Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02427047 2003-04-25
WO 02/36147 PCT/USO1/42477
MINERALIZED COLLAGEN-POLYSACCHARIDE MATRIX FOR BONE
AND CARTILAGE REPAIR
FIELD OF THE INVENTION
The present invention is directed to crosslinked mineralized collagen-
polysaccharide matrices for the therapeutic repair of tissue, such as, bone,
cartilage and
soft tissue; methods of producing such matrices; and methods of using the
matrices to
repair tissue. The present invention provides a crosslinked mineralized
collagen-
polysaccharide matrix that is administered by implantation or injection alone
or in
combination with other therapeutics, such as growth factors, for tissue
repair.
BACKGROUND OF THE INVENTION
There is a clinical demand for a bone grafting matrix that offers
osteoconductive
properties equal to autogenous bone and that can be produced in unlimited
supply.
Although some bone substitutes are available, many consist of materials that
have poor
physical handling and resorption characteristics that complicate their use and
radiographic evaluation.
Similarly, there is no consistently effective commercial product that supports
the
differentiation or maintenance of the chondrocyte phenotype of cartilage
tissue, despite
years of extensive research. Prior strategies to facilitate the repair of
damaged cartilage
have included the transplantation of existing host cartilage and/or the
implantation of
prosthetic devices. Limitations of these methods are the availability of donor
tissue and
the limited lifespan of prosthetic implants. More recently, the ex vivo
cultivation of
mature chondrocytes on polymeric scaffolds has been used in an attempt to
generate
cartilage graft material but this has not yet been widely accepted in part
because it
involves two surgical procedures: one to harvest chondrocytes and the second
to implant
them after expansion ih vitro.
CA 02427047 2003-04-25
WO 02/36147 PCT/USO1/42477
Collagens and glycosaminoglycans are two classes of biomaterials suited for
use
in bone and cartilage regeneration. Collagen based matrices have been used in
bone
grafting. Type I collagen has good cell adhesive properties, in particular,
for bone
forming osteoblast cells. Collagen has the capacity to serve both as an active
or inert
scaffold material for growth.
Bone is characteristically composed of type I collagen fibrils intimately
associated
in an orderly manner with calcium phosphate crystals. Minor constituents
include an
array of macromolecules as well as a series of small molecules associated
mainly with the
mineral phase.
One feature of bone is the exceedingly small size of the crystals. Bone
crystals are
among the smallest biologically formed crystals known and, in fact,
crystallographers
would intuitively not expect crystals just a few unit cells thick to be stable
at all.
Therefore, the collagen bone structure has unique characteristics as to its
formation,
components, and properties.
Mineralized collagen having calcium phosphate stably dispersed in an ordered
manner associated with collagen fibrils is disclosed in U.S. 5,231,169, which
is
incorporated-by-reference herein in its entirety.
Hyaluronic acid is a natural component of the cartilage extracellular matrix,
and it
is readily sterilized, is biodegradable and can be produced in a wide range of
consistencies and formats. It is generally biocompatable and its resorption
characteristics
can be controlled by the manipulation of monomers to polymer forms, most
commonly
through the esterification of the carboxylic groups of the glucuronic acid
residues.
Biological glue comprising fibrin has a long history as a tissue adhesive
medical
device and is believed to be commercially available in Europe (United States
patent No.
5,260,420, issued November 9, 1993). One obstacle that limits its application
is the short
turn over and residence time which ranges from a few days to a few weeks
depending on
the site of implantation. The incorporation of collagen fibers into fibrin
glue has been
_2_
CA 02427047 2003-04-25
WO 02/36147 PCT/USO1/42477
reported (Sierra et al., 1993, Trans. Soc. Biomater., vol. 16:257 and United
States Patent
No. 5,290,552). However, longer coagulation times are required for the
collagen/fibrin
compositions compared to fibrin alone.
There remains a need for biodegradable, biocompatable matrices which maintain
structural integrity and which can be used to repair tissues without resorting
to
undesirable ex vivo cultivation methods.
SUMMARY OF THE INVENTION
The present invention provides crosslinked mineralized collagen-polysaccharide
matrices, methods for preparing such matrices, and methods of using the
matrices by
implantation or injection in the repair of tissue, such as, bone, cartilage
and other soft
connective tissue. The mineralized collagen may be formed from purified,
native,
modified or recombinant collagen of any type. The type of polysaccharides
which can be
used include hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan
sulfate,
heparan, heparan sulfate, dextran, dextran sulfate, alginate, and other long
chain
polysaccharides. In a preferred embodiment, the polysaccharide is hyaluronic
acid.
A crosslinked mineralized collagen-polysaccharide matrix of the present
invention may be used alone to conduct the growth of tissue; or in combination
with
growth factor.
Growth factors which can be used with a matrix of the present invention
include,
but are not limited to, members of the TGF-13 superfamily, including TGF-131,2
and 3, the
bone morphogenetic proteins (BMP's), the growth differentiation
factors(GDF's), and
ADMP-1; members of the fibroblast growth factor family, including acidic and
basic
fibroblast growth factor (FGF-1 and -2); members of the hedgehog family of
proteins,
including Indian, sonic and desert hedgehog; members of the insulin-like
growth factor
(IGF) family, including IGF-I and -II; members of the platelet-derived growth
factor
(PDGF) family, including PDGF-AB, PDGF-BB and PDGF-AA; members of the
-3-
CA 02427047 2003-04-25
WO 02/36147 PCT/USO1/42477
interleukin (IL) family, including IL-1 thru -6; and members of the colony-
stimulating
factor (CSF) family, including CSF-l, G-CSF, and GM-CSF.
