U.S. patent application number 11/776997 was filed with the patent office on 2008-01-10 for methods of repairing bone.
This patent application is currently assigned to Nanotherapeutics, Inc.. Invention is credited to James F. Kirk, James D. Talton.
Application Number | 20080008766 11/776997 |
Document ID | / |
Family ID | 34919419 |
Filed Date | 2008-01-10 |
United States Patent
Application |
20080008766 |
Kind Code |
A1 |
Talton; James D. ; et
al. |
January 10, 2008 |
Methods of Repairing Bone
Abstract
This invention relates to an improved method of preparing an
implantable gel or paste for placement between injured bones or
placement in bony voids to induce regeneration, and the
compositions produced thereby. Specifically, mineral, ceramic, or
processed bone particles are coated with a high molecular weight
polymer capable of forming a viscous gel when reconstituted with
water, saline, autologous blood, sera, or other medically
acceptable solution. This high molecular weight polymer coating
material may be a natural or synthetic polymeric material,
producing a wettable gel upon exposure to water, saline, or another
solution. In storage, the composition will be granular and dry but
easily wetted. In use, the material is reconstituted to a viscous
malleable paste by the simple addition of water or other medically
acceptable solution without the need for aggressive mixing. The
paste may be delivered by syringe or manually deposited yet will be
resistant to lavage or to displacement by gravity induced flow.
Inventors: |
Talton; James D.; (Alachua,
FL) ; Kirk; James F.; (Del Mar, CA) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
Nanotherapeutics, Inc.
Alachua
FL
|
Family ID: |
34919419 |
Appl. No.: |
11/776997 |
Filed: |
July 12, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11070413 |
Mar 2, 2005 |
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11776997 |
Jul 12, 2007 |
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60548945 |
Mar 2, 2004 |
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Current U.S.
Class: |
424/496 ;
424/489; 424/490; 424/549 |
Current CPC
Class: |
A61P 9/00 20180101; A61L
27/365 20130101; A61K 9/0024 20130101; A61L 2430/02 20130101; A61L
24/02 20130101; A61K 9/5078 20130101; A61L 27/34 20130101; A61L
27/3608 20130101; A61P 19/00 20180101; A61L 27/222 20130101; A61L
24/001 20130101; A61K 35/32 20130101; A61L 24/0047 20130101; A61L
27/34 20130101; C08L 89/06 20130101 |
Class at
Publication: |
424/496 ;
424/489; 424/490; 424/549 |
International
Class: |
A61K 35/32 20060101
A61K035/32; A61K 9/14 20060101 A61K009/14; A61K 9/16 20060101
A61K009/16; A61P 9/00 20060101 A61P009/00 |
Claims
1-18. (canceled)
19. A method of repairing bone comprising: (a) providing a dry
composition comprising bioactive particles coated with at least one
high molecular weight polymer; (b) contacting the dry composition
with a solution, thereby to convert the dry composition into a wet
composition; and (c) placing the wet composition in contact with a
bone defect on an internal or external bone surface, wherein the
wet composition is capable of repairing bone.
Description
[0001] This is a divisional of application Ser. No. 11/070,413,
filed Mar. 2, 2005 which claims the benefit of U.S. Provisional
Application No. 60/548,945, filed Mar. 2, 2004, both of which are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
A. GOVERNMENT INTERESTS
[0002] None
B. RELATED APPLICATIONS
[0003] The present application claims priority under 35 U.S.C.
.sctn. 119 to U.S. Provisional Patent Application Ser. No.
60/548,945, filed Mar. 2, 2004. The entire contents of the
aforementioned application are specifically incorporated herein by
reference in its entirety.
C. FIELD OF THE INVENTION
[0004] This invention relates to an improved method of preparing an
implantable paste for placement between injured bones or placement
in bony voids to induce regeneration, and the compositions produced
thereby. Specifically, mineral, ceramic, or processed bone
particles, so-called bioactive particles (BP), are coated with a
matrix material (MM) capable of forming a viscous gel when
reconstituted with water, saline, autologous blood, sera, or other
medically acceptable solution. This MM may be a natural or
synthetic polymeric material, producing a wettable paste upon
exposure to water, saline, or another aqueous solution. In storage,
the composition will be granular and dry, but easily wetted during
reconstitution. To prepare the composition for administration, the
material is reconstituted to a viscous malleable paste by the
simple addition of water or other medically acceptable solution
without the need for aggressive mixing. The paste may be delivered
by syringe or manually deposited yet will be resistant to lavage or
to displacement by gravity induced flow.
