salt
of low endotoxin alginic acid and (b) SDF-1 are used in
combination, to a cartilage injury lesion. More specifically, the
present invention provides a cartilage regeneration method
comprising the step of applying a therapeutically effective amount
of a composition wherein (a) a monovalent metal salt of low
endotoxin alginic acid and (b) SDF-1 are used in combination, to a
cartilage injury lesion of a subject in need thereof.
[0104] The phrases "used in combination" and "apply to a cartilage
injury lesion" are as described above.
[0105] A "subject" refers to human or an organism other than human,
for example, a non-human mammal such as bovine, monkey, avian, cat,
mouse, rat, guinea pig, hamster, pig, dog, rabbit, sheep or horse.
A method for applying a composition for cartilage regeneration of
the present invention to a cartilage injury lesion is not
particularly limited. For example, the composition may directly be
injected into a cartilage defect lesion under an arthroscope or an
endoscope, with a syringe, a gel pipette, a specialized filling
instrument or the like. Alternatively, an affected area may be
exposed, for example, by a known surgical procedure such as
arthrotomy by medial parapatellar approach or the like and then the
composition can directly be injected into the cartilage defect
lesion with a syringe, a gel pipette, a specialized filling
instrument or the like.
[0106] Moreover, a co-administered drug, for example, an antibiotic
such as streptomycin, penicillin, tobramycin, amikacin, gentamicin,
neomycin or amphotericin B or an anti-inflammation drug such as
aspirin, a non-steroidal analgesic antipyretic drug (NSAID) or
acetaminophen may be administered before, simultaneously or after
applying the composition of the present invention to the cartilage
injury lesion. These drugs may also be used by being mixed with the
composition of the present invention.
[0107] One or more holes may be formed in a cartilage injury lesion
so that the composition of the present invention is injected into
the holes. Furthermore, one or more holes may further be formed
into a cartilage defect lesion for the same purpose.
[0108] For example, in the case where an affected area is exposed
by a surgical procedure, a power drill, steel wire or the like may
be used to create a plurality of defects (full-thickness defects)
that reach the subchondral bone with a relatively small diameter
of, for example, about 1.5 mm in a cartilage defect lesion having
remaining cartilage before injecting the composition of the present
invention. Creation of full-thickness defects allows the cartilage
precursor cells in the bone marrow to easily migrate toward the
cartilage defect lesion. Cell migration and cartilage regeneration
are promoted due to the effect of the composition of the present
invention and thus the cartilage regeneration effect can be
enhanced.
[0109] The cartilage injury lesion as the affected area is
preferably subjected to a necessary pretreatment before being
applied with the composition of the present invention. If
necessary, the affected area is washed. The phrase "to wash the
affected area" refers to removal of blood components, other
unwanted tissues and the like, for example, with physiological
saline from the site where the composition of the present invention
is to be applied. After washing the affected area, the remaining
unwanted fluid component are preferably dried by wiping off or the
like before applying the composition of the present invention.
[0110] After applying the composition of the present invention to
the cartilage injury lesion, a cross-linking agent is preferably
applied to the surface of said composition. By applying the
cross-linking agent to the surface of the composition, the
composition of the present invention is cured in the affected area.
Furthermore, the excessive amount of cross-linking agent remaining
in the affected area may be washed and removed with physiological
saline or the like.
[0111] All of these steps can be performed under an
arthroscope.
[0112] According to a preferred embodiment of a cartilage
regeneration method of the present invention, a cartilage injury
lesion or a cartilage disease can be treated.
