Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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DEVICE AND METHOD FOR REGENERATION AND REPAIR
OF CARTILAGE LESIONS
FIELD OF THE INVENTION
S The present invention relates to a cartilage regeneration and repair product
that
induces cell ingrowth into a bioresorbable material and cell differentiation
into cartilage
tissue, and to methods of using such a product to repair cartilage lesions.
BACKGROUND OF THE INVENTION
Articular cartilage, an avascular tissue found at the ends of articulating
bones, has
limited natural capacity to heal. During normal cartilage ontogeny,
mesenchymal stem
cells condense to form areas of high density and proceed through a series of
developmental stages that ends in the mature chondrocyte. The final hyaline
cartilage
tissue contains only chondrocytes that are surrounded by a matrix composed of
type II
collagen, sulfated proteoglycans, and additional proteins. The matrix is
heterogenous in
structure and consists of three morphologically distinct zones: superficial,
intermediate,
and deep. Zones differ among collagen and proteoglycan distribution,
calcification,
orientation of collagen fibrils, and the positioning and alignment of
chondrocytes (Archer
et al., 1996, J. Anat. 189(1):23-35; Morrison et al.,1996, J. Anat. 189(1): 9-
22; and Mow
et al.,1992, Biomaterials 13(2): 67-97). These properties provide the unique
mechanical
and physical parameters to hyaline cartilage tissue.
The meniscus, a C-shaped cartilaginous tissue, performs several functions in
the
knee including load transmission from the femur to the tibia, stabilization in
the anterior-
posterior position during flexion, and joint lubrication. Damage to the
meniscus results
in reduced knee stability and knee locking. Over 20 years ago, meniscectomies
were
performed which permitted immediate pain relief, but were subsequently found
to induce
the early onset of osteoarthritis (Fairbank, J. Bone Joint Surg. 34B: 664-670;
Allen et al.,
1984, J. Bone Joint Surg. 66B:666-671; and Roos et al., 1998, Arth. Rheum.
41:687-
693). More recently, partial meniscectomies and repair of meniscal tears have
been
performed (Fig. 9A-D; Jackson, D., ed., 1995, Reconstructive Knee Surgery
Master
Techniques in Orthopedic Surgery, ed. R. Thompson, Raven Press: New York).
However, partial resection results in the loss of functional meniscus tissue
and the early
onset of osteoarthritis (Lynch et al., 1983, Clin. Ortlrop. 172:148-153; Cox
et al., 1975,
Clin.Orthop. 109:178-183;King,1995,J.BoneJointSurg.77B:836-837). Additionally,
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repair of meniscal tears is limited to tears in the vascular 1/3 of the
meniscus; tears in the
semivascular to avascular 2/3 are not repairable (Fig. 9A-D; Jackson, ibid.).
Of the
approximately, 560,000 meniscal injuries that occur annually in the United
States, an
estimated 80% of tears are located in the avascular, irreparable zone.
Clearly, a method
S that both repairs "non-repairable" tears or that can induce regeneration of
resected menisci
would be valuable for painless musculoskeletal movement and prevention of the
early
onset of osteoarthritis in a large segment of the population.
The proximal, concave surface of the meniscus contacts the femoral condyle and
the distal, flat surface contacts the tibial plateaus. The outer one-third
ofthe meniscus is
highly vascularized and contains dense, enervated, connective tissue. In
contrast, the
remaining meniscus is semivascular or avascular, aneural tissue consisting of
fibrochondrocytes surrounded by abundant extracellular matrix (McDevitt et
al., Clin.
Orthop. Rel. Res. 252:8-17). Fibrochondrocytes are distinctive in both
appearance and
function compared to undifferentiated fibroblasts. Fibroblasts are elongated
cells
containing many cellular processes and produce predominantly type I collagen.
The
matrix produced by fibroblasts does not produce a sufficient mechanical load.
In contrast,
fibrochondrocytes produce type I and type II collagen and proteoglycans. These
matrix
components support compressive forces that are commonly exerted on the
meniscus
during musculoskeletal movement.
In the 1960's, demineralized bone matrix was observed to induce the formation
of
new cartilage and bone when implanted in ectopic sites (Urist, 1965, Science
150:893-
899). The components responsible for the osteoinductive activities were termed
Bone
Morphogenetic Proteins (BMP). At least seven individual BMP proteins were
subsequently identified from bone (BMP 1-7) and amino acid analysis revealed
that six of
the seven BMPs were related to each other and to other members of the TGF-~i
superfamily. During endochondral bone formation, TGF-~i family members direct
a
cascade of events that includes chemotaxis, differentiation of pluripotential
cells to the
cartilage lineage, maturation of chondrocytes to the hypertrophic stage,
mineralization of
cartilage, replacement of cartilage with bone cells, and the formation of a
calcified matrix
(Reddi, 1997, Cytokine & Grawth Factor Reviews 8:11-20). Although individual,
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recombinant BMPs can induce these events, the prevalence of multiple TGF-~i
family
members in bone tissue underlies the complexity involved in natural
osteogenesis.
Bone Protein (Sulzer Orthopedics Biologics, Wheatridge, CO), also referred to
herein as BP, is a naturally derived mixture of proteins isolated from
demineralized bovine
bones that has osteogenic activity in vitro and in vivo. In the rodent ectopic
model, BP
induces endochondral bone formation or bone formation through a cartilage
intermediate
(Damien, C. et al., 1990, J. Biomec~ Mater. Res. 24:639-654). BP in
combination with
calcium carbonate promotes bone formation in the body (Poser and Benedict, PCT
Publication No. W095/13767). In vitro, BP has been shown to promote
differentiation
to cartilage of murine embryonic mesenchymal stem cells (Atkinson et al.,
1996, In
"Molecular and Developmental Biology of Cartilage", Bethesda, MD, Annals New
York
Acad. Sci. 785:206-208; Atkinson et al., 1997,.1. Cell. Biochem. 65:325-339)
and ofadult
myoblast and dermal cells (Atkinson et al., 1998, 44th Annual Meeting,
Orthopaedic
Research Society, abstract). To ensure chondrogenesis in these in vitro
systems, however,
culture conditions must be tightly controlled throughout the culture period,
including by
controlling cellular organization within the culture, optimizing media
formulations, and
adding exogenous factors that must be carefully established to maximize
chondrogenesis
over mitogenesis. Such optimization of conditions makes the application of the
disclosed
in vitro methods to an in vivo system unrealistic and unpredictable. In
addition, although
in vitro cultures ofadult myoblast and dermal cells initially resulted in
chondrogenesis, the
effect was only transient and over time, the cultures reverted to their
original phenotype.
Although certain embryonic and precursor cell types showed prolonged
chrondrogenesis
in vitro in these studies, it would be unpredictable or even impossible in the
case of
embryonic cells that these specific cell types could be recruited to a site in
vivo in an adult
patient.
Atkinson et al., in PCT Application No. PCT/EP/05100, incorporated herein by
reference in its entirety, describe a delivery system for osteoinductive
and/or
chondroinductive mixture of naturally derived factors for the induction of
cartilage repair.
Hunziker (U.S. PatentNos. 5,368,858 and 5,206,023) describes a cartilage
repair
composition consisting of a biodegradable matrix, a proliferation and/or
chemotactic
agent, and a transforming factor. A two-stage approach is used where each
component
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has a specific function over time. First, a specific concentration of
proliferation/chemotactic agent fills the defect with repair cells. Second, a
larger
transforming factor concentration, preferably provided in conjunction with a
delivery
system, transforms repair cells to chondrocytes. The second stage delivery of
a high
concentration of transforming factor in a delivery system (i.e., liposomes)
was required
to obtain formation of hyaline cartilage tissue at the treatment site.
Chen and Jeffries (U.S. Patent No. 5,707,962) describe osteogenic compositions
consisting of collagen and sorbed factors to enhance osteogenesis.
Valee and King (LT. S. Patent No. 4,952,404) describe healing ofinjured,
avascular
meniscus tissue by release of the angiogenic factor, angiogenin, over at least
3 weeks.
Previously, Amoczky et al. described a method using an autogenous fibrin clot
to
repair an avascular, circular lesion in canine menisci (Amoczky et al., 1988,
J. Bone Joint
Surg. 70A:1209-1217). This approach enhanced repair of meniscal tissue
compared to
controls lacking the fibrin clot. However, the repair tissue was not meniscus-
like tissue,
but rather connective scar tissue.
Hashimoto et al. described a method using fibrin sealant with or without
endothelial cell growth factor in avascular, circular meniscal defects in the
canine model
(Hashimoto et al., 1992, Am. J. SportsMed. 20:537-541). The growth factor
added a
modest benefit compared to healing with fibrin sealant alone and this
additional effect was
not observed until three months after treatment, indicating an indirect
contribution of the
growth factor. In addition, the defect was filled with hyaline cartilage-like
cells, which are
not typically present in normal meniscus tissue.
Shirakura, et al. describe the use of an autogenous synovium graft sutured
into
meniscal tears. While the synovium did enhance healing in 1/3 of the animals,
the grafts
healed with fibrous tissue, not fibrocartilaginous tissue normally observed in
meniscus
tissue (Shirakura, 1997, Acta. Orthop. Scand. 68:51-54). Furthermore, 2/3 of
the grafts
did not heal.
The molecular mechanism for cartilage and bone formation has been partially
elucidated. Both bone morphogenetic proteins (BMP) and transforming growth
factor
~i (TGF(3) molecules bind to cell surface receptors (i.e., TGF~3BMP receptors)
to initiate
a cascade of signals to the nucleus that promotes proliferation,
diil'erentiation to cartilage,
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S
and/or differentiation to bone (Massague, 1996, Cell 85:947-950). In 1984,
Urist
described a substantially pure, but not recombinant, BMP combined with a
biodegradable
poly(lactic acid) polymer delivery system for bone repair (U.S. Patent No.
4,563,489).
This system blends together equal quantities ofBMP and poly(lactic acid) (PLA)
powder
(100 pg of each) and decreases the amount of BMP required to promote bone
repair.
Hattersley et al. (WO 96/39170) disclose a two factor composition for inducing
cartilaginous tissue formation using a cartilage formation-inducing protein
and a cartilage
maintenance-inducing protein. Specific recombinant cartilage inducing proteins
are
specified as BMP-13, MP-52 and BMP-12, and specific cartilage maintenance-
inducing
proteins are specified as BMP-9. In one embodiment, BMP-9 is encapsulated in a
resorbable polymer system and delivered to coincide with the presence of
cartilage
formation inducing protein(s).
Laurencin et al. (U.S. Patent No. 5,629,009) disclose a chondrogenesis-
inducing
device, consisting of a polyanhydride and polyorthoester, that delivers water
soluble
proteins derived from demineralized bone matrix, TGF~i, epidermal growth
factor (EGF),
fibroblast growth factor (FGF) or platelet-derived growth factor (PDGF).
Bentz et al. (PCT Publication No. WO 92/09697) have described the use of a
bone
morphogenetic protein (BMP) with a TGF~i protein for bone repair. The ratio of
BMP
to TGF(3 in the mixture is in the range of 10:1 to 1:10. The addition of TGF-
~i with either
BMP-2 or BMP-3 results in increased osteoinductive activity and an increased
ratio of
cartilage to bone when compared to either factor alone (Bentz et al., Matrix
11:269-275
(1991); Ogawa et al., J. Biol. Chem., 267(20):14233-7 (1992); W092/09697).
However,
this composition produced substantial bone in the rodent subcutaneous assay.
Bulpitt and Aeschlimann found that TGF(3-2 and BMP-2 led to accelerated bone
formation and decreased cartilage formation in the rat ectopic bone formation
assay
(Bulpitt et al., Tissue Engineering, pp. 162-169 (1999)).
Other studies demonstrate no or little effect of the combination of TGF(3-1 or
-2
with BMP. In vitro, the combination of TGF(3-1 and porcine BMP demonstrated no
synergistic effects on collagen production (Kim et al., Biochem. Mol. Biol.
Int'l,
33(2):253-261 (1994)). Similarly, Ballock et al., demonstrated no synergy
between
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TGF~i-1 and BMP-3 for collagen production in periosteum derived cells in vitro
(Ballock
et al., J .. Ortho. Res., 15:463-7 (1997)).
In the Rosen modified Sampath-Reddi rodent assay (Sampath et al., Proc. Nat'1
Acac~ Sci. USA, 80(21):6591-5 (1983)), BMP-2 containing implants showed
cartilage and
bone formation after ten days and mostly bone (no cartilage) after 21 days
(U.S. Patent
No. 5,658,882).
Previously, Li and Stone (IJ.S. Patent No. 5,681,353) have described a
Meniscal
Augmentation Device that consists of biocompatible and bioresorbable fibers
that acts as
a scaffold for the ingrowth ofmeniscal fibrochondrocytes, supports normal
meniscal loads,
and has an outer surface that approximates the natural meniscus contour. After
partial
resection of the meniscus to the vascular zone, this device is implanted into
the resulting
segmental defect. The results have been described in both canines and humans
(Stone et
al., 1992, Am. J. Sports Med. 20:104-111; and Stone et al., 1997, J. Bone
Joint Surg.
79:17701777).
The Meniscus Augmentation Device, the research reports and patent disclosures
described above, and current repair surgeries provide encouraging results in
the area of
cartilage repair, but are not satisfactory to induce repair of "non-
repairable" avascular
tears in which the repair tissue is meniscus tissue, and are not satisfactory
to produce short
patient rehabilitation times and regenerated meniscus tissue in the vascular
zone.
Furthermore, no reports have been described that demonstrate enhanced healing
rates of
"repairable" meniscal tears in vivo.
SUNINIARY OF THE INVENTION
The present invention relates to a product and method for repairing and/or
regenerating cartilage lesions. The product and method ofthe present invention
are useful
for repairing a variety of cartilage lesions, including articular and mensical
lesions,
including vascular, semivascular and avascular lesions. Moreover, the product
and
method of the present invention can be used to repair different sizes and
shapes of
cartilage lesions, including radial tears, bucket handle tears, and segmental
defects.
A first embodiment of the product of the present invention relates to a
product for
repair of cartilage lesions. Such a product includes: (a) a cartilage repair
matrix; and, (b)
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a cartilage-inducing composition associated with the matrix for provision of a
mixture of
proteins. In one embodiment of the product of the present invention, a
cartilage-inducing
composition includes a mixture ofproteins which includes: transforming growth
factor X31
(TGF~i 1), bone morphogenetic protein (BMP)-2, BMP-3, and BMP-7. The quantity
of
the TGF~i 1 in the mixture is from about 0.01% to about 99.99% of total
proteins in the
mixture; the quantity of the BMP-2 in the mixture is from about 0.01% to about
10% of
total proteins in the mixture; the quantity of the BMP-3 in the mixture is
from about 0.1
to about 15% of total proteins in the mixture; and, the quantity of the BMP-7
in the
mixture is from about 0.01% to about 10% of total proteins in the mixture.
In second embodiment of the product ofthe present invention, a cartilage-
inducing
composition includes a mixture of proteins which includes (a) a bone-derived
osteogenic
or chondrogenic formulation; and, (b) an exogenous TGF~3 protein. The
exogenous
TGF~i protein is present in an amount su~cient to increase cartilage induction
by the
composition over a level of cartilage induction by the bone-derived osteogenic
or
chondrogenic protein formulation in the absence of the exogenous TGF~3
protein. In one
aspect of this embodiment, the exogenous TGF~3 protein is TGF(31. In this
aspect, the
ratio of TGF~31 to all other proteins in the mixture of proteins is at least
about 1:10, at
least about 1:3, at least about 1:1, or at least about 10:1.
In a third embodiment ofthe product of the present invention, a cartilage-
inducing
composition includes a mixture of proteins comprising: (a) a TGF~3 protein;
and, (b) at
least one bone morphogenetic protein (BMP), wherein the ratio of the TGF~3
protein to
the BMP protein is greater than about 10:1. In this embodiment, the TGF~3
protein can
be any TGF~3 protein, including TGF(31, TGF(32, TGF(33, TGF(34, TGF~iS, or
mixtures
thereof. In a preferred embodiment, the TGF~i protein is TGF(31 or TGF(32,
with TGF~31
being most preferred. The BMP protein can be any BMP protein, including, but
not
limited to, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, CDMP,
and mixtures thereof.
In one aspect of the any of the above-described embodiments of the present
invention, the mixture of proteins includes TGF~3 superfamily proteins: TGF~i
1, bone
morphogenetic protein (BMP)-2, BMP-3, and BMP-7, wherein the TGF(3 superfamily
proteins comprise from about 0.5% to about 99.99% of the mixture of proteins.
In one
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aspect, the TGF(3 superfamily proteins comprise from about 0.5% to about 25%
of the
mixture of proteins; in another aspect, the TGF(3 superfamily proteins
comprise from
about 1% to about 10% of the mixture of proteins.
In one aspect of each of the above-referenced embodiments, the quantity of the
TGF(31 in the mixture is from about 0.01% to about 75% of total proteins in
the mixture;
in another aspect, the quantity of the TGF~31 in the mixture is from about
0.01 % to about
SO% of total proteins in the mixture; in another aspect, the quantity of the
TGF~31 in the
mixture is from about 0.01% to about 25% of total proteins in the mixture; in
another
aspect, the quantity of the TGF~i 1 in the mixture is from about 0.01% to
about 10% of
total proteins in the mixture; in another aspect, the quantity of the TGF~i 1
in the mixture
is from about 0.1% to about 1% of total proteins in the mixture; in another
aspect, the
quantity ofthe TGF~i 1 in the mixture is from about 33% to about 99.99%
oftotal proteins
in the mixture; in another aspect, the quantity of the TGF(31 in the mixture
is from about
50% to about 99.99% of total proteins in the mixture.
In one aspect of each of the above-referenced embodiments, the quantity of the
BMP-2 in the mixture is from about 0.1% to about 5% of total proteins in the
mixture.
In one aspect of each of the above-referenced embodiments, the quantity of the
BMP-3
in the mixture is from about 0.1% to about 5% of total proteins in the
mixture. In one
aspect of each of the above-referenced embodiments, the quantity of the BMP-7
in the
mixture is from about 0.1% to about 5% of total proteins in the mixture. In
the first
embodiment of the product of the present invention, the quantity ofBMP-3 in
the mixture
is from about 0.1% to about 10% of total proteins in the mixture.
In one aspect of each of the above-referenced embodiments, the mixture of
proteins further comprises a protein selected from the group consisting of
TGF(32,
TGF~33, BMP-4, BMP-5, BMP-6, BN1P-8, BMP-9, and cartilage-derived
morphogenetic
protein (CDMP). In one aspect, the TGF(32 comprises from about 0.5% to about
12%
ofthe mixture of proteins; in another aspect, the TGF~i3 comprises from about
0.01% to
about 15% ofthe mixture ofproteins; in another aspect, the BMP-4 comprises
from about
0.01% to about 1% of the mixture of proteins; in another aspect, the BMP-5
comprises
from about 0.01% to about 1% of the mixture of proteins; in another aspect,
the BMP-6
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comprises from about 0.01% to about 1% of the mixture of proteins; in another
aspect,
the CDMP comprises from about 0.01% to about 1% of the mixture of proteins.
In another aspect of each of the above-referenced embodiments, the mixture of
proteins further comprises at least one bone matrix protein. The bone matrix
protein can
include, but is not limited to, osteocalcin, osteonectin, bone sialoprotein
(BSP),
lysyloxidase, cathepsin L pre, osteopontin, matrix GLA protein (MGP),
biglycan, decorin,
proteoglycan-chondroitin sulfate III (PG-CS III), bone acidic glycoprotein
(BAG-75),
thrombospondin (TSP) and flbronectin. Typically, the bone matrix protein
comprises from
about 20% to about 98% of the mixture of proteins. In one aspect, the bone
matrix
proteins comprise: osteocalcin, osteonectin, bone sialoprotein (BSP),
lysyloxidase, and
cathepsin L pre. In another aspect, the bone matrix protein comprises from
about 40%
to about 98% of the mixture of proteins.
In another aspect of each of the above-referenced embodiments, the mixture of
proteins further comprises at least one growth factor protein. The growth
factor protein
can include, but is not limited to, fibroblast growth factor-I (FGF-I), FGF-
II, FGF-9,
leukocyte inhibitory factor (LIF), insulin, insulin growth factor I (IGF-I),
IGF-II, platelet-
derived growth factor AA (PDGF-AA), PDGF-BB, PDGF-AB, stromal derived factor-2
(SDF-2), pituitary thyroid hormone (PTIT), growth hormone, hepatocyte growth
factor
(HGF), epithelial growth factor (EGF), transforming growth factor-a (TGFa) and
hedgehog proteins. Typically, the growth factor protein comprises from about
0.01 % to
about SO% of the mixture of proteins. In one aspect, the growth factor protein
comprises
from about 0.05% to about 25% of the mixture of proteins; in another aspect,
the growth
factor protein comprises from about 0.1% to about 10% of the mixture of
proteins.
Preferably, the growth factor protein is fibroblast growth factor-I (FGF-I).
In this aspect,
the FGF-I comprises from about 0.001% to about 10% of the mixture of proteins.
In yet another aspect of each of the above-identified embodiments of the
present
invention, the composition further comprises one or more serum proteins. The
serum
proteins can include, but are not limited to, albumin, transferrin, a2-Hs
GlycoP, IgG, al-
antitrypsin, (32-microglobulin, Apo A1 lipoprotein (LP) and Factor XIIIb. In
one aspect,
the serum proteins are selected from the group consisting of albumin,
transferrin, Apo Al
LP and Factor XIIIb.