D. DESCRIPTION OF RELATED ART
[0005] Bone pastes, such as REGENAFIL.TM. or OSTEOFIL.TM. produced
by Regeneration Technologies, Inc., comprise particles of allograft
demineralized bone matrix (DBM) and gelatin (U.S. Patent
Applications 20020098222, 20020018796, and 20020076429). As taught
by Scheicher (U.S. Pat. No. 4,191,747), suspending osteoinductive
and/or osteoconductive materials in gelatin solutions provides an
implantable composition with temperature dependant flow properties.
Above the gel transition temperature, the composition is free
flowing while below that temperature, i.e. when at body
temperature, it forms a stable mass resistant to deformation and
dissolution.
[0006] However, there are some drawbacks to such compositions. If
provided as a pre-mixed suspension of DBM and hydrated gelatin, the
product must be stored frozen to prevent degradation of the
osteoinductive capability of the DBM. Prior to use, frozen material
must not only be thawed but raised above the gel transition
temperature in order to yield a free-flowing paste.
[0007] Regeneration Technologies, Inc. produces a version of
OSTEOFIL.TM. that can be stored at room temperature. This version
is comprised of a mixture of DBM particles and gelatin particles.
The drawback of this paste composition and method of preparation is
that heated liquid and/or aggressive mixing is required to produce
a uniform and free-flowing suspension (U.S. Patent Applications
20010016703, 20010037091, 20030180262).
[0008] It is conventional to store drugs, vaccines, medicaments,
and solutions in a sealed vial or other container for later use.
Drugs, vaccines, medicaments, and solutions may be stored in a dry
or powdered form to increase the shelf life and reduce inventory
space. Such dry or powdered materials are typically stored in a
conventional sealed vial having a puncturable closure, such as an
elastomeric stopper, and reconstituted in liquid form for later
use, such as administration to a patient, by adding a diluent or
solvent.
[0009] Dry materials available for reconstitution may also be
stored directly in a syringe. For example, as described in U.S.
Pat. No. 6,773,714 (Dunn, 2004), leuprolide acetate, a peptide
drug, is lyophilized directly in a syringe prior to use. A
biodegradable polymer and solvent solution is filled into another
syringe. The two syringes are coupled together and the contents are
drawn back and forth between the two syringes until the
polymer/solvent solution and the leuprolide acetate are effectively
mixed together, forming a flowable composition. The flowable
composition is drawn into one syringe and the two syringes then
disconnected. A needle is inserted onto the syringe containing the
flowable composition and then injected through the needle into the
body. Other flowable compositions are described in U.S. Pat. Nos.
5,324,519; 4,938,763; 5,702,716; 5,744,153; and 5,990,194. None of
these techniques, though, anticipate the preparation of an easily
dispersible bioactive particle composition, prepared from the
precipitation of a high molecular-weight polymer onto the bioactive
particle, with the described characteristics in the present
invention.
SUMMARY OF THE INVENTION
A. Features and Advantages of the Invention
[0010] The present invention overcomes these and other inherent
deficiencies in the prior art by providing novel bioactive
particles and preparation methods for use in preparing improved
therapeutic products. The described processes involve forming
bioactive particles in a solution and/or microencapsulating
particles, and compositions produced thereby. The process utilizes
alcohol precipitation under mechanical stirring at controlled
temperatures, which provides the proper forces during precipitation
to control the particle growth and mixing properties. Preferential
precipitation of the higher molecular weight fraction of the matrix
material produces desirable final compositions with high surface
area, controllable gelation, and shelf-life stability. Bioactive
particles may be (1) formed in a solution to obtain a particle
suspension and then (2) dried in an oven or fluid bed to control
the structure of the particle or particle surface. In general, the
process can be used to microencapsulate bioactive particles by
suspending the drug particles in a solution including a coating
material (such as a biodegradable polymer) and adding alcohol to
the solution under controlled process conditions. The bioactive
particle compositions produced thereby possess improved properties
including, but not limited to, improved flow and syringability,
controlled adhesion, stability, and/or resistance to moisture. This
process, and the compositions produced, also provide significant
advantages in the manufacture of bioactive particulate
formulations, as well as biomedical particulate compositions, where
sensitive macromolecules, such as proteins or DNA, are involved
that would be degraded using more rigorous processing conditions or
temperatures.
