Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS FOR
THE TREATMENT AND REPAIR OF DEFECTS OR
hESIONS IN CA_RTII,AGE OR BONE USING FL1NCTIONAT BARRIER
TECHNICAL FIELD OF THE TNVENTION
This invention relates to the treatment and repair of defects or
lesions in cartilage and full-thickness defects or lesions in cartilage and
bone. More
specifically, this invention relates to methods for treating defects or
lesions (used
interchangeably herein) in cartilage and bone and to cartilage repair
compositions
comprising a matrix containing an anti-angiogenic agent as a "functional
barrier" to
prevent ingrowth of blood vessels from the underlying bone tissue into the new
cartilage tissue. The cartilage repair composition may also contain one or
more
proliferating agents and a transforming factor to promote proliferation and
transformation of cartilage repair cells to form new stable cartilage tissue.
Bone
repair compositions comprising a matrix containing an angiogenic factor to
stimulate blood vessel formation and an osteogenic factor to stimulate
formation of
bone may also be used to treat the bone portion of full-thickness defects. The
compositions and methods of this invention are particularly useful in the
treatment
of full-thickness defects found in severe osteoarthritis, and in other
diseases and
traumas that produce cartilage and bone injury. Other compositions and methods
of
this invention, using other anti-tissue factors as functional barriers, are
useful in
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treating other injuries and defects such as in periodontal disease, where it
is desired
to inhibit or delay growth of certain tissue.
Joints are one of the common ways bones in the skeleton are
connected. The ends of normal articulated bones are covered by articular
cartilage
tissue, which permits practically frictionless movement of the bones with
respect to
one another [L. Weiss, ed., t~ell and Tissue BioloQV (Munchen: Urban and
Schwarzenburg, 1988) p. 247].
Articular cartilage is characterized by a particular structural
organization. It consists of specialized cells (chondrocytes) embedded in an
intercellular material (often referred to in the literature as the "cartilage
matrix")
which is rich in proteoglycans, collagen fibrils of predominantly type II,
other
proteins, and water [Buckwalter et al., "Articular Cartilage: Injury and
Repair," in
jniurv and Repair of the Musculoskeletal Soft Tissues (Park Ridge, Ill.:
American
1 S Academy of Orthopaedic Surgeons Symposium, 1987) p. 465]. Cartilage tissue
is
neither innervated nor penetrated by the vascular or lymphatic systems.
However,
in the mature joint of adults, the underlying subchondral bone tissue, which
forms a
narrow, continuous plate between the bone tissue and the cartilage, is
innervated
and vascularized. Beneath this bone plate, the bone tissue forms trabeculae,
containing the marrow. In immature joints, articular cartilage is underlined
by only
primary bone trabeculae. A portion of the meniscal tissue in joints also
consists of
cartilage whose make-up is similar to articular cartilage fBeaupre, A. et al.,
Orthop. Rel. Res., pp. 72-76 (1986)].
Two types of defects are recognized in articular surfaces, i.e., full-
thickness defects and superficial defects. These defects differ not only in
the extent
of physical damage to the cartilage, but also in the nature of the repair
response
each type of lesion can elicit.
_ __m_._._.. . _.__._._____ ___..~.__.. .. _~._... __.._ ...
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Full-thickness defects of an articular surface include damage to the
hyaline cartilage, the calcified cartilage layer and the subchondral bone
tissue with
its blood vessels and bone marrow. The damage to the bone tissue may range
from
a fissure or crack to an enlarged gap in the bone tissue. Full-thickness
defects can
cause severe pain since the bone plate contains sensory nerve endings. Such
defects
generally arise from severe trauma or during the late stages of degenerative
joint
disease, such as osteoarthritis. Full-thickness defects may, on occasion, lead
to
bleeding and the induction of a repair reaction from the subchondral bone
[Buckwalter et al., "Articular Cartilage: Composition, Structure, Response to
Injury, and Methods of Facilitating Repair," in Articular Cartilage and Knee
Jninr
Function: Basic Science and Arthroscoy (New York: Raven Press, 1990) pp. 19-
56]. The repair tissue formed is a vascularized fibrous type of cartilage with
insufficient biomechanical properties, and does not persist on a long-term
basis
[Buckwalter et al. (1990), supra].
Superficial defects in the articular cartilage tissue are restricted to
the cartilage tissue itself. Such defects are notorious because they do not
heal and
show no propensity for repair reactions.
Superficial defects may appear as fissures, divots, or clefts in the
surface of the cartilage, or they may have a "crab-meat" appearance in the
affected
tissue. They contain no bleeding vessels (blood spots) such as are seen in
full-
thickness defects. Superficial defects may have no known cause, but often they
are
the result of mechanical derangements which lead to a wearing down of the
cartilaginous tissue. Mechanical derangements may be caused by trauma to the
joint, e.g., a displacement of torn meniscus tissue into the joint,
meniscectomy, a
Taxation of the joint by a torn ligament, malalignment of joints, or bone
fracture, or
by hereditary diseases. Superficial defects are also characteristic of early
stages of
degenerative joint diseases, such as osteoarthritis. Since the cartilage
tissue is not
innervated [Ham's HistoloQV (9th ed.) (Philadelphia: J.B. Lippincott Co.
1987),
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pp. 266-272] or vascular-ized, superficial defects are not painful. However,
although painless, superficial defects do not heal and often degenerate into
full-
thickness defects.
It is generally believed that because articular cartilage lacks a
vasculature, damaged cartilage tissue does not receive suffrcient or proper
stimuli to
elicit a repair response [Webber et al., "Intrinsic Repair Capabilities of
Rabbit
Meniscal Fibrocartilage: A Cell Culture Model", (30th Ann. Orthop. Res. Soc.,
Atlanta, Feb. 1984); Webber et al., ,1. Orthon Res., 3_, pp. 36-42 (1985)]. It
is
theorized that the chondrocytes in the cartilaginous tissue are normally not
exposed
to su~cient amounts of repair-stimulating agents such as growth factors and
fibrin
clots typically present in damaged vascularized tissue.
One approach that has been used to expose damaged cartilage tissue
to repair stimuli involves drilling or scraping through the cartilage into the
subchondral bone to cause bleeding [Buckwalter et al. (1990), ].
Unfortunately, the repair response of the tissue to such surgical trauma is
usually
comparable to that observed to take place naturally in full-thickness defects
that
cause bleeding, viz., formation of a fibrous type of cartilage which exhibits
insui~rcient biomechanical properties and which does not persist on a long-
term
basis [Buckwalter et al. (1990), supra].
A variety of growth factors have been isolated and are now available
for research and biomedical applications [see e.g., Rizzino, A., Dev. Biol.,
~,
pp. 411-422 (1988)]. Some of these growth factors, such as transforming growth
factor beta (TGF-l3), have been reported to promote formation of cartilage-
specific
molecules, such as type II collagen and cartilage-specific proteoglycans, in
embryonic rat mesenchymal cells in vitro [e.g., Seyedin et al., Proc. Natl.
Acad. Sri.
USA; $~, pp. 2267-71 (1985); Seyedin et al., J. Biol. Chem., ~6 , pp. 5693-95
(1986); Seyedin et al., J. Biol. Chem., ~ø.~, pp. 1946-1949 (1987)].
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Furthermore, a number of protein factors have been identified that
apparently stimulate formation of bone. Such osteogenic factors include bone
morphogenetic proteins, osteogenin, bone osteogenic protein (BOP), TGF-f3s,
and
recombinant bone inducing proteins.
Millions of patients have been diagnosed as having osteoarthritis,
i.e., as having degenerating defects or lesions in their articular cartilage.
Nevertheless, despite claims of various methods to elicit a repair response in
damaged cartilage, none of these treatments has received substantial
application
[Buckwalter et al. (1990), supra; Knutsor, et al., J. Bone and 3oint Sure., ~8-
~,
p. 795 (1986); Knutson et al., J. Bone and Joint Sure., ~7-~, p. 47 (1985);
Knutson
et al., Clin. Orthon., ,1~, p. 202 (1984); Marquet, Clin. Orthon., ~, p. 102
( 1980)]. And such treatments have generally provided only temporary relief.
Systemic use of "chondroprotective agents" has also been purported to arrest
the
progression of osteoarthritis and to induce relief of pain. However, such
agents
have not been shown to promote repair of lesions or defects in cartilage
tissue.
To date, treatment of patients suffering from osteoarthritis has been
directed largely to symptomatic relief through the use of analgesics and anti-
inflammatory agents. Without a treatment that will elicit repair of
superficial
defects in articular cartilage, the cartilage frequently wears down to the
subchondral
bone plate. At this phase of the disease, i.e., severe osteoarthritis, the
unremitting
nature of the pain and the significant compromise of function often dictates
that the
entire joint be excised and replaced with an artificial joint of metal and/or
plastic.
Some one-half million procedures comprising joint resection and replacement
with
an artificial joint are currently performed on knees and hips each year. [See
e.g.,
Graves, E. J., "1988 Summary; National Hospital Discharge Survey", v n
Data From Vit2~and Health Statistics, 1~, pp. 1-12 (June 19, 1990)].
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There is, therefore, a need for a reliable treatment for cartilage tissue
in superficial cartilage defects and for cartilage and bone tissue in full
thickness
defects, e.g., as found in cases of severe osteoarthritis.
In addition to cartilage tissue defects, there are other defects for
which improved treatment is required. One area in which improved treatment
methods are needed is in periodontal repair and regeneration. Currently,
physical,
usually membrane-based, barriers are used to prevent unwanted tissue ingrowth
between compartments in, for example, cases of severe paradontitis. [See,
e.g.,
Robert, P.M., and Frank, R.M., "Peridontal guided tissue regeneration with a
new
resorbable polyactic acid membrane," J. Periodonto, 65.5, pp. 414-422
(1994).].
Physical membranes are also used in orthopedic guided tissue regenerations.
[See,
e.g., Farso, R, et al., "Guided tissue regeneration in long bone defects in
rabbits,"
t~cta Orthoi?, ~3, pp. 66-69 (1992)]. However, these procedures are not
desirable
because the membranes are usually not biodegradable and a second surgical
intervention is thus necessary. In addition, the physical membranes that are
biodegradable are often associated with long-lasting adverse effects,
including
inflammation, and chronic foreign body reaction because the degradation
products
of the membrane lead to local chronic inflammatory responses, and in
association
with this, lead to inhibition of surrounding tissue differentiation processes.
There is
therefore a need for an improved method of assisting in bone regeneration for
periodontal and orthopedic repair.