[0113] 10. Kit for Cartilage Regeneration
[0114] The present invention further provides a kit for cartilage
regeneration. The kit comprises a formulation containing at least
component (a) of the composition for cartilage regeneration of the
present invention. The kit may further include a cross-linking
agent, a solvent, a syringe, a gel pipette, a specialized filling
instrument, instruction and the like. A preferable example of such
a kit has an alginic acid solution enclosed in one chamber and
SDF-1 powder in the other chamber of the integrally molded two
chambers of a syringe which are separated by a partition. The
partition between the two chambers is made such that it can readily
be opened upon use so that both contents can be mixed/dissolved
upon use. Other exemplary kit has a composition for cartilage
regeneration enclosed in a pre-filled syringe so that the
composition can directly be used upon administration without the
need of manipulation for preparation. Other exemplary kit has an
alginic acid solution and a cross-linking agent enclosed in
separate syringes, which are included in one package. The kit may
further include a co-administered drug, for example, an antibiotic
such as streptomycin, penicillin, tobramycin, amikacin, gentamicin,
neomycin or amphotericin B or an anti-inflammation drug such as
aspirin, a non-steroidal analgesic antipyretic drug (NSAID) or
acetaminophen.
[0115] By using this kit, cartilage regeneration treatment can be
carried out smoothly.
[0116] All publications cited herein, such as prior art documents,
unexamined patent applications, patent publications and other
patent documents, are incorporated in their entirety herein by
reference. In addition, the present specification incorporates the
disclosed contents of the claims, specification and drawings of
Japanese Patent Application No. 2011-181662 (filed on Aug. 23,
2011), which serves as the basis for claiming priority of the
present application.
[0117] Hereinafter, the present invention will be specifically
described by means of examples, although the present invention is
not limited to these examples.
Example 1
Expression of SDF-1 Protein in Osteochondral Defect Models
[0118] An osteochondral defect model of a rabbit knee joint was
used to examine change in the expression of SDF-1 protein at a
injury site with time by immunohistostaining
[0119] 15-week-old female Japanese white rabbits (weight 2.6 to 2.9
kg) were used to prepare osteochondral defect models. Anesthesia
was performed by intravenous administration of 0.05 mg/kg
pentobarbital and inhalation anesthesia of isoflurane. An
antibiotic (penicillin G, Meiji Co., Ltd.) was intramuscularly
administered. After incision in the skin of about 2 cm, patella was
turned over by medial parapatellar approach to expand the patellar
surface of the femur. A power drill (Rexon, Kawasaki) was used to
create full-thickness osteochondral defects (diameter of 4.5 mm and
depth of 3 mm) that reach the subchondral bones in the patellar
surfaces of femurs of both knees. A sham surgery group (control)
received the same surgery except the creation of the full-thickness
osteochondral defect. The full-thickness osteochondral defects
provide a favorable model of a cartilage injury lesion. Additional
defect in the bone layer allows observation of the effects on the
subchondral bone formation and the tide mark (that forms boundary
between cartilage and bone).
[0120] At each of the time points, i.e., three hours, one week, two
weeks and four weeks following the surgery, the rabbits as the
subjects were euthanized by intravenous injection of an excessive
dose of pentobarbital. The limbs were resected with a power saw to
obtain knee specimens.
[0121] In order to confirm the expression of SDF-1 protein at the
cartilage injury lesion, immunohistostaining was performed using a
mouse anti-SDF-1 monoclonal antibody (R&D Systems, Inc.). The
specimen was immobilized with a 10% formalin solution containing 4%
phosphate buffer and embedded in paraffin. A sample was prepared
with a section from the center of the osteochondral defect created
part with a thickness of 5 .mu.m.
[0122] The specimens were freeze-fractured for Western blotting,
homogenized in 8M urea, 50 mM phosphate and 10 mM Tris (pH 8.0)
buffer, and added with EDTA (50 mM). After 24 hours of incubation
at room temperature, the homogenates were centrifuged to obtain
supernatants. The expression level of SDF-1 protein was assessed by
Western blotting using a mouse anti-SDF-1 monoclonal antibody.