B. Summary of the Invention
[0011] The present invention provides compositions for repairing
bone that comprise: bioactive particles; and a coating on the
bioactive particles comprising at least one high molecular weight
polymer, wherein the composition is in a form capable of repairing
bone. The bioactive particles may be chosen from mineral particles,
ceramic particles and processed bone particles. The processed bone
particles may be derived from human, bovine, ovine or porcine
sources. The processed bone particles may be derived from a
demineralized bone matrix and range in size from about 100 to about
800 microns. Alternatively, the processed bone particles may
comprise ground cortical and cancellous bone chips ranging in size
from about 1.0 to about 3.0 millimeters. In some embodiments, one
or more compositions described herein are in paste form. The paste
may be prepared by contacting the bioactive particles coated with
at least one high molecular weight polymer with a solution. The
high molecular weight polymer may be a naturally occurring high
molecular weight polymer. The high molecular weight polymer may be
a synthetic polymer. The high molecular weight polymer may be
chosen from gelatin, pectin, hydrogel polymers, polycarbophils,
polyanhydrides, polyacrylic acids, alginates, and gums. The
composition may further comprise a drug. The composition for
repairing bone may comprise: bloactive particles; and a coating on
the bioactive particles that may comprise at least one at least one
high molecular weight polymer, wherein the composition may be in a
form capable of repairing bone; wherein the bioactive particles may
be derived from a demineralized bone matrix and range in size from
about 100 to about 800 microns; wherein the high molecular weight
polymer may be gelatin.
[0012] The present invention provides a method of preparing a
composition for repairing bone comprising: providing a solution
comprising at least one high molecular weight polymer; adding
bioactive particles to the solution; and precipitating the at least
one high molecular weight polymer, thereby forming bioactive
particles coated with the at least one high molecular weight
polymer. The high molecular weight polymer may be precipitated
using a solvent chosen from 1-propanol, 2-propanol, ethanol,
hexanol, and acetone. The method may involve drying the bioactive
particles coated with the at least one high molecular weight
polymer, thereby producing a granular preparation suitable for
reconstitution. The bioactive particles may be dried in a
convection oven, a vacuum oven, or a fluidized bed apparatus. The
bioactive particles may be processed bone particles and the polymer
may be gelatin ranging in amounts from about 1:2 to about 10:1
weight fraction. The present invention provides a composition for
repairing bone prepared according to this method.
[0013] The present invention provides a method of repairing bone
comprising: providing a dry composition comprising bioactive
particles coated with at least one high molecular weight polymer;
contacting the dry composition with a solution, thereby to convert
the dry composition into a wet composition; and placing the wet
composition in contact with a bone defect on an internal or
external bone surface, wherein the wet composition may be capable
of repairing bone.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The drawings form part of the present specification and are
included to further demonstrate certain aspects of the present
invention. The invention may be better understood by reference to
one or more of these drawings in combination with the detailed
description of specific embodiments presented herein.
[0015] FIG. 1 is a schematic of the mixing process. A vessel, 13,
which may be heated or cooled, 3, has a cover, 21, through which
components of the composition may be added, 23, and through which
end products and byproducts may be removed, 25. The composition may
be stirred with an impeller, 31.
[0016] FIG. 2A is a scanning electron micrograph of Bioglass.RTM.
45S5 bioactive glass particles coated with porcine gelatin at 500
times magnification.
[0017] FIG. 2B is a scanning electron micrograph of Bioglass.RTM.
45S5 bioactive glass particles coated with porcine gelatin at 50
times magnification.