SUMMARY OF THE INVENTION
The present invention solves the problems referred to above by
providing effective therapeutic methods and compositions to induce the repair
of
lesions in cartilage or bone of humans and other animals. Use of the methods
and
compositions of this invention also promote the healing of traumatic lesions
and
_. _. . ~ .. __...._.. _. ~_ ._..~.._.._.__.._ T
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forms of osteoarthritis which would otherwise lead to loss of effective joint
function
leading to probable resection and replacement of the joint.
- In general outline, the methods and compositions of this invention
for treating superficial cartilage defects or the cartilage portion of full-
thickness
S defects comprise filling the cartilage portion of the defect with a
cartilage repair
matrix containing an anti-angiogenic agent for inhibiting vascular ingrowth
such as
anti-invasive factor, metalloprotease inhibitor or antibodies against
angiogenesis
inducing factors. The cartilage repair matrix will be incorporated into the
animal
tissue and is generally biodegradable; it may also contain a proliferation
agent and a
transforming factor. The cartilage repair matrices of this invention are
particularly
useful for treating full-thickness defects and apparent superficial cartilage
defects
where there is a possibility of a crack or fissure in the bone below.
In another embodiment, the methods of this invention comprise heat-
treating the areas in a full thickness defect where bleeding has occurred to
create a
transient tissue barrier and then filling the defect with a cartilage repair
matrix. In
this embodiment, the anti-angiogenic agent may be omitted from the matrix.
The methods of this invention for repairing full-thickness defects in
joints also comprise, where necessary or desirable due to extensive bone
injury,
filling the defect in the bone portion of a full-thickness defect up to the
level of the
bone-cartilage interface with a matrix that will be incorporated into the
animal tissue
and is generally biodegradable. The bone repair matrix may contain angiogenic
and
osteogenic factors. The remaining cartilage portion of the defect is filled to
the top
of the cartilage surface with a cartilage repair matrix containing an anti-
angiogenic
agent for inhibiting vascular ingrowth. The cartilage repair matrix may also
contain
a proliferation agent and a transforming factor.
Treatment of superficial and full-thickness defects can be effected
during arthroscopic, open surgical or percutaneous procedures using the
methods of
this invention. According to certain methods of this invention, after
identification of
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_g_
a full thickness defect, the defect is treated by the steps of ( 1 ) filling
the bone
portion of the defect with a composition comprising a matrix containing an
angiogenic factor and an osteogenic factor packaged in an appropriate delivery
system, e.g., liposomes; and (2) filling the cartilage portion of the defect
with a
composition comprising a matrix, preferably biodegradable, containing an anti-
angiogenic agent and a transforming factor which is packaged in an appropriate
delivery system. In this second step, the matrix may be bonded to the surface
of the
cartilage portion of the full-thickness defect, for example, by using an
adhesion-
promoting factor, such as transglutaminase.
Treatment of periodontal disease may be also aided with the
methods and compositions of this invention. For example, in the treatment of
disease such as paradontitis, in which the periodontal ligament recesses and
toothnecks are exposed, the regeneration of periodontal connective tissue can
be
aided by filling the periodontal connective tissue space with a matrix
containing one
or more factors to stimulate periodontal tissue formation and one or more
inhibitors
of epithelium formation.
Bone regeneration following loss of teeth can also be aided by the
methods and compositions of this invention. In particular, the bone of the
maxillar
or mandibular ridge can be built up by filling the defect area with a bone-
inducing
biodegradable matrix containing angiogenic and osteogenic factors and
inhibitors
for migration and/or proliferation and/or differentiation of connective tissue
cells to
assist in the re-growth of bone tissue. Within the periodontal connective
tissue
compartment a biodegradable matrix containing one or more factors to stimulate
periodontal tissue formation can be used.
Methods and compositions of this invention can also be used to
prevent periarticular calcification and ossification following, e.g., joint
replacement,
and to prevent bone growth into cartilagenous growth plates in young animals
and
children with distal end bone fractures. In particular, the periarticular
connective
T
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_ g -
tissue space or cartilagenous growth space can be filled
with a matrix containing an anti-angiogenic factor or anti-
bone factors to prevent calcification and bony tissue
formation.
According to one aspect of the present invention,
there is provided use of a matrix containing an effective
amount of an anti-angiogenic agent for the manufacture of a
medicament for the treatment of full-thickness defects in
cartilage in animals.
According to another aspect of the present
invention, there is provided use of a matrix containing:
(a) an effective amount of a proliferation agent to
stimulate proliferation of repair cells and (b) an effective
amount of a transforming factor associated with a delivery
system that releases the transforming factor at a
concentration sufficient to transform repair cells into
chondrocytes, for the manufacture of a medicament for
treating full-thickness defects in cartilage in an animal,
wherein locations of bleeding in the animal have been heat
treated to create a transient biological membrane.
According to still another aspect of the present
invention, there is provided use of a matrix containing:
(a) an effective amount of anti-angiogenic agent to prevent
ingrowth of blood vessels into the cartilage, an effective
amount of a chemotactic agent to attract repair cells, (b)
an effective amount of a proliferation agent to stimulate
proliferation of repair cells, and (c) an effective amount
of a transforming factor associated with a delivery system
that releases the transforming factor at a concentration
sufficient to transform repair cells into chondrocytes for
the manufacture of a medicament for the treatment of defects
in cartilage in animals.
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According to yet another aspect of the present
invention, there is provided a composition for the treatment
of defects in cartilage comprising: (a) a biodegradable
matrix or matrix-forming material used to dress the area of
the defect or lesion in the cartilage; and (b),an effective
amount of an anti-angiogenic agent to prevent ingrowth of
blood vessels into the cartilage.
According to a further aspect of the present
invention, there is provided a composition for promoting
regeneration of connective tissue in an animal comprising a
matrix containing an effective amount of a factor to
stimulate connective tissue formation and an effective
amount of an anti-epithelial factor associated with a
delivery system to provide sustained release to inhibit
epithelial formation.
According to yet a further aspect of the present
invention, there is provided a composition for promoting
regeneration of bone in an animal comprising a matrix
containing effective amounts of an angiogenic agent, an
osteogenic agent associated with a delivery system, and an
anti-connective tissue factor to inhibit connective tissue
formation.
According to still a further aspect of the present
invention, there is provided a composition for the
prevention of periarticular calcification and ossification
of the connective tissue in a joint area of an animal
comprising a matrix containing an effective amount of an
anti-bone factor associated with a delivery system to
provide sustained release to inhibit bone formation.
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According to another aspect of the present
invention, there is provided use for preventing bone tissue
ingrowth through a fracture site into a cartilaginous growth
plate of a matrix containing an effective amount of an
anti-bone factor associated with a delivery system to
provide sustained release to inhibit bone formation.
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T)ETATI.R DESCRIPTION OF INVENTION
In order that the invention may be more fully understood, the
following detailed description is provided. In the description the following
terms
are used.
~tg~enic Factor -- as used herein, refers to any peptide,
polypeptide, protein or any other compound or composition which induces or
stimulates the formation of blood vessels and associated cells (such as
endothelial,
perivascular, mesenchymal and smooth muscle cells) and blood vessel-associated
basement membranes. In vivo and in vitro assays for angiogenic factors are
well-
~o~ in the art [e.g., Gimbrone, M. A., et al., J. Natl. Cancer Inst., Sue, pp.
413-
419 (1974); Klagsbrun, M. et al., Cancer Res., 36, pp. 110-113 (1976); Gross
et al.,
Proc. Natl. Acad. Sci. fUSAI, 80, pp. 2623-2627 (1983); Gospodarowicz et al.,
Proc N~11 Acad. Sci. ~f~JSA), 73, pp. 4120-4124 (1976); Folkman et al., Proc.
Natl. Acad. Sci. (USA), 76, pp. 5217-5221 (1979); Zetter, B. R.,
Nature~London),
~~ PP~ 41-43 (1980); Azizkhan, R. G. et al., ~ Ex,~. Med., ].~, pp. 931-944
( 1980)].
Anti-Angiogenic Agent -- as used herein, refers to any compound or
composition with biological activity that prevents ingrowth of blood vessels
from
the underlying bone tissue into the cartilage tissue, such as anti-invasive
factors,
cartilage-derived angiogenesis inhibitors, angiostatin, metalloprotease
inhibitors,
antibodies against angiogenesis-inducing factors (including bFGF and
endothelial
cell stimulating angiogenic factor (ESAF)), Suramin (Germanin~, Bayer Co.,
Germany), fumagillin, fumagillin analogues and AGM-1470 [Peacock, D.J. et al.,
Cellular Immunology, .LO, pp. 178-84 (1995)]. In vivo and in vitro assays to
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determine anti-angiogenic agents are well-known in the art [e.g., Moses, M.A.,
Clinical & Ex~l. Rheumatoloev, l llSu~pl. 8), pp. 567-69 (1993); Moses, M.A.
et
al., I. Cell Bio., 119121, pp. 475-82 (1992); Moses, M.A. et al., Science,
2~$,
pp. 1408-10 (1990); Ingber, D. et al., j~ature, 348 , pp. 555-57 (1990)].
Anti-Tissue Factors -- as used herein, refers to any compound or
composition with biological activity that selectively prevents unwanted growth
of
particular tissues. For example, anti-epithelial factors to selectively
inhibit
epithelium formation include anti-epithelial antibodies, vitamin A inhibitors,
anti-
retinol, anti-basement membrane antibodies, epidermal growth factor
inhibitors,
matrices enriched with fibronectin, and any other factors that inhibit
epithelial cell
proliferation, growth or differentiation or epithelium formation. [See, e.g.,
Adams,
J.C. and Watt, F.M., "Fibronectin inhibits the terminal differentiation of
human
keratinocytes", Nature, 340, pp. 307-09 ( 1989). J Anti-connective tissue
factors to
selectively inhibit connective tissue formation include anti-connective tissue
I S antibodies, antibodies against connective tissue-specific growth factors,
antibodies
against mesenchymal cell surface proteins, and factors inhibiting mesenchymal
cell
proliferation. Anti-bone factors to selectively inhibit bone tissue formation
include
anti-angiogenic factors, and monoclonal or polyclonal antibodies, or
combinations
thereof, against members of the TGF-(3 superfamily.
ArthroscoRY -- as used herein, refers to the use of an arthroscope to
examine or perform surgery on a joint.
Bone -- as used herein, refers to a calcified connective tissue
primarily comprising a network of deposited calcium and phosphate in the form
of
hydroxyapatite, collagen (predominantly type I collagen) and bone cells, such
as
osteoblasts and osteoclasts.
done Repair Cell -- as used herein, refers to a cell which, when
exposed to appropriate stimuli, will differentiate and be transformed into a
bone
cell, such as an osteoblast or an osteocyte, which forms bone. Bone repair
cells
____.~_~
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include perivascular cells, mesenchymal cells, fibroblasts, fibroblast-like
cells and
dedifferentiated chondrocytes.
