Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE lNv~NllON
MODIFIED OSTEOGENIC MATERIA~S
PRIOR ~ PLICATIONS
S This application is a continuation-in-part of
U.S. patent application Serial N~mber 08/422,745 filed
April 14, 1995 which is a continuation of application
serial number 08/057,951 filed January 29, 1993, which is
a continuation of application serial num.ber 07/892,646
filed June 2, 1992, which is a continuation of application
serial number 07/718,914 filed of June 24, 1991, which is
a continuation of application serial number 07/119,916
filed November 13, 1987 which is a continuation-in-part of
application serial number 80,145 filed July 30, 1987 which
is a continuation of application serial number 844,886
filed March 27, 1986.
FIELD OF THE lNV~;N'l'lON
The present invention relates to bone repair
materials with improved cohesive and physical strength for
use in stress-bearing defects or where the ability to
produce and maintain the specific shape of an implant is
important. The principle of creating a stable interface
and conjugate between a protein-based particle and an
organic matrix is also applicable to drug delivery
materials and devices. This invention also relates to
osteogenic bone repair compositions having enhanced
osteogenic potential. In particular, to compositions of
~m;ne~alized bone and soluble calcium or mineral salts
and to methods for preparing these bone repair
compositions having enhanced osteogenic potential and to
therapeutic uses for these compositions.
BACKGROUND ART
The repair of osseous defects involves either
non-resorbable or resorbable prosthetic structures. The
resorbable structures or materials either support the
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ingrowth of adjacent bone and soft tissue or actively
induce the formation of new bone. This active formation
of new bone, termed osteoinduction, occurs only in the
presence of demineralized bone matrix or in the presence
of protein extracts from such matrix, or a combination of
both materials. Particles or powders produced from
demineralized bone matrix possess greater osteogenic
potential per unit weight due to their increased surface
area, than blocks or whole segments of demineralized bone.
Other methods of repairing damaged or missing
osseous tissue or bone have also been explored.
Replacement or support with nonresorbable materials, such
as biocompatible metals, ceramics, or composite
metal-ceramic materials, offers one method of clinical
treatment. Some of these materials, such as metal grade
titanium, can promote osteocoinduction at their surface,
thus leading to a stable, continuous interface with bone.
Caffessee et al. Journal of Periodontology, Feb. 1987
utilizing a "window" implantation technique, established
that nonabsorbable ceramics, such as hydroxyapatite, fail
to stimulate tissue, even when placed in osseous defects.
Resorbable ceramics, such as tricalcium phosphate, display
better conduction of mineralized tissue into the resorbing
graft material when placed in osseous defects. Unlike
demineralized bone matrix, tricalcium phosphate or
hydroxyapatite fail to stimulate induction of new bone
when placed in non-osseous tissue. The addition of
tricalcium phosphate or hydroxyapatite to demineralized
bone matrix or to the extracted bone-inducing proteins
actually inhibits the osteogenic potential of these
established osteoinductive compositions (see Yamazaki et
al. Experimental Study On the Osteoinduction Ability of
Calcium Phosphate Biomaterials with added Bone
Morphogenetic Protein Transactions of the Society For
Biomaterials pg 111, 1986.)
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Aside from the documented inability of
hydroxyapatite and tricalcium phosphate ceramic materials
to independently induce osteogenesis, recent clinical
findings indicate that osteointegration of inorganic
particles is highly dependent on the ability o~ those
S particles to remain fixed in a definite position,
preferably near a bony interface. Hence, the immobility
o~ the parties is a prere~uisite ~or involvement with new
bone formation (See Donath, et al., A Histologic
Evaluation of a ~n~; hular Cross Section One Year A~ter
Augmentation with Hydroxyapatite Particles Oral Surgery,
Oral Medicine. Oral Pathology vol 63 No. 6 pp. 651-655,
1987.
Nevertheless, numerous compositions have been
derived to create clinically useful bone replacement
materials. Cruz U.S. Patent No. 3,767,437 describes
arti~icial ivory or bone-like structures which are ~ormed
from a complex partial salt of collagen with a metal
hydroxide and an ionizable acid, such as phosphoric acid.
With regard to the metal hydroxide, this composition
stresses the use of a polyvalent metal cation in the metal
hydroxide, such as calcium hydroxide. Calcium phosphate
may be added to the complex collagen salt. Cruz also
recites the addition of fibers and ions to increase
hardness and structural strength, but does not document or
make claims with regard to these specific improvements.
Cruz does not mention or claim these compositions to be
osteoinductive or osteoconductive, nor does he mention
their behavior in-vivo.
Thiele, et al., U.S. Patent No. 4,172,128,
recites a process of degrading and regenerating bone and
tooth material and products. This process involves r~irst
- demineralizing bone or dentin, converting the
~m; n~ralized material into a mucopolysaccharide-free
colloidal solution by extraction with sodium hydroxide
~;ng to the resultant solution a physiologically inert
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foreign mucopolysaccharide, gelling the solution, and then
remineralizing the resulting gel. Thiele et al. indicate
this material to be biocompatible and totally resorbable,
thus replaced by body tissue as determined by histologic
analysis the gel material produced by this process is
S reported to completely replace destroyed bone sections
created in experimental ~n;m~ls. The patentees do not
indicate any ability by the material to induce new bone.
The ultimate fate of these materials in-vivo-, or their
ability to stimulate the formation of new bone in non-
osseous implant sites is not described. The patentees do
not describe or quantify the strength properties of thesematerial. Nevertheless, since they are described as gels,
one can assume their strength to be low.
Urist In U.S. Patent No. 4,294,753, describes a
process of extracting and solubilizing a Bone
Morphogenetic Protein (BMP). This is a glycoprotein
complex which induces the formation of endochon~ral bone
in osseous and non-osseous sites. This partially purified
glycoprotein, which is derived from demineralized bone
matrix by extraction, is lyophilized in the form of a
powder. Urist describes the actual delivery of BMP in in-
vivo testing via direct implantation of the powder,
implantation of the powder contained within a diffusion
chamber, or coprecipitation of the BMP with calcium
phosphate. While Urist describes the induction of new
bone after the implantation of one of these forms of BMP
in either osseous or non-osseous sites, Urist fails to
address the intrinsic physical strength properties of any
of these delivery forms. Lyophilized powders and calcium
phosphate precipitates, however, possess little if any,
physical strength. Furthermore, more recent investigators
(see aforementioned y~m~ aki, et al.) indicate that
calcium phosphate ceramics, such as tricalcium phosphate
and hydroxyapatite, when present in high concentrations
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relative to the BMP present, may actually inhibit the
osteogenic action of the BMP.
~effries in U.S. Patent No. 4,394,370 and
4,472,840 describes bone graft materials composed of
collagen and demineralized bone matrix, collagen and
extracted Bone Morphogenetic Proteins (BMP). Also
described is a combination of collagen, ~m; n~ralized bone
matrix, plus extracted bone morphogenetic proteins.
~effries describes an anhydrous lyophilized sponge
conjugate made from these compositions which when
implanted in osseous and non-osseous sites, is able to
induce the formation of new bone. The physical strength
of these sponges is not specific in the disclosure,
however, reports of the compressive strength of other
collagen sponges indicates these materials to be very weak
and easily compressible (much less than 1 kilogram load
needed to affect significant physical strain in
compression or tension).
Smestad in U.S. Patent No. 4,430,760 assigned to
Collagen Corporation, describes a nonstress-bearing
implantable bone prosthesis consisting of ~m; n~ralized
bone or dentin placed within a collagen tube or container.
As the patentee indicates, this bone prosthesis can not be
used in stress-bearing locations clinically.
Glowacki et al., in U.S. Patent No. 4,440,750
apparently assigned to Collagen Corporation and Harvard
University describe plastic dispersions of a~ueous
collagen m;~e~ with d~m;neralized bone particles for use
in inducing bone in osseous defects. This graft material,
as described exists in a gel state and possess little
- 30 physical strength of its own. It use, therefore, must be
restricted to defects which can maintain sufficient form
- and strength throughout the healing process. Furthermore,
with time, the demineralized bone particle suspended
within the aqueous collagen sol-gel begin to settle under
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gravitational forces, thus producing an nonhomogeneous or
stratified graft material.
Seyedin, et al., in U.S. Patent No. 4,434,094,
describes the purification of a protein factor, which is
claimed to be different than Urist's BMP molecule,
S responsible for the induction of chondrogenic activity.
Bell, in U.S. Patent 4,485,097, assigned to
Massachusetts Institutes of Technology, describes a bone
equivalent, useful in the fabrication of prostheses, which
is composed from a hydrated collagen lattice contracted by
fibroblast cells and cont~n~ng ~l~m~neralized bone powder.
As this prosthetic structure is also a hydrated collagen
gel, it has little strength of its own. The patentee
mentions the use of synthetic meshes to give support to
the hydrated collagen lattices to allow handling.
Nevertheless, there is no indication of the clinical use
of the material or measurement of its total physical
strength.
Reis, et al., in U.S. Patent No. 4,623,553,
describes a method for producing a bone substitute
material consisting of collagen and hydroxyapatite and
partially crosslinked with a suitable crosslinking agent,
such as glutaraldehyde or formaldehyde. The order of
addition of these agents is such that the crosslinking
agent is added to the aqueous collagen dispersion prior to
the addition of the hydroxyapatite or calcium phosphate
particulate material. The resultant dispersion is mixed
and lyophilized. The patent lacks any well known
components which are known osteogenic inducers, such as
demineralized bone matrix or extracted bone proteins.
Caplan, et al., in U.S. Patent No. 4,620,327, ~,
describes a method for treating implants such as a
biodegradable masses, xenogeneic bony implants,
allografts, and prosthetic devices with soluble bone
protein to enhance or stimulate new cartilage or bone
formation. These structures may then be crosslinked to
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immobilize the soluble bone protein or retard its release.
While the osteogenic activity of these implants are
described in detail, their physical strength is not
mentioned.
The above review of the prior art reveals that
none of the bone prosthetic materials which claim the
ability to induce new bone formation (osteoinductive
materials) possess high strength characteristics.
Furthermore, o~ those materials which are described with
enhanced strength, these materials consist solely of a
crosslinked conjugates of collagen and inorganic mineral,
which lacks the ability to stimulate the induction of new
bone.
It is especially relevant that none of the above
references address the need to bind the dispersed
particulate or inorganic phase to the organic carrier
matrix (i.e. collagen). As will be described below, the
treatment of ~m;n~ralized bone matrix or particles or
inorganic particles, prior to complexation with an organic
biopolymer, such as collagen, is extremely important in
determ;n;ng the physical strength characteristics of the
bioimplant. Furthermore, the ability to orient protein or
peptide particles in a stable fashion within inorganic or
natural polymeric matrixes permitS the ability to release
drugs, bioactive proteins, and bioactive peptides in a
controlled fashion.
As discussed above a variety of bone graft
materials are available to repair, replace or regenerate
bone lost to disease or injury. Bone grafts may be
allografts, mPAn;ng processed biologic bone material
derived from donors of the same species; or alloplastic,
meAn;ng not derived from biologic materials and composed
solely of inorganic or synthetic polymeric materials.
Bone graft materials can also be classified as
osteoinductive or osteoconductive. Osteoinductive
materials are capable of inducing the formation of new
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bone in both hard tissue defects and, uniquely, in defects
created in non-bony soft tissue sites in either muscle or
subcutaneous tissue. Osteoconductive biomaterials cannot
induce the formation of new bone via differentiation of
undifferentiated cell types, but do provide a scaffolding
S promote the migration of viable bone tissue from the
margins of the bony defect along the contacting surfaces
of the graft material. Because osteoinduction can produce
new bone even without any available viable bone adjacent
to he graft material, osteoinductive grafts may be
preferred to osteoconductive materials. Examples of
osteoinductive grafts materials include demineralized bone
powder and dPm;nPralized bone strips or plugs of cortical
or cancellous bone.
