Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
X027259
BONE COLLAGEN MATRIX FOR IMPLANTS
Background of the Invention
This invention relates to a biocompatible,
implantable material which is absorbed naturally in
vivo with minimal immunological reaction, and to the
methods for its production. More particularly, this
invention relates to a novel collagenous bone matrix
useful as an allogenic or xenogenic implant for use
as an osteogenic device, as a bone particle coating
for implantable prostheses, as a delivery vehicle for
the in vivo sustained release of protein, and as a
substratum for growth of anchorage-dependent cells.
A biocompatible, implantable material that
can be resorbed in viv could be used to promote
conductive bone growth, induce osteogenesis when
combined with an osteoinductive protein, provide a
substratum for in vivo or in vi r growth of
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anchorage-dependent cells, or serve as a carrier for
the sustained release of, for example, a therapeutic
drug or antibiotic. Such a material must be
biocompatible, that is, must not induce an
immunogenic or continued inflammatory response in
vivo Its physical structure must allow cell
infiltration, and it must have an in vivo resorption
time appropriate for its function.
The potential utility of an osteogenic
device capable of inducing endochondral bone
formation in vivo has been recognized widely. It is
contemplated that the availability of such devices
would revolutionize orthopedic medicine, certain
types of plastic surgery, and various periodontal and
craniofacial reconstructive procedures.
The developmental cascade of bone
differentiation induced by the implantation of
demineralized bone matrix is well documented in the
art (Reddi, 1981, Collagen Rel Res 1:209-226).
Although the precise mechanisms underlying the
phenotypic transformations are unclear, it has been
shown that the natural endochondral bone
differentiation activity of bone matrix can be
dissociated into two principle components: a soluble
proteineous components responsible for osteogenic
activity, and an insoluble collagenous matrix
(residue serves as a carrier for bone induction).
The soluble osteoinductive protein components are
then reconstituted with inactive residual collagenous
matrix to restore full bone inducing activity
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(Sampath et al., Proc. Natl. Acad. Sci. USA 78:7599-7603
(1981). Recently, the protein factors hereafter referred to
as osteogenic protein (OP) responsible for inducing
osteogenesis have been purified, expressed in recombinant host
cells, and shown to be truly osteoinductive when appropriately
sorbed onto allogenic demineralized bone powder. (U. S. Patent
No. 4,968,590 issued on November 6, 1990).
Studies have shown that while osteoinductive
proteins are useful cross species, the collagenous bone matrix
generally used for inducing endochondral bone formation is
species specific (Sampath and Reddi (1983) PNAS 80:6591-6594).
Implants of demineralized, delipidated, extracted xenogenic
bone matrix as a carrier in an in vivo bone induction system
invariably has failed to induce osteogenesis, presumably due
to inhibitory or immunogenic components in the bone matrix.
However, even the use of allogenic bone matrix in osteogenic
devices may not be sufficient for osteoinductive bone
formation in many species. For example, allogenic,
subcutaneous implants of demineralized, delipidated monkey
bone matrix is reported not to induce bony formation in the
monkey. (Asperberg et al., J. Bone Joint Surq. (Br) 70-8:625-
627 (1988)).
U.S. 4,563,350, published January 7, 1986, discloses
the use of trypsinized bovine bone matrix as a xenogenic
matrix to effect osteogenic activity when implanted with
extracted partially purified bone inducing protein
preparations. Bone formation is
l..i...
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said to require the presence of at least 5%, and
preferably at least 10%, non-fibrillar collagen in
the disclosed matrix. The authors claim that removal
of telopeptides which are responsible in part for the
immunogenicity of collagen preparations is more
suitable for xenogenic implants.
EPO 309,241 (published 3/29/89. filed
9/22/88, priority 9/25/87) discloses a device for
inducing endochondral bone formation comprising an
osteogenic protein preparation, and a matrix carrier
comprising 60-98% of either mineral component or bone
collagen powder and 2-40% atelopeptide
hypoimmunogenic collagen.
Deatherage et al., (1987) Collagen Rel. Res.
7:2225-2231, purport to disclose an apparently
xenogenic implantable device comprising a bovine bone
matrix extract that has been minimally purified by a
one-step ion exchange column and reconstituted highly
purified human Type-I placental collagen.
In order to repair bone defects in
orthopedic reconstructive surgery, biomaterials based
on collagens, minerals/ceramics, polymers and metal
implants are being used as implants. These
biomaterials are known to support healing by
conduction but do not induce new bone. The current
state of the art of materials used in surgical
procedures requiring conductive bone repair, such as
the recontouring or filling in of osseous defects, is
disclosed by Deatherage (1988) ~. Oral Maxillofac.
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Surg- 17:395-359. All of the known implant materials
described (hydroxylapatite, freeze-dried bone, or
autogenous bone grafts) have little or no
osteoinductive properties. The ability to induce
osteogenesis is preferred over bone conduction for
most procedures.
U.S. Patent No. 4,795,467 discloses a bone
repair composition comprising calcium phosphate
minerals (preferable particle size of 100-2,OOOU) and
atelopeptide, reconstituted, crosslinked, fibrillar
collagen. It purports to be a non-antigenic,
biocompatible, composition capable of filling bony
defects and promoting bone growth xenogenically.
U.S. 4,789,663 discloses using xenogenic
collagen from bone and/or skin to effect conductive
bone repair by exposing the defect to fresh bone,
wherein the collagen is enzymatically treated to
remove telopeptides, and is artificially crosslinked.