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In one aspect of any of the above-referenced embodiments of the present
invention, the mixture of proteins comprises TGF~i 1, TGF~32, TGF(33, BMP-2,
BMP-3,
BMP-4, BMP-S, BMP-6, BMP-7, CDMP, FGF-I, osteocalcin, osteonectin, BSP,
lysyloxidase, cathepsin L pre, albumin, transferrin, Apo A1 LP and Factor
XIIIb. In
5 another aspect, the mixture of proteins comprises Bone Protein (BP). In yet
another
aspect, the cartilage inducing composition has an identifying characteristic
selected from
the group consisting of an ability to induce cellular infiltration, an ability
to induce cellular
proliferation, an ability to induce angiogenesis, and an ability to induce
cellular
differentiation to type II collagen-producing chondrocytes.
10 In one aspect of any of the above-referenced embodiments of the present
invention, the cartilage-inducing composition is at a concentration offrom
about 0.5% to
about 33% by weight of the product. In another aspect, the cartilage-inducing
composition is at a concentration of from about 1% to about 20% by weight of
the
product.
In the first embodiment of the product of the present invention, the mixture
of
proteins, when used at a concentration of at least about 10 pg per 6.5-7.3 mg
of bovine
tendon collagen in a rat subcutaneous assay, induces a bone score of from
about 1.0 to
about 3.5, using a bone grading scale set forth in Table 8, and induces a
cartilage score
of at least about 1.2, using a cartilage grading scale set forth in Table 9.
In the second and third embodiments of the product of the present invention,
the
composition, when used at a concentration of at least about 10 pg per 6.5-7.3
mg of
bovine tendon collagen in a rat subcutaneous assay, induces a bone score of
less than
about 2.0, using a bone grading scale set forth in Table 8, and induces a
cartilage score
of at least about 2.0, using a cartilage grading scale set forth in Table 9.
Preferably, in the
second and third embodiments, the composition, when used at a concentration of
at least
about 10 pg per 6.5-7.3 mg of bovine tendon collagen in a rat subcutaneous
assay,
induces a bone score of less than about 2.0, using a bone grading scale set
forth in Table
8, and induces a cartilage score of at least about 2.5, using a cartilage
grading scale set
forth in Table 9. More preferably, in the second and third embodiments, the
composition,
when used at a concentration of at least about 10 pg per 6.5-7.3 mg of bovine
tendon
collagen in a rat subcutaneous assay, induces a bone score of less than about
2.0, using
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a bone grading scale set forth in Table 8, and induces a cartilage score of at
least about
3.0, using a cartilage grading scale set forth in Table 9.
In one aspect of the first embodiment of the present invention, the ratio of
TGF~i 1
to all other proteins in the mixture of proteins is at least about 1:10; in
another aspect, the
ratio of TGF~i 1 to all other proteins in the mixture of proteins is at least
about 1:3; in
another aspect, the ratio of TGF~il to all other proteins in the mixture of
proteins is at
least about 1:1; in another aspect, the ratio of TGF~i 1 to all other proteins
in the mixture
of proteins is at least about 10:1.
In the second or third embodiment of the product of the present invention, the
TGF~3 protein can be a recombinant TGF~i protein, or can be purified from a
bone-derived
protein mixture. In one aspect, the ratio of the TGF(3 protein to the BMP
protein is
greater than about 100:1; in another aspect, the ratio of the TGF(3 protein to
the BMP
protein is greater than about 1000:1; in another aspect, the ratio of the
TGF(3 protein to
the BMP protein is greater than about 10,000:1.
In the third embodiment referenced above, in a preferred aspect, the TGF(3
protein
is TGF~i 1. In another preferred aspect of this embodiment, the BMP protein is
selected
from the group consisting ofBMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8,
BMP-9 and CDMP.
Various other aspects of each of the above-identified embodiments of the
product
of the present invention are described in detail below.
The product of the present invention can also be formulated to include: (a) a
cartilage repair matrix; and (b) a cartilage-inducing composition associated
with the
matrix, which includes cells that have been cultured with the above-described
mixture of
chondrogenesis-enhancing proteins.
The cartilage repair matrix of a shape and size that conforms to the cartilage
defect
such that the defect is repaired. As such, the matrix can be configured as a
sheet, which
is most suitable for repairing cartilage tears, or the matrix can be
configured to repair a
segmental defect, which can include a tapered shape. The cartilage repair
matrix can be
formed of any suitable material, including synthetic polymeric material and
ground
substances. In one embodiment, the matrix is bioresorbable. In another
embodiment, the
matrix is porous. When the matrix is configured as a sheet, the matrix is
preferably not
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cross-linked, and when the matrix is configured to repair a segmental defect,
the matrix
is preferably cross-linked.
The cartilage-inducing composition can be associated with the matrix by any
suitable method, including, but not limited to freeze-drying the composition
onto a surface
of said matrix and suspension within said cartilage repair matrix of a
delivery formulation
containing said composition. Additionally, the composition can be associated
with the
matrix ex vivo or in vivo.
Another embodiment of the present invention relates to a method for repair of
cartilage lesions, which includes the steps of implanting and fixing into a
cartilage lesion
a cartilage repair product of the present invention, as described above,
including a
cartilage repair product including an of the above-referenced embodiments of a
cartilage-
inducing composition. The method of the present invention can be used to
enhance the
rate and/or quality of repair of vascular cartilage tears and segmental
defects, and can
provide the ability to repair semivascular and avascular tears and segmental
defects that,
prior to the present invention, were typically considered to be irreparable.
When the
lesion is in semivascular or avascular cartilage, the product can additionally
include a time
controlled delivery formulation.
In one aspect, the method ofthe present invention includes the use of two
cartilage
repair products to repair a segmental defect. The first product includes a
cartilage repair
matrix, which is configured as a sheet, is associated with the chondrogenesis-
inducing
composition as described above. The second product includes a cartilage repair
matrix
configured to replace cartilage removed from the segmental defect, which may
or may not
be associated with the chondrogenesis-inducing composition of the present
invention.
BRIEF DESCRIPTION OF THE FIGURES OF THE INVENTION
Fig. lA shows a meniscal radial tear.
Fig. 1B shows a conventional suture repair and resection ofthe meniscal radial
tear
illustrated in Fig. lA.
Fig. 1 C shows a meniscal triple bucket handle tear.
Fig. 1D shows a conventional suture repair and resection of the meniscal
triple
bucket handle tear illustrated in Fig. 1C.
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13
Fig. 2A illustrates implantation of a cartilage repair product of the present
invention to a meniscal segmental lesion.
Fig. 2B illustrates fixation of a cartilage repair product of the present
invention to
a meniscal segmental lesion.
S Fig. 3A is a diagram illustrating a meniscus cross section having vascular,
semi-
vascular and avascular zones.
Fig. 3B illustrates one approximate shape of a cartilage repair product of the
present invention.
Fig. 4A is an illustration of a meniscus having a longitudinal tear in the
avascular
region as viewed from the femur towards the tibia.
Fig. 4B is a diagram illustrating a cross section of the meniscus depicted in
Fig. 4A
containing a cartilage repair product of the present invention.
Fig. 5 is a line graph showing quantitation of Alcian Blue staining of ATDCS
micromass cultures.
Fig. 6 is a bar graph showing quantitation of Alcian Blue staining of ATDCS
micromass cultures in Nutridoma-containing media at 7 and 14 days.
Fig. 7 is a bar graph showing quantitation of Alcian Blue staining of ATDCS
micromass cultures containing HPLC fractions of proteins isolated from
demineralized
bovine bones.
Fig. 8 is a diagram illustrating a cross-section view of a combination
collagen
meniscus implant (CMI) and sheet cartilage repair product of the present
invention used
to repair a meniscal defect.
DETAILED DESCRIPTION OF THE INVENTION
The present application generally relates to a product for repairing and/or
regenerating cartilage lesions, and methods of repairing or regenerating
cartilage lesions
using such a product. The product and methods of the present invention are
particularly
useful for repairing defects (i.e., lesions) in articular cartilage (e.g.,
hyaline cartilage) and
meniscal cartilage (e.g., fibrocartilage). When used to repair meniscal
cartilage, the
product and method ofthe present invention are effective for repairing both
vascular and
avascular meniscal cartilage lesions. In particular, the product and method of
the present
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14
invention increase the rate of meniscus repair and induce more normal (i.e.,
endogenous-
type) meniscal tissue than is commonly observed during the conventional repair
practiced
currently. The cartilage repair product of the present invention can also
induce meniscus
repair of avascular, "irreparable" tears and, furthermore, fill the defect
with meniscus-like
tissue. Moreover, the product and method are useful for repairing and
regenerating
meniscal tissue which has been removed by partial or complete meniscectomy.
The
product and method ofthe present invention can enhance blood vessel formation,
produce
fibrochondrocytes, induce cellular infiltration into the product, induce
cellular
proliferation, and produce cellular and spatial organization to form a three-
dimensional
meniscus tissue.
The ability of the product of the present invention to repair and/or
regenerate both
vascular and avascular cartilage in vivo has not been achieved by any of the
presently
known cartilage repair devices/compositions or methods. Moreover, previous
devices and
methods have been primarily directed to the repair of very small defects and
have not been
successful in solving problems associated with repair of large, clinically
relevant defects.
Without being bound by theory, the present inventors believe that the reason
that these
previous approaches failed to adequately repair cartilage is that they were
not able to
recapitulate natural cartilage ontogeny faithfully enough, this natural
ontogeny being based
on a very complicated system of different factors, factor combinations and
factor
concentrations with temporal and local gradients. A single recombinant factor
or two
recombinant factors may lack the inductive complexity to mimic cartilage
development to
a sufficient degree. Moreover, prior investigators had not discovered how to
manipulate
various combinations of osteogenic/chondrogenic factors in order to limit bone
growth
and enhance cartilage growth in vivo. Similarly, the system used to provide
one or two
recombinant factors may not have been able to mimic the gradient complexity of
the
natural system to a satisfactory degree or to maintain a factor concentration
for a time that
is sufficient to allow a full and permanent differentiation of precursor cells
to
chondrocytes. Without being bound by theory, the present inventors believe
that the
repair of certain defects, particularly large defects, requires the
maintenance of a sufficient
concentration of a particular complex mixture of repair factors at the site
for a time
sufficient to induce the proper formation of cartilage.
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As discovered by the present inventors and as described herein, Bone Protein
(BP)
and mixtures derived from BP are chondrogenic in avascular environments (e.g.
articular
cartilage and in vitro), with limited osteogenic activity. However, although
BP is
chondrogenic in other, vascularized environments (lumbar spine, subcutaneous,
etc.), it
5 has significant osteogenic activity as well. Certain cartilage regeneration
applications,
such as the meniscus, require cartilaginous repair in a vascular environment.
Because the
meniscus is vascularized, it is therefore expected that BP and other bone-
inducing
molecules will induce bone formation during repair despite also inducing
cartilage
formation, which is not desirable for clinical applications. The present
invention describes
10 the identification of a novel mixture of factors derived from bovine bone
to induce
cartilage without bone formation. The cartilage induction activity was
observed in a
permissive bone-forming environment: the vascularized, rodent subcutaneous
model.
Specifically, this new mixture combines unexpectedly high concentrations of
TGF~3
proteins with the cartilage-inducing compositions disclosed herein, including
mixtures that
15 also have osteoinductive properties, to produce this novel chondrogenic
activity with
significantly reduced, or eliminated, osteogenic activity. To the present
inventors'
knowledge, prior to the present invention, the combination of high
concentration TGF~i
protein plus osteogenic and/or chondrogenic proteins) to promote only
cartilage
formation in the absence of bone formation in a vascularized (permissive bone
forming)
environment, has not been described.
One aspect ofthe present invention is directed to a product for repair of
cartilage
lesions. In one embodiment, such a product includes: (a) a cartilage repair
matrix; and,
(b) a cartilage-inducing composition associated with the matrix for provision
of a mixture
of proteins, which can be referred to herein as chondrogenesis-enhancing
proteins
(described in detail below). According to the present invention, the phrase
"cartilage-
inducing composition" refers to a formulation which contains a mixture of
dii~erent
chondrogenesis-enhancing proteins and which enhances (i.e., augments,
amplifies,
improves, increases, or supplements) cartilage growth in vivo. In a preferred
embodiment,
the cartilage-inducing composition useful in the product of the present
invention has an
identifying characteristic which includes: an ability to induce cellular
infiltration, an ability
to induce cellular proliferation, an ability to induce angiogenesis, and/or an
ability to
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16
induce cellular differentiation to type II collagen-producing chondrocytes, in
vivo or under
appropriate in vitro conditions.
More specifically, the cartilage-inducing composition of the present invention
provides a mixture of proteins which includes proteins that have osteogenic-
and/or
S chondrogenic-enhancing activities, particularly when combined into mixtures
as described
in detail herein. According to the present invention, the term "enhancing",
particularly
with regard to enhancing chondrogenesis, refers to any measure of augmenting,
amplifying, improving, increasing, or supplementing a biological activity
associated with
chondrogenesis such that cartilage forms in a manner that more closely mimics
the natural
ontogeny of cartilage formation, as compared to cartilage formation that would
occur in
the absence of the product, or in the absence of the composition portion of
the product.
The term enhancing also means that endochondral maturation to mineralized
cartilage and
bone may be prevented or delayed.
In one embodiment of the present invention, a mixture of chondrogenesis-
enhancing proteins that are included in a chondrogenesis-inducing composition
can be
characterized as being capable, when cultured together with ATDCS cells for
seven days
at a concentration of about 100 ng/ml or less, of inducing a statistically
significant increase
m As9s in an Alcian Blue assay performed with the ATDCS cells. The specific
conditions
associated with such an ATDCS/Alcian Blue assay are described in detail below.
It is
noted that although the mixture of chondrogenesis-enhancing proteins has the
above-
described characteristic, an individual chondrogenesis-enhancing protein, when
isolated
from the other proteins in the mixture, is not necessarily chondrogenic. For
example, as
described below, a bone matrix protein such as osteocalcin is a chondrogenesis-
enhancing
protein according to the present invention, because when such protein is
combined with
other suitable proteins, such as combinations of TGF~3 superfamily proteins as
described
herein, the mixture of proteins is capable of inducing a significant increase
in As9s in an
ATDCS Alcian Blue assay. Osteocalcin is not, however, chondrogenic in the
absence of
such TGF~i superfamily proteins.
Various embodiments ofthe chondrogenesis-inducing composition ofthe present
invention are described in detail below. For general reference, however, the
following
description is provided. According to the present invention, the
chondrogenesis-
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17
enhancing proteins in the cartilage-inducing composition ofthe present
invention typically
include at least two different members of the TGF(3 superfamily proteins. In
other
embodiments, the chondrogenesis-enhancing proteins include at least three
different
members of the TGF~i superfamily proteins, and in alternative embodiments, at
least four,
five, six, seven, eight, nine, and most preferably ten different members of
the TGF(3
superfamily proteins. As used herein, a "TGF~i superfamily protein" can be any
protein
of the art-recognized superfamily of extracellular signal transduction
proteins that are
structurally related to TGF~i 1-S. Preferably, a TGF(3 superfamily protein
suitable for use
in the present invention includes, but is not limited to the following
proteins: TGF~i 1,
TGF~i2, TGF(33, TGF~34, TGF(35, bone morphogenetic protein (BMP)-2, BMP-3, BMP-
4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, cartilage-derived morphogenetic protein
(CDMP)-1, CDMP-2, and/or CDMP-3. More preferably, the chondrogenesis-enhancing
proteins usefi~l in the composition of the present invention include, but are
not limited to:
TGF~il, TGF~32, TGF(33, BMP-2, BMP-3, BMP-4, BMP-S, BMP-6, BMP-7, and/or
CDMP (CDMP-1, CDMP-2, and/or CDMP-3).
In some aspects ofthe present invention, the cartilage-inducing composition
ofthe
present invention can include at least one bone matrix protein and/or at least
one growth
factor protein. In a preferred aspect, the mixture of proteins includes at
least one bone
matrix protein and at least one growth factor protein. In a more preferred
embodiment,
the chondrogenesis-enhancing proteins include, in increasing preference, at
least two,
three, four, and most preferably five different bone matrix proteins, and/or
at least two
growth factor proteins.
As used herein, "bone matrix proteins" are any of a group of proteins known in
the art to be a component of or associated with the minute collagenous fibers
and ground
substances which form bone matrix. As used herein, a bone matrix protein is
not a
member of the TGF~i superfamily as described herein, nor a growth factor
protein as
described herein. Borie matrix proteins can include, but are not limited to,
osteocalcin,
osteonectin, bone sialoprotein (BSP), lysyloxidase, cathepsin L pre,
osteopontin, matrix
GLA protein (MGP), biglycan, decorin, proteoglycan-chondroitin sulfate III (PG-
CS III),
bone acidic glycoprotein (BAG-75), thrombospondin (TSP) and/or fibronectin.
Preferably, bone matrix proteins suitable for use with the product of the
present invention
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18
include one or more of osteocalcin, osteonectin, MGP, TSP, BSP, lysyloxidase
and
cathepsin L pre. In one embodiment, the at least one bone matrix protein
includes at least
osteocalcin, osteonectin, B SP, lysyloxidase and cathepsin L pre. A
particularly preferred
bone matrix protein is MGP, and more preferred is osteonectin, and most
preferred is
TSP.
As used herein, "growth factor proteins" are any of a group of proteins
characterized as an extracellular polypeptide signaling molecule that
stimulates a cell to
grow or proliferate. Such growth factors may also have other actions besides
the
induction of cell growth or proliferation. As used herein, a growth factor is
not a member
of the TGF~i superfamily as defined herein nor is it a bone matrix protein as
defined herein.
Preferably, growth factor proteins suitable for use with the product of the
present
invention include one or more of fibroblast growth factor I (FGF-I), FGF-II,
FGF-9,
leukocyte inhibitory factor (LIF), insulin, insulin growth factor I (IGF-I),
IGF-II, platelet-
derived growth factor AA (PDGF-AA), PDGF-BB, PDGF-AB, stromal derived factor-2
(SDF-2), pituitary thyroid hormone (PTH), growth hormone, hepatocyte growth
factor
(HGF), epithelial growth factor (EGF), transforming growth factor-a (TGFa) and
hedgehog proteins. A most preferred growth factor protein for use with the
product of
the present invention is FGF-I.
In one aspect of the present invention, the mixture of proteins in the
chondrogenesis-inducing composition can also include one or more serum
proteins. As
used herein, serum proteins are any of a group of proteins that is a component
of serum.
A serum protein is not a member of the TGF~3 superfamily, a bone matrix
protein or a
growth factor, as described herein. In one embodiment, chondrogenesis-inducing
compositions include, in increasing preference, at least one, two, three, and
most
preferably four different serum proteins. Serum proteins suitable for use with
the product
of the present invention include one or more of albumin, transferrin, a2-Hs
GlycoP, IgG,
a 1-antitrypsin, ~i2-microglobulin, Apo A1 lipoprotein (LP) and Factor XIIIb.
Preferably,
serum proteins suitable for use with the product of the present invention
include one or
more of albumin, transferrin, Apo A1 LP and Factor XIIIb.
In one aspect of the present invention, the relative proportions of the
proteins in
the mixture of proteins are any proportions which are sufficient for the
mixture, at a
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19
concentration of 100 ng/ml or less, to induce a statistically significant
increase in As9s in
an Alcian Blue assay performed with ATDCS cells as described below. In one
embodiment, the percentage of TGF~3 superfamily members within the mixture
ranges
between about 0.1% to about 99.99% ofthe total mixture, and preferably between
about
0.5% to about 99.99% of the total mixture, and more preferably between about
0.1% to
about 50% of the total mixture, and more preferably between about 0.5% to
about 50%
of the total mixture, and more preferably, between about 0.5% and about 25%,
and even
more preferably, between about 1% and about 10% ofthe total mixture. The
percentage
of growth factors within the mixture ranges between about 0.01% to about 50%
of the
total mixture, and preferably, between about 0.05% and about 25%, and even
more
preferably, between about 0.1% and about 10% of the total mixture. The
percentage of
serum and bone matrix protein components, either separately or combined,
ranges
between about 20% to about 98%, and preferably between about 40% to about 98%,
and
even more preferably between about 80% to about 98%. In one embodiment of the
invention, the mixture of chondrogenesis-inducing proteins contains at least
BMP-3,
BMP-2 and TGF~i 1, wherein the quantity of BMP-3 in the mixture is about 2-6
fold
greater than the quantity of BMP-2 and about 10-30 fold greater than the
quantity of
TGF~31 in the mixture. Unless otherwise specified, reference to percentages of
proteins
or ratios of proteins either to the mixture of proteins or to specific
proteins is based on
weight to weight (w/w).
Having generally discussed certain aspects of mixtures of proteins which may
be
present in a chondrogenesis-inducing composition of the present invention,
particular
preferred embodiments of such a composition are described below.
In a first embodiment of the present invention, a cartilage-inducing
composition
includes a mixture of proteins which includes: transforming growth factor ~i 1
(TGF(31),
bone morphogenetic protein (BMP)-2, BMP-3, and BMP-7. The quantity ofthe
TGF~31
in the mixture is typically from about 0.01% to about 99.99% of total proteins
in the
mixture. The quantity ofthe BMP-2 in the mixture is typically from about 0.01%
to about
10% of total proteins in the mixture, or from about 0.01 to about 1 %, or from
about 0.01
to about 0.1%, or from about 0.1 to about 1%, or from about 0.1 to about 10%
of total
proteins. The quantity of the BMP-3 in the mixture is typically from about 0.1
% to about
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15% of total proteins in the mixture, or from about 0.1 to about 1%, or from
about 0.01
to about 15%, or from about 0.01 to about 1%, or from about 0.01 to about
0.1%. The
quantity of the BMP-7 in the mixture is typically from about 0. 01 % to about
10% of total
proteins in the mixture, or from about 0.01 to about 1%, or from about 0.01 to
about
5 0.1%, or from about 0.1 to about 1%, or from about 0.1 to about 10% oftotal
proteins.
The amino acid and nucleic acid sequence for each of the above-referenced
proteins are
known in the art and can be publicly accessed, for example, through a database
such as
GenBank. Additionally, these proteins can be purified, if desired, from an
appropriate
source, such as demineralized bone. For example, high purity TGF~i-1 can be
isolated
10 from bovine bone using methods disclosed by Seyedin (Ogawa et al., Meth.
