U.S. patent application number 10/219027 was filed with the patent office on 2003-01-02 for reinforced matrices.
Invention is credited to Asculai, Samuel, Giannetti, Bruno.
Application Number | 20030003153 10/219027 |
Document ID | / |
Family ID | 25309587 |
Filed Date | 2003-01-02 |
United States Patent
Application |
20030003153 |
Kind Code |
A1 |
Asculai, Samuel ; et
al. |
January 2, 2003 |
Reinforced matrices
Abstract
A reinforced matrix membrane containing one or more
scaffold-forming proteins suitable for cell growth and for use in
chondrocyte cell transplantation, and method of making same. The
scaffold is incubated with the collagen matrix in solutions,
colloidal dispersions, or suspensions of stabilizing proteins. The
reinforced matrix may be used in tissue engineering, cartilage
transplantation, bone and cartilage grafting, healing, joint repair
and the prevention of arthritic pathologies.
Inventors: |
Asculai, Samuel; (Toronto,
CA) ; Giannetti, Bruno; (Bonn, DE) |
Correspondence
Address: |
MORGAN, LEWIS & BOCKIUS LLP
1701 MARKET STREET
PHILADELPHIA
PA
19103-2921
US
|
Family ID: |
25309587 |
Appl. No.: |
10/219027 |
Filed: |
August 14, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10219027 |
Aug 14, 2002 |
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09850966 |
May 8, 2001 |
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6444222 |
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Current U.S.
Class: |
424/484 ;
514/16.7; 514/17.1; 514/9.4 |
Current CPC
Class: |
A61L 27/26 20130101;
A61L 27/26 20130101; A61L 27/26 20130101; A61K 38/012 20130101;
A61K 35/32 20130101; A61L 27/3817 20130101; A61L 27/26 20130101;
A61L 27/3843 20130101; A61K 35/32 20130101; C08L 89/06 20130101;
A61K 2300/00 20130101; A61L 27/227 20130101; A61L 27/38 20130101;
C08L 5/08 20130101; C08L 89/06 20130101 |
Class at
Publication: |
424/484 ;
514/21 |
International
Class: |
A61K 009/14; A61K
038/39 |
Claims
What is claimed:
1. A method for making a collagen-based reinforced matrix
comprising incubating collagen with a scaffold-forming protein to
form a mixture; lyophilizing the mixture to form a fleece-like
material; and pressing the fleece-like material into sheets to form
the matrix.
2. The method of claim 1 wherein the collagen is Type II
collagen.
3. The method of claim 1 wherein the collagen is Type I/III
collagen.
4. The method of claim 1 wherein the scaffold-forming protein is
hydrophobic non-glycosylated protein.
5. The method of claim 4 wherein the scaffold-forming protein is
elastin.
6. The method of claim 1 wherein the scaffold-forming protein
comprises elastin fibers.
7. The method of claim 1 wherein the scaffold forming protein is
elastin-like peptide.
8. The method of claim 1 wherein the scaffold forming protein is
soluble elastin.
9. The method of claim 1 with pH limit/temperature/time
limitations.
10. A collagen-based reinforced membrane comprising a collagen
matrix and a scaffold-forming protein.
11. The membrane of claim 10 wherein the collagen is Type II
collagen
12. The membrane of claim 10 wherein the collagen is Type I/III
collagen.
13. The membrane of claim 10 wherein the collagen is
non-cross-linked.
14. The membrane of claim 10 wherein the collagen is
cross-linked.
15. The membrane of claim 10 wherein the collagen is natural
collagen.
16. The membrane of claim 10 wherein the scaffold-forming protein
is hydrophobic non-glycosylated protein.
17. The membrane of claim 16 wherein the scaffold-forming protein
comprises elastin.
18. The membrane of claim 10 wherein the scaffold-forming protein
comprises elastic fibers.
19. The membrane of claim 10 wherein the scaffold forming protein
is elastin-like peptide.
20. The membrane of claim 10 wherein the scaffold forming protein
is soluble elastin.
21. A composition suitable for use as an implantation article or
for arthroscopic surgery comprised of chondrocytes and a
collagen-based membrane comprising a collagen matrix and a
scaffold-forming protein.
Description
FIELD OF INVENTION
[0001] The present invention relates to a reinforced matrix, and a
method to stabilize and reinforce matrices.
BACKGROUND OF THE INVENTION
[0002] Injuries to the cartilage of the knee or other joints often
result from abnormal mechanical loads which deform the cartilage
matrix. The loads applied to the joint can rupture the collagen
network in the matrix and decrease the stiffness of the cartilage
matrix.
[0003] Cartilage injuries are difficult to treat because human
articular cartilage has a limited capacity for regeneration once it
has been damaged. Type II collagen is the main structural protein
of the extracellular matrix in articular cartilage. Type II
collagen, similar to other types of collagen, is comprised of three
collagen polypeptides which form a triple helix configuration. The
polypeptides are intertwined with each other and possess at each
end telopeptide regions that provide the cross-linking between the
collagen polypeptides. Collagen matrices in their natural state
contain numerous cross-linked triple helices and the individual
molecules have a molecular weight of about 300,000 daltons. Type II
collagen is found almost exclusively in animal cartilage, while
other types of collagen are found in animal hides, membranes, and
bones.