[0018] FIG. 3A is a scanning electron micrograph of processed
bovine cortical bone chips coated with porcine gelatin at 50 times
magnification.
[0019] FIG. 3B is a scanning electron micrograph of demineralized
bone matrix coated with porcine gelatin at 50 times
magnification.
[0020] FIG. 4 is an example of size exclusion chromatography
spectra for the porcine gelatin at different stages of the process.
The top trace (A) shows the initial autoclaved porcine gelatin
solution before coating. The top middle trace (B) is from gelatin
found on the coated particles from Example 4. The bottom middle
trace (C) is from gelatin found on the coated particles from
Example 3. The bottom trace (D) is for the molecular weight
standards used to calibrate the runs. The shifting to the left of
the dominant peaks seen in the two middle traces relative to the
top and bottom traces indicate the coating process excludes lower
molecular weight fractions of the gelatin.
[0021] FIG. 5A is an x-ray radiograph, taken at day 28, of coated
bovine bone chips implanted in the mouse. The bright area indicates
mineralization is still present.
[0022] FIG. 5B is an x-ray radiograph, taken at day 28, of coated
bioactive glass particles implanted in the mouse. The bright area
indicates mineralization is still present.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The invention is directed to methods of forming
microencapsulated bioactive particles and wettable pastes, and the
compositions produced thereby. "Bioactive particles" to be produced
in accordance with this invention are typically, but not limited
to, those 750 micrometers to 1.5 millimeters in size particles.
Such bioactive particles and wetted paste compositions include, but
are not limited to, allograft bone, xenograft bone, processed bone,
natural bone substitutes, calcium salts, bioactive glasses, for
human or animal use, cosmetics, as well as inert particles for
which a biogels or pastes for bone void-filling or regeneration, as
well as hard and soft tissue augmentation. The possibilities and
combinations are numerous.
A. COMPOSITIONS OF COATED BIOACTIVE PARTICLES
[0024] The present invention relates to compositions of implantable
pastes and methods for incorporating (1) mineral, ceramic, or
processed bone particles as the BP and additionally (2) binders,
bulking agents, excipients, and/or surface modifiers as the MM into
easily wetting granular product that provides the same or improved
shelf-life stability of current dry compositions but much simpler
reconstitution. This is achieved by coating the component or
components selected from group (1) with one or more of the
components from group (2) to yield a dry, granular composition that
wets easily to form a cohesive yet malleable paste.
[0025] For example, demineralized bone matrix (DBM) particles might
be coated with a thin layer of gelatin and subsequently dried. The
resulting dry granular composition will easily wet when an aqueous
solution such as physiologic saline is added. The thin gelatin
layer around each particle of DBM will absorb water from the saline
and become adhesive. The coated particles will form a cohesive and
malleable paste even though a) the gelatin coating may not be
completely rehydrated and b) the paste may not be at a temperature
above the gel transition temperature.
[0026] For use in so-called bone pastes, a variety of matrix
materials have been tested including hydrogel polymers,
polycarbophils, polyanhydrides, polyacrylic acids, alginates,
gelatins, gums, and pectin. Adhesion may be affected by physical or
mechanical bonds; secondary chemical bonds; and/or primary, ionic
or covalent bonds, which can improve the adhesion of -delivery
system or viscosity. The disclosed method may be used to coat
mineral, ceramic, and/or processed bone particles with such
materials to produce an implantable composition with good
shelf-life stability, ease of storage, and ease of reconstitution.
Those coating materials that do not possess a gel transition
temperature may lack the resistance to lavage that a gelatin based
composition would possess. However, such compositions would still
possess the ease and rapidity of reconstitution afforded by this
method.
[0027] When delivering a non-soluble particulate material or
composition to a site in the body for the purposes of promoting
acceleration, delay, or enhancement of the healing process, it is
desirable to use a paste. Pastes are desirable because these are
easily formed to fill irregular voids or volumes, yet tend to
remain where they are placed.
[0028] When the implanted material is organic or tissue-derived,
long-term storage of the material becomes an issue. In the case of
so-called bone pastes, the DBM in these pastes is subject to
hydrolytic degradation if stored hydrated at room temperature.
