U.S. patent application number 15/705826 was filed with the patent office on 2018-03-08 for cartilage regenerative material.
This patent application is currently assigned to FUJIFILM Corporation. The applicant listed for this patent is FUJIFILM Corporation, JAPAN TISSUE ENGINEERING CO., LTD.. Invention is credited to Satoko HADA, Hayato MIYOSHI, Kentaro NAKAMURA, Masatoki WATANABE.
Application Number | 20180064849 15/705826 |
Document ID | / |
Family ID | 56920108 |
Filed Date | 2018-03-08 |
United States Patent
Application |
20180064849 |
Kind Code |
A1 |
NAKAMURA; Kentaro ; et
al. |
March 8, 2018 |
CARTILAGE REGENERATIVE MATERIAL
Abstract
An object of the invention is to provide a cartilage
regenerative material that is capable of regenerating bone and
cartilage using cells. Provided is a cartilage regenerative
material including a cell construct, which includes biocompatible
polymer blocks and stem cells, in which a plurality of the
biocompatible polymer blocks are disposed in gaps between a
plurality of the stem cells.
Inventors: |
NAKAMURA; Kentaro;
(Ashigarakami-gun, JP) ; MIYOSHI; Hayato;
(Ashigarakami-gun, JP) ; HADA; Satoko;
(Gamagori-shi, JP) ; WATANABE; Masatoki;
(Gamagori-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
FUJIFILM Corporation
JAPAN TISSUE ENGINEERING CO., LTD. |
Tokyo
Gamagori-shi |
|
JP
JP |
|
|
Assignee: |
FUJIFILM Corporation
Tokyo
JP
JAPAN TISSUE ENGINEERING CO., LTD.
Gamagori-shi
JP
|
Family ID: |
56920108 |
Appl. No.: |
15/705826 |
Filed: |
September 15, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/JP2016/058540 |
Mar 17, 2016 |
|
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15705826 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 27/222 20130101;
A61P 19/00 20180101; A61L 27/3852 20130101; A61F 2002/30766
20130101; A61K 35/28 20130101; A61K 35/32 20130101; A61L 27/3834
20130101; A61L 27/54 20130101; A61L 27/58 20130101; A61L 27/3654
20130101; A61K 38/17 20130101; A61L 2430/06 20130101; A61L 27/56
20130101; A61K 38/39 20130101; A61K 35/28 20130101; A61K 38/39
20130101 |
International
Class: |
A61L 27/36 20060101
A61L027/36; A61L 27/56 20060101 A61L027/56; A61L 27/54 20060101
A61L027/54; A61K 35/32 20060101 A61K035/32; A61L 27/38 20060101
A61L027/38 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 18, 2015 |
JP |
2015-054874 |
Claims
1. A cartilage regenerative material comprising: a cell construct
that includes biocompatible polymer blocks and stem cells, the cell
construct having a plurality of the biocompatible polymer blocks
disposed in gaps between a plurality of the stem cells.
2. The cartilage regenerative material according to claim 1, for
use in regeneration of cartilage and bone.
3. The cartilage regenerative material according to claim 1,
wherein the stem cells are mesenchymal stem cells.
4. The cartilage regenerative material according to claim 1,
wherein the cell construct includes the biocompatible polymer
blocks in an amount of from 0.0000001 .mu.g to 1 .mu.g per stem
cell.
5. The cartilage regenerative material according to claim 1,
wherein the size of each of the biocompatible polymer blocks is
from 10 .mu.m to 300 .mu.m.
6. The cartilage regenerative material according to claim 1,
wherein the cell construct has a thickness or diameter of from 100
.mu.m to 1 cm.
7. The cartilage regenerative material according to claim 1,
wherein the biocompatible polymer blocks are formed from a
recombinant peptide or a chemically synthesized peptide.
8. The cartilage regenerative material according to claim 1,
wherein the biocompatible polymer blocks are formed from a
recombinant gelatin or a chemically synthesized gelatin.
9. The cartilage regenerative material according to claim 8,
wherein the recombinant gelatin or the chemically synthesized
gelatin is represented by Formula 1, A-[(Gly-X-Y).sub.n].sub.m-B
Formula 1: in Formula 1, n units of X each independently represent
any amino acid residue; n units of Y each independently represent
any amino acid residue; m represents an integer from 2 to 10; n
represents an integer from 3 to 100; A represents an arbitrary
amino acid residue or amino acid sequence; and B represents an
arbitrary amino acid residue or amino acid sequence.
10. The cartilage regenerative material according to claim 8,
wherein the recombinant gelatin or the chemically synthesized
gelatin is any one of the following: a peptide comprising the amino
acid sequence set forth in SEQ ID NO:1; a peptide having
biocompatibility and comprising an amino acid sequence obtained by
modifying the amino acid sequence set forth in SEQ ID NO:1 by
deletion, substitution or addition of one or several amino acid
residues; and a peptide having biocompatibility and comprising an
amino acid sequence having at least 80% sequence identity with the
amino acid sequence set forth in SEQ ID NO:1.
11. The cartilage regenerative material according to claim 1,
wherein biocompatible polymers in the biocompatible polymer blocks
are crosslinked by heat, ultraviolet radiation, or an enzyme.
12. The cartilage regenerative material according to claim 1,
wherein the biocompatible polymer blocks are in the form of
granules obtainable by pulverizing a porous body of a biocompatible
polymer.
13. A cartilage regenerative material comprising: the cartilage
regenerative material according to claim 1; and a biocompatible
polymer film.
14. The cartilage regenerative material according to claim 13,
wherein the biocompatible polymer film is a film for isolating a
portion or the entirety of the transplant face of the cell
construct from the site of transplantation.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of PCT International
Application No. PCT/JP2016/058540 filed on Mar. 17, 2016, which
claims priority under 35 U.S.C .sctn. 119(a) to Japanese Patent
Application No. 2015-054874 filed on Mar. 18, 2015. Each of the
above application(s) is hereby expressly incorporated by reference,
in its entirety, into the present application.
REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB
[0002] This application includes an electronically submitted
sequence listing in .txt format. The .txt file contains a sequence
listing entitled "2017-11-16_2870-0673PUS1_ST25.txt" created on
Nov. 16, 2017 and is 31,850 bytes in size. The sequence listing
contained in this .txt file is part of the specification and is
hereby incorporated by reference herein in its entirety.
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0003] The present invention relates to a cartilage regenerative
material including a cell construct that includes biocompatible
polymer blocks and stem cells.
2. Description of the Related Art
[0004] Generally, articular osteochondral defects are not likely to
be accompanied by spontaneous regeneration, and thus, regenerative
medicine based on cell transplantation therapy has been actively
attempted. Cell transplantation therapy has been attempted in many
cases by administering cells in the form of cell aggregates. For
example, it is described in JP4122280B that a tissue plug is
produced by introducing a cell mass of cells derived from a tissue
collected from a test animal or a patient into a chamber having
micropores through which culture fluid can pass; and culturing the
cell mass in an excess amount of culture fluid compared to the
amount of the culture fluid in the chamber by introducing the
culture fluid into the chamber in an amount such that a portion of
the cell mass is in contact with the gas phase, and the tissue plug
thus produced is transplanted. It is described in J. I. Lee,
"Transplantation of scaffold-free spheroids composed of
synovium-derived cells and chondrocytes or the treatment of
cartilage defects of the knee," European Cells and Materials, Vol.
22, 2011, p 275-290, that spheroids that are composed of
synovium-derived cells and chondrocytes and do not include a
scaffold are transplanted for the treatment of knee cartilage
loss.
[0005] Meanwhile, WO2011/108517A describes a cell construct that
includes polymer blocks having biocompatibility and cells, in which
a plurality of the polymer blocks are disposed in gaps between a
plurality of the cells. In regard to the cell construct described
in WO2011/108517A, delivery of nutrients from the outside to the
inside of the cell construct is enabled, the cell construct has a
sufficient thickness, and cells are uniformly distributed within
the construct. In the Examples of JP4122280B, high cell survival
activity was verified by using polymer blocks formed from a
recombinant gelatin or naturally occurring gelatin material. In
Example 11 of WO2011/108517A, it is described that the cell
construct thus produced produces a large amount of
glycosaminoglycan (GAG) and promotes chondrocyte
differentiation.
SUMMARY OF THE INVENTION
[0006] As described above, regenerative medicine based on cell
transplantation therapy has been attempted for articular
osteochondral defects; however, simple administration of cells does
not lead to engraftment of the cells onto the site of loss, and a
sufficient regeneration effect is not obtained. Therefore,
administration of cells in the form of cell aggregates has been
attempted on numerous occasions (JP4122280B; J. I. Lee,
"Transplantation of scaffold-free spheroids composed of
synovium-derived cells and chondrocytes or the treatment of
cartilage defects of the knee," European Cells and Materials, Vol.
22, 2011, p 275-290; and the like). However, even in a case in
which cells are administered in the form of cell aggregates, it is
difficult to simultaneously regenerate desired bone and cartilage
while preventing the penetration of fibrous soft tissue. Also, it
is described in WO2011/108517A that the cell construct produces a
large amount of glycosaminoglycan (GAG); however, it has not been
verified whether cartilage and bone can be regenerated
simultaneously. In a case in which regenerative therapy for
osteochondral defects is performed using cell aggregates as
described above, the effects of regenerative therapy and the like
are enhanced compared to the case of using isolated single cells;
however, the effects are not necessarily satisfactory. Thus, there
is a demand for a cell construct that exhibits a superior
osteochondral regeneration effect.
[0007] It is an object of the invention to provide a cartilage
regenerative material that can regenerate bone and cartilage using
cells.
[0008] The inventors of the present invention conducted a thorough
investigation in order to solve the problems described above, and
as a result, the inventors found that a cell construct including
biocompatible polymer blocks and stem cells, in which a plurality
of the biocompatible polymer blocks are disposed in gaps between a
plurality of the stem cells, has excellent cartilage regenerative
capacity and excellent bone regenerative capacity. Thus, this
invention was completed based on these findings.
[0009] That is, according to the invention, the following
inventions are provided.
[0010] (1) A cartilage regenerative material comprising a cell
construct that includes biocompatible polymer blocks and stem
cells, the cell construct having a plurality of the biocompatible
polymer blocks disposed in gaps between a plurality of the stem
cells.
[0011] (2) The cartilage regenerative material according to (1),
for use in regeneration of cartilage and bone.
[0012] (3) The cartilage regenerative material according to (1) or
(2), in which the stem cells are mesenchymal stem cells.
[0013] (4) The cartilage regenerative material according to any one
of (1) to (3), in which the cell construct includes the
biocompatible polymer blocks in an amount of from 0.0000001 .mu.g
to 1 .mu.g per stem cell.
[0014] (5) The cartilage regenerative material according to any one
of (1) to (4), in which the size of each of the biocompatible
polymer blocks is from 10 .mu.m to 300 .mu.m.
[0015] (6) The cartilage regenerative material according to any one
of (1) to (5), in which the thickness or the diameter of the cell
construct is from 100 .mu.m to 1 cm.
[0016] (7) The cartilage regenerative material according to any one
of (1) to (6), in which the biocompatible polymer blocks are formed
from a recombinant peptide or a chemically synthesized peptide.
[0017] (8) The cartilage regenerative material according to any one
of (1) to (7), in which the biocompatible polymer blocks are formed
from a recombinant gelatin or a chemically synthesized gelatin.
[0018] (9) The cartilage regenerative material according to (8), in
which the recombinant gelatin or the chemically synthesized gelatin
is represented by Formula 1,
A-[(Gly-X-Y).sub.n].sub.m-B Formula 1:
[0019] in Formula 1, n units of X each independently represent any
amino acid residue; n units of Y each independently represent any
amino acid residue; m represents an integer from 2 to 10; n
represents an integer from 3 to 100; A represents an arbitrary
amino acid residue or amino acid sequence; and B represents an
arbitrary amino acid residue or amino acid sequence.
[0020] (10) The cartilage regenerative material according to (8) or
(9), in which the recombinant gelatin or the chemically synthesized
gelatin is any one of the following:
[0021] a peptide comprising the amino acid sequence set forth in
SEQ ID NO:1;
[0022] a peptide having biocompatibility and comprising an amino
acid sequence obtained by modifying the amino acid sequence set
forth in SEQ ID NO:1 by deletion, substitution or addition of one
or several amino acid residues; and
[0023] a peptide having biocompatibility and comprising an amino
acid sequence having at least 80% sequence identity with the amino
acid sequence set forth in SEQ ID NO:1.
[0024] (11) The cartilage regenerative material according to any
one of (1) to (10), in which biocompatible polymers in the
biocompatible polymer blocks are crosslinked by means of heat,
ultraviolet radiation, or an enzyme.
[0025] (12) The cartilage regenerative material according to any
one of (1) to (11), in which the biocompatible polymer blocks are
in the form of granules obtainable by pulverizing a biocompatible
polymer in the form of a porous body.