Enrymol.,
198:317-327 (1991); Seyedin et al., PNAS, 82:2267-71 (1985)).
In one aspect of this embodiment of the present invention, the mixture of
proteins
comprises TGF(3 superfamily proteins: TGF(31, bone morphogenetic protein (BMP)-
2,
BMP-3, and BMP-7, wherein the TGF(3 superfamily proteins comprise from about
0.5%
15 to about 99.99% ofthe mixture of proteins. Alternatively, the TGF(3
superfamily proteins
can be present at a percentage of from about 0.5% to about 25% of the mixture
of
proteins, or from about 1% to about 10% of the mixture of proteins.
As discussed above, mixtures of proteins according to the present invention
that
are capable of inducing significant chondrogenesis in vivo may have a
relatively low
20 percentage of TGF~i proteins, and particularly TGF~i 1, relative to the
total amount of
protein in the mixture of proteins (e.g., from about 0.01 to about 1%).
However, the
present inventors have discovered that the use of unexpectedly high
concentrations of a
TGF(3 protein, and particularly, TGF~i 1, relative to the total mixture of
proteins of the
present invention, results in enhanced induction of chondrogenesis in vivo,
with
significantly reduced osteogenesis. This discovery is particularly important
for in vivo
chondrogenesis-induction in a vascular, or bone-forming, environment, and will
significantly improve the clinical performance of compositions of the present
invention in
such in vivo environments. A mixture of proteins in this embodiment of the
present
invention can therefore include a quantity of TGF(31 which is from about 0.01
% to about
99.99% of the total quantity of proteins in the mixture, with increasing
TGF(31 relative
to the total amount of protein resulting in enhanced chondrogenesis. In other
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21
embodiments, the quantity of TGF~i 1 is from about 0.01% to about 75% of total
proteins
in the mixture, from about 0.01% to about 50% of total proteins in the
mixture, from
about 0.01% to about 25% of total proteins in the mixture, from about 0.01% to
about
10% of total proteins in the mixture, or from about 0.1% to about 1% of total
proteins
in the mixture. In a preferred embodiment, the quantity of TGF(31 is at least
about 33%
of total proteins in the mixture (up to a maximum of about 99.99%). In another
preferred
embodiment, the quantity of TGF(31 is at least about 50% of total proteins in
the mixture
(up to a maximum of about 99.99%).
Alternatively, the amount of TGF~i 1 to be added to the mixture of proteins
can be
determineu as a ratio. In one embodiment, the ratio of TGF~i 1 to all other
proteins in the
mixture of proteins is at least about 1:10. Preferably, the ratio of TGF(31 to
all other
proteins in the mixture of proteins is at least about 1:3, and more
preferably, at least about
1:1, and even more preferably, at least about 10:1. Examples of the use of
TGF~31 at a
ratio of 1:10, 1:3 and 1:1 relative to the total quantity of protein in the
mixture in vivo is
demonstrated in Example 15. Example 15 demonstrates that unexpectedly high
concentrations of TGF(3 protein relative to the total protein results in
significantly
enhanced chondrogenesis and significantly decreased osteogenesis. The optimal
amount
of TGF~i 1 to be added for a given mixture of proteins, cartilage repair
matrix, and in vivo
environment (i. e., vascular or avascular) can be determined, for example, by
using a simple
rat subcutaneous assay as described in detail in the examples section (e.g.,
see Example
14).
In yet another embodiment, the amount of TGF(i proteins such as TGF(31 to be
included in the mixture of proteins can be determined as an amount of TGF(3
protein in
excess of one or the total of BMP proteins in the mixture. In a preferred
embodiment, the
amount of TGF(3 protein should be greater than l OX higher than the amount
ofBMP (one
or a combination of BMPs in the mixture), but less than 100X higher than the
amount of
BMP in the mixture.
The quantity of the BMP-2 in the mixture is typically from about 0.01% to
about
10% of total proteins in the mixture, or from about 0.01 to about 1%, or from
about 0.01
to about 0.1%, or from about 0.1 to about 1%, or from about 0.1 to about 10%,
and in
one embodiment, is present in a quantity offrom about 0.1% to about S% oftotal
proteins
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22
in the mixture. The quantity of BMP-3 in the mixture is typically from about
0.01% to
about 15% of total proteins in the mixture, or from about 0.01 to about 1 %,
or from about
0.01 to about 0.1%, or from about 0.1 to about 1%, or from about 0.1 to about
15%, and
in one embodiment, is present in a quantity of from about 0.1% to about 10% of
total
proteins in the mixture, and in another embodiment, is present in a quantity
of from about
0.1 % to about 5% of total proteins in the mixture. The quantity of BMP-7 in
the mixture
is typically from about 0.01 % to about 10% of total proteins in the mixture,
or from about
0.01 to about 1%, or from about 0.01 to about 0.1%, or from about 0.1 to about
1%, or
from about 0.1 to about 10%, and in one embodiment, can be present in a
quantity of from
about 0.1% to about S% of total proteins in the mixture.
In one aspect of this embodiment of the present invention, the mixture of
proteins
further includes one or more of the following TGF(3 superfamily proteins:
TGF~i2,
TGF~33, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, and cartilage-derived morphogenetic
protein (CDMP), which can include one or more of CDMP-1, CDMP-2 or CDMP-3. The
quantity of TGF~32 in such a mixture is typically from about 0.5% to about 12%
of the
total mixture of proteins, although additional TGF(32 can be added to enhance
the
chondrogenic activity of the mixture of proteins, if desired, up to as much as
about
99.99% of the total mixture of proteins. The quantity of TGF(33 in such a
mixture is
typically from about 0.01% to about 15% of the total mixture of proteins,
although
additional TGF(33 can be added to enhance the chondrogenic activity of the
mixture of
proteins, if desired, up to as much as about 99.99% of the total mixture of
proteins. The
quantity of BMP-4 in such a mixture typically comprises from about 0.01% to
about 1%
of the total mixture of proteins, although additional BMP-4 can be added to
enhance the
chondrogenic activity of the mixture of proteins, if desired, up to as much as
about 10%
of the total mixture of proteins. The quantity of BMP-5 in the mixture of
proteins is
typically from about 0.01% to about 1% of the total mixture of proteins,
although
additional BMP-5 can be added to enhance the chondrogenic activity of the
mixture of
proteins, if desired, up to as much as about 10% of the total mixture of
proteins. The
quantity of BMP-6 in the mixture of proteins is typically from about 0.01% to
about 1%
of the total mixture of proteins, although additional BMP-6 can be added to
enhance the
chondrogenic activity of the mixture of proteins, if desired, up to as much as
about 10%
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23
of the total mixture of proteins. The quantity of CDMP in the mixture of
proteins is
typically from about 0.01% to about 1% of the total mixture of proteins,
although
additional CDMP can be added to enhance the chondrogenic activity of the
mixture of
proteins, if desired, up to as much as about 10% of the total mixture of
proteins.
In one aspect of this embodiment, the mixture of proteins can additionally
include
at least one bone matrix protein. Bone matrix proteins are generally described
above.
Preferred bone matrix proteins for use in this mixture of proteins include,
but are not
limited to, osteocalcin, osteonectin, bone sialoprotein (BSP), lysyloxidase,
cathepsin L
pre, osteopontin, matrix GLA protein (MGP), biglycan, decorin, proteoglycan-
chondroitin
sulfate III (PG-CS III), bone acidic glycoprotein (BAG-75), thrombospondin
(TSP) and
fibronectin. More preferably, bone matrix proteins suitable for use in this
mixture of
proteins include, but are not limited to, osteocalcin, osteonectin, bone
sialoprotein (BSP),
lysyloxidase, and cathepsin L pre. The bone matrix proteins are typically
present in the
mixture in a quantity from about 20% to about 98% of the total mixture of
proteins. In
one embodiment, the bone matrix proteins are present in the mixture in a
quantity from
about 40% to about 98% of the total mixture of proteins.
In another aspect of this embodiment, the mixture of proteins can additionally
include at least one growth factor protein. Growth factor proteins are
generally described
above. Preferred growth factor proteins for use in this mixture of proteins
include, but
are not limited to, fibroblast growth factor-I (FGF-I), FGF-II, FGF-9,
leukocyte inhibitory
factor (LIF), insulin, insulin growth factor I (IGF-I), IGF-II, platelet-
derived growth
factor AA (PDGF-AA), PDGF-BB, PDGF-AB, stromal derived factor-2 (SDF-2),
pituitary thyroid hormone (PTITj, growth hormone, hepatocyte growth factor
(HGF),
epithelial growth factor (EGF), transforming growth factor-a (TGFa) and
hedgehog
proteins. A particularly preferred growth factor for use in this mixture of
the present
invention is FGF-I. Typically, the growth factor protein is present in the
mixture of
proteins at a quantity from about 0.01% to about SO% of the total mixture of
proteins.
In other embodiments, the quantity of growth factor proteins in the mixture is
from about
0.5% to about 25% of the total mixture of proteins; or from about 0.1% to
about 10% of
the total mixture of proteins. When the growth factor is FGF-I, the quantity
of FGF-I in
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24
the mixture of proteins is typically from about 0.001% to about 10% of the
total mixture
of proteins.
In yet another aspect of this embodiment, the mixture of proteins can include
one
or more serum proteins. Serum proteins have been generally described above.
Preferably,
serum proteins useful in this mixture include, but are not limited to,
albumin, transferrin,
a2-Hs GIycoP, IgG, al-antitrypsin, ~i2-microglobulin, Apo A1 lipoprotein (LP)
and/or
Factor 3GIIb. More preferably, serum proteins useful in this mixture include,
but are not
limited to, albumin, transferrin, Apo A1 LP and/or Factor ~OIIb.
In one aspect of this embodiment of the present invention, a mixture of
proteins
suitable for use in a chondrogenesis-inducing composition portion of a
cartilage repair
product ofthe present invention includes the following proteins: TGF~i 1,
TGF(32, TGF~33,
BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, CDMP, FGF-I, osteocalcin,
osteonectin, BSP, lysyloxidase, cathepsin L pre, albumin, transfernn, Apo A1
LP and
Factor~b. Inyet another embodiment, a suitable mixture ofchondrogenesis-
enhancing
proteins includes the mixture of proteins referred to herein as Bone Protein
(BP), which
is defined herein as a partially-purified protein mixture from bovine long
bones as
described in Poser and Benedict, WO 95/13767, incorporated herein by reference
in its
entirety. In another aspect of this embodiment of the present invention, the
cartilage
inducing composition has an identifying characteristic selected from the group
consisting
of an ability to induce cellular infiltration, an ability to induce cellular
proliferation, an
ability to induce angiogenesis, and an ability to induce cellular
differentiation to type II
collagen-producing chondrocytes. In yet another aspect ofthis embodiment ofthe
present
invention, the mixture of proteins, when used at a concentration of at least
about 10 ltg
per 6.5-7.3 mg of bovine tendon collagen in a rat subcutaneous assay, induces
a bone
score of from about 1.0 to about 3.5, using a bone grading scale set forth in
Table 8
(Example 10), and induces a cartilage score of at least about 1.2, using a
cartilage grading
scale set forth in Table 9 (Example 10). A rat subcutaneous assay suitable for
determining
bone and cartilage scores according to this aspect, and the grading scales of
Tables 8 and
9 are described in detail in the Examples Section.
In a second embodiment ofthe present invention, a cartilage-inducing
composition
includes a mixture of proteins which includes (a) a bone-derived osteogenic or
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chondrogenic formulation; and, (b) an exogenous TGF~3 protein. The exogenous
TGF(3
protein is present in an amount sufficient to increase cartilage induction by
the
composition over a level of cartilage induction by the bone-derived osteogenic
or
chondrogenic protein formulation in the absence of the exogenous TGF(i
protein.
5 According to the present invention, a "bone-derived osteogenic or
chondrogenic
formulation" refers to any mixture of proteins containing a complex mixture of
proteins
which is isolated, or derived, (e.g., by at least one, and typically, multiple
purification
steps) from a starting material of bone, and which is osteogenic or
chondrogenic in vivo.
A bone-derived osteogenic or chondrogenic formulation contains at least one
bone
10 morphogenetic protein (BMP) and the ratio of exogenous TGF(3 to the BMP (or
more
than one BMP) in the mixture is greater than about 10:1. The BMP protein can
include
any BMP protein, including, but not limited to, BMP-2, BMP-3, BMP-4, BMP-5,
BMP-6,
BMP-7, BMP-8, BMP-9, CDMP, and mixtures thereof. Preferably, the bone-derived
formulation is capable of inducing bone and/or cartilage formation in an in
vivo rat
15 subcutaneous assay such as that described in the Examples section of the
Rosen modified
Sampath-Reddi rodent assay (Sampath et al., Proc. Nat'1 Acad. Sci. USA,
80(21):6591-5
(1983)). More preferably, the bone-derived formulation is capable of inducing
a bone
score of at least about 1.0 when used at a concentration of at least about 10
pg per 6.5-
7.3 mg of bovine tendon collagen in a rat subcutaneous assay as set forth in
Example 10
20 using a bone grading scale set forth in Table 8 (Example 10), and/or
induces a cartilage
score of at least about 1.0 under the same conditions, using a cartilage
grading scale set
forth in Table 9 (Example 10). Without being bound by theory, the present
inventors
believe that given their discovery described herein of the enhanced
chondrogenic effects
on osteogenic or chondrogenic protein mixtures of significantly high levels of
TGF~3
25 protein, it is likely that even protein mixtures that have strong
osteogenic activity and little
or no chondrogenic activity in the absence of excess (exogenous) TGF~3 protein
can be
converted to chondrogenic mixtures by the addition of high dose TGF(3 protein,
and
particularly, TGF~31. Particularly preferred bone-derived osteogenic or
chondrogenic
formulations for use in this embodiment of the present invention include Bone
Protein
(BP) and subfractions and related derivatives thereof. The Examples Section
describes
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26
examples of BP (Example 9), subfractions thereof (Example 12) and related
derivatives
thereof (Example 11).
The starting material of bone can be a bone sample from any source, including,
but
not limited to, bovine bone and human bone. The bone can be processed by any
of a
number of methods known in the art for producing compositions which have
osteogenic
activity, alone or in combination with some level of chondrogenic activity
(See for
example, U.S. Patent No. 4,563,489 to Urist; U.S. Patent No. 5,629,009 to
Laurencin et
al.; PCT Publication No. WO 92/09697 to Bentz et al.; and Poser and Benedict,
PCT
Publication No. W095/13767). According to the present invention, a "bone-
derived
osteogenic or chondrogenic formulation" is not used to refer to mixtures of
one or more
recombinant proteins, since recombinant proteins are not produced using bone
as a
starting material.
For example, one method for producing Bone Protein according to the present
invention, and as described, for example, in U.S. Patent No. 5,290,763,
entitled
"Osteoinductive Protein Mixtures and Purification Processes", incorporated
herein by
reference in its entirety, typically includes the steps of conducting anion
exchange
chromatography on a demineralized bone extract solution, a cation exchange
procedure,
and reverse phase HPLC procedure.
According to the present invention, an "exogenous TGF~i protein" refers to a
TGF~i protein that is in substantially pure form and which is not a part of
the bone-derived
osteogenic or chondrogenic formulation of proteins (i.e., the exogenous TGF(3
protein
was not isolated from with the bone-derived osteogenic or chondrogenic
formulation of
proteins). The exogenous TGF(3 protein is instead added to the formulation as
an
additional protein from a different source. It is to be understood that the
bone-derived
osteogenic or chondrogenic formulation of proteins can contain TGF(3 proteins
(i.e.,
"endogenous" proteins), since such mixtures typically do contain at least
TGF~il and
TGF~32. However, the second component in the composition of exogenous TGF(3
protein
is intended as a means of increasing the total amount of TGF(3 protein in the
composition
beyond what is naturally found in bone and mixtures derived therefrom.
Specifically, the
TGF(3 protein is provided in an amount that is sufficient to increase
cartilage induction
(i.e., in vivo) by the composition over a level of cartilage induction (i.e.,
in vivo under the
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27
same conditions) by the bone-derived osteogenic or chondrogenic protein
formulation in
the absence of the exogenous TGF~i protein. The exogenous TGF(3 protein can be
recombinant TGF~i protein or substantially purified TGF(3 protein from any
suitable
source, such as bone. Recombinant TGF~i proteins are publicly available and
TGF(3
proteins can be purified to high purity from bone, for example, using
previously described
methods (Ogawa et al., Meth. Enzymol., 198:317-327 (1991); Seyedin et al.,
PNAS,
82:2267-71 (1985). The exogenous TGF~i protein can be any TGF~i protein,
including
TGF(31, TGF(32, TGF~i3, TGF(34, TGF(35, or mixtures thereof. In a preferred
embodiment, the TGF~3 protein is TGF(31 or TGF(32, with TGF(31 being most
preferred.
Preferably, the ratio of the TGF(3 protein to the at least one BMP protein in
the
mixture (w/w), is greater than about 10:1, and in one aspect, is greater than
about 100:1,
and in another aspect, is greater than about 1000:1, and in another aspect, is
greater than
about 10,000:1. In one embodiment, the ratio of TGF(3 protein to total BMP
proteins in
the mixture of proteins is greater than 10: l, and in another aspect, is
greater than about
100:1, and in another aspect, is greater than about 1000:1, and in another
aspect, is
greater than about 10,000:1. It is noted that the percentages of various
components in a
mixture of proteins will adjust according to the amount of excess TGF~3 added,
and in
some embodiments, including where very high concentrations of TGF~i are added,
the
percentage of a BMP as a total of the composition, for example, may fall
significantly
below the more typical amount of BMP in the mixture.
In one aspect ofthis embodiment ofthe present invention, the mixture of
proteins,
which includes both the bone-derived osteogenic or chondrogenic formulation of
proteins
and the exogenous TGF~i protein, comprises TGF~i superfamily proteins: TGF(31,
bone
morphogenetic protein (BMP)-2, BMP-3, and BMP-7, wherein the TGF(3 superfamily
proteins comprise from about 0.5% to about 99.99% of the mixture of proteins.
Alternatively, the TGF(3 superfamily proteins can be present at a percentage
of from about
0.5% to about 25% of the mixture of proteins, or from about 1% to about 10% of
the
mixture of proteins. In this embodiment of the present invention, a TGF~i
protein can be
provided by the bone-derived osteogenic or chondrogenic formulation and/or the
exogenous source of TGF~3. All other proteins are typically provided by the
bone-derived
osteogenic or chondrogenic formulation, although other exogenous proteins may
be added
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28
to the mixture to further enhance the chondrogenic properties of the
composition, if
desired.
A mixture of proteins in this embodiment of the present invention can
therefore
include a quantity of TGF~i 1 which is from about 0.01% to about 99.99% of the
total
quantity of proteins in the mixture, with increasing TGF~31 relative to the
total amount of
protein resulting in enhanced chondrogenesis. In other embodiments, the
quantity of
TGF~31 is from about 0.01% to about 75% of total proteins in the mixture, from
about
0.01% to about 50% of total proteins in the mixture, from about 0.01% to about
25% of
total proteins in the mixture, from about 0.01% to about 10% of total proteins
in the
mixture, or from about 0.1% to about 1% of total proteins in the mixture.
Typically, the
bone-derived osteogenic or chondrogenic formulation will contribute from about
0.01%
to about 1 % TGF ~i 1 protein to the mixture, with higher amounts being
contributed by the
exogenous TGF~31. In a preferred embodiment, the quantity of TGF~31 is at
least about
33% of total proteins in the mixture (up to a maximum of about 99.99%). In
another
preferred embodiment, the quantity of TGF(31 is at least about 50% of total
proteins in
the mixture (up to a maximum of about 99.99%).
Alternatively, the amount of TGF~31 to be added to the mixture of proteins can
be
determined as a ratio. In one embodiment, the ratio of TGF(31 to all other
proteins in the
mixture of proteins is at least about 1:10 (with the ratio of TGF~i protein to
at least one
BMP being greater than about 10:1) . Preferably, the ratio of TGF(31 to all
other proteins
in the mixture of proteins is at least about 1:3, and more preferably, at
least about 1:1, and
even more preferably, at least about 10:1.
In yet another embodiment, the amount of TGF~i proteins such as TGF~i 1 to be
included in the mixture of proteins can be determined as an amount of TGF~i
protein in
excess of one or the total of BMP proteins in the mixture. In a preferred
embodiment, the
amount of TGF ~i protein should be greater than 1 OX higher than the amount of
BMP (one
or a combination of BMPs in the mixture), but less than 100X higher than the
amount of
BMP in the mixture.
The quantity of the BMP-2 in the mixture is typically from about 0.1% to about
5% of total proteins in the mixture, or from about 0.01 to about 1%, or from
about 0.01
to about 0.1 %, or from about 0.1 to about 1%, although the percentage ofBMP-2
can be
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29
less than 0.01% of the total proteins when high concentrations of TGF~i
proteins are
added (e.g., when the ratio of TGF(3 protein to BMP is greater than 10,000:1).
The
quantity of BMP-3 in the mixture is typically from about 0.1% to about S% of
total
proteins in the mixture, or from about 0.01 to about 1%, or from about 0.01 to
about
0.1%, or from about 0.1 to about 1%, although the percentage ofBMP-3 can be
less than
0.01% of the total proteins when high concentrations of TGF~i proteins are
added. The
quantity of BMP-7 in the mixture is typically from about 0.1% to about 5% of
total
proteins in the mixture, or from about 0.01 to about 1%, or from about 0.01 to
about
0.1 %, or from about 0.1 to about 1 %, although the percentage of BMP-7 can be
less than
0.01% of the total proteins when high concentrations of TGF~i'proteins are
added.