Frozen pastes require low-temperature freezers for transportation
and storage, as well as time to thaw prior to use. Current dry
compositions of pastes require aggressive mixing and warm liquids
to reconstitute and, in the case of gelatin based pastes, must be
maintained at a temperature above body temperature to be easily
flowable.
B. METHODS FOR FORMING COATED BIOACTIVE PARTICLES
[0029] In the current invention, the inventors found that it is
possible to produce porous compositions that wet easily and
reconstitute to an implantable paste without the need of aggressive
mixing. It is the object of this invention to produce such paste
compositions consisting of bioactive particles of minerals,
ceramics, and/or processed bone, and coating these bioactive
particles with a wettable material or materials capable of forming
a viscous gel upon exposure to water, such as gelatin, pectin,
hydrogel polymers, polycarbophils, polyanhydrides, polyacrylic
acids, alginates, and gums; with or without the inclusion of drugs,
binders, wetting agents, and/or bulking agents.
[0030] One particular embodiment of this invention is to produce a
dry granular composition comprised of DBM coated with gelatin in
the approximate mass ratio of 8 parts DBM to 3 parts gelatin. Such
a composition is reconstituted to a malleable paste by exposing a
densely packed plug of the composition to water in the approximate
mass ratio of 7 parts dry composition to 4 parts water and allowing
the mixture to sit for approximately 30 seconds, or such time
sufficient for the gelatin coating on the DBM particles to
rehydrate. Other aqueous solutions, such as saline, sera, or whole
blood, could be substituted for water.
[0031] To produce this dry composition, damp DBM particles taken at
the end of the demineralization process would be assayed for water
content. Once the dry mass of DBM particles is calculated by known
methods, these damp DBM particles would be added to a sufficient
quantity of 3% m/m gelatin solution in deionized water to produce
an approximate mass ratio of 2 parts DBM to 1 part gelatin. The
excess of gelatin is required because some of the gelatin will be
lost during the coating process through incomplete precipitation.
The gelatin/DBM suspension starts out above its gelation
temperature. The suspension is agitated or rapidly stirred while a
quantity of isopropanol, ethanol, and/or acetone, for example, is
added slowly to the mix. A simple schematic of a mixing apparatus
is depicted in FIG. 1. Low-frequency sonication may be used in
addition to stirring, as described in PCT application WO 03/090717.
The isopropanol, ethanol, and/or acetone acts as a non-solvent for
the gelatin in solution. Similarly, a metal ion complexing agent,
such as zinc, or pH shift may be used to precipitated the gelatin.
The quantity of nonsolvent needed to drive gelatin out of solution
depends on the non-solvent chosen, its temperature, and the
concentration of gelatin in solution.
[0032] It can be observed by those skilled in the art that the
cloudiness of the supernatant during the coating process is a
result of less than all of the gelatin coming out of the solution
at once. The least soluble (generally higher molecular weight)
traction of gelatin will be precipitated first. Thus, this process
and composition differs from that taught by Scheicher (U.S. Pat.
No. 4,191,747) and others in so far as not only are the particles
pre-coated with gelatin prior to creating an implantable paste, but
the resulting intermediate composition is comprised of DBM
suspended in gelatin of higher average molecular weight than other
methods, because of how the gelatin was attached onto the surface
of the DBM particles. The resulting compositon is easily dried and
demonstrates improved stability and wettability compared to other
compositions.
[0033] Once sufficient gelatin has been deposited onto the DBM
particles the mixer can be stopped and the supernatant decanted. In
the case of isopropanol used as the non-solvent, a volume is
chilled to 15 degrees Celsius and approximately 5 parts added to 1
part initial gelatin solution as a volume ratio and agitated for
about 3 minutes. The remaining material is filtered through a 270
mesh seive and washed again in isopropanol with agitation for about
2 minutes. The resulting particulate mass is then dried overnight
in an oven at 35 degrees Celsius, yielding a granular powder that
wets easily to form a cohesive, malleable paste.