[0026] (13) A cartilage regenerative material comprising the
cartilage regenerative material according to any one of (1) to (12)
and a biocompatible polymer film.
[0027] (14) The cartilage regenerative material according to (13),
in which the biocompatible polymer film is a film for isolating a
portion or the entirety of the transplant face of the cell
construct from the site of transplantation.
[0028] (15) A cell construct for use in cartilage regeneration
therapy, the cell construct comprising biocompatible polymer blocks
and stem cells, the cell construct having a plurality of the
biocompatible polymer blocks disposed in gaps between a plurality
of the stem cells.
[0029] (16) A method for regenerating cartilage, the method
comprising a step of transplanting a cell construct that includes
biocompatible polymer blocks and stem cells to a patient in need of
cartilage regeneration, in which the cell construct has a plurality
of the biocompatible polymer blocks disposed in gaps between a
plurality of the stem cells.
[0030] (17) Use of a cell construct for the production of a
cartilage regenerative material, the cell construct comprising
biocompatible polymer blocks and stem cells, in which the cell
construct has a plurality of the biocompatible polymer blocks
disposed in gaps between a plurality of the stem cells.
[0031] The cartilage regenerative material of the invention has
excellent cartilage regenerative capacity and excellent bone
regenerative capacity, and is useful for cell transplantation
therapy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 illustrates a liquid temperature profile obtained
under Condition A.
[0033] FIG. 2 illustrates a liquid temperature profile obtained
under Condition B.
[0034] FIG. 3 illustrates a liquid temperature profile obtained
under Condition C.
[0035] FIG. 4 illustrates a liquid temperature profile obtained
under Condition AA.
[0036] FIG. 5 illustrates a liquid temperature profile obtained
under Condition BB.
[0037] FIG. 6 shows the results of staining of a tissue onto which
only a sponge was transplanted (without film).
[0038] FIG. 7 shows the results of staining of a tissue onto which
a sponge (without cells) and a film were transplanted.
[0039] FIG. 8 shows the results of staining of a tissue onto which
a cell culture sponge and a film have been transplanted.
[0040] FIG. 9 shows the results of staining of a tissue onto which
a cell mass and a film have been transplanted.
[0041] FIG. 10 shows the results of staining of a tissue onto which
a mosaic cell mass and a film have been transplanted.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0042] Hereinafter, embodiments of the invention will be explained
in detail.
[0043] The cartilage regenerative material of the invention is a
material comprising a cell construct that includes biocompatible
polymer blocks and stem cells, the cell construct having a
plurality of the biocompatible polymer blocks disposed in gaps
between a plurality of the stem cells. The cell construct used in
the invention may also be referred to as mosaic cell mass (cell
mass in a mosaic state) in the present specification.
[0044] Since the cartilage regenerative material of the invention
has excellent cartilage regenerative capacity and excellent bone
regenerative capacity, in addition to the use for regenerating
cartilage, the cartilage regenerative material can also be used in
order to regenerate cartilage and bone. The cartilage regenerative
material of the invention can be used as, for example, a transplant
material to be transplanted into a cartilage defect site.
[0045] It is a completely unexpected, remarkable effect that a cell
construct including biocompatible polymer blocks and stem cells, in
which a plurality of the biocompatible polymer blocks are disposed
in gaps between a plurality of the stem cells, has excellent
cartilage regeneration action as well as excellent bone
regeneration action. In JP4122280B and J. I. Lee, "Transplantation
of scaffold-free spheroids composed of synovium-derived cells and
chondrocytes or the treatment of cartilage defects of the knee,"
European Cells and Materials, Vol. 22, 2011, p 275-290, it is
neither disclosed nor suggested to use biocompatible polymer
blocks. The technologies disclosed in JP4122280B and J. I. Lee,
"Transplantation of scaffold-free spheroids composed of
synovium-derived cells and chondrocytes or the treatment of
cartilage defects of the knee," European Cells and Materials, Vol.
22, 2011, p 275-290, are characterized in that a scaffold such as
biocompatible polymer blocks is not included, and these
technologies are different from the present invention from this
point of view. It is described in WO2011/108517A that the cell
construct produces a large amount of glycosaminoglycan (GAG);
however, the amount of GAG production is irrelevant to the
capability of simultaneously regenerating cartilage and bone.
Chondrocyte differentiation and cartilage regeneration are
different phenomena, and the cartilage regenerative capacity and
the bone regenerative capacity are conceptually completely
different. The GAG production in WO2011/108517A was achieved in an
ex vivo experiment using a particular medium (chondrocyte
differentiation medium) that promotes chondrocyte differentiation,
and the conditions for this experiment are significantly different
from the environment in vivo. Therefore, according to the findings
of WO2011/108517A, cartilage regeneration in the in vivo
environment cannot be expected, and particularly, it cannot be
expected at all from WO2011/108517A that cartilage and bone can be
regenerated simultaneously.
[0046] (1) Biocompatible Polymer Blocks
[0047] The cell construct used in the invention includes
biocompatible polymer blocks. The biocompatible polymer blocks will
be explained below.
[0048] (1-1) Biocompatible Polymer
[0049] Biocompatibility means that in a case in which the material
is brought into contact with a living body, the material does not
give a rise to a noticeably harmful reaction such as a long-term
and chronic inflammation reaction. Whether the biocompatible
polymer used in the invention is decomposed in vivo is not
particularly limited, as long as the polymer has biocompatibility;
however, it is preferable that the polymer is a biodegradable
polymer. Specific examples of a non-biodegradable polymer include
polytetrafluoroethylene (PTFE), polyurethane, polypropylene,
polyester, vinyl chloride, polycarbonate, acryl, stainless steel,
titanium, silicone, and MPC
(2-methacryloyloxyethylphosphorylcholine). Specific examples of a
biodegradable polymer include polypeptides such as a recombinant
peptide and a chemically synthesized peptide (for example, gelatin
that will be explained below), polylactic acid, polyglycolic acid,
a lactic acid-glycolic acid copolymer (PLGA), hyaluronic acid,
glycosaminoglycan, proteoglycan, chondroitin, cellulose, agarose,
carboxymethyl cellulose, chitin, and chitosan. Among the compounds
described above, a recombinant peptide is particularly preferred.
These biocompatible polymers may be devised in order to increase
the cell adhesiveness. Specifically, methods such as "coating of a
base material surface with a cell adhesion matrix (fibronectin,
vitronectin, or laminin) or a cell adhesion sequence (an RGD
sequence, a LDV sequence, a REDV (SEQ ID NO: 2) sequence, a YIGSR
(SEQ ID NO: 3) sequence, a PDSGR (SEQ ID NO: 4) sequence, a RYVVLPR
(SEQ ID NO: 5) sequence, a LGTIPG (SEQ ID NO: 6) sequence, a
RNIAEIIKDI (SEQ ID NO: 7) sequence, an IKVAV (SEQ ID NO: 8)
sequence, a LRE sequence, a DGEA (SEQ ID NO: 9) sequence, or a HAV
sequence; all indicated by one-letter codes of amino acids)
peptide", "amination or cationization of the base material
surface", or "hydrophilic treatment of the base material surface by
a plasma treatment or corona discharge" can be used.
[0050] The type of the polypeptide such as a recombinant peptide or
a chemically synthesized peptide is not particularly limited as
long as the polypeptide has biocompatibility; however, for example,
gelatin, collagen, elastin, fibronectin, pronectin, laminin,
tenascin, fibrin, fibroin, entactin, thrombospondin, and
retronectin are preferred, while gelatin, collagen, and
atelocollagen are most preferred. Gelatin that is intended to be
used in the invention is preferably naturally occurring gelatin, a
recombinant gelatin, or a chemically synthesized gelatin, and more
preferred is a recombinant gelatin. The term naturally occurring
gelatin as used herein means a gelatin produced from naturally
occurring collagen.
[0051] The term chemically synthesized peptide or chemically
synthesized gelatin means a peptide or gelatin that has been
artificially synthesized. Synthesis of a peptide such as gelatin
may be solid-phase synthesis or liquid-phase synthesis; however,
solid-phase synthesis is preferred. Solid-phase synthesis of
peptides is well known to those ordinarily skilled in the art, and
examples include a Fmoc group synthesis method of using a Fmoc
group (Fluorenyl-Methoxy-Carbonyl group) as a protective group for
an amino group; and a Boc group synthesis method of using a Boc
group (tert-ButylOxyCarbonyl group) as a protective group for an
amino group. Regarding preferred embodiments of the chemically
synthesized gelatin, the matters described in section (1-3)
Recombinant gelatin given below in the present specification can be
applied.
[0052] Recombinant gelatin will be explained below in the present
specification.
[0053] The hydrophilicity value "1/IOB" value of the biocompatible
polymer used in the invention is preferably from 0 to 1.0. The
hydrophilicity value is more preferably from 0 to 0.6, and even
more preferably from 0 to 0.4. IOB is an index of
hydrophilicity/hydrophobicity based on an organic conceptual
diagram showing the polarity/non-polarity of organic compounds
suggested by FUJITA, Atsushi, and the details thereof are explained
in, for example, "Pharmaceutical Bulletin", Vol. 2, 2, pp. 163-173
(1954), "Kagaku no Ryoiki (Domain of Chemistry)", Vol. 11, 10, pp.
719-725 (1957), and "Fragrance Journal", Vol. 50, pp. 79-82 (1981).
To describe briefly, the root of all organic compounds is
considered to be methane (CH.sub.4), and other compounds are all
regarded as derivatives of methane. Certain values are set
respectively for the number of carbon atoms, substituents, modified
parts, rings, and the like of the compounds, and the scores are
added to determine the organic values (OV) and the inorganic values
(IV). These values are plotted on a graph, with the X-axis
representing the organic values and the Y-axis representing the
inorganic values. The IOB in the organic conceptual diagram means
the ratio of the inorganic value (IV) with respect to the organic
value (OV) in the organic conceptual diagram, that is, "inorganic
value (IV)/organic value (OV)". Regarding the details of the
organic conceptual diagram, reference can be made to "Shinpan Yuki
Gainenzu--Kiso to Oyo--(New Edition Organic Conceptual
Diagram--Fundamentals and Applications--)" (written by KODA,
Yoshio, et al., Sankyo Shuppan Co., Ltd., 2008). In the present
specification, hydrophilicity and hydrophobicity is indicated with
the "1/IOB" value, which is the reciprocal of JOB. As the "1/IOB"
value is smaller (closer to 0), this indicates that the compound is
hydrophilic.
[0054] By adjusting the "1/IOB" value of the biocompatible polymer
used in the invention to the range described above, the
biocompatible polymer has higher hydrophilicity and has enhanced
water absorbing properties. Accordingly, it is speculated that the
high hydrophilicity acts effectively on the retention of nutrient
components, and consequently contributes to the stabilization and
ease of survival of cells in the cell construct (mosaic cell mass)
according to the invention.
[0055] In a case in which the biocompatible polymer used in the
invention is a polypeptide, the hydrophilicity/hydrophobicity index
represented by the Grand average of hydropathicity (GRAVY) value of
the polypeptide is preferably 0.3 or lower and -9.0 or higher, and
more preferably 0.0 or lower and -7.0 or higher. The Grand average
of hydropathicity (GRAVY) value can be obtained by the method
described in "Gasteiger E., Hoogland C., Gattiker A., Duvaud S.,
Wilkins M. R., Appel R. D., Bairoch A.; Protein Identification and
Analysis Tools on the ExPASy Server; (In) John M. Walker (ed): The
Proteomics Protocols Handbook, Humana Press (2005). pp. 571-607"
and "Gasteiger E., Gattiker A., Hoogland C., Ivanyi I., Appel R.
D., Bairoch A.; ExPASy: the proteomics server for in-depth protein
knowledge and analysis.; Nucleic Acids Res. 31: 3784-3788
(2003)".
[0056] By adjusting the GRAVY value of the biocompatible polymer
used in the invention to the range described above, the
biocompatible polymer has higher hydrophilicity and has enhanced
water absorbing properties. Accordingly, it is speculated that the
high hydrophilicity acts effectively on the retention of nutrient
components, and consequently contributes to the stabilization and
ease of survival of cells in the cell construct (mosaic cell mass)
according to the invention.
[0057] (1-2) Crosslinking
[0058] The biocompatible polymer used in the invention may be a
crosslinked polymer, or may be a polymer that is not crosslinked;
however, a crosslinked polymer is preferred. By using a crosslinked
biocompatible polymer, there is obtained an effect that in a case
in which the cartilage regenerative material of the invention is
cultured in a medium, and in a case which the cartilage
regenerative material is transplanted into a living body, the
cartilage regenerative material being instantaneously decomposed is
prevented. Regarding general crosslinking methods, thermal
crosslinking, crosslinking by means of an aldehyde (for example,
formaldehyde or glutaraldehyde), crosslinking by means of a
condensing agent (carbodiimide, cyanamide, or the like), enzymatic
crosslinking, photocrosslinking, ultraviolet crosslinking,
hydrophobic interaction, hydrogen bonding, ionic interaction, and
the like are known. The crosslinking method used in the invention
is preferably thermal crosslinking, ultraviolet crosslinking, or
enzymatic crosslinking, and particularly preferably thermal
crosslinking.