[0004] Excessive degradation of Type II collagen in the outer
layers of articular surfaces ofjoints is also caused by
osteoarthritis. The collagen network is accordingly weakened and
subsequently develops fibrillation whereby matrix substances, such
as proteoglycans, are lost and eventually displaced entirely. Such
fibrillation of weakened osteoarthritic cartilage can reach down to
the calcified cartilage and into the subchondral bone (Kempson, G.
E. et al., Biochim. Biophys. Acta 1976, 428, 741; Roth, V. and Mow,
V. C., J. Bone Joint Surgery, 1980, 62A, 1102; Woo, S. L.-Y. et
al., in Handbook of Bioengineering (R. Skalak and S. Chien Eds),
McGraw-Hill, New York, 1987, pp. 4.1-4.44).
[0005] A method for regeneration-treatment of cartilage would be
useful for treating arthritis and otherjoint conditions and could
be performed at an earlier stage ofjoint damage, thus reducing the
number of patients needing more extensive procedures, such as
artificial joint replacement surgery. With such preventive methods
of treatment, the number of patients developing osteoarthritis
would also decrease.
[0006] Methods for growing and using chondrocyte cells are
described by Brittberg, M. et al. (New Engl. J. Med. 1994, 331,
889). Autologous transplants using cells grown with these methods
are also disclosed. Additionally, Kolettas et al. examined the
expression of cartilage-specific molecules, such as collagens and
proteoglycans, under prolonged cell culturing (J. Cell Science
1995, 108, 1991). They found that, despite morphological changes
during culturing in monolayer cultures (Aulthouse, A. et al., In
Vitro Cell Dev. Biol., 1989, 25, 659; Archer, C. et al., J. Cell
Sci. 1990, 97, 361; Hanselmann, H. et al., J. Cell Sci. 1994, 107,
17; Bonaventure, J. et al., Exp. Cell Res. 1994, 212, 97), when
compared to suspension cultures grown over agarose gels, alginate
beads or as spinner cultures (which retain a round cell morphology)
tested by various scientists, such morphologies did not change the
chondrocyte--that is, expressed markers such as types II and IX
collagens and the large aggregating proteoglycans, aggrecan,
versican and link protein did not change (Kolettas, E. et al., J.
Cell Science 1995, 108, 1991).
[0007] In addition, chondrocyte cells from donors have been grown
in vitro to form neocartilage which has been implanted into animals
(Adkisson et al., "A Novel Scaffold-Independent Neocartilage Graft
for Articular Cartilage Repair," ICRS 2nd Symposium International
Cartilage Repair Society, Nov. 16-18, 1998). Further, chondrocyte
cells have been seeded onto the cartilage surface of osteochondral
cores to attempt cartilage regeneration (Albrecht et al.,
"Circumferential Seeding of Chondrocytes: Towards Enhancement of
Integrative Cartilage Repair," ICRS 2.sup.nd Symposium
International Cartilage Repair Society, Nov. 16-18, 1998).
Articular surface defects in knee joints have been treated with
various cultured chondrocytes (Stone et al., Operative Techniques
in Orthopaedics 7(4), pp. 305-311, Oct. 1997 and Minas et al.,
Operative Techniques in Orthopaedics 7(4), pp. 323-333, Oct.
1997).
[0008] U.S. Pat. No. 5,007,934 to Stone is directed to a prosthetic
resorbable meniscus formed from biocompatible and bioresorbable
fibers. The fibers include natural fibers or analogs of natural
fibers. The natural fibers useful in the invention include
collagen, elastin, reticulin, analogs thereof, and mixtures
thereof. The fibers are oriented in the matrix circumferentially or
radially, or alternatively, the fibers may have random
orientations. The fiber may be cross-linked, and the matrix
optionally may include glycosaminoglycans.
[0009] U.S. Pat. No. 5,837,278 --Geistlich et al. describe a
collagen-containing membrane which is resorbable and is used in
guided tissue regeneration. The membrane has a fibrous face which
allows cell growth thereon and a smooth face opposite the fibrous
face which inhibits cell adhesion thereon. The membrane product is
derived from a natural collagen membrane (that is, from the hide or
tendons of calves or piglets) and, although treated, it is
described as maintaining its natural structural features. The
collagen is purified with alkaline agents to defat the collagen and
degrade substances, and then the purified collagen is acidified,
washed, dried, degreased, and optionally cross-linked. The fats are
saponified. The membrane is described as containing about 95% by
weight native collagen. The collagen does not appear to contain a
reinforcing protein.
[0010] PCT WO 96/25961 --Geistlich et al. describe a matrix for
reconstructing cartilage tissue which consists of Type II collagen,
optionally including crosslinking. In producing the matrix,
cartilage is taken from an animal and frozen, subjected to size
reduction, dewatered, defatted, washed, and treated with alkaline
materials. Non-collagen alkaline soluble proteins are denatured,
destroyed, dissolved, and eliminated. Dialysis and freeze-drying
are mentioned as possible treatment steps. The matrix material is
stamped to form a required shape and then it is sterilized.