The method of making a mineralized collagen-polysaccharide matrix of the
present invention comprises the steps of oxidizing an exogenous polysaccharide
to form a
modified exogenous polysaccharide having aldehyde groups, and reacting the
modified
exogenous polysaccharide with mineralized collagen under conditions such that
the
aldehyde groups covalently react with mineralized collagen to form a
crosslinked matrix.
The method may further comprise the step of adding a growth factor to the
matrix. A
growth factor can be added before or after the step of reacting the modified
polysaccharide with the mineralized collagen. The mineralized collagen is
prepared from
dispersed or solubilized collagen according to the method of U.S. Patent
5,231,169.
The present invention provides methods of using. a crosslinked mineralized
collagen-polysaccharide matrix to conduct the growth of tissue by
administering the
matrix at the sites of desired tissue repair. The matrix in combination with a
growth
factor may be administered by implantation or injection to induce the growth
of tissue at
sites of desired repair. A matrix further comprising fibrin may be
administered to anchor
the matrix into desired sites, such as, tissue defect sites.
The prepared matrices may be implanted at a site of desired tissue growth. The
polysaccharide and mineralized collagen starting materials may also be
separately
injected into the site of desired tissue growth, along with any growth factor,
and the like.
Upin mixing at the site, the matrix will form ih situ, conforming to the shape
of the site.
As used in this discussion, "repair" is defined as growth of new tissue. The
new
tissue may or may not be phenotypically or genotypically identical to the
original lost
tissue. As used herein, "regeneration of tissue" means that the new tissue
grown is
identical to the lost tissue. Tissue repair can also be the result of
replacing lost tissue
with non-identical tissues, e.g., for example, the replacement of hyaline
articular cartilage
-4-
CA 02427047 2003-04-25
WO 02/36147 PCT/USO1/42477
with fibrocartilage in a joint defect. The basic cellular properties involved
in repair
include adhesion, proliferation, migration and differentiation.
By "conduction", it is meant that the host tissue, e.g.,bone, cartilage or
soft tissue
grows by extension of existing tissue onto or into the crosslinked collagen-
polysaccharide
matrix. In conduction, repair cells move onto and into the matrix to
synthesize and
remodel new tissue identical to the surrounding host tissue. By induction, it
is meant that
the growth and differentiation of progenitor repair cells is stimulated. These
progenitor
cells go on to synthesize and remodel new tissue to be continuous with the
surrounding
host tissue.
As used herein, a tissue defect can be the result of a congenital condition,
trauma,
surgery, cancer or other disease.
As used in this discussion, an exogenous polysaccharide refers to a free
polysaccharide.
The ratios of the mineralized collagen to polysaccharide can be varied to
change
both the physical and biological properties of the matrix. A higher proportion
of
mineralized collagen will result in a more porous sponge-like matrix. A higher
proportion of polysaccharide will result in a more gel-like matrix.
BRIEF DESCRIPTION OF THE DRAWING
The Figure is a graph of the comparative release rates of GDF-5 from three
matrices as described in Example 9.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The method of preparing a matrix of the present invention comprises the steps
of
opening sugar rings on an exogenous polysaccharide and oxidizing terminal
hydroxyl
groups to aldehydes using, for example, sodium or potassium periodate as a
selective
oxidizing agent. The amount of aldehyde groups produced in this manner can be
-5-
CA 02427047 2003-04-25
WO 02/36147 PCT/USO1/42477
stoichiometrically controlled. Typically, from about 1% to 50% of the rings
can be
opened in this manner. More preferably about 1% to 5% of the rings are opened
to form
the aldehyde groups. These aldehyde groups can form covalent crosslinks with
the
collagen at amine sites on the collagen peptide chains. Since the aldehyde
groups are
formed in situ without the addition of a separate cross-linking compound, the
intermolecular distance between the backbone of the polysaccharide chain and
the
collagen fibrils which is crosslinked to it is believed to be less than the
corresponding
distance using a crosslinking compound. Accordingly, the polysaccharide and
collagen
backbones are relatively closely bound, which produces an advantageous
structure for the
purpose of providing a matrix that supports, conducts or induces the growth of
bone,
cartilage or soft connective tissue.
The starting material for producing the collagen may be purified, native
collagen,
modified or recombinant collagen of any type. A preferred collagen for bone
growth is
Type I collagen, whereas a preferred collagen for cartilage growth is Type II
collagen.
The collagen may be crosslinked or non-cross-linked, but it is preferred that
the collagen
be non-crosslinked to provide more accessibility to side groups for
crosslinking to the
polysaccharide aldehyde groups. If Type I collagen is used for tissue repair
where it is
desired to mask the inherent cell adhesion sites, such as cartilage repair,
the adhesion
sites can be masked by the use of non cell-adhesive polysaccharides to support
the
increased cell-to-cell interaction and adhesion.