[0059] In a case in which enzyme-induced crosslinking is carried
out, the enzyme is not particularly limited as long as the enzyme
has an effect of crosslinking between polymer molecules; however,
preferably a transglutaminase and a laccase, and most preferably a
transglutaminase, can be used. There are no particular limitations
on specific examples of the polymer that is enzymatically
crosslinked by a transglutaminase as long as the polymer is a
protein having a lysine residue and a glutamine residue. The
transglutaminase may be a mammal-derived enzyme or a microbially
derived enzyme, and specifically, ACTIVA series manufactured by
Ajinomoto Co., Inc., and mammal-derived transglutaminases that are
released as reagents, for example, Guinea pig liver-derived
transglutaminase, goat-derived transglutaminase, and rabbit-derived
transglutaminase, which are products of Oriental Yeast Co., Ltd.;
Upstate USA, Inc.; Biodesign International, Inc.; and the like, and
human-derived blood coagulation factor (Factor XIIIa, Haematologic
Technologies, Inc.).
[0060] The reaction temperature in the case of performing
crosslinking (for example, thermal crosslinking) is not
particularly limited as long as crosslinking is enabled; however,
the reaction temperature is preferably -100.degree. C. to
500.degree. C., more preferably 0.degree. C. to 300.degree. C.,
even more preferably 50.degree. C. to 300.degree. C., still more
preferably 100.degree. C. to 250.degree. C., and even more
preferably 120.degree. C. to 200.degree. C.
[0061] (1-3) Recombinant Gelatin
[0062] The recombinant gelatin as used herein means a polypeptide
or protein-like substance having an amino acid sequence similar to
that of gelatin, which is produced by a gene recombination
technology. It is preferable that the recombinant gelatin that can
be used in the invention has repeats of a sequence represented by
Gly-X-Y (where X and Y each independently represent any amino acid
residue), which is characteristic to collagen. Here, a plurality of
the Gly-X-Y sequences may be identical to or different from one
another. Preferably, two or more sequences of cell adhesion signals
are included in one molecule. Regarding the recombinant gelatin
that is used in the invention, a recombinant gelatin having an
amino acid sequence derived from a partial amino acid sequence of
collagen can be used. For example, the recombinant gelatins
described in EP1014176B, U.S. Pat. No. 6,992,172B, WO2004/85473A,
and WO2008/103041A can be used; however, the examples are not
limited to these. Preferred examples of the recombinant gelatin
that is used in the invention are recombinant gelatins of the
following embodiments.
[0063] A recombinant gelatin has the original properties of
naturally occurring gelatin and thus has excellent
biocompatibility. Also, since it is not a substance derived from a
natural source, a recombinant gelatin has no risk of bovine
spongiform encephalopathy (BSE) or the like, and has an excellent
characteristic of being non-infectious. Since a recombinant gelatin
is homogeneous compared to naturally occurring gelatin and has a
predetermined sequence, it is possible to precisely design a
recombinant gelatin with fewer fluctuations, in connection with
strength and degradability, through crosslinking or the like.
[0064] The molecular weight of the recombinant gelatin is not
particularly limited; however, the molecular weight is preferably
from 2,000 to 100,000 (from 2 kDa to 100 kDa), more preferably from
2,500 to 95,000 (from 2.5 kDa to 95 kDa), even more preferably from
5,000 to 90,000 (from 5 kDa to 90 kDa), and most preferably from
10,000 to 90,000 (from 10 kDa to 90 kDa).
[0065] It is preferable that the recombinant gelatin has repeats of
a sequence represented by Gly-X-Y, which is characteristic to
collagen. Here, a plurality of the Gly-X-Y sequences may be
identical to or different from one another. In regard to the
sequence Gly-X-Y, Gly represents glycine, and X and Y each
represent an arbitrary amino acid (preferably, an arbitrary amino
acid other than glycine). The sequence represented by Gly-X-Y
characteristic to collagen is a highly specific partial structure
present in the amino acid compositions and sequences of gelatin and
collagen, compared to other proteins. In this partial structure,
glycine accounts for about one-third of the whole composition, and
in the amino acid sequence, glycine repeatedly appears at a rate of
one in every three amino acid residues. Glycine is the simplest
amino acid, and there are fewer restrictions to the arrangement in
a molecular chain. Thus, glycine greatly contributes to
regeneration of the helix structure in the case of gelation. It is
preferable that the amino acids represented by X and Y include a
large proportion of imino acids (proline and oxyproline), and imino
acids account for 10% to 45% of the total amount of the amino
acids. Preferably, amino acids that account for 80% or more, more
preferably 95% or more, and most preferably 99% or more, of the
sequence of the recombinant gelatin, constitute the repeating
structure of Gly-X-Y.
[0066] In general gelatins, polar amino acids that have an electric
charge and polar amino acids that are uncharged exist at a ratio of
1:1. Here, the term polar amino acid specifically refers to
cysteine, aspartic acid, glutamic acid, histidine, lysine,
asparagine, glutamine, serine, threonine, tyrosine, or arginine,
and among these, polar uncharged amino acids include cysteine,
asparagine, glutamine, serine, threonine, and tyrosine. In regard
to the recombinant gelatin used in the invention, the proportion of
polar amino acids among all the amino acids that constitute the
recombinant gelatin is 10% to 40%, and preferably 20% to 30%.
Meanwhile, the proportion of uncharged amino acids in the polar
amino acids is preferably 5% or more and less than 20%, and more
preferably 5% or more and less than 10%. It is also preferable that
any one amino acid, and preferably 2 or more amino acids, of
serine, threonine, asparagine, tyrosine, and cysteine are not
included in the amino acid sequence.
[0067] Generally, in regard to polypeptides, minimal amino acid
sequences that function as cell adhesion signal sequences are known
(for example, "Byotai Seiri (Pathophysiology)", Vol. 9, No. 7
(1990), p. 527, published by Nagai Shoten Co., Ltd.). It is
preferable that the recombinant gelatin used in the invention
contains two or more such minimal amino acid sequences that
function as cell adhesion signals in one molecule. Regarding
specific sequences, from the viewpoint of being applicable to many
kinds of adhering cells, an RGD sequence, a LDV sequence, a REDV
(SEQ ID NO: 2) sequence, a YIGSR (SEQ ID NO: 3) sequence, a PDSGR
(SEQ ID NO: 4) sequence, a RYVVLPR (SEQ ID NO: 5) sequence, a
LGTIPG (SEQ ID NO: 6) sequence, a RNIAEIIKDI (SEQ ID NO: 7)
sequence, an IKVAV (SEQ ID NO: 8) sequence, a LRE sequence, a DGEA
(SEQ ID NO: 9) sequence, and a HAV sequence, which are expressed in
one-letter codes of amino acids, are preferred. More preferred
sequences include an RGD sequence, a YIGSR (SEQ ID NO: 3) sequence,
a PDSGR (SEQ ID NO: 4) sequence, a LGTIPG (SEQ ID NO: 6) sequence,
an IKVAV (SEQ ID NO: 8) sequence, and a HAV sequence, and
particularly preferred is an RGD sequence. Among RGD sequences, an
ERGD (SEQ ID NO: 10) sequence is preferred. When a recombinant
gelatin having cell adhesion signal sequences is used, the amount
of cell matrix production can be increased. For example, in a case
in which mesenchymal stem cells are used as cells, the production
of glycosaminoglycans (GAG) in chondrocyte differentiation can be
increased.
[0068] In regard to the disposition of RGD sequences in the
recombinant gelatin used in the invention, it is preferable that
the number of amino acids between RGD sequences is between 0 and
100, and preferably between 25 and 60, and is not uniform.
[0069] The content of these minimal amino acid sequences is
preferably 3 to 50, more preferably 4 to 30, even more preferably 5
to 20, and most preferably 12, in one molecule of protein, from the
viewpoints of cell adhesion and proliferation properties.
[0070] In regard to the recombinant gelatin used in the invention,
the proportion of the RGD sequences (motifs) with respect to the
total number of amino acid residues is preferably at least 0.4%. In
a case in which a recombinant gelatin includes 350 or more amino
acid residues, it is preferable that each stretch of 350 amino acid
residues includes at least one RGD motif. The proportion of the RGD
motif with respect to the total number of amino acid residues is
more preferably at least 0.6%, even more preferably at least 0.8%,
still more preferably at least 1.0%, even more preferably at least
1.2%, and most preferably at least 1.5%. The number of RGD motifs
within a recombinant peptide is preferably at least 4, more
preferably at least 6, even more preferably at least 8, still more
preferably from 12 to 16, per 250 amino acid residues. The
proportion of 0.4% of the RGD motifs corresponds to at least one
RGD sequence per 250 amino acid residues. Since the number of the
RGD motifs is an integer, in order to satisfy the characteristic
requirement of 0.4%, a gelatin molecule containing 251 amino acid
residues must include at least two RGD sequences. Preferably, the
recombinant gelatin of the invention includes at least two RGD
sequences per 250 amino acid residues; more preferably includes at
least three RGD sequences per 250 amino acid residues; and even
more preferably includes at least four RGD sequences per 250 amino
acid residues. According to another embodiment of the recombinant
gelatin of the invention, the recombinant gelatin includes at least
four RGD motifs, preferably at least six RGD motifs, more
preferably at least eight RGD motifs, and still more preferably
from 12 to 16 RGD motifs.
[0071] The recombinant gelatin may be partially hydrolyzed.
[0072] Preferably, the recombinant gelatin used in the invention is
represented by Formula 1: A-[(Gly-X-Y).sub.n].sub.m-B. n units of X
each independently represent any one amino acid residue, and n
units of Y each independently represent any one amino acid residue.
m represents an integer from 2 to 10, and preferably 3 to 5. n
represents an integer from 3 to 100, preferably 15 to 70, and more
preferably 50 to 65. A represents an arbitrary amino acid residue
or amino acid sequence, and B represents an arbitrary amino acid
residue or amino acid sequence.
[0073] More preferably, the recombinant gelatin used in the
invention is represented by formula (SEQ ID NO: 11):
Gly-Ala-Pro-[(Gly-X-Y).sub.63].sub.3-Gly (in the formula, 63 units
of X each independently represent any one amino acid residue; 63
units of Y each independently represent any one amino acid residue;
and 63 units of Gly-X-Y may be identical to or different from one
another).
[0074] It is preferable that a plurality of the sequence units of
naturally occurring collagen are bonded to the repeating unit. The
naturally occurring collagen as used herein may be any collagen
substance that exists in nature; however, the collagen is
preferably type I, type II, type III, type IV, or type V collagen.
The collagen is more preferably type I, type II, or type III
collagen. According to another embodiment, the source of the
above-mentioned collagens is preferably human, cow, pig, mouse, or
rat, and more preferably a human source.
[0075] The isoelectric point of the recombinant gelatin used in the
invention is preferably 5 to 10, more preferably 6 to 10, and even
more preferably 7 to 9.5.
[0076] Preferably, the recombinant gelatin is not deaminated.
[0077] Preferably, the recombinant gelatin does not have a
telopeptide.
[0078] Preferably, the recombinant gelatin is a substantially pure
polypeptide produced from a nucleic acid that encodes an amino acid
sequence.
[0079] The recombinant gelatin used in the invention is
particularly preferably:
[0080] (1) a peptide comprising the amino acid sequence set forth
in SEQ ID NO:1;
[0081] (2) a peptide having biocompatibility and comprising an
amino acid sequence obtained by modifying the amino acid sequence
set forth in SEQ ID NO:1 by deletion, substitution or addition of
one or several amino acid residues; or
[0082] (3) a peptide having biocompatibility and comprising an
amino acid sequence having at least 80% (preferably at least 90%,
more preferably at least 95%, and most preferably at least 98%)
sequence identity with the amino acid sequence set forth in SEQ ID
NO:1.
[0083] The term "one or several" in the phrase "amino acid sequence
obtained by modifying . . . by deletion, substitution or addition
of one or several amino acid residues" means preferably 1 to 20,
more preferably 1 to 10, even more preferably 1 to 5, and
particularly preferably 1 to 3.
[0084] The recombinant gelatin used in the invention can be
produced by a gene recombination technology that is known to those
ordinarily skilled in the art, and the recombinant gelatin can be
produced according to the methods described in, for example,
EP1014176A2, U.S. Pat. No. 6,992,172B, WO2004/85473A, and
WO2008/103041A. Specifically, a gene that encodes the amino acid
sequence of a predetermined recombinant gelatin is obtained, this
is incorporated into an expression vector to produce a recombinant
expression vector, and this is introduced into an appropriate host.