While a wide variety of osteoinductive
compositions have been used in bone repair and
regeneration there is always a need in the art for
improvements or enhancements of existing technologies
which would accelerate and enhance bone repair and
regeneration allowing for faster recovery and enhanced
healing for the patient receiving the osteogenic implants.
SI ~ RY OF THE lN\T~;N'l'lON
Currently available or described compositions
which contain dPm;neralized bone matrix particles or
conjugates of inorganic particles plus reconstituted
structural or matrix proteins exhibit poor physical
stability or physical strength when subjected to load of
any magnitude. Furthermore, due to the poor structural
integrity of these materials, further processing into
alternative shapes or sizes for actual clinical use to
induce new bone formation in osseous defects is limited.
One of the major objects of this invention is to describe
a method of producing an osteogenic, biocompatible,
composite which possesses unique strength properties
and/or osteogenic properties. While many disclosures in
the art describe the use of crosslinking agents to enhance
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the physical integrity o~ protein-based, conjugate,
osteoinductive materials, this disclosure documents a
precise method and procedure application which produce~
osteogenic graft materials of exceptional strength and
physical integrity.
Furthermore, the basic concept described in this
application may be adapted to create con~ugates of natural
biopolymers and inorganic bone minerals which display
exceptional bonds between the inorganic particles and the
polymeric matrix. The spacial stability of these
particles is critical to their success~ul use clinically.
A further object is the creation of protein
based structures which may release drugs or others agents
in a controlled and stable fashion. The ~;m~n~ional and
physical stability of these conjugate material plays a
significant role in the p~rm~cologic release properties
o~ these materials. Hence, the physical strength and drug
delivery capabilities are interrelated.
Two elements are germ~n~ to the observed
properties o~ these novel compositions. First, the
sur~ace activation and partial crosslinking o~ the
proteinaceous particles forms a reactive interface such
that these particles bind in a stable fashion to the
organic matrix, i.e. reconstituted collagen. This step is
important with respect to enhanced physical properties.
Second, inorganic particles may be bound to and stabilized
within an organic or protein-based polymer by first
creating a bound interface o~ calcium-binding protein or
peptide to the particle. The modified particle is then
bound to the matrix proteins via chemical crosslinking or
activation methods. This method, as in the first case,
significantly enhances the physical properties of these
conjugates.
In summary, primary objects o~ this application
are:
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(1) A method for surface activating and/or
partially crosslinking protein-based
or protein coated particles to enhance
their binding and reactivity to
organic matrixes, including serum,
plasma, naturally occurring proteins,
and bone substrates.
(2) To disclose a method and composition
which induces bone when implanted in
an An;m~l or hllm~n and has early on
stress-bearing properties not
described in the prior art.
(3) To disclose a method and composition
of binding inorganic particles or
particles which contain inorganic,
mineral elements to a surrounding
organic matrix such that a stable,
stress-bearing conjugate results. The
inorganic particles in such a
conjugate are not easily displaced or
dislodged from the matrix, as can be
the case when the particles are simply
added to the matrix without
appropriate surface treatment.
(4) Applying one of the above methods to
stabilize drug-cont~;n;ng, protein-
based particles within an organic or
polymer matrix to effect a delayed or
controlled release of the drug from
conjugate material.
(5) A method and composition comprising a ',
biocompatible implantable sponge which
contains a filler component at a
weight ratio sufficient to enhance the
resilience of the composite sponge,
under both dry and wet conditions, and
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also to permit maintenance of sponge
shape, ~;m~nRions, and form, even
under wet conditions.
This invention also relates to methods of
surface modification of demineralized bone resulting in
bone gra~t materials or compositions having enhanced
osteogenic potential. The osteogenic bone repair
compositions of this invention having enhanced osteogenic
potential are used as implants in the treatment of bone or
periodontal defects. The improved osteogenic compositions
provided herein comprise demineralized bone and at least
one added calcium or mineral salt. The osteogenic bone
graft material of this invention, produced by methods
described herein, exhibit enhanced osteogenic activity
relative to other bone repair compositions.
It is a general object of this invention to
provide improved osteogenic bone graft materials
comprising ~m; n~ralized bone and at least one calcium or
mineral salt, wherein said calcium salt or mineral has
been sorbed onto or into or within the mass of the
~l~m;neralized bone or distributed onto the surface or
within the mass of the demineralized bone.
It is an object of this invention to provide an
osteogenic bone graft material having enhanced activity
comprising tl~m~n~ralized freeze-dried bone powder and at
least one calcium salt or mineral salt wherein said
calcium or mineral salt has been sorbed onto or into the
surface of the cl~m;n~ralized bone or distributed onto the
surface or within the mass of the demineralized bone.
It is yet another object of this invention to
provide osteogenic bone graft materials having enhanced
activity comprising ~Pm;n~ralized freeze-dried bone powder
and at least one calcium or mineral salt and at least one
drug, antibiotic, nutrient, growth factor or blood
protein.
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It is a further object of this invention to
provide methods of making these improved osteogenic
compositions.
It is a further object of this invention to
provide therapeutic uses for these improved osteogenic
compositions in the repair or replacement of bone or
periodontal defects.
It is yet another object of this invention to
provide osteogenic compositions comprising about 60
percent to about 95 percent demineralized bone which
exhibit enhanced osteogenic potential and other unique
properties.
It is yet another object of this invention to
provide compositions capable of inducing the formation o~
mineralized bony like structures comprising a carrier and
alkaline phosphatase.
It is also an object of this invention to
provide compositions capable of enhancing induction of
vital new bone in both osseous and non osseous sites
comprising an osteogenic carrier and alkaline phosphatase.
Further objects and advantages of the present
invention will become apparent from the description that
follows.
DETAILED DESCRIPTION OF THE
PREFERRED EMBODIMENTS OF THE lNV~;N'l'lON
When particles which contain protein or amino
acid components, such as protein microcapsules, finely
divided particles of reconstituted collagen, demineralized
bone matrix, or demineralized bone matrix extracted in
chaotropic agents are partially crosslinked in a low
concentration solution of glutaraldehyde, the surface of
these particles become highly reactive, thus allowing an
increased degree of bonding between the particle and an
organic matrix or polymer, in which the particles may be
dispersed. These structures, when dehydrated into a solid
mass, display internal cohesive strength properties not
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found in simple combinations of the particles dispersed
within the matrix component. If the glutaraldehyde is
added directly to the matrix prior to addition of the
particles and subsequent dehydration, very low levels of
cohesive strength are developed. This is also true if the
S entire dehydrated conjugate matrix is crosslinked. The
critical element to increasing the strength and internal
cohesiveness protein-based particle/biopolymer matrix
conjugate appears to be the partial crosslinking or
surface activation of only the particles prior to
complexation with the biopolymer organic matrix.
Alternatively, or additionally, critically controlling the
actual weight percent of the particle component as weight
percent of the total conjugate implant can enhance the
physical properties of sponge configurations as well the
shape and spacing maint~;n;ng functions of the implant or
drug delivery device.
If bioactive particles, such a demineralized
bone matrix, or drug cont~;n;ng particles are to be
complexed, the conditions of surface activation and
partial crosslinking are material. For example,
crosslinking of ~m;n~ralized bone particles above .25
weight percent glutaraldehyde destroys most of the
osteoinductive capacity of the particles. At higher
crosslinking levels, the particles will mineralized by the
uptake of calcium phosphate, but will not induce new bone.
Thus, the use of glutaraldehyde below .25 weight percent
and, preferably, below .l weight percent, is a material
condition in this invention.
The nature of the matrix effects the ultimate
strength properties of the conjugate biomaterial, which is
critical in clinical stress-bearing applications. For
~ example, reconstituted collagen provides a matrix which
demonstrates the uni~ue and unexpected strength properties
of this material. The method in which the collagen is
reconstituted, however, can have a direct effect on the
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magnitude of the increased cohesive strength. This will
be illustrated in the Examples which follow.
Agents other than glutaraldehyde may be used to
enhance the surface binding of protein-based particles
within a biocompatible matrix. For example, free and
S available carboxyl groups on the protein particle may be
converted to amine groups via reaction with a water
soluble carbodiimide in the presence of a diamine. These
additional available amine groups can then react with
glutaraldehyde in the particle crosslinking reaction.
Alternatively, demineralized bone matrix particles can be
immersed in solutions of tetracycline which, will enhance
binding an organic biopolymer matrix. In addition, bone
particles or partially demineralized bone particles may be
~m; neralized in solutions of tetracycline.
Particles with inorganic components may be added
to these osteogenic stress-bearing compositions, provided
these particles makeup no more than twenty percent of the
total weight of the particles. These inorganic component
particles are bound to the biopolymeric organic matrix via
functional molecules with calcium or hydroxyapatite
binding functionality. In one embodiment, all the
particles may be inorganic in nature and bound to the
matrix in this fashion. The advantage here is enhanced
strength as well as limiting the loss of particles from
the matrix itself.
The increased binding between the particle and
matrix constituents can also be advantageous in drug
delivery. The method of dispersing a drug, protein, or
peptide within the particle prior to crosslinking and
surface activation and permits the use of drug cont~;n;ng ,
particles with reduced solubility to act as drug
reservoirs within a biocompatible matrix. The nature of
the matrix can regulate the rate of drug release from the
conjugate material.
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The matrix biopolymer can be modified in a
number o~ ways. For example, the hydrophilic or
hydrophobic nature of the matrix may be altered by the
addition of carbohydrates or lipids. The addition of
acidic phospholipids to the matrix enhances the calcium
binding capacity to the matrix. Additional macromolecules
may be added to the matrix to achieve a particular
biologic response. The addition o~ calcium hydroxide
whether in a soluble form or as part of a protein-based
particle, was found to increase the pH of matrix such that
in-vitro bone collagen synthesis was increased in such an
environment. Heparin may also be added.
Furthermore, crosslinking agents may be added to
the matrix or subjected to the entire conjugate to further
retard the degradation of the matrix and decrease it
solubility. The degree of matrix degradation and its
inflammatory response can also be controlled by the
stabilizing affect of alkaline phosphatase.
Finally, a decided advantage of these
compositions is their ability to be cast into definite
shapes with good registration of surface detail. Due to
their structure, there is much greater uniformity in these
compositions than is found in allogenic tissue.
Furthermore a significant finding is the ability of these
conjugates structures to be ground or milled by
conventional means without gross breakdown of the entire
matrix or the development of severe surface de~ects. This
~inding is significant since diagnostic techniques now
allow the accurate three-~tm~n~ional representation of
bony defects with the resultant milling of a graft
material via CAD/CAM technology. There is no other
processed, truly osteogenic, graft material which can be
- ground to precise speci~ications for insertion in a bony
defect.
The present invention also relates to bone graft
material having enhanced osteogenic potential. The
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compositions having enhanced osteogenic potential provided
herein are based on an observation by the inventor that
the osteoinductive ability o~ demineralized bone is
dramatically enhanced by the addition of at least one
calcium or mineral salt to the demineralized bone.
S Furthermore, the composition and method of this disclosure
greatly increases the speed of bone and mineral formation
with demineralized bone.
Material to this invention is a method and
resultant composition which enhances the mineral content
of demineralized bone by the sorption of a soluble or
saturated calcium or mineral salt solution, thereby
producing the unexpected result of enhancing the rate and
probability of bone formation by osteoinduction as well as
the quantity of bone induced by a given mass or volume of
~m;n~ralized bone matrix. The osteogenic bone graft
materials provided herein and having enhanced osteogenic
potential are comprised of demineralized bone and at least
one soluble calcium or mineral salt. Examples of types of
d~m;neralized bone that may be used include, but are not
limited to, d~m;n~ralized bone matrix or partially
~m;neralized bone matrix or ~m;neralized or partially
demineralized freeze-dried bone powder allograft (DFDBA)
or matrix (DFDBM). By way of example, the degree of
dPm;neralization as measured by the weight percent of
calcium r~mA;n;ng in the bone, may range from about 10
percent to about 0 weight percent (less than about 0.1
weight percent), most preferably, less than about 3 weight
percent to about 0 weight percent calcium r~m~;n;ng in the
bone, and most preferred less than about 1 weight percent
to about 0 weight percent calcium r~m~;n;ng in the bone
after ~Pm;neralization~ Less than about 3 weight percent
calcium after ~m;n~ralization is preferred and most
preferred is less than about 1 weight percent calcium
r~m~;n;ng after ~m;nPralization~ A wide range of sizes
and shapes of demineralized bone matrix, ranging from fine
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powders to coarse powders, to chips, strips, rings, match-
sticks, wedges, small bone segments a large bone segments,
may be used in this invention. In a preferred embodiment
DFDBA is used in the composition.