Cartilage -- as used herein, refers to a type of connective tissue that
contains chondrocytes embedded in an intercellular material (often referred to
as the
"cartilage matrix") comprising fibrils of collagen (predominantly type II
collagen
along with other minor types, e.g., types IX and XI), various proteoglycans
(e.g.,
chondroitinsulfate-, keratansulfate-, and dermatansulfate proteoglycans),
other
proteins, and water. Cartilage as used herein includes articular and meniscal
cartilage. Articular cartilage covers the surfaces of the portions of bones in
joints
and allows movement in joints without direct bone-to-bone contact, and thereby
prevents wearing down and damage to apposing bone surfaces. Most normal
healthy articular cartilage is also described as "hyaline", i.e., having a
characteristic
frosted glass appearance. Meniscal cartilage is usually found in joints which
are
exposed to concussion as well as movement. Such locations of meniscal
cartilage
include the temporo-mandibular, sterno-clavicular, acromio-clavicular, wrist
and
knee joints [Gra3r's Anatomv (New York: Bounty Books, 1977)].
Cartilage Repair Cell -- as used herein, refers to a cell which, when
exposed to appropriate stimuli, will differentiate and be transformed into a
chondrocyte. Cartilage repair cells include mesenchymal cells, fibroblasts,
fibroblast-like cells, macrophages and dedifferentiated chondrocytes.
Cell Adhesion Promoting Factor -- as used herein, refers to any
compound or composition, including fibronectin and other peptides as small as
tetrapeptides which comprise the tripeptide Arg-Gly-Asp, which mediates the
adhesion of cells to extracellular material [Ruoslathi et al., ~g~_l, 44, pp.
517-518
( 1986)].
Chemotactic Agent -- as used herein, refers to any compound or
composition, including peptides, proteins, glycoproteins and glycosaminoglycan
chains, which is capable of attracting cells in standard in vitro chemotactic
assays
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[e.g., Wahl et al., Proc Natl Acad Sci USA; $~, pp. 5788-92 (198?);
Postlewaite
et al., J. Exp. Med., 6~5, pp. 251-56 (1987); Moore et al., Int. J. Tiss.
Reac., ~,
pp. 301-07 (1989)].
Chondrocvtes -- as used herein, refers to cells which are capable of
producing components of cartilage tissue, e.g., type II cartilaginous fibrils
and fibers
and proteoglycans.
Fihroblast growth factor lFGFI -- any member of the family of FGF
polypeptides [Gimenez-Gallego et al., Biochem. Bio~hvs. Res. Commun., X35,
pp. 541-548 (1986); Thomas et al., Trends Biochem. Sci.,1_l, pp. 81-84 (1986)]
or
derivatives thereof, obtained from natural, synthetic or recombinant sources,
which
exhibits the ability to stimulate DNA synthesis and cell division in vitro
[for assays
see, e.g., Gimenez-Gallego et al., 1986, supra; Canalis et al., J. Clin.
Invest., 81,
pp. 1572-1577 (1988)] of a variety of cells, including primary fibroblasts,
chondrocytes, vascular and corneal endothelial cells, osteoblasts, myoblasts,
smooth
muscle and glial cells [Thomas et al., 1986, supra]. FGFs may be classified as
acidic
(aFGF) or basic (bFGF) FGF, depending on their isoelectric points (pI).
miatrix -- as used herein, refers to a porous composite, solid or semi-
solid substance having pores or spaces sufficiently large to allow cells to
populate
the matrix. The term matrix includes matrix-forming materials, i.e., materials
which
can form matrices within a defect site in cartilage or bone. Matrix-forming
materials may require addition of a polymerizing agent to form a matrix, such
as
adding thrombin to a solution containing fibrinogen to form a fibrin matrix.
Other
matrix materials include collagen, combinations of collagen and fibrin,
agarose,
(e.g., Sepharose~), and gelatin. Calcium phosphates, such as tricalcium
phosphate,
hydroxyapatite or other calcium salts that form solid matrices may be used
alone or
in combination with other matrix materials in treating defects in bones.
embrane -- as used herein, refers to any material which can be
placed between the bone defect portion and the cartilage defect portion of a
full
T
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- thickness defect and which prevents cell migration and blood vessel
infiltration from
the bone defect portion into the cartilage defect portion of the full
thickness defect.
The membranes used in the methods and compositions of this invention for the
repair of full thickness defects are preferably biodegradable.
OsteQgenic Factor -- as used herein, refers to any peptide,
polypeptide, protein or any other compound or composition which induces or
stimulates the formation of bone. The osteogenic factor induces
differentiation of
bone repair cells into bone cells, such as osteoblasts or osteocytes. This
process
may be reached via an intermediary state of cartilage tissue. The bone tissue
formed
from bone cells will contain bone specific substances such as type I collagen
fibrils,
hydroxyapatite mineral and various glycoproteins and small amounts of bone
proteoglycans.
Proliferation (,mitogeni~~gent -- as used herein, refers to any
compound or composition, including peptides, proteins, and glycoproteins,
which is
capable of stimulating proliferation of cells in vitro. In vitro assays to
determine the
proliferation (mitogenic) activity of peptides, polypeptides and other
compounds are
well-known in the art [see, e.g., Canalis et al., J. Clin. Invest., pp. 1572-
77 (1988);
Gimenez-Gallego et al., Biochem. Bionh_'rs. Res. Commun., X35, pp. 541-548
( 1986); Rizzino, "Soft Agar Growth Assays for Transforming Growth Factors and
Mitogenic Peptides", in Methods Enz~mol., 46 (New York: Academic Press,
1987), pp. 341-52; Dickson et al., "Assay of Mitogen-Induced Effects on
Cellular
Incorporation of Precursors for Scavengers, ~g Novo, and Net DNA Synthesis",
in
Methods Enz~mol., 146A (New York: Academic Press, 1987), pp. 329-40]. One
standard method to determine the proliferation (mitogenic) activity of a
compound
or composition is to assay it in vitro for its ability to induce anchorage-
independent
growth of nontransformed cells in soft agar [e.g., Rizzino, 1987, supra].
Other
mitogenic activity assay systems are also known [e.g., Gimenez-Gallego et al.,
1986, supra; Canalis et al., 1988, supra; Dickson et al., 1987, su~a].
Mitogenic
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effects of agents are frequently very concentration-dependent, and their
effects can
be reversed at lower or higher concentrations than the optimal concentration
range
for mitogenic effectiveness.
Transformin Fg actor -- as used herein, refers to any peptide,
polypeptide, protein, or any other compound or composition which induces
differentiation of a cartilage repair cell into a chondrocyte. The ability of
the
compound or composition to induce or stimulate production of cartilage-
specific
proteoglycans and type II collagen by cells can be determined by in vitro
assays
known in the art [Seyedin et al., Proc. Natl. Acad. Sci. USA, 82, pp. 2267-71
(1985); Seyedin et al., Path. Immunol. Res., Z, pp 38-42 (1987)].
Transforming~Growth Factor Beta (TGF-f3) -- any member of the
family of TGF-(3 polypeptides [Derynck, R. et al., Nature, 33 I6, pp. 701-705
(1985);
Roberts et al., "The transforming growth factor-f3's", In Pe tn idegrowth
factors and
their receptors I (Berlin: Springer Verlag, 1990), p. 419)] or derivatives
thereof,
obtained from natural, synthetic or recombinant sources, which exhibits the
characteristic TGF-13 ability to stimulate normal rat kidney (NRK) cells to
grow and
form colonies in a soft agar assay [Roberts et al., "Purification of Type f3
Transforming Growth Factors From Nonneoplastic Tissues", in Methods for
Preparation of Media. Sunnlements_ and Substrata for Serum-Free Animal dell
Culture (New York: Alan R. Liss, Inc., 1984)] and which is capable of inducing
transformation of cartilage repair cells into chondrocytes as evidenced by the
ability
to induce or stimulate production of cartilage-specific proteoglycans and type
II
collagen by cells in vitro [Seyedin et al., 1985, supra].
This invention relates to compositions and methods for treating
defects or lesions in cartilage and bone. The compositions of this invention
comprise matrices having pores su~ciently large to allow cells to populate the
matrices.
. _. .T
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For use in the repair of cartilage as in superficial defects or the
cartilage layer in a full-thickness defect, the matrix contains an anti-
angiogenic
agent which has biological activity that prevents blood vessel growth into the
cartilage tissue, thereby preventing bone formation and inadequate repair of
the
cartilage tissue. The matrix may also contain a proliferation agent to
stimulate the
proliferation of cartilage repair cells in the matrix. Preferably, the
proliferation agent
also serves as a chemotactic agent.to attract cartilage repair cells to the
matrix.
Alternatively, the matrix may contain a chemotactic agent in addition to the
proliferation agent. In one preferred embodiment of this invention, the matrix
also
contains an appropriate concentration of a transforming factor, the
transforming
factor being contained within or in association with a delivery system which
erects
release of the transforming factor at the appropriate time to transform the
proliferated cartilage repair cells in the matrix into chondrocytes which
produce
stable cartilage tissue. The matrix may also contain a cell adhesion promoting
factor.
For cartilage repair matrices to be used in the repair of fixll-thickness
defects a chemotactic or proliferation agent may not be required and it may
not be
necessary to substantially delay the release of the transforming factor. In
firll
thickness defects, adequate access exists to the repair cells in the bone
underneath
and there is no need to recruit synovial cells for this purpose. The repair
cells from
the bony space will migrate quickly into the cartilage defect site.
Proliferation
agents and chemotactic factors may be included, however, if desired,
especially
where the defect area is large. Because repair cells will quickly populate the
defect
site, substantially delayed exposure to transforming factor is not as
important as in
superficial defects where more time is required to attract and proliferate
repair cells.
However, if the defect area is large, the transforming factor may be
sequestered to
ensure sufficient proliferation of repair cells throughout the defect area
prior to
exposure to the transforming factor. In addition, for the treatment of full-
thickness
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defects the anti-angiogenic agent and the transforming factor may be contained
in
the matrix both in free form and associated with a delivery system to provide
sustained concentrations over time.
In the case of full-thickness defects that extend significantly into the
underlying bone, the bone portion of the defect in preferably filled with a
bone
repair matrix prior to filling the cartilage portion of the defect with a
cartilage repair
matrix of this invention.