Thus, a transformant is produced. The transformant thus obtained is
cultured in an appropriate medium, and thereby, a recombinant
gelatin is produced. Then, the recombinant gelatin thus produced is
collected from the culture product. Thereby, the recombinant
gelatin used in the invention can be produced.
[0085] (1-4) Biocompatible Polymer Blocks
[0086] According to the invention, blocks (masses) comprising the
above-described biocompatible polymer are used.
[0087] The shape of the biocompatible polymer blocks according to
the invention is not particularly limited. For example, the shape
is an irregular shape, a spherical form, a particulate (granular)
form, a powder form, a porous form (porous body), a fibrous form, a
spindle shape, a flat shape, and a sheet form, and preferred
examples of the shape include an irregular shape, a spherical form,
a particulate (granular) form, a powder form, and a porous form. An
irregular shape implies that the surface shape is not uniform, and
for example, it is implied that the shape has concavities and
convexities such as a rock. Examples of the shape mentioned above
are not separate and isolated, and for example, the polymer block
may have an irregular shape as an example of a subordinate concept
of the particulate (granular) form.
[0088] The size of one biocompatible polymer block according to the
invention is not particularly limited; however, the size is
preferably from 1 .mu.m to 1,000 .mu.m, more preferably from 10
.mu.m to 1,000 .mu.m, even more preferably from 10 .mu.m to 700
.mu.m, still more preferably from 10 .mu.m to 300 .mu.m, even more
preferably from 10 .mu.m to 200 .mu.m, still more preferably from
20 .mu.m to 200 .mu.m, particularly preferably from 20 .mu.m to 150
.mu.m, and most preferably from 50 .mu.m to 110 .mu.m. It is
preferable from the viewpoint of cartilage regeneration that the
size of one biocompatible polymer block is adjusted to be in the
range described above. The size of one biocompatible polymer block
is not intended to mean that the average value of the size of a
plurality of the biocompatible polymer blocks is in the range
described above, but is intended to mean the size of each of
individual biocompatible polymer blocks obtainable by sieving a
plurality of the biocompatible polymer blocks.
[0089] The size of one block can be defined by the size of the
sieve used in the case of classifying the blocks. For example, in a
case in which blocks that have passed through a sieve having a mesh
size of 180 .mu.m are sieved through a sieve having a mesh size of
106 .mu.m, the blocks remaining on the sieve can be defined as
blocks having a size of 106 to 180 .mu.m. Next, in a case in which
the blocks that have passed through the sieve having a mesh size of
106 .mu.m are sieved through a sieve having a mesh size of 53
.mu.m, the blocks remaining on the sieve can be defined as blocks
having a size of 53 to 106 .mu.m. Next, in a case in which the
blocks that have passed through the sieve having a mesh size of 53
.mu.m are sieved through a sieve having a mesh size of 25 .mu.m,
the blocks remaining on the sieve can be defined as blocks having a
size of 25 to 53 .mu.m.
[0090] (1-5) Method for Producing Biocompatible Polymer Blocks
[0091] The method for producing biocompatible polymer blocks is not
particularly limited; however, for example, irregularly shaped
biocompatible polymer blocks, which constitute an example of a
granular form, can be obtained by pulverizing a porous body of a
biocompatible polymer using a pulverizing machine (NEW POWER MILL
or the like).
[0092] The method for producing a porous body of a biocompatible
polymer is not particularly limited; however, for example, there
may be mentioned a production method including:
[0093] (a) a step of cooling a solution of a biocompatible polymer
to an unfrozen state at a temperature at which the difference
between the temperature of a part having the highest liquid
temperature in the solution and the temperature of a part having
the lowest liquid temperature in the solution is 2.5.degree. C. or
less, and the temperature of the part having the highest liquid
temperature in the solution is lower than or equal to the melting
point of the solvent;
[0094] (b) a step of freezing the solution in an unfrozen state of
the biocompatible polymer obtained in Step (a); and
[0095] (c) a step of freeze-drying the frozen solution of the
biocompatible polymer obtained in Step (b).
[0096] In a case in which the biocompatible polymer solution is
cooled to an unfrozen state, as the difference between the
temperature of a part having the highest liquid temperature in the
solution and the temperature of a part having the lowest liquid
temperature in the solution is adjusted to be 2.5.degree. C. or
less (preferably 2.3.degree. C. or less, and more preferably
2.1.degree. C. or less), that is, as the difference in temperature
is adjusted to be smaller, the difference in the size of the pores
in the porous body thus obtainable is made smaller. The lower limit
of the difference between the temperature of a part having the
highest liquid temperature in the solution and the temperature of a
part having the lowest liquid temperature in the solution is not
particularly limited, and the temperature difference may be
0.degree. C. or more, and for example, may be 0.1.degree. C. or
more, 0.5.degree. C. or more, 0.8.degree. C. or more, or
0.9.degree. C. or more. Thereby, an effect that the cell construct
obtained by using the biocompatible polymer blocks produced using a
porous body of the biocompatible polymer thus produced presents a
high cell population, is achieved.
[0097] In regard to the cooling of Step (a), it is preferable to
perform cooling by means of, for example, a material having a
thermal conductivity lower than that of water (preferably, TEFLON
(registered trademark)), and the part having the highest liquid
temperature in the solution can be assumed to be a part remotest
from the cooling surface, and the part having the lowest liquid
temperature in the solution can be assumed to be the liquid
temperature at the cooling surface.
[0098] Preferably, in Step (a), the difference between the
temperature of a part having the highest liquid temperature in the
solution and the temperature of a part having the lowest liquid
temperature in the solution immediately before the generation of
the heat of solidification is 2.5.degree. C. or less, more
preferably 2.3.degree. C. or less, and even more preferably
2.1.degree. C. or less. Here, the "temperature difference
immediately before the generation of the heat of solidification"
means the temperature difference at the time when the temperature
difference becomes the largest in a time period between 1 second
and 10 seconds before the generation of the heat of
solidification.
[0099] Preferably, in Step (a), the temperature of a part having
the lowest liquid temperature in the solution is (melting point of
the solvent-5.degree. C.) or lower, more preferably (melting point
of the solvent-5.degree. C.) or lower and (melting point of the
solvent-20.degree. C.) or higher, and even more preferably (melting
point of the solvent-6.degree. C.) or lower and (melting point of
the solvent -16.degree. C.) or higher. The solvent of the "melting
point of the solvent" is the solvent of the solution of the
biocompatible polymer.
[0100] In Step (b), the solution of the biocompatible polymer in an
unfrozen state obtained in Step (a) is frozen. The cooling
temperature for freezing in Step (b) is not particularly limited
and may vary depending on the cooling equipment. Preferably, the
cooling temperature is a temperature lower by 3.degree. C. to
30.degree. C., more preferably a temperature lower by 5.degree. C.
to 25.degree. C., and even more preferably a temperature lower by
10.degree. C. to 20.degree. C., than the temperature of the part
having the lowest liquid temperature in the solution.
[0101] In Step (c), the frozen solution of the biocompatible
polymer obtained in Step (b) is freeze-dried. Freeze-drying can be
carried out by a conventional method, and for example,
freeze-drying can be carried out by performing vacuum drying at a
temperature lower than the melting point of the solvent, and
further performing vacuum drying at room temperature (20.degree.
C.).
[0102] (2) Stem Cells
[0103] Regarding the stem cells to be used in the invention, any
arbitrary stem cells can be used as long as the stem cells are
capable of cell transplantation and are capable of exhibiting
cartilage regenerative capacity, and the type of the cells is not
particularly limited. One type of stem cells may be used, or a
combination of multiple types of stem cells may also be used. The
stem cells to be used are preferably animal cells, more preferably
vertebrate-derived cells, and particularly preferably human-derived
cells. The type of the vertebrate-derived cells (particularly,
human-derived cells) may be any of pluripotent cells and somatic
stem cells. Regarding the pluripotent cells, for example, embryonic
stem cells (ES cells), germline stem cells (GS cells), or induced
pluripotent stem cells (iPS cells) can be used. Regarding the
somatic stem cells, for example, mesenchymal stem cells (MSC),
amniotic cells, cord blood-derived cells, bone marrow-derived
cells, or adipose-derived stem cells can be used, and particularly
preferred are mesenchymal stem cells (MSC). The origin of the cells
may be any of autologous cells and heterologous cells.
[0104] (3) Cell Construct
[0105] According to the invention, a cell construct is produced
using the biocompatible polymer blocks and the stem cells described
above, by disposing a plurality of the biocompatible polymer blocks
in gaps between a plurality of the stem cells three-dimensionally
in a mosaic pattern. As the biocompatible polymer blocks and the
stem cells are disposed three-dimensionally in a mosaic pattern, a
cell construct in which the stem cells are uniformly distributed in
the cell construct is formed, and delivery of nutrients such as
medium components from the outside to the interior of the cell
construct is enabled.
[0106] In the cell construct used in the invention, a plurality of
biocompatible polymer blocks are disposed in gaps between a
plurality of stem cells, and here, the "gaps between stem cells"
need not be spaces closed by the constituting stem cells, and may
be spaces sandwiched between the stem cells. Furthermore, it is not
necessary that gaps should be provided everywhere in between the
stem cells, and there may be sites where the stem cells are in
contact. The distance of a gap between stem cells that sandwich the
biocompatible polymer blocks therebetween, that is, the distance of
the gap in the case of selecting a certain stem cell and another
stem cell that exists in the shortest distance from the foregoing
stem cell, is not particularly limited. However, it is preferable
that the distance is equal to the size of the biocompatible polymer
block, and a suitable distance is also in the range of a suitable
size of the biocompatible polymer block.
[0107] The biocompatible polymer blocks are configured to be
interposed between stem cells; however, it is not necessary that
stem cells should be present between all the biocompatible polymer
blocks, and there may be sites where the biocompatible polymer
blocks are in contact. The distance between the biocompatible
polymer blocks sandwiching stem cells therebetween, that is, the
distance in the case of selecting a biocompatible polymer block and
another biocompatible polymer block that exists in the shortest
distance from the foregoing biocompatible polymer block, is not
particularly limited. However, the distance is preferably the size
of a mass of the stem cells obtainable in the case of gathering one
to several stem cells that are used, and for example, the distance
is from 10 .mu.m to 1,000 .mu.m, preferably from 10 .mu.m to 100
.mu.m, and even more preferably from 10 .mu.m to 50 .mu.m.
[0108] In the present specification, the expression "uniformly
distributed" is used in the phrase "cell construct in which stem
cells are uniformly distributed in the cell construct" and the
like; however, this does not mean perfect uniformity, and it is
meant that delivery of nutrients such as medium components from the
outside to the interior of the cell construct is enabled.
[0109] The thickness or diameter of the cell construct can be
adjusted to any desired thickness; however, as the lower limit, the
thickness is preferably 215 .mu.m or more, more preferably 400
.mu.m or more, and even more preferably 500 .mu.m or more. The
upper limit of the thickness or diameter is not particularly
limited; however, as a general range for practical use, the upper
limit is preferably 3 cm or less, more preferably 2 cm or less, and
even more preferably 1 cm or less. The range of the thickness or
diameter of the cell construct is preferably from 400 .mu.m to 3
cm, more preferably from 500 .mu.m to 2 cm, and even more
preferably from 500 .mu.m to 1 cm. By having the thickness or
diameter of the cell construct adjusted to be in the range
described above, the cell construct can easily manifest the
cartilage regenerative capacity.
[0110] In the cell construct, preferably, regions comprising
biocompatible polymer blocks and regions comprising stem cells are
arranged in a mosaic pattern. The "thickness or diameter of the
cell construct" according to the present specification is intended
to represent the following. In a case in which a certain point A
inside the cell construct is selected, among straight lines that
pass through the point A, a line segment that divides the cell
construct such that the distance from the outside the cell
construct becomes the shortest, is selected, and the length of this
line segment is designated as line segment A. A point A at which
the line segment A becomes the longest inside the cell construct is
selected, and the length of the line segment A in this case is
designated as the "thickness or diameter of the cell
construct".
[0111] The ratio of the stem cells and the biocompatible polymer
blocks in the cell construct is not particularly limited; however,
the mass of the biocompatible polymer block per stem cell is
preferably from 0.0000001 .mu.g to 1 .mu.g, more preferably from
0.000001 .mu.g to 0.1 .mu.g, even more preferably from 0.00001
.mu.g to 0.01 .mu.g, and most preferably from 0.00002 .mu.g to
0.006 .mu.g. As the ratio between the stem cells and the
biocompatible polymer blocks is adjusted to the range described
above, the stem cells can be distributed more uniformly. By
adjusting the lower limit to the range described above, the effects
of the stem cells can be manifested in a case in which the cell
construct is used for the above-described applications, and by
adjusting the upper limit to the range described above, any
components that optionally exist in the biocompatible polymer
blocks can be supplied to the stem cells. Here, the components in
the biocompatible polymer blocks are not particularly limited, and
the components may be the components included in the medium that
will be described below.
