Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS FOR PROGRAMMING AN ORGANIC
MATRIX FOR REMODELING INTO A TARGET TISSUE
Background of the Invention
The ability to selectively promote tissue regrowth in vivo would greatly
facilitate
wound healing and post-surgical recovery of patients who have suffered tissue
damage
or destruction due to accident or disease. Recent studies have found that
certain matrix
compositions can promote bone growth when implanted into damaged bone. thereby
stabilizing the damaged bone and providing a means for speeding healing.
However,
generalized methods for promoting regrowth or repair of a variety of tissues
have been
elusive.
Summary of the Invention
This invention provides methods and compositions for promoting regrowth or
repair of a variety of tissues.
In one aspect, the invention provides a method for programming a non-
immunogenic matrix for remodeling into a target biomorphic form, i.e., for
preparing a
target biomorphic form. The method includes the steps of providing a non-.
immunogenic matrix, e.g., by demineralizing a collagen source to form a
demineralized
organic matrix; selecting a treatment step for programming the non-immunogenic
matrix
for remodeling into a target biomorphic form; and treating the non-immunogenic
matrix
such that remodeling into the target biomorphic form occurs.
In preferred embodiments, the treatment step is selected such that the target
biomorphic form is a cartilage-forming composition, a bone-forming composition
or a
muscle-forming composition.
In another aspect, the invention provides a method for preparing an organic
material for promoting tissue growth or repair. The method includes the steps
of
demineralizing ground bone to provide a demineralized organic matrix; and
treating the
demineralized organic matrix with hyaluronic acid (HA) or a glycosaminoglycan
(GAG)
to prepare an organic material for promoting tissue growth or repair.
In preferred embodiments, the method comprises the further step of contacting
the demineralized bone matrix with a growth factor in an amount effective to
promote
tissue growth. In certain embodiments, the demineralized organic matrix is
treated with
about 1-5% by weight of HA or a glycosaminoglycan. In certain embodiments, the
growth factor is selected from the group consisting of osteopontin, bone
morphogenic
protein, and bone sialoprotein. In certain embodiments, the step of
demineralizing
ground bone includes contacting the ground bone with at least one chelating
agent. In
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another aspect, the invention provides an injectable, non-immunogenic
composition for
promoting tissue growth or repair, prepared by this method.
In another aspect, the invention provides a method for preparing an organic
material for promoting tissue growth or repair. The method includes the steps
of
demineralizing ground bone to provide a demineralized organic matrix; and
treating the
demineralized organic matrix with a mineral acid under conditions such that a
muscle
growth-promoting factor is activated.
In another aspect, the invention provides an injectable, non-immunogenic
composition for promoting tissue growth or repair. The composition comprises
at least
about 80% collagen matrix by weight; and a growth factor in an amount
effective for
promoting tissue growth. The composition is preferably substantially free of
endogenous growth factors.
In certain embodiments, the composition further comprises hyaluronic acid or a
pharmaceutically effective salt thereof. In certain embodiments, the growth
factor is
osteopontin or bone sialoprotein. In certain embodiments, the composition
further
comprises a glycosaminoglycan. In certain embodiments, the collagen matrix is
substantially pure Type I collagen. In certain embodiments, the matrix is
substantially
non-migratory when injected into a living subject. In certain embodiments, the
composition comprises particles between about 75 microns and about 200 microns
in
size. In certain embodiments, the composition comprises at least about 85%
collagen by
weight. In certain embodiments, the composition comprises at least about 90%
collagen
by weight.
In yet another aspect, the invention provides a method for promoting tissue
growth in a living subject without causing inflammation in the subject. The
method
includes the steps of injecting into the subject an injectable, non-
immunogenic
composition, the composition including at least about 80% collagen matrix, and
a
growth factor in an amount effective for promoting tissue growth; such that
tissue
growth is promoted in the living subject without causing inflammation in the
subject. In
certain embodiments, muscle growth, bone growth, or cartilage growth is
promoted.
In still another aspect, the invention provides a method for promoting the
differentiation of mesenchymal cells. The method comprises contacting the
mesenchymal cells with a matrix; the matrix includes an injectable, non-
immunogenic
composition which includes at least about 80% collagen matrix; and a growth
factor in
an amount effective for promoting tissue growth. The matrix contacts the
mesenchymal
cells under conditions such that the mesenchymal cells become differentiated.
In another aspect, the invention provides a pharmaceutical preparation,
including
an injectable, non-immunogenic composition for promoting tissue growth or
repair, and
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a pharmaceutically-acceptable carrier. The injectable non-immunogenic
composition
includes at least about 80% collagen matrix by weight; and a growth factor in
an amount
effective for promoting tissue growth, and preferably is substantially free of
endogenous
growth factors.
In another aspect, the invention provides a method for promoting attachment
and
fusion of mesenchymal cells. The method includes the steps of implanting a
matrix into
a tissue containing mesenchymal cells, under conditions such that the
mesenchymal cells
attach to the matrix and become fused. The matrix includes means for
attracting
mesenchymal cells to the matrix; means for attaching mesenchymal cells to the
matrix;
and means for promoting fusion of mesenchymal cells.