[0011] U.S. Pat. No. 4,424,208 --Wallace et al. describe an
injectable collagen implant material comprising particulate
cross-linked atelopeptide collagen and reconstituted atelopeptide
collagen fibers dispersed in an aqueous carrier. The atelopeptide
form of collagen lacks the native telopeptide crosslinking. In the
method described in the '208 patent, collagen obtained from bovine
or porcine corium (sub-epithelial skin layer) is softened by
soaking in a mild acid; depiliated; comminuted by physical
treatment, such as grinding; solubilized by treatment with acid and
a proteolytic enzyme; treated with an alkaline solution; and freed
of enzyme. The cross-linked gel form of collagen is formed by
radiation-induced or chemical-induced crosslinking, such as by
addition of glutaraldehyde. The fibrous form of collagen is
produced by neutralizing the solution with a buffer, such as
Na.sub.2HPO4. Collagen content of the injectable implant comprises
5-30% fibrous collagen and 70-98% of the cross-linked gel form of
collagen.
[0012] U.S. Pat. No. 4,488,911 --Luck et al. describe the formation
of collagen fibers free of the immunogenic, telopeptide portion of
native collagen. The telopeptide region provides points of
crosslinking in native collagen. The fibers, which may be
cross-linked, are described for use as sponges, prosthetic devices,
films, membranes, and sutures. In the method described in the '911
patent, (non-Type II; Type I and others), collagen obtained from
tendons, skin, and connective tissue of animals, such as a cow, is
dispersed in an acetic acid solution, passed through a meat
chopper, treated with pepsin to cleave the telopeptides and
solubilize the collagen, precipitated, dialyzed, cross-linked by
addition of formaldehyde, sterilized, and lyophilized. The '911
patent indicates that its disclosed method obtains the
atelocollagen form of collagen, free from noncollagen proteins,
such as glycosaminoglycans and lipids. Further, it describes that
the collagen may be used as a gel to make, for example, a membrane,
film, or sponge and that the degree of crosslinking of the collagen
can be controlled to alter its structural properties.
BRIEF SUMMARY OF THE INVENTION
[0013] In one embodiment, the present invention provides a method
for the reinforcement of matrices with an internal scaffold. One
embodiment of the present invention is directed to a method for
making a collagen-based matrix comprising incubating collagen with
one or more scaffold-forming proteins to form a collagen-protein
suspension, lyophilizing the suspension to form a fleece-like
material, and pressing the fleece-like material into sheets to form
a matrix. In one embodiment, the collagen is Type II or Type I/III
collagen. Collagen matrices for use in the present invention
include those produced from animal sources such as pig, calf,
chicken, sheep, goat, kangaroo and others. In one preferred
embodiment, the scaffold-forming protein is a hydrophobic
non-glycosylated protein, such as elastin or elastin-like
peptide.
[0014] In another aspect, the present invention includes
chondrocytes seeded on a protein reinforced collagen matrix.
BRIEF DESCRIPTION OF THE DRAWING
[0015] FIG. 1 shows an exemplary pressing device for shaping a
reinforced matrix into the sheet-like configuration according to
the present invention;
[0016] FIG. 2 shows the two components of the exemplary pressing
device shown in FIG. 1; and
[0017] FIG. 3 shows an apparatus for measuring mechanical strength
of reinforced matrices according to the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0018] Currently available matrices and membranes having different
chemical moieties show a very limited mechanical strength. Most of
these matrices or membranes disintegrate after a short period of
exposure to cells. Mechanical stability is essential for
manipulation of cell-loaded matrices for tissue implantations. In
one embodiment, the present invention provides a protein scaffold
effective and efficient to reinforce matrices to create new
matrices suitable for arthroscopic or other minimal invasive
transplantations of chondrocytes or other cells, such as
osteoblasts, or mesenchymal stem cells into an area to be treated.
The present invention is also directed to polylactic acid and
polyglycolic acid reinforcement scaffolds useful in reinforcing
matrices.
[0019] While analyzing the degradation behavior of different types
of collagen materials, it was surprisingly discovered that certain
matrix structures were easily degraded by collagenase, but not by
trypsin. Other matrix structures demonstrated a significant loss of
mechanical strength after treatment with trypsin, but not after
treatment with collagenase. In some cases, a combined or subsequent
treatment of collagenase and trypsin did not show any significant
effect on mechanical strength of the membrane. A subsequent
systematic analysis showed that natural, synthetic or
semi-synthetic membranes consisting of pure collagen Type I, Type
I/III, or Type II structures without or with only a partial
cross-linking were susceptible to degradation with collagenase.
[0020] Some natural membranes from peritoneum or skin from
different animals, such as pigs, sheep, goats, cows, horses,
chicken, kangaroos, as well as some commercially available
membranes (such as Chondro-Gide.RTM. or Chondro-Cell,.RTM. from Ed
Geistlich Sohne, Switzerland) were found to be quite resistant to
collagenase although they contained collagen. Treatment of these
membranes with trypsin or trypsin/collagenase, however, showed
complete degradation within a certain period of time, that varied
in connection with the origin and thickness of the material. These
findings suggested the existence of an additional protein scaffold
that is not degraded by collagenase and significantly contributes
to the mechanical strength and stability of the respective
material.