In order to enhance bone ingrowth in
fixation of orthopedic prostheses, porous coatings on
metallic implants are being employed. Although the
surface chemistry of porous coatings plays a role in
bone conduction, it appears bone has higher tensile
and shear strength and higher stiffness at the porous
coating - bone interface. The need to provide a
"biological anchor" for implanted prostheses,
particularly metallic implants, is well documented in
the art. The state of the art of prosthetic
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implants, disclosed by Specter (1987) J. Arthroplasty
2:163-177, generally utilizes porous coated devices,
as these coats have been shown to promote cellular
ingrowth significantly.
Recently the art also has sought to increase
cellular ingrowth of implanted prostheses by coating
their surfaces with collagen preparations. For
example, EPO 169,001 (published 1/22/86) claims a
collagen-coated prosthesis wherein the coat comprises
a purified, sterile, non-immunogenic xenogenic
collagen preparation from bone or skin. The collagen
is preferably atelopeptide collagen. The coating is
formed by dipping the prosthesis into a suspension of
collagen, or forming a collagen sheet that is wrapped
about the prosthesis.
U.S. 4,812,120 discloses a prosthetic device
comprising a metal core over which are applied
successive polymer layers. The outer layer comprises
a biopolymer having protruding collagen fibrils. The
protruding fibrils are subject to damage upon
implantation of the device. Increased surface area
and pore size in a matrix has been shown to enhance
cell attachment and growth of anchorage-dependent
cells in vi r
Efficient in vi r growth of mammalian cells
is often limited by the materials used as the
substratum or "scaffold" for anchorage-dependent
cells. An optimal matrix for this purpose must be
physiologically acceptable to the anchorage dependent
-.027259
cells, and it must also provide a large available
surface area to which the cells can attach. GB
2,178,447, published 02/11/87, discloses a fibrous or
porous foam matrix comprising open or closed form
fibers, with a pore size on the order of 10-100um
(matrix height is 50-500u). The fiber network is
generated as a sheet which must then be modified if
different scaffold shapes are desired. Strand et al.
(Biotechnology and Bioengineering, V. 26, 503, 1984)
disclose microcarrier beads for use as a matrix for
anchorage dependent cells in a matrix perfusion cell
culture. Bead materials tested were DEAF or
polyacrylamide. Surface area available was 250-300
cm2/g and required a cell innoculaton of 106
cells/ml. U.S. Patent 4,725,671 claims collagen
fiber membranes suitable for cell culture, comprising
soluble atelopeptide collagen fibers that are dried
and preferably cross-linked.
The art has sought sustained release
vehicles with known, reliable "release" rates.
Effective carriers must be biocompatible,
water-insoluble, capable of trapping or otherwise
holding the therapeutic agent of interest for a
required time, and must have a resorption time in
vivo that mimics the desired release rate of the
agent. Collagens are attractive carriers for
clinical use, primarily because of their
biocompatible and biodegradable properties. The
carriers are generally formulated into "sponge-like"
structures by solubilizing or dispersing collagen and
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then solidifying the solution so that monofilments
are captured in a generally, random, open-structured
array. The solvent is then removed, and the
molecules chemically crosslinked to maintain the
open-structure and render the carrier water
insoluble. The therapeutic compound is preferably
mixed with the collagen in solution prior to
solidification.
The structures can be made in a variety of
shapes. For example, EPO 230,647, published
08/05/87, discloses structures formed as
micropellets. The structures can also be made in
sheet form (U. S. 4,703.108, published 10/27,87), rods
or tubes (see, for example, EPO 069,260, published
1/12/83, EPO 170,979, published 02/12/86, and U.S
4,657,548, published 4/14/87), or beads. U.S. No.
4,837,285, published 6/6/89, discloses a composition
for wound dressings or drug delivery systems made of
porous beads formed by freeze-drying microdroplets
containing the agent of interest and solubilized or
dispersed Type I or Type III collagen. The
microdroplets are then slowly lyophilized or
air-dried to form beads which are then crosslinked.
Unfortunately, the high "fiber-forming"
property of collagen can interfere with the formation
of uniform and homogeneous solutions, making
efficient synthesis of appropriate carrier matrices
difficult. In addition, the use of crosslinking
agents (e. g., glutaraldehyde) may have adverse
biological effects, such as cell cytotoxicity (Cooke,
et al. British J. Ex~. Path. ~4, 172, 1983).
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It is an object of this invention to provide
a biocompatible, in viv biodegradable bone matrix,
implantable in a mammalian host with little or no
significant inhibitory or immunogenic response.
Another object is to provide a biocompatible, in vivo
biodegradable matrix capable in combination with an
osteoinductive protein of producing endochondral bone
formation in mammals, including humans. Still other
objects are to provide a superior material for
coating implantable prothetic devices, to increase
the cellular ingrowth into such devices, to provide a
biocompatible, in viv biodegradable matrix for use
as a carrier of sustained-release pharmaceutical
compositions, wherein the resorption rate of the
matrix can be adjusted to match that of the
pharmaceutical agent, and to provide a biocompatible,
in viv biodegradable matrix capable of acting as a
scaffold or substratum for anchorage-dependent cells,
wherein the surface area available for cell
attachment can be adjusted. Yet another object of
the invention is to provide a method for the
production of such matrix material.
These and other objects and features of the
invention will be apparent from the description,
drawings, and claims that follow. As used herein,
"bone collagen matrix" or "bone matrix" is intended
to mean stripped, cleaned and demarrowed, pulverized,
delipidated bone that has been demineralized and
protein extracted with guanidine hydrochloride or an
equivalent extractant. "Implantable", as used
herein, includes surgical introduction as well as
topical application, and introduction by injection.