Matrix materials useful in the methods and compositions of this
invention for filling or otherwise dressing the cartilage or bone defects
include
fibrinogen (activated with thrombin to form fibrin in the defect or lesion),
collagen,
agarose, gelatin and any other biodegradable material which forms a matrix
with
pores sufl'lciently large to allow cartilage or bone repair cells to populate
and
proliferate within the matrix and which can be degraded and replaced with
cartilage
or bone during the repair process. In some instances, calcium phosphate
containing
compounds, such as tricalcium phosphate, and hydroxyapatite, as well as other
calcium salts that form solid matrices, may be used alone or in combination
with
other biodegradable matrix materials in treating bone defects.
The matrices usefial in the compositions and methods of this
invention may be preformed or may be formed in situ, for example, by
polymerizing
compounds and compositions such as fibrinogen to form a fibrin matrix.
Matrices
that may be preformed include collagen (e.g., collagen sponges and collagen
fleece),
chemically modified collagen, gelatin beads or sponges, a gel-forming
substance
such as agarose, and any other gel-forming or composite substance that is
composed of a matrix material that will fill the defect and allow cartilage or
bone
repair cells to populate the matrix, or mixtures of the above.
In one embodiment of this invention, the matrix is formed using a
solution of fibrinogen, to which is added thrombin to initiate polymerization
shortly
before use. A fibrinogen concentration of 0.5-5 mg/ml of an aqueous buffer
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solution may be used. Preferably, a fibrinogen solution of 1 mg/ml of an
aqueous
buffer solution is used. Polymerization of this fibrinogen solution in the
defect area
yields a matrix with a pore size sufficiently large (e.g., approximately 50-
200 pm)
so that cartilage or bone repair cells are free to populate the matrix and
proliferate
in order to fill the volume of the defect that the matrix occupies.
Preferably, a
sufficient amount of thrombin is added to the fibrinogen solution shortly
before
application in order to allow enough time for the surgeon to deposit the
material in
the defect area prior to completion of polymerization. Typically, the thrombin
concentration should be such that polymerization is achieved within a few to
several
(2-4) minutes since exposure of cartilage to air for lengthy periods of time
has been
shown to cause damage [Mitchell et al., J. Bone Joint Surd, 71~, pp. 89-95
(1989)]. Excessive amounts of thrombin should not be used since thrombin has
the
ability to cleave growth factor molecules and inactivate them. Thrombin
solutions
of 10-500 units per ml, and preferably 100 units per ml, of an aqueous buffer
solution may be prepared for addition to the fibrinogen solution. In a
preferred
embodiment of this invention, approximately 20 a 1 of thrombin ( 100 U/ml) are
mixed with each ml of a fibrinogen solution (1 mg/ml) approximately 200
seconds
before filling the defect. Polymerization will occur more slowly if a lower
concentration of thrombin is added. It will be appreciated that the amount of
thrombin solution needed to achieve fibrin polymerization within 2-4 minutes
can be
given only approximately, since it depends upon the environmental temperature,
the
temperature of the thrombin solution, the temperature of the fibrinogen
solution,
etc. Alternatively, where convenient, the thrombin may be added by placing it
on
top of the matrix solution after the solution has been placed in the defect
site and
allowing it to diffuse through the solution. The polymerization of the
thrombin-
activated matrix solution filling the defect is easily monitored by observing
the
thrombin-induced polymerization of an external sample of the fibrinogen
solution.
Preferably, in the compositions and methods of this invention, fibrin matrices
are
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formed from autologous fibrinogen molecules, i.e., fibrinogen molecules
derived
from the blood of the same mammalian species as the species to be treated. Non-
immunogenic fibrinogen from other species may also be used.
Matrices comprising fibrin and collagen or, more preferably, fibrin
and gelatin may also be used in the compositions and methods of this
invention.
Collagenous matrices may also be used in repairing cartilage defects,
including full
thickness defects. In a preferred embodiment of this invention, more solid
matrices,
such as those containing hydroxyapatite or tricalcium phosphate, are used in
repairing the bone portion of deep full-thickness defects.
When collagen is used as a matrix material, sufficiently viscous
solutions can be made, e.g., using Collagen-Vliess~ ("fleece"), Spongostan~,
or
gelatine-blood-mixtures, and there is no need for a polymerizing agent.
Collagen
matrices may also be used with a fibrinogen solution activated with a
polymerizing
agent so that a combined matrix results.
Polymerizing agents may also be unnecessary when other
biodegradable compounds are used to form the matrix. For example, Sepharose~
solutions may be chosen that will be liquid matrix solutions at 39-42°C
and become
solid (i.e., gel-like) at 35-38°C. The Sepharose should also be at
concentrations
such that the gel filling the defect has a mesh size to allow bone or
cartilage repair
cells to freely populate the matrix and defect area.
In the compositions of this invention used in cartilage repair, one or
more anti-angiogenic agents is added to the matrix solution in an appropriate
concentration range to prevent blood vessel growth into the cartilage tissue.
Anti-
angiogenic agents that may be used include any agent with biological activity
that
prevents ingrowth of blood vessels from the underlying bone tissue into the
cartilage tissue. Some examples of anti-angiogenic agents that may be useful
for
this invention are set forth above. The anti-angiogenic agent should be freely
available to provide immediate activity in the matrix and may also be present
in a
__ ~_
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sustained-release form, e.g., associated with a delivery system as described
below,
for prolonged activity.
One or more proliferation (mitogenic) agents may also be added to
the matrix solution used in cartilage repair. The proliferation agent or
agents should
be present in an appropriate concentration range to have a proliferative
effect on
cartilage repair cells in the matrix filling the defect. Preferably, the same
agent
should also have a chemotactic effect on the cells (as in the case of TGF-f3);
however, a factor having exclusively a proliferative effect may be used.
Alternatively, to produce chemotactic cell immigration, followed by induction
of
cell proliferation, two different agents may be used, each one having just one
of
those specific effects (either chemotactic or.proliferative).
Proliferation (mitogenic) agents useful in the compositions and
methods of this invention for stimulating the proliferation of cartilage
repair cells
include transforming growth factors ("TGFs") such as TGF-as and TGF-13s;
insulin-
1 S like growth factor ("IGF I"); acidic or basic fibroblast growth factors
("FGFs");
piatelet-derived growth factor ("PDGF"); epidermal growth factor ("EGF");
hemopoietic growth factors, such as interleukin 3 ("IL-3") and bone
morphogenic
proteins ("BMPs"), such as bone morphogenic protein-2 ("BMP-2") [Rizzino,
1987, supra; Canalis et al., su~r_a, 1988; Growth factors in bioloav and
medicine,
Ciba Foundation SX~ro~_ium, ~ (New York: John Wiley & Sons, 1985); Baserga,
R., ed., Cell ;growth and division (Oxford: IRL Press, 1985); Sporn, M.A. and
Roberts, A.B., eds., Pe tp ide growth factors and their rece~ t~ ors, Vols. I
and II
(Berlin: Springer-Verlag, 1990)]. However, these particular examples are not
limiting. Any compound or composition which is capable of stimulating the
proliferation of cells as demonstrated by an in vitro assay for cell
proliferation is
useful as a proliferation agent in this invention. Such assays are known in
the art
(e.g.; Canalis et al., 1988, ~uora; Gimenez-Gallego et al., 1986, ; Dickson
et al., 1987, bra; Rizzino, 1987, supra].
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Chemotactic agents useful in' the compositions and methods of this
invention for attracting cartilage repair cells to the cartilage defect
include, for
example, TGF-(3s, FGFs (acid or basic), PDGF, tumor necrosis factors (e.g.,
TNF-
a, TNF-13) and proteoglycan degradation products, such as glycosaminoglycan
chains [Roberts et al. (1990), supra; Growth factors in biol_o;"~",r and
medicine,
Foundation ~,ymposium, ~6_ (New York, 3ohn Wiley & Sons, 1985); R. Baserga,
ed., Cell grovv~~, and division (Oxford: IRL Press, 1985)]. Assays to
determine the
chemotactic ability of polypeptides and other compounds are known in the art
(e.g.,
Postlewaite et al., 1987, supra; Wahl et al., 1987, supra; Moore et al., 1989,
sulk].
In a preferred embodiment of this invention, the matrix used in
cartilage repair contains TGF-f3 as the proliferation agent and as the
chemotactic
agent. In particular, TGF-13I or TGF-f3II may be used as the proliferation and
chemotactic agent. Other TGF-l3 forms (e.g., TGF-f~III, TGF-f3IV, TGF-f3V,
etc.)
or polypeptides having TGF-(3 activity [see Roberts, 1990, supra] may also be
useful for this purpose, as well as other forms of this substance to be
detected in the
future, and other growth factors. For use as the proliferation agent and
chemotactic
agent, TGF-f3 molecules are dissolved or suspended in the matrix at a
concentration
of preferably 2-50 ng/ml of matrix solution, and most preferably, 2-10 ng/ml
of
matrix solution. Alternatively, BMP-2 may be used at a concentration of less
than
I ng/ml as a proliferation agent. It will be appreciated that the preferred
concentration of TGF-(3 or BMP-2 that will stimulate proliferation of
cartilage
repair cells may vary with the particular animal to be treated.
A transforming factor or factors may also be present in the matrix
solution used in cartilage repair so that after cartilage repair cells have
populated
the.matrix, the transforming factor will be released into the defect site in a
concentration sufficient to promote differentiation (i.e., transformation)
ofthe
cartilage repair cells into chondrocytes which form new stable cartilage
tissue.
Proper timing of the release of the transforming factor is particularly
important if
.. . . ...._._... . . _..._...T
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the transforming.factor can inhibit or interfere with the effectiveness of the
proliferation agent [see Roberts et al. (1990), supra].
Transforming factors useful in the compositions and methods of this
invention to promote cartilage repair include any peptide, polypeptide,
protein or
any other compound or composition which induces differentiation of cartilage
repair
cells into chondrocytes which produce cartilage-specific proteoglycans and
type II
collagen. The ability of a compound or composition to induce or stimulate
production of cartilage-specific proteoglycans and type II collagen in cells
can be
determined using assays tcnown in the art (E.g., Seyedin et al., 1985, supra;
Seyedin
et al., 1987, ~,~ra]. The transforming factors useful in the compositions and
methods of this invention include, for example, TGF-f3s, TGF-as, FGFs (acid or
basic) and BNlPs, including BMP-2. These transforming factors may be used
singly
or in combination. Dimers and multimers of these factors may also be used. In
addition, TGF-13 may be used in combination with EGF.