The collagen to be mineralized will normally be dispersed or solubilized
collagen
where solubilization is achieved by dispersing the collagen source in a medium
at an
elevated pH, using at least about pH 8, more usually about pH 11-12, and
generally less
than about 1 N. Commonly, sodium hydroxide is employed, although other
hydroxides
may find use, such as other alkali metal hydroxides or ammonium hydroxide.
The concentration of collagen will generally be in the range of about 1 to 10
weight percent, more usually from about 1 to 5 weight percent. The collagen
medium
-6-
CA 02427047 2003-04-25
WO 02/36147 PCT/USO1/42477
will generally be at a concentration of the base in the range of about 0.0001
to 0.1 N. The
pH is generally maintained during the course of the reaction in the range of
about 10-13,
preferably about 12.
The phosphate and calcium are added as solutions, generally at a concentration
in
the range of about 0. 010.2 M, preferably about 0.025-0.075 M. The volume of
the
solutions added to the collagen medium will generally increase the collagen
medium
volume by at least 10 percent, usually at least 25 percent and not more than
about 400
percent, generally in the range of about 50 to 150 percent. Thus, the collagen
solution
will generally not be diluted by more than four-fold.
The rate of addition is relatively slow, generally requiring at least about
one hour
and not more than about 72 hours, generally being in the range of about 2 to
18 hours,
more usually in the range of about 4 to 16 hours. For example, with one liter
of a
collagen dispersion, where about a total of about one liter of reagents is
added, the rate of
addition will generally be in the range of 50 to 150 ml per hour.
The addition of the reagents can be provided in a stoichiomefiric ratio,
although
stoichiometry is not required, variations from stoichiometry of up to about 50
percent,
preferably not more than about 25 percent are preferred. Thus, where the
stoichiometry
of addition is not maintained, one of the components may be exhausted, while
addition of
the other components continue.
During the course of the reaction, mild agitation is maintained, so as to
ensure
substantially uniform mixing of the collagen fibrils and the calcium phosphate
mineral.
Mild temperatures are employed, usually not less than about 4°C and not
more than
about 40 °C preferably in the range of about 15 °C to
30°C. The weight ratio of the
collagen to calcium phosphate mineral will generally be in the range of about
8:2 to 1:l,
more usually about 7:3.
After completion of the addition, agitation, e.g., stirring, will normally be
continued, usually at least about 1 h, more usually about 2 h and agitation
may
_7_
CA 02427047 2003-04-25
WO 02/36147 PCT/USO1/42477
continue even more. The amount of continued agitation is not critical to the
preparation
of the product.
Upon completion of the reaction, the mineralized collagen may be treated in a
variety of ways. The product may be washed repeatedly to remove any unbound
minerals or other components of the medium, as well as provide a more neutral
pH.
Washing may be readily accomplished with water, saline, or the like.
The mineralized collagen may be further treated in a variety of ways. The
subject
compositions may be cross-linked using a variety of cross-linking agents, such
as
formaldehyde, glutaraldehyde, chromium salts, di-isocyanates or the like.
It is a feature of the invention that the polyaldehyde polysaccharide and
mineralized collagen are separately injectable materials which can react when
contacted
to form the matrix ivc situ at the site of desired tissue growth. The
advantages are that no
operative implantation procedures are necessary and the flowable starting
materials
conform to the shape of the site before the reaction is complete. The result
is a matrix
that conforms in shape to the site without the need for cutting and shaping of
a pre-
formed solid matrix.
The type of polysaccharides which may be utilized include hyaluronic acid,
chondroitin sulfate, dermatan, dextran sulfate, alginate, and other long chain
polysaccharides. Typically, the polysaccharide will have an average molecular
weight of
about 1,000 to 10,000,000 DA.
The reagents for opening sugar rings on the exogenous polysaccharide may be
any selective oxidizing agent which oxidizes a terminal hydroxyl group to an
aldehyde,
such as potassium or sodium periodate. Other reagents include specific sugar
oxidases.
The preferred polysaccharide is hyaluronic acid. The relative proportion of
polysaccharide to mineralized collagen will impart various physical and
biological
characteristics to the matrix. The proportion of polysaccharide to mineralized
collagen
may be characterized on a molar ratio basis or on a weight ratio basis.
Typically, the
_g_
CA 02427047 2003-04-25
WO 02/36147 PCT/USO1/42477
ratio by weight of mineralized collagen to polysaccharide is from 99:1 to
about 1:99.
This represents an approximate molar ratio of 99.9:0.1 to 1:9, respectively,
assuming an
average molecular weight of 1,000,000 daltons for hyaluronic acid and 100,000
daltons
for the collagen (based on non-mineralized weight). The molar ratio may vary
depending
on the actual molecular weight of the polysaccharide and collagen used. In a
preferred
embodiment disclosed herein, the ratio by weight of collagen to polysaccharide
is from
9:1 to about 1:9.
The ratios of the mineralized collagen to polysaccharide can be varied to
change
both the physical and biological properties of the matrix. Biologically, a
higher
proportion of Type I collagen will more closely mimic the composition and
architecture
of bone, whereas a higher proportion of Type II collagen will more closely
mimic the
composition of cartilage. Bone forming cells will interact with specific cell
adhesion
sites on collagen and will divide, migrate and differentiate to form new bone.