In one aspect ofthis embodiment ofthe present invention, the mixture of
proteins
further includes one or more of the following TGF(3 superfamily proteins:
TGF~i2,
TGF~i3, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, and cartilage-derived morphogenetic
protein (CDMP), which can include one or more of CDMP-1, CDMP-2 or CDMP-3. The
quantity of TGF~32 in the bone-derived osteogenic or chondrogenic formulation
is
typically from about 0.5% to about 12-1 S% of the total formulation, although
additional
TGF(32 can be added as an exogenous TGF~i protein to enhance the chondrogenic
activity
of the mixture of proteins, if desired, up to as much as about 99.99% of the
total mixture
of proteins. The quantity of TGF(33 in the bone-derived osteogenic or
chondrogenic
formulation is typically from about 0.01 % to about 15% of the total
formulation, although
additional TGF(33 can be added to enhance the chondrogenic activity of the
mixture of
proteins, if desired, up to as much as about 99.99% of the total mixture of
proteins. The
quantity of BMP-4 in the bone-derived osteogenic or chondrogenic formulation
typically
comprises from about 0.01% to about 1% of the total formulation, although
additional
BMP-4 can be added to enhance the chondrogenic activity of the mixture of
proteins. In
some embodiments, the percentage of BMP-4 can be less than 0.01% of the total
proteins
when high concentrations of TGF~3 proteins are added (e.g., when the ratio of
TGF~i to
BNIP is greater than 10,000:1). The quantity ofBMP-5 in the bone-derived
osteogenic
or chondrogenic formulation is typically from about 0.01% to about 1% of the
total
formulation, although additional BMP-S can be added to enhance the
chondrogenic
activity of the mixture of proteins. In some embodiments, the percentage of
BMP-S can
CA 02362600 2001-08-14
WO 00/48550 PCT/US00/03972
be less than 0.01% of the total proteins when high concentrations of TGF~i
proteins are
added (e.g., when the ratio of TGF~3 to BMP is greater than 10,000:1). The
quantity of
BMP-6 in the bone-derived osteogenic or chondrogenic formulation is typically
from
about 0.01% to about 1% of the total formulation, although additional BMP-6
can be
5 added to enhance the chondrogenic activity of the mixture of proteins. In
some
embodiments, the percentage ofBMP-6 can be less than 0.01% ofthe total
proteins when
high concentrations of TGF~i proteins are added (e.g., when the ratio of TGF(3
to BMP
is greater than 10,000:1). The quantity of CDMP in the bone-derived osteogenic
or
chondrogenic formulation is typically from about 0.01% to about 1% of the
total
10 formulation, although additional CDMP can be added to enhance the
chondrogenic
activity of the mixture of proteins. In some embodiments, the percentage of
CDMP can
be less than 0.01% of the total proteins when high concentrations of TGF(3
proteins are
added (e.g., when the ratio of TGF~i to BMP is greater than 10,000:1).
In one aspect of this embodiment, the mixture of proteins can additionally
include
15 at least one bone matrix protein, typically provided by the bone-derived
osteogenic or
chondrogenic formulation. Bone matrix proteins are generally described above.
Preferred
bone matrix proteins for use in this mixture of proteins include, but are not
limited to,
osteocalcin, osteonectin, bone sialoprotein (BSP), lysyloxidase, cathepsin L
pre,
osteopontin, matrix GLA protein (MGP), biglycan, decorin, proteoglycan-
chondroitin
20 sulfate III (PG-CS III), bone acidic glycoprotein (BAG-75), thrombospondin
(TSP) and
fibronectin. More preferably, bone matrix proteins suitable for use in this
mixture of
proteins include, but are not limited to, osteocalcin, osteonectin, bone
sialoprotein (BSP),
lysyloxidase, and cathepsin L pre. The bone matrix proteins are typically
present in the
mixture in a quantity from about 20% to about 98% of the total mixture of
proteins. In
25 one embodiment, the bone matrix proteins are present in the mixture in a
quantity from
about 40% to about 98% of the total mixture of proteins.
In another aspect of this embodiment, the mixture of proteins can additionally
include at least one growth factor protein. Growth factor proteins are
generally described
above. Preferred growth factor proteins for use in this mixture of proteins
include, but
30 are not limited to, fibroblast growth factor-I (FGF-I), FGF-II, FGF-9,
leukocyte inhibitory
factor (I,IF), insulin, insulin growth factor I (IGF-I), IGF-II, platelet-
derived growth
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31
factor AA (PDGF-AA), PDGF-BB, PDGF-AB, stromal derived factor-2 (SDF-2),
pituitary thyroid hormone (PTH), growth hormone, hepatocyte growth factor
(HGF),
epithelial growth factor (EGF), transforming growth factor-a (TGFa) and
hedgehog
proteins. A particularly preferred growth factor for use in this mixture of
the present
invention is FGF-I. Typically, the growth factor protein are present in the
mixture of
proteins at a quantity from about 0.01% to about 50% of the total mixture of
proteins.
In other embodiments, the quantity of growth factor proteins in the mixture is
from about
0.5% to about 25% of the total mixture of proteins; or from about 0.1% to
about 10% of
the total mixture of proteins. When the growth factor is FGF-I, the quantity
of FGF-I in
the mixture of proteins is typically from about 0.001 % to about 10% of the
total mixture
of proteins.
In yet another aspect of this embodiment, the mixture of proteins can include
one
or more serum proteins. Serum proteins have been generally described above.
Preferably,
serum proteins useful in this mixture include, but are not limited to,
albumin, transferrin,
a2-Hs GlycoP, IgG, al-antitrypsin, (32-microglobulin, Apo A1 lipoprotein (LP)
and/or
Factor XIIIb. More preferably, serum proteins usefizl in this mixture include,
but are not
limited to, albumin, transferrin, Apo A1 LP and/or Factor XIIIb.
In one aspect of this embodiment of the present invention, a mixture of
proteins
suitable for use in a chondrogenesis-inducing composition portion of a
cartilage repair
product ofthe present invention includes the following proteins: TGF~31,
TGF~i2, TGF~33,
BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, CDMP, FGF-I, osteocalcin,
osteonectin, BSP, lysyloxidase, cathepsin L pre, albumin, transfernn, Apo Al
LP and
Factor XIIIb. In yet another embodiment, a suitable mixture of chondrogenesis-
enhancing
proteins includes the mixture of proteins referred to herein as Bone Protein
(BP), which
is defined herein as a partially-purified protein mixture from bovine long
bones as
described in Poser and Benedict, WO 95/13767, incorporated herein by reference
in its
entirety. In another aspect of this embodiment of the present invention, the
cartilage
inducing composition has an identifying characteristic selected from the group
consisting
of an ability to induce cellular infiltration, an ability to induce cellular
proliferation, an
ability to induce angiogenesis, and an ability to induce cellular
differentiation to type II
collagen-producing chondrocytes.
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32
Preferably, in this embodiment, the composition, when used at a concentration
of
at least about 10 pg per 6.5-7.3 mg of bovine tendon collagen in a rat
subcutaneous assay,
induces a bone score of less than about 2.0, using a bone grading scale set
forth in Table
8, and induces a cartilage score of at least about 2.0, using a cartilage
grading scale set
forth in Table 9. More preferably, the composition, when used at a
concentration of at
least about 10 ~g per 6.5-7.3 mg of bovine tendon collagen in a rat
subcutaneous assay,
induces a bone score of less than about 2.0, using a bone grading scale set
forth in Table
8, and induces a cartilage score of at least about 2.5, using a cartilage
grading scale set
forth in Table 9. Even more preferably, the composition, when used at a
concentration
of at least about 10 pg per 6.5-7.3 mg of bovine tendon collagen in a rat
subcutaneous
assay, induces a bone score of less than about 2.0, using a bone grading scale
set forth in
Table 8, and induces a cartilage score of at least about 3.0, using a
cartilage grading scale
set forth in Table 9.
In a third embodiment of the product of the present invention, a cartilage-
inducing
composition includes a mixture of proteins comprising: (a) a TGF~3 protein;
and, (b) at
least one bone morphogenetic protein (BMP), wherein the ratio of the TGF(3
protein to
the BMP protein is greater than about 10:1. In this embodiment, the TGF~3
protein can
be any TGF(3 protein, including TGF~i 1, TGF~32, TGF~i3, TGF~i4, TGF~iS, or
mixtures
thereof. In a preferred embodiment, the TGF(3 protein is TGF(31 or TGF(32,
with TGF~i 1
being most preferred. The BMP protein can be any BMP protein, including, but
not
limited to, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, CDMP,
and mixtures thereof.
Preferably, the ratio of the TGF~i protein to the at least one BMP protein in
the
mixture (w/w), is greater than about 10:1, and in one aspect, is greater than
about 100:1,
and in another aspect, is greater than about 1000:1, and in another aspect, is
greater than
about 10,000:1. In one embodiment, the ratio of TGF~3 protein to total BMP
proteins in
the mixture of proteins is greater than 10:1, and in another aspect, is
greater than about
100:1, and in another aspect, is greater than about 1000:1, and in another
aspect, is
greater than about 10,000:1. It is noted that the percentages of various
components in a
mixture of proteins will adjust according to the amount of excess TGF~3 added,
and in
some embodiments, including where very high concentrations of TGF(3 are added,
the
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33
percentage of a BMP as a total of the composition, for example, may fall
significantly
below the more typical amount of BMP in the mixture.
In yet another embodiment, the amount of TGF~i proteins such as TGF~iI to be
included in the mixture of proteins can be determined as an amount of TGF~i
protein in
S excess of one or the total ofBMP proteins in the mixture. In a preferred
embodiment, the
amount of TGF ~i protein should be greater than l OX higher than the amount of
BMP (one
or a combination of BMPs in the mixture), but less than 100X higher than the
amount of
BMP in the mixture.
According to this embodiment ofthe present invention, the TGF~3 protein and
the
at least one BMP protein can be provided as a recombinant protein, as a
substantially pure
protein, or as a component of a mixture of proteins, such as a component of a
bone
derived osteogenic or chondrogenic formulation as described in the embodiment
above.
Although in this embodiment, the mixture of proteins can include as few as one
TGF~i protein and one BMP protein, in one aspect of this embodiment of the
present
invention, the mixture of proteins comprises TGF~3 superfamily proteins: TGF~i
l, bone
morphogenetic protein (BMP)-2, BMP-3, and BMP-7, wherein the TGF~i superfamily
proteins comprise from about 0.5% to about 99.99% of the mixture of proteins.
Alternatively, the TGF~3 superfamily proteins can be present at a percentage
of from about
0.5% to about 25% of the mixture of proteins, or from about 1% to about 10% of
the
mixture of proteins.
A mixture of proteins in this embodiment of the present invention can include
a
quantity of TGF~31 which is from about 0.01% to about 99.99% of the total
quantity of
proteins in the mixture, as long as the ratio of TGF~3 to at least one BMP
protein in the
mixture is greater than 10:1. In other embodiments, the quantity of TGF~i 1 is
from about
0.01% to about 75% of total proteins in the mixture, from about 0.01% to about
50% of
total proteins in the mixture, from about 0.01% to about 25% of total proteins
in the
mixture, from about 0.01% to about 10% of total proteins in the mixture, or
from about
0.1% to about 1% oftotal proteins in the mixture. In a preferred embodiment,
the quantity
of TGF(31 is at least about 33% of total proteins in the mixture (up to a
maximum of
about 99.99%). In another preferred embodiment, the quantity of TGF(31 is at
least about
50% of total proteins in the mixture (up to a maximum of about 99.99%).
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34
The quantity of the BMP-2 in the mixture is typically from about 0.1% to about
5% of total proteins in the mixture, or from about 0.01 to about 1%, or from
about 0.01
to about 0.1%, or from about 0.1 to about 1%, although the percentage ofBMP-2
can be
less than 0.01% of the total proteins when high concentrations of TGF(i
proteins are
added (e.g., when the ratio of TGF(3 protein to BMP is greater than 10,000:1).
The
quantity of BNIP-3 in the mixture is typically from about 0.1% to about 5% of
total
proteins in the mixture, or from about 0.01 to about 1%, or from about 0.01 to
about
0.1%, or from about 0.1 to about 1%, although the percentage ofBMP-3 can be
less than
0.01% of the total proteins when high concentrations of TGF(3 proteins are
added. The
quantity of BMP-7 in the mixture is typically from about 0.1% to about 5% of
total
proteins in the mixture, or from about 0.01 to about 1%, or from about 0.01 to
about
0.1%, or from about 0.1 to about 1%, although the percentage ofBMP-7 can be
less than
0.01% of the total proteins when high concentrations of TGF~i proteins are
added.
In one aspect of this embodiment of the present invention, the mixture of
proteins
further includes one or more of the following TGF~i superfamily proteins:
TGF~i2,
TGF~33, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, and cartilage-derived morphogenetic
protein (CDMP), which can include one or more of CDMP-l, CDMP-2 or CDMP-3. The
quantity of TGF~i2 in such a mixture is typically from about 0.5% to about 12%
of the
total mixture of proteins, although additional TGF(32 can be added to enhance
the
chondrogenic activity of the mixture of proteins, if desired, up to as much as
about
99.99% of the total mixture of proteins. The quantity of TGF~33 in such a
mixture is
typically from about 0.01% to about 15% of the total mixture of proteins,
although
additional TGF~i3 can be added to enhance the chondrogenic activity of the
mixture of
proteins, if desired, up to as much as about 99.99% of the total mixture of
proteins. The
quantity of BMP-4 in the bone-derived osteogenic or chondrogenic formulation
typically
comprises from about 0.01% to about 1% of the total formulation, although
additional
BMP-4 can be added to enhance the chondrogenic activity of the mixture of
proteins. In
some embodiments, the percentage of BMP-4 can be less than 0.01 % of the total
proteins
when high concentrations of TGF(i proteins are added (e.g., when the ratio of
TGF(3 to
BMP is greater than 10,000:1). The quantity of BMP-5 in the bone-derived
osteogenic
or chondrogenic formulation is typically from about 0.01% to about 1% of the
total
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formulation, although additional BMP-5 can be added to enhance the
chondrogenic
activity of the mixture of proteins. In some embodiments, the percentage of
BMP-5 can
be less than 0.01% of the total proteins when high concentrations of TGF~3
proteins are
added (e.g., when the ratio of TGF(3 to BMP is greater than 10,000:1). The
quantity of
5 BMP-6 in the bone-derived osteogenic or chondrogenic formulation is
typically from
about 0.01% to about 1% of the total formulation, although additional BMP-6
can be
added to enhance the chondrogenic activity of the mixture of proteins. In some
embodiments, the percentage ofBMP-6 can be less than 0.01 % of the total
proteins when
high concentrations of TGF(3 proteins are added (e.g., when the ratio of TGF~3
to BMP
10 is greater than 10,000:1). The quantity of CDMP in the bone-derived
osteogenic or
chondrogenic formulation is typically from about 0.01% to about 1% of the
total
formulation, although additional CDMP can be added to enhance the chondrogenic
activity of the mixture of proteins. In some embodiments, the percentage of
CDMP can
be less than 0.01% of the total proteins when high concentrations of TGF~3
proteins are
15 added (e.g., when the ratio of TGF~i to BMP is greater than 10,000:1).
In one aspect of this embodiment, the mixture of proteins can additionally
include
at least one bone matrix protein. Bone matrix proteins are generally described
above.
Preferred bone matrix proteins for use in this mixture of proteins include,
but are not
limited to, osteocalcin, osteonectin, bone sialoprotein (BSP), lysyloxidase,
cathepsin L
20 pre, osteopontin, matrix GLAprotein (MGP), biglycan, decorin, proteoglycan-
chondroitin
sulfate III (PG-CS III), bone acidic glycoprotein (BAG-75), thrombospondin
(TSP) and
fibronectin. More preferably, bone matrix proteins suitable for use in this
mixture of
proteins include, but are not limited to, osteocalcin, osteonectin, bone
sialoprotein (BSP),
lysyloxidase, and cathepsin L pre. The bone matrix proteins are typically
present in the
25 mixture in a quantity from about 20% to about 98% of the total mixture of
proteins. In
one embodiment, the bone matrix proteins are present in the mixture in a
quantity from
about 40% to about 98% of the total mixture of proteins.
In another aspect of this embodiment, the mixture of proteins can additionally
include at least one growth factor protein. Growth factor proteins are
generally described
30 above. Preferred growth factor proteins for use in this mixture of proteins
include, but
are not limited to, fibroblast growth factor-I (FGF-I), FGF-II, FGF-9,
leukocyte inhibitory
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36
factor (LIF), insulin, insulin growth factor I (IGF-I), IGF-II, platelet-
derived growth
factor AA (PDGF-AA), PDGF-BB, PDGF-AB, stromal derived factor-2 (SDF-2),
pituitary thyroid hormone (PTH), growth hormone, hepatocyte growth factor
(HGF),
epithelial growth factor (EGF), transforming growth factor-a (TGFa) and
hedgehog
S proteins. A particularly preferred growth factor for use in this mixture of
the present
invention is FGF-I. Typically, the growth factor protein are present in the
mixture of
proteins at a quantity from about 0.01% to about 50% of the total mixture of
proteins.
In other embodiments, the quantity of growth factor proteins in the mixture is
from about
0.5% to about 25% ofthe total mixture ofproteins; or from about 0.1% to about
10% of
the total mixture of proteins. When the growth factor is FGF-I, the quantity
of FGF-I in
the mixture of proteins is typically from about 0.001% to about 10% of the
total mixture
of proteins.
In yet another aspect of this embodiment, the mixture of proteins can include
one
or more serum proteins. Serum proteins have been generally described above.
Preferably,
serum proteins useful in this mixture include, but are not limited to,
albumin, transfernn,
a2-Hs GlycoP, IgG, al-antitrypsin, (32-microglobulin, Apo A1 lipoprotein (LP)
andlor
Factor XIIIb. More preferably, serum proteins useful in this mixture include,
but are not
limited to, albumin, transfernn, Apo Al LP and/or Factor XIIIb.
In one embodiment of the present invention, a mixture of chondrogenesis-
enhancing proteins suitable for use in a chondrogenesis-inducing composition
portion of
a cartilage repair product ofthe present invention includes the following
proteins: TGF~31,
TGF(32, TGF~33, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, CDMP, FGF-I,
osteocalcin, osteonectin, MGP, BSP, lysyloxidase, and cathepsin L pre, wherein
the ratio
of TGF~31 to at least one, and preferably all, BMP proteins (including CDMP)
is greater
than 10:1. In another embodiment, a suitable mixture of chondrogenesis-
enhancing
proteins includes the following proteins: TGF~i 1, TGF~i2, TGF(33, BMP-2, BMP-
3, BMP-
4, BMP-5, BMP-6, BMP-7, CDMP, FGF-I, osteocalcin, osteonectin, BSP,
lysyloxidase,
cathepsin L pre, albumin, transfernn, Apo Al LP and Factor XIIIb, wherein the
ratio of
TGF~31 to at least one, and preferably all, BMP proteins (including CDMP) is
greater than
10:1. In yet another embodiment, a suitable mixture of chondrogenesis-
enhancing
proteins includes the mixture of proteins referred to herein as Bone Protein
(BP; described
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37
above), wherein the ratio of TGF(31 in the mixture to at least one, and
preferably all, BMP
proteins in the mixture, including in BP, is greater than 10:1. An example of
a mixture of
proteins which contained TGF(31 and at least one BMP protein (as present in
BP) at a
ratio of greater than from about 10:1 to 10,000:1 is illustrated in Example
14.
S In one embodiment of the cartilage repair product of the present invention,
each
of the chondrogenesis-enhancing proteins in the cartilage-inducing composition
is
provided by the composition either: (1) directly as a protein that is
associated with the
matrix, or (2) as a recombinant nucleic acid molecule associated with the
matrix, such
recombinant nucleic acid molecule encoding the protein and being operatively
linked to
a transcription control sequence such that the protein can be expressed under
suitable
conditions. Therefore, a cartilage-inducing composition of the present
invention can
include proteins, recombinant nucleic acid molecules, or a combination of
proteins and
recombinant nucleic acid molecules, such composition providing the
chondrogenesis-
enhancing proteins described above.
According to the present invention, a chondrogenesis-enhancing protein can be
obtained from its natural source, produced using recombinant DNA technology,
or
synthesized chemically. As used herein, a chondrogenesis-enhancing protein can
be a full-
length protein (i.e., in its full-length, naturally occurnng form), any
homologue of such a
protein, any fusion protein containing such a protein, or any mimetope of such
a protein.
The amino acid sequences for chondrogenesis-enhancing proteins disclosed
herein,
including the TGF(3 superfamily proteins described herein, as well as nucleic
acid
sequences encoding the same are known in the art and are publicly available,
for example,
from sequence databases such as GenBank. Such sequences can therefore be
obtained and
used to produce proteins and/or recombinant nucleic acid molecules of the
present
invention.
A homologue is defined as a protein in which amino acids have been deleted
(e.g.,
a truncated version of the protein, such as a peptide or fragment), inserted,
inverted,
substituted and/or derivatized (e.g., by glycosylation, phosphorylation,
acetylation,
myristoylation, prenylation, palmitation, amidation and/or addition
ofglycosylphosphatidyl
inositol). A homologue of a chondrogenesis-enhancing protein is a protein
having an
amino acid sequence that is sufficiently similar to a naturally occurnng
chondrogenesis-
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38
enhancing protein amino acid sequence that the homologue has substantially the
same or
enhanced biological activity compared to the corresponding naturally occurnng
protein.