[0123] Results from the immunohistostaining indicated that
expression of SDF-1 protein at the injury site was found a week
following the creation of the full-thickness osteochondral defects
(FIGS. 1B and 1I). On the other hand, expression of SDF-1 protein
was not confirmed three hours, two weeks and four weeks following
the injuries (FIGS. 1A, 1C and 1D). In the sham operation group,
expression of SDF-1 protein was not observed at any time point
(FIGS. 1E-1H). Western blotting also indicated that expression of
SDF-1 protein was observed only in the tissues of a week following
the injuries, which coincide with the results from the
immunohistostaining (FIG. 1J). This experiment revealed expression
of SDF-1 protein at a osteochondral injury lesion for the first
time
Example 2
Application of Alginic Acid Gel to Rabbit Osteochondral Defect
Models
[0124] (2-1) Alginic Acid
[0125] A low endotoxin sodium alginate (Sea Matrix (sterilized),
with a molecular weight of about 1,700,000 Da, distributed from
Mochida International Ltd.) was used. 12.5 ml of deionized water
sterilized by a filter procedure was added to 0.25 g of sodium
alginate (lyophilized product) to obtain a 2% sodium alginate
solution that is to be used in the experiment. The endotoxin level
of the low endotoxin sodium alginate was 5.76 EU (endotoxin
unit)/g. The endotoxin level of food grade (commercial grade)
sodium alginate (Wako Pure Chemical Industries, Ltd., sodium
alginate 500, 199-09961) is 75,950 EU/g, and thus sodium alginate
with an extremely low endotoxin level is used in this
experiment.
[0126] (2-2) Preparation of Models
[0127] Rabbit osteochondral defect models were prepared by the
procedure described in Example 1. The prepared full-thickness
osteochondral defects were washed with physiological saline so that
no hematoma exists at the defect lesion. Thereafter, the defect
lesion was filled with a 2% sodium alginate solution. The
preparation was carried out for the following four groups where
n=10.
[0128] 1) Untreated group (without administration of alginic acid;
indicated as "Defect" in the figure)
[0129] 2) Alginic acid group
[0130] (a 2% sodium alginate solution containing 10 .mu.g/ml of BSA
as a protein control for SDF-1; indicated as "Vehicle" in the
FIG.
[0131] 3) Alginic acid+SDF-1 administration group
[0132] (a 2% sodium alginate solution containing 10 .mu.g/ml of
SDF-1 (Human SDF-1.alpha.; Miltenyi Biotec Inc., Auburn, Calif.);
indicated as "SDF-1" in the figure)
[0133] 4) Alginic acid+AMD3100 administration group
[0134] (a 2% sodium alginate solution containing 250 .mu.g/ml of
AMD3100 (CXCR4 antagonist, Sigma-Aldrich, Saint Louis, Mo.);
indicated as "AMD3100" in the figure)
[0135] Since the sodium alginate solution has high viscoelasticity,
it did not flow out from the defect lesion. A 100 mM calcium
hydrochloride (Wako Pure Chemical Industries, Ltd.) solution was
applied onto the surface of the sodium alginate solution injected
into the defect lesion, left to stand for 10 seconds to confirm
curing of the alginic acid. No other anchoring was added to the
site for injecting the sodium alginate solution. Articular capsule,
fascia and skin were sutured with a 4-0 nylon thread. No anchor
splint was used and the rabbits were allowed to freely move in the
cage.
[0136] (2-3) Macroscopic Findings, Histological Assessment,
Immunohistological Assessment
[0137] The rabbits were sacrificed under high concentration
intravenous anesthesia 4 and 16 weeks after the surgery. The limbs
were resected with a power saw to obtain knee specimens. Then,
their pictures were taken with a digital camera for assessment for
macroscopic findings. The specimens for histological assessment and
immunohistological assessment were prepared in the same manner as
Example 1. Samples were prepared using sections from the center of
the osteochondral defect created part with a thickness of 5 .mu.m.
The samples were stained with Safranin-O and H-E.
Immunohistostaining was carried out by staining with an anti-type I
collagen antibody and an anti-type II collagen antibody (Fuji
Pharma Co. Ltd.). The macroscopic and histological findings were
scored according to the method of Niederrauer et al. (Biomaterial
21 (2000) 2561-2574). Scoring was carried out by an independent
blind observer.