Alternatively, increasing the proportion of polysaccharide, preferably
hyaluronic
acid, will more closely mimic a natural cartilage matrix. In addition, a
higher proportion
of polysaccharide will mask some specific cell adhesive sites on collagen and
will favor
other cell-cell interactions and aggregation important in the development of
cartilage
tissue.
Growth factors which can be used with a matrix of the present invention
include,
but are not limited to, members of the TGF-13 superfamily, including TGF-131,2
and 3, the
bone morphogenetic proteins (BMP's), the growth differentiation
factors(GDF's), and
ADMP-1; members of the fibroblast growth factor family, including acidic and
basic
fibroblast growth factor (FGF-1 and -2); members of the hedgehog family of
proteins,
including Indian, sonic and desert hedgehog; members of the insulin- like
growth factor
(IGF) family, including IGF-I and -II; members of the platelet-derived growth
factor
(PDGF) family, including PDGF-AB, PDGF-BB and PDGF-AA; members of the
interleukin (IL) family, including IL-1 thru -6; and members of the colony-
stimulating
-9-
CA 02427047 2003-04-25
WO 02/36147 PCT/USO1/42477
factor (CSF) family, including CSF-1, G-CSF, and GM-CSF. Growth factor
preparations
are obtained either commercially or isolated and purified from tissue or from
recombinant
sources. Growth factors can be loaded into the collagen/HAlfibrin matrices
across a wide
dose range (fentogram to millgram range). Factors such as cost, safety and the
desired
growth factor release profile will dictate the amount of growth factor that is
loaded onto
the matrix.
Thrombin acts as a catalyst for fibrinogen to provide fibrin. In the present
invention, the fibrinogen and thrombin are added individually to a reaction
mixture
containing oxidized exogenous polysaccharide and mineralized collagen. In this
embodiment, it is desired to keep the fibrinogen and thrombin separated and
the oxidized,
exogenous polysaccharide and mineralized collagen separated until final
reaction is
desired.
The concentration of fibrinogen used in forming the matrix is preferably 10
mg/ml or greater. The thrombin is added to the fibrinogen in a concentration
of from
about O.O1 NIH units to about l00 NIH units/ml and preferably from about 0.1 -
2.0 NIH
unitslml. The thrombin is commercially available from a variety of sources.
Fibrinogen
may be derived from autologous patient plasma or from commercial sources. The
matrices according to the present invention may be formed into any shape by
lyophilization, or wet-laying and air drying in molds of the desired shape.
The wet-laid
material having a high proportion polysaccharides may also be formed into
viscous gels
for injection or direct application into a fracture or joint. As described
above, the starting
materials are also injectable and may be separately injected to mix at the
site of desired
tissue growth to react without formation of undesired side products.
The usefulness of the matrices according to the present invention can be shown
by
both ih vitro and i~ vivo tests. For the i~ vitro tests, primary fetal rat
calvarial cells,
harvested by a series of collagenase digestions, according to the method of
Wong and
Cohn (PNAS USA 72:3167-3171, 1975), or primary rat epiphyseal cartilage
Thyberg and
-10-
CA 02427047 2003-04-25
WO 02/36147 PCT/USO1/42477
Moskalewski, (Cell Tissue Res. 204:77-94, 1979) or rabbit articular
chondrocytes,
harvested by the method of Blein-Sella O. et al., (Methods Mol. Biol., 43:169-
175,1995),
are seeded into the matrices and cultured under conventional conditions for 1-
4 weeks.
Cultures are then processed and evaluated histologically.
A particular advantage of the polysaccharide mineralized collagen matrices
according to the invention is that they have comparable growth factor binding
ability to
cross-linked mineralized collagen but much better osteoconductivity.
Furthermore, while
the polysaccharide-mineralized collagen matrices have comparable
osteoconductivity to
polysaccharide-nonmineralized collagen, they have slower growth factor release
kinetics,
which is an advantage for growth factor retention within the matrix.
The chondroconductive capability of the matrices of the present invention can
be
determined by successful support of adhesion, migration, proliferation and
differentiation
of primary rat bone marrow and stromal cells as well as retinoic acid-treated
primary rat
or rabbit chondrocytes or human mesenchyme stem cells. Bone marrow and bone
marrow stromal cells closely approximate the early chondroprogenitor cells
found in the
subchondral bone marrow of full-thickness defects. Bone marrow are harvested
from the
long bones of 2-3 week-old inbred Lewis rats and added directly to a matrix
and cultured
for 2 weeks under standard conditions. The adherent stromal cell population
that grows
out of these cultures are passaged and frozen for use. Cells from up to six
passages are
used for culturing or seeding on the matrix.
Retinoic acid-treated chondrocytes represent the latter stages of
chondrogenesis.
Retinoic acid treatment of primary is performed prior to culturing or seeding
the cells on
a candidate matrix (Dietz, U. et al., 1993, J. Cell Biol. 52(1):57-68).
In an alternative method, in vitro studies of the early and late stage
chondrocytes
are merged to allow stromal cells to condition the matrices and then to
replace them with
more mature chondrocytes. In this way, evolution of the matrices during the
early phases
of chondrogenesis may be tested for effects on the later stages of the
process.