[0034] This granular composition can be loaded into appropriate
containers and sterilized via ionizing radiation (X-ray, e-beam, or
gamma). It will be recognized by one skilled in the art that the
amount of material required for the desired effect on
administration will, of course, vary with the composition and the
nature and severity of the condition and size of the person or
animal undergoing treatment, and is ultimately at the discretion of
the physician.
[0035] While various embodiments of the present invention have been
described above, it should be understood that they have been
presented by way of example only, and not limitation. The present
invention is not limited to the described compositions and methods,
nor is it limited to a particular composition or material, nor is
the present invention limited to a particular scale or batch size
of production. Thus, the breadth and scope of the present invention
should not be limited by any of the above-described exemplary
embodiments, but should be defined only in accordance with the
following claims and their equivalents.
C. USES FOR COATED BIOACTIVE PARTICLES
[0036] Bioactive particles coated with matrix material or matrix
materials may be used to produce easily reconstituted and malleable
pastes. Such compositions may be placed in contact with an internal
or external bone surface for the purposes including, but not
limited to, void filling, e.g. inducing or conducting the regrowth
of bony tissue in a surgically or traumatically induced void in
boney tissue. Such pastes may also be used to form drug depots,
e.g. bioactive particles with chemotherapy agents may be injected
into a surgically induced void from which cancerous tissue was
removed.
D. EXAMPLES
[0037] The following examples are included to demonstrate example
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventors to
function well in the practice of the invention, and thus can be
considered to constitute relevant examples for its practice.
However, those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
[0038] A solution of porcine gelatin (12 g) in water (388 g) was
stirred in a polypropylene pitcher using a Lightnin Mixer with a
two-blade impeller. To this was added bovine tendon collagen (15.8
g). To this was added a total of 1900 ml of isopropyl alcohol.
After precipitate formed, the stirring was stopped and the
particles were allowed to settle. Supernatant was decanted and the
particulate material was rinsed with 300 ml of isopropyl alcohol.
Decanting and rinsing were repeated again. After final decant, the
curd-like particles were dried at room temperature under vacuum
overnight. A total of 22.1 g of finely divided dry curd-like
particles were collected. Moisture content, as determined by loss
on heating, was 7.8% mass/mass. Approximately 1.4 g was placed in a
10 ml syringe and wetted with 1 ml saline, easily syringable after
reconstitution.
Example 2
[0039] A solution of porcine gelatin (6 g) in water (194 g) was
stirred in a polypropylene pitcher using a Lightnin Mixer with a
two-blade impeller. To this was added calcium sulfate dihydrate
particles (12.35 g; sieved to 1-3 mm size range). To this was added
a total of 900 ml of isopropyl alcohol. After precipitate formed,
the stirring was stopped and the particles were allowed to settle.
Supernatant was decanted and the particulate material was rinsed
with 200 ml of isopropyl alcohol. Decanting and rinsing were
repeated again. After final decant, the curd-like particles were
dried at room temperature under vacuum for 5.5 hours. A total of
22.1 g of finely divided dry curd-like particles were collected.
Moisture content, as determined by loss on heating, was 15.8%
mass/mass.
Example 3
[0040] A solution of porcine gelatin (3 g) in water (100 g) was
stirred in a 600 ml pyrex beaker using a magnetic stir bar. To this
was added Bioglass.RTM. 45S5 bioactive glass particles (6.0 g;
sieved to 90 to 710 micron size range). To this was added a total
of 350 ml of isopropyl alcohol. After precipitate formed, the
stirring was stopped and the particles were allowed to settle.
Supernatant was decanted and the particulate material was rinsed
with 150 ml of isopropyl alcohol. Decanting and rinsing were
repeated again. After final decant, the curd-like particles were
dried at room temperature under vacuum overnight. A total of 8.3 g
of finely divided dry curd-like particles were collected. Moisture
content, as determined by loss on heating, was 6.1% mass/mass.
[0041] The particles are shown in FIG. 2A and B and appeared
several hundred microns in size, with regions of incomplete gelatin
coating attached to the surface. The particles mixed easily in a
plastic weighboat to form a sticky clump of putty-like material.