In preferred embodiments, the means for attracting mesenchymal cells to the
matrix comprises a chemotactic peptide. In preferred embodiments, the means
for
attaching mesenchymal cells to the matrix comprises a spreading domain of a
growth
factor. In preferred embodiments, the means for promoting fusion of
mesenchymal cells
comprises hyaluronic acid or a glycosaminoglycan.
Detailed Description of the Invention
This invention provides compositions and methods for selectively promoting the
growth of tissues in vivo.
In general, the compositions of the invention include a particulate "scaffold"
which serves to stabilize the site of a tissue defect, and which can be
infiltrated by cells
and remodeled into a target tissue. In certain preferred embodiments, the
scaffold
comprises demineralized matrices derived from cartilage or bone.
Alternatively, the
scaffold can include a (synthetic) polymeric matrix suitable for supporting
the growth of
cells. The scaffold can include growth factors and other materials which
promote the
formation of the desired tissue type when the scaffold is implanted in the
subject.
Methods for preparing the compositions of the invention are described in more
detail
below. Additional information on collagen-based matrix preparations can be
found in
U. S. Patent No. 5,516,532, issued May 14, 1996, herein incorporated by
reference.
The term "biomorphic composition," as used herein, refers to a composition
which, when implanted in the body of a living subject, promotes the growth of
non-
inflammatory tissue of a pre-determined tissue type. Examples of tissue which
can be
formed by injection or implantation of the biomorphic compositions of the
invention
include bone, muscle, cartilage, skin, fat, tendon, and the like. In preferred
embodiments, the biomorphic compositions of the invention can be used to
promote
formation of tissues which are derived from mesenchymal cells.
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The term "target biomorphic form," as used herein, refers to a composition
capable of selectively promoting the growth of a target tissue, e.g., bone,
muscle,
cartilage, and the like.
An "endogenous" growth factor, as used herein, refers to a growth factor
present
in a naturally-occurring matrix without addition of additional growth factors
from an
external source. For example, whole natural bone can contain endogenous growth
factors, which can be removed by extraction, proteolysis, and the like.
The term "subject" is intended to include vertebrates, more preferably warm-
blooded animals, preferably mammals, including cats, dogs, horses, cattle,
swine, and
humans.
In the discussion which follows, the biomorphic compositions of the invention
are described for use in promoting tissue formation in the body of a living
subject.
However, it will be understood that the compositions can be employed to
promote tissue
growth in vitro, e.g., in cell culture. Thus, the compositions of the
invention can be
employed to grow tissues, e.g., tissue suitable for implantation or
transplantation, e.g.,
grafting, into a host animal.
Biomorphic Compositions
The inventions provides biomorphic compositions which promote the formation
of a pre-selected tissue type when implanted in the body of a living subject.
The tissue
type promoted by a particular biomorphic composition will be related, at least
in part, to
the environment for cell growth that is provided by the biomorphic
composition.
Without wishing to be bound by theory, it is believed that the biomorphic
compositions
of the invention can promote recruitment of pluripotent (non-differentiated)
cells from
the tissue surrounding the implant, thereby providing cells which can grow and
differentiate within the implant to form a target tissue. Accordingly,
chemoattractants
which can attract cells of an appropriate type can be employed in the
biomorphic
compositions of the invention to attract the correct cell types from the
surrounding tissue
into the implant, as described in more detail, infra. The matrix of the
biomorphic
composition can also be selected to prevent invasion of the implant by
differentiated
cells.
The biomorphic compositions of the invention preferably provide an
environment conducive to differentiation of pluripotent cells which infiltrate
the
implant. In a preferred embodiment, the cells are mesenchymal cells. It will
be
appreciated by the skilled artisan, however, that the differentiation of cells
should
generally be balanced with the growth and multiplication of established cells
to provide
new tissue. Thsu, the biomorphic compositions of the invention, when implanted
into a
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living subject, preferably provides a structured environment which allows
ordered
differentiation of cells within the implant. For example, without wishing to
be bound by
theory, it is believed that, in certain embodiments, pluripotent cells can
form an
aggregate within the implant, in which cells near the center of the aggregate
remain
undifferentiated, while secreting growth factors which promote the
differentiation of
cells at the periphery of the implant, thereby producing a target tissue.
Other considerations inlude the size of the matrix particles (discussed
further
infra), and the spacing of the particles. It is believed that the interstitial
space between
particles of the matrix can be important in excluuding certain large cells
(such as
keratinocytes or lymphocytes) from entering the implant. In addition, in
certain
embodiments in which vascularization of the implant is desired (e.g., when the
target
tissue type is bone or muscle), it is preferable to employ a matrix which
provides
sufficient interstitial space to permit the formation of vascularization in
the implant (e.g.,
70-100 microns between particles). Conversely, vascularization is inhibited by
interstitial spaces less than about 70 microns in size; thus, for formation of
tissues such
as cartilage in which vascularization is not desired, smaller interstitial
spaces can be
employed by using smaller matrix particles and/or higher densities of matrix.