-11-
CA 02427047 2003-04-25
WO 02/36147 PCT/USO1/42477
Cell adhesion and proliferation on the matrix are monitored using an MTS assay
that can measure cell number and viability based on mitochondrial activity.
Stromal cells
or chondrocytes are cultured on matrices for 6-18 hrs. in the presence or
absence of
serum for adhesion analysis and for 1-2 weeks for proliferation assessment.
For cell migration testing, matrices are coated or fitted onto porous Trans-
well
membrane culture inserts (Corning). Stromal cells are seeded on top of the
matrices in
the upper chamber of the Trans-well and a chemoattractant (growth factor,
PDGF) placed
in the bottom chamber. After 12-18 hrs of culture the cells that have migrated
through
the matrix to the bottom side of the Trans-well membrane are quantitated by
the MTS
assay. Matrices are removed from the upper chamber and processed
histologically to
assess degree of infiltration.
The analysis of differentiation markers relevant to chondrogenesis and
osteogenesis are evaluated at both the protein and transcriptional level. The
specific
markers that may be analyzed include: 1) Type II collagen and IIA, IIB
isoforms; 2)
Aggrecan proteoglycan; 3) Type IX, X and XI collagen; 4) Type I collagen; 5)
Cartilage
matrix protein (CMP); 6) Cart-1 transcription factor; 7) Fibronectin (EDA, EDB
isoforms); 8) Decorin proteoglycan; 9) Link protein; 10) NG-2 proteoglycan;
11)
Biglycan proteoglycan; 12) Alkaline phosphatase. Differentiation may be
measured by
Northern/PCR analysis, Western blotting or by metabolic cell labeling.
For Northern/PCR analysis, RNA are isolated by standard procedures from
stromal cells or chondrocytes that have been cultured on composite matrices.
Time
course tests may be used to determine optimal culture periods that range from
1 to 6
weeks depending on the cell type. The isolated RNA is analyzed by Northern gel
and
hybridization techniques with specific cDNA or PCR amplified probes. Northern
analysis is quantified by densitometric scanning of autoradiographs and
normalization to
housekeeping gene signals (G3PDI~. Northern analysis may be supplemented with
-12-
CA 02427047 2003-04-25
WO 02/36147 PCT/USO1/42477
quantitative PCR analysis using primers generated from the published cDNA
sequences
of the genes to be analyzed.
For Western blotting, solubilized protein lysates are isolated from cells
cultured
on composite matrices by standard techniques (Spiro R.C., et al., 1991, J.
Cell. Biol.,
115:1463-1473). After the lysis of cells the matrices are extracted in
stronger denaturants
(8 M urea, GnHCL) to remove and examine matrix-bound or incorporated proteins.
Protein samples are analyzed by standard Western blotting techniques using
specific
polyclonal or monoclonal antibodies.
For metabolic cell labeling, cells cultured on a composite matrix are
metabolically
radiolabeled with 3sSO4, ssS_methionine or 3H/~4C-labeled amino acids by
standard
techniques (Spiro et al., su ra . Solubilized cellular and matrix-associated
proteins are
quantitatively immunoprecipitated with antibodies specific for the protein of
interest and
analyzed by SDS-PAGE (Spiro et al., su ra . Quantitation of results are
performed by
densitometric scanning of autoradiographs and signals will be normalized to
either cell
equivalents or to a house-keeping protein such as actin.
Additionally, the ability of a matrix of the present invention to support
chondrogeneic differentiation i~ vivo may be tested in an inbred rat soft
tissue implant
model. Rat bone marrow or stromal cells described above are seeded onto
matrices at
high density, cultured overnight in MEM medium containing 10% FBS serum and
antibiotics, then transferred into Millipore diffusion chambers and implanted
intraperitoneally or subcutaneously into 8 week-old recipients. Chambers are
harvested
after 3 weeks and evaluated histologically for cartilage formation.
A transplantation model in outbred rats is used to evaluate the ability of the
composite matrices to maintain the cartilage phenotype in vivo. Rib costal
cartilage
chondrocytes are seeded onto matrices at high density and cultured overnight
in Ham's F-
12 containing 1 % rat serum and antibiotics. The seeded matrices are then
implanted into
posterior tibial muscle pouches created by blunt dissection in 8 week-old male
Sprague-
-13-
CA 02427047 2003-04-25
WO 02/36147 PCT/USO1/42477
Dawley rats. Explants are taken at 14 and 28 days and evaluated histologically
for matrix
compatibility, cartilage growth, and maintenance of the differentiated
phenotype based on
staining for aggrecan and type II collagen.
In addition, the ability of a matrix of the present invention to interact with
extracellular matrix proteins (proteoglycans, proteins and growth factors)
found in the
surrounding serum, tissue fluid, or in the secretion products of
chondroprogenitor cells
correlate with the chondroconductive potential of a matrix. The interaction of
the
matrices of the present invention with extracellular matrix proteins may be
measured by
means known to those of skill in the art such as, Western blotting, affinity
co-
electrophoresis techniques and binding characteristics.