[0112] (4) Method for Producing Cell Construct
[0113] A cell construct can be produced by mixing biocompatible
polymer blocks and stem cells. More specifically, a cell construct
can be produced by alternately disposing the biocompatible polymer
blocks and the stem cells. The production method is not
particularly limited; however, a method of forming the
biocompatible polymer blocks and then inoculating the stem cells is
preferred.
[0114] Specifically, a cell construct can be produced by incubating
a mixture of biocompatible polymer blocks and a stem
cell-containing culture fluid. For example, in a container, stem
cells and biocompatible polymer blocks that have been produced in
advance are disposed in a mosaic pattern in a liquid that is
retained in the container. Regarding the means for disposition, it
is preferable to promote or control the formation of the
mosaic-patterned arrangement formed from stem cells and
biocompatible polymer blocks by using spontaneous aggregation,
gravity drop, centrifugation, or stirring.
[0115] Regarding the container used, a container formed from a
low-cell-adhesive material or a non-cell-adhesive material is
preferred, and a container formed from polystyrene, polypropylene,
polyethylene, glass, polycarbonate, or polyethylene terephthalate
is more preferred. The shape of the bottom face of the container is
preferably a flat bottom type, a U-shaped form, or a V-shaped
form.
[0116] In regard to the cell construct having a mosaic-patterned
arrangement that is obtained by the method described above, a cell
construct having a desired size can be produced by, for example, a
method such as:
[0117] (a) integrating mosaic-patterned cell masses that have been
produced separately, or
[0118] (b) increasing the volume in a differentiation medium or a
proliferation medium. The method of integration and the method of
increasing the volume are not particularly limited.
[0119] For example, during the process of incubating a mixture of
biocompatible polymer blocks and a stem cell-containing culture
fluid, the volume of the cell construct can be increased by
replacing the medium with a differentiation medium or a
proliferation medium. Preferably, during the process of incubating
a mixture of biocompatible polymer blocks and a stem
cell-containing culture fluid, a cell construct having a desired
size, in which stem cells are uniformly distributed in the cell
construct, can be produced by further adding biocompatible polymer
blocks.
[0120] The method of integrating mosaic-patterned cell masses that
have been produced separately is specifically a method for
producing a cell construct, the method including a step of
integrating a plurality of cell constructs, each of the cell
constructs including a plurality of biocompatible polymer blocks
and a plurality of stem cells, in which one or a plurality of the
biocompatible polymer blocks are disposed in some or all of a
plurality of gaps formed by a plurality of the stem cells.
[0121] It is preferable that the thickness or diameter of each cell
construct before integration or volume increase is from 10 .mu.m to
1 cm, and the thickness or diameter after integration or volume
increase is from 100 .mu.m to 3 cm. Here, the thickness or diameter
of each cell construct before integration is more preferably from
10 .mu.m to 2,000 .mu.m, even more preferably from 15 .mu.m to
1,500 .mu.m, and most preferably from 20 .mu.m to 1,300 .mu.m. The
range of the thickness or diameter after integration is more
preferably from 100 .mu.m to 2 cm, even more preferably from 100
.mu.m to 1 cm, still more preferably from 200 .mu.m to 1 cm, and
particularly preferably from 400 .mu.m to 1 cm.
[0122] It is preferable that the cell constructs that need to be
integrated are disposed at a distance of from 0 .mu.m to 50 .mu.m,
and the distance is more preferably from 0 .mu.m to 20 .mu.m, and
even more preferably from 0 .mu.m to 5 .mu.m. In a case in which
the cell constructs are integrated, it is considered that as a
result of proliferation and extension of the cells, the cells or
the matrix produced by the cells accomplishes the role as an
adhesive, and the cell constructs join together. Thus, adhesion
between cell constructs is facilitated by adjusting the distance to
the range described above.
[0123] A cell construct having a desired size can also be produced
by further adding biocompatible polymer blocks. Specifically,
second biocompatible polymer blocks can be further added to a cell
construct including a plurality of first biocompatible polymer
blocks and a plurality of stem cells, the cell construct having one
or a plurality of the biocompatible polymer blocks disposed in some
or all of a plurality of gaps formed by a plurality of the stem
cells, and the mixture can be incubated.
[0124] It is preferable that the rate at which the second
biocompatible polymer blocks are added in a case in which the
second biocompatible polymer blocks are further added to the cell
construct and incubated together, is appropriately selected in
accordance with the rate of proliferation of the stem cells that
are used.
[0125] Specifically, if the rate at which the second biocompatible
polymer blocks are added is fast, the stem cells move to the
outside of the cell construct, and the uniformity of the stem cells
is decreased. If the rate of addition is slow, there occur sites
where the proportion of the stem cells increases, and the
uniformity of the stem cells is decreased. Therefore, the rate of
addition is selected in consideration of the rate of proliferation
of the stem cells used.
[0126] (5) Method of Using Cartilage Regenerative Material
[0127] According to the invention, the cell construct described
above is used as a cartilage regenerative material. The cartilage
regenerative material of the invention can be used for the purpose
of cell transplantation to a diseased site of cartilage defect.
Examples of the disease associated with cartilage defect include,
but are not particularly limited to, arthrosis deformans,
osteochondral defect, osteochondritis dissecans, traumatic
cartilage injury, osteoarthritis, relapsing polychondritis,
achondroplasia, injury of intervertebral discs, and hernia of
intervertebral discs.
[0128] Examples of the transplantation method include methods using
incision, injection, an arthroscope, and an endoscope. Regarding
the cell construct of the invention, unlike cell transplants such
as a cell sheet, the size of the construct can be made small, and
therefore, a less invasive transplantation method such as
transplantation by injection is enabled.
[0129] The amount used in the case of transplanting the cartilage
regenerative material of the invention can be appropriately
selected in accordance with the disease state or the like; however,
the number of cells to be transplanted is preferably
1.0.times.10.sup.5 cells/cm.sup.3 to 1.0.times.10.sup.10
cells/cm.sup.3, and more preferably 1.0.times.10.sup.6
cells/cm.sup.3 to 1.0.times.10.sup.9 cells/cm.sup.3.
[0130] Regarding the number of times of transplantation of the
cartilage regenerative material of the invention, transplantation
may be performed only once, or transplantation may be performed two
or more times as necessary.
[0131] (6) Biocompatible Polymer Film
[0132] The cartilage regenerative material of the invention as
described above may be used alone as a cartilage regenerative
material; however, the cartilage regenerative material can also be
used in combination with a biocompatible polymer film as a
cartilage regenerative material. The cartilage regenerative
material of the invention and the biocompatible polymer film
described above may be supplied separately in the form of kits, or
the cartilage regenerative material of the invention and the
biocompatible polymer film may also be supplied in the form of a
product bonded together. In a case in which the cartilage
regenerative material and the biocompatible polymer film are
supplied in the form of separate kits, the user can bond the
cartilage regenerative material and the biocompatible polymer film
together and then transplant the resultant. Alternatively, the user
may transplant the biocompatible polymer film and then transplant
the cartilage regenerative material.
[0133] In a case in which a biocompatible polymer film is used, it
is preferable that the biocompatible polymer film is used as a film
for isolating a portion or the entirety of the transplant face of
the cell construct from the site of transplantation. For example,
it is preferable that the biocompatible polymer film is
transplanted first to the site of transplantation, and
subsequently, the cell construct is transplanted on the top surface
of the biocompatible polymer film (the surface on the opposite side
of the surface that is in contact with the site of
transplantation). Alternatively, in a case in which a cartilage
regenerative material including the cell construct of the invention
is bonded together with the biocompatible polymer film and then the
resultant is transplanted, it is preferable that the biocompatible
polymer film is transplanted so as to be brought into direct
contact with the site of transplantation.
[0134] Specific examples and preferred ranges of the biocompatible
polymer that constitutes the biocompatible polymer film are the
same as those in the case of the biocompatible polymer that
constitutes the biocompatible polymer blocks, and specifically, the
specific examples and the preferred ranges are as described above
in sections (1-1) Biocompatible polymer, (1-2) Crosslinking, and
(1-3) Recombinant gelatin in the present specification. The
biocompatible polymer that constitutes the biocompatible polymer
film may be the same as, or may be different from, the
biocompatible polymer that constitutes the biocompatible polymer
blocks.
[0135] The method for producing a biocompatible polymer film is not
particularly limited, and the production can be carried out by a
conventional method. For example, a biocompatible polymer film can
be produced by causing an aqueous solution of a biocompatible
polymer to flow into a plastic tray, and drying the aqueous
solution at low temperature (for example, in a refrigerator).
[0136] The biocompatible polymer film can be crosslinked. In a case
in which the polymer film is crosslinked, the degree of
crosslinking is not particularly limited; however, the degree of
crosslinking is generally 4 to 15, and more preferably 6 to 13. The
degree of crosslinking is the number of crosslinks per molecule.
Measurement of the degree of crosslinking can be carried out using
the TNBS (2,4,6-trinitrobenzenesulfonic acid) method described in
section [7] Method for measuring degree of crosslinking in the
Examples.
[0137] The rate of decomposition of the biocompatible polymer film
varies depending on the degree of crosslinking. The rate of
decomposition of the biocompatible polymer film can be measured and
evaluated by the method described below in section [8] Method for
measuring rate of decomposition in the Examples. The rate of
decomposition of the biocompatible polymer film measured by the
method is not particularly limited; however, the rate of
decomposition is generally 0.1 to 10 [mass %/hour], and more
preferably 0.5 to 6.9 [mass %/hour].
[0138] (7) Use and Cartilage Regeneration Method
[0139] According to the invention, there is provided a cell
construct for use in the treatment of cartilage regeneration, the
cell construct including biocompatible polymer blocks and stem
cells, in which a plurality of the polymer blocks are disposed in
gaps between a plurality of the stem cells. In addition to the cell
construct, the biocompatible polymer film can also be used in
combination. Preferred ranges of the biocompatible polymer blocks,
the stem cells, the cell construct, and the biocompatible polymer
film are the same as described above in the present
specification.
[0140] According to the invention, there is provided a cartilage
regeneration method including a step of transplanting the
above-described cell construct to a patient in need of cartilage
regeneration. In the cartilage regeneration method of the
invention, the cell construct described above is used as a
cartilage regenerative material. In the case of transplanting the
cell construct, the biocompatible polymer film may be transplanted.
Preferred ranges of the biocompatible polymer blocks, the stem
cells, the cell construct, and the biocompatible polymer film are
the same as described above in the present specification.
[0141] Furthermore, according to the invention, use of the cell
construct for the production of a cartilage regenerative material
is provided. In addition to the cell construct, the biocompatible
polymer film can also be used in combination. Preferred ranges of
the biocompatible polymer blocks, the stem cells, the cell
construct, and the biocompatible polymer film are the same as
described above in the present specification.
[0142] The invention will be explained more specifically by way of
the following Examples; however, the invention is not intended to
be limited by the following Examples.
EXAMPLES
[0143] [1] Recombinant Peptide (Recombinant Gelatin)
[0144] As a recombinant peptide (recombinant gelatin), the
following CBE3 was prepared (described in WO2008/103041A).
[0145] CBE3:
[0146] Molecular weight: 51.6 kD
[0147] Structure: GAP[(GXY).sub.63].sub.3G (SEQ ID NO: 11)
[0148] Number of amino acid residues: 571
[0149] RGD sequence: 12 sequences
[0150] Imino acid content: 33%
[0151] Almost 100% of the amino acid residues constitute a
repeating structure of GXY. Serine, threonine, asparagine,
tyrosine, and cysteine were not included in the amino acid sequence
of CBE3. CBE3 comprises an ERGD (SEQ ID NO: 10) sequence.
[0152] Isoelectric point: 9.34
[0153] GRAVY value: -0.682
[0154] 1/IOB value: 0.323
[0155] Amino acid sequence (SEQ ID NO:1 in the Sequence Listing)
(Identical to SEQ ID NO:3 disclosed in WO2008/103041A. However, X
at the end was corrected to "P")
TABLE-US-00001 GAP(GAPGLQGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGAPG
LQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGER
GAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGPAGAPGAPGL
QGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPP)3G
[0156] Furthermore, a porous body and a sponge according to the
present specification are synonyms.
[0157] [2] Production of Recombinant Peptide Porous Body
[0158] [PTFE Thick Cylindrical Container]
[0159] A cylindrical cup-shaped container made of
polytetrafluoroethylene (PTFE) and having a bottom face thickness
of 3 mm, a diameter of 51 mm, a lateral face thickness of 8 mm, and
a height of 25 mm was prepared. The cylindrical cup was such that
when the curved face was erected as the lateral face, the lateral
face was closed with a PTFE plate having a thickness of 8 mm, and
the bottom face (circular-shaped flat plate) was also closed with a
PTFE plate having a thickness of 3 mm. Meanwhile, the cylindrical
cup had an open top face. Therefore, the inner diameter of the
cylindrical cup was 43 mm. Hereinafter, this container will be
referred to as PTFE thick cylindrical container.