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Brief Description of the Drawings
The foregoing and other objects of this
invention, the various features therof, as well as
the invention itself, may be more fully understood
from the following description, when read together
with accompanying drawings, in which:
FIGURES lA through 1F are, respectively,
scanning electron micrographs (< 500X) of: (lA) ,
Demineralized, guanidine-extracted rat bone matrix,
(498X); (1H) Demineralized, guanidine-extracted
bovine bone matrix, (480X); (1C) Demineralized,
guanidine-extracted bovine bone matrix, further
treated with hydrogen fluoride (HF), and washed,
(350X); (1D) Demineralized, guanidine-extracted
bovine bone matrix, further treated with 99.9%
dichloromethane and 0.1% trifluoroacetic acid
(DCM/TFA), and washed, (441X); (lE) Demineralized,
guanidine-extracted bovine bone matrix, further
treated with 99.9% acetonitrile and 0.1%
trifluoroacetic acid (ACN/TFA), and washed, (429X);
(1F) Demineralized, guanidine-extracted monkey bone
matrix, further treated with hydrogen fluoride, and
washed, (350X).
FIGURES 2A through 2E are respectively,
Scanning electron micrographs (_> 5000X) of: (2A)
Demineralized, guanidine-extracted rat bone matrix,
(9800X); (2B) Demineralized, guanidine-extracted
bovine bone matrix, (8700X); (2C) Demineralized,
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202725 9
guanidine-extracted bovine bone matrix, further
treated with hydrogen fluoride, and washed, (5000X);
(2D) Demineralized, guanidine-extracted bovine bone
matrix, further treated with DCM/TFA, and washed,
(8000X); and (2E) Demineralized, guanidine-extracted
bovine bone matrix, further treated with ACN/TFA, and
washed, (11000X).
FIGURES 3A and 3B are, respectively,
scanning electron micrographs (5000X) of: (3A)
Demineralized, guanidine-extracted bovine bone
matrix, further treated with dichloromethane, but not
washed; and (3B) Demineralized, guanidine-extracted
bovine bone matrix, further treated with
dichloromethane, and washed.
FIGURE 4A is a bar graph of alkaline
phosphatase activity as a measure of osteogenesis in
the presence of variously-treated bovine bone matrix
materials using DCM and DCM/TFA, and differing
amounts of osteogenic protein.
FIGURE 4B is a bar graph of alkaline
phosphatase activity as a measure of osteogenesis in
the presence of temperature treated bovine bone.
matrix materials.
FIGURES 5A-5D are, respectively, scanning
electron micrographs (approx. 1000X) of bovine matrix
heat treated in water for one hour at (5A) 37°C, (5B)
45°C, (5C) 55°C, and (5D) 65°C. Compare FIGURE 1B,
untreated bovine matrix at 480X.
s
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Summary of the Invention
This invention involves a matrix for
implantation in a mammalian host comprising
biocompatible, mineral-free, delipidated, insoluble
Type-I bone collagen which may be allogenic or
xenogenic to the host, and which, when implanted in
the host, is biodegradable and can serve as a
substratum to support mesenchymal cell migration and
proliferation. As disclosed herein, the matrix may
be combined with osteogenic protein to induce
endochondral bone formation reliably and reproducibly
in a mammalian body. It may also be used as a
surface coat around implantable prosthetic devices to
promote cellular ingrowth. It can act as a carrier
for the sustained release of various compositions in
the mammalian body, and can provide a biocompatible
substrate for anchorage-dependent cells.
The development of this matrix material
resulted from the discovery of key features required
for successful implantation of xenogenic bone matrix
and osteogenic protein. Studies indicated that
osteogenic devices comprising substantially pure
osteogenic protein and allogenic demineralized,
delipidated guanidine-extracted bone matrices must
have interstices dimensioned to permit the influx,
proliferation and differentiation of migratory
cells. It was also observed that osteogenic devices
comprising xenogenic bone matrices induce little or
no endochondral bone formation in viv The absence
of bone formation by xenogenic matrices generally has
202725 9
been thought to be due to an immunogenic or
inhibitory response to protein components still
present in the matrix (either the collagen
telopeptides or associated non-collagenous
glycoproteins.)
It has now been discovered that the overall
specific surface area (surface area/unit mass),
degree of porosity and micropitting, and the size of
the micropits and pores of the matrix is important
for successful xenogenic implants, and even for
allogenic implants of certain species
Panels A and H of FIGURES 1 and 2 are
scanning electron micrographs showing the particle
structure of demineralized, guanidine-extracted bone
matrix from rat and calf, respectively. As can be
seen from the SEMs, there is a significantly greater
inherent porosity, or surface area, in rat bone
matrix than in bovine bone matrix. It has been
discovered that increasing porosity and intraparticle
surface area of bone matrix can promote osteogenic
induction as evidenced by rat collagenous bone matrix
implants. This is achieved by treating collagenous
bone matrix with certain solvents or heat so as to
alter its morphology. Agents suitable for this
purpose are disclosed herein and are termed collagen
fibril modifying agents.
In one aspect, this invention comprises a
matrix for implantation in a mammalian host
comprising biodegradable, biocompatible mineral-free,
delipidated, insoluble Type-I bone collagen,
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allogenic or xenogenic to the host, depleted in
non-collagenous proteins, preferably in the form of
particles having a mean diameter within the range of
70~un-850~un, and having an increased surface area
relative to untreated material.
The treated matrix material has an increased
intrusion volume, preferably at least 25% greater,
and often 50% or more of untreated material; an
increased number of pores and micropits; and surface
area which is at least doubled as measured by the BET
method. The preferred particulate material, which
may be used as a shaped, interadhered particle mass
or a simple agglomeration of close-packed particles,
has a particle size within the range of 150~un to
420utn.