To assay serum protein binding to a matrix of the present invention, the
matrix is
incubated in culture media containing increasing amounts of serum (various
species and
sources). After washing, bound proteins are eluted by boiling in SDS-PAGE
sample
buffer and unsolubilized matrix will be removed by centrifugation. SDS-PAGE
analysis
is used to initially document the binding pattern of the matrices. Western
blotting is then
performed to identify specifically bound components such as fibronectin and
vitronectin.
Affinity coelectrophoresis is used to analyze proteoglycan binding to a matrix
of
the present invention. 35504-labeled or iodinated proteoglycan (aggrecan)
isolated from
bovine and rat (or other sources) is loaded into ACE gels (Lee, M.K. et al.,
1991,
88:2768-2772) containing composite matrices or collagen scaffolds alone. The
binding
affinity of aggrecan for collagen scaffolds plus and minus hyaluronic acid or
dextran
sulfate are taken as a measure of the ability of composite matrices to
organize a cartilage
matrix.
An evaluation of protein interactions with mineralized collagen-based
composite
matrices can potentially be hindered by the large excess of collagen protein.
The
mineralized collagen scaffolds have enough inherent structural integrity and
are
crosslinked to an extent that will prevent their complete solubilization, but
some collagen
-14-
CA 02427047 2003-04-25
WO 02/36147 PCT/USO1/42477
protein may become solubilized in the SDS-PAGE sample buffer. Thus, this could
obscure the visualization of other bound proteins, particularly the cell-
synthesized
collagens, and may also cause high background in Western blot analysis.
Therefore, an
alternative approach is to use radiolabeled or biotinylated proteins for the
binding
analysis. Serum proteins may be biotinylated prior to incubation with the
composite
matrices and then developed with avidin-based reagents. Both approaches allow
the
visualization of matrix-associated components without the interference of the
scaffold
collagen protein.
Alternatively, the shift in expression from Type I to Type II collagen and the
splicing of the Type II collagen transcript from the Type IIA to the Type IIB
isoform
(Sandell, L.J. et al., 1991, J. Cell Biol. 114:1307-1319) are measured by
means known to
those of skill in the art to determine differentiation down a chondrogenic
pathway. Also,
the expression of the cartilage-associated proteoglycan, aggrecan (Schmid,
T.M., et al.,
1985, J. Cell Biol. 100:598-605 and Kuettner K.E. 1992, Clin. Biochem. 25:155-
163) and
a cartilage homeoprotein transcription factor (Cart-1) appear to be markers
for cells
committed to the chrondrocytic lineage.
For the in vivo tests, the matrices are evaluated for the capabilities for
supporting
osseous healing in a rat cranial defect model by implantation into a 5 mm by 3
mm defect
created in the parietal bone of 6 weeks old male Sprague-Dawley rats. The
defects are
evaluated at 28 days by radiographic and histologic analysis.
The in vivo model for cartilage repair is a full-thickness articular cartilage
defect
in the rabbit (Amiel et al., 1985, J. Bone Joint Surg. 67A:911). Defects
measuring
approximately 3.7 mm in diameter and 5 mm deep defect are created in the
center of the
medial femoral condyles of adult male New Zealand white rabbits. The defects
are then
either filled with matrix or left unfilled as controls. The defects are
evaluated
morphologically and histologically at 6 and 12 weeks.
-15-
CA 02427047 2003-04-25
WO 02/36147 PCT/USO1/42477
The matrices of the present invention may be used for the treatment of bone
and/or cartilage defects associated with surgical resection, such as spinal
fusions; trauma;
disease; infection; cancer or genetic defects. The matrices according to the
present
invention may be administered through implantation, direct application or
injection
depending on the intended application of the matrix, the physical properties
of the matrix
and the ratio by weight of mineralized collagen to polysaccharide in the
matrix.
In one aspect of the present invention, the matrix is provided having a higher
proportion of mineralized collagen compared to polysaccharide, is in a sponge-
like form
and is surgically implanted at a site where growth of new bone tissue is
desired, such as
in spinal fusions. In one aspect, the matrix further comprises a growth
factor, such as
BMP-2. In another aspect, the matrix further comprises fibrin to facilitate
anchoring of
the matrix into the desired site. In another aspect of the invention the
starting
polyaldehyde polysaccharide and mineralized collagen are separately injected
into the
site of the desired tissue growth along with any desired growth factors. The
materials
react i~ situ to form the matrix at the desired site.
In another aspect of the present invention, the matrix has a higher proportion
of
polysaccharide compared to mineralized collagen, is formed into a viscous gel
and is
either directly applied or injected into a site where growth of new bone
tissue is desired,
such as in filling bone defects, fracture repair and grafting periodontal
defects. In yet
another aspect of the present invention, the matrix is provided with a higher
proportion of
polysaccharide, is formed into a viscous gel and is injected directly or
delivered through
an arthoscopic procedure into a site where growth of cartilage tissue is
desired, such as in
injury induced cartilage damage or disease-induced cartilage damage such as
in,
osteoarthritis or rheumatoid arthritis.
As will be understood by those of skill in the art, the amount of matrix to be
administered to conduct growth of bone or cartilage tissue depends upon the
extent of the
bone or cartilage defect to be treated. As will also be understood by those of
skill in the
-16-
CA 02427047 2003-04-25
WO 02/36147 PCT/USO1/42477
art, the cost, safety, and desired growth factor release profile will dictate
the type and
amount of growth factor that is loaded onto the matrix.