[0160] [Aluminum Glass Plate Cylindrical Container]
[0161] A cylindrical cup-shaped container made of aluminum and
having a thickness of 1 mm and a diameter of 47 mm was prepared.
The cylindrical cup was such that when the curved face was erected
as the lateral face, the lateral face was closed with an aluminum
plate with a thickness of 1 mm, and the bottom face
(circular-shaped flat plate) was also closed with an aluminum plate
having a thickness of 1 mm. Meanwhile, the cylindrical cup had an
open top face. A TEFLON (registered trademark) plate having a
thickness of 1 mm was uniformly lined over the entire surface on
the inner side of the lateral face, and as a result, the inner
diameter of the cylindrical cup was 45 mm. The bottom face of this
container was in a state of being joined with a glass plate having
a thickness of 2.2 mm on the outside of aluminum. Hereinafter, this
container will be referred to as an aluminum glass cylindrical
container.
[0162] [Freezing Step with Small Temperature Difference, and Drying
Step]
[0163] An aqueous solution of CBE3 was poured respectively into the
PTFE thick cylindrical container and the aluminum glass plate
cylindrical container, and the aqueous solution of CBE3 was cooled
through the bottom face using a cooling shelf board inside a vacuum
freeze-drying machine (TF5-85ATNNN: Takara Co., Ltd.).
[0164] The container, the final concentration of the aqueous
solution of CBE3, the liquid amount, and the setting of the shelf
board temperature employed in this case were as described
below.
[0165] Condition A:
[0166] PTFE thick cylindrical container, final concentration of the
aqueous solution of CBE3: 4 mass %, amount of the aqueous solution:
4 mL. Regarding the setting of the shelf board temperature, cooling
was performed until the temperature reached -10.degree. C., and
freezing was performed for 1 hour at -10.degree. C., subsequently
for 2 hours at -20.degree. C., for 3 hours at -40.degree. C., and
lastly for 1 hour at -50.degree. C. Subsequently, the shelf board
temperature was returned to the setting of -20.degree. C., and then
the present frozen product was subjected to vacuum drying for 24
hours at -20.degree. C. After 24 hours, while vacuum drying was
still continued, the shelf board temperature was raised to
20.degree. C., and vacuum drying was performed for another 48 hours
at 20.degree. C. until the degree of vacuum sufficiently decreased
(1.9.times.10.sup.5 Pa). Subsequently, the frozen product was
removed from the vacuum freeze-drying machine. Thus, a porous body
was obtained.
[0167] Condition B:
[0168] Aluminum glass plate cylindrical container, final
concentration of aqueous solution of CBE3: 4 mass %, amount of the
aqueous solution: 4 mL. Regarding the setting of the shelf board
temperature, cooling was performed until the temperature reached
-10.degree. C., and freezing was performed for 1 hour at
-10.degree. C., subsequently for 2 hours at -20.degree. C., for 3
hours at -40.degree. C., and lastly for 1 hour at -50.degree. C.
Subsequently, the shelf board temperature was returned to the
setting of -20.degree. C., and then the present frozen product was
subjected to vacuum drying for 24 hours at -20.degree. C. After 24
hours, while vacuum drying was still continued, the shelf board
temperature was raised to 20.degree. C., and vacuum drying was
performed for another 48 hours at 20.degree. C. until the degree of
vacuum sufficiently decreased (1.9.times.10.sup.5 Pa).
Subsequently, the frozen product was removed from the vacuum
freeze-drying machine. Thus, a porous body was obtained.
[0169] Condition C:
[0170] PTFE thick cylindrical container, final concentration of the
aqueous solution of CBE3: 4 mass %, amount of the aqueous solution:
10 mL. Regarding the setting of the shelf board temperature,
cooling was performed until the temperature reached -10.degree. C.,
and freezing was performed for 1 hour at -10.degree. C.,
subsequently for 2 hours at -20.degree. C., for 3 hours at
-40.degree. C., and lastly for 1 hour at -50.degree. C.
Subsequently, the shelf board temperature was returned to the
setting of -20.degree. C., and then the present frozen product was
subjected to vacuum drying for 24 hours at -20.degree. C. After 24
hours, while vacuum drying was still continued, the shelf board
temperature was raised to 20.degree. C., and vacuum drying was
performed for another 48 hours at 20.degree. C. until the degree of
vacuum sufficiently decreased (1.9.times.10.sup.5 Pa).
Subsequently, the frozen product was removed from the vacuum
freeze-drying machine. Thus, a porous body was obtained.
[0171] [3] Measurement of Temperature Difference in Various
Freezing Steps
[0172] In regard to each of Condition A to Condition C, the liquid
temperature of the liquid surface at the circle center in the
container was measured as the liquid temperature at the remotest
place from the cooling side (non-cooling surface liquid
temperature) within the solution, and the liquid temperature at the
bottom in the container was measured as the liquid temperature
closest to the cooling side (cooling surface liquid temperature)
within the solution.
[0173] As a result, the profiles of the respective temperatures and
the temperature differences were obtained as shown in FIG. 1 to
FIG. 3.
[0174] From these FIG. 1, FIG. 2, and FIG. 3, it can be seen that
under Condition A, Condition B, and Condition C, the liquid
temperature was below the melting point, 0.degree. C., in the
section with the shelf board temperature set at -10.degree. C.
(before lowering to -20.degree. C.), and that state was a state in
which freezing had not occurred (unfrozen/overcooled). In this
state, the temperature difference between the cooling surface
liquid temperature and the non-cooling surface liquid temperature
was 2.5.degree. C. or less. Subsequently, as the shelf board
temperature was further lowered to -20.degree. C., a time point at
which the liquid temperature rapidly increased to near 0.degree. C.
was confirmed. Thus, it is understood that the heat of
solidification was generated here, and freezing was initiated. It
could also be confirmed that ice formation had actually started at
that time point. Subsequently, a certain time elapsed while the
temperature remained at near 0.degree. C. Here, a state in which
water and ice existed as a mixture was maintained. Lastly,
temperature drop started again from 0.degree. C.; however, at this
time, the liquid portion had disappeared, and only ice was left.
Therefore, the temperature that was measured was the solid
temperature inside the ice, and this was not a liquid
temperature.
[0175] In the following description, the temperature difference at
the time when the non-cooling surface liquid temperature reached
the melting point (0.degree. C.), the temperature difference
immediately before lowering of the shelf board temperature from
-10.degree. C. to -20.degree. C., and the temperature difference
immediately before the generation of the heat of solidification
will be described in conjunction with Condition A, Condition B, and
Condition C. The "temperature difference immediately before" as
used in the present specification means the largest temperature
difference among the temperature differences detectable in a period
between 1 second and 20 seconds before the main event.
[0176] Condition A
[0177] Temperature difference at the time when the liquid
temperature of the non-cooling surface reached the melting point
(0.degree. C.): 1.1.degree. C.
[0178] Temperature difference immediately before lowering from
-10.degree. C. to -20.degree. C.: 0.2.degree. C.
[0179] Temperature difference immediately before the generation of
the heat of solidification: 1.1.degree. C.
[0180] Condition B
[0181] Temperature difference at the time when the liquid
temperature of the non-cooling surface reached the melting point
(0.degree. C.): 1.0.degree. C.
[0182] Temperature difference immediately before lowering from
-10.degree. C. to -20.degree. C.: 0.1.degree. C.
[0183] Temperature difference immediately before the generation of
the heat of solidification: 0.9.degree. C.
[0184] Condition C
[0185] Temperature difference at the time when the liquid
temperature of the non-cooling surface reached the melting point
(0.degree. C.): 1.8.degree. C.
[0186] Temperature difference immediately before lowering from
-10.degree. C. to -20.degree. C.: 1.1.degree. C.
[0187] Temperature difference immediately before the generation of
the heat of solidification: 2.1.degree. C.
[0188] Hereinafter, these will be referred to as "freezing step
with small temperature difference/porous body".
[0189] [4] Freezing Step with Small Temperature Difference in 1
Mass % Ethanol-Containing Solution, and Drying Step
[0190] A 1 mass % (w/w) ethanol-containing aqueous solution of CBE3
was respectively poured into the PTFE thick cylindrical container
and the aluminum glass plate cylindrical container, and the aqueous
solution of CBE3 was cooled through the bottom face using a cooling
shelf board inside a vacuum freeze-drying machine (TF5-85ATNNN:
Takara Co., Ltd.). Since an ethanol-containing aqueous solution at
a final concentration of 1 mass % was used, the melting point was
-0.4.degree. C. The melting point change at the ethanol/water
concentration ratio was calculated from literature "Pickering S.
U.: A Study of the Properties of Some Strong Solutions. J. Chem.
Soc. London, 63 (1893), 998-1027".
[0191] The container, the final concentration of the aqueous
solution of CBE3, the liquid amount, and the setting of the shelf
board temperature employed in this case were as described
below.
[0192] Condition AA:
[0193] PTFE thick cylindrical container, final concentration of the
aqueous solution of CBE3: 4 mass %, final ethanol concentration: 1
mass %, amount of the aqueous solution: 4 mL. Regarding the setting
of the shelf board temperature, cooling was performed until the
temperature reached -10.degree. C., and freezing was performed for
1 hour at -10.degree. C., subsequently for 2 hours at -20.degree.
C., for 3 hours at -40.degree. C., and lastly for 1 hour at
-50.degree. C. Subsequently, the shelf board temperature was
returned to the setting of -20.degree. C., and then the present
frozen product was subjected to vacuum drying for 24 hours at
-20.degree. C. After 24 hours, while vacuum drying was still
continued, the shelf board temperature was raised to 20.degree. C.,
and vacuum drying was performed for another 48 hours at 20.degree.
C. until the degree of vacuum sufficiently decreased. Subsequently,
the frozen product was removed from the vacuum freeze-drying
machine. Thus, a porous body was obtained.
[0194] Condition BB:
[0195] Aluminum glass plate cylindrical container, final
concentration of the aqueous solution of CBE3: 4 mass %, final
ethanol concentration: 1 mass %, amount of the aqueous solution: 4
mL. Regarding the setting of the shelf board temperature, cooling
was performed until the temperature reached -10.degree. C., and
freezing was performed for 1 hour at -10.degree. C., subsequently
for 2 hours at -20.degree. C., for 3 hours at -40.degree. C., and
lastly for 1 hour at -50.degree. C. Subsequently, the shelf board
temperature was returned to the setting of -20.degree. C., and then
the present frozen product was subjected to vacuum drying for 24
hours at -20.degree. C. After 24 hours, while vacuum drying was
still continued, the shelf board temperature was raised to
20.degree. C., and vacuum drying was performed for another 48 hours
at 20.degree. C. until the degree of vacuum sufficiently decreased.
Subsequently, the frozen product was removed from the vacuum
freeze-drying machine. Thus, a porous body was obtained.
[0196] [Measurement of Temperature Difference of 1 Mass %
Ethanol-Containing Solution in Freezing Step]
[0197] In regard to Condition AA and Condition BB, the liquid
temperature of the liquid surface at the circle center in the
container was measured as the liquid temperature at the remotest
place from the cooling side (non-cooling surface liquid
temperature) within the solution, and the liquid temperature at the
bottom in the container was measured as the liquid temperature
closest to the cooling side (cooling surface liquid temperature)
within the solution. Here, since 1 mass % ethanol was used as the
solvent, the solvent melting point was -0.4.degree. C. The melting
point change at the ethanol/water concentration ratio was
calculated from literature "Pickering S. U.: A Study of the
Properties of Some Strong Solutions. J. Chem. Soc. London, 63
(1893), 998-1027".
[0198] As a result, the profiles of the respective temperatures and
the temperature differences were obtained as shown in FIG. 4 to
FIG. 5. From these FIG. 4 and FIG. 5, it can be seen that under
Condition AA and Condition BB, the liquid temperature was below the
melting point, -0.4.degree. C., in the section with the shelf board
temperature set at -10.degree. C., and that state was a state in
which freezing had not occurred (unfrozen/overcooled). In this
state, the temperature difference between the cooling surface
liquid temperature and the non-cooling surface liquid temperature
was 2.degree. C. or less. Subsequently, as the shelf board
temperature was further lowered to -20.degree. C., a time point at
which the liquid temperature rapidly increased to near -0.4.degree.
C. was confirmed. Thus, it is understood that the heat of
solidification was generated here, and freezing was initiated. It
could also be confirmed that ice formation had actually started at
that time point. Subsequently, a certain time elapsed while the
temperature remained at near -0.4.degree. C. Here, a state in which
water and ice existed as a mixture was maintained. Lastly,
temperature drop started again from 0.degree. C.; however, at this
time, the liquid portion had disappeared, and only ice was left.