Where necessary, the properly timed release of the transforming
factor may be achieved by packaging the transforming factor in or with an
appropriate delivery system. Delivery systems useful in the compositions and
methods of this invention include liposomes, bioerodible polymers,
carbohydrate-based corpuscles, water-oil emulsions, fibers such as collagen
which
may be chemically linked to heparin sulfate proteoglycans or other such
molecules
to which transforming factors bind spontaneously, and osmotic pumps. Delivery
systems such as liposomes, bioerodible polymers, fibers with bound
transforming
factors and carbohydrate-based corpuscles containing the transforming agent
may
be mixed with the matrix solution used to fill the defect. These systems are
known
and available in the art [see P. Johnson and J. G. Lloyd-Jones, eds., Drug
Delivery
hems (Chichester, England: Ellis Horwood Ltd., 1987)]. Liposomes may be
prepared according to the procedure of Kim et al., Biochem. Bioy~h_ys. Acta,
~,
pp. 339-348 (1983). Other liposome preparation procedures may also be used.
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Additional factors for stimulating chondrocytes to synthesize the cartilage
tissue
components may be included with the transforming factor in the delivery
system.
The timing of transforming factor availability should be coordinated with the
speed
in which repair cells will proliferate and fill the defect site to be treated.
Where
substantial delay of the release of the transforming factor is not required,
the
transforming may be included in the matrix in both a freely available form and
associated with a delivery system to provide sustained release at the
appropriate
concentration.
In a preferred embodiment of this invention, the matrix used in
cartilage repair contains an anti-angiogenic agent, TGF->3 or BMP as the
proliferation and chemotactic agent, and TGF-(3 or BMP packaged in a delivery
system as the transforming factor. In particular, TGF-f3I or TGF-(3II or BMP-2
may be used as the proliferation and chemotactic agent and as the transforming
factor. Other TGF-f3 forms (e.g., TGF-f3III, TGF-fiIV, TGF-f3V, or any member
of
the TGF-~3 superfamily) or polypeptides having TGF-f3 activity (see Roberts,
1990,
may also be useful for this purpose, as well as other forms of this substance
to be detected in the future, and other growth factors. The anti-angiogenic
agent is
preferably contained within the delivery system containing the transforming
concentration of TGF-~i or BMP as well as in free form in the matrix.
In a preferred embodiment for cartilage repair, a TGF-f3
concentration of preferably 2-50 ng/ml of matrix solution, and most
preferably, 2-10
ng/ml of matrix solution, is used as a proliferation agent and as a
chemotactic agent.
A substantially higher concentration of TGF-f3 is also present in a
subsequently
releasable form in the matrix composition as a transforming factor.
Preferably, the
subsequent concentration of TGF-f3 is greater than 200 ng/ml of matrix and,
most
preferably, is greater than or equal to 500 ng/ml of matrix. Alternatively,
BMP may
be used as a transforming factor at a preferable concentration of 100-2000 ng
per
ml. It will be appreciated that the preferred concentration of TGF-f3 or BNiP
to
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induce differentiation of cartilage repair cells may vary with the particular
animal to
be treated.
It is necessary to stagger the exposure of the cartilage repair cells to
the two concentration ranges of TGF-(3, since TGF-f3 at relatively high
concentrations (e.g., greater than 200 ng/ml of matrix solution) may not only
transform cartilage repair cells into chondrocytes, but also will inhibit
chemotactic
attraction of cartilage repair cells; whereas at relatively low concentrations
(e.g., 2-
IO ng/ml), TGF-13 attracts cartilage repair cells and stimulates their
proliferation, but
will not induce transformation of cartilage repair cells into chondrocytes
which
produce cartilage tissue.
In a preferred embodiment of this invention, where necessary to
obtain the sequence of chemotaxis and proliferation, followed by
transformation,
TGF-fi is present both in a free, unencapsulated form and in an
e'r~capsulated, or
otherwise sequestered, form in the matrix. Preferably, for the purpose of
attracting
IS and inducing proliferation of cartilage repair cells in the matrix and
defect area,
TGF-13 molecules are dissolved or suspended in the matrix at a concentration
of 2-
10 ng/ml of matrix solution. To promote transformation of cartilage repair
cells in
the matrix into chondrocytes, TGF-fi molecules are also present in the matrix
sequestered in multi vesicular liposomes according to the method of Kim et
al.,
1983, sera, at a concentration of greater than 200 nglml of matrix solution,
and
preferably at a concentration of 500-800 ng/ml. The TGF-f3-loaded liposomes
are
disrupted when the attracted cartilage repair cells have populated the matrix
and
have started to degrade the matrix. During the degradation of the matrix, the
cartilage repair cells ingest and/or degrade the liposomes, resulting in the
release of
TGF-(3 at concentrations sufficient to induce the transformation of cartilage
repair
cells into chondrocytes.
The required two-stage delivery of chemotactic and proliferating
versus transforming concentrations of TGF-f3 may also be achieved by combining
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transforming concentrations of TGF-f3 with a bioerodible polymer.
Alternatively, a
pump, and preferably an implanted osmotic pump, may be used to control the
concentration of TGF-f3 in the defect and matrix. In this embodiment of the
invention, the pump controls the concentration of TGF-f3 in the matrix, i.e.,
the
pump may release TGF-13 at an initial chemotactic and proliferation
stimulating
concentration and at a subsequent transforming concentration. Preferably, the
transforming concentration of TGF-f3 is delivered by the pump approximately 1
to 2
weeks post-operatively. Delivery of the transforming factor into the defect
volume
is preferably localized to the matrix in the defect site.
The proliferation agents and, when used, the transforming factors in
the compositions of this invention are applied in the defect site within the
matrix.
Their presence is thus restricted to a very localized site. This is done to
avoid their
free injection or infusion into a joint space. Such free infusion may produce
the
adverse effect of stimulating the cells of the synovial membrane to produce
joint
1 S effusion.
In certain embodiments of this invention for treating full-thickness
defects, delayed exposure to transforming factor is not necessary. In many
full-
thickness defects, adequate access to repair cells exists and delayed exposure
to
transforming factor is less critical than with superficial defects where more
time is
required to attract and proliferate repair cells. However, in deep defects, it
may be
desirable to delay the exposure to transforming factor to allow repair cells
to
populate the entire defect site.
In the compositions of this invention used for bone repair, one or
more angiogenic factors is may be added to the matrix solution to stimulate
the
formation and ingrowth of blood vessels and associated cells (e.g.,
endothelial,
perivascular, mesenchymal and smooth muscle cells) and of basement membranes
in
the area of the bone defect. Angiogenic factors useful in the compositions and
methods of this invention for stimulating vascularization throughout the
deposited
T
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matrix in the area~of the bone defect include bFGF, TGF-l3, PDGF, TNF-a,
angiogenin or angiotropin. Heparin sulfate has been found to enhance the
angiogenic activity of bFGF. In a preferred embodiment of this invention, bFGF
is
dissolved, suspended or bound in a matrix at a concentration of 5-10 ng/mi of
matrix solution along with an amount of heparin sulfate sufficient to enhance
the
angiogenic activity of bFGF. The preferred concentrations for other angiogenic
factors are: 5 ng/ml of matrix solution for TGF-f3, 10 ng/ml of matrix
solution for
TNF-a, and 10 ng/ml of matrix solution for PDGF. However, bFGF in combination
with heparin sulfate is the most preferred angiogenic factor among the above
named
angiogenic factors.
An osteogenic factor may also be present in the matrix solution used
for bone repair so that after blood vessels and associated cells have
populated the
matrix, the osteogenic factor is released into the bone defect site as the
matrix is
degraded in a concentration sufFrcient to promote a process leading to the
eventual
development of osteoblasts and osteocytes. The osteogenic factor is
sequestered or
packaged in an appropriate delivery system within the matrix and is released
as the
matrix is degraded. The delivery systems used in the cartilage repair
compositions
are useful in the bone repair compositions of this invention, e.g., liposomes
or
carbohydrate-based corpuscles (see supra). In one embodiment of this
invention,
the matrix used in bone repair contains TGF-f3 packaged in a delivery system
as the
osteogenic factor, at a preferable concentration of 100 ng/m1 of matrix
solution.
Lower and higher concentrations of TGF-(3 may be used. In another embodiment,
the matrix used for bone repair contains BMP-2 packaged in a delivery system
as
the osteogenic factor, at a preferable concentration of 100-2000 ng/ml of
matrix
solution. In still another embodiment, the matrix contains FGF at an
appropriate
concentration packaged in a delivery system as an osteogenic factor.
Osteogenic factors useful in the bone repair compositions of this
invention include any peptide, polypeptide, protein or any other compound or
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composition which induces differentiation of bone repair cells into bone
cells, such
as osteoblasts and osteocytes, which produce bone tissue. The osteogenic
factors
useful in this invention include proteins such as TGF-f3 [Sampath, T. R. et
al., ,L,,
Biol. Chem., 265 ;~, pp. 13198-13205 ( 1990)], osteogenin [Luyten, F. P. et
al., ,~
Biol. Chem., _2,64151, pp. 13377-80 (1989)], bone morphogenic protein (BMP)
[Wang, E. et al., Proc. Natl. Acad. Sci. USA ~, pp. 2220-24 (1990)], FGF, and
TGF-f3 combined with epidermal growth factor (EGF).
The differentiation of mesenchymal cells induced by an osteogenic
factor may include the formation of intermediary tissues such as fibrous,
hyaline and
calcified cartilage; and endochondral ossification, which leads to the
formation of
woven bone tissue, which will become remodeled and transformed into mature
lamellar bone tissue. In some instances, bone may be formed directly from
mesenchymal cells without the appearance of an intermediary tissue. Within the
matrix, the process of bone tissue formation usually occurs 3 to 4 weeks after
blood
vessels have formed and infiltrated the matrix in response to the angiogenic
factor
present in the matrix. Although bone will grow into the bone defect site in
the
absence of added angiogenic and osteogenic factors (use of at least a matrix
material is desirable in large defects), the use of such factors substantially
speeds up
the repair process.
The matrix compositions described in this invention for repairing the
bone portion of a full-thickness defect in joints are also useful in treating
any defect
in bone tissue as is desirable. Such defects include bone fractures, joint
fractures,
non-unions and delayed unions, percutaneous arthrodesis, pseudo-arthrosis and
bone defects resulting from congenital defects, trauma, tumor infection,
degenerative disease and other causes of loss of skeletal tissue. The bone
repairing
matrix compositions are also useful for prosthesis implantation and
enhancement of
prosthesis stability, enhancement of ossec~integration of implant materials
used for
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internal fixation procedures, stabilization of dental implant materials,
healing
acceleration of ligament insertion, and spine or other joint fusion
procedures.