Formation of inert matrix
The compositions of the invention include an inert matrix which functions as a
"scaffold" for the biomorphic composition. Inert matrices suitable for use in
the present
invention generally are substantially non-immunogenic, that is, the inert
matrix does not
provoke a substantial immunogenic response, such as inflammation, when
injected or
implanted in a living subject. Suitable inert matrices are known in the art,
and include,
e.g., particles of inert, non-immunogneic substances such as silicone, Teflon.
and
collagen, e.g., from demineralized bone powder. An inert matrix preparation is
preferably sized to permit easy handling (e.g., by injection)) while being
resistant to
migration after placement at a target site in vivo, as described in more
detail infra. An
inert matrix is preferably flexible enough to permit cell growth and
attachment to the
implant.
A particularly preferred inert matrix is derived from bone by demineralization
of
bone powder. Such inert matrices can be prepared according to several methods.
Two
methods for producing an inert matrix are described in Examples 1 and 2,
below. In
general, the methods involve treating bone with chelating or leaching agents
to remove
minerals from the bone, preferably without significantly disrupting the triple-
helical
nature of the collagen fibers present in the bone. It will be understood that
other sources
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of triple-helical Type I collagen can be used in the compositions and methods
of the
invention.
Inert matrices prepared by the methods described herein, and useful for the
preparation of the bone- and muscle-forming matrices described below, can be
characterized in several ways. In preferred embodiments, the inert matrix is
prepared
from demineralized bone, and has a calcium concentration of less than about
100
mg/gm, more preferably less than about 50 mg/gm, less than about 20 mg/gm,
less than
about 10 mg/gm, or less than about 1 mg/gm of the matrix (w/w).
Chelating agents useful in demineralizing bone are known in the art. Exemplary
chelating agents include chelators of Ca(II), including, for example, EDTA,
EGTA,
citrate, and the like. The bone, preferably ground bone, is treated with
chelating
reagents in an amount and for a time sufficient to remove calcium from the
bone. The
residual calcium present in the inert matrix is preferably present at a level
not greater
than about 100 mg/gm matrix, more preferably less than about 50 mg/gm, less
than
about 20 mg/gm, less than about 10 mg/gm, or less than about 1 mg/gm of the
matrix
(w/w).
If desired, the phosphate concentration of bone can be further lowered by
treatment with agents such as phosphatase, and other agents known to the
ordinarily
skilled artisan.
It is frequently advantageous to perform repeated extractions and washings of
the
ground matrix to reduce the amount of calcium, phosphate, and other mineral
matter to
an acceptable level, and to remove any components of the matrix which could
otherwise
provoke an inflammatory response. As described in the Examples, below,
repeated
and/or prolonged washing of the matrix is effective in producing an inert, non-
immunogenic matrix having a low level of minerals.
Washing or leaching solutions can comprise protease inhibitors, if desired, to
prevent proteolysis of matrix components. Such protease inhibitors are not
required,
however, and fully active biomorphic compositions can be prepared without use
of
protease inhibitors. In embodiments in which protease inhibitors are present,
such
inhibitors will generally be selected to inhibit enzymes such as
metalloproteases, serine
proteases, cysteine proteases, cathepsins, and phosphatases. Exemplary enzyme
inhibitors include the following: phenylmethylsulfonyl fluoride, benzamidine,
epsilon-
amino caproic acid, ~i-hydroxy mercuribenzoate, pyrophosphate, sodium
fluoride,
sodium orthovanadate, levamisole, and pepstatin A (all available from Sigma
Chemical
Co, St. Louis, MO).
In preferred embodiments, the inert matrix comprises at least about 80%
protein
by weight, more preferably at least about 85% protein by weight, more
preferably at
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least about 90% protein by weight, and most preferably at least about 95%
protein by
weight.
In certain preferred embodiments, the total protein of the matrix comprises at
least about 80% collagen by weight, more preferably at least about 85%
collagen by
S weight, more preferably at least about 90% collagen by weight and most
preferably at
least about 95% collagen by weight.
The presence of Type I triple-helical collagen can be detected by examining a
collagen sample under a polarizing light microscope. Triple-helical collagen
has a
distinctive birefringence diagnostic of the undenatured state. Thus, an inert
matrix (or a
biomorphic matrix) prepared according to the methods described herein can be
assayed
for the presence of triple-helical collagen by examination of the material
under polarized
light. Also, triple-helical collagen is highly resistant to gelatinases.
In preferred embodiments, the collagen is substantially pure Type I collagen.
The hydroxyproline/proline ratio of pure Type I collagen is about 0.6.
Accordingly, the
hydroxyproline/proline ratio of the protein of the inert matrix is at least
about 0.4, more
preferably at least about 0.50, and most preferably at least about 0.55.
The inert matrix is preferably prepared in the form of particles. In preferred
embodiments, the particles are sized so as to permit injection of the inert
matrix particles
through a needle, e.g., a hypodermic needle, e.g., a 28-gauge needle. Thus, in
preferred
embodiments, the particles are not larger than about 200 microns mean
diameter. The
particles are preferably sized to prevent significant migration in the
subject's body.
Migration is a function of several factors, including the ability of cells to
infiltrate or
engulf the particles. The ability of cells to engulf the particles can depend
upon the
"effective size" of the particles, i.e., the ability of the particles to pass
through cell or
tissue pores (e.g. interstitial spaces). Such pores can be charged; particles
of the same
charge will be repelled by the pore, and will therefore have a larger
effective size, that is,
will be hindered in the ability to pass through pores (cell or tissue pore.