[0021] One protein which formed such a scaffold was identified as
elastin, however other non-soluble polymeric biodegradable proteins
will also work. One example of elastin that can be used in
accordance with the present invention is the elastin fractions
described by Partridge et al. (Biochem. J. 61: 11-21, 1954), which
is hereby incorporated by reference. Specifically, elastin is
extracted and purified from the ligamentum nuchae of cattle to
yield a soluble and substantially pure elastin powder.
[0022] In addition, different elastin structures, such as those
described by Debelle and Alix (Biochimie 81: 981-994, 1999), which
is hereby incorporated by reference, can also be used in accordance
with the present invention. Elastin is constituted of globular
tropoelastin monomers with substantial amounts of irregular and
distorted .beta.-structures. Further, elastin structures are mobile
and influenced by the presence of water.
[0023] Elastin from different animal species can also be employed.
Elastin may be obtained from human, bovine, porcine chicken, sheep,
goat, and kangaroo sources.
[0024] Elastin-like peptides can also be used, such as those
prepared and described by Davril and Han (FEBS Letters 43: 331-336,
1974), which is hereby incorporated by reference. In particular,
these porcine elastin-like peptides are enriched desmosine or
isodesmosine by enzymatic and chemical digestion of porcine aorta
with elastase, themolysin and pancreatic proteases followed by gel
filtration, electrophoresis and paper chromatography.
[0025] Additionally, salt-soluble elastin can also be used in
accordance 15 with the present invention, such as that described by
Smith et al. (J. Biol. Chem. 8: 2427-2432, 1972) and Manning et al.
(Connective Tissue Res. 13: 313-322, 1985), both of which are
hereby incorporated by reference. Salt-soluble elastin can be
prepared and purified from porcine aorta by extraction,
precipitation and sequential centrifugation or from sheep vascular
tissue by hydrophobic interaction chromatography on a column of
decyl-agarose.
[0026] Treatment of some of the analyzed membranes with different
concentrations of elastase lead also to a significant degradation
of the analyzed product. Membranes resistant to trypsin,
collagenase and elastase were either fully synthetic, such as
polyethylenglycol or polyethylenoxide/polybutyleneterephtalate
co-polymers, or were natural and contained additional chemical
crosslinking agents.
[0027] We demonstrated that incubation of collagen matrices with
different quantities of a scaffold forming protein such as elastin
significantly increased the mechanical stability of the matrix
without affecting the biodegradability of the matrix. Thus, in one
aspect, the present invention teaches methods to increase the
mechanical strength and stability of collagen matrices and
materials using a scaffold forming protein. These reinforced
collagen matrices may then be used for a variety of purposes,
including cell (e.g., chondrocyte cells) cultivation and
implantation.
[0028] Suitable matrix materials according to the present invention
are characterized as having the ability to enable the growth and
attachment of cells such as chondrocytes, and provide a system
similar to the natural environment of the cartilage cells. The
matrix material is stable for a sufficient period of time to allow
full cartilage repair and to be reabsorbed or broken down over
time.
[0029] Suitable matrix materials include collagen, hyaluronic acid
and its derivatives, homologs and analogs; polylactic and
polyglycolic acids; polyethylene oxide; and mixtures thereof;
fibrin; proteoglycans; proteins and sugars.
[0030] Matrix materials for the present invention are prepared from
natural sources such as animal skin, peritoneum, or animal
cartilage, according to generally accepted and described methods,
such as those described in U.S. patent application Ser. No.
09/467,584; U.S. Pat. Nos. 5,201,745 and 5,837,278; and PCT WO
96/25961, all of which are hereby incorporated by reference.
[0031] In one such method, the cartilage tissue obtained from the
animal is solubilized by physical and/or chemical treatment as
described in U.S. patent application Ser. No. 09/467,584, which is
hereby incorporated by reference. The solubilization process
includes treatment with various buffers to remove impurities and to
separate solid and liquid phases; physical treatment to separate
solid and liquid phases, such as by centrifugation; and treatment
with a proteolytic enzyme to break the crosslinking of the collagen
in its telopeptide region into its virtually non-cross-linked
atelocollagen, triple helix form.
[0032] By reconstituting, it is meant that the non-cross-linked,
atelocollagen form of collagen reestablishes its crosslinking
between the variable regions along the collagen molecule, including
some remaining residues in the telopeptide region. As a result, the
collagen loses its liquid or gel-like consistency and becomes more
rigid with a higher degree of structural integrity such that cells
may be grown upon it.
[0033] In another embodiment of the present invention, the collagen
matrix is prepared by incubating a matrix with a suspension of
elastin, under conditions that will not break down the protein
structure. The matrix is prepared under temperature conditions in
the range of ambient to 80.degree. C. and under pH conditions in
the range of 4-9.
[0034] In another embodiment, the matrix composition is formed from
recombinantly produced Type II collagen. The substantially pure,
recombinantly produced Type II collagen is not cross-linked.
However, it can have telopeptide regions. In an embodiment, it is
soluble and can be formed into a fleece-like structure.