[0138] (2-3-1) Macroscopic Findings
[0139] Infection or inflammation reaction such as foreign-body
reaction was found in none of the knees. Four weeks after the
surgery, the implanted alginic acid gel remained at the defect
lesions for the alginic acid group and the alginic acid+AMD3100
administration group (FIGS. 2B and 2D). For the untreated group and
the alginic acid+AMD3100 administration group, roughness and mild
dents on the surface of the repaired tissues and incomplete fusion
with the adjacent cartilage were found 16 weeks after the surgery
(FIGS. 2A, 2D, 2E and 2H).
[0140] Meanwhile, for the alginic acid group and the alginic
acid+SDF-1 administration group, the surface was smooth and fusion
with the adjacent cartilage was improved 16 weeks after the surgery
(FIGS. 2F and 2G). For the alginic acid+SDF-1 administration group,
the defect lesion was partially filled with white glossy solid
hyaline cartilage-like tissues four weeks after the surgery (FIG.
2C), and was almost replaced by hyaline cartilage-like tissues 16
weeks after the surgery (FIG. 2G).
[0141] With respect to the average macroscopic scores, significant
improvement was observed 16 weeks than 4 weeks after the surgery in
all of the groups (FIGS. 21 and 2J). Regarding comparison between
the groups, macroscopic scores of the alginic acid+SDF-1
administration group 4 weeks after the surgery were significantly
higher than those of the other groups (FIG. 2I). The macroscopic
scores of the alginic acid+AMD3100 administration group 4 weeks
after the surgery was significantly lower than those of the alginic
acid group (FIG. 2I). The macroscopic scores of the alginic
acid+SDF-1 administration group 16 weeks after the surgery were
significantly higher than those of the other groups (FIG. 2J).
[0142] (2-3-2) Histological and Immunohistological Findings (4
Weeks after Surgery)
[0143] The repaired tissues of the untreated group and the alginic
acid+AMD3100 administration group were fibrous tissues mainly
consisting of type I collagen (FIGS. 3A, 3D, 3E, 3H, 3I and 3L).
Meanwhile, in the alginic acid group and the alginic acid+SDF-1
administration group, the defect lesions were partially repaired
with hyaline cartilage-like tissues that contain glycosaminoglycan
and type II collagen (FIGS. 3B, 3C, 3J and 3K). In both groups, the
repaired tissue partially contained type I collagen (FIGS. 3F and
3G). In the alginic acid group, crack was found in the defect
lesion (FIG. 3B). None of the groups showed formation of normal
subchondral bone, smooth cartilage surface or complete tide mark
(that forms boundary between cartilage and bone) 4 weeks after the
surgery (FIGS. 3A-D).
[0144] The average histological score of the alginic acid+SDF-1
administration group was significantly higher than those of other
groups (FIG. 3M).
[0145] (2-3-3) Histological and Immunohistological Findings (16
Weeks after Surgery)
[0146] The untreated group resulted in repair with only fibrous
tissue, with multiple cracks and severe breakage in the surface
(FIGS. 4A, 4E and 4I). The alginic acid group and the alginic
acid+AMD3100 administration group gave repaired images showing weak
staining of type I collagen and type II collagen (FIGS. 4B, 4D, 4F,
4H, 4J and 4L). Regeneration of the subchondral bone was promoted
in the alginic acid group than in the alginic acid+AMD3100
administration group (FIG. 4B). While crack was observed at the
boundary with cartilage adjacent to the defect lesion in the
alginic acid+AMD3100 administration group (FIG. 4D), no crack was
observed in the alginic acid group (FIG. 4B). Meanwhile, almost
normal hyaline cartilage regeneration was observed in the alginic
acid+SDF-1 administration group, which was associated with abundant
glycosaminoglycan content, strong type II collagen staining,
formation of normal subchondral bone, smooth cartilage surface and
complete tide mark (FIGS. 4C and 4K). Type I collagen staining was
not found in the cartilage layer (FIG. 4G). Neoplastic cartilage
tissue showed good fusion with the adjacent cartilage and bone
(FIG. 4C).