(interstitial
space). In general, particles larger than about 75 microns are not migratory
when
implanted. Smaller particles, e.g., particles between about 50 microns and
about 75
microns, may not be migratory where the particles are charged, especially
where the
charge is the same as the charge on cell or tissue pores.
Matrix particles of any desired size can be prepared according to the methods
described herein, or according to methods known in the art. For example,
ground
particles of matrix can be passed through sieves of decreasing size, until a
suitable
particle size is reached. The matrix material can be sized at any time during
the
preparation of the inert matrix. Conveniently, the particles are screened
prior to
treatment with chelating agents. The process of removing calcium and other
minerals
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from bone is substantially faster when the bone is first ground into
particles, as
compared to whole bone.
The inert matrix preferably is substantially non-immunogenic, that is, does
not
produce a substantial inflammatory response when implanted, and is not
rejected by the
S host animal. Importantly, the matrix is inert and non-immunogenic when the
matrix is
prepared from bone obtained from a species different from the host animal.
Thus, inert
matrix can be prepared from readily available sources of bone, such as bovine
bone,
regardless of the species of the subject. The inert matrix also does not
substantially
promote the formation of any tissue when the matrix alone is implanted (e.g.,
the matrix
without any additional growth factors), although some capsule formation may be
noted
after implantation. The inert matrix is also preferably stable (e.g., is not
resorbed) after
extended time periods, as evidenced by stable size and mass of implanted inert
matrix
after one year in test subjects.
Formation of Biomorphic Compositions
Biomorphic compositions are preferably formed by treatment of an inert matrix,
e.g., an inert matrix as described herein, with growth factors or other
substances which
promote the recruitment, growth, or differentiation of cells appropriate for
formation of
the pre-selected target tissue.
Such factors can be added to an inert matrix by methods which will be routine
for one of ordinary skill in the art. For example, as described infra, the
inert matrix can
be stirred with a solution or suspension of a growth factor and then
lyophilized to
provide a dried matrix which includes the growth factor. In preferred
embodiments,
growth factors and other materials added to the inert matrix substrate are
physically
trapped within the matrix, or adsorbed into or onto the matrix, but are not
covaiently
linked to the matrix. Thus, for example, growth factors can be immobilized
within the
matrix through interactions such as ionic and hydrophobic interactions, rather
than
covalent bonds.
Bone-forming Compositions
In one embodiment, a matrix useful for promoting the growth of bone in a
subject can be prepared from demineralized bone, e.g., the inert matrix
described above.
For example, bone (e.g., mammalian bone, e.g., bovine or human bone) can be
treated
with reagents to demineialize the bone without substantially denaturing the
collagen
matrix present in the bone. Selection of appropriate extracting and washing
steps can
provide a matrix which contains the growth factors necessary for the formation
of new
bone in a subject, as can be determined by use of assays as described below.
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Alternatively, a bone-forming matrix can be prepared from an inert
demineralized
matrix, as described above and in Example 4, below, by addition of appropriate
growth
and attachment factors to the inert demineralized matrix.
Thus, as described in more detail below, an inert matrix (e.g., prepared from
bone by treatment with chelating agents, followed by washing to remove
unwanted
impurities, as described herein), is treated with bone growth factors, such as
osteopontin,
bone sialoprotein (BSP) and hyaluronic acid (see, e.g., U.S. Patent No.
5,340,934 to
Termine et al., and references cited therein). A preferred bone growth factor
is
osteopontin. Osteopontin (OPN) is a cell adhesion protein first identified in
bone, but
now associated with other tissues as well. Osteopontin is a phosphorylated
glycoprotein
containing an RGD cell-binding sequence. In mineralized tissues, OPN is
expressed
prior to mineralization and regulated by osteotropic hormones, binds to
hydroxyapatite,
and enhances osteoclast and osteoblast adhesion. Although the exact function
of OPN is
yet unknown, possibilities include a role in the recruitment of bone precursor
cells to a
site of mineralization, and a role in protection against bacterial infection
(Butler WT,
Connect. Tissue Res. 23,123-136, l989).
The resulting matrix can promote the growth of bone in vivo. Other bone growth
factors such as bone morphogenetic protein (BMP) can also be provided to
promote
bone growth (see, e.g., U.S. Patent No. 5,670,336 to Oppermann et al., and
references
cited therein). Additional compounds such as decorin (biglycan) can be
provided in the
biomorphic composition to regulate the rate of mineral growth in the newly-
formed
bone. Addition of thrombospondin to the biomorphic composition permits the
rate of
vascularization to be slowed, if desired. For other references to growth
factors (e.g.,
cytokines) which may be useful in the present invention, see, e.g., U.S.
Patent No.
5,667,810 to Levin, and references cited therein.