As used herein, a mimetope (also referred to as a synthetic mimic) of a
chondrogenesis-enhancing protein according to the present invention refers to
any
compound that is able to mimic the activity of such a chondrogenesis-enhancing
protein,
often because the mimetope has a structure that mimics the chondrogenesis-
enhancing
protein. Mimetopes can be, but are not limited to: peptides that have been
modified to
decrease their susceptibility to degradation; anti-idiotypic and/or catalytic
antibodies, or
fragments thereof; non-proteinaceous immunogenic portions of an isolated
protein (e.g.,
carbohydrate structures); and synthetic or natural organic molecules,
including nucleic
acids. Such mimetopes can be designed using computer-generated structures of
naturally
occurnng chondrogenesis-enhancing protein. Mimetopes can also be obtained by
generating random samples of molecules, such as oligonucleotides, peptides or
other
organic or inorganic molecules, and screening such samples by affinity
chromatography
techniques using the corresponding binding partner.
According to the present invention, a fusion protein is a protein that
includes a
chondrogenesis-enhancing protein-containing domain attached to one or more
fusion
segments. Suitable fission segments for use with the present invention
include, but are not
limited to, segments that can: enhance a protein's stability; enhance the
biological activity
of a chondrogenesis-enhancing protein; and/or assist purification of a
chondrogenesis-
enhancing protein (e.g., by amity chromatography). A suitable fizsion segment
can be
a domain of any size that has the desired function (e.g., imparts increased
stability, imparts
enhanced biological activity to a protein, and/or simplifies purification of a
protein).
Fusion segments can be joined to amino and/or carboxyl termini of the
chondrogenesis-
enhancing protein-containing domain of the protein and can be susceptible to
cleavage in
order to enable straight-forward recovery of a chondrogenesis-enhancing
protein. Fusion
proteins are preferably produced by culturing a recombinant cell transformed
with a fizsion
nucleic acid molecule that encodes a protein including the fission segment
attached to
either the carboxyl and/or amino terminal end of a chondrogenesis-enhancing
protein-
containing domain. Preferred fission segments include a metal binding domain
(e.g., a
poly-histidine segment); an immunoglobulin binding domain (e.g., Protein a;
Protein G;
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39
T cell; B cell; Fc receptor or complement protein antibody-binding domains); a
sugar
binding domain (e.g., a maltose binding domain); and/or a "tag" domain (e.g.,
at least a
portion of (3-galactosidase, a strep tag peptide, other domains that can be
purified using
compounds that bind to the domain, such as monoclonal antibodies).
In one embodiment, a mixture of chondrogenesis-enhancing proteins according to
the present invention is capable, when cultured together with ATDCS cells for
seven days
at a concentration of about 100 ng/ml or less, ofinducing a statistically
significant increase
in A59s in an Alcian Blue assay performed with the ATDCS cells. In a preferred
embodiment, a mixture of chondrogenesis-enhancing proteins is capable of
inducing a
significant increase in A59s in an Alcian Blue assay performed with the ATDCS
cells when
cultured under the above conditions at a concentration of about 50 ng/ml or
less, and
more preferably, about 25 ng/ml or less, and even more preferably, about 10
ng/ml or less.
As used herein, a statistically significant increase is defined as an increase
in A59s as
compared to a control, in which the probability of such an increase being due
to chance
is p<0.05, and more preferably, p<0.001, and even more preferably, p<0.005.
According to the present invention, an ATDCS Alcian Blue assay is known in the
art and is described, for example, in von Schroeder et al., 1994, Teratology
50:54-62. For
the purposes of determining whether a mixture of chondrogenesis-enhancing
proteins
meets the requirements of being capable of inducing a significant increase in
A59s in an
Alcian Blue assay performed with the ATDCS cells when cultured with such cells
at a
given concentration (e.g., 100 ng/ml), the following protocol can be used.
Murine ATDCS cells were deposited by T. Atsumi (Deposit No. RCB0565) and
are publicly available from the Riken Cell Bank, 3-1-1 Koyadai, Tsukuba
Science City,
305 Japan. The ATDCS cells are maintained in 100 X 20 mm standard tissue
culture
plates in Dulbecco's modified Eagle's medium (DMEM):Ham's F-12 (1:1) media
that
contains 5% fetal bovine serum, penicillin (50 U/ml), and streptomycin (50
mg/ml).
Cultures are incubated in a humidified incubator at 37°C and S% CO2.
Passages 3-8 can
be used to assay the activity of the mixture of proteins to be evaluated. To
perform the
Alcian Blue assay, first, the micromass culture technique is performed as
described in
Atkinson et al., 1997, ibid., with minor alterations. Briefly, trypsinized
cells are
resuspended in the ATDCS culture medium described above at a concentration of
about
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100,000 cells/25 pl. The 25 pl spot of cells is placed in the center of a 24
well polystyrene
microtiter tissue culture dish. After 1.5 hours, 1 ml of the culture media
described above
is added to the dish. After overnight incubation at 37°C and 5% CO2,
media containing
various concentrations of the mixture of chondrogenesis-inducing proteins
(e.g., 100
5 ng/ml, 50 ng/ml, 25 ng/ml, 10 ng/ml), 5% FBS, 50 pg/ml ascorbic acid, and 10
mM (3-
glycerophosphate are added (Day 0), and the incubation is continued. This
latter media
is then replaced every 3-4 days (for a total of 2 more additions of BP). After
incubation
with the mixture of proteins to be tested, on Day 7, the culture media is
removed and the
cultures are washed three times with 1 ml of PBS. The cultures are then fixed
with 10%
10 neutral buffered formalin for 15 hours and washed twice with 0.5 N HCI.
Cultures are
stained for one hour at room temperature with a 0.5% Alcian Blue solution (pH
1.4). The
stain is then removed and the cultures are washed with PB S to remove unbound
stain.
The blue stain is then extracted with guanidium HCl (4M, pH 1.7) at 70
° C for 18 hours,
followed by measurement of absorption at 595 nm.
15 It is noted that those of skill in the art will be able to make minor
modifications to
the above protocol and obtain a similar outcome. Such modifications include
seeding the
bottom of a well with ATDCS cells (i.e, about 25,000-50,000, not in micromass
culture),
using a serum substitute in the media, altering the concentration of serum,
and/or omitting
ascorbic acid and/or ~3-glycerophosphate from the media. Minor modifications
to the
20 Alcian Blue assay itself can include altering the pH of the Alcian Blue
solution within the
range from about pH 1 to about pH 1.4 and altering the concentration of the
Alcian Blue
solution within the range from about 0.05% to about 0.5%.
As discussed above, one or more of the chondrogenesis-enhancing proteins in
the
cartilage-inducing composition can be provided by the composition as a
recombinant
25 nucleic acid molecule associated with the cartilage repair matrix, such
recombinant nucleic
acid molecule encoding a chondrogenesis-enhancing protein and being
operatively linked
to a transcription control sequence such that the protein can be expressed
under suitable
conditions. A recombinant nucleic acid molecule useful in the present
invention can
include an isolated natural gene encoding a chondrogenesis-enhancing protein
or a
30 homologue of such a gene, the latter ofwhich is described in more detail
below. A nucleic
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41
acid molecule useful in the present invention can include one or more
regulatory regions,
full-length or partial coding regions, or combinations thereof.
In accordance with the present invention, an isolated nucleic acid molecule is
a
nucleic acid molecule that has been removed from its natural milieu (i.e.,
that has been
S subject to human manipulation) and can include DNA, RNA, or derivatives of
either DNA
or RNA. As such, "isolated" does not reflect the extent to which the nucleic
acid
molecule has been purified. An isolated nucleic acid molecule encoding a
chondrogenesis-
enhancing protein can be isolated from its natural source or produced using
recombinant
DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning)
or
chemical synthesis. Isolated nucleic acid molecules can include, for example,
natural
allelic variants and nucleic acid molecule homologues modified by nucleotide
insertions,
deletions, substitutions, and/or inversions in a manner such that the
modifications do not
substantially interfere with the nucleic acid molecule's ability to encode a
chondrogenesis-
enhancing protein of the present invention or to form stable hybrids under
stringent
conditions with natural gene isolates. An isolated nucleic acid molecule can
include
degeneracies. As used herein, nucleotide degeneracies refers to the phenomenon
that one
amino acid can be encoded by dii~erent nucleotide codons. Thus, the nucleic
acid
sequence of a nucleic acid molecule that encodes a chondrogenesis-enhancing
protein of
the present invention can vary due to degeneracies.
A nucleic acid molecule homologue can be produced using a number of methods
known to those skilled in the art (see, for example, Sambrook et al., ibid. ).
For example,
nucleic acid molecules can be modified using a variety of techniques
including, but not
limited to, by classic mutagenesis and recombinant DNA techniques (e.g., site-
directed
mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of
nucleic acid
fragments and/or PCR amplification), or synthesis of oligonucleotide mixtures
and ligation
of mixture groups to "build" a mixture of nucleic acid molecules and
combinations thereof.
Nucleic acid molecule homologues can be selected by hybridization with a
naturally
occurnng gene or by screening for the function of a protein encoded by the
naturally
occurring nucleic acid molecule. Although the phrase "nucleic acid molecule"
primarily
refers to the physical nucleic acid molecule and the phrase "nucleic acid
sequence"
primarily refers to the sequence of nucleotides on the nucleic acid molecule,
the two
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42
phrases can be used interchangeably, especially with respect to a nucleic acid
molecule,
or a nucleic acid sequence, being capable of encoding a chondrogenesis-
enhancing protein.
Knowing the nucleic acid sequence encoding a naturally occurring
chondrogenesis-
enhancing protein according to the present invention allows one skilled in the
art to, for
example, (a) make copies of those nucleic acid molecules, and (b) obtain
nucleic acid
molecules including at least a portion of such nucleic acid molecules (e.g.,
nucleic acid
molecules including full-length genes, full-length coding regions, regulatory
control
sequences, truncated coding regions). Such nucleic acid molecules can be
obtained in a
variety of ways including screening appropriate expression libraries with
antibodies;
traditional cloning techniques using oligonucleotide probes to screen
appropriate libraries
or DNA; and PCR amplification of appropriate libraries or DNA using
oligonucleotide
primers. Techniques to clone and amplify genes are disclosed, for example, in
Sambrook
et al., ibid.
According to the present invention, a nucleic acid molecule encoding a
chondrogenesis-enhancing protein is operatively linked to one or more
transcription
control sequences to form a recombinant molecule. The phrase "operatively
linked" refers
to linking a nucleic acid molecule to a transcription control sequence in a
manner such that
the molecule is able to be expressed when transfected (i.e., transformed,
transduced or
transfected) into a host cell.
Transcription control sequences are sequences which control the initiation,
elongation, and termination of transcription. Particularly important
transcription control
sequences are those which control transcription initiation, such as promoter,
enhancer,
operator and repressor sequences. Suitable transcription control sequences
include any
transcription control sequence that can function in at least one of the
recombinant cells
useful in the product and method of the present invention. A variety of such
transcription
control sequences are known to those skilled in the art. Preferred
transcription control
sequences include those which function in mammalian, bacterial, insect cells,
and
preferably in mammalian cells.
One or more recombinant nucleic acid molecules encoding a chondrogenesis-
enhancing protein can be used to produce the protein. In one embodiment, the
protein is
produced by expressing a recombinant nucleic acid molecule under conditions
effective
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43
to produce the protein. A preferred method to produce an encoded protein is by
transforming a host cell with one or more recombinant molecules to form a
recombinant
cell. Suitable host cells to transform include any mammalian cell that can be
transformed.
Host cells can be either untransfected cells or cells that are already
transformed with at
least one nucleic acid molecule. Host cells useful in the present invention
can be any cell
capable of producing a chondrogenesis-enhancing protein. In a preferred
embodiment,
the host cell itself is useful in enhancing chondrogenesis. A particularly
preferred host cell
includes a fibrochondrocyte, a chondrocyte, and a mesenchymal precursor cell.
According to the method of the present invention, a host cell can be
transformed
with a recombinant nucleic acid molecule encoding a chondrogenesis-enhancing
protein
in vitro or in vivo. Transformation of a recombinant nucleic acid molecule
into a cell in
vitro can be accomplished by any method by which a nucleic acid molecule can
be inserted
into the cell. Transformation techniques include, but are not limited to,
transfection,
electroporation, microinjection, lipofection, adsorption, and protoplast
fusion. The
resulting recombinant cell can then be associated with the cartilage repair
matrix of the
present invention by any suitable method to provide the chondrogenesis-
enhancing
proteins.
Recombinant nucleic acid molecules can be delivered in vivo and associated
with
the cartilage repair matrix in a variety of methods including, but not limited
to, (a)
administering a naked (i. e., not packaged in a viral coat or cellular
membrane) nucleic acid
molecule (e.g., as naked DNA or RNA molecules, such as is taught, for example
in Wolff
et al., 1990, Science 247,1465-1468); (b) administering a nucleic acid
molecule packaged
as a recombinant virus or a recombinant cell (i.e., the nucleic acid molecule
is delivered
by a viral or cellular vehicle), whereby the virus or cell is associated with
the cartilage
repair matrix; or (c) administering a recombinant nucleic acid molecule
associated with
the cartilage repair matrix via a delivery vehicle such as a liposome or
nanosphere delivery
system described herein.
As discussed above, a recombinant nucleic acid molecule encoding a
chondrogenesis-enhancing protein can be associated with the cartilage repair
matrix as a
recombinant virus particle. A recombinant virus includes a recombinant
molecule that is
packaged in a viral coat and that can be expressed in an animal after
administration.
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44
Preferably, the recombinant molecule is packaging-deficient. A number of
recombinant
virus particles can be used, including, but not limited to, those based on
alphaviruses,
poxviruses, adenoviruses, herpesviruses, and retroviruses. When administered
to an
animal, a recombinant virus infects cells at the site of administration ofthe
cartilage repair
product and directs the production of a chondrogenesis-enhancing protein.
Suitable liposomes for use as a delivery vehicle for a recombinant nucleic
acid in
vivo include any liposome. Preferred liposomes of the present invention
include those
liposomes commonly used in, for example, gene delivery methods known to those
of skill
in the art. Methods for preparation of liposomes and complexing nucleic acids
with
liposomes are well known in the art.
As described in detail below, a cartilage-inducing composition is associated
with
a cartilage repair matrix, such that the cartilage repair matrix serves, in
one capacity, as
a delivery vehicle for the composition to be delivered to the site of a
cartilage lesion.
Suitable methods for associating a cartilage-inducing composition containing
chondrogenesis-enhancing proteins and/or recombinant nucleic acid molecules
encoding
such proteins with a cartilage repair matrix include any method which allows
the proteins
and/or recombinant nucleic acid molecules to be delivered to a site of
cartilage repair
together with a cartilage repair matrix such that the cartilage repair product
is effective
to repair and/or regenerate cartilage at the site. Such methods of association
include, but
are not limited to, suspension of the composition within the cartilage repair
matrix, freeze-
drying of the composition onto a surface of the matrix and suspension within
the matrix
of a carner/delivery formulation containing the composition. Additionally, the
cartilage-
inducing composition can be associated with the matrix prior to placement of
the product
into a cartilage lesion (i. e., the association of the composition with matrix
occurs ex vivo)
or alternatively, a cartilage repair matrix can first be implanted into a
lesion, followed by
association ofthe cartilage-inducing composition with the matrix, such as by
injection into
or on top of the matrix (i.e., the association of the composition with matrix
occurs in
vivo). A cartilage-inducing composition can contain additional delivery
formulations or
Garners which enhance the association of the composition with the matrix,
which enhance
the delivery of the composition to the appropriate cells and tissue at the
site of the lesion,
and which assist in controlling the release of the factors in the composition
at the site of
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the lesion. Suitable delivery formulations include carriers, which, as used
herein, include
compounds that increase the half life of a cartilage-inducing composition in
the treated
animal. Suitable carriers include, but are not limited to, polymeric
controlled release
vehicles, biodegradable implants, liposomes, bacteria, viruses, oils, cells,
esters, and
5 glycols.
One embodiment of the present invention is a controlled release formulation
that
is capable of slowly releasing a composition of the present invention into an
animal. As
used herein, a controlled release formulation comprises a cartilage-inducing
composition
of the present invention in a controlled release vehicle. Suitable controlled
release vehicles
10 include, but are not limited to, biocompatible polymers, other polymeric
matrices,
capsules, microcapsules, microparticles, bolus preparations, osmotic pumps,
diffusion
devices, liposomes, lipospheres, and transdermal delivery systems. Other
controlled
release formulations of the present invention include liquids that, upon
association with
the matrix or upon administration to an animal, form a solid or a gel in situ.
Such
15 controlled release vehicles are preferably associated with the cartilage
repair matrix by one
of the above-described methods. Preferred controlled release formulations are
biodegradable (i.e., bioerodible).
A preferred controlled release formulation of the present invention is capable
of
releasing a composition of the present invention at the site of a cartilage
lesion of a treated
20 animal at a constant rate sufficient to attain therapeutic dose levels of
the chondrogenesis-
enhancing proteins provided by the composition to result in enhancement of
chondrogenesis at the lesion. A particularly preferred controlled release
vehicle according
to the present invention is a nanosphere delivery vehicle.
A nanosphere delivery vehicle according to the present invention includes the
25 nanosphere delivery vehicle described in copending PCT Application No.
PCT/EP
98/05100, which is incorporated herein by reference in its entirety. In a
preferred
embodiment, such a delivery vehicle includes polymer particles having a size
of less than
1000 nm and being loaded with between 0.001% and 17% by weight of the
cartilage-
inducing composition. The nanospheres have an in vitro analytically determined
release
30 rate profile with an initial burst of about 10% to about 20% of the total
amount of the
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46
composition over a first 24 hour period, and a long time release rate of a
least 0.1% per
day during at least seven following days.
A cartilage-inducing composition useful in the cartilage repair product of the
present invention can also include one or more pharmaceutically acceptable
excipients.
As used herein, a pharmaceutically acceptable excipient refers to any
substance suitable
for associating a cartilage-inducing composition with a cartilage repair
matrix and
maintaining and delivering the components of the composition (e.g., proteins
and/or
recombinant nucleic acid molecules) to the appropriate cells at a suitable in
vivo site (i.e.,
a cartilage lesion). Preferred pharmaceutically acceptable excipients are
capable of
maintaining a nucleic acid molecule in a form that, upon arrival of the
nucleic acid
molecule at the delivery site, the nucleic acid molecule is capable of
expressing a
chondrogenesis-enhancing protein either by being expressed by a recombinant
cell or by
entering a host cell at the site of the lesion and being expressed by the
cell. A suitable
pharmaceutically acceptable excipient is capable of maintaining a protein in a
form that,
upon arrival of the protein at the delivery site, the protein is biologically
active such that
chondrogenesis at the site is enhanced. Examples of pharmaceutically
acceptable
excipients include, but are not limited to water, phosphate buffered saline,
Ringer's
solution, dextrose solution, serum-containing solutions, Hank's solution,
other aqueous
physiologically balanced solutions, oils, esters and glycols. Aqueous Garners
can contain
suitable auxiliary substances required to approximate the physiological
conditions of the
recipient, for example, by enhancing chemical stability and isotonicity.
Particularly
preferred excipients include non-ionic diluents, with a preferred non-ionic
buffer being 5%
dextrose in water (DWS). Suitable auxiliary substances include, for example,
sodium
acetate, sodium chloride, sodium lactate, potassium chloride, calcium
chloride, and other
substances used to produce phosphate buffer, Tris buffer, and bicarbonate
buffer.
Auxiliary substances can also include preservatives, such as thimerosal, - or
o-cresol,
formalin and benzol alcohol. Cartilage-inducing compositions ofthe present
invention can
be sterilized by conventional methods and/or lyophilized.
A cartilage-inducing composition is present in the cartilage repair product of
the
present invention at a concentration that is effective to induce, at the site
of a cartilage
lesion, one or more of cellular infiltration, cellular proliferation,
angiogenesis, and cellular
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differentiation to type II collagen-producing chondrocytes. Preferably, a
cartilage-
inducing composition is present in the cartilage repair product of the present
invention at
a concentration that is effective to induce cartilage repair and/or
regeneration at the site
of a cartilage lesion. When the chondrogenesis-enhancing proteins are provided
by the
cartilage-inducing protein directly as a protein, the cartilage-inducing
composition is
typically provided at a concentration of from about 0.5% to about 33% by
weight ofthe
cartilage repair product. More preferably, the cartilage-inducing composition
is provided
at a concentration of from about 1% to about 20% by weight of the cartilage
repair
product. When one or more of the chondrogenesis-enhancing proteins are
provided by
the composition as a recombinant nucleic acid molecule, an appropriate
concentration of
a nucleic acid molecule expressing one chondrogenesis-enhancing protein is an
amount
which results in at least about 1 pg of protein expressed per mg of total
tissue protein at
the site of delivery per pg of nucleic acid delivered, and more preferably, an
amount which
results in at least about 10 pg of protein expressed per mg of total tissue
protein per p.g
of nucleic acid delivered; and even more preferably, at least about 50 pg of
protein
expressed per mg of total tissue protein per pg of nucleic acid delivered; and
most
preferably, at least about 100 pg of protein expressed per mg of total tissue
protein per
pg of nucleic acid delivered. One of skill in the art will be able to adjust
the concentration
of proteins and/or nucleic acid molecules in the composition depending on the
types and
number of proteins to be.provided by the composition, and the delivery vehicle
used.
In another embodiment of the cartilage repair product of the present
invention, a
cartilage-inducing composition can also contain a factor that non-covalently
attaches to
one or more of any ofthe chondrogenesis-enhancing proteins or recombinant
nucleic acid
molecules in the composition and thus, modify the release rate of the factor.
Such factors
include, but are not limited to, any ground substance or other polymeric
substance. As
used herein, a ground substance is defined as the non-living matrix of
connective tissue,
which includes natural polymers and proteoglycans. Natural polymers include,
but are not
limited to collagen, elastin, reticulin and analogs thereof. Proteoglycans
include, but are
not limited to any glycosaminoglycan-containing molecules, and include
chondroitin
sulfate, dermatan sulphate, heparan sulphate, keratan sulphate and hyaluronan.
Preferred
ground substances include, but are not limited to, type I collagen, type II
collagen, type
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III collagen, type IV collagen and hyaluronic acid. Preferred other polymeric
substances
include, but are not limited to, poly(lactic acid) and poly(glycolic acid).