[0035] Matrices for use in the present invention are also
commercially available. One material identified as suitable is
Chondro-Cell.RTM. (a type II collagen matrix pad, Geistlich und
Sohne, Switzerland). Another material which may be used in the
present invention is Chondro-Gide.RTM. (a type I collagen matrix
pad, Geistlich und Sohne, Switzerland). Additional matrices for use
in the present invention are bovine collagen I/III matrix (Immedex,
France) and other matrices such as Permacol.TM. and various
uncross-linked or cross-linked versions thereof, available from
Tissue Science Laboratories (UK), and Antema.RTM. from Opocrin
S.p.A. (Italy) and various universities and institutes. In one
embodiment, the matrix has two smooth surface sides, or one smooth
surface and one textured or rough surface. A smooth surface on the
matrix typically impedes tissue ingrowth, while a textured or rough
surface typically promotes cell ingrowth. The surface properties of
the matrix may be altered by slowly adding an alcohol, such as
ethanol (in a 10-30% solution) in the lyophilization mixture as
described in U.S. patent application Ser. No. 09/467,584.
[0036] In another embodiment, the consistency of the matrix is
fleece-like, and is formed by treating it with one or more
cross-linking agents. Cross-linking us also accomplished by heating
or subjecting the composition to radiation. The resulting
properties of the matrix will vary, but the matrix preferably has a
strength in the range of 0.1 to 20 kp, and most preferably in the
range of 1.5 to 5 kp.
[0037] In one embodiment, the crosslinking agent is an
aldehyde-based biocompatible crosslinking agent or a polyvalent
aldehyde, such as glutaraldehyde. Also, the crosslinking agent can
be a bifunctional agent with two moieties reacting with the support
matrix and its components. Examples of the moieties are aldehydes;
ketones; acetals; half acetals; moieties which are available for
oxidative coupling, such as phenolic groups; quinones, such as
flavoids; carboxylic groups; and activated carboxylic acids. Also,
ethyl-dimethyl-aminopropylcarbodiimide (EDC) may be used as a
crosslinking agent. Preferred crosslinking agents are chemical
compounds containing two aldehyde groups, such as bioflavonoid or
cyanidanol, which promote crosslinking by bridging lysine residues
on Type II collagen. The type of crosslinking agent to be used is
determined by evaluating its effect on the consistency and physical
properties of the matrix and its physiological compatibility with
the area of the body in which the matrix and cells are to be
implanted.
[0038] By the term protein scaffold, it is meant an inter-linked,
fibrous texture supporting structure such as a three dimensional
porous structure comprised of structural proteins. Examples of
acceptable scaffold-forming proteins for the present invention
include hydrophobic non-glycosylated proteins such as elastin,
either in soluble or insoluble form, or elastin-like peptides.
Elastin-like peptides include peptides isolated by partial
exhaustive hydrolysis of elastin or soluble elastin with different
types of elastases, such as pancreatic sputum. In one embodiment of
the present invention, the protein reinforced matrix according to
the present invention has cells (such as chondrocyte cells) grown
thereon to form an implantable article for implanting in animals
for repair of an injury or defect, such as cartilage damage.
[0039] Chondrocyte cells, which may be autologous or homologous,
can be cultured and/or retained on the protein reinforced matrix
for use in the treatment of cartilage defects in joints.
Chondrocyte cells can be grown directly on the support matrix in
standard dishes and/or loaded onto the matrix before use. In use,
the chondrocyte cell-loaded protein reinforced matrix, i.e., an
implantable article, according to the present invention, preferably
is introduced into the joint through an arthroscope, or by
minimally invasive or open joint surgery technique. The
implantation method of the invention also contemplates the use of
suitable allogenic and xenogenic chondrocyte cells for the repair
of a cartilage defect.
[0040] The cell-loaded protein reinforced matrix is incorporated
into various other techniques for effecting or stimulating repair
of a bodily defect or damage using various placement and securing
devices for implantation. Certain of these techniques and devices
are shown in the U.S. patent application of Behrens et al. entitled
"METHODS, INSTRUMENTS AND MATERIALS FOR CHONDROCYTE CELL
TRANSPLANTATION," Ser. No. 09/373,952, filed Aug. 13, 1999; in U.S.
Provisional Application No. 60/096,597, filed Aug. 14, 1998; and
U.S. Provisional Application No. 60/146,683, filed Aug. 2, 1999,
the entire disclosures of which are incorporated herein by
reference.
[0041] Thus, the present invention teaches methods and systems for
the effective repair or treatment of defects in articular cartilage
and bone; osteochondral defects; skin and wound defects; and
defects of ligaments, menisci, and vertebral discs. These methods
and systems involve the use of an implantable article comprising a
protein reinforced matrix of the present invention along with
cells, such as chondrocyte cells.
[0042] For these purposes, the reinforced matrix of the present
invention has a sufficient physical integrity such that it holds a
stable form for a period of time to be manipulated for its intended
purpose. This strength allows for the growth of cells on the
reinforced matrix both before transplant and after transplant, and
to provide a system similar to the natural environment of the cells
to optimize cell growth differentiation. Over time, perhaps within
two to three months, the reinforced matrix is expected to be
resorbed in a body of a patient receiving the implant without
leaving any significant traces and without forming toxic
degradation products. The term "resorbed" is meant to include
processes by which the reinforced matrix is broken down by natural
biological processes, and the broken down reinforced matrix and
degradation products thereof are disposed, for example, through the
lymphatics or blood vessels.