The salt may be a calcium or other mineral salt.
By way of example other mineral salts that may be used
include, but are not limited to, salts such as sodium
hydroxide, sodium chloride, and magnesium salts, such as
magnesium chloride or magnesium hydroxide or other
biocompatible salts. Examples of calcium salts that may
10 be used in the methods and compositions described herein
include, but are not limited to, calcium acetate, calcium
citrate, calcium chloride, calcium formate, calcium
glycerophophosphate, calcium lactate, calcium lacerate,
calcium oleate, calcium oxide, calcium palistate, calcium
15 salicylate, calcium stearate, calcium succinate or calcium
sulfate. In a preferred embodiment calcium hydroxide is
used. The salt solutions used in the methods and
compositions disclosed herein may be at neutral or
alkaline pH. In a preferred e-m-bodiment alkaline pH is
20 preferred. A soluble or saturated calcium or mineral salt
solution may be used in the methods described herein.
By way of example, concentrations Qf soluble
salt solution that may be used may range from about 100~
to about 0.001~ of the salt by weight, or may range from
25 about 10~ to about 0.01~ of the salt by weight. By way of
example, for calcium hydroxide, suggested concentrations
of the solution that may be used may range from about 3
to about 0.001~ of the salt by weight.
This invention relates to bone graft
30 compositions having enhanced osteogenic potential. By way
of example the weight proportions (weight of the salt
~ divided by the pretreatment weight of the demineralized
bone) of added calcium salt or mineral salt to
~m;n~ralized bone may range from about 0.0001 percent to
35 about 20 percent or about 0.0010 percent to about 10
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percent. In a preferred embodiment the composition
comprises calcium hydroxide to DFDBA at weight proportions
ranging from about 0.001 percent to about 10 percent.
This invention also relates to a method for
producing the osteogenic bone graft compositions having
S enhanced osteogenic activity comprising exposing the
demineralized bone to at least one soluble or saturated
solution of calcium or other mineral salt, for a time
sufficient for the ions in the solution to be sorbed into
or onto the bone matrix or distributed onto the surface or
within the mass of ~m;nPralized bone. In a preferred
embodiment calcium is sorbed onto or into the
demineralized bone, preferably DFDBA, by sorption of a
saturated calcium hydroxide solution onto or into the
structure of the d~m;neralized bone material or
distributed onto the surface or within the mass of the
demineralized bone. The saturated solution may be at an
alkaline pH. Alternative methods may be used to prepare
the compositions of this invention having enhanced
osteogenic potential. By way of example such methods may
include, but are not limited to, depositing the calcium or
mineral salt to the demineralized bone by electrical
current or plasma discharge.
Also, intended to be encompassed by this
invention are functionally equivalent compositions to the
bone repair compositions of this invention having enhanced
osteogenic potential.
In an alternative embodiment the demineralized
bone composition comprising demineralized bone which has
been exposed to at least one soluble calcium or other
mineral salt can further comprise demineralized bone that ',
has not been exposed to calcium or other mineral salts.
In addition, if large bone segment or segments are used in
the methods described herein, the complete bone segment or
segments used need not be ~m;neralized completely.
Alternatively, only the exposed outer surface of the bone
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segment or segments may be demineralized, and then treated
with calcium or other mineral salt.
The composition may be dehydrated by
conventional methods under ambient or elevated temperature
conditions or may be lyophilized in a commercial range
drier under a wide range of conditions. The composition
may be in the form of a powder or in the ~orm of
demineralized bone strips, chips, segments, assays or
other sizes and geometries larger and distinct form
demineralized bone powder. The composition comprising
demineralized bone and added soluble calcium salts or
mineral salts may be partially activated with a cross
linking agent by the methods described herein. In yet
another embodiment of this invention, the calcium or
mineral salt modified d~m;n~ralized bone may be ~m; X~
with demineralized bone which has not be modified, or
alternative, or in addition to, ~m;xed with ~em;neralized
bone which has been partially activated with a
crosslinking agent by the methods described herein. The
weight ratio of each of these various types of powders can
range from about 5 to about 95 percent of the total blend.
Further, all three types of demineralized bone matrix
particles can be ~m;~ed at a wide variety of ratios to
create the powder-blend admixture.
The sorption of a soluble calcium solution or
mineral solution onto and into or within the mass of the
d~m;n~ralized bone matrix or distributed onto the surface
or within the mass of the demineralized bone results in a
significant increase in both the rate and frequency of
osteoinduction, when compared to untreated demineralized
bone matrix. The soluble calcium/d~m;neralized bone
complex also significantly increases the size of induced
- calcified viable bone when compared to equivalent amounts
of non-calcium enriched demineralized bone as assessed by
radiograph analysis of mineral formation and histological
analysis of induced bone treated by the compositions and
,
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o
methods described herein. By way of example, the
compositions and methods disclosed herein may increase the
predictability of induction by demineralized bone, to a
level of about 75~, and more preferably about 90 to 100
osteoinduction and mineralization in an ~n;m~l model.
This invention also relates to osteogenic
compositions comprised of about 60 weight percent to about
95 weiyht percent of demineralized bone, preferably about
60 weight percent to about 90 weight percent. Examples of
types of demineralized bone that may be used in these
compositions include, but is not limited to, demineralized
bone matrix or partially demineralized bone matrix,
demineralized or partially demineralized freeze-dried bone
powder or particles. Examples of materials that may make
up the r~m~;n;ng about 40 weight percent to about 5 weight
percent or the r~m~;n;ng about 40 weight percent to about
10 weight percent of the osteogenic composition include,
but are not limited to, collagen, gelatin, growth factors,
bone morphogenetic protein (BMPs), blood proteins such as
fibrin, albumin or other biocompatible excipients such as
methycellulose or hydroxymethyl cellulose. Preferably
reconstituted collagen is used. The osteogenic
composition comprising about 60 weight percent to about 95
weight percent, preferably about 60 weight percent to
about 90 weight percent demineralized bone described
herein may be fabricated in the form of a dehydrated form
of a sponge, powder, particles, membrane, fleece or fibers
by st~n~rd methods known to one of skill in the art.
Sponges can be made by lypholization or controlled
dehydration under ambient or other control conditions. If
the composition is produced in the form of a sponge, the
sponge may be ground into particles, powder, or fleece by
conventional methods. If the composition is in the form
of a sponge, preferably it is characterized by a density
of about 0.1 grams/cubic centimeter (cc) or greater than
o.l grams/cubic centimeters (cc). By way of example, the
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range of sponge density may be from about O.l grams/cc to
about 0.5 grams/cc, preferably having a density ~rom
about O.ll to about 0.35 grams/cubic centimeter. To
fabricate sponges with about 90 weight percent or above of
demineralized bone an acid or alkaline material may be
used to form the r~m~;n;ng balance of the composition. If
the material to be used is in the acid range, the pH is
pre~erably about 5, and most pre~erably, about 4.5 or
below. If the material to be used is in the alkaline
range, the pH is pre~erably about 9 or above. The
demineralized bone may be combined with the material, when
the material is in the form of an aqueous solution or a
dried or lyophilized powder. The lyophilized powder would
preferably be in the form of an acid or alkaline salt. By
way of example, collagen or gelatin may be the material
used to ~orm the r~m~;n;ng balance of the compositions.
Any collagen may be used, preferably m~mm~lian collagen,
including, but not limited to, hnm~n or bovine.
Yet another embodiment of this invention relates
to compositions capable of inducing the formation of
mineralized bone-like structures or boney-like structures.
Such compositions comprise a carrier and alkaline
phosphatase. Examples of a carrier that may be used
include, but are not limited to collagen, d~m;neralized
bone, gelatin, antigen extracted ~mineralized bone or
~5 ~m; n~ralized bone matrix extracted with chaotropic agents
to remove most or all non-collagen proteins.
Examples of collagen that may be used include,
but are not limited to, reconstituted collagen, partially
demineralized collagen, enzyme extracted collagen or
collagen treated with proteolytic enzymes such as facin or
pepsin. The collagen may be at neutral acid or alkaline
~ pH. The d~m;n~ralized bone may be in the form of powder
or particles. By way of example ranges of alkaline
phosphatase to carrier that may be are about lO
units/milligram carrier to about 5000 units/milligram
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carrier, preferably about 100 units/milligram carrier to
about 1000 units/milligrams carrier. Examples of alkaline
phosphatase that may be used in these compositions
includes any mAmmAlian alkaline phosphatase, such as, but
not limited to, bovine or human. One of skill in the art
S will appreciate that the actual weight of alkaline
phosphatase used in these compositions will vary depending
on the specific activity of the alkaline phosphatase.
These compositions may be fabricated in the form of a
sponge, powder, particle, fleece, or fiber or membrane by
conventional methodology. Also intended to be encompassed
by this invention are functionally equivalent compositions
comprising a carrier and alkaline phosphatase capable o~
inducing bone-like or boney-like structures.
These materials can be used therapeutically as a
grafting implant in plastic and reconstructive surgery,
periodontal bone grafting, and in endodontic procedures
and implanted by stAn~Ard surgical procedures. The
osteogenic implants o~ this invention having enhanced
osteogenic potential are suitable for both human and
veterinary use.
All books, articles, or patents referenced
herein are incorporated by reference. The following
examples are by way of illustrative aspects of the
invention but are in no way intended to limit the scope
thereof.
EXAMPLE ONE
Ten grams of demineralized bone matrix are
milled in an A-10 mill to a uniform particle size ranging
from 75 to 400 microns. The ~m; neralized bone matrix
particles are sieved to eliminate particles above 400 ',
microns. Controlling the concentration of glutaraldehyde
is material to maint~;n;ng sufficient osteoinductive
activity of demineralized bone matrix particles. For
example, glutaraldehyde crosslinking solutions of as low
as 1.0 to 1.5 weight percent can reduce the residual
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osteoinductive activity of demineralized bone matrix to
10~ or less. Glutaraldehyde crosslinking in aldehyde
concentrations of .08 or 0.2 weiyht percent, however, only
reduce the residual osteoinductive activity of
demineralized bone matrix by 30 to 35 percent, leaving
from a background osteoinductive activity of from 65 to 70
percent of uncrosslinked demineralized bone m.atrix
particles. Therefore, control of the glutaraldehyde
concentration used in this procedure is material to
maintaining the biologic activity of processed
demineralized bone matrix particles.
The range of glutaraldehyde used to partially
crosslink and surface activate the ~mi neralized bone
matrix particle may range from .002 to .25 weight percent
glutaraldehyde. The preferred range is from .005 to .09
lS weight percent glutaraldehyde. The partial crosslinking
of demineralized bone matrix retards the resorption of the
matrix in a non-inflammatory fashion, enhances the
attachment of plasma proteins to the surface of
demineralized bone matrix, and facilitates the attachment
of the d~m;n~ralized bone matrix to the organic collagen
matrix of the bony surface o~ the osseous defect.
In this example, the d~m,neralized bone
particles are immersed in a .05 weight percent
glutaraldehyde aqueous solution buffered with phosphate
buffer to a pH of from 7.0 to 7.6. The glutaraldehyde
solution is made isotonic by adding NaCl to a final
concentration of approximately 0.9 weight percent.
Alternatively, the glutaraldehyde solution may be buffered
in the acid or the alkaline range. The glutaraldehyde
solution may be unbuffered consisting of only sterile
distilled deionized water or sterile isotonic saline.