Without wishing to be bound by theory, it is believed that the formation of
bone
by the compositions of the invention proceeds by infiltration of cells into
the implanted
composition, attachment of the cells to the matrix of the implant, and fusion
(or
aggregation) of the cells to form bone. The infiltration of cells, e.g.,
mesenchymal cells,
can be promoted by the presence in the composition of a factor known to
promote bone
growth, e.g., as described herein. Attachment of such cells to the matrix can
also be
promoted by addition of a suitable factor. It is believed that Type I collagen
provides a
suitable environment for attachment of cells, and Type I collagen (preferably
a
substantially purified, non-immunogenic Type I collagen) is accordingly a
preferred
matrix for a bone-forming biomorphic composition. Fusion of cells can be
promoted by
use of a suitable factor in the composition {e.g., hyaluronic acid (HA) or
glycosaminoglycans (GAGs), as described below). HA is also believed to provide
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additional spacing between particles of the matrix, e.g., when the matrix
particles are
coated with a layer of HA. It will be appreciated that each of the steps can
be promoted
by addition of a factor specific for that step; alternatively, one factor can
provide more
than one function. For example, it is believed that osteopontin promotes both
attraction
of cells to the implant (infiltration) and attachment of cells to the matrix.
In preferred
embodiments, a bone-forming composition of the invention comprises one or more
factors which promote cell infiltration, cell attachment to the matrix, and
cell fusion. It
is believed that bone formation requires the infiltration of macrophages into
the implant;
thus, an implant which permits infiltration of macrophages is preferred.
Osteopontin is a
specific recruiter of macrophages; therefore, a bone-forming composition
preferably
includes osteopontin in an amount effective to recruit macrophages into the
implant
from the surrounding tissue.
In one embodiment, the inert matrix is treated with osteopontin, BSP, and
hyaluronic acid or a glycosaminoglycan. The matrix can be suspended in buffer,
and the
growth factors then added to the buffer, followed by lyophilization of the
suspension to
yield a dry matrix. In preferred embodiments, osteopontin is added to the
matrix in the
range of about 0.05% to about 0.5% (w/w when dry), more preferably about 0.1 %
w/w.
When BSP is added to the matrix suspension, BSP is preferably added to the
matrix in
the range of about 0.001 % to about 0.1 % (w/w when dry), more preferably
about 0.01
w/w. Interestingly, when the composition is made without osteopontin, little
or no bone
formation occurs. Accordingly, osteopontin is a preferred bone growth factor.
BSP,
although preferred, is not required for bone formation. However,
mineralization is faster
in the presence of BSP.
Hyaluronic acid (HA) or a glycosaminoglycan (GAG, e.g., dermatan or
chondroitan sulfate) can provide a modified surface conducive to tissue
formation.
Accordingly, the bone-forming composition preferably comprises HA. In
preferred
embodiments, HA is added to the matrix in the range of about 0.05% to about
0.5%
(w/w when dry), more preferably about 0.1 % w/w. Similarly, when GAGS are used
in
addition to, or instead of, HA, the GAG (or GAGs) can be added to the matrix
the range
of about 0.05% to about 0.5% (w/w when dry), more preferably about 0.1 % w/w.
It will be appreciated that the inert matrix can be introduced into the body
of a
subject, and the above-identified factors (i.e., osteopontin, BSP, and HA or a
GAG) can
be introduced (e.g., by injection) into the implanted matrix. This in situ
formation of a
biomorphic device is suitable for, e.g., optimizing the activity of the
implant after
implantation, thereby permitting the biomorphic composition to be tailored to
any
application. Also, while the examples below describe the formation of
biomorphic
matrices which are dried to produce a material suitable for resuspension in a
solvent, the
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inert matrix., as a suspension, can be combined, e.g., ex vivo, with any
factors necessary
to the function of the biomorphic composition, and the in situ constituted
composition
injected or implanted as described herein.
In general, the bone-forming compositions of the invention are infiltrated by
macrophages within one day after implantation. Angiogenesis of the implant
generally
occurs in about one week, followed by mineralization of the infiltrated
implant.
Mineralization can occur, after about three weeks, although the time course of
mineralization can vary depending upon the composition of the implant. For
example. a
composition comprising osteopontin, BSP, and HA can, after implantation,
become
mineralized more rapidly than a similar composition which does not include
BSP.
Cartilage-forming Compositions
Compositions which promote the growth of cartilage tissue can be prepared by
methods similar to the methods described above for bone-forming compositions,
with
the difference that no bone-forming factors are added to (or substantially
present in) the
composition. Cartilage-forming compositions are preferably formulated to
promote the
growth of chondrocytes. A preferred matrix for a cartilage-forming implant is
Type I or.
more preferably, Type II collagen. Addition of HA or GAGs to an inert matrix
as
described above, provides a composition which promotes the formation of
cartilage
when implanted in a subject. For example HA can be added at a concentration of
about
0. 5 - 5 .0 mg/ml or GAG at a concentration of about 0.1-1 mg/m 1 can be added
to
promote cartilage formation. A preferred GAG is chondroitan sulfate.
In general, cartilage-forming compositions will be formulated to avoid or
prevent
angiogenesis in the implant. If substantial angiogenesis occurs) the initially-
formed
cartilage tissue can be converted to bone, which can be a disadvantage in
certain
applications. As described above, the addition of inhibitors of angiogenesis,
or selection
of appropriately-sized matrix particles, can slow or inhibit angiogenesis.
The cartilage formed by the inventive compositions can be fibrous cartilage,
but
more preferably is hyaline cartilage.
The assay methods described above can be employed to determine whether a
particular composition possesses cartilage-forming activity when implanted.
For
example, histological examination of an implant after a period of, e.g., seven
days, will
reveal the presence or absence of cartilaginous tissue.