Fibronectin or any other compound, including peptides as small as
tetrapeptides, that contain the amino acid sequence Arg-Gly-Asp, may be used
as
S cell adhesion promoting factors [Ruoslathi et al., ~l , 44, pp. S I7-18
(1986)] in
order to enhance the initial adhesion of cartilage or bone repair cells to a
matrix
deposited in a defect site. Fibrin and certain collagen matrices already
contain this
sequence [Ruoslathi et al., 1986, supra]. When other biodegradable matrices
are
used, such cell adhesion promoting factors may be mixed with the matrix
material
before the matrix is used to fill or dress the defect. Peptides containing Arg-
Gly-
Asp may also be chemically coupled to the matrix material (e.g., to its fibers
or
meshes) or to a compound added to the matrix, such as albumin.
The compositions hereinbefore described are useful in methods to
induce cartilage or bone formation at a selected site of defect in cartilage
or bone
tissue of an animal.
T.he methods of this invention allow for a treatment of cartilage and
bone defects in animals, including humans, that is simple to administer and is
restricted in location to an affected joint area. The entire treatment may be
carned
out by arthroscopic, open surgical or percutaneous procedures.
To carry out the methods of treating defects or lesions in cartilage
and bone according to this invention, a defect or lesion is identified,
prepared, and
filled with the matrix compositions according to this invention.
For cartilage repair, the matrix composition may contain an anti-
angiogenic agent to prevent ingrowth of blood vessels. A proliferation
(mitogenic)
agent may also be present in the matrix composition at an appropriate
concentration
to stimulate the proliferation of cartilage repair cells in the matrix and
defect or
lesion The same agent may also, at this concentration, serve as a chemotactic
agent to attract cartilage repair cells, provided that the factor used has a
combined
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effect with respect to cell proliferation and chemotaxis (as does TGF-fi at 2-
ng/ml of matrix). Alternatively, two different agents may be present in the
matrix, one with a specific proliferative effect, and the other with a
specific
chemotactic effect. In an alternative embodiment, after the defect area is
dressed
5 with the matrix, the anti-angiogenic agent and, if desired, a proliferation
agent and a
chemotactic agent, may be injected directly into the matrix-filled defect
area.
Injection should be localized to the matrix and filled defect area to avoid
exposure
of cells of the synovial membrane to growth factors which could lead to cell
proliferation and joint effusion.
10 After the defect site is dressed with the matrix composition (and, in
the case of fibrin matrices, once the matrix has solidified) and, if required,
the anti-
angiogenic agent or the proliferation agent has been injected into the matrix-
filled
defect site, the joint capsule and skin incisions may be closed and the
arthroscopy or
open surgery terminated.
In a subsequent step of cartilage repair, the cartilage repair cells in
the matrix are exposed to a transforming factor at the appropriate time at a
concentration sufficient to transform the cartilage repair cells into
chondrocytes
which produce stable cartilage tissue. This may be accomplished by including
an
appropriate delivery system containing the transforming factor within the
matrix
composition as described above. Alternatively, the transforming agent may be
delivered by injection directly or by an osmotic pump into the matrix-filled
defect
area at the appropriate time. In a superficial cartilage defect with no access
to repair
cells from bone tissue, the transforming concentration should be made
available to
the cells approximately 1 to 2 weeks following the initial implantation of the
matrix
into the defect area. In a fiall-thickness cartilage defect, depending on the
size of the
defect, the transforming factor may be made available earlier. Additional
factors
may be added to the delivery system or directly injected in order to better
promote
.._ .____. ~
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synthesis of the cartilage matrix components at this time point. Also,
additional anti-
angiogenic agent may be included in the delivery system or directly injected.
Cartilage or bone defects in animals are readily identifiable visually
during arthroscopic examination of the joint or during simple examination of
the
lesion or defect during open surgery. Cartilage or bone defects may also be
identified inferentially by using computer aided tomography (CAT scanning) X-
ray
examination, magnetic resonance imaging (MRI) analysis of synovial fluid or
serum
markers, or by any other procedure known in the art.
Once a detect has been ident~ned, the surgeon may elect to surgically
modify the defect to enhance the ability of the defect to physicatfy retain
the
solutions and matrix material that are added in the treatment methods
described
herein. Preferably, instead of having a flat or shallow concave geometry, the
defect
has or is shaped to have vertical edges or is undercut in order to better
retain the
solutions and matrix materials added in the treatment methods described
herein.
According to the methods of this invention, the bone defect site of a
full-thickness defect may be filled up to the calcified cartilage layer at the
bone-
cartilage interface with a bone repair matrix composition such that a flat
plane is
formed. Filling the bone defect with a bone repair matrix is particularly
useful for
defects several millimeters or more deep. The bone repair matrix composition
may
contain an angiogenic factor and an osteogenic factor packaged in an
appropriate
delivery system.
The remaining cartilage portion of the defect is completely filled with
a matrix composition used to stimulate cartilage repair. The composition for
cartilage repair comprises a matrix material containing an anti-angiogenic
agent and,
if desired, a proliferation agent and a chemotactic agent. Anti-angiogenic
agents
useful in the compositions and methods of this invention include any agents
with
biological activity capable of inhibiting vascutarization. This invention
contemplates
that the anti-angiogenic agent may comprise one or more molecules capable of
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inhibiting angiogenesis. The composition used in this step may also contain a
transforming factor packaged in a delivery system and, if appropriate, in free
form
as well. In the most preferred method of cartilage repair of the invention,
the matrix
contains an anti-angiogenic factor (in free form and packaged in or associated
with
S a delivery system for sustained release), a proliferation agent, a
chemotactic agent
(which may be identical to the proliferation agent), and a transforming factor
that is
packaged in or associated with a delivery system that releases the
transforming
factor at a time that the repair cells populating the matrix have begun
remodeling
the intercellular substance. Preferred compositions are described above.
As described in U.S. Patent No. 5,270,300, the bone-cartilage
interface of a full-thickness defect may be separated with a physical
membrane,
preferably a biodegradable membrane, which is impermeable to cells (e.g., pore
sizes less than 5 pm), prior to filling the cartilage portion of a full
thickness defect.
The membrane is placed over the matrix-filled bone defect, and the edges of
the
membrane must be sealed to the perimeter of the defect in the region of the
cartilage-bone junction to prevent vascular ingrowth into the cartilage defect
area.
In this method, repair cells from the bone area are not readily available to
populate
the cartilage defect area and a proliferation agent and/or chemotactic agent
is
therefore necessary in the cartilage repair matrix to attract and stimulate
proliferation of repair cells from the synovium.
In a preferred embodiment of this invention for treating full-
thickness defects, no membrane is placed at the bone-cartilage interface. The
bone
portion of the defect may or may not be filled with a hone repair composition,
as
desired. The cartilage portion of the defect is filled with a matrix
composition
containing an anti-angiogenic agent and a transforming factor and, if desired,
a
proliferation agent, and/or a chemotactic agent, as discussed above. The anti-
angiogenic agent in the cartilage repair matrix composition acts as a
functional
barrier to prevent the ingrowth of blood vessels and the formation of bone in
the
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cartilage area, avoiding the necessity of a physical membrane and allowing the
migration of repair cells from the bone area.
In another embodiment of the methods of this invention for treating
full-thickness defects, the bone-cartilage interface is separated by a
transient
biological membrane, created at the bone-cartilage interface by a heated
instrument.
A heated instrument may be applied to locations of bleeding to coagulate the
blood
and form a layer of precipitated protein, thus resulting in a biological
physical
barner that prevents ingrowth of blood vessels and bone tissue formation in
the
defect site. Examples of heated instruments include, but are not limited to
heated
scalpel blade, heated scissors or heated forceps. The instrument should be
heated to
a temperature of about 200°C. The heated instrument should be applied
to the base
of the full thickness defect. In another embodiment, heat may be applied by a
C02,
N2 or Neodyruum-Yag laser. This heat-created transient biological membrane at
the bone-cartilage interface may be employed in addition to or in lieu of
including
an anti-angiogenic agent in the cartilage repair matrix. When the heat
treatment
method is used, a proliferation agent and/or chemotactic agent should be
included in
the cartilage repair matrix.
Chemical measures may enhance matrix adhesion. Such measures
include degrading the superficial layers of cartilage proteoglycans on the
defect
surface to expose the collagen fibrils of the cartilage so that they may
interact with
the collagen fibrils of the matrix (when a collagenous matrix is used) or with
the
fibrin fibrils of the matrix (when a fibrin matrix issued). The proteoglycans
on the
surface of the cartilage not only tend to interfere with adherence of a fibrin
or other
biodegradable matrix to the cartilage, but also inhibit thrombin activity
locally.
Advantageously, proteoglycan degradation products may also have a chemotactic
effect on repair cells [Moore, A.R. et al., Int. J. Tiss. Reac., XI(6), pp.
301-307
(1989)].
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According to one embodiment of the methods of this invention, the
surface of the defect is dried by blotting the area using sterile absorbent
tissue, and
the defect volume is filled with a sterile enzyme solution for a period of 2-
10
minutes to degrade the proteoglycans present on the surface of the cartilage
and
S locally within approximately 1 to 2 ,um deep from the surface of the defect.
Various enzymes may be used, singly or in combination, in sterile buffered
aqueous
solutions to degrade the proteoglycans. The pH of the solution should be
adjusted
to optimize enzyme activity.
Enzymes usefial to degrade the proteoglycans in the methods of this
invention include chondroitinase ABC, chondroitinase AC, hyaluronidase,
pepsin,
trypsin, chmotrypsin, papain, pronase, stromelysin and Staph V8 protease. The
appropriate concentration of a particular enzyme or combination of enzymes
will
depend on the activity of the enzyme solution.
In a preferred embodiment of this invention, the defect is filled with
a sterile solution of chondroitinase AC at a concentration of 1 U/ml and
digestion is
allowed to proceed for 4 minutes. The preferred concentration of
chondroitinase
AC is determined according to the procedure described in Example 1. Any other
enzyme used should be employed at a concentration and for a time period such
that
only superficial proteoglycans down to a depth of about 1-2 pm are degraded.
The amount of time the enzyme solution is applied should be kept to
a minimum to effect the degradation of the proteoglycans predominantly in the
repair area. For chondroitinase ABC or AC at a concentration of 1 U/ml, a
digestion period longer than 10 minutes may result in the unnecessary and
potentially harmful degradation of the proteoglycans outside the defect area.