- The demineralized bone matrix (DBM) particles
are immersed in the solution of .05 weight percent
glutaraldehyde in neutral phosphate buffered isotonic
saline for 12 hours with constant agitation at 4 degrees
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centigrade. At the end of the incubation period, the
particles are filtered from the crosslinking solution and
washed once with phosphate-buffered isotonic saline. The
DBM particles prepared are dried under sterile conditions
and then sterilized by an appropriate method, such as
S ethylene oxide, gamm~a, radiation, or electron beam
sterilization.
These activated particles may be placed directly
in an osseous defect or alternatively, complexed with an
organic biopolymer as described in later Examples.
EXAMPLE TWO
The d~m;neralized bone matrix particles are
extracted with a chaotropic agent to remove all bioactive
or ;mmllnologic elements. Allogenic or heterogenic
particles treated in this fashion make excellent delivery
lS particles for the complexation of drugs, peptides, or
proteins. After swelling in acid or alkaline solutions
the extracted dPm;neralized bone particles are immersed in
the agent to be bound and released from the particle. The
particle is then dried and crosslinked in a controlled
fashion as described in Example One. The specific
illustration below describes the use of this method.
Ten grams of demineralized bone matrix
particles, with a particle size of from 75 to 400 microns
(preferably from 150 to 400 microns), are immersed in
guanidinium hydrochloride buffered with 50 millimolar
phosphate buffer, pH 7.4. The particles are maintained in
this extraction medium at 4 degrees centigrade for 10 to
15 hours with gentle agitation. Optionally, protease
inhibitors such as 0.5-millimolar phenylmethyl-sulfonyl
fluoride, 0.1 molar 6-aminohexanoic acid, are added to the ,
extraction medium.
At the end of the extraction period, the
extracted demineralized bone matrix particles are removed
from the extraction ~olution by vacuum filtration or
centrifugation at 800 to 1000 rpm. The extracted
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demineralized bone matrix particles (EDBMP) are washed 10
to 20 times with neutral sterile phosphate buffered
saline. The particles are then dialyzed against several
changes o~ neutral phosphate bu~ered saline to remove any
r~m~in;ng amounts of the chaotropic agent.
S A ~uitable bioactive peptide or protein may be
absorbed onto EDMB particles. In this Example
thyrocalcitonin is used in this fashion. A one gram
~raction o~ the EDBM particles are immersed in a 100 ppm
solution of thyrocalcitonin in sterile normal saline. The
particles are maintained in this solution for 24 to 72
hours with periodic gentle agitation.
The complex EDBM-thyrocalcitonin particles are
separated by vacuum filtration and rinsed once to remove
any excess peptide. The EDMB-thyrocalcitonin particles
are immersed in a low concentration glutaraldehyde
crosslinking solution as described in Example One. The
particles are dried and sterilized as described in that
example. When tested in-vitro and in-vivo, particles
showed a time dependent release of the peptide.
Other peptides and proteins, such as Bone
Morphogenetic Protein, Insulin-like growth factor,
Epidermal Growth Factor, Nerve Growth Factor, Human Growth
Hormone, Bovine Growth Hormone, or Porcine Growth Hormone,
are several examples of peptides or proteins that can be
carried by the EDBM matrix particles. Conventional drugs,
such as tetracycline or other antibiotics, may also be
delivered via this system.
EXAMPLE THREE
Protein-based microcapsules can be fabricated
and then partially crosslinked under controlled conditions
so that they become reactive and bind to an organic
- biopolymer matrix under controlled conditions. As an
illustration, a gelatin-protein microcapsule is ~abricated
and partially crosslinked to surface activate the
microcapsule.
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Two and one-half grams of U.S.P. gelatin and 25
milligrams of Bone Morphogenetic Protein (purified as
described by Urist in the above) are mixed in 8
milliliters of sterile distilled water at 60 degrees
centigrade. Following solubilization of the gelatin and
S complexation with Bone Morphogenetic protein (BMP), 2
milliliters of 1 millimolar phosphate buffer, pH 7.4 is
added to the gelatin-BMP solution with constant stirring.
This solution is maintained at 55 to 60 degrees
centigrade. In a separate container, one hundred
milliliters of an oil phase is prepared by combining 20
milliliters of petroleum either with 80 milliliters of
mineral oil. This solution is heated to 55 to 60 degrees
centigrade.
The gelatin-BMP solution is added to the oil
phase with rapid stirring over a 15 second period leading
to the formation of gelatin-BMP microspheres. Upon
chilling to 2 to 4 degrees centigrade, the gelatin-BMP
spheres jelled into beads. The oil phase of the solution
is removed by vacuum filtration. The beads were washed
with petroleum ether and diethyl ether.
The microspheres so obtained are then
crosslinked as described in Example One. In this Example,
the microspheres are crosslinked in .03 weight percent
glutaraldehyde in neutral phosphate buffered isotonic
saline. The microspheres are filtered by vacuum
filtration and rinsed once with neutral sterile isotonic
saline. The spheres are dehydrated and stored dry.
Alternatively, the spheres may be complexed with an
organic biopolymer matrix to form a stress-bearing
bioprosthesis.
EXAMPLE FOUR
Ten grams of milled bone powder (not
d~m;neralized), which has been defatted and extracted with
an organic solvent, such as diethyl ether, is immersed in
a solution of tetracycline HCl at a concentration of from
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5 micrograms per milliliter to 50 milligrams per
milliliter. Alternatively, the milled bone powder or
particles is first partially demineralized in a .05 to 0.3
molar solution of HCl at 4 degrees centigrade for ~rom 30
minutes to 5 hours. These partially ~m;neralized bone
S particles are then contacted in a solution of tetracycline
HCl as speci~ied above.
The particles are immersed in a l0 microgra-m-s
per milliliter solution of tetracycline HCl ~or ~rom l to
24 hours at 4 degrees centigrade. At the end o~ the
immersion period, the particles are rinsed once in neutral
buffered isotonic saline. The particles are collected and
dried or lyophilized. The particles in this instance are
collected, dried under ambient conditions and lyophilized.
As an additional procedure, the dried particles
are partially cro~slinked with glutaraldehyde as described
in Example One. As will be described in Example 6, these
tetracycline treated ~m;n~ralized bone matrix particles
are subjected to other means of chemical group activation
such as via carbodiimide activation of surface carboxyl
groups and reaction with an amine or diamine.
EXAMPLE FIVE
Other protein contA; n; ng particles are
~abricated from pulverized reconstituted collagen
particles. As an example, collagen-tetracycline
2~ conjugates sponges are fabricated by adding tetracycline
HCl to an acid solubilized reconstituted collagen
dispersion. The final tetracycline concentration is l0 to
50 micrograms per milliliter and the collagen
concentration is ~rom a .5 weight percent dispersion to a
3.5 weight percent dispersion. The collagen is
solubilized with acetate or hydrochloric acid in the acid
~ range or sodium hydroxide in the alkaline range. The pH
of the collagen dispersion is adjusted to neutrality or
near neutrality by repeated dialysis against sterile
distilled water or phosphate bu~fered saline.
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After the collagen dispersion is adiusted to
near neutrality, the appropriate drug, peptide, or protein
is added to the collagen dispersion and agitated to assure
complex m; ~; ng In this example the collagen-tetracycline
composition is poured into a cylindrical mold and allowed
S to stand for 24 hours in a sterile l~m; n~r flow box to
allow initial gellation. After gellation, the dispersion
is placed on the minus 60 degree shelf of a lyophilizer
and freeze-dried to form a sponge material. The sponge
conjugate material is removed from the lyophilizer and
placed in a controlled dry-heat oven at a temperature of
from 45 to 80 degrees centigrade. The heat stability of
the molecule conjugated to the collagen determines the
appropriate temperature. The dried sponge is removed and
milled to a powder in an A-20 mill. The collagen-
tetracycline particles produced are then surface activated
and partially cross linked.
EXAMPLE SIX
The binding and covalent attachment of protein-
based particles protein microcapsules, demineralized bone
matrix particles, or protein conjugated inorganic
particles, are enhanced by increasing the number of
surface binding sites. This increase in binding sites
accomplished by the following procedure.
Ten grams of demineralized bone matrix particles
are obt~;nP~ with a particle size of from 50 to 400
microns. The particles are immersed in a water soluble
carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide is varied between 0.005 molar to about 0.1
molar preferably about 0.05 molar to about 0.1 molar
preferably about 0.05 molar in a isotonic salt solution. ',
The pH of the carbodiimide solution was maintained between
about 4.7 and about 5.2 by the addition of HCl. Ethanol
and other organic compounds, such as mannitol are added
from time to time to alter the dielectric constant of the
crosslinking solution. Alternatively, the ionic strength
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o
is increased by the addition of NaCl ~rom about .1 molar
to 1.0 molar. Similar modification is undertaken from
time to time with the glutaraldehyde crosslinking
procedures.
The reaction with the carbodiimide proceeds from
about 20 minutes up to 12 hours or more. In this
particular example, the reaction time is 2 hours and the
reaction is carried out at four ~C, the surface activated
~m; n~ralized bone particles are then contacted with an
amine or ~;Am;ne Materials with amine functional groups
include amino acids, polyamino acids, globular proteins
such as albumin and gelatin, ~ibrillar proteins such as
collagen and elastin. Alternatively, in this instance a
~;Am;ne, namely hexane~;Am;ne, is used to react with the
carbodiimide activated particles. The h~ne~; ~m~ n~
permits the increa~e o~ free available amine binding sites
for activation by glutaraldehyde. The h~x~n~ m; ne
solution contains ~rom .01 weight percent to about 2.0
weight percent diamine. The optimal ~;Am;ne concentration
is approximately .1 to .5 weight percent in a neutral
bu~fered saline solution at pH 7.4. The contact time is
from 2 to 10 hours with the usual time being four hours.
The particles are removed ~rom the ~; ~m; ne
solution by filtration and are rinsed several times with
neutral buffered saline to remove excess ~; ~m; ne The
demineralized bone particles are added to a crosslinking
solution of glutaraldehyde with an aldehyde concentration
of from .001 weight percent to .25 weight percent. The
method used is identical to Example One and the
concentration of glutaraldehyde is .05 weight percent.
The partial cross-linking occurs at 4~ C in a neutral
buffered isotonic saline solution. The crosslinking
~ solution time is 8 to 12 hours. The particles filtered
from the solution and are washed once with buffered
neutral isotonic saline. The particles are dried and at
this point can be used for binding in an organic
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biopolymer matrix to produce a stress-bearing bone gra~t,
as described herein. Alternatively, the particles are
lyophilized and sterilized by either ethylene oxide,
liquid sterilizing solution, gamma radiation, or electron
beam sterilization.
S EXAMPLE SEVEN
An aqueous collagen dispersion is made ~rom a
high purity, medical grade, sterile powdered collagen.
The constituted collagen dispersion is made at 2.5 weight
percent collagen by solubilizing the collagen powder in a
.01 N acetic acid buffer. The collagen powder is added,
from time to time in concentrations ranging from 0.5
weight percent to 2.5 weight percent. Other organic
acids, such as lactic acid or inorganic acids, such as
hydrochloric acid, are also used from time to time to
facilitate the swelling of the collagen matrix.
The acid dispersion of the collagen is mixed
with moderate agitation and stored overnight to permit
thorough swelling of the collagen gel. The collagen
dispersion is vigorously agitated and sheared in a Waring
Blender under medium to high speed using 3 to 5
intermittent, 30 second m;x; ng periods. The collagen
dispersion is then poured into an appropriately sized
centrifuge tubes and centri~uged at 800 rpm to remove
entrained air within the collagen dispersion. The
dispersion is then dialyzed against a solution of sterile
distilled water. The collagen dispersion is repeatedly
dialyzed against fresh ~Xch~nges of sterile distilled
water until the pH of the collagen dispersion is in the
range of pH 5. 3 to 7Ø On occasion to obtain a
dispersion with a pH of from 6.8 to 7.6 in an efficient ,
m~nn~r, the collagen dispersion is dialyzed against a
buffer solution such as neutral phosphate buffer. The
dialyzed collagen dispersion is collected and placed in a
cont~;n~r at 4 degrees centigrade. The dispersion serves
as a matrix material.