Muscle-formine Compositions
Muscle-forming compositions preferably include Type I collagen as the inert
matrix. Muscle-forming compositions can be prepared from ground demineralized
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bone, which can be prepared as described above and in Examples 1 and 2 for the
inert
matrix. However, if the procedure of Example 1 is followed, a preferred
preparation
omits the final high-salt ( 1 M NaCI) washing steps. It has now been found
that omitting
the high-salt wash results in higher muscle-formation activity when the matrix
is treated
as described below and implanted into a subject.
The inert matrix, formed as just described, is then treated with a mineral
acid,
e.g., HCI, e.g., at a concentration of from about 0.1 N to about 2 N, for a
time from
about 1 hour to about 48 hours. Acids other than HCl have been found to be
considerably less effective at producing a muscle-forming composition. The
skilled
artisan, in view of the teachings herein, will be able to select appropriate
conditions
which result in muscle-forming activity of the composition, without
substantially
degrading the triple-helical nature of the collagen of the matrix.
After the acid-treatment step, the resulting composition is treated, e.g.,
repeatedly
washed or neutralized with a base, e.g., ammonium carbonate, to remove traces
of acid.
The material can then be lyophilized and stored prior to implantation, or can
be
implanted directly after neutralization.
Without wishing to be bound by theory, it is believed that acid treatment of
the
inert matrix may release or activate an endogenous muscle-forming factor
present, but
not active, in the inert matrix. Although in certain embodiments, a muscle-
forming
composition can include a growth factor such as muscle morphogenic protein
(see, e.g.,
U.S. Patent No. 5,328,695 to Lucas et al.), in a preferred embodiment, a
muscle-forming
composition contains no exogenous growth factors.
Preparations of Biomorphic Compositions
Biomorphic compositions can be prepared as suspensions of matrix particles
suspended in a pharmaceutically acceptable vehicle. The vehicle can be a
solvent or
dispersion medium containing, for example, water) ethanol, polyol (for
example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the like),
suitable
mixtures thereof, and vegetable oils. The proper fluidity can be maintained,
for
example, by the use of a coating such as lecithin, by the maintenance of the
required
particle size in the case of dispersion and by the use of surfactants.
Prevention of the
action of microorganisms can be achieved by various antibacterial and
antifungal agents,
for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and
the like. In
many cases, it will be preferable to include isotonic agents, for example,
sugars, sodium
chloride, or polyalcohols such as mannitol and sorbitol, in the composition.
Alternatively, biomorphic compositions can be prepared as gels, pastes,
putties,
semi-solids or solids, which can be shaped, formed, extruded, or otherwise
processed
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before implantation, and which can be shaped or formed after implantation to
conform
to a desired shape or size, e.g., of a tissue defect.
Accordingly, biomorphic compositions can be introduced into the body of a
subject by injection or by surgical implantation at a target site. The
compositions can be
constituted so as to occupy a defined space or cavity in the body (e.g., to
fill a cavity left
by the surgical removal of tissue), or can be sufficiently fluid to occupy any
space,
whether regular or irregular, into which the composition is placed.
Uses for Biomorphic Compositions
The biomorphic compositions of the invention can be implanted or injected into
the body of a subject to promote the growth of a variety of tissues. Thus,
biomorphic
compositions are useful in a variety of procedures for repairing, replacing,
or
augmenting tissues of the body.
For example, a bone-forming biomorphic composition can be used to promote
healing of surgically-altered bone (e.g., after removal of osseous tumors or
extraction of
teeth); or to promote healing, e.g., in peridontitis or of fractures {e.g.,
non-union
fractures) or to form new bony structures. Bone-forming implants are thus
useful in
many applications for which autologous bone transplants are currently
performed.
Similarly, muscle-forming biomorphic compositions can be used to replace
muscle, e.g., muscle tissue removed by surgery or damaged through accident.
Biomorphic compositions which form cartilage can be used for replacement or
repair of
cartilage, e.g., cartilage removed in surgical procedures (e.g., arthroscopic
removal of
torn cartilage) or cartilage damaged, e.g., by tearing. Biomorphic
compositions which
form skin have applications to healing of wounds and to skin grafts, e.g., to
assist burn
healing.
Biomorphic compositions can also be useful in plastic surgery applications,
e.g.,
for lip augmentation, or for surgical reconstruction, e.g., of cartilaginous
structures such
as ears or nose.
Assays for Biomorphic Activity of Compositions
It is important to be able to determine whether a given composition has
activity
as a biomorphic composition, e.g., for quality control in the preparation of
biomorphic
materials. The activity of a given composition can readily be determined by
assays
which will be routine to the skilled artisan.
Thus, for example, a biomorphic composition can be implanted in a test animal,
and the effect of the implanted composition assayed at one or more time points
to
determine the in vivo efficacy of the composition. Assays can be performed in
vivo or ex
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vivo, as described herein, or according to known methods of diagnosis,
histology or
pathology.
For example, a bone-forming biomorphic composition can be assayed by
subcutaneous implantation in a test animal such as a mouse. After a selected
time
period, e.g., 24 hours or one week, the implant can be removed and assayed to
determine
the infiltration of cells into the implant. For example, the implant can be
digested with a
neutral protease or a collagenase to degrade the collagenous matrix and
release any cells
that have infiltrated the implant. The released cells can then be sorted and
counted, e.g.,
using FAGS, to determine the type and number of cells present in the implant.