The following examples are provided for purposes of illustration and are not
intended to limit the invention in any way.
Example 1
Implantable Matrices of Amine-linked Mineralized Collagen and Polysaccharides
The raw materials, mineralized Type I collagen and polysaccharide-
polyaldehyde,
were prepared by the methods disclosed in U.S. Patent No. 5,231,169 and U.S.
Patent No.
5,866,165, respectively. Mineralized sewed F collagen (63 mg/ml) was mixed
with a
hyaluronate-polyaldehayde solution (7 mg/ml, 5% repeating units were oxidized;
pH 7.5-
9.0) at the equal volume ratio in the container of a heavy duty blender.
Sodium
cyanoborohydride (NaCNBH3 5.0 M in 1.0 M NaOH) was added to the mixture to the
final concentration of 10 mM. The mixture was then blended 3 times at low
speed for 10
seconds. The reaction was continued carrying on by pouring the slurry into a
heavy-wall
bottle incorporated with a tight-fitting polypropylene screw cap. The bottle
was rotated
at the speed of 100 rotes/min. at ambient temperature in dark for 24 hr. The
slurry was
then poured into a mold and lyophilized. This formed a matrix, which was
washed with
D.I. water to removed NaCNBH3 and re-lyophilized. This procedure was followed
to
make a series of matrices from mCOL with other oxidized polysaccharides. The
surface
property, structures and biological activity of the matrices were controlled
by altering the
ratio of mCOL to the polysaccharides, the type of polysaccharides, the density
of
aldehyde groups generated on the polysaccharides, the density of matrix, as
well as the
process of lyophilization.
Example 2
Implantable Matrices of Imine-linked Mineralized Collagen and Polysaccharides
-17-
CA 02427047 2003-04-25
WO 02/36147 PCT/USO1/42477
The matrices were prepared by the procedure as described in Example 1, except
that no NaCNBH3 was used.
Example 3
Injectable Gel-Matrices with Fibrin
The gel-matrices were prepared using a FibrinJet~'"~ surgical sealant delivery
system. The typical procedure in detail is described in following:
The mCOL fibers with the diameter of about 100 pm and fibrinogen (mCOL, 42
mg/ml; fibrinogen, 21 mg/ml; pH 7.5) were loaded in one syringe, an equal
volume of
activated hyaluronate solution (7 mg/ml, pH 7.5) containing thrombin (1-5
U/ml) was
placed in another syringe. Both of the syringes were mounted onto the
FibrinJet~
surgical sealant delivery system connected with a 18 G needle. Gel-matrix was
formed in
less than three minutes at the exit site of the needle upon pushing the two
parts of
composition to flow through the syringes simultaneously.
The above procedure was followed to make a series of injectable gel-matrices
from mCOL, fibrinogen, and other oxidized polysaccharides. The surface
property, the
porous structures and biological activity of the gel-matrices were controlled
by altering
the ratio of mCOL and polysaccharides to fibrinogen, the type of
polysaccharides, the
density of aldehyde groups, the density of the final matrix and the
concentration of
thrombin. The gelation time and the gel hardness were controlled by amount of
thrombin
added.
Example 4
Injectable Gel-Matrices with Plasma
The gel-matrices were prepared by the method described in Example 3, except
that blood plasma was used instead of fibrinogen. The blood plasma was
prepared from
citrate (10 w%) added whole blood, which was centrifuged at 3,000 rpm for 15
minutes.
-18-
CA 02427047 2003-04-25
WO 02/36147 PCT/USO1/42477
Example 5
Injectable Gel-Matrices with Plasma
The gel-matrices were prepared by the method described in Example 4, except
that 50 mM calcium chloride solution was used instead of thrombin.
Example 6
Injectable Gel-Matrices with Whole Blood
The gel-matrices were prepared by the method described in Example 3, except
that whole blood was used instead of blood plasma.
Example 7
Implantable Matrices with Growth Factors
The typical procedure to load growth factors onto the matrices is described as
follows:
Matrices prepared from mCOL and active polysaccharides as prepared in
Examples l and 2 were used. Pre-dried matrices were cut to cubes with the size
of 5 x 4
x 2 mm. The water uptake of the cubes was measured and found to be 85 ~ 5 g,
per piece,
Growth and differentiation factor-5 (GDF-5, 0.588 mg/ml, 20 mM acetic acid)
was drop-
wise added to the matrix specimens at 85 g,1 for each piece. The GDF-5 loaded
matrix
specimens were allowed to stand at ambient temperature in hood for 5 minutes,
then
froze at -78°C, and lyophilized.
The above procedure was followed to load a series of growth factors with
various
concentrations, such as bone morphogenic proteins, transferin growth factor-
(3, and
insulin-like growth factors to matrices prepared from mCOL with other oxidized
polysaccharides.
-19-
CA 02427047 2003-04-25
WO 02/36147 PCT/USO1/42477
The above procedure was followed to load DNA, hormones, and cytokines to
matrices prepared from mCOL with other oxidized polysaccharides.