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Two types o~ demineralized bone matrix particles
are utilized in this procedure. The ~irst type are normal
demineralized bone particles without surface activation
with glutaraldehyde. The second type are particles o~
demineralized bone matrix identical to the first group
S except they are activated by partial crosslinking in
glutaraldehyde as described in Example One. These two
systems are describes as ~ollows:
(1) D~m;neralized bone particles at 85 weight
percent are dispersed in the aqueous collagen matrix;
placed in a cylindrical mold and cast by forced air
dehydration at ambient conditions. The conjugate
cylinders are retained ~or physical testing.
(2) D~m;nPralized bone particles, identical to
about (1) are activated in glutaraldehyde as described in
Example One. These particles are then dispersed at 85
weight percent in the aqueous collagen matrix. The
conjugate is placed in a cylindrical mold and cast by
forced air dehydration at ambient conditions. The
conjugate cylinders are retained for physical testing.
To better understand the action o~
glutaraldehyde in these matrix particle conjugates, three
other methods of addition of 0.5 weight percent
glutaraldehyde are also employed. These are
(3) D~m;neralized bone particles at 85 weight
percent are dispersed in the collagen matrix. Neutral
buf~ered glutaraldehyde is added to the aqueous dispersion
so that the final concentration is 0.5 weight percent.
The conjugate is placed in a cylindrical mold and cast by
~orced air dehydration at ambient conditions. The
conjugate cylinders are retained for physical testing.
(4) Neutral buffered glutaraldehyde is added to
~ the collagen dispersion prior to the addition of
~m;n~ralized bone matrix particles (unactivated). The
glutaraldehyde is added so that its concentration with
respect to the total weight of the conjugate would be 0.5
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~ weight percent. The demineralized bone matrix particles
are then added with mixing at a weight ratio of 85 weight
percent. The conjugate is placed in a cylindrical mold
and cast by forced air dehydration at ambient conditions.
The conjugate cylinders are retained for physical testing.
(5) Conjugate cylinders are fabricated as
described for System (l) above, but are then immersed in a
neutral buffered solution of 0.5 weight percent
glutaraldehyde at 4 degrees centigrade for 72 hours. The
cylinders are removed and washed repeatedly in neutral
phosphate-buffered isotonic saline. The cylinders are
replaced in their original molds and dried by forced air
dehydration under ambient conditions. The conjugate
cylinders are retained for physical testing.
The following table displays the results
obtained with the physical testing of the different
systems. The cylinders are tested for diametrial tensile
strength in an Instron Tester at constant loads 5 or 20
kilograms, depending on the strength of the material. The
dimensions of the cylinders are measured prior to testing
and all cylinders are tested on their sides as is usual
for the diametrial internal cohesive strength of a
material.
SYSTEM
} ~ 3 4 5
Force 5Kg 2OKg 5Kg 5Kg 5Kg
A~lied
Strain Sponge- Resistant Sponge- Sponge- Sponge-
Profile like to load like like like
with yield
point
Diametrial
Tensile c2.5 p8i 90 pSi <2.5 p6i <2.5 p8i <2.5 p8i
Strenqth
Note: Collagen-demineralized bone particle
compositions at or above 90 weight percent bone
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particles to collagen fail to aggregate into a
cohesive mass and spontaneous disintegrate under
any degree o~ ~orce.
EXAMPLE EIGHT
The nature of the matrix biopolymer also has a
definite ef~ect on the internal cohesive strength of the
material and its ultimate strength properties. The
procedure below illustrates the fabrication o~ a collagen-
based material which is adhesive to itsel~ or other bone
compositions, is hemostatic, and is osteogenic.
Ten (10) grams of sterile collagen powder
(Collastat) is mixed in 100 milliliters of .1 N HCl with
stirring-bar agitation. After 15 minutes of agitation,
collagen dispersion is diluted ~rom 10 weight percent to 5
weight percent by a two-fold dilution with sterile
lS distilled water. This results in a final acid
concentration of .05 N HCl and a final pH of 4.1 to 4.3.
Four point three (4.3) grams o~ milled
~m; nPralized bone powder (particle size 125 microns or
less; MW O.250 sieve) are added to the collagen mixture.
After thorough stirring the 5 percent dispersion is mixed
in a Waring Blender for 5 to 10, 20 second agitations to
increase the dispersion viscosity. The thickened solution
i8 poured into centrifuge tubes and spun in a table-top
centrifuge at 400-600 rpm for 5 minutes to remove air and
concentrate the collagen.
Excess fluid supernatant is removed by pipetting
and the collagen conjugate fraction is collected into a
single volume (approximately 170 milliliters). This
collagen-demineralized bone dispersion is stored at 4
degrees centigrade for at least one hour to check for
consistency and the presence of phase separation. The pH
- of the mixture is 4.50 to 4.57.
The collagen mixture is transferred to dialysis
tubing (Spectrapor. 12,000 to 14,000 molecular weight
cut-off) and dialyzed overnight against sodium phosphate
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buffer .02 molar pH 7.4. The collagen-DBP dispersion is
removed from the dialysis tubing using aseptic technique.
The dispersion is homogeneous and shows no evidence of
separation. The pH of the dialyzing solution is 6. 5.
The pH of the collagen dispersion is 5.00 to 5.12.
S The dialyzed collagen-DBP dispersion is
collected, placed in a 250 milliliter centrifuge bottle,
then spun at 800 rpm for 10 minutes. The clear
supernatant is collected and checked for pH which is 5.10.
The collagen-DBP dispersion is placed in sterile
petri dishes and frozen, under aseptic conditions, at
minus 4~C under vacuum, the vacuum is maintained for 18 to
24 hours to assure complete dehydration. The resultant
foam-like sponge material is placed in an A-10 mill and
milled into a powder. The powder is divided into equal
lS aliquots and bottled. The bottles of collagen-DBP powder
are sterilized under ethylene oxide for 2 and 1/2 hours.
The bottles are aerated under vacuum for at least 24 hours
and then sealed under vacuum.
The resultant material is hemostatic in that it
promotes the clotting of blood.
EXAMPLE NINE
The collagen-~m;n~ralized bone particle powder,
as described in Example Eight is reconstituted in a 5 mM
solution of sodium phosphate buffer, pH 8Ø
Approximately .2 grams of the powder is hydrated with 1
milliliter of the buffer and m;~eA to assure complete
m;~;ng. Demineralized bone particles, average particle
size 250 microns are activated and partially crosslinked
as described in Example One. A weight of .10 grams of
these particles are added to the buffer-collagen conjugate
dispersion with gentle m;~;ng. The mixture is placed in a
cylindrical mold and dehydrated by forced air under
ambient conditions. The resultant disc dried very
rapidly, i.e., within 4 to 10 hours. If the mass is
lyophilized, a more porous structure results. The detail
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of the mold is well reproduced on the cylinder. Cylinders
demonstrate a smooth surface appearance and have
suf~icient integrity to be milled or ground to precise
shapes with surgical burs or grinding wheels in low or
int~rme~;ate speed handpieces. The cylinders so produced
are tested for diametrial tensile strength at 20 kilogram
constant load. The results are as follows:
S~ ~ 6
Force Applied 20 kg load
~train Profile T.; n~r, elastic
behavior with
increased module
in tension
Diametrial Tensile
Strength (PSI) 279 to 320 psi
EXAMPLE TEN
Other drugs, proteins, or peptides are added to
the matrix phase of these compositions which contain ac-
tivated particles. For example, a purified or recombinant
bone morphogenetic protein, as described by Urist in U.S.
Patent 4,294,753 is added to the matrix prior to the
addition of activated particles or microcapsules. As the
stability of the conjugate does not rely on addition of
glutaraldehyde to the bone matrix, the chance of
inactivating the BMP molecular is reduced. The conjugate
material can be used in its aqueous form, however, in this
instance the activated d~m;n~ralized bone
particles-collagen-BMP conjugate is dehydrated under
ambient conditions, as described earlier. Another sample
is dehydrated and then lyophilized at minus 40 to minus 60
degrees centigrade.
Another conjugate, made in identical fashion
with respect to order of addition of components, consist
of activated demineralized bone particles-collagen and
tetracycline HCL. This conjugate is dehydrated and
lyophilized. Other proteins and peptide growth factors
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are evaluated when complexed with the matrix phase of this
novel, cohesive compositions.
EXAMPhE EhEVEN
The activated and partially crosslinked protein
particles, microcapsules or demineralized bone matrix
particles whose methods of surface activation were
described in above Examples, are added to viscous mixtures
of blood proteins, glycoproteins, or cell component
fractions.
Specifically, 0.5 grams of activated
demineralized bone matrix or bone matrix particles are
removed from the cont~;ner in which they are sterilized.
In this instance, the bone is being used to fill an
osseous defect in a laboratory ~n;m~l Five milliliters
of the An;m~l~s blood is withdrawn by venipuncture. The
blood is spun at 800 to lO00 rpm in a table-top centrifuge
to spin-down platelets, white blood cells and red blood
cells. The blood is drawn into a plain vial which does
not contain any type of anticoagulant. After the cellular
components of the blood are pelleted, the supernatant
cont~;n;ng serum is withdrawn carefully with a pipette.
The serum is added to the activated demineralized bone
particles so that the particles are evenly coated. The
ratio of activated bone particle to serum or plasma can
vary from 20 to 95 percent by weight. The conjugate is
placed into the bony defect such that it is filled
completely. The defect is gradually replaced with new bone
over a period of 6 to 12 weeks.
The identical procedure is undertaken with
another research ~n;m~l except this time the blood is
drawn into a heparinized tube and plasma is obtained after
centrifugation. This blood plasma is combined with the
activated blood particles in a m~nner identical to the
above.
In certain instances, such as large osseous
defects or non-unions, it is beneficial to add bioactive
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molecules or antibiotics to the serum or plasma ~raction.
Rabbit bone morphogenetic protein is purified from rabbit
demineralized bone matrix, using a method described by
Urist in U.S. Patent 4,294,753. The puri~ied BMP is added
to the plasma so as to constitute about .5 to 3 percent by
weight. After m;~;ng the lyophilized protein into the
plasma and dispersing it thoroughly, the activated
demineralized bone particles are mixed into the BMp-plas-m--a
at a weight ratio of 80 to 90 parts of particles to 10 to
20 parts of plasma.
Another laboratory ~n;m~l is presented with a
bone injury with possible bacterial contAm;n~tion. Blood
is drawn and plasma obtained as previously mentioned. To
the plasma is added a powder tetracycline hydrochloride
salt at a concentration of 5 to 25 micrograms per
milliliter. The antibiotic is mixed thoroughly in the
plasma and the plasma m;~e~ with activated ~m;n~ralized
bone particles at a weight ratio of 80 to 90 parts
particles to 10 to 20 parts plasma-tetracycline.
EXAMPLE TWELVE
The proteins which constitute the matrix can be
further modified by the addition of phospholipids. In
particular, reconstituted collagen and acidic
phospholipids demonstrate together an enhanced uptake of
calcium as compared to collagen matrixes without
conjugated acidic phospholipids.
A 2.5 weight percent collagen dispersion at a pH
of 5.0 to 5.5 was used for the addition of an acidic
phospholipid, L-alpha-phosphatidic acid, dipalmitoyl, is
added to the above reconstituted collagen dispersion at
from .01 milligrams per milliliter collagen to 10
milligrams per milliliter collagen. The conjugate
dispersion is dehydrated at ambient temperatures and
lyophilized. Alternatively, activated protein particles,
microcapsules, or demineralized bone matrix particles are
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added to the conjugate aqueous dispersion as described
within this disclosure.