Therefore, the temperature that was measured was the solid
temperature inside the ice, and this was not a liquid
temperature.
[0199] In the following description, the temperature difference at
the time when the non-cooling surface liquid temperature reached
the melting point (-0.4.degree. C.), the temperature difference
immediately before lowering of the shelf board temperature from
-10.degree. C. to -20.degree. C., and the temperature difference
immediately before the generation of the heat of solidification
will be described in conjunction with Condition AA and Condition
BB.
[0200] Condition AA
[0201] Temperature difference at the time when the liquid
temperature of the non-cooling surface reached the melting point
(-0.4.degree. C.): 0.8.degree. C.
[0202] Temperature difference immediately before lowering from
-10.degree. C. to -20.degree. C.: 0.3.degree. C.
[0203] Temperature difference immediately before the generation of
the heat of solidification: 0.8.degree. C.
[0204] Condition BB
[0205] Temperature difference at the time when the liquid
temperature of the non-cooling surface reached the melting point
(-0.4.degree. C.): 1.3.degree. C.
[0206] Temperature difference immediately before lowering from
-10.degree. C. to -20.degree. C.: 0.0.degree. C.
[0207] Temperature difference immediately before the generation of
the heat of solidification: 1.3.degree. C.
[0208] As a result, it was found that even under Condition AA and
Condition BB, the porous body can be produced as "freezing step
with small temperature difference/porous body", similarly to
Condition A and Condition B.
[0209] [5] Production of Petaloid Blocks from Freezing Step with
Small Temperature Difference/Porous Body (Pulverization and
Crosslinking of Porous Body)
[0210] Each of the CBE3 porous bodies of Condition A and Condition
B obtained in the above section [2] (for the measurement of
temperature difference, section [3]) was pulverized with a NEW
POWER MILL (Osaka Chemical Co., Ltd., NEW POWER MILL PM-2005).
Pulverization was carried out for 1 minute.times.5 times at the
maximum rotation speed, for 5 minutes in total. The pulverization
product thus obtained was classified by size with sieves made of
stainless steel, and uncrosslinked blocks having sizes of 25 to 53
.mu.m, 53 to 106 .mu.m, and 106 .mu.m to 180 .mu.m were obtained.
Subsequently, the uncrosslinked blocks were subjected to thermal
crosslinking at 160.degree. C. under reduced pressure (regarding
the crosslinking time, six different times such as 8 hours, 16
hours, 24 hours, 48 hours, 72 hours, and 96 hours were employed),
and thus sample CBE3 blocks were obtained. Hereinafter, the blocks
originating from the porous body of Condition A, which were
crosslinked for 48 hours, will be referred to as E, and the blocks
originating from the porous body of Condition B, which were
crosslinked for 48 hours, will be referred to as F. That is, E and
F are small temperature difference blocks produced from freezing
step with small temperature difference/porous bodies. Regarding the
difference in the crosslinking time, since no influence on the
performance was recognized in the evaluation of the present
invention, products obtained by crosslinking for 48 hours were used
herein as representative products. Furthermore, since consequently
there was no difference observed between E and F in terms of
performance, these were collectively used as petaloid blocks
later.
[0211] [6] Method for Producing Recombinant Peptide Film
[0212] An aqueous solution of CBE3 at a concentration of 4 mass %
was prepared, and 5.4 ml of this aqueous solution of CBE3 was
caused to flow into a plastic tray provided with a silicon frame (8
cm.times.3.5 cm). This plastic tray was transferred into a
refrigerator, and the aqueous solution was dried until no moisture
left. Thus, a recombinant peptide film was obtained. The
recombinant peptide film was taken out from the plastic
tray/silicon frame, and was subjected to thermal crosslinking at
160.degree. C. under reduced pressure (crosslinking time was 48
hours or 72 hours). Thus, samples for an animal test were
obtained.
[0213] [7] Method for Measuring Degree of Crosslinking
[0214] The degree of crosslinking (number of crosslinks per
molecule) of the film produced in the above section [6] was
calculated. For the measurement, a TNBS
(2,4,6-trinitrobenzenesulfonic acid) method was used.
[0215] <Preparation of Sample>
[0216] A sample (about 10 mg), a 4 mass % aqueous solution of
NaHCO.sub.3 (1 mL), and a 1 mass % aqueous solution of TNBS (2 mL)
were introduced into a glass vial, and the mixture was shaken for 3
hours at 37.degree. C. Subsequently, 37 mass % hydrochloric acid
(10 mL) and pure water (5 mL) were added thereto, and then the
mixture was left to stand for 16 hours or longer at 37.degree. C.
The resultant was used as a sample.
[0217] <Preparation of Blank>
[0218] A sample (about 10 mg), a 4 mass % aqueous solution of
NaHCO.sub.3 (1 mL), and a 1 mass % aqueous solution of TNBS (2 mL)
were introduced into a glass vial, 37 mass % hydrochloric acid (3
mL) was added thereto immediately thereafter, and the mixture was
shaken for 3 hours at 37.degree. C. Subsequently, 37 mass %
hydrochloric acid (7 mL) and pure water (5 mL) were added thereto,
and then the mixture was left to stand for 16 hours or longer at
37.degree. C. The resultant was used as a blank.
[0219] The light absorbance (345 nm) of a dilution of the sample
obtained by diluting 10 times with pure water, and the light
absorbance of the blank were measured, and the degree of
crosslinking (number of crosslinks per molecule) was calculated
from (Formula 2) and (Formula 3).
(As-Ab)/14600.times.V/w (Formula 2)
[0220] (Formula 2) represents the amount of lysine (molar
equivalent) per gram of the recombinant peptide.
[0221] In Formula 2, As represents the light absorbance of the
sample; Ab represents the light absorbance of the blank; V
represents the amount of the reaction liquid (g); and w represents
the mass (mg) of the recombinant peptide.
1-(Sample (Formula 1)/uncrosslinked recombinant peptide (Formula
1)).times.34 (Formula 3)
[0222] (Formula 3) represents the number of crosslinks per
molecule.
[0223] As a result, the film obtained by crosslinking for 48 hours
in the above section [6] had a degree of crosslinking of 6, and the
film obtained by crosslinking for 72 hours in the above section [6]
had a degree of crosslinking of 13.
[0224] [8] Method for Measuring Rate of Decomposition
[0225] The rate of decomposition of the film produced in the above
section [6] was evaluated.
[0226] 5 mg of a sample produced in the above section [6] was
introduced into a plastic tube, the mass of which had been measured
in advance, and the actual amount of addition was recorded.
[0227] 2.5 mg of Actinomyces-derived collagenase was dissolved in
50 ml of phosphate buffered saline (PBS), and a collagenase
solution was obtained. 1 ml of this collagenase solution was added
to the tube containing the sample, and the content was mixed by
vortexing. Subsequently, the mixture was shaken for 5 hours at
37.degree. C. Subsequently, the tube was centrifuged for 1 minute
at 10,000 G, and the supernatant was removed using a pipette. 1 ml
of ultrapure water was added to the tube, and the content was mixed
by vortexing. Subsequently, the tube was centrifuged for 1 minute
at 10,000 G, and the supernatant was removed using a pipette. This
operation was repeated one more time. Subsequently, the sample was
freeze-dried, and the mass of the tube containing the sample was
recorded.
[0228] The rate of decomposition of the film was calculated from
the following formula (Formula 4).
Rate of decomposition=((W-We)-wo)/wo/T (Formula 4)
[0229] In Formula 4, W represents the mass of the tube containing
the sample, which was recorded after freeze-drying; and We
represents the blank mass of the tube that was recorded in advance.
wo represents the actual amount of addition of the sample. T
represents the time taken for shaking in the collagenase solution,
and in this test, T was 5 hours.
[0230] As a result, the film of the above section [6] resulted in a
rate of decomposition of 6.9 [mass %/hour] under crosslinking for
48 hours, and a rate of decomposition of 0.5 [mass %/hour] under
crosslinking for 72 hours.
[0231] [9] Collection of Rabbit Mesenchymal Stem Cells (MSC)
[0232] The bone marrow aspirates of five Japanese white rabbits
(3-week old male) were collected from 10 femurs and 10 tibias.
First, the bones were disinfected with an isodine dilution and were
washed with DULBECCO's phosphate buffered saline (DPBS). The bones
were transferred onto a 10-cm dish, and both ends of each bone were
cut with bone clippers. 5 mL of a medium dispensed in a 10-mL
syringe equipped with an 18G needle was collected, a femur was
taken using a Dispin, and the needle was pierced into the bone
marrow on a 50-mL tube.
[0233] Subsequently, a medium was caused to flow into the bone
marrow, and the bone marrow was collected into the 50-mL tube. The
bone marrow aspirate thus collected was carefully pipetted and
passed through a cell strainer. Subsequently, the bone marrow
aspirate was centrifuged for 5 minutes at 1,000 rpm, subsequently
the supernatant was removed, and the residue was suspended in a
medium. Then, the suspension was inoculated into a flask. The
medium was exchanged the next day after the inoculation, and
adhered cells were collected 5 days after the inoculation. Thereby,
collection of rabbit MSC cells was completed. Thereafter, the cells
were subcultured for proliferation as appropriate for use.
Regarding the medium used in the above procedure, a medium of
DULBECCO's modified Eagle medium/high glucose (DMEM high glucose),
10 vol % fetal bovine serum (FBS), and penicillin/streptomycin
(50,000 U) was used in all cases.
[0234] [10] Production of Mosaic Cell Mass Using Petaloid Blocks
(Rabbit MSC)
[0235] The rabbit bone marrow-derived mesenchymal stem cells
(rabbit MSC) collected in the above section [9] were prepared into
a suspension at a concentration of 1.times.10.sup.5 cells/mL or
4.times.10.sup.5 cells/mL using a medium, and the petaloid blocks
53-106 .mu.m produced in the above section [5] were added thereto
at a concentration of 0.1 mg/mL. Subsequently, 200 .mu.L of the
cell suspension thus obtained was inoculated onto a SUMILON
CELL-TIGHT X96U plate (Sumitomo Bakelite, with a U-shaped bottom),
and the cell suspension was centrifuged (600 g, 5 minutes) using a
tabletop plate centrifuge and left to stand for 24 hours. Thus, a
spherical mosaic cell mass having a diameter of about 1 mm or a
diameter of about 1.3 mm and formed from petaloid blocks and rabbit
MSC cells was produced (0.001 .mu.g of blocks per cell). Since the
cell mass was produced in a U-shaped plate, this mosaic cell mass
was spherical in shape. The mosaic cell mass produced at a density
of 1.times.10.sup.5 cells/mL is referred to as small mosaic cell
mass, and the mosaic cell mass produced at a density of
4.times.10.sup.5 cells/mL is referred to as large mosaic cell
mass.
[0236] [11] Production of Cell Mass (Rabbit MSC)
[0237] The rabbit bone marrow-derived mesenchymal stem cells
(rabbit MSC) collected in the above section [9] were prepared into
a suspension at a concentration of 1.times.10.sup.5 cells/mL or
4.times.10.sup.5 cells/mL using a medium. 200 .mu.L of the cell
suspension thus obtained was inoculated onto a SUMILON CELL-TIGHT
X96U plate (Sumitomo Bakelite, with a U-shaped bottom), and the
cell suspension was centrifuged (600 g, 5 minutes) using a tabletop
plate centrifuge and left to stand for 24 hours. Thereby, a
spherical cell mass having a diameter of about 400 .mu.m or a
diameter of about 1 mm was produced. The cell mass produced at a
density of 1.times.10.sup.5 cells/mL is referred to as small cell
mass, and the cell mass produced at a density of 4.times.10.sup.5
cells/mL is referred to as large cell mass.
[0238] [12] Production of Cell Culture Sponge (Rabbit MSC)
[0239] A specimen having a diameter of 5 mm and a thickness of 1 mm
was cut out from the CBE3 sponge produced under Condition AA in the
above section [4], and the rabbit bone marrow-derived mesenchymal
stem cells (rabbit MSC) collected in the above section [9] were
inoculated into the sponge. Thus, a cell culture sponge was
prepared.
[0240] [13] Production of Rabbit Osteochondral Defect Model
[0241] In a 22-week old male Japanese white rabbit (Kitayama Labes
Co., Ltd., SPF), an osteochondral defect having a size with a
diameter of 5 mm and a depth of about 1 mm was produced at a knee
joint site.
[0242] [14] Sample Transplantation into Rabbit Osteochondral
Defect
[0243] First, the film (having a degree of crosslinking of 6)
prepared in the above section [6] and cut out into the bottom area
size (diameter 5 mm) of the defect, was placed at the rabbit
osteochondral defect site produced in the above section [13]. The
following was transplanted thereon.