In a further embodiment, the cartilage-inducing composition can include one or
more types of cells which are provided to further enhance chondrogenesis at
the site of
the cartilage lesion. Such cells include, but are not limited to,
fibrochondrocytes,
chondrocytes, mesenchymal precursors, and any other cell that can serve as a
chondrocyte
precursor. Such cells can be associated with the composition and the matrix by
any of the
methods described above. In one embodiment, at least some of the cells are
transformed
with a recombinant nucleic acid molecule encoding a chondrogenesis-enhancing
protein
to form a recombinant cell.
The cartilage repair product ofthe present invention also includes a cartilage
repair
matrix. The cartilage repair matrix is the component of the cartilage repair
device which
provides a vehicle for delivery of the cartilage-inducing composition to the
site of a
cartilage lesion and a suitable scaffold upon which cartilage repair and
regeneration can
occur. In a preferred embodiment, the cartilage repair matrix is
bioresorbable.
According to the present invention, a cartilage repair matrix can be formed of
any
material that is suitable for in vivo use, and which provides the above-
described
characteristics of a cartilage repair matrix for use with a cartilage-inducing
composition
of the present invention. The matrix can be formed of materials which include,
but are not
limited to, synthetic polymers and/or a ground substance. Preferred ground
substances
include natural polymers and proteoglycans. Natural polymers include, but are
not limited
to collagen, elastin, reticulin and analogs thereof. Proteoglycans include,
but are not
limited to, any glycosaminoglycan-containing molecules. Particularly preferred
glycosaminoglycans include chondroitin sulfate, dermatan sulphate, heparan
sulphate,
keratan sulphate and hyaluronan. Other preferred ground substances include,
but are not
limited to, type I collagen, type II collagen, type III collagen, type IV
collagen and
hyaluronic acid. Preferred synthetic polymers include poly(lactic acid) and
poly(glycolic
acid).
In one embodiment of the present invention, the cartilage repair matrix
includes
collagen. Preferably, the matrix contains from about 20% to about 100%
collagen by dry
weight of the matrix, and more preferably, from about 50% to about 100%
collagen by
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dry weight of the matrix, and even more preferably, from about 75% to about
100%
collagen by dry weight of the matrix. In one embodiment, a suitable cartilage
repair
matrix includes collagen from bovine tendon.
A cartilage repair matrix suitable for use in the present invention can
include a
material as described above which is in any suitable form for use in repairing
a cartilage
lesion, including a sponge, a membrane, a film or a gel. In one embodiment, a
suitable
cartilage repair matrix includes autograft tissue, allograft tissue and/or
xenograft tissue.
A cartilage repair product of the present invention is usefi~l for repairing a
variety
of defects in cartilage, including both tears and segmental defects in both
vascular and
avascular cartilage tissue. The product is particularly useful for repairing
defects in
hyaline (e.g., articular) and/or fibrocartilage (e.g., meniscal). Examples of
various types
of cartilage tears and segmental defects for which the cartilage repair
product of the
present invention can be used are illustrated in Figs. 1-4. Briefly, Fig. 1
shows a meniscal
radial tear (Fig. 1 A); a meniscal triple bucket handle tear (Fig. 1 C); and a
longitudinal tear
in the avascular area of a meniscus (Fig. 4A) . A meniscal segmental lesion is
illustrated
in Figs. 2A and 2B. Fig. 3A additionally schematically illustrates a cross
section of the
meniscus, which includes vascular, semi-vascular and avascular regions for
which, prior
to the present invention, only tears in the vascular region were repairable.
Therefore, since cartilage defects (i.e., lesions) can occur in a variety of
shapes,
sizes, and locations, a cartilage repair matrix suitable for use in a
cartilage repair product
of the present invention is of a shape and size sufficient to conform to a
specific defect in
the cartilage of the patient to be treated. Preferably, the cartilage repair
matrix, when used
in the repair of a cartilage defect, achieves a geometry at the defect site
that is suitable to
provide a therapeutic benefit to the patient. Such a therapeutic benefit can
be any
improvement in a patient's health and well being that is related to a
correction of the
cartilage defect, and preferably, the therapeutic benefit includes the repair
of the defect
such that the natural configuration of the cartilage is at least partially
restored.
In the case of a cartilage tear, the cartilage repair matrix is typically
configured as
a sheet. The sheet is preferably of a shape and size suitable for insertion
into the tear and
to cover the entire tear surface. One embodiment of a sheet type matrix is
schematically
illustrated in Fig. 3B. The use of such a matrix to repair an avascular
longitudinal tear is
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schematically illustrated in Fig. 4B. Preferably, the matrix provides an
immediate
mechanical repair of the tear, a surface for interacting the cartilage-
inducing composition
with the natural cartilage tissue, and a scaffold upon which chondrogenesis
can occur.
Additionally, the matrix preferably imparts upon the product a mechanical
stability
5 sufficient to allow the product to be anchored into the lesion. Prior to the
present
invention, a tear in meniscal vascular cartilage as shown in Figs. lA and 1C
was repaired
by suture repair and resection as illustrated in Fig. 1B and 1D, respectively.
Additionally,
prior to the present invention, if the tear occurred in avascular cartilage
tissue (or in semi-
vascular tissue as shown in Fig. 3A), the tear would have been considered
"irreparable".
10 The cartilage repair product of the present invention advantageously allows
for the repair
of tears in both avascular and vascular cartilage and when used in vascular
cartilage, the
product enhances the rate and quality of the repair as compared to previously
used
products and methods, such as shown in Figs. 1B and 1D.
In the embodiment in which the cartilage repair matrix is configured as a
sheet, the
15 matrix preferably has a thickness of from about 0.1 mm to about 3 mm, and
more
preferably, from about 0.5 mm to about 2 mm. The thickness can, of course, be
varied
depending on the configuration of the tear which is to be repaired. In this
embodiment,
the matrix can be prepared by applying an aqueous dispersion of matrix
material into a
mold, for example, wherein the mold sets the appropriate thickness for the
sheet. Such
20 a method is described in Example 4. In a preferred embodiment, the matrix
is prepared
from an aqueous dispersion of from about 0.2% to about 4% collagen by weight,
and
more preferably, the matrix is prepared from an aqueous dispersion of from
about 0.5%
to about 3% collagen by weight.
In the case of a segmental defect in cartilage (i.e., any defect that is
larger and of
25 a different shape than a tear), the cartilage repair matrix is typically
configured to achieve
a suitable geometry that repairs the defect, and includes matrices which are
configured to
replace damaged cartilage which has been removed. Prior to the present
invention, repair
of a segmental defect or indeed, any defect which occurred in the avascular
region of
meniscal tissue, typically involved the removal of the damaged tissue, such as
by partial
30 or complete excision of the meniscus (i.e., a meniscectomy). Excision was
then
sometimes followed by a replacement prosthetic meniscus, but until the present
invention,
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such methods were unable to regenerate endogenous-type cartilage in the
avascular region
of the meniscus. The cartilage repair matrix useful for segmental cartilage
defects (i.e.,
"non-tear" defects) is preferably of a shape and size suitable for providing
an immediate
mechanical repair of the defect, a surface for interacting the cartilage-
inducing
composition with the natural cartilage tissue, and a scaffold upon which
chondrogenesis
can occur. Additionally, the matrix preferably imparts upon the product a
mechanical
stability sufficient to allow the product to be anchored into the lesion. A
cartilage repair
matrix suitable for use for repairing segmental defects in the cartilage
repair product of
the present invention is described in detail in U.S. Patent 5,681,353 to Li et
al. which is
incorporated herein by reference in its entirety. Other preferred cartilage
repair matrices
are described in the Examples section. In one embodiment, a cartilage repair
matrix used
to repair a segmental defect in meniscal cartilage contains a porous ground
substance
composite which includes collagen and has the shape and mechanical
characteristics
suitable to repair a meniscus lesion.
In one embodiment, a cartilage repair matrix suitable for use in the repair of
segmental defects has a tapered shape. Such a matrix typically varies in
thickness from
about 0.5 mm to about 3 mm at its thinnest region to from about 4 mm to about
10 mm
at its thickest region. Such a matrix typically has a density of from about
0.07 to about
0.5 grams matrix per cm3, and more preferably, from about 0.1 to about 0.25
grams matrix
per cm3, wherein g/cm3 represents the number of grams in a cubic centimeter of
matrix.
Figs. 2A and 2B illustrate a cartilage repair matrix configured to repair a
segmental defect
in a meniscus.
In a preferred embodiment, the cartilage repair matrix of the present
invention is
porous, which enhances the ability of the matrix to serve as a delivery
vehicle for the
cartilage-inducing composition and particularly, as a scaffold for
chondrogenesis, such as
by allowing for the ingrowth of cells into the matrix. Preferably, the pore
size is sufficient
to maintain the desired mechanical strength of the matrix, while allowing
sufficient
ingrowth of cells for regeneration of cartilage at the lesion. The porosity of
the matrix can
vary depending on the configuration of the matrix, but typically, the matrix
has a pore size
of from about 10 ~.m to about 500 pm. When the matrix is configured to repair
a tear
defect, the pore size is typically from about 10 pm to about 100 pm. When the
matrix is
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52
configured to repair a segmental defect, the pore size is typically from about
50 pm to
about S00 pm.
When the cartilage repair matrix is configured as a sheet, it is preferably
not cross
linked. When the cartilage repair matrix is configured to repair a segmental
defect,
however, the matrix can be cross-linked, such as by artificial cross-linking
methods,
although such cross-linking is not required. a cartilage repair matrix can be
cross-linked
by any suitable agent which includes, but is not limited to, formaldehyde,
glutaraldehyde,
dimethyl suberimidate, carbodiimides, mufti-functional epoxides,
succinimidyls, Genipin,
poly(glycidyl ether), diisocyanates, acyl azide, ultraviolet irradiation,
dehydrothermal
treatment, tris(hydroxymethyl) phosphine, ascorbate-copper, glucose-lysine and
photo-
oxidizers. In one embodiment, a cartilage repair matrix is cross-linked with
an aldehyde.
Another embodiment of the present invention relates to a product for the
repair
of cartilage lesions which includes: (a) a cartilage repair matrix as
described in detail
above; and, (b) a cartilage inducing composition associated with the matrix
which includes
cells that have been cultured with a mixture of the chondrogenesis-enhancing
proteins as
previously described herein. The cartilage-inducing composition can be any
ofthe above-
described cartilage-inducing compositions useful in a product of the present
invention.
Preferably, the cells to be cultured with the mixture of proteins are cells
which are
involved in chondrogenesis, and include, but are not limited to,
fibrochondrocytes,
chondrocytes, mesenchymal precursors, and any other cell that can serve as a
chondrocyte
precursor. Such cells are preferably cultured in vitro prior to their
association with a
cartilage repair matrix, under conditions effective to allow the cells to
interact with the
proteins and initiate chondrogenesis by the cells. Effective culture
conditions include, but
are not limited to, effective media, bioreactor, temperature, pH and oxygen
conditions that
permit interaction of the proteins and cells and initiation of chondrogenesis
processes by
the cells. An effective, medium refers to any medium in which a cell is
cultured provide
such a result. Such medium typically comprises an aqueous medium having
assimilable
carbon, nitrogen and phosphate sources, and appropriate salts, minerals,
metals and other
nutrients, such as vitamins. Cells can be cultured in conventional
fermentation
bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates.
Culturing can be
carried out at a temperature, pH and oxygen content appropriate for a the
cell. Such
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53
culturing conditions are within the expertise of one of ordinary skill in the
art. In another
aspect of this embodiment of the present invention, a cartilage repair matrix
is cultured in
vitro together with the cells and mixture of proteins prior to implantation
into a cartilage
lesion in vivo. In a further embodiment, the cells that have been cultured
with the mixture
of proteins can be associated with the cartilage repair matrix in conjunction
with additional
chondrogenesis-enhancing proteins and/or recombinant nucleic acid molecules
encoding
such proteins as described above.
Another embodiment of the present invention relates to a product for the
repair
of vascular and avascular meniscus tears. Such a product includes: (a) a
cartilage repair
matrix comprising collagen and configured as a sheet; and, (b) a cartilage-
inducing
composition associated with the matrix. The cartilage-inducing composition can
be any
of the above-described cartilage-inducing compositions useful in a product of
the present
invention. Each of the chondrogenesis-enhancing proteins is provided as a
protein or by
a recombinant nucleic acid molecule encoding the protein operatively linked to
a
transcription control sequence. In one embodiment, the cartilage repair matrix
is formed
of a collagen sponge. When the lesion to be repaired is in the avascular
region, the
product can additionally include a time controlled delivery formulation as
described in
detail above.
Yet another embodiment of the present invention relates to a method for repair
of
cartilage lesions. The method includes the steps of implanting and fixing into
a cartilage
lesion a product which includes: (a) a cartilage repair matrix; and (b) a
cartilage-inducing
composition associated with the matrix. The cartilage-inducing composition can
be any
of the above-described embodiments of a cartilage-inducing composition
suitable for use
with a product of the present invention. In one embodiment, a cartilage-
inducing
composition useful in the method of the present invention includes a mixture
of proteins
including: transforW ing growth factor ~i 1 (TGF~31), bone morphogenetic
protein (BMP)-
2, BMP-3, and BMP-7, wherein the quantity of the TGF(31 in the mixture is from
about
0.01 % to about 99.99% of total proteins in the mixture; wherein the quantity
of the BMP-
2 in the mixture is from about 0.01% to about 10% of total proteins in the
mixture;
wherein the quantity of the BMP-3 in the mixture is from about 0.1% to about
15% of
total proteins in the mixture; and, wherein the quantity of the BMP-7 in the
mixture is
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from about 0.01% to about 10% of total proteins in the mixture. Various
alternate and
additional aspects of this embodiment of a cartilage-inducing composition have
been
described in detail above. In particular, in one aspect of this embodiment,
the ratio of
TGF~31 to all other proteins in the mixture of proteins is at least about
1:10, and more
S preferably, at least about 1:3, and more preferably, at least about 1:1, and
even more
preferably, at least about 10:1, weight for weight (w/w).
In another embodiment, the cartilage-inducing composition suitable for use in
the
method of the present invention includes a mixture of proteins including: (a)
a bone-
derived osteogenic or chondrogenic formulation of proteins; and, (b) an
exogenous TGF(i
protein, wherein the exogenous TGF(3 protein is present in an amount
sufficient to
increase cartilage induction by the composition over a level of cartilage
induction by the
bone-derived osteogenic or chondrogenic protein formulation in the absence of
the
exogenous TGF~3 protein. Various alternate and additional aspects of this
embodiment
of a cartilage-inducing composition have been described in detail above. In
particular, in
one aspect of this embodiment, the TGF(3 protein is TGF/31, and the ratio of
TGF~i 1 to
all other proteins in the mixture of proteins is at least about 1:10, and more
preferably, at
least about 1:3, and more preferably, at least about 1:1, and even more
preferably, at least
about 10:1, weight for weight (w/w).
In another embodiment, the cartilage-inducing composition suitable for use in
the
method of the present invention includes a mixture of proteins including: (a)
a TGF(i
protein; and, (b) at least one bone morphogenetic protein (BMP), wherein the
ratio of the
TGF~i protein to the BMP protein is greater than about 10:1. Various alternate
and
additional aspects of this embodiment of a cartilage-inducing composition have
been
described in detail above. In particular, in one aspect of this embodiment,
the TGF~i
protein is TGF~31, and the ratio of TGF~31 to the BMP protein is greater than
about 100:1,
and more preferably, greater than about 1000:1, and even more preferably,
greater than
about 10,000:1, weight for weight (w/w).
The method of the present invention is useful for repairing any of the
cartilage
lesions described above, including both articular and meniscal cartilage
lesions, and both
avascular and vascular defects. The step of implanting is performed using
surgical
techniques known in the art, and typically involves inserting the repair
product directly
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into the tear when the matrix is configured as a sheet, and involves a more
complex
process of removing damaged tissue and implanting the repair product when the
matrix
is configured to repair a segmental defect. The step of fixing can include
attaching the
product to the cartilage at the site of the lesion by any means suitable for
attaching a
S matrix as described herein to cartilage or tissue surrounding the cartilage
in vivo. Such
a means for attaching can include, but is not limited to application of
bioresorbable
sutures, application of non-resorbable sutures, press-fitting, application of
arrows,
application of nails, or application of a T-fix suture anchor device. Examples
5 and 7-9
describe the method ofthe present invention when the cartilage repair matrix
is configured
10 as a sheet and used to repair meniscal and articular cartilage. Example 6
describes the
method of the present invention which is used to repair meniscal and articular
cartilage
when the repair matrix is configured to repair a segmental defect. Figs. 2A
and 2B
illustrate the implantation and fixation of a cartilage repair product into a
segmental defect
in meniscal cartilage. Figs. 4A and 4B schematically illustrate the repair of
a longitudinal
15 tear in avascular meniscal tissue using a cartilage repair product
configured as a sheet.
In one embodiment ofthe method ofthe present invention, a cartilage lesion
which
is a segmental defect is repaired by using two cartilage repair products of
the present
invention. In this embodiment, a segmental defect, and preferably a meniscal
segmental
defect, is repaired by trimming damaged cartilage tissue away to form a
suitable interface
20 for implantation of the repair devices. A first cartilage repair product
having a matrix
configured as a sheet is implanted and fixed along the defect (e.g., along the
meniscal rim
when the defect is in vascular cartilage and this cartilage has been removed).
A second
cartilage repair matrix configured to replace the segmental defect, or a
cartilage repair
product having a matrix configured to replace the segmental defect, is
implanted and fixed
25 to the first product configured as a sheet. The sheet provides an interface
in which cells
can quickly infiltrate and react with the cartilage repair composition. In
this embodiment,
the cartilage repair product configured as a sheet contains a cartilage repair
composition
as described herein, and the cartilage repair product configured to repair the
segmental
defect may or may not be associated with the cartilage repair composition, as
deemed
30 necessary by the surgeon. Fig. 8 schematically illustrates the concurrent
use of both a
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56
cartilage repair product configured as a sheet and a cartilage repair product
configured to
repair a segmental defect.
Another embodiment of the method of the present invention relates to a method
for repair of avascular meniscus lesions. The method includes the steps of
implanting and
fixing into a cartilage lesion of the avascular region of a meniscus a
cartilage repair
product as described herein, wherein the cartilage repair matrix is configured
as a sheet,
and wherein the cartilage repair product further includes a time controlled
delivery
formulation which is associated with the matrix in conjunction with the
cartilage-inducing
composition.
Another embodiment of the method of the present invention is a method for
enhanced repair ofvascular meniscus lesions. The method includes the steps
ofimplanting
and fixing into a cartilage lesion in the vascular region of a meniscus a
cartilage repair
product as described herein, wherein the cartilage repair matrix includes
collagen and is
configured as a sheet. The present inventors have discovered that the use of
the cartilage
1 S repair product of the present invention to repair vascular lesions
measurably enhances the
rate of repair of the vascular lesion as compared to the rate of repair of a
meniscus lesion
repaired in the absence of the product. According to the present invention, a
measurable
enhancement of the rate of repair is any measurable improvement in the time
between Day
0 of the repair (i. e., the day the product is implanted into the patient) and
the day on which
it is determined that suitable cartilage tissue growth has occurred at the
lesion as
compared to a vascular lesion that is repaired in the absence of the product
of the present
invention. Suitable cartilage tissue growth is defined as an initial
indication of enhanced
blood vessel formation, production of fibrochondrocytes, induction of cellular
infiltration
into the product, induction of cellular proliferation, and production of
cellular and spatial
organization to form a three-dimensional tissue that more nearly represents
endogenous
cartilage tissue from the site of the lesion. In a preferred embodiment, the
product of the
present invention measurably enhances the rate of repair of a vascular
cartilage lesion, as
compared to a vascular lesion that is repaired in the absence of the product
of the present
invention, by at least 25%, and more preferably, by at least about 50%, and
more
preferably by at least about 100%.
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57
In addition, the use of the cartilage repair product of the present invention
to
repair vascular cartilage lesions results in a measurable enhancement of the
quality of
repair of the vascular lesion as compared to the quality of repair of a lesion
repaired in the
absence of the product. A measurable enhancement in the quality of repair of
the vascular
lesion is defined as any measurable improvement in quality of cartilage
formation at the
site of the lesion, with an improvement being defined as development of a more
normal
cartilage tissue, which can be indicated by enhanced blood vessel formation,
production
of fibrochondrocytes, induction of cellular infiltration into the product,
induction of
cellular proliferation, and production of cellular and spatial organization to
form a three-
dimensional tissue that more nearly represents endogenous cartilage tissue
from the site
of the lesion.
More particularly, the quality of repair of cartilage tissue according to the
present
invention can be evaluated as follows.
The quality of meniscal cartilage repair (i.e., fibrocartilage cartilage) can
be
evaluated by histological analysis of the tissue at the repair site on a scale
of 0 to 4 based
on the following parameters: I. Histological cartilage staining in the defect,
II. Cellular
infiltration into the collagen implant, and III. Integration into the
endogenous meniscus.
These scales according to the present invention are defined as follows:
Score % of cells within implant
that stain with
Azure at pH = 1
4 75-100
3 50-75
2 25-50
1 10-25
0 ~ 0_~0
Score % of cells that have infiltrated
the
collagen implant
4 75-100
3 50-75
2 25-50
1 10-25
0 0-10
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58
Score % of the repaired site
that integrates
with the endogenous meniscus
4 75-100
3 50-75
2 25-50
1 10-25
0 0_10
Preferably, a measurable enhancement in the quality of repair of a meniscal
lesion
is defined as a higher score in at least one of the parameters defined above
as I, II and III,
with a "4" being the highest score in each parameter, than a lesion repaired
in the absence
of the product of the present invention. More preferably, a measurable
enhancement in
the quality of repair of a meniscal lesion is defined as a score of at least
1, and more
preferably at least 2 and more preferably at least 3, and most preferably at
least 4 in at
least one of the parameters defined above as I, II and/or III, as compared to
a lesion
repaired in the absence of the product of the present invention.