EXAMP~E THIRTEEN
A reconstituted collagen matrix can be further
modified by the addition of an alkaline source of calcium
S ions. For example a reconstituted collagen dispersion
with a collagen composition of 0.5 to 2.5 percent by
weight and a pH of 5.0 to 5.5 is dialyzed against a
saturated solution of calcium hydroxide in sterile
distilled water. When the pH of the collagen dispersion
reaches 10 to 10.5 the collagen dispersion is removed from
the alkaline solution, placed in an appropriate sized mold
and lyophilized to form a sponge. Another aliquot of the
collagen-calcium hydroxide is combined with activated
~Pm;neralized bone particles and m;xe~ to thoroughly
disperse the particles in the alkaline matrix. The
conjugate is dehydrated and lyophilized to form a
stress-bearing sponge material.
These collagen-calcium hydroxide conjugates
~mo~trate rapid release of the calcium and hydroxide
ions and load only sufficient amounts of hydroxide ions to
slightly adjust the pH.
EXAMPLE FOURTEEN
A calcium hydroxide (CaOH)/ collagen-gelatin
microbead is fabricated using the following method. A
reconstituted collagen dispersion at neutral or acidic pH
is made as described in prior Examples. Powdered calcium
hydroxide is slowly added to the dispersion until a pH
such that a collagen to gelatin conversion was evident.
The pH necessary to effect this conversion is
approximately 11.0 or above. The visual effect at this ,
conversion was quite noticeable, as the collagen
dispersion loses all its translucency and becomes opaque
and chalky.
The colloidal dispersion can be formed into
microbeads by immersion in an oil phase, as described in
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Example Three. Nevertheless, in this example, the
collagen-CaOH gelatin dispersion may be dried by
lyophilization at minus 40 minus 60 degrees centigrade.
Dehydration at ambient temperatures also yields a solid
mass.
S This mass is milled and pulverized is into fine
particles. The particles are partially cross-linked in a
.05 weight percent glutaraldehyde solution at a pH o~ 7.8.
After rinsing once, the activated collagen/gelatin-CaOH
particles are added to an alkaline collagen dispersion
cont~;n~ng calcium hydroxide. This mixture may be
lyophilized or dehydrated. However, activated
d~m;neralized bone particles may be added in a weight
percent range o~ from lO to 85 weight percent.
EXAMPLE FIFTEEN
A collagen-calcium phosphate conjugate is
derived as described by Cruz in U.S. Patent 3,767,437. A
reconstituted collagen dispersion at a pH of 3.5 to 4.5 in
sodium acetate is dialyzed ~irst against 3 to 7 changes of
deionized water and then dialyzed against a saturated
solution of calcium hydroxide for 2 to 5 changes. The
collagen-CaOH solution is then dialyzed against a solution
of phosphoric acid adjusted to pH 3.0 to 4Ø The
dialysis for 2 to 6 changes resulted in a Collagen-Calcium
Phosphate conjugate. The dispersion is lyophilized or
dehydrated under an ambient conditions. The resultant
mass is pulverized under moderate force. The resultant
particles are sieved to a uniform particle size of 50 to
lOOO millimicrons. The particles are dried and placed in
a .08 glutaraldehyde solution also contains 8 mM calcium
phosphate buffer. The particles are filtered and rinsed
once with sterile distilled water.
The partially crosslinked, activated particles
are added to a reconstituted collagen dispersion wi~
moderated m; ~; ng and agitation. The dispersion can be
left in a viscous gel-state, lyophilized, or dehydrated at
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ambient conditions. The resultant dried mass has a
diametrical tensile strength greater than one hundred PSI.
EXAMPLES Sl~l~
Collagen-calcium phosphate particles, prepared
and activated as described in Example Fifteen, are added
to a composition derived as described in Example Seven,
System No. 2. Inorganic particles are added to collagen
matrix phase, so that no more than 20 weight percent of
the entire conjugate is composed of the protein/inorganic
particles. The entire mass is cast and dehydrated as
described in the earlier Examples.
EXAMPLE S ~; \T~;N'l'~;~!;N-
Collagen-calcium phosphate particles, prepared
and activated as described in Example Fifteen are added to
a composition derived as described in Example Nine. The
inorganic particles are added so that no more than 20
weight percent of the entire conjugate is composed of the
protein/inorganic particles. The entire mass is cast and
dehydrated as described in the above Examples.
EXAMPLE EI~l~N
Collagen-calcium phosphate particle conjugate
derived from either hydroxyapatite or tricalcium phosphate
particles even when crosslinking agents such as
glutaraldehyde in low concentrations are added to the
collagen matriX~ ~mon~trate very low tensile strengths
i.e., on the order of 30 psi or less. A method is
described in this example to provide
collagen-hydroxyapatite or collagen-tricalcium phosphate
conjugates with enhanced strength and reduced plucking of
the inorganic particles from the matrix.
An acid dispersion of reconstituted collagen is ',
made in the acid pH range using 0.05 acetic acid as de-
scribed earlier. The collagen dispersion is made at .75
weight percent collagen sheared in a Waring Blender and
dialyzed against sterile isotonic saline until the pH of
the dispersion reaches a range of 4.0 to 5.5. Tricalcium
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phosphate particles medical grade and sterile with a
particle size of 50 to 150 millimicrons are added to the
dispersion with moderate m;~ng The dispersion is
degassed under vacuum with moderate agitation. The
dispersion is placed in a dialysis tube and dialyzed
against .01 molar phosphate buffer at pH 8Ø The
dialysis tube is periodically removed aseptically and
inverted several times to prevent separation of the
mineral phase. After 24 to 48 hours of dialysis the
dispersion is removed from the dialysis tubing, poured
into a stainless steel mold and lyophilized at between
minus 40 and minus 60~C.
At the conclusion of lyophilization the sponge
like mass is cut into about .5 cm square cubes and milled
carefully at low settings in an A-10 mill so as to provide
a group of collagen-mineral particles on order of about
250 to 550 microns. The particles are activated in a
m~nner consistent with one of the e-m-bodiments of the
invention. Specifically, in this example, the conjugate
particles are immersed in a neutral buffered isotonic
solution of bout 0.08 weight percent glutaraldehyde. The
concentration of the glutaraldehyde was varied from .001
to .25 weight percent glutaraldehyde. The conjugate
particles are activated for about 8 to 12 hours at 4
degree centigrade. The particles are removed by vacuum
filtration and washed once in neutral buffered isotonic
saline.
The activated protein-coated mineral particles
are added to a reconstituted collagen dispersion of one to
2.5 percent by weight collagen, with a pH of from 3.5 to
5Ø The activated particles are added to the dispersion
in a weight range of from 25 to 85 percent by weight. The
preferred range is from 40 to 75 percent by weight. The
activated protein-mineral particle/reconstituted collagen
conjugate is poured into a stainless steel mold and dehy-
drated at am~bient temperatures with forced recirculated
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air. The conjugate, once dehydrated may be lyophilized at
minus 40 to minus 60~C.
Another conjugate of this type is cast except
that prior to dehydration, a bioactive protein, peptide,
or drug is added to the matrix, as has been described in
S earlier Examples.
EXAMPLE NI~l~N
While a stable coating of reconstituted collagen
can be formed in a continuous adherent layer on the
surface of an inorganic particle a preferred method is to
form multiple chelation links between the calcium, rich
surface and the protein-based surface layer.
Particles of a calcium phosphate ceramic
material, namely tricalcium phosphate particles with a
size of about 100 millimicrons are immersed in a 10 ppm
solution of L-y-carboxyglutamic acid. The particles are
incubated in this solution for 24 to 48 hours 4~C. The
particles are removed from the solution dried under
ambient conditions and immersed in about a 0.5 to 1 weight
percent collagen dispersion cont~; n; ng about 10 to 50 ppm
of L-y-carboxyglutamic acid. The particles are agitated
gently in this dispersion filtered from the dispersion
then placed in a .15 molar NaCl solution cont~;n;ng .05
molar sodium phosphate buffer adjusted to pH 7.4 with
dibasic and tribasic sodium phosphate. After 15 minutes
to one hour in this solution. The collagen coated
particle is partially crosslinked in a .075 weight percent
solution of glutaraldehyde for 8 to 10 hours.
The particles are removed from the
glutaraldehyde solution by filtration then rinsed once in
sterile saline solution. Once activated some of these ~,
particles are used directly is osseous defects.
Alternatively, some of the activated particles are mixed
into a 1 weight percent dispersion of the reconstituted
collagen. The particles are mixed and agitated to assure
a uniform dispersion. The gel so obtained is used in
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certain osseous defects. Alternatively, the collagen-
particle dispersion is lyophilized or dehydrated under
forced air under ambient conditions. The resultant
material is sterilized with ethylene oxide, gamma
radiation, and/or by immersion in a .2 percent buffered
glutaraldehyde solution.
EXAMPhE TWENTY
In place of the L-y-carboxyglutamic acid
disclosed in Example Nineteen, the sodium salt of poly- L-
glutamic acid or the random copolymer of L-glutamic acid,
which contains at least one lysine in its repeating
structure, may be used to coat the calcium phosphate
particle prior to complexation with reconstituted
collagen. In this procedure, the particles are mixed and
agitated within the polyamino acid solution, then under
lS ambient conditions the particles are dehydrated or
alternatively, lyophilized. The coated particles are
mixed in a reconstituted collagen dispersion and again
dried to provide a uniform coating. The coated particles
so produced are partially crosslinked in .05 weight
percent neutral buffered glutaraldehyde for about 10 to 12
hours at 4~C. The particles are vacuum filtered from the
activating solution and dried. The particles are then
used as described within the embodiments of the invention.
Alternatively, the polyamino acid coated particles once
dried may be added to a reconstituted collagen dispersion
which contains about .05 to .1 weight percent
glutaraldehyde. The entire conjugate may be dehydrated or
lyophilized, then milled to a powder if further
complexation is intended.
~MPLE TWENTY-ONE
System No. 2 of Example Seven described the
fabrication of a reconstituted collagen/activated
~m~ n~ralized bone matrix conjugate with improved internal
cohesive strength. The weight percentage of activated
particles is demonstrated to be useful in the range of 5
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to 85 weight percent of the conjugate. Nonactivated
particles can be added to matrix in weight percent ranging
from 0 to 95 percent of the total conjugate weight. If
the non-activated or activated particles are inert,
inorganic particles, speci~ically, tricalcium phosphate
hydroxyapatite, their weight percent does not exceed 20
weight of the total conjugate mass.
EXAMPLE 'l'W~N'l'~!-TWO
Example Nine described a cohesive stress-bearing
conjugate which is composed of an adhesive
collagen-demineralized powder which is hydrated and
~m; xPd with an additional 20 weight percent of activated
demineralized bone particles. This composition is
comprised of 30 weight percent original unactivated
particles plus twenty weight percent activated
demineralized bone particles (average particle size 150
microns). The percentage of activated ~pm;neralized bone
particles is from time to time, increased up to 50 weight
percent of the total mass. Other conjugates are ~m; ~Pd
to contain up to 20 weight percent (with respect to the
total conjugate mass) of activated or non-activated inert
inorganic particles consisting o~ particles of tricalcium
phosphate or hydroxyapatite with a particle size range of
20 to 750 millimicrons, with the preferred range being 20
to 150 millimicrons the total weight percent of particles~5 of any type greater than 85 percent of the total mass.
EXAMPLE TWENTY-THREE
The matrix component of the above examples may
contain from a non-fibrillar collagen group, such as gela-
tin. Sufficient gelatin with a Bloom strength of at least
200 is added to the reconstituted collagen so that no more
then 10 weight percent of matrix consists of gelatin.
EXAMPLE TWENTY FOUR
Polyamino acid microcapsules may be used to form
protein-based, partially crosslinked particles as
described in Example Three. The same procedure is
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~ollowed except that a viscous solution of poly-l-lysine
is used instead of gelatin. The other exception to the
procedure is that the poly-L-lysine is used instead o~
gelatin. The other exception to the procedure is that the
poly-L-lysine is warmed only to 37 to 43 degrees
centigrade.