Furthermore, digestion times longer than 10 minutes contribute excessively to
the
overall time of the procedure. The overall time for the procedure should be
kept to
a minimum especially during open arthrotomy, because cartilage may be damaged
by exposure to air [Mitchell et al., ( 1989), supra]. For these reasons, in
the
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embodiments of the methods of this invention that include the step of
degradation
of proteoglycans by enzymatic digestion, digestion times of less than 10
minutes are
preferred and digestion times of less than 5 minutes are most preferred.
According to the methods of this invention, after the enzyme has
S degraded the proteoglycans from the surface of the defect, the enzyme
solution
should be removed from the defect area. Removal of the enzyme solution may be
effected by using an aspirator equipped with a fine suction tip followed by
sponging
with cotonoid. Alternatively, the enzyme solution may be removed by sponging
up
with cotonoid alone.
Following removal of the enzyme solution, the defect should be
rinsed thoroughly, preferably three times, with sterile physiologic saline
(e.g., 0.15
M NaCI). The rinsed defect site should then be dried. Sterile gauze or
cottonoid
may be used to dry the defect site.
The adhesion of the matrix to the cartilage of the defect can also be
enhanced by using fibrin glue (i.e., blood factor XIII or fibrin stabilization
factor) to
promote chemical bonding (cross-linking) of the fibrils of the matrix to the
cartilage
collagen fibrils on the defect surface [see Gibble et al., Transfusion, 30(8),
pp. 741
47 (1990)]. The enzyme transglutaminase may be used to the same effect [see
e.g.,
Ichinose et al., J. Biol. Chem., 265(23), pp. 13411-14 ( 1990);
"Transglutaminase,"
Eds: V.A. Najjar and L. Lorand, Martinus NijhoffPublishers (Boston, 1984)].
Other compounds that can promote adhesion of extraceilular materials may also
be
used.
Treatment of periodontal disease may be also aided with the
methods and compositions of this invention. For example, in the treatment of
disease such as paradontitis, in which the periodontal ligament recesses and
toothnecks are exposed, the regeneration of periodontal connective tissue can
be
aided. In particular, the periodontal connective tissue, i.e., ligament, space
is filled
with a biodegradable matrix containing one or more factors to stimulate
periodontal
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tissue formation, such as IGF-1 or FGF, and one or more anti-epithelial
factors to
inhibit epithelium formation, such as an inhibitor for epidermal growth factor
(EGF), anti-basement membrane antibodies, vitamin A inhibitors, anti-retinol,
and
matrices enriched with fibronectin.. The anti-epithelial factor should be in
free form
and packaged in or associated with a delivery system to provide sustained
release in
the area filled by the matrix. Preferable matrix materials are crosslinked
gelatin or
collagen matrices with suffcient ability to adhere to the tooth and gum.
Bone regeneration, e.g., following loss of teeth can also be aided by
the methods and compositions of this invention. When a tooth is lost, the bony
compartment in which the tooth was anchored is usually atrophic and recessed.
This area, i.e., the maxillar or mandibular ridge, must be built up and
reformed prior
to positioning of a dental implant. However, bony regrowth is usually
prevented by
the rapidly growing connective tissue around it. This can be prevented by
using a
bone repair matrix such as those described above, and including in the matrix
an
effective amount of one or more inhibitors of migration and/or proliferation
and/or
differentiation of connective tissue cells. For example, a matrix containing
an
angiogenic factor, an osteogenic factor associated with a delivery system, and
an
anti-connective tissue factor, such as factors inhibiting mesenchymal cell
proliferation, may be placed in the area to be built up with bone tissue. To
promote
the re-formation of connective tissue in the surrounding periodontal
connective
tissue compartment a biodegradable matrix containing factors to stimulate the
migration, proliferation and differentiation of connective tissue stem and
precursor
cells, such as IGF-1 and FGF, may be placed in the spaces within the
peridontium.
The connective tissue matrix may also contain an anti-epithelial factor.
Another aspect of this invention is the prevention of periarticular
calcification and ossification of the connective tissue compartments around
bone
which occurs, for example, after joint replacements such as total hip
replacements.
A matrix containing anti-bone factors such as anti-angiogenic agents,
antibodies to
T
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TGF-f3 and BMP compounds, or combinations thereof, in free form and associated
with or packaged in a delivery system to provide sustained release, may be
applied
in the periarticular connective tissue space to prevent calcification and the
formation
of bony tissue.
A further application of this invention is the prevention of bone
tissue ingrowth into a fracture site which extends through the cartilaginous
growth
plate in young animals and children, which may occur when distal ends of long
bones are fractured. As with the prevention of ossification of connective
tissue
compartments around bone, a matrix containing anti-bone factors such as an
anti-
angiogenic factor, antibodies to 'fGF-~i and BMP compounds, or combinations
thereof can be used to fill the fracture gap in the area of the cartilaginous
growth
plate.
In order that the invention described herein may be more fully
understood, the following examples are set forth. It should be understood that
these examples are for illustrative purposes and are not to be construed as
limiting
this invention in any manner.
E~~AMPLE 1
Mme Testine for Proteoe~can Removal
In order to promote and improve matrix adherence along superficial
defect surfaces of articular cartilage tissue, proteoglycan molecules within
the
superficial cartilage matrix may be removed enzymatically, in order to expose
the
collagen fibrillar network to externally applied matrices and to migrating
repair
cells. Various proteases and glycosaminoglycan-degrading enzymes are suitable
to
be used for this purpose, but pH conditions should be controlled to provide
maximal activity for each enzyme.
In this example, we tested chondroitinase ABC (0.5-5 U/ml) and
trypsin (0. S-4%) for their ability to effect proteoglycan removal. Knee
joints from
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freshly slaughtered rabbits, obtained from a local butcher, were employed.
Mechanically-created superficial cartilage defects were exposed to the enzyme
solutions for a period of 4 minutes. Solutions were then removed with
absorbent
tissue and the defect sites rinsed thoroughly with physiologic saline.
Following this
procedure, cartilage tissue was fixed immediately in 2% (v/v) glutaraldehyde
solution (buffered with 0.05 M sodium cacodylate, pH 7.4) containing 0.7%
(w/v)
ruthenium hexamine trichloride (RHT) for histological examination. The post-
fixation medium consisted of a 1% RHT-osmium tetroxide solution (buffered with
0:1 M sodium cacodylate). Tissue was dehydrated in a graded series of ethanol
and
embedded in Epon 812* Thin sections were cut, stained with uranyl acetate and
lead citrate, and examined in an electron microscope. In these sections, RHT-
fixed
(i.e., precipitated) proteo~lycans appeared as darkly-staining granules.
Enzyme
concentrations removing a superficial layer of proteoglycans no more than 1-2
pm
in thickness were defined as optimal (deeper penetration of enzymes could
affect
the underlying chondrocytes). Chondroitinase ABC was found to be optimally
active at a concentration of approximately 1 U/ml. Trypsin was found to be
optimally active at a concentration of approximately 2.5%. The optimal
activity
range for other glycosaminoglycanases or proteases may be determined in a
similar
manner. Any buffer may be used in conjunction with the enzyme provided that it
is
nontoxic and that its maximal buffering capacity occurs at a pH value close to
that
required for maximal enzyme activity.
~,?~AMPLE 2
Matrix Adherence to Superficial Defects
The possibility of promoting matrix adhesion along defect surfaces
by controlled enzyme digestion of superficial cartilage proteoglycans was
investigated. Defects were created in the knee joints of three mature rabbits
by
cutting with a planing knife. These defects were not enzyme treated. The
defects
*Trade-mark
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were filled with a fibrin matrix, formed by mixing 20, p1 of a thrombin
solution (100
U/ml aqueous buffer) with each ml of fibrinogen solution (1 mg/m) aqueous
buffer)
approximately 200 seconds before filling the defect. The rabbits were
sacrificed
after 1 month and the knee joints examined to determine the extent to which
the
S fibrin matrix had adhered to the defect site. The results were compared to
those
achieved in rabbits whose defects had been treated with chondroitinase ABC
(1 U/ml for 4 minutes) before the defect was filled with fibrin matrix (see
Examples
3, 4 and 5).
The fibrin matrices deposited in defect areas left untreated with an
enzyme exhibited low affinity to adhere to the defect surface. Following
enzyme
treatment, the sticking capacity of the fibrin matrices (determined indirectly
by
measuring mechanical strength to adhere, i.e., by testing the easiness with
which the
matrix could be pushed away manually with the tip of a forceps, and indirectly
by
noting the number of defects in which the matrix successfully remained
sticking
throughout the experiment) was significantly increased. The low affinity of
matrices
for the defect surfaces in the absence of enzyme treatment probably is due to
a local
inhibition of matrix adhesion by proteoglycan molecules and an inhibition of
fibrin
polymerization. Both of these effects are prevented by enzymatic removal of
superficial proteoglycans along the defect surface area.
EXAMPLE 3
Application of Growth Factors to Defect Sites
to Provide Chemotactic Stimulation of
Repair Cell Migration into Defect Areas
and Induction of Repair Cell Proliferation
Various growth factors were tested for their usefulness in
stimulating chemotactic migration of repair cells to the defect area in order
to
accomplish healing of the defect.
The growth factors employed included
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a) epidermal growth factor (EGF), b) basic fibroblast growth factor (bFGF), c)
insulin-like growth factor I (IGF I), d) human growth hormone (hGH) and
e) transforming growth factor-~i (TGF-(3) at concentrations ofbetween 5-10
ng/ml.
Each of these factors was applied locally to defects produced in the
knee following chondroitinase ABC treatment and rinsing as described in
Example
2. A total of ten anirrials (two per growth factor) were utilized. Each growth
factor
was able to chemotactically attract or locally stimulate proliferation of
repair cells to
the defect surfaces sutl7ciently to completely cover the defect surfaces.
However,
the cells were only present on the surfaces of the defects, and in no instance
was
proliferation of the repair cells adequate to fill the defect volume.
(It is believed that the proteoglycan degradation products by
themselves, i.e., without the addition of any other agent, exert a sufficient
chemotactic effect to attract repair cells to the defect. Moore, A.R. et al.
,(,ant-J.
Tiss. Reac., XI(b), pp. 301-107, 1989] have shown that proteoglycan
degradation
products have chemotactic effects per se. )
Application to Defect Sites of Growth
Factors Entrapped in Biodegradable Matrices to
Provide Chemotactic stimulation of Repair
Cell Migration into Defect Areas and
induction of Repair Cell Proliferation
Since local application of a growth factor under the conditions of
Example 3 in no instance induces repair cell proliferation adequate to fill
the defect
volume, the experiment was repeated using the same growth factors, but this
time
the growth factors were entrapped in biodegradable matrices. The biodegradable
matrices used were fibrin, collagen and Sepharose. Sufficient quantities of
matrices
containing growth factor were applied to fill the defect volumes completely.