Another aspect of this invention involves
methods of treating demineralized, delipidated
guanidine extracted collagenous bone matrix with a
collagen fibril modifying agent so as to increase the
porosity of the collagen matrix to achieve the
desired increase in surface area. The matrix
treatments herein described increases the porosity
and micropitting of the matrix particles, thereby
altering the morphology of the particles. In the
preferred embodiment the surface takes on the
appearance at high magnification resembling an oyster
shell with superposed layers with average pore size
range on the order of 1.0 to 100 microns. The effect
can be achieved by treatment with acids (for example
trifluoroacetic acid (TFA) or hydrogen fluoride HF)
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20225 g
alone or preferably used to acidify organic solvents,
for example, dichloromethane (DCM), acetonitrile
(ACN), isopropanol (IP) and/or chloroform. All these
agents are known to have protein denaturation
properties and to swell insoluble collagenous protein
by modifying collagen fibrils. Among these,
preferred agents are DCM and ACN. The currently most
preferred agents are DCM or ACN acidified with a
small amount, e.g., 0.1%, of TFA. This alteration
can have the effect potentially of increasing or
decreasing the resorption time of the matrix in
vivo Thus, one can extend treatment times to
shorten the matrix resorption rate in vivo (longer
treatment times yield faster resorption rates).
The surface morphology also can be achieved
by heating demineralized delipidated guanidine
extracted bone collagen in water at high temperature
(37°-65°, preferably 45°-60°C) for one hour or
other
time sufficient to achieve the desired surface
morphology. Although the mechanism is not yet clear,
it is hypothesized that the heating of collagen
alters collagen fibrils to result in increase in
surface area. Thus bone matrix may be treated at
various elevated temperatures in water (lg/30m1) with
stirring and then filtered. Treatment of insoluble
collagen in water by increasing temperature results
initially in a melting transition (Tm), the
temperature required to go from one-quarter to three
quarters of the total transition from helical
structure to non-helical. Thereafter the fibrils
will abruptly shrink a fraction of length at some
..
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202759
higher temperature, designated as the shrinkage
temperature (Ts). Ts is normally higher then Tm,
reflecting the added stability contributed by
molecular packing. Heating collagen at pHs below
approximately 5, both Tm and Ts temperature will
decrease.
Examination of solvent treated bone
collagenous matrix shows that demineralized
guanidine-extracted xenogenic bovine bone has a
mixture of additional materials associated with it,
and that extracting these materials plays a part in
improving the biological properties of the matrices.
The collagen of the matrix also may be deglycosylated.
Treatment of the matrix with a fibril
modifying agent should be followed by an appropriate
wash. Bone matrices that have been treated but left
unwashed are generally less osteoinductive when
implanted with osteogenic protein in a mammalian
host. Panels A and B in FIGURE 3 show the dramatic
effect the wash step has on intraparticle surface
area. Currently preferred washes include
urea-containing buffer and water, or alternatively, a
saline buffer.
Mammalian bone tissue growth requires the
influx, proliferation and differentiation of
migratory progenitor cells. Accordingly, in one
aspect, the invention comprises packed matrix
particles defining interstices dimensioned to permit
the influx, proliferation and differentiation of
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2Q~7259
migratory cells, preferably having a particle
diameter that is in the range of 150-420 dun. The
matrix particles also may be deglycosylated. The
matrix comprises dispersed protein, e.g., osteogenic
protein, and is capable of inducing endochondral bone
formation when implanted in a mammalian host.
Preferred means of adsorbing the substantially pure
osteogenic protein onto the matrix particles include
precipitation in cold ethanol from guanidine HC1
solution, or incubation in an
acetonitrile/trifluoroacetic acid solution or in PBS,
followed by lyophilization. The matrix may be shaped
to span a non-union fracture or to fill in a defect
in bone of a mammalian host.
The biocompatible and in vivo biodegradable
nature of the matrix also make it suitable for use as
a delivery vehicle for the 'fin vivo sustained release
of therapeutic drugs. The increased porosity can
increase the matrix's ability to trap and absorb
therapeutics. Moreover, it has been discovered that
varying the treatment time, solvent concentration,
and related treatment parameters can serve to alter
the resorption rate of the matrix in vivo Thus,
this invention provides an easily generated carrier
source material of great versatility. In view of
this disclosure, those skilled in the art easily can
create a carrier matrix having a specific, desired,
reliable resorption time. They can then adsorb the
agent of interest onto the matrix using one of the
methods disclosed herein, or any of the techniques
known in the art, to provide a sustained release
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202759
vehicle with improved reliability in release of the
therapeutic compound.
The particulate and porous nature of the
material of this invention, along with its
biocompatibility in mammalian hosts, permit its use
at the interface of an implanted prosthetic device
and the surrounding mammalian tissue to promote
cellular ingrowth. Moreover, the matrix structure
lends itself to increased durability during
implantation when compared with the collagen fibrils
commonly used in such compositions. In view of this
disclosure, those skilled in the art easily can
create a surface coating for prosthetic devices
having a specific, predetermined porosity or
micropitting and increased durability. They can then
attach the coat to the prosthetic core using any of
the techniques known in the art. See, for example,
Cook et al., Clin. Ortho. Rel Res No 232, p. 225,
1988. The matrix further can comprise osteogenic
protein if endochondral bone induction is desired.