EXAMP~E TWENTY-FIVE
Other types of inorganic particles can be
activated and reacted with collagen, gelatin, polyamino
acid or polyalkenoic acids to form rigid, stress-bearing
implants and cements. Aluminosilicate glasses, which
contain varying amounts o~ calcium fluoride, are used for
stress-bearing cements and implantable bone replacement
structures.
These hard-setting cements formed from the
reaction of powders and liquids. Specifically, milled
aluminosilicate glass, designated G-309 or G-385 are
provided. The reactant liquid consists of from 35 to 55
percent polyacrylic acid, molecular weight from 15,000 to
60,000 and ~rom 2 to 35 weight percent reconstituted
collagen and the balance distilled, deionized water.
The powder and liquid are mixed at a powder to
liquid ratio of from 1.4 to 3 grams per milliliter liquid.
The working time for the cement is about 1 minute 45
seconds to 2 minutes 45 seconds and the final set from 5
minutes 30 seconds to 6 minutes 45 seconds.
EXAMPLE TWENTY SIX
The reconstituted collagen-glass ionomer cements
are varied by the addition of from .01 to 3 percent
glutaraldehyde into the liquid component as described in
Example Twenty-Six. The inclusion of glutaraldehyde
shortens the working/setting time and produces a stronger
cement as det~rm;ne~ by physical testing.
EXAMPLE 'l'Wlt:N'l'Y SEVEN
The liquid component as described in Examples
Twenty-Fi~e and Twenty Six can be further modified by the
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addition or substitution of polyamino acids for the
polyalkenoic acids in the liquid component. For the
entire polyacid component of the liquid may be replaced
with poly-L-glutamic acid. Alternatively, from 5 to 45
weight percent of the liquid component may consist of a
polyamino acid, namely, poly-L-glutamic acid,
poly-L-asparatic acid, poly-L-lysine, homopolymers or
random co-polymers of these or any polyamino acid may be
added to the liquid component combinations of these
polyamino acids polymers vary the setting time and the
ultimate physical strength of the cement or implant.
EXAMPLB TWENTY EIGHT
Bone Morphogenetic Protein and/or bone proteins
extracted from demineralized bone matrix may be
incorporated into uniform unilamellar liposomes for
controlled delivery to osseous defects. The procedure for
incorporation of the bioactive proteins onto and into the
membrane bilayer is described below.
A phospholipid,
1-palmitoyl-2-oleoyl-phosphatodylchlorine, is dispersed in
an aqueous (sterile distilled water) phase by sonication
and then mixed with lyophilized BMP such that the protein
to lipid mass ratio to produce unilamellar BMP liposomes
of optimal size (high encapsulation efficiency) is in the
range of 1:2 to 1:3 with the optimal ratio being 1:2.5.
The resultant mixture is dried under nitrogen in
a rotating flask. The dried sample is then rehydrated in
aqueous medium under nitrogen with gentle rotation of the
flask. The resulting unilamellar liposomes where
separated from the free morphogenetic protein by
chromatography through a B-4 or G200 Sephadex column. f,
The BMP-liposomes are stored at 4~C or
alternatively, lyophilized. Prior to implantation
reconstituted collagen sponges allogenic bone autogenous
bone grafts or ~m;neralized bone matrix can be soaked in
the liposome preparation to stimulate osteogenesis.
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Alternatively, the BMP-liposome can be mixed with an
a~ueous collagen dispersion for direct placement or
injection to the wound site, or added to the matrix phase
described in embodiments of this invention.
E~AMPLE TWENTY-NINE
Bone morphogenetic protein and/or extracted bone
proteins can be entrapped in the patient's own red blood
cells by resealing the cell ghosts in the presence of the
bioactive proteins. This permits a highly biocompatible
delivery system for BMP delivery to a wound site.
Fresh heparin-treated whole blood (about 50
milliliters) is centrifuged at 1000 gs for 10 minutes.
The plasma and buffy coat is removed and the cells are
washed three times in cold (4 degrees centigrade) Hanks
Basic Salt Solution (HBSS). The packed cells are mixed
rapidly with twice their volume of cold hemolysing
solution consisting of distilled water contA;n~ng
approximately .5 milligram per milliliter BMP. After 5
minutes equilibration in the cold, sufficient concentrated
cold HBSS is added to restore isotonicity. This
suspension is warmed to 37~C and incubated at that
temperature for 45 minutes. The resealed cells are
collected by centrifugation at 1000 gs for 15 minutes and
washed three times with isotonic HBSS to remove any
untrapped enzyme.
The encapsulated BMP/RBC conjugate may be
pelleted and the pellet placed directly into an osseous
defect. The conjugate RBCs may be surface activated and
partially crosslinked and incorporated into an osteogenic
and/or stress-bearing implant. Monoclonal antibodies, to
bone tissue antigenic markers, may be attached to the
surface of the cells so that the osteogenic proteins can
be directed, parenterally, to an osseous defect to promote
heating.
-
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EXAMPhE THIRTY
The method of Example Twenty such that a calcium
binding protein or peptide is used to create a bond
between the inorganic particle and the matrix. A calcium
binding peptide of molecular weight of 5,000 to 7,000,
namely, osteocalcin, which binds to hydroxyapatite may be
used as the calcium binding interface in this method. The
particle is immersed in a 1 to 1000 ppm solution of
osteocalcin prior to drying to affect this bound. The
procedure in Example Twenty is then followed.
I~ E~AMPLE THIRTY-ONE
The substrate or matrix for the novel bone graft
material of this invention may be, demineralized, freeze-
dried bone allograft or matrix (DFDBA or DFDBM), is
processed by procedures well known in the art. By way of
example, the process may include all or some the following
steps, as described by Mellonig (see "Freeze-Dried Bone
Allografts in Periodontal Reconstructive Surgery," Dental
Clinics of North America, Vol. 35, No. 3, July 1991.):
1. Sterile harvesting of cortical bone. This bone
material is sometimes placed in an antibiotic
solution.
2. The cortical bone is grossly cut to particle of
500 microns to 5 mm. Strips, wedges, chips, or
other shapes may also be fabricated.
3 The graft material is immersed in 100~ ethyl
alcohol for 1 hour to remove fat and to
inactivate virus.
4. The bone is then frozen at - 80 degrees
Centigrade for 1 to 2 weeks to inhibit
degradation. During this time period, test ',
results from serologic tests, antibody and
direct antigen assays, and bacterial cultures
are obtained and bone is retained, discarded, or
sterilized by additional methods.
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5. The bone is freeze-dried to remove more than 95
of its water content.
6. The cortical bone may be ground and sieved to a
finer particle size. By way of example, about
250 to about 750 microns.
7. The bone graft material is again immersed in
100~ ethyl alcohol and then washed repeatedly in
distilled water to remove all prior chemicals
used in the processing.
8. The bone graft material is decalcified in 0.6 N
HCl to remove virtually all of the mineral
calcium, leaving the organize bone matrix.
9. The bone is washed in sterile water and/or
sodium phosphate buffer to remove residual acid.
10. The demineralized bone matrix is refreeze dried
IS and vacuum sealed in sterile containers.
Those skilled in the art will realized that the
actual sequence of procedures and steps used in this
process may vary among different tissue banks that process
the bone graft material. This bone graft is further
processed by methods and materials described below to
produce the enhanced, osteogenic bone graft material
described in this disclosure.
EXAMPLE THIRTY-TWO
D~m;nPralized, freeze-dried, bone matrix powder
(DFBM) is obtained from a tissue bank. In this example,
DFBM powder was obtained from Musculoskeletal Transplant
Foundation (Homdel, N.J.). By way of example, the
procedure for processing of freeze-dried, demineralized
bone matrix may involve some or all of the following
steps: antibiotic soak, grinding of the bone matrix,
washing of the ground bone matrix in sterile water and/or
100~ ethyl alcohol, demineralization in 0.5 to 0.6 N HCl,
a sterile water rinse to remove residual HCl acid,
followed by ethanol wash, and the final step of freeze
drying the demineralized bone matrix powder. This
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material obtained was provided in the form of a sterile
powder.
The sterile freeze-dried demineralized bone
powder was removed ~rom its sterile glass bottle
container, placed in a covered sterile plastic well. A
S saturated solution of USP calcium hydroxide is prepared in
sterile distilled water solution. After the insoluble
portion of the CaOH solution has s~;m~nted to the bottom
of the solution container, the supernatant is removed with
a pipette and suction and placed in a separate sterile
container.
The saturated calcium hydroxide supernatant is
removed with a sterile syringe cont~;n;ng a 27 gauge
needle. The calcium hydroxide concentration of a
saturated calcium hydroxide solution is, according to
Lange's Handbook of Chemistry, 11th edition, approximately
0.19 parts of calcium hydroxide to 100 parts of water at 0
degrees Centigrade, or approximately 19 mg/100 mls of
water. One of skill in the art will understand that the
calcium concentration of this saturated solution, however,
is variable, and is dependent on the temperature. The
calcium hydroxide solution is dispensed onto the sterile
freeze-dried demineralized bone powder until the bone
powder matrix is visibly saturated with the solution. The
saturated DFBPM material is permitted to dry under ambient
conditions in a ~terile hood. The dried, calcium-enriched
demineralized bone matrix is lightly re-ground into a fine
powder and replaced in a sealed sterile glass container
until needed for implantation. By way of example the
weight proportions or weight ratios of added salt to bone
can vary from about 0.001~ to about 20~ by weight. By way r
of example, the weight proportion or weight ratio ratio of
added calcium to bone can vary from about 0.001~ to about
10~ by weight.
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EXA~PLE THIRTY-THREE
The saturated calcium hydroxide supernatant is
diluted in a series of serial dilutions to achieve varying
dilutions of calcium salt concentration. Two solutions,
one representing a two-fold dilution (2 to 1 dilution) of
S the saturated calcium hydroxide supernatant, the 8econd
representing a four-fold dilution (4 to 1 dilution) of
that same saturated solution, are prepared in a sterile
water solution.
Again using a syringe with a 27 gauge needle,
each of the two diluted calcium hydroxide solutions are
added, respectfully, to separate 1 cc portions of freeze-
dried d~m;neralized bone powder until the bone powder mass
appeared fully wetted and saturated with the respective
solutions.
~MPLE THIRTY-FOUR
The ~m~ n~ralized bone matrix powder
compositions described in Examples Thirty Two and Thirty
Three, together with corresponding demineralized bone
matrix powders which are from the same lot as each
experimental batch (thus serving as the corresponding
control groups), were implanted intramuscularly in the
hind thighs of laboratory mice. Each experimental batch
of DFDBA was paired with its corresponding control
material in a paired grouping in each ~n;m~l After
intramuscular implantation, the paired sites were
radiographed at 8, 29, and 46 days to assess the presence
and size of mineralized bony masses produced through
osteoinduction by the intramuscular implants. The ~n;m~ls
were sacrificed at the prescribed time frame and implants
with surrounding tissues were dissected and prepared for
histologic evaluation and analysis.
The results of the radiographic and histologic
analysis of these DFDBA implants are described in Table I
below:
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o
TABLE I. FRliQUENCY OF BONE INDUCTION IN INI~MIJSCULAR
IMPLANTS
Experiment One
System Tested 8 Days 29 Days 46 Days
Freq. Percent ~ Percent Freq. Percent
Control D~L)BA 0/28 0 % 0/28 0 % 0/28 0 %
Experimlont~l 10/10100 % 10/10 100% 10/10100 %
DFDBA (sat.)
Experim-~nt~l 0/10 0 % 0/10 0 % 1/1010 %
DFDBA (1:2 dil.)
~cperim~:nt~l 0/10 0 % 0/10 0 % 0/10 0 %
D~BA (1:4 dil.)
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A ~econd implantation experiment was undertaken
to assess both the reproducibility of the first
experiment, and to det~r~;ne both the frequency and size
of bone induction and mineralization in the experimental
and control DFDBA intramuscular implants. The results are
depicted in Table II:
TABT~P ~. FREQUn3NCY A*~DSIZE OF BONE ~NDUCIION rN
INTRAMUSCULAR IMPLANTS
Experim~nt Two
System Tested 8 Days 15 Days
Freq. Size of Cal. * Freq. Size of Cal. *
Control DFDBA 0/100.0 0/10 0.0
Experim~nt~l 10/10 2.45 10/10 2.45
DFDBA
(sat. ~lcillm salt)
* Size of c~ fic-~tinn del~ Y1 by grading scale r~n~in~ from "1" to "4", with
20 grade of "4" being the largest mass.