General Example
[0043] In one embodiment, the reinforced matrix of the present
invention is prepared by the following method. Six pieces, each
approximately 1 cm.sup.2 in size of a collagen membrane are
incubated with a suspension of elastin in the range of 0.1 mg/100
ml to lOOg/100 ml in a suitable buffer, such as phosphate buffer.
The final concentration of elastin in the membrane is between 0.1
mg/100 g to 50 g/100 g. Elastin for use in the present invention is
available from EPC, Inc. (USA), specifically sold under the
following product numbers: E60, ES60, F65, E61, ES61, E70, ES70,
SB77, SB87, SP46, SC55, MT65, ME15, LK215, KE57, K267, ES12, TB872,
AE17, BE73, AC27, RA50, MT60, SH476, HA587, HS395, HL457, and
HT754. In a preferred embodiment, the phosphate buffer is
KH.sub.2PO.sub.4. Acceptable pH ranges for the mixture are between
about 4.0 and 9.0.
[0044] Incubation is performed with suspensions, colloidal
dispersions or solutions of a scaffold protein, such as elastin, in
different buffers (for example 0.2 m Tris pH 8.8 with 0.1%
NaN.sub.3 or 0.02M KH.sub.2PO.sub.4, pH 7.4) at temperatures
between 1 and 102.degree. C. with concentrations of the protein
between 0.5 mg/ml and 100 mg/ml.
[0045] The above mixture is allowed to coacervate, such as soak,
incubate or mix, for 0.05 to 80 hours, preferably about 2 to
8hours.
[0046] The suspension is then lyophilized to obtain a solid. An
acceptable temperature range for lyophilization is between about
20.degree. C. and about 60.degree. C., preferably at about
25.degree. C. and at a pressure of about 0.01 to 10 mbar.
Lyophilization may be repeated after soaking the product in an
aqueous solution, such as with between 10-20 ml of distilled water.
The amount of water used will depend upon the size of the
lyophilized collagen pellet.
[0047] Lyophilization yields a fleece-like material which is then
pressed mechanically into sheets for use with cells as an
implantation article. The fleece-like material is pressed for a
time period of one minute to 48 hours at a pressure of 500 to 1000
grams, preferably at a pressure of 750 grams. A suitable pressing
machine for the matrix contains two non-textured stainless steel
pieces with bonding material implanted in the pieces. The material
is pressed until a sheet-like material that resists tearing upon
being handled is obtained.
[0048] FIGS. 1 and 2 depict an exemplary pressing apparatus which
is in the form of two parts and is similar to a mortar 10 and
pestle 12. In this embodiment, the pressing apparatus is a
stainless steel device with a mortar-like receptacle of
approximately 2.5 cm diameter into which a stainless steel
pestle-like stamp exactly fits. The matrix material is placed in
the mortar-like part and the pestle-like stamp is inserted into
opening 14 to apply a mechanical pressing force on the matrix. The
pressing device may be any suitable device with enough weight to
continually apply force to the matrix. The pressing device
preferably is made of stainless steel; however, metals and other
materials, for example, plastic, glass, or ceramic, may also be
used.
[0049] Mechanical strength of a reinforced matrix according to the
present invention, optionally with cells such as chondrocytes grown
thereon, is tested by using an in vitro system to study the
behavior of the chondrocytes when in contact with the reinforced
material. The strength of the chondrocyte-seeded reinforced
matrices was then tested against chondrocyte-seeded commercial
matrices. In particular, the apparatus shown in FIG. 3 is a
preferred method to measuring mechanical strength of the seeded
chondrocyte matrices. In use, a reinforced matrix 26 produced
according to the present invention is attached to two smooth clamps
16 and 18. Clamp 18 is attached to an immovable surface 20. A
calibrated caliper 22 (Ericsen, model number 391-100 II) is
attached to clamp 16. Caliper 22 includes a display dial 24 that
displays a measure of mechanical resistance at least in the range
of 0 to 15 kp. Mechanical strength of reinforced matrix 26
according to the present invention is obtained by applying a
pulling force on caliper 22 and observing the level of mechanical
resistance reinforced matrix 26 sustains before tearing or breaking
down. This in vitro method tests the point of mechanical breakdown
of the matrices and predicts the ability of certain materials to
mechanically withstand the arthroscopic procedure.
[0050] In a preferred embodiment, chondrocytes are grown in culture
medium containing one or more suitable buffers and approximately 5
to 7.5% autologous serum in an incubator at 37.degree. C. After a
suitable period of time, for example 1 to 14 days, cells are
removed from culture and assessed for viability before placing them
directly on top of the reinforced matrix material and dispersed
over the surface of a cell culture tray. The reinforced matrix is
then tested for strength characteristics as described above.
[0051] Certain aspects of the instant invention will be better
understood as illustrated by the following examples, which are
meant by way of illustration and not limitation.