[0147] The histological score of the alginic acid group and the
alginic acid+SDF-1 administration group significantly improved 16
weeks than 4 weeks after the surgery (FIGS. 3M and 4M). The
histological scores of the alginic acid+SDF-1 administration group
16 weeks after the surgery were significantly higher than those of
the other groups (FIG. 4M). The histological scores of the alginic
acid group were significantly superior over those of the untreated
group (FIG. 4M). No significant difference was found in the scores
between the untreated group and the alginic acid+AMD3100
administration group.
[0148] An alginic acid gel was applied to rabbit osteochondral
defect models in the same method as described in (2-1) and (2-2)
except that an aqueous solution was prepared by adding 6.25 mL of
water to 0.25 g of low endotoxin sodium alginate in (2-1), and that
a serum collected from the treated rabbit was added to this aqueous
solution in a 1:1 volume to obtain a 2% sodium alginate solution
and this was administered to the rabbit osteochondral defect models
in (2-2) (alginic acid+autologous blood group). As a result, the
macroscopic findings, histological findings and immunohistological
findings of the alginic acid+autologous blood group were comparable
to those of the alginic acid group.
[0149] (2-4) Mechanical Assessment
[0150] The knee specimens 4 and 16 weeks after the surgery from
each group were used to assess the mechanical strength of the
repair tissue. Comparison was made to normal knee specimens as a
control group. A rod with a hemispherical chip having a diameter of
2.5 mm was vertically pressed against the surfaces of the samples
at a speed of 10 mm/min to determine the compression moduli thereof
based on the initial slope of the stress-strain curve. The results
are shown in Table 1.
TABLE-US-00001 TABLE 1 4 weeks 16 weeks Group (MPa) (MPa) Defect
0.66 .+-. 0.08.sup..dagger. 0.59 .+-. 0.11.sup..dagger. Vehicle
0.50 .+-. 0.16.sup..dagger. 1.82 .+-. 0.28**.sup.,.dagger..dagger.
SDF-1 0.89 .+-. 0.05.sup..dagger. 2.34 .+-. 0.38* AMD3100 0.60 .+-.
0.93.sup..dagger. 1.89 .+-. 0.18**.sup.,.dagger..dagger. Normal
cartilage 2.89 .+-. 0.25 Mean .+-. SEM. *p < 0.001 vs. Defect at
the same time, **p < 0.01 vs Defect at the same time.
.sup..dagger.p < 0.001 vs. Normal knee, .sup..dagger..dagger.p
< 0.05 vs. Normal knee
[0151] Four weeks after the surgery, the compression moduli of the
repair tissues of all of the groups were lower than the compression
modulus of the cartilage tissue of the normal knee specimen (each
group n=5, Table 1). No significant different was found among the
untreated group, the alginic acid group, the alginic acid+SDF-1
administration group and the alginic acid+AMD3100 administration
group. Other than the untreated group, the compression moduli
significantly improved 16 weeks after the surgery than 4 weeks
after the surgery. The compression moduli of the alginic acid group
and the alginic acid+AMD3100 administration group 16 weeks after
the surgery were significantly superior over that of the untreated
group. The compression modulus of the alginic acid+SDF-1
administration group significantly improved as compared to that of
the untreated group, reached about 80% of the normal cartilage and
no significant difference was found with the normal cartilage.
[0152] (2-5) Assessment of SDF-1 Dosage
[0153] In order to examine the SDF-1 dosage, three alginic
acid+SDF-1 administration groups with the SDF-1 concentrations of
1, 10 and 100 .mu.g/ml were prepared in the same manner as in (2-2)
and they were compared to the alginic acid group. In the same
manner as in (2-3), a macroscopic assessment, a histological
assessment and an immunohistological assessment were carried out.