Approximately 1.7 g was placed in a 10 ml syringe and wetted with 1
ml saline, easily syringable after reconstitution. Samples were
placed into leg muscle ectopic sites of athymic mice and 28 day
radiographs are shown in FIG. 5A, demonstrating sites of mineral
deposits. Additional samples were packed into syringes and
sterilized by gamma-irradiation. Further, reconstituted samples
were injected into critical-size drilled defects in rabbits and
demonstrated osteoconductive responses by radiography.
Example 4
[0042] A solution of porcine gelatin (34.5 g) in water (1,115 g)
was stirred in a polypropylene pitcher using a Lightnin Mixer with
a two-blade impeller. To this was added demineralized bovine bone
(bovine DBM) suspension (35 g) taken right after the acid
neutralization step (i.e. 53.5 g moist demineralized bone). To this
was added a total of 3000 ml of isopropyl alcohol. After
precipitate formed, the stirring was stopped and the particles were
allowed to settle. Supernatant was decanted and the particulate
material was rinsed with ca. 400 ml of isopropyl alcohol. Decanting
and rinsing were repeated two more times. After final decant, the
particulate curds were dried at room temperature under vacuum for
17 hours. After sieving, 96.5 g of spongy dry curd-like particles
were obtained with approximately 35% moisture content. The
particles are shown in FIG. 3A and appeared several hundred microns
in size with a roughened surface indicating a gelatin coating.
Approximately 1.3 g was placed in a 10 ml syringe and wetted with
0.8 ml saline and 1ml rat blood, both samples easily syringable
after reconstitution.
[0043] In addition, the molecular weight of (A) autoclaved gelatin
starting solution (1% in water) was compared to (B) gelatin diluted
(1%) from the above Example as well as (C) Example 3. Size
Exclusion Chromotography (SEC) chromatograms, including the
molecular weight marker (D) shows thyroglobulin (bovine, MW670,000)
at 6.919 minutes, g-globulin (bovine, MW=158,000) at 8.101 minutes,
ovalbumin (chicken, MW44,000) at 9.108 minutes, myoglobin (horse,
MW=17,000) at 11.212 minutes, and vitamin B12 (MW1,350) at 14.440
minutes. These SEC profiles display clearly that the described
method selectively coats the bioactive particles with the high
molecular weight fraction of the polymer, in this case gelatin, and
the low molecular weight fraction stays in solution and is decanted
off before drying.
Example 5
[0044] A solution of porcine gelatin (25 g) in water (808 g) was
stirred in a polypropylene pitcher using a Lightnin Mixer with a
two-blade impeller. To this was added ground bovine cortical bone
particles (50 g; cleaned; moist; sieved to 150-1,000 micron size
range). To this was added a total of 2400 ml of isopropyl alcohol.
After precipitate formed, the stirring was stopped and the
particles were allowed to settle. Supernatant was decanted and the
particulate material was rinsed with 600 ml of isopropyl alcohol.
Decanting and rinsing were repeated two more times. After final
decant, the curd-like particles were dried at room temperature
under vacuum overnight. A total of 71 g of dry curd-like particles
were collected. The particles are shown in FIG. 3B and appeared
several hundred microns in size and gelled upon mixing with
saline.
Example 6
[0045] A solution of hyaluronic acid (4.5 g) in water (300 g) was
stirred in a polypropylene pitcher using a Lightnin Mixer with a
two-blade impeller. To this was added demineralized bovine cortical
bone (11.2 g). To this was added a total of 600 ml of isopropyl
alcohol. After precipitate formed, the stirring was stopped and the
particles were allowed to settle. Supernatant was decanted and the
particulate material was rinsed with 300 ml of isopropyl alcohol.
Decanting and rinsing were repeated again. After final decant, the
curd-like particles were dried at room temperature under vacuum
overnight. Large clumped curd-like particles were produced; some
clear indicating hyaluronic acid was not thoroughly dissolved.
Example 7
[0046] A solution of 100,000 molecular weight polyethylene oxide
(ca 2.5 g) in water (50 ml) was stirred for 30 seconds after the
addition of Bioglass.RTM. bioactive glass (2.5 g; sieved to 90-710
micron size range). To this was added a solution of isopropyl
alcohol and hexanol (120 ml, 1:5 ratio) while stirring for another
minute. Solution was placed in the freezer (-5 degrees C.) under
stirring where precipitate formed after 1 hour. Supernatant was
decanted and the particulate material was rinsed with 100% hexanol.