EXAMPLE 1
[0052] Chondrocytes were grown in minimal essential culture medium
containing HAM F 12, 15mM Hepes buffer and 5 to 7.5% autologous
serum in a CO.sub.2 incubator at 37.degree. C. and handled in a
Class 100 laboratory at Verigen Transplantation Service ApS,
Copenhagen, DK. Other compositions of culture medium may be used
for culturing the chondrocytes.
[0053] The cells were trypsinised using trypsin EDTA for 5 to 10
minutes and counted using Trypan Blue viability staining in a
Burker-Turk chamber. The cell count was adjusted to
7.5.times.10.sup.5 cells per ml. One NUNCLON.TM. plate was
uncovered in the Class 100 laboratory.
[0054] Six Pieces of a size of 1 cm.sup.2 each of commercially
available collagen I/III fleece (Chondro-Gideg.RTM., Geistlich,.
CH) were placed under aseptic conditions into the bottom of the
well in the NUNCLON.TM. cell culture tray.
[0055] Approximately 5.times.10.sup.6 of the chondrocytes in 5 ml
of the culture medium were placed directly on top of the carrier
material and dispersed over the surface. The plate was incubated in
a CO.sub.2 incubator at 37.degree. C. for three days. After this
period the chondrocytes were arranged in clusters and started to
grow on the carrier. The chondrocytes could not be removed from the
carrier by rinsing it with medium or even by mechanically exerting
mild pressure on the matrix. At the end of the incubation period
the medium was decanted.
[0056] Mechanical resistance of the seeded membrane was tested
manually under standard conditions by using a calibrated caliper
such as the one shown in FIG. 3 to test the point of mechanical
breakdown of the fleece. In this example, the breakdown of the
membranes occurred at an average traction of 5.4 kp.
[0057] The remaining pieces were incubated with cold refrigerated
2.5% glutaraldehyde containing 0.1 M sodium salt of dimethylarsinic
acid. The matrix was stained with Safranin O for histological
evaluation.
[0058] The breakdown traction measured in this experiment was
considered as a baseline for comparison for the other experiments
as described below.
EXAMPLE 2
[0059] Chondrocytes were grown in minimal essential culture medium
containing HAM F 12, 15 nM Hepes buffer and 5 to 7.5% autologous
serum in a CO.sub.2 incubator at 37.degree. C. and handled in a
Class 100 laboratory at Verigen Transplantation Service ApS,
Copenhagen, DK. Other compositions of culture medium may be used
for culturing the chondrocytes.
[0060] The cells were trypsinised using trypsin EDTA for 5 to 10
minutes and counted using Trypan Blue viability staining in a
Burker-Turk chamber. The cell count was adjusted to
7.5.times.10.sup.5 cells per ml. One NUNCLON.TM. plate was
uncovered in the Class 100 laboratory.
[0061] Six Pieces of a size of 1 cm.sup.2 each of a collagen I/III
matrix (Immedex, France) was cut to a suitable size fitting into
the bottom of the well in the NUNCLON.TM. cell culture tray and
placed under aseptic conditions on the bottom of the well.
[0062] Approximately 5.times.10.sup.5 of the chondrocytes in 5 ml
of culture medium were placed directly on top of the carrier
material and dispersed over the surface. The plate was incubated in
a CO.sub.2 incubator at 37.degree. C. for 3 days. At the end of the
incubation period the medium was decanted.
[0063] Mechanical resistance of the seeded membrane was tested
manually under standard conditions by using a calibrated caliper as
shown in FIG. 3 to test the point of mechanical breakdown of the
fleece. In this example breakdown of the membranes occurred at an
average traction of 0.3 kp. Mechanical resistance was very low
compared to Chondro-Gide.RTM. making this material not suitable for
arthroscopic surgery purposes.
[0064] The remaining pieces were incubated with cold refrigerated
2.5% glutaraldehyde containing 0.1 M sodium salt of dimethylarsinic
acid was added as fixative. The matrix was stained with Safranin O
for histological evaluation.
EXAMPLE 3
[0065] Six pieces, 1 cm.sup.2 each in size, of the collagen I/III
matrix of Example 2 were incubated for 2 hours at a temperature of
50.degree. C. under gentle stirring with a solution of soluble
elastin (EPC Inc., USA) in a suitable buffer such as phosphate
buffer (0.02M KH.sub.2PO.sub.4, pH 7.4) and the pH value was then
brought down to 5.0 by adding acetic acid under gentle stirring.
The coacervation reaction was allowed to occur for 5 hours.
[0066] The suspension was then lyophilized at a temperature of
25.degree. C. and a pressure of 0.05 mbar.
[0067] The lyophilization yielded a fleece-like material which was
pressed mechanically using the apparatus in FIGS. 1 and 2 into
sheets for use with cells as an implantation article. The material
was pressed for about 24 hours until a sheet-like material which
resisted tearing upon being handled was obtained.
[0068] Six Pieces, each 1 cm.sup.2 in size, of the fleece matrix
were cut to a suitable size fitting into the bottom of the well in
the NUNCLON.TM. cell culture tray and placed under aseptic
conditions on the bottom of the well.
[0069] Approximately 5.times.10.sup.5 of the chondrocytes in 5 ml
culture medium were placed directly on top of the carrier material
and dispersed over the surface. The plate was incubated in a
CO.sub.2 incubator at 37.degree. C. for 3 days. At the end of the
incubation period the medium was decanted.