The nature of the matrix of this invention
also makes it a superior substratum for in v' r
growth of anchorage-dependent cells. The matrix
itself provides a physiologically acceptable surface
for cell attachment, and the particle interstices and
intraparticle porosity and micropitting provides
significant increases in the surface area available
for cell attachment over other known matrices. A
surface area on the order of 3000 cm2/g or higher can
be achieved readily. Moreover, the structure of the
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2A27~59
matrix of this invention allows one to vary the
particle porosity as desired. The cascade of pores
present in this matrix promotes efficient nutrient
access to cells, and increases the surface area
available for cell attachment, thereby lowering the
cell innoculant concentration required in a cell
perfusion system (See GB 2,178,447). In view of this
disclosure, one skilled in the art efficiently can
create a biocompatible matrix of choice. having a
specific, known, desired porosity or surface
microtexture.
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Detailed Description
Practice of the invention requires the
availability of bone, preferably mammalian bone,
e.g., bovine. The bone is cleaned, demarrowed,
delipidated, demineralized, reduced to particles of
an appropriate size, extracted to remove soluble
proteins, sterilized, and otherwise treated as
disclosed herein to produce an implantable material
useful in a variety of clinical settings.
Matrices of various shapes fabricated from
the material of the invention may be implanted
surgically for various purposes. Chief among these
is to serve as a matrix for bone formation in various
orthopedic, periodontal, and reconstructive
procedures, as a sustained release carrier, or as a
collagenous coating for implants. The matrix may be
shaped as desired in anticipation of surgery or
shaped by the physician or technician during
surgery. Thus, the material may be used for topical,
subcutaneous, intraperitoneal, or intramuscular
implants; it may be shaped to span a nonunion
fracture or to fill a bone defect. In bone formation
or conduction procedures, the material is slowly
absorbed by the body and is replaced by bone in the
shape of or very nearly the shape of the implant.
Various growth factors, hormones, enzymes,
therapeutic compositions, antibiotics, and other body
treating agents may be sorbed onto the carrier
material and will be released over time when
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implanted as the matrix material is slowly absorbed.
Thus, various known growth factors such as EGF, PDGF,
IGF, FGF, TGF alpha, and TGF beta may be released _in
vivo The material can be used to release
antibiotics, chemotherapeutic agents, insulin,
enzymes, or enzyme inhibitors.
Details of how to make and how to use the
materials of the invention are disclosed below.
A. Preparation of DemineraliTed Bon
Demineralized bovine bone matrix is prepared
by previously published procedures (Sampath and Reddi
(1983) Proc. Natl. Acad. Sci. USA $Q:6591-6595).
Bovine diaphyseal bones (age 1-10 days) are obtained
from a local slaughterhouse and used fresh. The
bones are stripped of muscle and fat, cleaned of
periosteum, demarrowed by pressure with cold water,
dipped in cold absolute ethanol, and stored at
-20°C. They are then dried and fragmented by
crushing and pulverized in a large mill. Care is
taken to prevent heating by using liquid nitrogen.
The pulverized bone is milled to a particle size in
the range of 70-850 um, preferably 150 yam-420 um, and
is defatted by two washes of approximately two hours
duration with three volumes of chloroform and
methanol (3:1). The particulate bone is then washed
with one volume of absolute ethanol and dried over
one volume of anhydrous ether yielding defatted bone
powder. The defatted bone powder is then
demineralized by four successive treatments with 10
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volumes of 0.5 N HC1 at 4°C for 40 min. Finally,
neutralizing washes are done on the demineralized
bone powder with a large volume of water.
B. Suanidine Extraction
Demineralized bone matrix thus prepared is
extracted with 5 volumes of 4 M guanidine-HC1, 50mM
Tris-HC1, pH 7.0 for 16 hr, at 4°C. The suspension
is filtered. The insoluble material is collected and
used to fabricate the matrix. The material is mostly
collagenous in nature. It is devoid of osteogenic or
chondrogenic activity.
C. Matrix Trea ments
The major component of all bone matrices is
Type-I collagen. In addition to collagen,
demineralized bone extracted as disclosed above
includes non-collagenous proteins which may account
for 5% of its mass. In a xenogenic matrix, these
noncollagenous components may present themselves as
potent antigens, and may constitute immunogenic
and/or inhibitory components. These components may
also inhibit osteogenesis in allogenic implants by
interfering with the developmental cascade of bone
differentiation. The treatments described below use
solvents to extract potentially unwanted components
from the matrix, and use solvents or heat treatments
to alter the surface structure of the matrix material.
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After contact with the solvents, the treated
matrix is washed to remove the extracted components,
following a form of the procedure set forth below:
1. Suspend in TBS (Tris-buffered saline)
lg/200 ml and stir at 4°C for 2 hrs; or in 6 M urea,
50 mM Tris-HC1, 500 mM NAC1, pH 7.0 (UTBS) or water
and stir at room temperature (RT) for 30 minutes
(sufficient time to neutralize the pH);
2. Centrifuge and repeat wash step; and
3. Centrifuge; discard supernatant; water
wash residue; and then lyophilize.
C1. Acid Treatments
1. Trifluoroacetic acid.
Trifluoroacetic acid is a strong
non-oxidizing acid that is a known swelling agent for
proteins which modifies collagen fibrils.
Bovine bone residue prepared as described
above is sieved, and particles of the appropriate
size are collected. These particles are extracted
with various percentages (1.0% to 100%) of
trifluoroacetic acid and water (v/v) at 0°C or room
temperature for 1-2 hours with constant stirring.
The treated matrix is filtered, lyophilized, or
washed with water/salt and then lyophilized.
2. Hydrogen Fluoride.
-24-2~~7259 ~__._.... .
Like trifluoroacetic acid, hydrogen fluoride
is a strong acid and swelling agent, and also is
capable of altering intraparticle surface structure.
Hydrogen fluoride is also a known deglycosylating
agent. As such, HF may function to increase the
osteogenic activity of these matrices by removing the
antigenic carbohydrate content of any glycoproteins
still associated with the matrix after guanidine
extraction.