Example 8
Injectable Gel-Matrices with Growth Factors
The typical procedure to load growth factors into injectable gel-matrices is
described as follows:
mCOL fibers (D.I. < 100 ~,m, 42 mg/ml), fibrinogen (21 mg/ml), and GDF-5 (100
~,g/ml) were loaded in one syringe, an equal volume of activated hyaluronate
solution (7
mg/ml) containing thrombin (1-5 U/ml) was placed in anther syringe. Both of
the
syringes were mounted onto a FibriJelTM surgical sealant delivery system
connected with
a 18 G needle. Gel-matrix containing GDF-5 was formed in less than three
minutes at
the exit site of the needle upon pushing the two parts of composition to
simultaneously
flow through the syringes.
The above procedure was followed to make a series of injectable gel-matrices
containing other growth factors, hormone, and cytokines.
Example 9
Comparative Sustained Release of GDF-5 from Matrices
Implantable mCOL/HA matrices (5 x 4 x 2 mm) with pre-loaded GDF-5 at the
ratio of 50 ~,g/piece were prepared as described in Example 7, using radio-
lebeled GDF-5
as a tracer. Glutaraldehyde cross-linked mCOL and hyaluronate polyaldehyde
cross-
linked COL (COL/HA) were also loaded with GDF-5 at the same ratio and served
as
controls. The release kinetics of GDF-5 from these matrices was investigated.
One piece
of total five pieces of each type of matrix was placed into a 2.0 ml
polypropylene tubes
containing 5.0 ml PBS, pH 4.0 adjusted by 30 mM acetic acid. The tubes were
shaked
gently at 37°C. The medium was refreshed at a designated time and the
radioactivity in
-20-
CA 02427047 2003-04-25
WO 02/36147 PCT/USO1/42477
each replaced medium was detected by scintillation counter. As shown in the
Figure, the
release of GDF-5 from both mCOL/HA and glutaraldehyde linked matrices showed a
longer duration than that from COL/HA matrix. Since both mCOL/HA and
glutaxaldehyde-linked matrices contained 90% of mCOL, while the COL/HA matrix
contained the same amount of non-mineralized COL, the results indicated that
GDF-5
was bound to mCOL fiber more effectively than bound to COL. Consequently,
mCOL/HA matrix is considered superior to COL/HA matrix for GDF-5 sustained
delivery.
The above procedure was followed to determine the kinetics of a series of
growth
factors, hormone, and cytokines released from mCOL/HA matrices.
Example 10
Osteoconductivity of Implanted Matrices
Matrices comprising 9 parts of mCOL and 1 part of HA polyaldehyde (5% repeat
units oxidized) were prepared as described in Example 2. Specimens of the
matrix with
the size of 5 mm x 3 mm x 2 mm were sterilized with ethanol and implanted into
the
defects created in parietal bone of 6 week old male Spregue-Dawley rats. The
defects
were evaluated at 28 days by radiographic and histologic analysis, and the
results
summarized in Table 1. Defects without implantation of matrix showed only a
little
radiographic reduction (20 ~ 2%), defects filled with glutaraldehyde cross-
linked mCOL
showed a higher reduction (55 ~ 3%), defects filled with mCOL/HA showed a
fiu~ther
increase in radiopacity (88 ~ 3%), which was similar to those of filled with
COL/HA
matrix (93 ~ 4%). The histolofic evaluation correlated with radiographic
results. Defects
filled with either mCOL/HA or COL/HA showed a higher bone formation score than
those filled with mCOL (Table 1). Since both the mCOLIHA and COLlHA matrices
contain 10% of HA, which made them different from mCOL prepared from 100%
-21-
CA 02427047 2003-04-25
WO 02/36147 PCT/USO1/42477
mCOL, these results demonstrated that the introduction of 10% HA to collagen-
based
matrices enhanced their osteoconductivity.
Table 1 Radiographic and histological scores of rat cranial defects
treated with mCOL/HA, mCOL, and COL/HA matrices
Implant % Radiographic Reduction in Histological bone score* (mean
defect Size (mean ~ SD) ~ SD)
Untreated 20 ~ 2 1.2 ~ 0.5
mCOL/HA 88 ~ 3 5
mCOL 55 ~ 3 2.7 ~ 0.5
COL/HA 93 ~ 4 5
*Healing of the defects was scored in the scale of 1-5, based on the width of
the defect bridged with reparative bone: 1= 0 to <20%; 2 = >20 <40%; 3 =
>40 <60%; 4 = >60 <80%; 5 = >80%.
Example 11
In Vitro Growth of Cells and Expression of Bone Phenotype
This example illustrates that the mCOL/HA matrix supports the growth of fetal
rat
calvarial cells (FRCS) and demonstrates the expression of bone phenotype in
vitro. FRCS
were prepared from a 19 day old fetus and expanded, seeded into the matrix
made by the
method described in Example 2 comprising 90% mCOL and 10% HA (5% repeat units
were oxidized) and cultured under standard conditions for 4 weeks. Cultures
were then
evaluated for cell growth and the express of alkaline phosphatase activity
(ALP). Results
showed that RFCs seeded on the matrix grew continually, and the cell number
was
increased by 9 fold at day 28, compared to day 1. The expression of ALP, a
marker for
bone formation, also increased with time and reached the highest value at day
21,
indicating the utility for bone formation of the mCOL/HA matrix to guide the
seed FRC
differentiation.
-22-