Comparative Example 2
[0244] Sponge without cells, obtained by excluding the process of
inoculating cells in the above section [12]
Comparative Example 3
[0245] Cell culture sponge produced in the above section [12]
Comparative Example 4
[0246] 144 units of small cell mass produced in the above section
[11]
Example 1
[0247] 144 units of small mosaic cell mass produced in the above
section [10]
[0248] Furthermore, as another Comparative Example, a group in
which only a sponge without cells was transplanted, without placing
the film prepared in the above section [6] (Comparative Example 1),
was also prepared.
[0249] [15] Bone/Cartilage Regeneration Effect in Rabbit
Osteochondral Defect Model
[0250] The rabbit that received transplantation in the above
section [14] was autopsied after 8 weeks, and osteochondral tissue
slices in the periphery of the transplantation site were produced.
The tissue was fixated with formalin and embedded in paraffin.
Thus, skin tissue slices including mosaic cell masses were
produced. Staining of the slices was carried out by HE staining
(hematoxylin-eosin staining) or safranin O staining.
[0251] The results of staining are shown in FIG. 6 to FIG. 10.
Cartilage regeneration, bone regeneration, suppression of fibrous
soft tissue, and formation of bone-cartilage interface were
evaluated according to the following criteria. The evaluation
criteria are presented in Table 1.
[0252] Cartilage Regeneration [0253] AA: Satisfactory cartilage
regeneration is recognized in general. [0254] A: Cartilage
regeneration is recognized in general. [0255] B: Slight cartilage
regeneration is recognized in some part. [0256] C: Cartilage
regeneration is not recognized.
[0257] Bone Regeneration [0258] A: Bone regeneration is recognized.
[0259] B: Slight bone regeneration is recognized in some parts.
[0260] C: Bone regeneration is not recognized.
[0261] Suppression of Infiltration of Fibrous Soft Tissue [0262] A:
Suppression of the infiltration of fibrous soft tissue is
recognized. [0263] B: Suppression of the infiltration of fibrous
soft tissue is slightly recognized. [0264] C: Suppression of the
infiltration of fibrous soft tissue is not recognized. [0265] D:
Infiltration of inflammation cannot be suppressed, and suppression
of the infiltration of fibrous soft tissue is poorly achieved.
[0266] Formation of Bone-Cartilage Interface [0267] A: A boundary
line (tidemark) between regenerated bone and regenerated cartilage
is formed at a right position. [0268] B: A boundary line (tidemark)
between regenerated bone and regenerated cartilage is formed to a
slight extent. [0269] C: A boundary line (tidemark) between
regenerated bone and regenerated cartilage is not formed.
TABLE-US-00002 [0269] TABLE 1 Suppression Formation of Cartilage
Bone of fibrous bone/cartilage regeneration regeneration soft
tissue interface Comparative Sponge only C C D C Example 1
transplanted (without film) (FIG. 6) Comparative Sponge (without C
C B C Example 2 cells) and film transplanted (FIG. 7) Comparative
Cell culture sponge B B D C Example 3 and film transplanted (FIG.
8) Comparative Cell mass and film B A C C Example 4 transplanted
(FIG. 9) Example 1 Mosaic cell mass and AA A A A (Present film
transplanted invention) (FIG. 10)
[0270] In the case in which only a sponge was transplanted (without
film) (FIG. 6) and in the case in which a sponge (without cells)
and a film were transplanted (FIG. 7), cartilage regeneration was
not recognized, and bone regeneration was also not recognized.
[0271] In the case in which a cell culture sponge and a film were
transplanted (FIG. 8), slight cartilage regeneration and bone
regeneration were recognized in some parts; however, most of the
tissue became a fibrous soft tissue, and cartilage regeneration and
bone regeneration were not successful.
[0272] In the case in which a cell mass and a film were
transplanted (FIG. 9), slight cartilage regeneration was recognized
in some parts, and bone regeneration was also recognized; however,
a fibrous tissue was generated in most of the positions where
cartilage should have been generated. Thus, cartilage regeneration
and bone regeneration were not successful.
[0273] In the case in which a mosaic cell mass and a film were
transplanted (FIG. 10), satisfactory cartilage regeneration was
recognized in general, and a boundary line (tidemark) between
regenerated bone and regenerated cartilage was formed at a right
position. Bone regeneration was also recognized at a right
position, hardly any fibrous soft tissue was formed, and
satisfactory cartilage regeneration and bone regeneration could be
achieved.
[Sequence List]
[0274] International Application Application 15F02882 Cartilage
Regenerative Material JP16058540 20160317----00110214551600554135
Normal 20160317153508201602221618251590_P1AP101_15_1.app Based on
International Reception Patent Cooperation Treaty
Sequence CWU 1
1
111571PRTArtificial SequenceSynthetic Recombinant 1Gly Ala Pro Gly
Ala Pro Gly Leu Gln Gly Ala Pro Gly Leu Gln Gly 1 5 10 15 Met Pro
Gly Glu Arg Gly Ala Ala Gly Leu Pro Gly Pro Lys Gly Glu 20 25 30
Arg Gly Asp Ala Gly Pro Lys Gly Ala Asp Gly Ala Pro Gly Ala Pro 35
40 45 Gly Leu Gln Gly Met Pro Gly Glu Arg Gly Ala Ala Gly Leu Pro
Gly 50 55 60 Pro Lys Gly Glu Arg Gly Asp Ala Gly Pro Lys Gly Ala
Asp Gly Ala 65 70 75 80 Pro Gly Lys Asp Gly Val Arg Gly Leu Ala Gly
Pro Ile Gly Pro Pro 85 90 95 Gly Glu Arg Gly Ala Ala Gly Leu Pro
Gly Pro Lys Gly Glu Arg Gly 100 105 110 Asp Ala Gly Pro Lys Gly Ala
Asp Gly Ala Pro Gly Lys Asp Gly Val 115 120 125 Arg Gly Leu Ala Gly
Pro Ile Gly Pro Pro Gly Pro Ala Gly Ala Pro 130 135 140 Gly Ala Pro
Gly Leu Gln Gly Met Pro Gly Glu Arg Gly Ala Ala Gly 145 150 155 160
Leu Pro Gly Pro Lys Gly Glu Arg Gly Asp Ala Gly Pro Lys Gly Ala 165
170 175 Asp Gly Ala Pro Gly Lys Asp Gly Val Arg Gly Leu Ala Gly Pro
Pro 180 185 190 Gly Ala Pro Gly Leu Gln Gly Ala Pro Gly Leu Gln Gly
Met Pro Gly 195 200 205 Glu Arg Gly Ala Ala Gly Leu Pro Gly Pro Lys
Gly Glu Arg Gly Asp 210 215 220 Ala Gly Pro Lys Gly Ala Asp Gly Ala
Pro Gly Ala Pro Gly Leu Gln 225 230 235 240 Gly Met Pro Gly Glu Arg
Gly Ala Ala Gly Leu Pro Gly Pro Lys Gly 245 250 255 Glu Arg Gly Asp
Ala Gly Pro Lys Gly Ala Asp Gly Ala Pro Gly Lys 260 265 270 Asp Gly
Val Arg Gly Leu Ala Gly Pro Ile Gly Pro Pro Gly Glu Arg 275 280 285
Gly Ala Ala Gly Leu Pro Gly Pro Lys Gly Glu Arg Gly Asp Ala Gly 290
295 300 Pro Lys Gly Ala Asp Gly Ala Pro Gly Lys Asp Gly Val Arg Gly
Leu 305 310 315 320 Ala Gly Pro Ile Gly Pro Pro Gly Pro Ala Gly Ala
Pro Gly Ala Pro 325 330 335 Gly Leu Gln Gly Met Pro Gly Glu Arg Gly
Ala Ala Gly Leu Pro Gly 340 345 350 Pro Lys Gly Glu Arg Gly Asp Ala
Gly Pro Lys Gly Ala Asp Gly Ala 355 360 365 Pro Gly Lys Asp Gly Val
Arg Gly Leu Ala Gly Pro Pro Gly Ala Pro 370 375 380 Gly Leu Gln Gly
Ala Pro Gly Leu Gln Gly Met Pro Gly Glu Arg Gly 385 390 395 400 Ala
Ala Gly Leu Pro Gly Pro Lys Gly Glu Arg Gly Asp Ala Gly Pro 405 410
415 Lys Gly Ala Asp Gly Ala Pro Gly Ala Pro Gly Leu Gln Gly Met Pro
420 425 430 Gly Glu Arg Gly Ala Ala Gly Leu Pro Gly Pro Lys Gly Glu
Arg Gly 435 440 445 Asp Ala Gly Pro Lys Gly Ala Asp Gly Ala Pro Gly
Lys Asp Gly Val 450 455 460 Arg Gly Leu Ala Gly Pro Ile Gly Pro Pro
Gly Glu Arg Gly Ala Ala 465 470 475 480 Gly Leu Pro Gly Pro Lys Gly
Glu Arg Gly Asp Ala Gly Pro Lys Gly 485 490 495 Ala Asp Gly Ala Pro
Gly Lys Asp Gly Val Arg Gly Leu Ala Gly Pro 500 505 510 Ile Gly Pro
Pro Gly Pro Ala Gly Ala Pro Gly Ala Pro Gly Leu Gln 515 520 525 Gly
Met Pro Gly Glu Arg Gly Ala Ala Gly Leu Pro Gly Pro Lys Gly 530 535
540 Glu Arg Gly Asp Ala Gly Pro Lys Gly Ala Asp Gly Ala Pro Gly Lys
545 550 555 560 Asp Gly Val Arg Gly Leu Ala Gly Pro Pro Gly 565 570
24PRTArtificial SequenceDescription of Artificial Sequence adhesive
sequence 2Arg Glu Asp Val 1 35PRTArtificial SequenceDescription of
Artificial Sequence adhesive sequence 3Tyr Ile Gly Ser Arg 1 5
45PRTArtificial SequenceDescription of Artificial Sequence adhesive
sequence 4Pro Asp Ser Gly Arg 1 5 57PRTArtificial
SequenceDescription of Artificial Sequence adhesive sequence 5Arg
Tyr Val Val Leu Pro Arg 1 5 66PRTArtificial SequenceDescription of
Artificial Sequence adhesive sequence 6Leu Gly Thr Ile Pro Gly 1 5
710PRTArtificial SequenceDescription of Artificial Sequence
adhesive sequence 7Arg Asn Ile Ala Glu Ile Ile Lys Asp Ile 1 5 10
85PRTArtificial SequenceDescription of Artificial Sequence adhesive
sequence 8Ile Lys Val Ala Val 1 5 94PRTArtificial
SequenceDescription of Artificial Sequence adhesive sequence 9Asp
Gly Glu Ala 1 104PRTArtificial SequenceDescription of Artificial
Sequence adhesive sequence 10Glu Arg Gly Asp 1 11571PRTArtificial
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Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly 1 5 10 15 Xaa Xaa Gly Xaa
Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa 20 25 30 Xaa Gly
Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa 35 40 45
Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly 50
55 60 Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly
Xaa 65 70 75 80 Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa
Gly Xaa Xaa 85 90 95 Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa
Xaa Gly Xaa Xaa Gly 100 105 110 Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly
Xaa Xaa Gly Xaa Xaa Gly Xaa 115 120 125 Xaa Gly Xaa Xaa Gly Xaa Xaa
Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa 130 135 140 Gly Xaa Xaa Gly Xaa
Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly 145 150 155 160 Xaa Xaa
Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa 165 170 175
Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa 180
185 190 Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa
Gly 195 200 205 Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa
Xaa Gly Xaa 210 215 220 Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly
Xaa Xaa Gly Xaa Xaa 225 230 235 240 Gly Xaa Xaa Gly Xaa Xaa Gly Xaa
Xaa Gly Xaa Xaa Gly Xaa Xaa Gly 245 250 255 Xaa Xaa Gly Xaa Xaa Gly
Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa 260 265 270 Xaa Gly Xaa Xaa
Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa 275 280 285 Gly Xaa
Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly 290 295 300
Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa 305
310 315 320 Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly
Xaa Xaa 325 330 335 Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa
Gly Xaa Xaa Gly 340 345 350 Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa
Xaa Gly Xaa Xaa Gly Xaa 355 360 365 Xaa Gly Xaa Xaa Gly Xaa Xaa Gly
Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa 370 375 380 Gly Xaa Xaa Gly Xaa Xaa
Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly 385 390 395 400 Xaa Xaa Gly
Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa 405 410 415 Xaa
Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa 420 425
430 Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly
435 440 445 Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa
Gly Xaa 450 455 460 Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa
Xaa Gly Xaa Xaa 465 470 475 480 Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa
Gly Xaa Xaa Gly Xaa Xaa Gly 485 490 495 Xaa Xaa Gly Xaa Xaa Gly Xaa
Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa 500 505 510 Xaa Gly Xaa Xaa Gly
Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa 515 520 525 Gly Xaa Xaa
Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly 530 535 540 Xaa
Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa 545 550
555 560 Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly 565 570
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