The quality of osteochondral repair (articular cartilage defects) can be
evaluated
by histological analysis of the tissue at the repair site on a scale of 0 to 4
based on the
following parameters: : I. defect filling with bone, II. thickness of the
repaired cartilage,
and III. repaired cartilage integration with the endogenous cartilage. These
scales
according to the present invention are defined as follows:
Score % of defect that is filled
with bone
4 75-100
3 50-75
2 25-50
1 10-25
0 0-10
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Score % of repaired cartilage
that is
thickness of endogenous
cartilage
4 75-100
3 50-75
2 25-50
1 10-25
0 0-10
III.
Score % of the repaired cartilage
that
integrates with the endogenous
cartilage
4 75-100
3 50-75
2 25-50
1 10-25
0 0-10
Preferably, a measurable enhancement in the quality of repair of an
osteochondral
lesion is defined as a higher score in at least one of the parameters defined
above as I, II
and III, with "4" being the highest score, than a lesion repaired in the
absence of the
product of the present invention. More preferably, a measurable enhancement in
the
quality of repair of an osteochondral lesion is defined as a score of at least
1, and more
preferably at least 2 and more preferably at least 3, and most preferably at
least 4 in at
least one of the parameters defined above as I, II or III, as compared to a
lesion repaired
in the absence of the product of the present invention.
The grading scale utilized for bone-inductive activity in the rodent
subcutaneous
assay, which was developed by Sulzer Biologics, Inc., is shown in Example 10,
Table 8.
The grading scale utilized for cartilage-inductive activity in the rodent
subcutaneous assay,
which was also developed by Sulzer Biologics, Inc., is shown in Example 10,
Table 9.
Such grading scales can also be used to optimize compositions for use in the
present
invention and to evaluate the predicted in vivo efficacy of such compositions.
It is to be noted that the term "a" or "an" entity refers to one or more of
that
entity; for example, a protein refers to one or more proteins, or to at least
one protein.
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As such, the terms "a" (or "an"), "one or more" and "at least one" can be used
interchangeably herein. It is also to be noted that the terms "comprising",
"including", and
"having" can be used interchangeably.
The following examples are provided for the purposes of illustration and are
not
S intended to limit the scope of the present invention.
EXAMPLES
Example 1
10 The following example demonstrates that a naturally derived mixture of
proteins
isolated from demineralized bovine bones (BP) induces spheroid formation and
chondrogenesis in vitro in the mesenchymal precursor cell lines, IOTl/2 and
CZC12.
Murine C3H/lOTl/2 (ATCC No. CCL-226) embryonic mesenchymal stem cells
and CZC12 adult myoblast cells (ATCC No. CRL-1772; derived from leg muscle)
were
15 obtained from the American Type Tissue Collection. IOTl/2 and CZC12 cells
were
proliferated in the presence of 10% and 15% FB S, respectively. Micromass
cultures were
performed as follows. Briefly, trypsinized cells were resuspended in media
containing
FB S at a concentration of 10' cells/ml, and 10 gl of cells were placed in the
center of a 24
well microtiter tissue culture dish. After 2-3 hours at 37 ° C, 1 ml of
DMEM (for l OT 1/2)
20 or 1:1 DMEM:F-12 (for CZC12) media containing 1% Nutridoma and various
concentrations ofBP (prepared as described in Poser and Benedict, WO 95/13767,
ibid.)
were added. BP was present for the initial 48 hours and was not subsequently
added.
The results presented in Table 1 show that BP concentrations greater than or
equal
to 20 ng/ml induced spheroid formation in >90% of l OT 1/2 micromass cultures.
In >90%
25 of CZC12 micromass cultures, BP concentrations greater than or equal to 100
ng/ml induce
spheroid formation.
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TABLE 1
BP Concentration (ng/ml)
Cell 0 10 20 50 100 >100
1 OT1 - - + + + +
/2
CZC~2 - - - +/- + +
- = spheroid formation in 0% of micromass cultures
+/- = spheroid formation in <90% of micromass cultures
+ = spheroid formation in >90% of micromass cultures
For CzC,2 cells, the resulting spheroids were placed in Bouin's fixative for
24 hours
and histology and immunocytochemistry was performed. The myosin F1.652
antibody
was purchased from the Iowa Hybridoma Bank.
More specifically, for histology, spheroids were dehydrated and fixed for 20
minutes in absolute methanol at 4° C. Fixed sections were infiltrated
and polymerized
using the glycol methacrylate embedding technique. The polymerized plugs were
then
sectioned at 5 mm thickness using a JB-4 Sorvall microtome. Sections were
mounted on
silate-coated slides and stained with 0.2% Azure II at pH 1.
For immunocytochemistry, spheroids were snap-frozen in a 100% isopentane/dry
ice solution, sectioned at 5 pm thickness using a Reichert-Jung cryostat, and
mounted on
silane-coated slides. Frozen sections were then fixed in 1 % paraformaldehyde
for 20 min.
rinsed in 0.05 M Tris-CI (pH 7.4), blocked with 1% BSA for 20 min. at room
temperature, and incubated with either goat or mouse primary antibody for 1
hour at room
temperature. After rinsing, the sections were blocked with 10% normal rabbit
serum for
20 min. at room temperature. The sections were then treated with 1:2,000
biotinylated,
rabbit anti-goat IgG followed by incubation with a 1:100 streptavidin-
conjugated alkaline
phosphatase. Each incubation was far 30 min. at room temperature. The mouse-
antibody
treated slides were incubated with an unlabeled rabbit anti-mouse (rat
absorbed) antibody
and then incubated with an alkaline phosphatase anti-alkaline phosphatase
antibody. Each
incubation was for 30 minutes at room temperature. The reaction was visualized
with an
alkaline phosphatase substrate, New Fuchsin. The slides were counterstained
with Gill's
# 1 hematoxylin for 10 sec., dehydrated through graded alcohols, and cleared
in Americlear
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xylene substitute. Polyclonal goat primary antibodies for type II collagen
were diluted
1:200 in 0.5 M Tris-HCl (pH 7.4), 1% BSA, and 1% sodium azide prior to use.
Chondrogenesis is indicated by positive staining of sulfated proteoglycans
with
Azure at pH 1, morphology of a rounded cell type encompassed by a terntorial
matrix,
S and the presence of type II collagen. The collagen quantity was subjectively
graded in
duplicate samples with a '-' representing no stain detected and increasing
'+'s reflecting
increasing amounts and intensity of stain. Samples lacking the primary
antibody received
a ' ' score.
Table 2 shows that BP (500 ng/ml) induces chondrogenic markers in l OT 1/2
cells
over a period of 28 days.
TABLE 2
Days in culture
Marker Correlated with 2 7 14 21 28
Azure (pH Cartilage/sulfated- ++ + ++ +++
1) proteoglycan
Morphology Cartilage/rounded- ++ ++ ++ +++
cells
and territorial
matrix
Type II CollagenCartilage - ++++ ++++ ++++ ++++
Table 3 shows that in CZC12 cells, BP (1000 ng/ml) inhibits myosin production
(an
indicator of muscle) and induces type II collagen production (an indicator of
cartilage)
after three days in culture.
TABLE 3
Marker Indicative Presence of
of: Marker
Myosin Muscle -
Type II collagenCartilage ++++
Example 2
The following example demonstrates that a naturally derived mixture of
proteins
isolated from demineralized bovine bones (BP) inhibits myogenesis in a dose
dependent
manner in CZC12 cells.
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For myogenesis inhibition experiments, 25,000 CZC,2 cells were seeded in
triplicate
to a 24 well plate in DMEM media that contained 15% FBS. The next day (day 0),
this
media was replaced with media containing 1% Nutridoma +/- various
concentrations of
BP. Media was replaced every 2-3 days.
BP concentrations (0, 10, 20, 60, 100, 400, 1000, or 3000 ng/ml) were tested
for
the effect on CZCIZ myotube formation. As shown in Table 4, a BP concentration
of 10
ng/ml produced no morphological differences when compared to cultures lacking
BP.
However, at 20 and 60 ng/ml, BP substantially decreased the number of
myotubes.
Complete myotube inhibition was observed at BP concentrations above or equal
to 100
ng/ml.
TABLE 4
BP concentration (ng/ml)
0 10 20 60 100 400 1000 3000
++++ ++++ ++ + _ - -
Example 3
The following example demonstrates that a naturally derived mixture of
proteins
isolated from demineralized bovine bones (BP) quantitatively induces
chondrogenesis of
ATDCS cells in a dose dependent manner.
Inthis experiment,100,000 ATDCS cells/25 ~1 media (DMEM:Ham's F-12 (l : l);
5% FBS; 50 U/ml penicillin; SO mg/ml streptomycin) were seeded in triplicate
to a 24 well
plate (micromass culture). ATDCS cells were deposited by T. Atusmi and are
publicly
available as Deposit No. RCB0565 from the Riken Cell Bank, 3-1-1 Koyadai,
Tsukuba
Science City, 305 Japan. After 1 '/z hour, 1 ml of the media was added. The
next day, the
same media containing various concentrations of BP, 50 pg/ml ascorbic acid, S%
FBS,
and 10 mM ~3-glycerophosphate were added (day 0). This latter media was
replaced every
3-4 days. To those skilled in the art, slight modifications can be made to
this protocol to
obtain a similar result. For example, fewer cells (e.g., 25,000-50,000) could
be seeded to
the bottom of the well (e.g., not in micromass culture). Media containing a
serum
substitute, such as 1% Nutridoma (Boehringer Mannheim) could be used in place
of
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64
serum. Also, the cultures may contain different concentrations of serum or may
contain
or lack the ascorbic acid and/or the ~3-glycerophosphate.
To measure cartilage matrix production, an Alcian Blue staining method was
used
as previously described (von Schroeder et al, 1993, ibid.) with minor
modifications.
Briefly, after incubation with BP as described above, the culture media was
removed and
the cultures were washed three times with 1 ml of PB S. The cultures were then
faced with
10% neutral buffered formalin for 15 hours and washed twice with 0.5 N HCI.
Cultures
were stained for 1 hour at room temperature with a 0.5% Alcian Blue solution
(pH 1.4).
The stain was removed and the cultures were washed with PBS to remove the
unbound
stain. The blue stain was then extracted with guanidium HCl (4M, pH 1.7) at
70°C for
18 hours, followed by measurement of absorption at 595 nm. The cultures were
performed in triplicate. Using this method, glycosaminoglycan quantification
was
demonstrated to be proportional to 35504 incorporation (Lau, et al., 1993,
Teratology
47:555-563).
To determine the effect of BP on chondrogenesis, 0, 10, 20, 60, 100, 400, and
1000 ng/ml BP were added to chondrogenic ATDCS micromass cultures and, after 7
days,
the cultures were stained with Alcian Blue. Qualitative, microscopic
evaluation showed
that cultures lacking BP contained no positive staining and cultures
containing low BP
concentrations (10, 20, and 60 ng/ml) showed diffuse staining. With higher
doses ofBP
( 100, 400 and 1000 ng/ml BP), both staining intensity and the number of
positively stained
focal cell areas increased in a dose dependent manner. Cultures treated with
400 and 1000
ng/ml BP contained positively stained focal clusters consisting of rounded
cells
encompassed by a territorial matrix (data not shown). Negatively stained areas
were also
present. Quantitation of Alcian Blue staining (Fig. 5) revealed that BP
concentrations as
low as 10 ng/ml significantly increased Alcian Blue content compared to
control cultures
lacking BP (p < 0.0005). Maximal Alcian Blue staining was observed at 400
ng/ml BP.
BP also stimulates chondrogenesis in cultures containing a serum substitute;
Nutridoma, and this stimulation occurs over 14 days. In the absence of BP,
very little
Alcian Blue staining was observed at days 7 and 14 (Fig. 6). However, cultures
containing 1000 ng/ml BP significantly (p < 0.001) stimulated chondrogenesis
8.5 and
11.2 fold at days 7 and 14, respectively, compared to cultures lacking BP
(Fig. 6).
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To determine whether other fractions isolated from bone have a similar
activity as
BP, the following experiment was performed. BP was purified as previously
described
(Poser and Benedict, PCT Publication No. W095/13767). However, after the BP
containing fractions were collected from the HPLC, two fractions, IBP and
PIBP, that
S were more hydrophobic than BP, were also collected. The chondrogenic
activity of BP,
IBP and PIBP was tested on ATCDS cells. Fig. 7 shows that both IBP and PIBP
induce
significantly less (p < 0.005) chondrogenesis than BP.
To determine whether different lots of BP differed significantly for
chondrogenesis, two lots were tested at two concentrations. Table S shows that
the
10 chondrogenic activity is not significantly different between two different
BP lots.
TABLE 5
Absorbance
BP Lot # 200 ng/ml 300 ng/ml
15 97182 0.364 +/- 0.0190.535 +/- 0:031
98001 0.353 +/- 0.0410.484 +/- 0.043
Example 4
The following example describes a procedure for preparation of a cartilage
repair
20 product of the present invention, configured as a sheet.
Bovine tendon type I collagen, obtained from ReGen Biologics, Inc., was placed
in one syringe. The appropriate volume of 10 mM acetic acid was placed in
another
syringe. For sponges that contain BP, the appropriate dose of BP was placed in
the
syringe containing 10 mM acetic acid. The syringes were coupled, and the
contents of
25 each syringe were mixed to produce a 2% collagen (w/w) slurry. After an
overnight
incubation, the preparation was placed in molds of appropriate thickness,
frozen at -20 ° C
for more than 4 hours, and lyophilized until dry. Approximate implant
dimensions for the
sheet are length: 15.5 mm; width: 4.8 mm; and thickness: 1.2 mm.
30 Example 5
The following example describes the procedure for surgical implantation of a
cartilage repair product of the present invention which is configured as a
sheet.
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Adult castrated male Capra hircus goats weighing 50-70 pounds were used in
this
study. After pre-anesthesia administration, the goats were anesthetized with
an inhalation
anesthetic (IsoFlurane). The hind limbs of each goat were clipped, scrubbed,
and draped
in preparation for the surgical procedure. An incision was made over the
medial aspect
ofthe knee (stifle) joint. The sartorial fascia was dissected to expose the
medial collateral
ligament (MCL). A wedge shaped femoral bone block centered on the MCL was
created
with an oscillating saw and osteotome. The block was then drilled and tapped
for later
reattachment using a 3.5 mm bicortical screw. The bone block was then elevated
to
expose the surface of the medial meniscus leaving the coronary ligaments
intact. A
longitudinal tear was placed in the central, avascular portion of the meniscus
using a
scalpel. Four treatments were tested in the defect as follows. Group I:
nothing was
placed in the defect, but the defect was repaired using 1-0 non-absorbable
sutures and a
horizontal mattress technique (i.e., a conventional repair method); Group II:
a collagen
sheet was sutured into the defect using the method of Group I; and, Group III:
a collagen
sheet containing 35 pg BP was sutured into the defect using the method of
Group I.
After reduction and fixation of the bone block with a screw and washer, the
sutures were tied outside the capsulate. The subcutaneous tissues were then
closed with
absorbable suture and subcuticular 3-0 prolene skin closure. After recovery
from
anesthesia, the goats were placed in holding pens and then placed in an
outdoor facility
with unrestricted activity.
After 8 weeks, the animals were euthanized with a pentobarbitol sodium
solution
(100 mg/kg IV) solution. The menisci were fixed in a 10% neutral buffered
formalin
solution. Tissue sections for the menisci were then stained with H + E.
Group III defects that contained 3 5 pg BP were filled with tissue from the
superior
to inferior sides of the meniscus and the reparative tissue was integrated
into the
endogenous meniscus tissue. In addition, the endogenous meniscus tissue
immediately
adjacent to the defect was more cellular than normal endogenous meniscus,
indicating a
reparative response. In contrast, Group I and II defects remained empty; no
evidence of
healing was observed.
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Example 6
The following example describes the procedure for surgical implantation of a
cartilage repair product of the present invention which is configured to
replace a
segmental meniscal cartilage defect.
If a meniscal repair can not be accomplished (i.e., such as by using a product
of
the present invention configured as a sheet) due to the severity of the tear
or poor quality
of the tissue, then preparation of the meniscal rim is undertaken by removing
the torn
portions of the cartilaginous tissue. A cartilage repair product of the
present invention can
be configured to replace the segmental meniscal cartilage defect, thereby
serving to both
regenerate and repair meniscal cartilage. The surgical procedure, in the
absence of the
cartilage repair product of the present invention, has been previously
described by Stone
and Rosenberg (cites). The following procedure describes a modification ofthe
procedure
of Stone and Rosenberg, incorporating the use of a cartilage repair product of
the present
invention.
Briefly, the torn, fragmented pieces of native meniscal cartilage are removed,
and
the attachment sites for meniscal horns are anatomically placed or the natural
peripheral
rim and horns are preserved. Using the surgical techniques described by Stone
and
Rosenberg, a cartilage repair matrix configured as a collagen meniscus implant
(CMI;
having the shape and mechanical characteristics ofthe meniscus), which is
associated with
a cartilage repair composition according to the present invention, is
implanted and fixed
to the meniscal rim. During the surgery, the periphery of the meniscal implant
must be
attached securely enough to permit axial and rotational loads, and the
surrounding capsule
and ligaments of the knee joint must be neither excessively violated nor
constrained by the
fixation technique.
In some cases, a cartilage repair product of the present invention which is
configured as a sheet can be fixed to the meniscus rim using the same surgical
procedures
as described for the CMI product above. This combination use of the cartilage
repair
product of the present invention is illustrated schematically in Fig. 8. Use
of such a sheet
optimizes the integration between the meniscal rim and the CMI by providing a
thin,
porous collagen containing the cartilage repair composition. The sheet
provides an
interface in which cells can quickly infiltrate and react with the cartilage
repair
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composition. In this embodiment, the cartilage repair product configured as a
sheet
contains the cartilage repair composition of the present invention, and the
cartilage repair
product configured as a CMI may or may not be associated with the cartilage
repair
composition.
Example 7
The following experiment demonstrates that a cartilage repair product of the
present invention enhances and induces meniscus regeneration in both vascular
and
avascular meniscal tissue in vivo.
One hind leg was operated on in 1-2 year old female sheep. Briefly, the
surgical
approach was to make an incision in the skin and subcutaneous tissues from the
distal
fourth of the femur distally to the proximal fourth of the tibia. A bone block
that
contained the origin of the medial collateral ligament was then removed from
the femoral
condyle. The tibia was abducted and externally rotated. Using a 3 mm dermal
biopsy
punch (Miltex), two defects were created in the vascular/avascular zone of the
medial
meniscus. The Collagen Meniscus Implant (CMI) was obtained from ReGen
Biologics.
Using a 4 mm dermal biopsy punch (Miltex), CMI sponges were cut to size. The 4
mm
implants were press-fit into the meniscal defects. CMI either lacked or
contained BP (69
pg/mg collagen). To produce CMI containing BP, the following protocol was
followed.
A saturating volume of 10 mM acetic acid solution that contained BP was added
to the
4 mm CMI sponges. The sponges were placed in a humidified environment at room
temperature for 30 minutes, frozen at -20 ° C for more than 4 hours,
and then lyophilized
until dry.
The animals were sacrificed after four weeks and the menisci were fixed in a
10%
neutral buffered formalin solution. Tissue sections were stained with Azure B
at pH 1 and
4.5. Cells infiltrated the CMI matrix both in the presence or absence of BP,
however,
histology results demonstrated that only undifferentiated fibroblast cells
were present in
the implant that lacked BP (data not shown). In contrast, the BP impregnated
implant
contained differentiated fibrochondrocytes (data not shown). Fibrochondrocytes
were
identified by rounded cell shape and cells surrounded by positively staining
lacunae. In
addition, CMI sponges that lacked BP contained cells that did not stain with
antibodies
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against type II collagen. In contrast, CMI sponges that contained BP contained
cells that
stained positively with antibodies against type II collagen.
Example 8
The following example demonstrates that a cartilage repair product of the
present
invention enhances and induces articular cartilage repair in vivo.
Five 8 month old (skeletally mature) New Zealand White Rabbits were used in
this
experiment. Bilateral defects, 8 mm long, 3 mm wide, and 3 mm deep, were
produced in
the trochlear groove of each knee. For each rabbit, a 2% collagen sponge 8 mm
long, 3
mm wide and 3 mm deep, was placed in each knee defect, with one knee receiving
the
sponge in the absence of BP, and one knee receiving the sponge containing 40-
45 ~g of
BP. Autologous fibrin was prepared from 50 ml of rabbit blood using the
alcohol
precipitation method according to Kjaergard et al., 1992, Surg. Gynecol. Obst.
175(1):
72-73. Approximately 20 pl of fibrinogen was placed on top of the collagen and
was
polymerized with 10 pl bovine thrombin (Gentrak). After 2 months, the animals
were
sacrificed.
A 500 ~m slab of the defect area was embedded in glycol methacrylate after
fixation in cold methanol and stained with azure B at pH 1 and 4.5. An
adjacent 500 pm
slab was fixed and decalcified with EDTA in 4% paraformaldehyde and embedded
in
paraffin. Serial sections were digested with chondroitinase ABC and stained
with
hemotoxilin and eosin.
Histologically, defects that contained the collagen sponge only produced more
immature tissue than defects that contained collagen + BP. Without added BP,
the bone
region was filled with fibrocartilage and fibroblastic tissue. In contrast,
defects containing
BP were filled with osteoblasts and contained a bone morphology. The cartilage
area, in
the absence of BP, was often 3 mm dep, rather than the normal 0.5-0.7 mm deep.
With
BP added, the cartilage surface thickness was nearly identical to the
endogenous cartilage
thickness. In addition, polarized light microscopy revealed that the BP
treated defects
contained a more mature collagen fiber architecture than defects that did not
contain BP.