[0070] Mechanical resistance of the seeded membrane was tested
manually under standard conditions by using a calibrated caliper as
shown in FIG. 3 to test the strength the fleece. In this example,
breakdown of the membranes occurred at an average traction of 4.8
kp. Mechanical resistance was comparable to Chondro-Gide.RTM., thus
making this material suitable for arthroscopic surgery
purposes.
[0071] The remaining pieces were incubated with cold refrigerated
2.5% glutaraldehyde containing 0.1 M sodium salt of dimethylarsinic
acid was added as fixative. The matrix was stained with Safranin O
for histological evaluation.
EXAMPLE 4
[0072] Six pieces, 1 cm.sup.2 each, of collagen II membrane
(produced according to U.S. patent application Ser. No. 09/467,584)
were incubated with a water suspension of insoluble elastin (EPC
Inc., USA) at 50.degree. C. with gentle stirring produced by
treating a suspension of 20 mg/ml micronised insoluble elastin in a
0.2 m Tris buffer (pH 8.8) with 0.1% Triton X and 0.01% NaN.sub.3
in a suitable buffer such as phosphate buffer (0.02M
KH.sub.2PO.sub.4, pH 7.4) and the pH value was then brought down to
5.0 by adding acetic acid under gentle stirring. The coacervation
reaction was allowed to occur for 5 hours.
[0073] The suspension was lyophilized. The lyophilization may be
repeated after re-soaking with an aqueous solution, such as 10-20
ml of distilled water (depending on the size of the lyophilized
collagen pellet) at a temperature between about 20.degree. C. and
about 60.degree. C., preferably at about 25.degree. C. and at a
pressure of about 0.05 mbar. The lyophilization yielded a
fleece-like material which was pressed mechanically using the
apparatus shown in FIGS. 1 and 2, into sheets for use with cells as
an implantation article. The additional working steps were
performed identically as described above in Example 2.
[0074] Mechanical resistance of the seeded membrane was tested
manually under standard conditions by using a calibrated caliper as
shown in FIG. 3 to test the point of mechanical breakdown of the
fleece. In this example, breakdown of the membranes occurred at an
average traction of 4.1 kp. Mechanical resistance was comparable to
Chondro-Gide.RTM., thus making this material as such suitable for
arthroscopic surgery purposes.
[0075] The remaining pieces were incubated with cold refrigerated
2.5% glutaraldehyde containing 0.1 M sodium salt of dimethylarsinic
acid was added as fixative. The matrix was stained with Safranin O
for histological evaluation.
EXAMPLE 5
[0076] Six pieces, 1 cm.sup.2 each, of the same material of Example
2 were incubated with a solution of soluble elastin-like peptides,
for example elastin peptides CB573, QP45, RY53 at a concentration
of 1 to 200 mg/ml from EPC Inc., USA in a suitable buffer such as
phosphate buffer (0.02M KH.sub.2PO4, pH 7.4) and then heated up to
50.degree. C. for four hours under gentle stirring. The suspension
was then cooled to 40 to 0.degree. C. and lyophilized. The
lyophilization may be repeated after re-soaking with an aqueous
solution, such as 10-20 ml of distilled water (depending on the
size of the lyophilized collagen pellet) at a temperature between
about 20.degree. C. and about 60.degree. C., preferably at about
25.degree. C. and at a pressure of about 0.05 mbar. The
lyophilization yielded a fleece-like material which was pressed
mechanically into sheets for use with cells as an implantation
article.
[0077] The sheets of material yielded were tested for tearing
resistance by a calibrated caliper as shown in FIG. 3. The membrane
was fixed in a frame with the calibrated caliper attached to one
side of the membrane. Tension was applied to the membrane by
pulling the caliper. Tension was continuously recorded with a
qualified gauge. Tensile force at which tearing occurred was
recorded. The force was detected to be between 3 and 12 kp
depending on the lyophilization conditions, the amount and the
properties of the added elastin or elastin-like peptides.
Commercially available collagen I/III membranes (Geistlich, CH or
Tissue Sciences Laboratories, UK) tore between 1 and 6 kp.
[0078] In summary, as shown in Table 1, the protein-reinforced
matrices of Examples 3, 4, and 5 exhibited a comparable mechanical
resistance to Chondro-Gide.RTM. and higher mechanical resistance
than the matrix of Example 2 that was not reinforced with a
protein.
[0079] It will be appreciated by persons skilled in the art that
numerous variations and/or modifications may be made to the
invention shown in the specific embodiments without departing from
the spirit and scope of the invention as described.
1TABLE 1 Example Protein Mechanical No. Matrix Conditions
Reinforcement Resistance (kp) 1 Chondrocytes on Chondro- No 5.4
Gide 2 Chondrocytes on Immedex No 0.3 membrane 3 Chondrocytes on
protein- Yes 4.8 reinforced Immedex membrane 4 Chondrocytes on
protein- Yes 4.1 reinforced collagen II membrane 5 Chondrocytes on
protein- Yes 3-12 reinforced Immedex membrane
* * * * *