The assessments took place 4 and 12 weeks after the surgery.
[0154] With respect to the macroscopic findings 4 weeks after the
surgery, good cartilage regeneration was observed for all of the
alginic acid+SDF-1 administration groups (1, 10 and 100 .mu.g/ml)
as compared to the alginic acid group (FIG. 5).
[0155] Referring to the immunohistological images 12 weeks after
the surgery (FIG. 6), regeneration by the hyaline cartilage-like
tissues was found in both of the alginic acid group and the SDF-1
administration group, while better cartilage regenerations were
observed in the alginic acid+SDF-1 administration groups
proportional to the SDF-1 concentrations. In the group containing
SDF-1 at a high concentration (100 .mu.g/ml), the cartilage tended
to become hyperplastic. Considering the regeneration of the
subchondral bone, an appropriate SDF-1 concentration for this model
seemed to be approximately 1 to 60 .mu.g/ml, and in particular 10
.mu.g/ml.
Example 3
Assessment of In Vivo Cell Homing
[0156] After applying the alginic acid+SDF-1 to the osteochondral
defect lesion, quantitative assessment was conducted to see how the
in vivo cell homing and migration to the injury lesion take place.
An alginic acid group, an alginic acid+SDF-1 administration group
and an alginic acid+AMD3100 administration group were prepared in
the same method as the rabbit osteochondral defect models described
in Example 2. Seven days after the surgery, the implanted alginic
acid gels were collected and the numbers of cells in the gels were
counted. The collected gel was immobilized with 4% phosphate
buffered paraformaldehyde for 24 hours and embedded in paraffin to
prepare a sample using a section from the center of the gel with a
thickness of 5 .mu.m. The samples were stained with H-E
(hematoxylin and eosin).
[0157] A week after the surgery, more cells were found in the gel
of alginic acid+SDF-1 administration group compared to other groups
(FIG. 7C). From the quantitative assessment of the cell counts, the
cell counts of the alginic acid+SDF-1 administration group were
found to be significantly higher than the cell counts of other
groups (each group n=5, FIG. 7E). The cell count of the alginic
acid group (FIG. 7E, Vehicle) tended to be higher than the cell
count of the alginic acid+AMD3100 administration group (FIG. 7E,
AMD3100), but it was not of a significant difference. These results
indicate that application of alginic acid+SDF-1 (FIG. 7E, SDF-1) to
a cartilage injury lesion promoted migration of the host-derived
cells to the injury lesion.
Example 4
In Vitro Assessment of Effect of SDF-1 on Behavior of BMSC
[0158] Bone marrows were collected from a tibia of a 15-week-old
Japanese white rabbit to isolate and culture bone marrow
mesenchymal stromal cells (BMSC). Isolation was carried out
according to the method of Wakitani et al. (Wakitani S et al., J
bone Joint Surg Am, Vol. 76, p 579, 1994). The isolated cells were
incubated at 37.degree. C. under humidified 5% CO.sub.2 and
maintained in monolayer culture.
[0159] (4-1) Test for BMSC Migration with SDF-1
[0160] A test for BMSC migration with SDF-1 was carried out using
CytoSelect.TM. 96-well cell migration assay (Cell Biolabs, San
Diego, Calif.). 100 .mu.l of BMSC culture solution containing
5.times.10.sup.5 cells/ml was added to the upper tray while the
lower tray was filled with 150 .mu.l of a SDF-1-free or SDF-1-added
(concentration 100 ng/ml) medium, incubated at 37.degree. C. under
5% CO.sub.2 for 8 hours and the number of the migrated cells were
detected by fluorescent labeling.
[0161] BMSC migration significantly increased in the SDF-1-added
medium than the SDF-1-free medium (n=16, FIG. 8A).