Alternatively, the implant can be dissected out of the test animal and then
sectioned and stained for microscopic evaluation, as is routine in pathology
laboratories.
A bone-forming composition will in general be infiltrated by macrophages
within one day after implantation in a mouse. Thus, a suitable screening assay
for
activity of a bone-forming composition is to implant the composition into a
mouse and
examine the implant for macrophage infiltration after 24 hours. Also, blood
vessels are
generally present in the implant after about three weeks; this process is
readily
determined under a microscope.
The activity of a biomorphic composition in a test animal can also be assayed
by
examining the animal without removing the implant.. For example, simple
palpation of
a subcutaneous implant can be sufficient to determine, e.g., whether
mineralization of a
bone-forming implant has occurred. Also, techniques such as magnetic resonance
imaging, bone scanning, or CAT scanning can be employed to examine the effect
of the
biomorphic composition in vivo. Bone formation can be readily monitored by X-
ray
imaging once mineralization of the implant has begun.
The invention will next be described in connection with certain non-limiting
examples:
Example 1: Preparation of an Inert Matrix: Procedure A
The cartilage and/or bone is cleaned, ground in a liquid nitrogen cooled mill,
passed through a sieve having a nominal size of 200 microns, and collected
with a sieve
having a nominal size of 100 microns. The particles were then washed four
times with
ice cold (0 to 4~C) HEPES buffer, pH 8.2, containing 0.5M KCl (Buffer A). l00
gm
(wet weight) of bone was demineralized with three changes of 4000 ml
prechilled (0 to 4
~C) 20 mM HEPES buffer, pH 8.2, containing 0.5M EGTA (Buffer B), at a
temperature
of 2~C, until the calcium concentration was below about 100 mg/gm bone. The
bone
particles were then collected by filtration or by centrifugation, for example
in a GSA
rotor at 4000 x g for 30 minutes. The pellet was then washed three times with
HEPES
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buffer, pH 8.2, containing 1 M NaCI (Buffer C), then resuspended in Buffer C
and
stirred overnight at 4~C. The matrix was collected by filtration and extracted
stirred
twice more with Buffer C. The matrix was collected and washed, then suspended
in 20
mM HEPES buffer, pH 8.2, containing 1 M ammonium bicarbonate (Buffer D) and
stirred overnight at 4~C. The matrix was collected and washed three times with
0.1 M
sodium bicarbonate solution, pH 7.4. Finally, the wet matrix was dried under
vacuum
and stored at -20~C until use.
The resulting material was substantially non-immunogenic and had the following
properties on analysis:
Total protein: 84% by weight
Total collagen: 80% by weight
Collagen as a percentage 95.2%
of total protein
Minerals: 12% by weight
Example 2: Preparation of an Inert Matrix: Procedure B
The inert matrix was made by the following procedure:
l00 gm ground bone, prepared as in example 1, was demineralized with 500 ml of
chilled buffer containing 0.2 M ED'rA, pH 8.2, at 2~C, for nine days. The
buffer was
changed every third day. After nine days, the residual matrix was collected
and
extracted with 500 ml of 0.2 M sodium citrate, pH 5.2, until the calcium
concentration
was below about 20 mg/gm matrix. The particles were then collected by
filtration and
washed three times with one liter of ice-cold water. The wet matrix was dried
under
vacuum and stored at -20~C.
The resulting material was substantially non-immunogenic and had the following
properties on analysis:
Total protein: 94% by weight
Total collagen: 96% by weight
Collagen as a percentage 100%
of total protein
Minerals: 4% by weight
Example 3: Preparation of Muscle-formin~~ Matrix
A demineralized matrix was prepared by the method described in Example 1,
supra, up to the final high-salt (1 M NaCI) washing step. At this point, the
matrix was
extracted with 1 N HCl at 4~C overnight. The matrix was then collected and
residual
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acid was neutralized with ammonium bicarbonate. Lyophilization yielded a dry
matrix,
which was stored at -20~C prior to use.
Example 4: Preparation of Bone-forming Matrix
The inert matrix prepared in Example 1, supra, was suspended in physiological
saline (PBS) with 0.1 % (w/w) osteopontin, 0.01 % (w/w) bone sialoprotein and
0.1
(w/w) of high-molecular-weight hyaluronic acid.. The suspension was dried down
to
yield a dry matrix, which stored at -20~C prior to use.
The inert matrix prepared in Example 2 was treated in the same way, and a
similar material resulted.
Example 5: Formation of Bone in vivo with Bone-forming Matrix
The bone-forming matrix of Example 4 (50 mg, suspended in saline (PBS) at a
concentration of 200 mg/ml) was injected subcutaneously over a shoulder blade
of 4
week old c57 blk mice. The implants were removed one week, four weeks, or six
months after implantation. The removed implants were fixed with 1 %
formaldehyde in
PBS, embedded in paraffin, and thinly sectioned. The sections were stained
with
Hematoxilin and eosin and examined under a microscope at 40x, 200x, or 400x
magnification. Control mice were injected with the inert matrix material of
Example 2.
The implanted inert matrix showed a thin capsule around the implant, and very
little cell infiltration into the implant, even after six months. The size and
mass of the
inert implant did not significantly change over the course of the experiment.