T
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Fibrin matrices were formed by mixing 20 p1 of a thrombin solution
(100 U/ml of an aqueous buffer solution: Veronal acetate buffer, pH 7.0) with
each
ml of fibrinogen solution ( 1 mg/ml of an aqueous buffer solution: O.OSM Tris,
pH
7.4, O.1M NaCI) approximately 200 seconds prior to filling the defect. For
collagen matrices, sui~ciently viscous solutions were made using Colagen-
Vliess~
or gelatine-blood-mixtures. For Sepharose matrices, defects were filled with
liquid
solutions of Sepharose at 39-42°C. Upon cooling (3538°C), a
Sepharose matrix
was formed in the defect.
Thirty rabbits (two far each type of matrix and growth factor) were
utilized for this experiment. In all cases where the deposited matrix remained
adherent to the defect, it became completely populated by fibroblast-like
repair
cells. This situation was found to exist as early as eight to ten days
postoperatively.
No further changes occurred in the structural organization of the repair
tissue up to
four weeks post-operatively, except that the biodegradable matrices became
remodeled by the repair cells and replaced by a loose, connective tissue type
of
extracellular matrix.
Transformation of this tissue to cartilage tissue did not occur.
Application to Defect Sites of Growth
Factors Entrapped in Biodegradable Matrices
to Provide Chemotactic Stimulation of
Repair Cell Migration into Defect Areas
and Induction of Repair Cell Proliferation
Followed by Timed, Local Release of a
Transforming Factor at a Secondary Stage
to Provide Transformation of the
Defect Site into HXaline Cartilage
The observation that matrices within the defect volume were
completely filled with repair cells following application of growth factor,
and that
these cells were able to remodel the deposited matrix (see Example 4),
prompted
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the investigation of the effects of introducing a transforming factor {such as
TGF-~3)
in an encapsulated form (e.g., liposomes) from which the transforming factor
would
be released when the matrix was completely populated with repair cells that
had
begun to remodel the intercellular structure.
TGF-(i was mixed into the fibrinogen solution ( I mg/ml) at a low
concentration (e.g., 2-10 ng/ml) for the purpose of promoting the initial
chemotactic and proliferative effects. TGF-~3 was also encapsulated in
liposomes
according to the method of Kim et al. ( 1983) . These TGF-~3 containing
liposomes were added to the same fibrinogen solution in a concentration
adequate
to provide, when the liposomes were ruptured and the TGF-(3 was released, the
higher concentration of 100-1000 ng of TGF-~3 per ml of fibrinogen for the
purpose
of promoting transformation of the repair cells into chondrocytes and
transformation of the matrix-filled defect into cartilage during a secondary
stage
when the repair cells populating the fibrin matrix have begun to remodel the
intercellular substance.
Ten mature rabbits, in which superficial knee joint articular cartilage
defects were produced as in Example 2, were treated by application of this
mixture
of fibrinogen containing free and liposome-encapsulated TGF-~i to the defect
site.
In the various experiments in this series of experiments, the concentration of
free
TGF-~i was maintained in the range from 2-10 ng/ml of fibrinogen while the
concentration of encapsulated TGF-~3 was varied to provide (upon release of
the
TGF-~3 from the liposomes) a concentration between 100 and 1000 ng TGF-(3/ml
fibrinogen in 100 ng steps. Formation of hyaline cartilage tissue occurred at
the
treatment sites in all cases. The most reproducible results were obtained at
concentrations of above 200 ng encapsulated TGF-~i/ml fibrinogen solution, and
preferably above 500 ng TGF-~3/ml of fibrinogen solution.
T
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EXAMPLE 6
Determination of the Time
Point of Tissue Transformation
In this experiment, a group of six mature rabbits were subjected lo
knee surgery to produce superficial defects as in Example 2. A full treatment
scheme for superficial defect repair was applied, i.e., treatment with
chondroitinase
ABC (1 U/ml for 4 minutes), followed by filling the defect site with fibrin
matrix (1
mg/ml fibrinogen solution, 20 p1 100 U/ml thrombin solution per ml of
fibrinogen
solution) containing free TGF-~i (-2-10 ng/ml) and liposome encapsulated TGF-
~i
(-800 ng/ml). Three rabbits were sacrificed at eight, ten and twelve days
postoperatively, the remaining three at twenty, twenty-four and twenty-eight
days.
Transformation of the primitive, fibroblast-like repair cell tissue into
hyaline
cartilage tissue occurred between days twelve and twenty in this animal model.
This was determined on the basis of hisfological examination. At days eight to
twelve, loose fibrous repair tissue was still present (the applied fibrin
matrix being
partially or completely remodeled), whereas at day twenty and subsequently,
the
defect space was partially or completely filled with hyaline cartilage tissue.
EXAMPLE 7
Application of Cartilage Repair
Procedures in a Mini-dig Model
The experimental procedures utilized in the rabbit model, ,
were applied to a larger animal model, the mini-pig. Superficial defects (0.6
mm
wide, 0.6 mm deep and approximately 10-15 mm long) were created in four mature
mini-pigs (2-4 years old, 80-110 Ibs.) by cutting with a planing knife in the
patellar groove and on the medial condyle. The defects were then treated with
chondroitinase ABC (1 U/ml for 4 minutes, as used for rabbits, supra). The
enzyme
solution was removed, the defect dried, rinsed with physiological saline, then
dried
again. The defect sites were then filled with a fibrinogen matrix solution.
The
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fibrinogen matrix solution used in this experiment contained 2-6 ng of free
TGF-~3
per ml, and 1500-2000 ng of liposome-encapsulated TGF-~3 per ml of fibrinogen
solution. Prior to filling the defects, thrombin was added to the matrix
solution as
described above in the rabbit experiment.
The mini-pigs were sacrificed 6 weeks postoperatively, and the sites
of the matrix-filled defects were examined histologically. All sites showed
healing,
i.e., formation of hyaline cartilage tissue at the treatment site.
EXAMPLE 8
Repair Of Full-Thickness
Defects In Articular Cartilage
Usin~~i-AnQLeenic Agent
Full-thickness articular cartilage defects, 1 mm deep and 10 mm
wide, were created in the medial condyles and patellar grooves of adult mini-
pig
knee joints. Five lesions were effected in each of two animals, using a
planing
instrument. In each patellar groove, two defects were made in the cranial
region,
two defects in the caudal region and one defect in the medial femoral condyle.
The
vertical extensions of each lesion into the subchondral bone (containing blood
vessels and bone marrow cells) was controlled macroscopically by the
occurrence of
bleeding to insure that a full-thickness lesion had been made in the joint.
The
defects were then treated with chondroitinase AC ( 1 Ulml for 4 minutes). The
enzyme solution was removed, the defect dried, rinsed with physiological
saline,
then dried again. The defect sites were then filled with a cartilage repair
matrix
solution. The matrix solution used in this experiment consisted of a copolymer
of
gelatin (Gelfoam;~ Upjohn) (used at 100 mg per ml) and fibrinogen (used at 20
mg
per ml). Thrombin (used at 50 LU.) was added to the top surface of the defect
after
the matrix was placed in the defect and was allowed to diffuse into the
matrix.
The cartilage repair matrix ;,ontained a free proliferation agent
insulin-like growth factor-1 (IGF-1 ) at a concentration of about 40 ng/ml of
matrix
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volume as a transforming factor, and liposome-encapsulated TGF-~i 1 at a
concentration of S00 ng/ml of matrix volume as a transforming factor. In
addition,
free Suramin was added at a concentration of 10 millimolar of matrix volume
and
liposome-encapsulated Suramin was added at a concentration of 10 millimolar of
matrix volume in the same liposomes that contained the TGF-(31. In control
lesions, defects were treated in the same manner, except that Suramin was not
added.
About 8 weeks after the operation and treatment, the animals were
sacrificed and the sites of the matrix-filled defects examinEd histologically.
The part
of the defect space adjacent to articular cartilage tissue, i.e., in the
region filled with
the matrix composition containing Suramin, was filled with articular cartilage
tissue.
The same part of the defect space in control lesions, i.e., those treated
without
Suramin, was filled with newly-formed bone tissue.
The above experiment was repeated with the substitution of BMP-2
for TGF-X31, at a concentration of 1000 ng/ml of matrix volume. The same
results
were obtained.
EXAMPLE 9
Repair Of Full-Thickness
Defects In Articular Cartilage
Using Heating Procedure
Full-thickness articular cartilage defects, I mm deep and 10 mm
wide, were created in the medial condyles and patellar grooves of adult mini-
pigs.
Five lesions were effected in each knee joint of two mini-pigs. At each
location
where bleeding occurred, coagulation was induced by applying a heated
instrument
to the floor ~of defects to form a biological physical barrier. We used a
heated
scalpel blade (heated to 220°C) to create the transient tissue barrier.
In one mini-pig, articular cartilage defects in one joint were filled
with a cartilage repair matrix containing IGF-1 at a concentration of about 40
ng/ml
CA 02258601 1998-12-17
WO 98/00183 PCTlUS97/11208
-44-
of matrix volume and liposome-encapsulated TGF-(33 at a concentration of 500
ng/ml of matrix volume. In the defect in the other joint, Suramin was included
in
the matrix as described in Example 8.
In the second mini-pig, the defects of one joint were filled with a
cartilage repair matrix containing IGF-I at a concentration of about 40 ng/ml
of
matrix volume and liposome-encapsulated BMP-2 at a concentration of 1000 ng/ml
of matrix volume. In the defects of the other joint of the second mini-pig,
Suramin
was included in the matrix as described in Example 8.
As in the previous experiment, the animals were sacrifced and
examined eight weeks after operation and treatment. No bone tissue formed in
the
defect space adjacent to articular cartilage tissue in either animal. Rather,
the defect
spaces were filled with articular cartilage tissue.
EXAMPLE 10
Repair Of Deep Full-Thickness
Defects In Articular Cartilage
Using Anti-Angi~genic Agent
Very deep full-thickness articular cartilage defects, up to S mm deep,
can be created in the medial condyles and patellar grooves of adult mini-pig
knee
joints. Lesions can be effected in animals maintained under general
anaesthesia,
using a planing instrument. The bone portion of the defect may be ftlled with
a
bone repair matrix composition such as those described above. The bone portion
of
the defect should be filled with matrix up to the cartilage-bone interface.
The
articular cartilage defect space can be filled with a cartilage repair matrix
containing
an anti-angiogeruc factor, such as those described above, e.g., at Examples 8-
9.
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