Bovine bone residue prepared as described
above is sieved, and particles of the appropriate
size are collected. The sample is dried ~n vacuo
over P205, transferred to the reaction vessel and
exposed to anhydrous hydrogen fluoride (10-20 ml/g of
matrix) by distillation onto the sample at -70°C.
The vessel is allowed to warm to 0°C and the reaction
mixture is stirred at this temperature for 120
minutes. After evaporation of the hydrogen fluoride
in vacuo, the residue is dried thoroughly in vacuo
over KOH pellets to remove any remaining traces of
acid. Extent of deglycosylation can be determined
from carbohydrate analysis of matrix sample taken
before and after treatment with hydrogen fluoride,
after washing the samples appropriately to remove
non-covalently bound carbohydrates. SDS-extracted
protein from HF-treated material is negative for
carbohydrate as determined by CON A blotting.
The deglycosylated bone matrix is next
washed twice in TBS (iris-buffered saline) or UTBS,
water-washed, and then lyophilized.
~~2~25 9 .~ . .. .
-25-
Other acid treatments are envisioned in
addition to HF and TFA. TFA is a currently preferred
acidifying reagent in these treatments because of its
volatility. However, it is understood that other,
potentially less caustic acids may be used, such as
acetic or formic acid.
C2. solvent Trea ment
1. Dichloromethane.
Dichloromethane (DCM) is an organic solvent
capable of denaturing proteins without affecting
their primary structure. This swelling agent is a
common reagent in automated peptide synthesis, and is
used in washing steps to remove components.
Bovine bone residue, prepared as described
above, is sieved, and particles of the appropriate
size are incubated in 100% DCM or, preferably, 99, g%
DCM/0.1% TFA. The matrix is incubated with the
swelling agent for one or two hours at 0°C or at room
temperature. Alternatively, the matrix is treated
with the agent many times (X3) with short washes (20
minutes each) with no incubation.
FIGURE 4A illustrates the effectiveness of
the presence of a small amount of acid in an organic
solvent swelling agent treatment in converting bovine
matrix to a material useful as a bone formation
matrix in rat.
-26-
2. Acetonitrile.
Acetonitrile (ACN) is an organic solvent,
capable of denaturing proteins without affecting
their primary structure. It is a common reagent used
in high-performance liquid chromatography, and is
used to elute proteins from silica-based columns by
perturbing hydrophobic interactions.
Bovine bone residue particles of the
appropriate size, prepared as described above, are
treated with 100% ACN (1.0 g/30 ml) or, preferably,
99.9% ACN/0.1$ TFA at room temperature for 1-2 hours
with constant stirring. The treated matrix is then
water-washed, or washed with urea buffer, or 4 M NAC1
and lyophilized. Alternatively, the ACN or ACN/TFA
treated matrix may be lyophilized without wash.
0 3. Isopropanol.
Isopropanol is also an organic solvent
capable of denaturing proteins without affecting
their primary structure. It is a common reagent used
to elute proteins from silica HPLC columns.
Bovine bone residue particles of the
appropriate size prepared as described above are
treated with 100% isopropanol (1.0 g/30 ml) or,
30 preferably, in the presence of 0.1% TFA, at room
temperature for 1-2 hours with constant stirring.
The matrix is then water-washed or washed with urea
buffer or 4 M NAC1 before being lyophilized.
_27_ 2 0 2 7 2 5 9
4. Chloroform
Chloroform also may be used to increase
surface area of bone matrix like the reagents set
forth above, either alone or acidified.
Treatment as set forth above is effective to
assure that the material is free of pathogens prior
to implantation.
C3. Heat Treatment
Various amounts of delipidated,
demineralized guanidine-extracted bone collagen was
heated in water (lg/30m1) under constant stirring in
a glass flask, water jacked, and maintained in a
given temperature for 1 hour. In some instances the
water is replaced with O.1M acetic acid to help
"swell" the collagen before heating. The temperature
employed is held constant at room temperature, and
about 37°C, 45°C, 55°, 65°, 75°. After the
heat
treatment, the matrix is filtered and lyophilized and
used for implant. The results are shown in Figure
4B. Figure 8 illustrates the morphology of the
successfully altered collagen surface treated at
37°C, 45°C, 55°C and 65°C.
The collagen matrix materials preferably
take the form of a fine powder, insoluble in water,
comprising nonadherent particles. It may be used
simply by packing into the volume where new bone
growth or sustained release is desired, held in place
-28-
227259 y
by surrounding tissue. Alternatively, the powder may
be encapsulated in, e.g., a gelatin or polylactic
acid coating, which is adsorbed readily by the body.
The powder may be shaped to a volume of given
dimensions and held in that shape by interadhering
the particles using, for example, soluble, species
biocompatible collagen. The material may also be
produced in sheet, rod, bead, or other macroscopic
shapes.
The functioning of the various matrices can
be evaluated with an in vivo rat bioassay. Studies
in rats show the osteogenic effect in an appropriate
matrix to be dependent on the dose of osteogenic
protein dispersed in the matrix. No activity is
observed if the matrix is implanted alone.
Demineralized, guanidine extracted xenogenic bone
matrix materials of the type described in the
literature are ineffective as a carrier, fail to
induce bone, and produce an inflammatory and
immunological response when implanted unless treated
as disclosed above. Many of the allogenic matrix
materials also are ineffective as carriers. The
following sets forth various procedures for preparing
osteogenic devices from control and matrix materials
prepared as set forth above, and for evaluating their
osteogenic utility.