[0162] (4-2) Effect of SDF-1 on BMSC Proliferation
[0163] Proliferation and differentiation of BMSC were tested by
embedding BMSC in alginic acid gel beads. The cells were suspended
in a 2% sodium alginate solution. The suspension was dripped in a
CaCl.sub.2 solution with a pipette for gelation. Ten minutes later,
the resulting microcapsules were washed twice with Ca/Mg-free PBS
and once with DMED-HG. The resulting beads contained
1.times.10.sup.6 cells per 40 .mu.l of beads.
[0164] The alginic acid gel beads containing 10 .mu.g/ml of SDF-1
or 250 .mu.g/ml of AMD3100 were cultured in DMED-HG containing 10%
FBS and 1% antibiotics for 3 hours, 1 day, 2 days, 3 days and 7
days. The cells collected from the alginic acid gel beads were
washed with PBS for three times, incubated in 50 mM EDTA (Gibco BRL
laboratories) at 37.degree. C. under 5% CO.sub.2. Ten minutes
later, the cells were centrifuged at 1,500 g for 5 minutes for cell
collection and the number of live cells was counted with Cell
Counting Kit-8 (Dojindo Laboratories, Tokyo, Japan).
[0165] In BMSC proliferation test, no significant difference was
found among the alginic acid only group (control), the 10 .mu.g/ml
SDF-1-added group and the 250 .mu.g/ml AMD3100-added group (n=5,
FIG. 8B).
[0166] (4-3) Effect of SDF-1 on BMSC Differentiation
[0167] Differentiation into chondrocytes in the alginic acid gel
beads was tested by culturing BMSC-containing beads in a DMED-HG
medium containing 100 .mu.g/ml sodium pyruvate (ICN Biomedicals,
Aurora, Ohio), 40 .mu.g/ml proline (ICN Biomedicals), 50 .mu.g/ml
ascorbate-2-phosphate (Wako, Osaka, Japan), 1.times.10.sup.-7M
dexamethasone (ICN Biomedicals), 1% ITS plus mix (Sigma-Aldrich,
St. Louis., MO), 1% antibiotics and 10 ng/ml recombinant human
transforming growth factor .alpha.3 (R&D Systems, Minneapolis,
Minn.) (dissolved with 4 mM HCl containing 1 mg/ml bovine serum
albumin) for 28 days. The medium was exchanged every three days.
The beads were washed with PBS, immobilized with 4% phosphate
buffered paraformaldehyde for 24 hours and embedded in paraffin to
prepare a sample using a section from the center of the gel with a
thickness of 5 .mu.m. The samples were stained with Safranin-O and
H-E. Immunohistostaining was carried out using an anti-type I
collagen antibody and an anti-type II collagen antibody (Fuji
Pharma Co. Ltd.).
[0168] In the test for differentiation of BMSC into chondrocytes,
no difference was found among the alginic acid only group, the 10
.mu.g/ml SDF-1-added group and the 250 .mu.g/ml AMD3100-added
group.
[0169] (4-4) Discussion
[0170] From the in vitro tests, considering that the BMSC migration
was promoted by SDF-1 and that the application of alginic
acid+SDF-1 to the cartilage injury lesion in Example 3 promoted the
migration of the host-derived cells to the injury lesion,
chemotaxis of SDF-1 seemed to promote migration of stem cells such
as host-derived BMSC to the osteochondral injury lesion. On the
other hand, SDF-1 was found to show no direct action on the BMSC
proliferation and differentiation into chondrocytes. The alginic
acid appears to play an important role in the cartilage
regeneration by the host-derived cells recruited to the cartilage
injury lesion. Specifically, it was found that when a composition
of an alginic acid containing SDF-1 is applied to a cartilage
injury lesion, SDF-1 promotes migration of host-derived stem cells
or the like to the affected area while the alginic acid provides
environment suitable for the stem cells or the like to
differentiate into chondrocytes and for cartilage regeneration,
thereby obtaining a good hyaline cartilage regeneration effect
without using graft cells.
* * * * *