As shown in Tables I and II, data obtained at
all time-frames evaluated revealed that the experimental
DFDBA complexed with the saturated calcium hydroxide
25 solution demonstrated 100~ induction and formation of new
vital bone in all intramuscular implants. The DFDBA which
was not treated with bone in all intramuscular implants.
The DFDBA which was not treated with the saturated calcium
hydroxide solution failed to produce radiographically
detectable bone, even at 6 weeks. The DFDBA bone powder,
which was treated with a 1 to 2 dilution of the saturated
calcium hydroxide solution, produced 1 out of ten implants
with radiographically evident bone formation at 6 weeks
evaluation. The DFDBA treated with the 1 to 4 dilution of
saturated calcium hydroxide did not produce any
radiographically detectable bone at the 6 week time point.
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Nevertheless, the histologic analysis of the DFDBA matrix
powder treated with the 1 to 2 and the 1 to 4 dilutions of
the saturated calcium hydroxide solution did provide
beneficial cellular responses with reduced inflammatory
cells and early evidence of an osteogenic response, when
compared with the inflammatory cellular response seen with
the untreated, st~nA~rd demineralized bone material.
EXAMPLE THIRTY-FIVE
The demineralized bone matrix material may be
rinsed with a variety of other buffers or salt solutions
prior to the exposure to the free calcium salt solution.
For example, the bone matrix may be demineralized in 0.5 N
or 0.6 N HCl for a sufficient time period to effect
sufficient mineral removal to demonstrate osteogenic
properties (as measured by a residual pH level of 1.0 or
less). After removal from the acid solution, and removal
of residual acid by washing in sterile distilled water,
the ~m;neralized bone matrix may be rinsed in various
concentrations of buffer solutions, adjusted to various
pHs as may be desired. Use of neutral or slightly
alkaline buffer systems can assist in neutralizing
residual acid left after water rinsing.
For example, the demineralized bone powder,
after d~m;neralization in 0.5 or 0.6 HCl and sterile water
rinsing, may then be rinsed in a phosphate buffer
solution, for example disodium phosphate buffer solution,
ranging from 0.001 M to 0.2 M (pH 7.5 to 9.0). After
buffer rinsing, the demineralized bone matrix is then
saturated with a solution cont~;n;ng soluble calcium, such
as a saturated solution of calcium hydroxide, and then
permitted to dry under ambient conditions or by ',
lyophilization.
EXAMPLE THIRTY-SIX
The calcium source may be delivered to the
surface of the d~m;n~ralized bone matrix by a means other
than aqueous or water solution. For example, the soluble
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calcium source may be applied in a water-soluble or water-
insoluble film former. Alternatively, the DFDBA treated
with the soluble calcium salt may be further co-mbined with
a water-soluble or water insoluble filming forming agent.
For example, the soluble calcium enhanced
S demineralized bone matrix may be complex with a aqueous
collagen dispersion of gelatin solution and, optionally,
~urther lyophilized or dehydrated into a sponge of
membrane configuration. Alternatively, the soluble
calcium solution may be added to collagen dispersion or
gelatin solution, a~ter which untreated demineralized bone
matrix powder then added to the calcium/collagen or
calcium/gelatin dispersions or solutions, and the entire
conjugate dehydrated or lyophilized into a solid form for
implantation.
EXAMPLE THIRTY-SEVEN
The calcium salt saturated bone mass, processed
as described in these examples, can be lyophilized rather
than allowed to dry under ambient conditions.
EXAMP~E THIRTY-EIGET
The demineralized bone matrix starting material
can be processed by alternative methods which extract the
bone matrix further to remove additional potential
antigens. First, the bone graft is placed in a 1:1
chloroform-methanol solution for 4 hours at 25 degrees C.
A solution of 100~ ethyl alcohol may be substituted for
the chloroform-methanol solution. The bone is then
immersed in a 0.1 M Phosphate buffer solution, pH 7.4,
cont~;n;ng 10 mM/L iodoacetic acid and 10 mM/L sodium
azide for 72 hours at 37 degrees C. After rinsing in
sterile distilled water, the bone graft is placed in 0.6 N
Hydrochloric acid for 24 hours at 2 degrees C to
facilitate ~m;n~ralization. After thorough rinsing in
sterile water or buffer, the d~m;n~ralized bone matrix
material is freeze-dried at -72 degrees C for 24 hours.
At this pc;nt, the antigen-extracted DFDBA material is
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saturated with the soluble calcium solution (such as the
saturated calcium hydroxide solution) and either allowed
to dry under sterile ambient conditions, or lyophilized
(freeze-dried). The calcium enriched DFDBA is then placed
in a sterile container for storage.
E ~ MPLE THIRTY-NINE
The demineralized bone matrix can be extracted
with bu~Eers cont~;n;ng lyotropic agents, such as 4 M
guanidine, 6 M Urea, or 1~ sodium dodecyl sulphate, prior
to treatment with the soluble calcium solution. Protease
inhibitors may also be added to these extracting bu~ers
to inhibit degradation of the demineralized bone matrix
and also the proteins extracted by this process.
Following extraction of the demineralized bone matrix, the
bone matrix is rinsed in sterile water and fresh phosphate
bu~er. A soluble calcium cont~;n;ng solution, such as a
saturated soluble solution of calcium hydroxide, is
applied to the extracted bone matrix, then the treated
bone matrix is allowed to dry under sterile ambient
conditions, or by lyophilization.
EXAMPLE FORTY
Other calcium containing salt solutions may be
used in this invention. For example, soluble or saturated
solutions cont~;n;ng calcium acetate, calcium citrate,
calcium chloride, calcium ~ormate, calcium
glycerophosphate, calcium lactate, calcium laurate,
calcium oleate, calcium oxide, calcium palmate, calcium
salicylate, calcium stearate, calcium succinate, or
calcium sulfate (anhydrous, hemihydrate, dihydrate) would
be examples of acceptable soluble calcium sources. The
solubility o~ these compounds in 100 parts of water range ',
from as low as 0.003 parts to as high as almost 100 parts.
Those skilled in the art will realize that other calcium
containing compounds, in addition to those listed above,
may be suitable for use in this invention.
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EXAMP~E FORTY-ONE
The calcium or mineral salt modified
demineralized bone matrix (see Examples Thirty One -
Thirty Nine) may be added to an organic fibrous or
nonfibrous material, such as a collagen or gelatin matrix,
in such a m~nner as to form a enhanced d~m'neralized bone
matrix-filled porous or semi-porous sponge material. Such
a sponge may be ~ormed by adding various proportions o~
calcium or mineral salt modified demineralized bone powder
or particles to either an aqueous or dry powder dispersion
o~ collagen or gelatin. By way o~ example, the
fabrication of such a enhanced-osteogenic sponge, the
following procedure serves to provide one such possible
example under the range of possible approaches re~erred to
above.
An aqueous collagen dispersion can be produced
from a puri~ied bovine collagen material by redispersing
the collagen powder or fleece in an acidic or alkaline
solution of either dilute hydrochloric acid or sodium
hydroxide. For example, the dried collagen material can
be incrementally added to a .01 N solution of HCl to
produce anywhere from about a 0.01~ to a 5~ collagen
dispersion. The dispersion is m;xe~ thoroughly with a
Waring Blender under refrigeration with short bursts of 5
to 10 seconds agitation. The mixed dispersion can be
dialyzed against sterile distilled water at 4 degrees C.
to reduce the acid concentration while gradually elevating
the pH of the collagen dispersion to approximately a range
of from pH 4 to 5.5. The calcium or mineral salt modified
demineralized bone may then be added incrementally to the
collagen dispersion at 4 degrees C. Depending on the
initial pH of the collagen dispersion, various weight
ratios of demineralized bone may be added to the collagen
dispersion, ranging anywhere ~rom about 5 weight percent
of bone matrix to approximately about 95 weight percent
bone matrix. By way of example, the added ~m~n~ralized
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bone may be demineralized bone comprising added calcium or
mineral salt, partially activated demineralized bone or
untreated demineralized bone or combinations thereo~. For
example, aqueous collagen/enhanced demineralized bone
matrix powder dispersion can then be lyophilized into a
S sponge and cut into the desired size and configuration
with a sharp-bladed instrument or a sponge cutting device
by conventional methods.
Alternatively, the acidic or alkaline collagen
dispersion can be lyophilized by conventional methods then
may be ground, under cooling with dry ice and/or liquid
nitrogen, into a powder. This collagen powder can then be
dry blended with various ratios of enhanced demineralized
bone matrix powder. After blending, the powder mixture
can be hydrated with sufficient sterile distilled water to
form a uniform dispersion. This blended collagen/enhanced
demineralized bone dispersion may then be lyophilized into
a sponge configuration. The source of the collagen may be
from a hllmAn or ~n;m~l origin.
EXAMoeLES FORTY-TWO
Demineralized bone matrix powder, which has not
been surface activated or modified with calcium or mineral
salts (other than phosphate buffer), may be added to
various forms of reconstituted collagen or gelatin as
described in Example Forty One. The weight ratio of
demineralized bone powder to collagen or gelatin matrix
material is from about 60 weight percent to about 90
weight percent of the ~m;ne~alized bone powder matrix
component. The resultant sponges have the following
unique properties which enhance their clinical utility:
1) Enhanced maintenance of shape, form, and ~,
resilience under moist conditions.
2) Enhanced resistance to compressibility in
the dry and/or moist conditions, while
maint~; n; ng an elastic, sponge-like
physical behavior.
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3) ~nh~nced space maint~;n;ng function.
4) ~nh~nced cellular in~iltration without a
significant increase in inflammatory cells,
such as macrophages.
If the composition is in the ~orm o~ a sponge,
preferably it is characterized by a density of about 0.1
grams/cubic centimeter (cc) or greater than 0.1
grams/cubic centimeters (cc). The range of sponge density
may be from about 0.1 grams/cc to about 0.5 grams/cc, with
the preferred density from about 0.11 to about 0.35
grams/cubic centimeter. Sponges with about 90 weight
percent or greater o~ ~Pm;n~ralized bone require a pH for
matrix collagen (or gelatin) o~ less than pH 5.0, and
preferably less than pH 4.5. If the collagen (or gelatin)
is provided as an acidic powder, for later blending with
the demineralized bone, the pH of the collagen dispersion
prior to lyophilization and milling into a powder for
blending, should be less than pH 5.0, and preferably below
pH 4.5. If the collagen is dispersed in the alkaline
range, the pH should be above 9Ø The source of the
collagen may be from human or ~n;m~l origin."
E~AMPLES FORTY-THREE
Demineralized bone matrix or reconstituted
collagen matrix may be treated with concentrations of
aqueous or soluble alkaline phosphatase ranging from as
low as 10 enzyme units per milligram bone or collagen up
to or greater than 100 units per milligram bone or
collagen. D~m;ne~alized bone matrix which was determined
to be inactivate (as tested in the mouse thigh ~n;m~l
model) can be converted to active, mineralizing bone by
pre-treatment with 100 units per milligram of alkaline
phosphatase, followed by dehydration or drying of the
treated bone. Reconstituted aqueous collagen, cont~;n;ng
approximately 18 to 20 units of alkaline phosphatase per
milligram of collagen (dry weight) were lyophilized and
then ground into a fine powder. This collagen-alkaline
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phosphatase powder was implanted subcutaneously in mice,
which resulted in mineralized masses which revealed bone-
like structures under histologic evaluation.
While this invention has been described with
reference to certain specific embodiments, it will be
S appreciated that various modifications of the invention in
addition to those shown and described herein will become
apparent to those skilled in the art from the foregoing
description. Such modifications are included to fall
within the scope of the claims appended hereto.