Decanting and rinsing were repeated again. After final decant, the
curd-like particles were dried at room temperature under vacuum
overnight.
Example 8
[0047] A solution of porcine gelatin (3 g) and polyhexamethylene
biguanide (PHMB, a biguanide antimicrobial, 100 mg) in water (100
g) was stirred in a 600 ml pyrex beaker using a magnetic stir bar.
To this was added Bioglass.RTM. 45S5 bioactive glass particles (6.0
g; sieved to 90 to 710 micron size range). To this was added a
total of 350 ml of isopropyl alcohol. After precipitate formed, the
stirring was stopped and the particles were allowed to settle.
Supernatant was decanted and the particulate material was rinsed
with 150 ml of isopropyl alcohol. Decanting and rinsing were
repeated again. After final decant, the curd-like particles were
dried at room temperature under vacuum overnight. A total of
approximately 8 g of finely divided dry curd-like particles were
collected. Moisture content, as determined by loss on heating, was
similar to Example 3 above and PHMB content, analyzed by HPLC, was
approximately 1%. After reconstitution, the antimicrobial
slow-releasing paste could be used to prevent infection for several
days following placement in the wound. Similar drug-releasing
systems incorporating drugs and/or bioactive proteins could be
prepared in a similar fashion by mixing during the precipitation
step or after the particles are dried. Finally, a drug or bioactive
protein could also be introduced in the reconstitution fluid to
produce a homogenous mixture.
Example 9
[0048] A solution of porcine gelatin (87 g) in water (2,813 g) was
stirred in a stainless steel bowl using a air-powered stirrer with
a two-blade impeller. To this was added Bioglass.RTM. 45S5
bioactive glass particles (174 g). To this was added a total of
9,000 ml of isopropyl alcohol. After precipitate formed, the
stirring was stopped and the particles were allowed to settle.
Supernatant was decanted and the particulate material was rinsed
with ca. 3,000 ml of isopropyl alcohol. Decanting and rinsing were
repeated two more times. After final decant, the particulate curds
were dried in a Glatt Uni-Glatt fluid bed dryer at 35.degree. C.
for 1 hour. After sieving, 230 g of dense dry curd-like particles
were obtained. Approximately 2.6 g was placed in a 10 ml syringe
and wetted with 2.0 ml saline, both samples easily syringable after
reconstitution.
CITED DOCUMENTS
[0049] 1. Wironen, J. F. and Grooms, J. M., "Bone paste", U.S. Pat.
Applic. 20020098222, submitted Mar. 13, 1997.
[0050] 2. Wironen, J. F., "Thermally sterilized bone paste", U.S.
Pat. Applic. 20020018796, submitted Jan. 28, 1998.
[0051] 3. Wironen, J., Felton, P., and Jaw, R. "Bone paste
subjected to irradiative and thermal Treatment", U.S. Pat. Applic.
20020076429, submitted Sep. 16, 1998.
[0052] 4. Scheicher, H., "Corrective agent for the covering and/or
filling of bone defects, method for the preparation of same and
method of using the same", U.S. Pat. No. 4,191,747, issued Mar. 4,
1980.
[0053] 5. Wironen, J., Kao, P., and Bernhardt, A., "System for
reconstituting pastes and methods of using same", U.S. Pat. Applic.
20010016703, submitted Dec. 29, 2000.
[0054] 6. Wironen, J., and Walpole, M., "System for reconstituting
pastes and methods of using same", U.S. Pat. Applic. 20010037091,
submitted Nov. 1, 2001
[0055] 7. Wironen, J., Kao, P., and Bernhardt, A., "System for
reconstituting pastes and methods of using same", U.S. Pat. Applic.
20030180262, submitted Oct. 11, 2001.
[0056] 8. Talton, J. and McConville, C, "Process for forming and
modifying particles and compositions produced thereby", PCT
application WO 03/090717, filed Apr. 23, 2002.
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