In contrast, the bone-forming matrix implants showed rapid infiltration of
mesenchymal cells and macrophages after only one week. Implants of the bone-
forming
matrix also showed rapid angiogenesis in the implant (visible after one week).
Some
inflammatory nodules were seen, but the implant did not provoke a generalized
inflammatory response.
For analysis of implants four weeks after injection, the implants of bone-
forming
matrix were removed, implanted in JB4 before sectioning, and stained with Van
Kossa
stain (or Safranine O with a fast green counter stain). Some implants were
demineralized with 1 % formic acid for three days before being embedded in
paraffin.
Van Kossa staining of the implants at four weeks showed abundant mineral
deposition
throughout the implant. Demineralized samples stained with Safranine O/fast
green
showed embedding of osteocytes at the periphery of newly-formed bone, and the
presence of osteoblasts within the newly-formed bone. Highly organized
collagen fibers
could be seen, forming a periosteal collar around the immature, less-developed
new
bone.
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Example 6: Formation of Muscle in vivo with Muscle-forming Matrix
The muscle-forming matrix of Example 3 (50 mg, suspended in saline (PBS) at a
concentration of 200 mg/ml) was injected subcutaneousiy over a shoulder blade
of 4
week old c57 blk mice. The implants were removed one week, two weeks, or four
weeks after implantation. The removed implants were fixed with 1 %
formaldehyde in
PBS, embedded in paraffin, and thinly sectioned. The sections were stained
with
Hematoxilin and eosin and examined under a microscope at 40x or 400x
magnification.
After one week, muscle cells were seen within the implant; the bulk of the
implant contained large numbers of undifferentiated mesenchyrnal cells. After
two
weeks, the majority of the implant was replaced by muscle tissue; Z bands were
visible
in many cells. After four weeks, muscle tissue had completely replaced the
matrix
material of the implant.
Example 7: Repair of Bone Defects with Bone-Forming Matrix
The ability of a bone-forming composition of the invention to repair bone
defects
was assessed using an animal model.
Bone defects were created in the jaws of male Sprague-Dawley rats. In each
animal, two 1.0 cm extraoral submandibular incisions were performed
bilaterally and
mucoperoteal flaps including the muscles were elevated. Two circular defects,
6.0 mm
in diameter were created using a round burr and a 6.0 mm rephine at low speed
under
vigorous irrigation with sterile saline. The defects extended the entire width
of the
ramus.
The defects were randomly treated with one of four treatments. In the first
(control) group, the surgical incision was closed without further treatment of
the bone
defect. In the second group, the bone defect was filled with an inert, non-
immunogenic
bone composition, prepared as described in Example 2, supra. In the third
group, the
bone defect was filled with rat autograftlallograft bone (obtained from
genetically-
identical twin litter mates). In the fourth group, the bone defect was filled
with the
bone-forming matrix prepared in Example 4, supra. For all animals, after
treatment (if
any), the muscular flap was repositioned and sutured with chromic gut sutures
and the
overlying skin was sutured with vicryl. Animals were sacrificed and the
mandibles
removed and split for separate analysis of each defect.
Results for animals sacrificed at two weeks after treatment are as follows:
The untreated control mandibles were found to have massive hematoma, and no
evidence of bone or cartilage formation as seen on histology slides. Scar
tissue and/or
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connective tissue healing appeared to have begun, and fibroblast invasion had
also
begun.
Bone defects treated with the inert, non-immunogenic bone composition had
little bone formation. The implant was found to be cellular, with bone
formation
beginning on the periphery of the implant. There was no evidence of scar or
fibrous
healing, and little invasion of fibroblastic or muscle cells was noted.
Bone defects treated with allograft bone showed that immature bone formation
had occurred; the implant was highly cellular. New bone formation was present
at the
periphery of the implanted graft material. No cartilage formation or
fibroblastic
invasion was seen.
Bone defects treated with the bone-forming matrix of the invention showed
extensive trabecular formation through bone appositional growth. New bone-
forming
cells (osteoblasts) were attached to the implanted matrix and deposited new
bone around
the matrix. There was also indication of new bone formation from the periphery
into the
interior of the implant. The implant was less cellular than the control-
treated defects.
No evidence of cartilage formation was seen. Several areas of trabecular bone
showed
the presence of blood vessels and distinct marrow spaces.
Further results were obtained by sacrifice of the animals at four weeks post-
treatment. At the four-week time point, defects filled with the bone-forming
matrix of
the invention were substantially indistinguishable from the surrounding bone.
Bone
defects treated with allograft bone showed considerable mature bone formation,
although the border of the defect was still evident. Bone defects treated with
the inert,
non-immunogenic bone composition had some bone formation, but less than was
seen
with the bone-forming matrix of the invention. Unfilled defects showed little
or no bone
formation but were filled with connective tissue.
The results of this experment show that the bone-forming compositions of the
invention can provide new bone formation in bone defects. It is believed that
the bone-
forming compositions of the invention provided results equal to, or superior
to, the
results seen with bone allograft treatment.
The contents of all references cited throughout this application are hereby
incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain using no more
than
routine experimentation, numerous equivalents to the specific procedures
described
herein. Such equivalents are considered to be within the scope of this
invention and are
covered by the following claims.