A. Fabrication of Osteoc~enic Device
The osteogenic protein may be obtained using
the methods disclosed in U.S. Patent No.
202725 9
-29-
4,968,590, issued November 6, 1990; WO 89/09788 published on
October 19, 1989 (entitled Biosynthetic Osteogenic Proteins
and Osteogenic Devices Containing Them), and WO 89/09787
published on October 19, 1989 (entitled Osteogenic Devices).
Both PCT applications were filed April 7, 1989.
Alternatively, extracts rich in osteogenic protein useful in
fabricating devices may be obtained as disclosed in U.S.
Patent No. 4,294,753 to Urist.
A1. Ethanol Precipitation
Matrix is added to osteogenic protein
dissolved in guanidine-HC1. Samples are vortezed and
incubated at a low temperature. Samples are then
further vorteged. Cold absolute ethanol is added to
the mixture which is then stirred and incubated.
After centrifugation (microfuge, high speed) the
supernatant is discarded. The matrix is washed with
cold concentrated ethanol in water and then
lyophilized. _
A2. Acetonitrile Trifluoroacetic Acid
Lyophilization
In this procedure, osteogenic protein in an
acetonitrile trifluroacetic acid (ACN/TFA) solution
was added to the carrier material. Samples were
vigorously vorteaed many times and then lyophilized.
Osteogenic protein was added in varying
concentrations, and at several levels of purity.
This method is currently preferred.
-30-
2~~7259
A3. Urea Lyophilization
For those osteogenic proteins that are
prepared in urea buffer, the protein is mined with
the matriz material, vortezed many times, and then
lyophilized. The lyophilized material may be used
"as is" for implants.
A4. Buffered Saline Lyophilization
OP preparations in physiological saline may
also be vortezed with the matrix and lyophilized to
produce osteogenically active material.
These procedures also can be used to adsorb
other active therapeutic drugs, hormones, and various
bioactive species for sustained release purposes.
B. Implantation
The bioassay for bone induction as described
by Sampath and Reddi (Proc. Natl. Acad. Sci. USA
(1983) 80: 6591-6595), may be used to monitor endochondral bone
differentiation activity. This assay consists of
implanting the bovine test samples zenogenically in
subcutaneous sites in recipient rats under ether
anesthesia. Male Long-Evans rats, aged 28-32 days,
were used. A vertical incision (1 crn) is made under
sterile conditions in the skin over the thoraic
region, and a pocket is prepared by blunt
_. ~0~~259
-31-
dissection. Approximately 25 mg of the test sample
is implanted deep into the pocket and the incision is
closed with a metallic skin clip. The day of
implantation is designated as day of the experiment.
Implants were removed on day 12. The heterotropic
site allows for the study of bone induction without
the possible ambiguities resulting from the use of
orthotropic sites.
C. Cellular Events
Successful implants exhibit a controlled
progression through the stages of matrix induced
endochondral bone development including: (1)
transient infiltration by polymorphonuclear
leukocytes on day one; (2) mesenchymal cell migration
and proliferation on days two and three; (3)
chondrocyte appearance on days five and six; (4)
cartilage matrix formation on day seven; (5)
cartiliage calcification on day eight; (6) vascular
invasion, appearance of osteoblasts, and formation of
new bone on days nine and ten; (7) appearance of
osteoblastic and bone remodeling and dissolution of
the implanted matrix on days twelve to eighteen; and
(8) hematopoietic bone marrow differentiation in the
ossicle on day twenty-one. The results show that the
shape of the new bone conforms to the shape of the
implanted matrix.
2027259
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D. Histological Evaluation
Histological sectioning and staining is
preferred to determine the extent of osteogenesis in
the implants. Implants are fixed in Bouins Solution,
embedded in paraffin, and cut into 6-8 dun sections.
Staining with toluidine blue or hemotoxylin/eosin
demonstrates clearly the ultimate development of
endochondral bone. Twelve day implants are usually
sufficient to determine whether the implants contain
newly induced bone.
E. Biological Markers
Alkaline phosphatase activity may be used as
a marker for osteogenesis. The enzyme activity may
be determined spectrophotometrically after
homogenization of the implant. The activity peaks at
9-10 days in viv and thereafter slowly declines.
Implants showing no bone development by histology
have little or no alkaline phosphatase activity under
these assay conditions. The assay is useful for
quantitation and obtaining an estimate of bone
formation quickly after the implants are removed from
the rat. Alternatively, the amount of bone formation
can be determined by measuring the calcium content of
the implant.
F. Results
The histological evaluation of implants made
using HF-, DCM-, DCM/TFA-, and ACN/TFA-treated bone
matrices is given in Table 1 and in FIGURE 4A. The
-33-
202,25s
osteogenic protein (OP) used in these experiments was
isolated by the method disclosed in U.S. Patent
No. 4,968,590. Experiments were performed
using highly pure (C-18) protein. The results
demonstrate unequivocally that zenogenic implants of
collagenous bovine bone matrix treated as disclosed
herein induces successful endochondral bone formation.
FIGURE 4A illustrates the osteoinductive
effect of water washed matrix treated with nanogram
quantities of purified OP, as indicated by specific
activity of alkaline phosphatase, for allogenic rat
matrix and xenogenic bovine matrix untreated, treated
with DCM alone, 99.9% DCM plus 0.1% TFA, and 90% DCM
plus 10% TFA. As illustrated, DCM with low acidified
concentrations of acid enhances bone formation.
The utility of the material of the invention
in its use as an osteogenic implant is believed to be
dependent in part on increases in intraparticle
surface area, including the formation of micropits
and pores with the size range 1-100um. The basis for
this conclusion is apparent from a review and
comparison of FIGURES lA through 2E. Untreated rat
matrix, shown in FIGURES lA and 2A, is active in rats
and has an obvious, open pore, high surface area
structure. The untreated bovine matrix of FIGURE 1B
and 2B has a lower surface area and is inactive in
rats. However, treatment of the bovine collagen with
HF (FIGURES 1C and 2C), DCM/TFA (FIGURE 1D and 2D),
or with ACN/TFA (FIG lE and 2E), produce an open
pore, high surface area structure which is active
s
.- -34' 20225 s
xenogenically. Pore number, average pore diameter, and
total surface area as measured by intrusion volume, all
are increased by the various treatments. Note the
typical appearance of pores within pores and micropits
within micropits resulting in an "oyster shell"
appearance of the surface. The number and size of such
increases may vary and generally are at least threefold
or at least tenfold. FIGURE 3 shows the appearance of
bovine bone matrix particles when
treated with DCM, with and without a subsequent wash
step (3A and 3B, respectively). As illustrated,
omission of the wash produces a low surface area
structure similar to untreated bovine collagen, and
results in an inactive matrix material. FIGURE 1F
shows the structure of monkey bone collagen after
treatment with HF as disclosed above. The bone
particles may be used genogenically to induce bone.
Demineralized, guanidine extracted monkey bone
reportedly is ineffective as a osteogenic matrix,
even as an allogenic implant.
A number of physical analyses were performed
on the treated matrices as a means of corroborating
the microscopic and physiological observations.
Mercury porosimetry was used to compare the degree of
porosity and deep micropitting of the various
matrices. This standard method depends on the
ability to intrude mercury into a material with
incremental increases in applied pressure. In the
case of an organic material such as treated bone
matrix this pressure must be carried only to the
point of bursting of the material or an error will be
introduced skewing the pore diameter information
towards the Angstrom level. The results of these
2027259
-35-
experiments are shown in Table II and indicate that
the treatment of bovine demineralized bone with
collagen fibril modifying agents significantly
increases the mercury intrusion volume, a parameter
directly related to the increase in pore and deep
micropit volume and area in the treated material,
making this treated bovine matrix similar in
intrusion volume to the active rat matrix material,
and strongly corroborating the visual evidence of the
scanning electron micrographs.
Specific surface area of each of the
matrices was measured by the BET method using Krypton
as the absorbate after outgassing of the material for
1 week at ambient temperature. Table II indicates
that the treated bovine matrices have a significantly
increased surface area when compared with untreated
material, and are similar to the active rat matrix
material, further corroborating the SEM data.
Skeletal density was measured for each of
the materials by helium pycnometry and was found as
would be expected to be about the same for each, so
that one may conclude that the differences in bulk
density as derived from mercury porosimetry data is
attributable entirely to increase in surface area and
porosity of the treated bovine matrices.
2Q27259
TABLE II
Matrix Intrusion Skeletal Bulk Surface Pore Size
Volume Density Density Area (SEM)(u)
(Hg porosimetry) (BET)
RAT 2.525 1.340 .278 .3227 1-100
UNTREATED
BOVINE 1.620 1.360 .420 .1566 0.01-10
BOVINE
ACN/TFA 2.290 1.345 .330 .3160 1-100
BOVINE
ACN/TFA 2.470 1.360 .300 .5724 1-100
BOVINE
DCM/TFA 2.550 1.340 .290 .2242 1-100
An amino acid composition analysis has also
been performed on the different matrices, in an
effort to determine what effect, if any, the collagen
fibril modifying agents have on non-collagenous
protein associated with the matrix. The tyrosine
content of the matrices is used as an indicator of
noncollagenous protein, as the helical domain of
collagen generally has only 2-3 tyrosine residues per
1000 residues. As indicated in Table III below, the
treatments do not significantly affect the tyrosine
(Y) content of the matrices, suggesting that the
guanidine extracted bone collagen contained low
concentrations of noncollagen proteins and the
treatment fails to extract non-collagenous protein
from the carrier matrix.
202725 9
-37-
TABLE III
AMINO ACID ANALYSIS OF UNTREATED AND TREATED MATRIX1
St 7
D 19 22 21 20 28 15 18
E 67 67 70 68 71 65 70
S 31 25 25 27 27 27 27
G 350 354 355 348 336 322 301
H 5 5 5 6 6 8 9
R 55 54 53 47 53 66 57
T 17 13 11 13 13 16 16
A 112 123 120 116 119 123 126
P 138 139 130 128 123 129 130
HYP 101 99 99 108 107 119 127
V 26 26 27 26 27 28 30
M 3 1 2 1 2 3 2
I 11 11 12 11 12 11 12
L 24 23 27 26 28 23 25
F 16 14 25 15 15 16 17
K 21 23 23 23 23 25 27
1 - Expressed as residues/1000 residues
1) RAT MATRIX
2) UNTREATED BOVINE
MATRIX
3) UNTREATED BOVINE
MATRIX, UREA
WASHED
4) 99.9% DCM/0.1% TFA BOVINE MATRIX, UREA WASH
5) 99.9% DCM/0.1% TFA BOVINE MATRIX, NO WASH
6) 99.9% ACN/0.1% TFA BOVINE MATRIX, NO WASH
7) 99.9% ACN/0.1% TFA BOVINE MATRIX, UREA WASH
-38- X027259
The invention may be embodied in other
specific forms without departing from the spirit or
essential characteristics thereof. The present
embodiments are therefore to be considered in all
respects as illustrative and not restrictive, the
scope of the invention being indicated by the
appended claims rather than by the foregoing
description, and all changes which come within the
meaning and range of equivalency of the claims are
therefore intended to be embraced therein.