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Example 9
The following example demonstrates that bone protein (BP) is a complex mixture
of proteins which includes at least: TGF~i 1, TGF(32, TGF~i3, BMP-2, BMP-3,
BMP-4,
BMP-5, BMP-6, BMP-7, CDMP, FGF-I, osteocalcin, osteonectin, BSP, lysyloxidase,
5 cathepsin L pre, albumin, transferrin, Apo A1 LP and Factor XIIIb.
The present inventors used standard techniques and reagents available in the
art
to identify these proteins within bone protein (See for example, "Current
Protocols in
Protein Science", Ed. JE Coligan et al.; 1995-1998, John Wiley and Sons, Inc.;
"Protein
Purification: Principles and Practice" Scopes and Verlas;1982). The proteins
which have
10 been identified as present in BP by at least one of these assays are
identified in Table 6.
TABLE 6
BP COMPOSITION
15 Compound Ma ss (kD) pl PresenceBP
in
Mass.
LiteratureExperimental ELISAImmunoblotSpec)
Sequencing
TGF~ Supertamily
20 TGFa~ 12.5b 12.5 YES YES
TGF(i2 12.7T 12 7.7T YES YES
TGFii3
YES
BMP-2 16-18b 16 7.9T NO YES
25 BMP-3 16-18b 31 8.5T YES YES
BMP-4 16-18b 7.7T YES
BMP-5 16-18b 8.3T YES
BMP-6 16-18b 1 ? 8.6T YES
BMP-~ 16-18b 17.5 8.1T YES YES
CDMP (pan) YES YES
Growth Factors
35 FGF-1 (acidic)16b 13 & 15 5.4 YES
Bone Matrix
Proteins
Osteocalcin 5,8 <10 YES YES
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Compound Ma ss (kD) pl PresenceBP
in
Mass.
LiteratureExperimental ELISAhrrnunoblotSpec)
Sequencing
osteonectin 32 33 NO YES
ssP ~ssP-u) 35T YES
Lysyloxidase
YES
Cathepsin L 37,4T 38 6.6 NO YES
Pre
Serum Proteins
Albumin 66T 68 5.6T YES YES
serotransferrin76T 80 6.5T YES YES
Precursor
Apo-A1-Lipoprotein28 36 5.6 YES
Factor Xlllb I $Q I YES
Precursor
I
Additionally, intracellular proteins identified in Bone Protein include
intracellular
proteins: Dynein associated protein, protamine II, histone-like protein, L6
(ribosomal
protein), and L32 (ribosomal protein). Other serum proteins that have been
identified in
BP include a2 microglobulin. Other extracellular matrix (bone matrix) proteins
that have
been identified include Frizzled related protein. All such proteins can be
included, if
desired, in a composition of the present invention.
Several preparations of protein mixtures or Bone Protein or derivatives
thereof
were examined to provide a gross estimate of the amount of several of the
proteins in
Table 6. Specifically, several different lots of BP and AX fractions that were
extracted
at pH 9.0, 9.5 and 10.0 (See Example 11) were examined by standard Western
blot
analysis using antibodies against TGF~i l, TGF~i2, BMP-3 and BMP-7. The
resulting
radiograph was scanned using a Sharp JX-330 scanner and the lane volume was
calculated
using ImageMaster ID software. To quantitate the amount of TGF~i 1 in BP and
its
derivatives, recombinant bovine TGF(31 was run as a standard (2, 4, 8 and 16
ng;
Promega) and anti-bovine TGF(31 was used. To calculate the amount of TGF~i 1,
TGF~i2,
BMP-3, or BMP-7 in any given sample, the following formula was utilized: (lane
volume
sample/lane volume standard TGF(31) X (quantity of standard TGF~31 loaded).
Anti-
bovine TGF(3 antibody was used. This method is a specific estimator of the
amount of
TGF~i 1 in the samples and a gross estimator of the quantities of TGF~32, BMP-
3 and
BMP-7 in the mixtures. Only gross estimates of TGF~i2, BMP-3 and BMP-7 can be
made, because bovine standards and bovine antibodies for each of these three
proteins are
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not available (human antibodies were used for these proteins, and quantities
were
estimated based on the TGF~i 1 standard). The low and high quantity estimates
for each
of the four proteins over all of the compositions tested is shown in Table 7.
For these
compositions, the ratio (w/w) of TGF~i 1 to all other proteins in the mixture
is from about
1:1000 to about 1:100.
Those skilled in the art recognize that bovine and human TGF~i-1 have
identical
amino acid sequences and, therefore, that bovine TGF(3-1 and rhTGF(3-1 should
have
identical activities. Those skilled in the art also recognize that high purity
TGF(3-1 can be
isolated from bovine bone using methods disclosed by Seyedin (Ogawa et al.,
Meth.
Enzymol., 198:317-327 (1991); Seyedin et al., PNAS, 82:2267=71 (1985)).
TABLE 7
TGFIi-1 TGFIi-2BMP-3 BMP-7
Low quantity estimate 0.9 8.4 3.8 1.5
(ng/Ng protein
in composition)
High quantity estimate 9.8 114.0 89.2 43.5
(ng/pg protein
in composition)
Low quantity estimate 0.09 0.84 0.38 0.15
(% of total
proteins)
High quantity estimate 0.98 11.4 8.9 4.3
(% of total
proteins)
Example 10
The following example demonstrates that the cartilage-inductive activity of
bone
protein (BP) and protein mixtures derived from the complex mixture of proteins
in BP is
superior to the cartilage-inductive activity of individual recombinant protein
components,
as determined using a standard in vivo rodent subcutaneous assay.
A. In the following experiment, a rodent subcutaneous assay and grading
system developed by Sulzer Biologics, Inc. (Denver, Colorado), was used to
evaluate the
cartilage- and bone-inductive capabilities of recombinant BMP-2 (provided to
Sulzer
Biologics, Inc. by Genetics Institute). The results were then compared to data
for BP
which was generated in the same assay and using the same grading system.
To perform the Sulzer Biologics, Inc. rat subcutaneous assay, a matrix is
prepared
from a suitable material. When the material was type I collagen, a dispersion
of 4% w/w
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73
collagen was prepared using bovine type I collagen and 1 % glacial acetic
acid. Collagen
discs/sponges were formed in molds, and then frozen and lyophilized. For the
assay, the
discs were loaded with the composition (e.g., bone protein, a subset of bone
protein, a
growth factor) by incubation of the disc with the composition at room
temperature for
about 30 minutes, followed by freezing and lyophilization of the discs. The
discs were
implanted into Long-Evans rats in subcutaneous positions. In all of the
experiments
described in this example and other examples below, 5-10 rats were used per
treatment
and the composition treated collagen disc/sponge (or other material in Example
14) was
implanted for three weeks. At the end of three weeks, the rats were
euthanized, and the
explants were surgically removed, histologically processed, and graded. The
grading scale
utilized for bone-inductive activity in the rodent subcutaneous assay, which
was developed
by Sulzer Biologics, Inc., is shown in Table 8. The grading scale utilized for
cartilage-
inductive activity in the rodent subcutaneous assay, which was also developed
by Sulzer
Biologics, Inc., is shown in Table 9.
TABLE 8
Zero No residual implanted sample found.
(0):
Section shows no silver stained deposits or those
deposits are associated with
acellular events, (e.g., dystrophic mineralization
of collagen fibrils).
Explants generally small, soft and avascular.
One Focal areas of silver stained mineralized tissues
(1): are of cellular origin. This may
include mineralized cartilage as well as mineralized
osteoid matrix.
Silver stained areas are randomly located throughout
the explant, and typically
encompass less than 50% of the explant.
Generally smaller than original implants.
Two Silver stained areas are mineralized cartilage or
(2): very early woven bone.
Osteoblasts appear in rows of only about 6 to 10
cells.
If osteoid is present, it is generally present on
less than 10% of the mineralizing
tissue in the section.
Small areas of hematopoietic marrow elements may
be visible (generally sinusoids
containing red blood cells).
Three Sheets of active osteoblasts, (e.g., cells are plump
(3): and cuboidal or polygonal)
generally consisting of 10 or more cells, appear
in less than 50% of the active
mineralized portion. They are generally not continuous.
Bone associated with osteoblasts is generally woven,
containing some osteocytes.
- Woven bone appears at outer regions of explant
and may have breaks of fibrous
tissue or mineralized cartilage <10~ of surface.
Some hematopoietic marrow elements may be visible.
(Hemopoietic cords and
sinusoids containing red blood cells.)
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Four Mineralized tissue at the periphery is generally
(4): not woven, but a mature band
containing lamellar bone.
Mature bone is associated with continuous osteoblast
surtaces in at least 50r6 of
bony area.
Osteoid contains active osteoblasts and a visible
osteoid matrix.
Bone marrow as evidenced by granulocytes, hemopoietic
cords and sinusoids is
common.
Evidence of osteoclastic resorption (presence of
osteoclasts and/or Howship's
lacunae).
Five Solid rim of mature bone with few breaks around
(5): outer edge of explant.
Mature bone contains osteocytes in organized patterns.
Mature bone contains wide dark staining (in TBO
stain) osteoid.
Osteoid seams are continuous with few breaks; very
tick with osteoblasts that may
be flattened.
Bone marrow contains hemopoietic cords packed with
cells, granulocytes,
sinusoids and adipocytes.
- Trabecular bone in marrow is resorbing and may
appear as focal areas with little
branching.
- Osteoclastic resorption is occurring on outer
edge of mature bone (presence of
osteoclasts and/or Howship's lacunae).
Explant center may contain mature woven bone or
be infarcted and largely
acellular.
No evidence of chondrocytes.
TABLE 9
A B C D E
0 No NIA NIA NIA
1 Yes No No No
2 Yes Yes No No
3 Yes Yes Yes No
4 Yes Yes Yes Yes
KEY: A=Score; B=Presence of mineralized tissue; C=some non-mineralized
cartilage;
D=non-mineralized cartilage area is comparable to bone area; cartilage is in
ring >50%
of circumference of explant; often heavy cartilage stain; E=non-mineralized
cartilage
area is greater than bone area; often very little or no mineralization
resulting from bone
or cartilage.
BP, administered at a dose of 10 pg on a collagen sponge, routinely scores
between 1.5 and 2.2 for cartilage, and between 2.0 and 2.5 for bone. In
contrast,
recombinant human BMP-2 gave the results shown in Table 10, revealing that BMP-
2 has
both a lower bone and cartilage score as compared to BP.
TABLE 10
BMP-2 Rat subcutaneous assay
1.0 Ng 3.5 Ng 10 Ng
Bone Score 1.210.1 1.310.1 1.710.2
Cartilage r 1.0 t 0.0 1.0 t 0.0 1.0 t 0.0
Score
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B. TGF(3-1 and -2 were initially identified by the ability to stimulate
chondrogenesis in vitro. However, it is known in the art that TGF(31-5, and
growth
factors such as FGF and PDGF, do not induce bone or cartilage in the rat
subcutaneous
5 assay, such as the in vivo rodent ectopic assay (e.g., both TGF~i-1 and -2
are unable to
initiate cartilage or bone formation; only fibrous tissue is observed). This
is confirmed by
the results shown below with recombinant TGF~31. The assay was performed using
Sulzer
Biologics, Inc. rat subcutaneous assay and grading system as described in
section (A)
above. Table 11 shows that recombinant human TGF~i 1 does not induce bone or
cartilage
10 in this assay. In contrast, Bone Protein (BP), administered at a dose of 10
pg, routinely
scores between 1.5 and 2.2 for cartilage, and between 2.0 and 2.5 for bone
(see examples
below).
TABLE 11
TGF(31 Rat subcutaneous assay
15 10 Ng 35 Ng
Bone Score 0.0 t 0.0 0.0 t 0.0
Cartilage Score 0.0 t 0.0 0.0 t 0.0
Example 11
20 The following example demonstrates that another mixture of proteins derived
from
a modified purification process for BP that contains BMP-2, BMP-3, BMP-7, and
TGF~i-
1 produces comparable bone, but greater quantities of cartilage as compared to
BP.
BP is normally purified using anion exchange (AX), cation exchange (CX) and
high performance liquid chromatography (HPLC) steps (PCT Publication No.
25 W095/13767, incorporated herein by reference in its entirety). Such a
method is described
in detail above and in U. S. Patent No. 5,290,763, incorporated herein by
reference in its
entirety.
Typically, proteins are eluted from the AX column at pH=8.5. For this
experiment, proteins were eluted from the AX column at pH 9.0, 9.5, and 10Ø
The
30 samples were then purified across the CX and HPLC columns as described
previously
(PCT Publication No. W095/13767). Each HPLC fraction that was extracted at the
above
pHs were assayed by Western blot for the presence ofBMP-2, BMP-3, BMP-7, TGF~i-
1,
and TGF~3-2.
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Extraction of the AX column with increasing pH resulted in increased quantity
of
BMP-2, BMP-3, BMP-7, and TGF(3-1 in BP. At a pH of 9.0, BMP-2 and BMP-7
quantities show the greatest increase, whereas TGF~3-1 and BMP-3 show limited
or no
increase compared to pH 8.5. Increasing quantities of these factors have no
significant
effect on the bone score and slightly increase the cartilage score (Table 12).
At pH 10,
the TGF(3-1 quantity increases much more than the increase for BMP-2, -3, and -
7, and
the pH 10 fraction shows the greatest cartilage score, indicating a specific
role for TGF~31
in cartilage induction within the mixture of proteins. The HPLC purified
fractions ( 10 ~ g)
were tested in the rat subcutaneous model, the results of which are shown in
Table 12.
TABLE 12
pH 8.5 9.0 9.5 10.0
Bone Score 2.0 t 0.0 2.2 t 0.2 2.0 t 0.0 2.1 t 0.3
Cartilage 1.5 t 0.2 2.0 t 0.1 1.9 t 0.1 2.3 t 0.1
Score
Example 12
The following example demonstrates that a composition purified from BP and
including BMP-2, Blue-3, BMP-7 and TGF~i-1, contains components required for
cartilage formation.
To obtain specific protein subsets of BP, proteins within BP were separated on
a
hydroxyapatite column. The void material contained proteins that eluted with
<120 mM
KP04 buffer. During the elution, the pH gradient ranged from 6.0-7.4. 10 ltg
of each
fraction was added to collagen sponges and the rodent subcutaneous assay was
performed
as described in Example 10 above. In addition, an equivalent quantity of each
fraction
was loaded onto a protein gel and a Western blot was performed with antibodies
against
BMP-2, BMP-3, BMP-7, and TGF~i-1 (Table 13). A (-) indicates no signal
detected and
a (+) indicates that a signal was detected.
TABLE 13
Marker Void Peak Wash BP
BMP-2 - + +
BMP-3 - + + +
BMP-7 - + + +
TGF~i-1 - + + +
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The data in Table 14 demonstrates that a BP fraction containing BMP-2, 3, 7
and
TGF~3-1, contains proteins which are required components for BP cartilage and
bone
inductive activity.
TABLE 14
Measure Void Peak Wash BP
Bone score 0.2 2.4 2.4 2.2 -
2.5
Cartilage O.O t 2.410.2 2.O t 2.210.2
score 0.0 0.0
Example 13
The following example demonstrates that increasing amounts of high levels of a
pure source of TGF(31, when added exogenously to a complex mixture of
osteogenic/chondrogenic proteins, leads to the progressive loss of bone and
mineralized
cartilage formation, and the progressive formation of non-mineralized
cartilage in vivo.
Bone Protein (BP) naturally contains TGF~31, in addition to a variety of other
proteins, as described in various examples above. Examples 11-12, however,
indicated
that TGF~i 1 may be particularly important for the chondrogenic abilities of
the
osteogenic/chondrogenic mixture. Therefore, the present inventors sought to
determine
whether an exogenous source of substantially pure TGF~i 1 (i. e., recombinant
or purified),
when provided in a high concentration relative to the amount of other
osteogenic/chondrogenic proteins in a mixture such as BP, would influence the
chondrogenic induction capabilities of the composition as a whole.
In this example, recombinant human TGF~31 (rhTGF(31) was added in increasing
amounts (0, 1 ~.g, 3. S pg, 10 fig) to 10 pg of Bone Protein (BP). The mixture
was then
placed on collagen sponges and the rodent subcutaneous assay was performed as
described above in Example 10. The results of the different ratios of rhTGF(31
to BP for
cartilage and bone induction are shown below in Table 15. It is noted that
since the
quantity of individual BMP proteins in BP ranges from about 0.01% to about 9%
(minimum and maximum quantities), the effective ratio of TGF~i 1 to at least
one BMP in
the mixture ranges from greater than 10:1 to greater than 1000:1.
Table 15
rh TGF(3-1:BP0:1 1:10 1:2.9 1:1
Bone Score 2.0 t 0.0 2.2 t 0.2 1.0 t 0.0 1.0 t 0.0
Cartilage 1.4 t 0.2 1.8 t 0.2 3.8 t 0.2 3.7 t 0.2
Score
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Table 15 shows that the osteogenic and chondrogenic activity previously
demonstrated herein for BP can be altered to progressively decrease bone
production and
increase cartilage production by the addition ofincreasing amounts of
exogenous TGF~i 1.
Surprisingly, at high concentrations of TGF~i 1, the composition is primarily
chrondrogenic, with very little osteogenic activity observed.
Example 14
The following example demonstrates that different cartilage repair matrices
combined with BP induce cartilage formation in vivo according to the present
invention.
A. This example demonstrates the use of a poly lactic acid:polyglycolic acid
material that contains BP for bone and cartilage formation.
A 60% (v/v) solution of polylactic acid: polyglycolic acid (PLGA) (50:50) in N-
Methyl Pyrrilidone was made. Collagen (6 mg) was pressed in a Delrin mold. The
PLGA
( 140 mg) was added and mixed. BP ( 100 fig) contained in a 10 mM HCl solution
( 100
microliters) was added and all components were mixed together to form a solid
disc. This
mixture was pressed into a mold and incubated at room temperature for one hour
and then
implanted into rats in the rat subcutaneous assay as described in section 5.
Histology was
performed after one month of implantation. Table 16 shows that BP combined
with the
PLGA matrix induces cartilage formation in this in vivo model.
TABLE 16
PLGA-collagen-BP
Bone score 2.6 t 0.7
Cartilage Score 1.2 t 0.5
B. In this assay, a 3% collagen Type I/ 1% collagen Type IV (Sigma)
composite sponge was made using the normal preparation method as described in
Example 4 of the application. This matrix, containing 10 ~g BP, was implanted
into rats
in the rat subcutaneous assay as described in section 5, and histology was
performed after
three weeks. Table 17 shows that BP combined with the matrix containing both
type I
and type IV collagen induces cartilage formation in this in vivo model.
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TABLE 17
Type I/IV
Bone Score 1.2 t 0.2
Cartilage Score 1.2 t 0.2
C. This experiment demonstrates the use of an injectable collagen gel
containing BP that induces bone and cartilage. A BP solution ( 1 mg/ml)
contained in 10
mM HCL (5 ml) was added to collagen (30 mg/ml). The solutions were mixed until
a
jelly-like consistency and were incubated at 4° C overnight. In the
absence of an incision,
100 microliters ofthe gel was injected subcutaneously into the rat. Table 18
demonstrates
that BP in a matrix in the form of a gel induces cartilage formation in this
in vivo model.
TABLE 18
Collagen gel + 100
Ng BP
Bone score 3.5 t 0.5
Cartilage Score 2.0 t 1.0
D. This experiment demonstrates that cartilage repair matrices containing
triethanolamine and collagen in the form of a gel (basic pH), combined with
BP, induce
cartilage formation in vivo.
For basic collagen preparation, 0.5 gram TEA was added to 99.5 gram dI water
to pH 9.9. To make a 2% collagen gel, 2.0 gram collagen was added to pH 8.8.
The
mixture was incubated at room temperature for one hour and then frozen and
lyophilized
48 hours. The mixture was reconstituted in 10 mM HCL (containing 35 micrograms
of
BP) as 3 and 6% collagen sponges. The gel was delivered through a #18 or #20
needle
in a 100 microliter volume. After drying, the resulting sponges were 80%
collagen and
20% TEA by weight. Table 19 demonstrates that both 3 and 6% collagen TEA
sponges
combined with BP induce cartilage in this in vivo system.
TABLE 19
6% collagen 3% collagen
TEA TEA
AED + 35 Ng BP + 35 Ng BP
Bone Score 3.0 t 0.8 2.3 t 1.0
Cartilage 2.3 t 0.3 2.3 t 0.3
Score
E. This experiment demonstrates that cartilage repair matrices containing
collagen at acidic pH in the form of a gel, combined with BP, induce cartilage
formation
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in vivo. To prepare the acidic pH collagen gel, to 995 ml dI water, 5.0 ml 1 M
H3P04 was
added, followed by 1.75 ml 1.00 N NaOH (mixture pH = 2.49). 10.0 g collagen
was
added to BP and mixed for 2 hours (final pH = 3.61). This makes a 1% collagen
gel,
which was frozen and lyophilized. A 60 mg collagen disc was loaded with 2 ml
dI water
5 and incubated 2 hours at room temperature. The resulting 3% collagen gel had
a pH of
3.7. The material was injectable using #18 and #20 gauge needles, but not with
#22 or
#25 gauge needles. A 100 microliter volume contained 3 5 gg BP. Table 20
demonstrates
that 3 % acidic collagen matrices in the form of a gel and combined with BP
induce
cartilage in this in vivo system.
TABLE 20
AED 3% collagen + 35
Ng BP
Bone score 3.0 t 0.0
Cartilage 1.8 t 0.3
score
While various embodiments of the present invention have been described in
detail,
it is apparent that modifications and adaptations of those embodiments will
occur to those
skilled in the art. It is to be expressly understood, however, that such
modifications and
adaptations are within the scope of the present invention, as set forth in the
following
claims:
