U.S. patent application number 10/739492 was filed with the patent office on 2004-04-29 for implantable putty material.
This patent application is currently assigned to Centerpulse Biologics Inc.. Invention is credited to Benedict, James J., Damien, Christopher J..
Application Number | 20040081704 10/739492 |
Document ID | / |
Family ID | 32106021 |
Filed Date | 2004-04-29 |
United States Patent
Application |
20040081704 |
Kind Code |
A1 |
Benedict, James J. ; et
al. |
April 29, 2004 |
Implantable putty material
Abstract
The present invention provides compositions for an implantable
putty material for delivery of active compounds to a patient. More
specifically, the present invention provides a material having a pH
of between about 3 and 6 and possessing putty-like physical
properties, wherein the composition of the material includes
collagen and water. The present invention also provides a method
for using the implantable putty material.
Inventors: |
Benedict, James J.; (Arvada,
CO) ; Damien, Christopher J.; (Denver, CO) |
Correspondence
Address: |
Gary J. Connell
SHERIDAN ROSS P.C.
Suite 1200
1560 Broadway
Denver
CO
80202-5141
US
|
Assignee: |
Centerpulse Biologics Inc.
|
Family ID: |
32106021 |
Appl. No.: |
10/739492 |
Filed: |
December 17, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10739492 |
Dec 17, 2003 |
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09023617 |
Feb 13, 1998 |
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6679918 |
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Current U.S.
Class: |
424/572 ;
424/549 |
Current CPC
Class: |
A61L 24/102 20130101;
A61L 27/24 20130101 |
Class at
Publication: |
424/572 ;
424/549 |
International
Class: |
A61K 035/32 |
Claims
What is claimed is:
1. A putty material comprising collagen and water, wherein the
putty material has a pH of between about 3.0 to about 6.0.
2. The putty material of claim 1, wherein said collagen is selected
from the group consisting of fibrillar collagen, atelopeptide
collagen, telopeptide collagen and tropocollagen.
3. The putty material of claim 1, wherein said collagen is bovine
tendon Type I collagen.
4. The putty material of claim 1, wherein the material is prepared
by a process comprising addition of an acid to said collagen.
5. The putty material of claim 4, wherein said acid is selected
from the group consisting of ascorbic acid, acetic acid, acetyl
salicylic acid, benzoic acid, citric acid, glutamic acid, glycolic
acid, lactic acid, malic acid, salicylic acid, and hydrochloric
acid.
6. The putty material of claim 4, wherein said acid is ascorbic
acid.
7. The putty material of claim 1, wherein said putty material
further comprises an active ingredient.
8. The putty material of claim 7, wherein said active ingredient is
selected from the group consisting of osteoinductive materials,
growth factors, cartilage inducing factors, angiogenic factors,
hormones, antibiotics, and antiviral compounds.
9. The putty material of claim 8, wherein said active ingredient is
an osteoinductive material.
10. The putty material of claim 9, wherein the osteoinductive
material comprises an osteoinductive protein or protein mixture
selected from the group consisting of BGP, BMP 1, BMP 2, BMP 3, BMP
4, BMP 5, BMP 6, BMP 7, BMP 8, BMP 9, BMP 10, BMP 11, BMP 12, BMP
13, and OP1.
11. The putty material of claim 10, wherein said osteoinductive
factor is BGP.
12. The putty material of claim 1, further comprising a
demineralized bone material.
13. A method for preparing a putty material comprising the step of
admixing collagen, an acid, an active ingredient and water to form
a gel.
14. The method of claim 13, further comprising the step of
sterilizing said gel.
15. The method of claim 14, wherein said step of sterilizing
comprises exposing said gel to g-radiation.
16. The method of claim 13, further comprising the step of
lyophilizing said gel.
17. The method of claim 13, further comprising the step of adding a
demineralized bone material to the gel to produce an osteogenic
putty, wherein said osteogenic putty has a pH of between about 3.0
to about 6.0.
18. The method of claim 17, further comprising sterilizing said
lyophilized gel by contacting it with ethylene oxide.
19. The method of claim 13, wherein said collagen is selected from
the group consisting of fibrillar collagen, atelopeptide collagen,
telopeptide collagen and tropocollagen.
20. The method of claim 19, wherein said collagen is bovine tendon
Type I collagen.
21. The method of claim 13, wherein said acid is selected from the
group consisting of ascorbic acid, acetic acid, acetyl salicylic
acid, benzoic acid, citric acid, glutamic acid, glycolic acid,
lactic acid, malic acid, salicylic acid, and hydrochloric acid.
22. The method of claim 13, wherein said acid is ascorbic acid.
23. The method of claim 13, wherein said step of admixing further
comprises admixing an osteoinductive material.
24. The method of claim 23, wherein said osteoinductive material
comprises an osteoinductive protein or protein mixture selected
from the group consisting of BGP, BMP 1, BMP 2, BMP 3, BMP 4, BMP
5, BMP 6, BMP 7, BMP 8, BMP 9, BMP 10, BMP 11, BMP 12, BMP 13, and
OP1.
25. The method of claim 24, wherein said osteoinductive material is
BGP.
26. The method of claim 13, wherein the active ingredient comprises
a protein or protein mixture selected from the group consisting of
BGP, BMP 1, BMP 2, BMP 3, BMP 4, BMP 5, BMP 6, BMP 7, BMP 8, BMP 9,
BMP 10, BMP 1, BMP 12, BMP 13, OP1, bFGF, and TGF-beta.
27. A process for making a dry osteoinductive composition
comprising the steps of: admixing collagen, an acid, an
osteoinductive material and water to form a gel; and lyophilizing
said gel.
28. The process of claim 27, wherein said acid comprises from about
0.05 mmol of said acid per 100 mg of said collagen to about 2.3
mmol of said acid per 100 mg of said collagen.
29. The process of claim 27, wherein said acid is selected from the
group consisting of ascorbic acid, acetic acid, acetyl salicylic
acid, benzoic acid, citric acid, glutamic acid, glycolic acid,
lactic acid, malic acid, salicylic acid, and hydrochloric acid.
30. The process of claim 27, wherein said acid is ascorbic
acid.
31. The process of claim 27, wherein said collagen comprises from
about 1.0% by weight to about 10.0% by weight of said dry
osteoinductive composition.
32. The process of claim 27, wherein said collagen is selected from
the group consisting of fibrillar collagen, atelopeptide collagen,
telopeptide collagen and tropocollagen.
33. The process of claim 27, wherein said collagen is bovine tendon
Type I collagen.
34. The process of claim 27, wherein said osteoinductive material
comprises from about 0.1% by weight to about 10.0% by weight of
said dry osteoinductive composition.
35. The process of claim 27, wherein said osteoinductive material
comprises an osteoinductive protein or protein mixture selected
from the group consisting of BGP, BMP 1, BMP 2, BMP 3, BMP 4, BMP
5, BMP 6, BMP 7, BMP 8, BMP 9, BMP 10, BMP 11, BMP 12, BMP 13, and
OP1.
36. The process of claim 27, wherein said osteoinductive material
is BGP.
37. A method for administering an active compound to a patient
comprising the steps of: preparing a delivery vehicle by admixing
collagen and an acid to form a composition having a pH of between
about 3.0 and about 6.0; incorporating an active compound into said
delivery vehicle; and implanting said delivery vehicle in a desired
portion of the patient's body.
38. The method of claim 37, wherein said step of implantation
comprise a surgery.
39. The method of claim 37, wherein said step of implantation
comprises an injection.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 09/023,617, filed Feb. 13, 1998, which is incorporated
herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to an implantable putty
material for delivery of active compounds to a patient.
BACKGROUND OF THE INVENTION
[0003] A wide variety of implantable materials have been used in
the delivery of active compounds to a patient. For example, such
materials can be used in the repair of bone defects. Typically,
these materials are implanted at a desired site to promote bone
growth. Ideally, such a material should have the ability to adhere
and conform to the implanted site and facilitate bone growth.
[0004] U.S. Pat. Nos. 5,314,476 and 5,073,373 disclose a
deformable, shape-sustaining osteogenic composition comprising
demineralized bone particles and a polyhydroxy compound such as
glycerol, or an oligosaccharide.
[0005] U.S. Pat. Nos. 5,405,390 and 5,236,456 disclose a
surface-adherent osteogenic composition derived from demineralized
and thermally modified bone tissue. The composition is administered
in the form of a powder, a viscous liquid, or by direct
injection.
[0006] U.S. Pat. No. 5,246,457 discloses a bone-repair composition
comprising a calcium phosphate salt and reconstituted fibrillar
atelopeptide collagen. It does not include any biologically active
ingredients. The physical and handling properties are improved by a
number of curing processes, including heat, maturation of the wet
mixture and/specific cross-linking of collagen.
[0007] U.S. Pat. No. 4,440,750 discloses an osteogenic composition
comprising demineralized bone powder and reconstituted native
atelopeptide collagen fibers in a continuous aqueous phase having a
substantially physiologic pH and ionic strength.
[0008] U.S. Pat. No. 4,975,526 discloses a matrix material
comprising protein-extracted demineralized bone powder and a
swelling agent to increase the intraparticle porosity of the
matrix.
[0009] U.S. Pat. No. 4,394,370 discloses a bone graft material for
treating osseous defects. The material comprises collagen and
demineralized bone particles and is sponge-like.
[0010] Currently known implantable materials, including those
discussed above, are lacking in acceptable texture properties, such
as cohesiveness, elasticity and the ability to be molded to a
selected shape. Moreover, other paste-like materials such as those
disclosed in U.S. Pat. Nos. 5,314,476 and 5,073,373 require an
organic solvent such as glycerol, as discussed above.
[0011] Therefore, there is a need for an osteoinductive material
which have an improved handling properties and which does not
require an organic solvent.
SUMMARY OF THE INVENTION
[0012] One embodiment of the present invention is a putty material
which includes collagen and water, wherein the putty material has a
pH of between about 3.0 to about 6.0. Materials of the invention
have excellent physical properties and handling characteristics.
The collagen of the putty material can be selected from the group
consisting of fibrillar collagen, atelopeptide collagen,
telopeptide collagen and tropocollagen. The putty material can be
formed by the addition of an acid selected from the group
consisting of ascorbic acid, acetic acid, acetyl salicylic acid,
benzoic acid, citric acid, glutamic acid, glycolic acid, lactic
acid, malic acid, salicylic acid, and hydrochloric acid. The putty
material can also include an active ingredient, such as an active
ingredient selected from the group consisting of osteoinductive
materials, growth factors, cartilage inducing factors, angiogenic
factors, hormones, antibiotics, and antiviral compounds.
[0013] Another embodiment of the present invention is an osteogenic
composition which includes collagen, an osteoinductive material,
and an acid, wherein the osteogenic composition includes between
about 0.05 mmol of acid per 100 mg of the collagen to about 2.3
mmol of acid per 100 mg of the collagen.
[0014] Another embodiment of the present invention is an osteogenic
composition which includes bovine tendon Type I collagen, ascorbic
acid, water, bone growth protein and a demineralized bone
material.
[0015] Another embodiment of the present invention is a composition
produced from a process including the steps of admixing collagen,
an acid, and water to form a gel; and adding a demineralized bone
material to said gel to produce an osteogenic putty, wherein the
osteogenic putty has a pH of about 6.0 or less.
[0016] Another embodiment of the present invention is a process for
making a dry osteoinductive composition comprising the steps of
admixing collagen, an acid, an osteoinductive material and water to
form a gel; and lyophilizing said gel.
[0017] Another embodiment of the present invention is a method for
administering an active compound to a patient comprising the steps
of preparing a delivery vehicle by admixing collagen and an acid to
form a composition having a pH of between about 3.0 and about 6.0,
incorporating an active compound into the delivery vehicle and
implanting the delivery vehicle in a desired portion of the
patient's body.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 illustrates volatility of ascorbic acid from an
implantable material during lyophilization process.
[0019] FIG. 2 illustrates volatility of malic acid from an
implantable material during lyophilization process.
[0020] FIG. 3 illustrates volatility of acetic acid from an
implantable material during lyophilization process.
[0021] FIG. 4 illustrates volatility of lactic acid from an
implantable material during lyophilization process.
[0022] FIG. 5 illustrates volatility of glycolic acid from an
implantable material during lyophilization process.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention is directed to a material composition
which includes collagen and water. The material of the present
invention has a putty consistency and can be molded to a desirable
shape. The present invention is also directed to a process for
implanting the material in the body for the purpose of stimulating
or causing a biological response or activity such as inducing bone
formation. Particularly, the material of the present invention is
suitable for implanting in humans and animals with an osseous
defect to induce the regeneration of osseous tissue to correct the
defect.
[0024] The collagen component of the present invention is
preferably fibrillar collagen, atelopeptide collagen, telopeptide
collagen or tropocollagen and can be collected from a variety of
mammalian sources. Methods for preparing atelopeptide collagen and
tropocollagen are described by Glowacki et al., U.S. Pat. No.
4,440,750, which is incorporated herein in its entirety.
Preferably, the collagen is a mammalian collagen. More preferably,
the collagen is selected from the group consisting of bovine Type I
collagen, and porcine Type I collagen, and most preferably from the
group consisting of purified fibrillar bovine tendon Type I
collagen. Preferably, the amount of collagen present in the
materials and compositions of the present invention is from about
1% by weight (not including any water that is added) to about 10%
by weight, more preferably from about 2% by weight to about 8% by
weight, and most preferably from about 3% by weight to about 5% by
weight.
[0025] Materials and compositions of the present invention have a
pH of between about 3 and about 6, more preferably between about
3.5 and about 5, and most preferably between about 3.8 and about
4.6. The pH of the material is measured by placing a flat pH
electrode on the surface of the material using Ross flat surface
electrode available from Orion Co. (Boston, Mass.). It has been
found that when the pH is within the limitations identified above,
the materials have excellent physical properties, such as a putty
consistency which is elastic and dough-like. At higher pH, the
materials become crumbly with the consistency of wet sand. A putty
consistency is desired because it provides many benefits such as
enhanced cohesiveness, ease of handling and moldability. Because
materials of the present invention are cohesive, they are also
believed to provide the benefit of maintaining an active compound
at the site of implantation longer than comparative materials with
less cohesiveness.
[0026] A desired pH of the material of the present invention can be
achieved by forming the material by adding an acid to collagen. As
used in this invention, the term "acid" refers to a compound which
has lower pKa than water, and the term "acidic proton" refers to a
proton whose pKa is lower than water. Suitable acids for use in the
present invention include organic acids, such as phenols and
carboxylic acids, and inorganic acids, such as hydrochloric acid,
phosphoric acid or sulfuric acid. Preferably, the acid is organic
acid, hydrochloric acid, or phosphoric acid. Preferably, the acid
is selected from the group consisting of acetic acid, ascorbic
acid, aspartic acid, benzoic acid, citric acid, glutamic acid,
glycolic acid, hydrochloric acid, lactic acid, malic acid,
phosphoric acid, salicylic acid, and tartaric acid. More
preferably, the acid is selected from the group consisting of
ascorbic acid (i.e., vitamin C), acetic acid, acetyl salicylic
acid, benzoic acid, citric acid, glutamic acid, glycolic acid,
lactic acid, malic acid, salicylic acid, and hydrochloric acid.
Most preferably, the acid is selected from the group consisting of
ascorbic acid, citric acid, malic acid and lactic acid.
[0027] The acid should be added in a sufficient amount to produce a
material with acceptable physical properties. Preferably, the
amount of acid present in the material is from about 0.05
equivalent mmole (eq. mmol) of acid per 100 mg of collagen to about
2.30 eq. mmol of acid per 100 mg of collagen, more preferably from
about 0.1 eq. mmol of acid per 100 mg of collagen to about 1.5 eq.
mmol of acid per 100 mg of collagen, and most preferably from about
0.2 eq. mmol of acid per 100 mg of collagen to about 1.5 eq. mmol
of acid per 100 mg of collagen. The term "equivalent mmole" refers
to the amount of acid, in mmole, divided by the number of acidic
protons present per molecule of the acid. For example, some acids
such as malic acid have two equivalent acidic protons per molecule;
therefore, the preferred amount of malic acid or any other acid
having two acidic protons per molecule of acid is one-half that of
acids having only one acidic proton. For example, 5 mmol of malic
acid and 10 mmol of acetic acid can both be expressed as 10 eq.
mmol of acid because malic acid has two acidic protons while acetic
acid has only one acidic proton.
[0028] Another way to characterize the amount of acid present in
the material is in terms of the amount of the acid per 100 mg of
collagen. Thus, for example, for a material composition including
ascorbic acid, it is preferred that from about 20 mg to about 200
mg of ascorbic acid is added per about 100 mg of collagen, more
preferably from about 26 mg to about 131 mg of ascorbic acid per
about 100 mg of collagen, and most preferably from about 65 mg to
about 131 mg of ascorbic acid per about 100 mg of collagen. It
should be appreciated that the amount of the acid will vary
depending on its molecular weight. In the event that material is
lyophilized, acid can be volatilized during lyophilization which
affects the pH and the consistency of the material when the dry
solid is reconstituted with water. Materials having, for example,
ascorbic acid or malic acid are particularly well suited for
lyophilization due to the low volatility of these acids during this
process. It is preferred that the amount of acid loss during
lyophilization process be less than about 30%, more preferably less
than about 15%, and most preferably less than about 5%.
[0029] As discussed above, materials and compositions of the
present invention have good physical properties, such as
cohesiveness and retention of shape after implantation. One measure
of such physical properties is that materials and compositions of
the present invention have a peak resistance force of at least
about 10 grams (g), preferably at least about 20 g, and more
preferably at least about 30 g. As used herein, a "peak resistance
force" (i.e., peak force) refers to a maximum force exerted by the
material when stretched to its breaking point using a TA.XT2
Texture Analyzer apparatus which is available from Texture
Technologies Corp. (Scarsdale, N.Y.) or some equivalent apparatus.
The material tested is prepared by a SMS/Kieffer molding form and
press (TA-105a Texture Technologies) or some equivalent apparatus
having a trapezoidal shape measuring 53 mm (1).times.4 mm
(h).times.4 mm (w) at one end and 2.5 mm (w) at the other end.
[0030] Another measure of such physical properties is that
materials and compositions of the present invention preferably have
an extensibility of from about 2 mm to about 25 mm, more preferably
from about 3 mm to about 25 mm, and most preferably from about 5 mm
to about 25 mm. The term "extensibility" refers to the distance a
probe that pulls the material travels until the material breaks
when using the same apparatus and the same material dimensions for
testing of peak resistance force.
[0031] Materials of the present invention can also include an
effective amount of an active ingredient. An "active ingredient"
refers to any compound or mixture of compounds that have a
biological activity. Exemplary active ingredients include
osteoinductive materials, growth factors, hormones, antibiotics,
and antiviral compounds. Osteoinductive materials are described in
detail below. Growth factors can include basic fibroblast growth
factor (bFGF) and transforming growth factor beta (TGF-beta) (See
Cuevas et al., Basic Fibroblast Growth Factor (FGF) Promotes
Cartilage Repair In Vivo, Biochem Biophys Res Commun
156:611-618,1988). These growth factors have been implicated as
cartilage stimulating and angiogenic agents. bFGF, for example, has
been shown to increase the rate of osteoblast replication while
simultaneously inhibiting their activity (Frenkel S, Singh I J; The
effects of fibroblast growth factor on osteogenesis in the chick
embryo. In: Fundamentals of bone growth: Methodology and
applications. Ed. A D Dixon, B G Samat, D. Hoyte, CRC Press, Boca
Raton, Fla., USA, pp. 245-259, 1990). This effect is dose
dependent, with higher and lower doses causing decreased activity
and middle range doses stimulating activity (Aspenberg P, Thorngren
K G, Lohmander L S; Dose-dependent stimulation of bone induction by
basic fibroblast growth factor in rats. Acta Orthop Scand
62:481-484, 1991).
[0032] The term "effective amount" refers to an amount of an active
ingredient sufficient to achieve a desired affect without causing
an undesirable side effect. In some cases, it may be necessary to
achieve a balance between obtaining a desired effect and limiting
the severity of an undesired effect. It will be appreciated that
the amount of active ingredient used will vary depending upon the
type of active ingredient and the intended use of the composition
of the present invention. When the material of the present
invention includes an osteogenic material, the amount of
osteoinductive material is preferably between about 0.1% by weight
and about 10% by weight of the total weight of the putty material,
more preferably between about 0.25% by weight and about 4% by
weight, and most preferably between about 0.35% by weight and about
1.6% by weight.
[0033] An "osteoinductive material" refers to any material that is
capable of inducing bone formation (i.e., a material having
osteogenic properties) when implanted in a body and includes
demineralized bone matrix and osteoinductive factors. An
"osteoinductive factor" refers to a natural, recombinant or
synthetic protein or mixture of proteins which are capable of
inducing bone formation. For example, the term osteoinductive
factor refers to the materials described as bone growth factors in
Damien et al., U.S. Pat. No. 5,563,124. It should be noted that
while most contemplated applications of the present invention are
concerned with use in humans, the products and processes of the
present invention work in animals as well. Induction of bone
formation can be determined by a histological evaluation showing
the de novo formation of bone with accompanying osteoblasts,
osteoclasts, and osteoid matrix. For example, osteoinductive
activity of an osteoinductive factor can be demonstrated by a test
using a substrate onto which material to be tested is deposited. A
substrate with deposited material is implanted subcutaneously in a
test animal. The implant is subsequently removed and examined
microscopically for the presence of bone formation including the
presence of osteoblasts, osteoclasts, and osteoid matrix. A
suitable procedure is illustrated in Example 5 of U.S. Pat. No.
5,290,763.
[0034] No generally accepted scale for evaluating the degree of
osteogenic activity exists, however, certain factors are widely
recognized as indicating bone formation. Such factors are
referenced in the scale of 0-8 which is provided in Table 3 of
Example 1 of U.S. Pat. No. 5,563,124. The 0-4 portion of this scale
corresponds to the scoring system described in U.S. Pat. No.
5,290,763, which is limited to scores of 0-4. The remaining portion
of the scale, scores 5-8, references additional levels of
maturation of bone formation. The expanded scale also includes
consideration of resorption of collagen, a factor which is not
described in U.S. Pat. No. 5,290,763.
[0035] Suitable osteoinductive factors of the present invention can
be produced by purification of naturally occurring proteins from
bone or by recombinant DNA techniques. As used herein, the term
recombinantly produced osteoinductive factors refers to the
production of osteoinductive factors using recombinant DNA
technology. For example, nucleic acids encoding proteins having
osteogenic activity can be identified by producing antibodies that
bind to the proteins. The antibodies can be used to isolate, by
affinity chromatography, purified populations of a particular
osteogenic protein. The amino acid sequence can be identified by
sequencing the purified protein. It is possible to synthesize DNA
oligonucleotides from the known amino acid sequence. The
oligonucleotides can be used to screen either a genomic DNA and/or
cDNA library made from, for example bovine DNA, to identify nucleic
acids encoding the osteogenic protein. The correct oligonucleotide
will hybridize to the appropriate cDNA thereby identifying the cDNA
encoding the osteogenic protein encoding gene.
[0036] Antibodies that bind osteogenic proteins can also be used
directly to screen a cDNA expression library. For example,
eukaryotic cDNA sequences encoding osteogenic proteins can be
ligated into bacterial expression vectors. The expression vectors
can be transformed into bacteria, such as E. coli, which express
the transformed expression vector and produce the osteogenic
protein. The transformed bacteria can be screened for expression of
the osteogenic protein by lysing the bacteria and contacting the
bacteria with radioactively-labelled antibody.
[0037] Recombinant osteoinductive factor can be produced by
transfecting genes identified according to the method described
above into cells using any process by which nucleic acids are
inserted into cells. After transfection, the cell can produce
recombinant osteoinductive factors by expression of the transfected
nucleic acids and such osteoinductive factors can be recovered from
the cells.
[0038] A number of naturally occurring proteins from bone or
recombinant osteoinductive factors have been described in the
literature and are suitable for the present invention.
Recombinantly produced osteoinductive factors have been produced by
several entities. Creative Biomolecules of Hopkinton, Mass., USA
produces a osteoinductive factor referred to as Osteogenic Protein
1 or OP1. Genetics Institute of Cambridge, Mass., USA produces a
series of osteoinductive factors referred to as Bone Morphogenetic
Proteins 1-13 (i.e., BMP 1-13), some of which are described in U.S.
Pat. Nos. 5,106,748 and 5,658,882 and in PCT Publication No. WO
96/39,170. Purified osteoinductive factors have been developed by
several entities. Collagen Corporation of Palo Alto, Calif., USA
developed a purified protein mixture which is believed to have
osteogenic activity and which is described in U.S. Pat. Nos.
4,774,228; 4,774,322; 4,810,691; and 4,843,063. Marshall Urist of
the University of California developed a purified protein mixture
which is believed to be osteogenic and which is described in U.S.
Pat. Nos. 4,455,256; 4,619,989; 4,761,471; 4,789,732; and
4,795,804. International Genetic Engineering, Inc. of Santa Monica,
Calif., USA developed a purified protein mixture which is believed
to be osteogenic and which is described in U.S. Pat. No. 4,804,744.
All of the foregoing patents are incorporated herein by
reference.
[0039] A preferred osteoinductive factor of the present invention
and process for making the same is described in detail in related
U.S. Pat. No. 5,290,763. This osteoinductive factor is particularly
preferred because of its high osteogenic activity and because it is
a purified osteoinductive factor. The osteoinductive factor of U.S.
Pat. No. 5,290,763 exhibits osteoinductive activity at about 3
micrograms when deposited onto a suitable carrier and implanted
subcutaneously into a rat. In one embodiment, the osteoinductive
factor is an osteoinductively active mixture of proteins which
exhibit the gel separation profile shown in FIG. 1 of U.S. Pat. No.
5,563,124. This gel separation profile was obtained using SDS-PAGE.
The first column is a molecular weight scale which was obtained by
performing SDS-PAGE on standards of known molecular weight. The
second column illustrates the SDS-PAGE profile for a mixture of
proteins in accordance with the present invention which have been
reduced with 2-mercaptoethanol. The third column illustrates the
SDS-PAGE profile for a non-reduced mixture of proteins in
accordance with the present invention. Although the mixture of
proteins which provide the SDS-PAGE profile illustrated therein
have been found to have high osteoinductive activity, it is
anticipated that mixtures of proteins having SDS-PAGE profiles
which differ slightly from that illustrated therein will also be
effective. For example, effective protein mixtures can include
proteins that differ in molecular weight by plus or minus 5 KD from
those shown therein, and can include fewer or greater numbers of
proteins than those shown. Therefore, mixtures of proteins having
profiles which comprise substantially all of the protein bands
detected in the reduced or nonreduced SDS-PAGE profiles therein
will be considered to be within the scope of the invention.
[0040] Yet another embodiment of the preferred osteoinductive
factor of the invention includes an osteoinductively active mixture
of proteins having, upon hydrolysis, an amino acid composition of
from about 20.7 to about 26.1 mole percent acidic amino acids,
about 11.3 to about 15.7 mole percent hydroxy amino acids, about
37.6 to about 42.4 mole percent aliphatic amino acids, about 5.8 to
about 7.9 mole percent aromatic amino acids and about 13.3 to about
19.9 mole percent basic amino acids. More particularly, the
preferred osteoinductive factor has an amino acid composition of
about 20.7 to about 26.1 (preferably about 23.4) mole percent of
ASP (+ASN) and GLU (+GLN); about 11.3 to about 15.7 (preferably
about 13.5) mole percent SER and THR; about 37.6 to about 42.4
(preferably about 40.0) mole percent ALA, GLY, PRO, VAL, MET, ILE,
and LEU; about 5.8 to about 7.9 (preferably about 6.8) mole percent
TYR and PHE; and about 13.3 to about 19.9 (preferably about 16.6)
mole percent HIS, ARG, and LYS. A further embodiment of the
preferred osteoinductive factor is a protein mixture having the
approximate amino acid composition shown in Table 1.
1 TABLE 1 Amino Acid Mole Percent Asp 11.14 Glu 12.25 Ser 9.48 Gly
8.50 His 2.28 Arg 7.19 Thr 4.03 Ala 8.05 Pro 7.16 Tyr 3.63 Val 3.79
Met 1.73 Ile 2.75 Leu 8.00 Phe 3.21 Lys 7.11
[0041] A still further embodiment of the preferred osteoinductive
factor is a protein mixture obtained by any of the purification
processes described in U.S. Pat. No. 5,290,763.
[0042] Materials of the present invention are typically derived by
admixing collagen, water and an acid. As discussed above, the
material can also include other substances such as an active
ingredient. The material can also be sterilized by dialysis,
irradiation (e.g. using g-radiation), filtration, chemical
treatment (e.g., using ethylene oxide), or other known
sterilization methods. Alternatively, the material which can be a
gel is lyophilized to a dry solid before being sterilized. When
sterilizing the material using a chemical treatment, it is
preferred that the material be lyophilized to a dry solid prior to
being sterilized. Lyophilization removes water and prevents any
chemical reaction which may occur between the chemical used for
sterilization (e.g., ethylene oxide) and water. Another alternative
method is to make the material of the present invention in an
aseptic environment, thereby eliminating the need for a separate
sterilization step.
[0043] Materials of the present invention can also include
demineralized bone material. A method for preparing demineralized
bone material in a particulate form is described by Glowacki et
al., U.S. Pat. No. 4,440,750. Alternatively demineralized bone
material can be prepared by grinding a bone, demineralizing it with
0.6 M HCl solution, washing with a phosphate buffered solution,
washing with ethanol and drying it. Demineralized bone material can
also be obtained from a commercial bone or tissue bank, for
example, from AlloSource (Denver, Colo.).
[0044] Materials of the present invention can be part of a kit
containing the components of the materials. Such kits are
particularly useful for health care professionals in preparing the
materials and compositions of the present invention immediately
before use. Such kits, in addition to including the component parts
of the various materials and compositions of the invention also
include one or more containers for mixing the components, along
with optional mixing devices such as stirrers. Further, such kits
can include the components in sealed, pre-measured packages. The
sealed packages can be sealed aseptically and the amounts of the
components can be pre-measured in relative amounts as described
elsewhere herein.
[0045] Another aspect of the present invention includes a process
of implanting a material or composition as broadly described above
into a body. As noted above, most uses of the present invention are
concerned with human application. The process, however, is
applicable to a wide variety of animals, particularly mammals. As
used in this invention, the term "implanting" refers to placing the
material or composition of the present invention in an area in
which it is desired to achieve the activity of the active
ingredient. In this embodiment of the present invention, the
materials function as a delivery vehicle for an active ingredient.
Such methods of implantation can involve a surgery or a simple
injection of the product using any of the known methods including a
use of syringe.
[0046] For the product of the present invention comprising an
osteogenic composition, the present material and process can be
used in a variety of application whenever there is a need to
generate bone. Such applications include induction of bone
formation for hip replacement operations, knee replacement
operations, spinal fusion procedures, repair of periodontal
defects, treatment of osteoporosis, repair of bone tumor defects,
dental procedures, repair of cranialmaxillafacial defects, and
repair of bone fractures.
[0047] In the case of hip-replacement operations, the ball and
socket joint of a hip is replaced when a person's hip is not
functioning properly. The ball portion of a joint is replaced by
surgical removal of the ball portion from the terminus of the
femur. The artificial ball portion has a functional ball end with
the opposite end being a spike which is inserted into the proximal
end of the femur from which the natural ball portion was removed.
The spike can have a porous surface so that bone growth around the
spike can anchor the spike in the femur. Materials of the present
invention can be layered or packed between the spike and the cavity
in the femur in which spike is to be inserted. The socket portion
of a joint is replaced by inserting an artificial socket into the
natural socket. The artificial socket is sized to fit with the
artificial ball. On the surface of the artificial socket which
contacts the natural socket, the artificial socket can have a
porous surface. Materials of the present invention can be placed in
the natural socket cavity so that upon placement of the artificial
socket, the material is between the natural and artificial socket.
In this manner, as bone is formed, the artificial socket is
anchored in the natural socket.
[0048] Materials of the present invention are also suitable for use
in knee replacement operations. Knee prostheses have a femoral and
a tibial component which are inserted into the distal end of the
femur and the surgically prepared end of the tibia, respectively.
Materials of the present invention can be layered or packed between
the femoral and/or tibial components of the prosthesis and the
respective portions of the femur and tibia. In this manner, as bone
formation is induced between the prosthesis and the bones, the
prosthesis becomes anchored.
[0049] Materials of the present invention are also suitable for use
in spinal fusion operations in which it is desired to substantially
immobilize two vertebrae with respect to each other. The material
can be applied, for example, between adjacent spinous and
transverse processes so that upon bone formation throughout the
material, two adjacent vertebrae are joined by fusion between the
respective spinous processes and transverse processes.
[0050] Materials of the present invention can also be used in
spinal fusion operations in which it is desired to substantially
immobilize two vertebrae with respect to each other by using metal
cages or equivalent implants. In this case, the cages are placed in
the disk space between two vertebral bodies, and the material of
the present invention is packed into and around the cages to obtain
bone formation through and around the cages thus fusing two
vertebrae and stabilizing the spine.
EXAMPLES
[0051] Unless otherwise stated, following general procedures were
used throughout the Examples.
[0052] Bovine demineralized bone material was prepared by grinding
bovine bone to a particle size of about 125 .mu.m to about 850
.mu.m. It was demineralized in 0.6 M HCl, washed with phosphate
buffered solution, rinsed with ethanol and dried.
[0053] The putty materials were tested using TA-XT2 Texture
Analyzer having following testing parameters: pre-test speed=2.0
mm/sec, test speed=3.3 mm/sec, post test speed=10.0 mm/s,
distance=30 mm, trigger force=3 g.
[0054] The pH of putty materials were measured using Ross flat
surface electrode available from Orion Co. (Boston, Mass.).
Example 1
[0055] This example illustrates the effect of acid on the
consistency of the material produced when combined with collagen
and demineralized bone material.
[0056] For each acid and molarity concentration tested, a gel was
prepared by mixing 100 mg of purified bovine tendon Type I collagen
and 7.4 mL of aqueous acid solution. The gel was lyophilized and
then mixed with water and about 2.1 g to about 2.4 g of
demineralized bone material. The resulting composition was
qualitatively evaluated for its physical properties by manual
examination for properties such as cohesiveness, elasticity and
moldability. Each gel was graded as having acceptable physical
properties or not. Some acids, such as ascorbic acid and benzoic
acid, showed a wide range of useful concentrations in producing a
composition having acceptable physical properties, while others
such as acetic acid and lactic acid showed a narrow range of amount
which is suitable for producing a composition with putty
consistency. If a gel was found to initially have acceptable
physical properties, it was then subjected to ethylene oxide
sterilization and re-evaluated. Table 2 summarizes the qualitative
evaluation of the compositions tested.
2TABLE 2 Post ethylene oxide Acceptable Acceptable mM Avg. pH
Physical Physical Acid (mequiv.) (w/DBM) Properties Properties
Acetic Acid 6.67 (0.007) 5.95 N N/A " 33.3 (0.037) 4.97 N N/A "
50.0 (0.056) N/R N N/A " 66.6 (0.074) 4.85 N N/A " 100 (0.111) N/R
Y N " 167 (0.185) 4.51 Y N " 333 (0.370) 4.09 Y N/A Ascorbic Acid
10 (0.074) N/R N N " 20 (0.148) N/R Y/N N " 30 (0.222) 4.33 Y Y "
50 (0.370) 4.10 Y Y " 100 (0.740) N/R Y Y " 50 (0.370) 4.46 Y N/A "
50 (0.370) 4.64 N N/A " 50 (0.370) N/R N N/A " 50 (0.370) N/R N N/A
Aspartic Acid 30 (0.444) N/R N N/A Benzoic Acid 10 (0.074) N/R N N
" 25 (0.185) N/R Y N Citric Acid 10 (0.148) N/R N " 20 (0.298) N/R
N N/A " 50 (0.740) N/R Y N " 100 (1.480) 3.58 Y N " 100 (1.480)
3.77 Y N/A " 100 (1.480) 3.95 Y/N N/A " 100 (1.480) 4.05 N N/A
Glutamic Acid 10 (0.148) N/R N N/A " 100 (1.480) N/R N N/A Glycolic
Acid 100 (0.659) N/R Y N Hydrochloric 10 (0.074) 5.76 Y/N N Acid "
100 (0.740) N/R N N/A Lactic Acid 1.9 (0.010) 6.50 N N/A " 9.4
(0.051) 6.17 N N/A " 10 (0.054) N/R N N/A " 24 (0.130) 5.18 Y/N N/A
" 30 (0.162) 4.09 Y N/A " 47 (0.254) 4.48 Y N/A " 60 (0.324) 3.94 Y
N " 100 (0.540) N/R N N/A " 100 (0.540) 4.38 N N/A " 100 (0.540)
4.55 N N/A " 100 (0.540) 4.85 N N/A " 100 (0.540) 5.17 N N/A " 100
(0.540) 5.56 N N/A " 188 (1.016) 3.75 Y/N N/A Malic Acid 10 (0.148)
N/R N N/A " 30 (0.444) 4.00 Y N/A " 50 (0.740) 3.77 Y N " 100
(1.48) N/R Y N/A Phosphoric Acid 5.4 (0.80) 6.11 N N/A " 10.8
(0.160) 5.91 N N/A " 50 (0.740) N/R Y N/A " 100 (1.480) N/R N N/A
Salicylic Acid 10 (0.074) N/R Y N Tartaric Acid 100 (1.480) N/R Y
N/A N/R = not recorded, N/A = not applicable (not tested) and Y/N =
marginally acceptable results.
Example 2
[0057] This example illustrates the amount of acid loss during
lyophilization of compositions and the physical properties of the
resulting material before and after ethylene oxide
sterilization.
[0058] A control solution of 500 .mu.L of a 1 M solution of the
acids tested in 100 mL of water was titrated with 1 N NaOH
solution. To test for the amount of acid loss during
lyophilization, samples were prepared using the procedure of
Example 1, except that no demineralized bone material was added
after lyophilization. The resulting material was dissolved in water
(at a ratio of 1 g of lyophilized material/100 mL), and titrated
with 1 N NaOH solution. The materials, except the one made with
acetic acid were subsequently treated with 2-propanol and titrated
as follows. The lyophilized samples of collagen/acid mixture were
chopped in 2-propanol and relyophilized to free much of the acid
which was then removed by the second lyophilization as evidenced
when the samples were titrated after this treatment. The results of
titrations curves for materials made with ascorbic acid, malic
acid, acetic acid, lactic acid and glycolic acid are shown in FIGS.
1-5, respectively. The amount of acid loss and the amount of acid
remaining in the samples are shown on Table 3.
3TABLE 3 % Acid mg Acid in wt % Acid mM loss sample of acid in
sample Acetic Acid 50 85 3.33 3.2 Ascorbic Acid 10 0 13.03 11.5 "
20 0 26.07 20.7 " 50 0 65.17 39.5 " 100 0 130.34 56.6 Citric Acid
10 0 14.22 12.4 " 20 0 28.43 22.1 " 50 0 71.08 41.5 " 100 0 142.17
58.7 Hydrochloric Acid 10 ? 2.70 2.6 Lactic Acid 50 27 24.33 19.6 "
100 27 48.66 32.7 Glycolic Acid 50 11 28.14 22.0 " 100 11 56.28
36.0 Malic Acid 50 0 49.61 33.2 " 100 0 99.23 49.8
[0059] These results indicate that ascorbic acid, citric acid and
malic acid were of relatively low volatility and acetic acid was
found to be the most volatile.
Example 3
[0060] This example illustrates bioactivity of the implantable
materials having various acids.
[0061] A gel was prepared by mixing 100 mg of collagen, 7.4 mL of
aqueous acid solution of a given molarity, and a given amount of
bone growth protein (BGP) as described in U.S. Pat. No. 5,290,763.
The gel was lyophilized and some of the lyophilized gels were
sterilized by contacting with ethylene oxide. About 15 mg of
lyophilized gel is mixed with 1.14 mL of water and 173 mg of rat
demineralized bone material. The resulting material was placed in a
mold and 12 to 15 disks of 7 mm diameter and 2 mm thickness was
formed. The disks were frozen, lyophilized overnight, and implanted
subcutaneously in rats. The animals were sacrificed after 28 days
and histological slides were made of the explanted tissues. Acid
fuchsin and Sanderson's Rapid Bone Stain or toluidine blue were
used to stain the explanted tissue slides to facilitate viewing of
bone and cartilage formation. The type and molarity of each acid
tested, the amount of BGP added, and whether the gel was exposed to
ethylene oxide are set forth in Table 4.
4TABLE 4 Acid mM BGP (.mu.g) Ethylene oxide exposure? H.sub.2O
(control) -- 20 Y Ascorbic Acid 10 20 Y " 20 20 Y " 30 20 Y " 50 0
N " 50 10 N " 50 20 Y " 50 3.5 N " 50 35 N " 50 8 N " 100 20 Y
Benzoic Acid 25 20 Y Citric Acid 50 20 Y Glycolic Acid 50 10 N " 50
20 Y Lactic Acid 50 20 Y " 100 10 N Malic Acid 50 10 N " 50 20
Y
[0062] Materials made with lactic acid at 50 mM and 100 mM, and
ascorbic acid at 10 mM, 20 mM, 30 mM, 50 mM and 100 mM showed good
complete ossicle formation. Bone was seen throughout the explanted
tissue. There was generally a mature rim of bone and an occasional
pocket of soft tissue. Results of materials made with malic acid at
50 mM, glycolic acid at 50 mM, citric acid at 50 mM and benzoic
acid at 25 mM varied from sparse islands to full ossicles. The
sensitivity of the osseoinductive response to BGP does was
evaluated with ascorbic acid at 50 mM. The results showed no bone
formation at 0 BGP, and at 35 .mu.g, significantly larger amounts
of bone growth occurred. The results also indicate that, after
ethylene oxide exposure, the osseoinductive response appears to be
inversely related to the molarity of the acid. Good bone formation
was observed where ascorbic acid at 30 mM and 50 mM was added to a
mixture of collagen and BGP that had not been exposed to ethylene
oxide, even where the ascorbic acid had itself been exposed
separately to ethylene oxide. Good bone formation was observed in
samples made using 10 mM and 20 mM ascorbic acid, regardless of
whether it was exposed to ethylene oxide or not.
Example 4
[0063] This example illustrates the effect of adding an acidic
buffer solution to a lyophilized collagen material just prior to
addition of the demineralized bone matrix.
[0064] Instead of preparing the collagen with the acid solution and
then lyophilizing the material as described in Experiment 1, a
sample of collagen was prepared with a dilute, volatile acid (e.g.,
acetic acid) and lyophilized. This material when reconstituted with
water resulted in subjectively poor handling properties and could
not be tested objectively using the methods described in the
following examples. When the same material was reconstituted by
adding an acid buffer solution instead of water, the resulting
material was in a putty form that was both subjectively adequate
and was testable using the experimental procedures described in the
following examples. A variety of acid buffer solutions, including
ascorbic acid at 20, 30, 50 and 100 mM, citric acid at 50 mM, malic
acid at 50 mM, and lactic acid at 50 mM, can be used as an acid
buffer solution in reconstituting the lyophilized collagen to
obtain a material having a desired physical properties.
Example 5
[0065] This example illustrates effect of different acids in
extensibility and peak resistance force (peak force) of a
material.
[0066] Gels were prepared and lyophilized using the procedure of
Example 4. Lyophilized gels were refrigerated until use.
[0067] Samples for physical property testing were prepared by
adding 6 mL of water to the lyophilized gel. About 1.75 g of bovine
demineralized bone material having a particle size of from 125
.mu.m to about 850 .mu.m was added, mixed and allowed to stand for
about 5 minutes, unless otherwise noted. The putty was then placed
in an SMS/Kieffer molding form and press (TA-105a Texture
Technologies). This produced test specimens having a trapozoidal
shape measuring 53 mm (l).times.4 mm (h).times.4 mm (w) at one end
and 2.5 mm (w) at the other end. Table 5 shows a peak force and a
distance to peak force measured using TA-XT2 texture analyzer with
test rate (i.e., rate of probe travel) of 2.0 mm/sec and trigger
force of 5.0 g for putty materials having a various acid
solutions.
5 TABLE 5 Acid Distance (mm) Peak Force (g) 50 mM Ascorbic Acid
-6.75 .+-. 0.48 58.21 .+-. 5.11 50 mM Acetic Acid -0.93 .+-. 0.07
6.65 .+-. 0.23 50 mM Citric Acid -10.83 .+-. 0.67 39.72 .+-. 5.41
10 mM HCl -4.25 .+-. 0.16 20.24 .+-. 2.54 50 mM Lactic Acid -7.50
.+-. 0.35 49.28 .+-. 4.45
[0068] Without being bound by a theory, the poor result of 50 mM
acetic acid sample is believed to be due to the volatility of
acetic acid during lyophilization process where about 85% of the
acetic acid is lost.
Example 6
[0069] This example illustrates the effect of different acid
concentration on the physical property of a putty material.
[0070] Samples using solution of a various concentrations of
ascorbic acid and citric acid were prepared and tested using the
procedure of Example 6. The results are shown in Table 5. In
general, as the concentration of acid increases the extensibility
of the putty material increases.
6TABLE 6 Acid pH Distance (mm) Peak Force (g) 10 mM Ascorbic Acid
5.7 -1.74 .+-. 0.49 38.84 .+-. 20.69 20 mM Ascorbic Acid 5.0 -3.58
.+-. 0.18 80.31 .+-. 24.65 50 mM Ascorbic Acid 4.6 -6.21 .+-. 0.34
47.55 .+-. 12.09 100 mM Ascorbic Acid 4.1 -6.96 .+-. 0.81 40.28
.+-. 8.82 10 mM Citric Acid 5.0 -3.25 .+-. 1.37 26.39 .+-. 11.15 20
mM Citric Acid 4.6 -4.78 .+-. 0.46 71.49 .+-. 15.98 50 mM Citric
Acid 4.1 -11.74 .+-. 0.89 88.71 .+-. 11.19 100 mM Citric Acid 3.3
-11.55 .+-. 0.76 42.41 .+-. 3.36
Example 7
[0071] This example illustrates the effect of preparation time on
the pH, distance to peak force and peak force of a putty
material.
[0072] Samples using 50 mM ascorbic acid, 50 mM citric acid and 100
mM citric acid solutions were prepared and tested using the
procedure of Example 5. Stand times for the putty materials were
varied at 2, 5, 10 and 20 minutes. The results are shown on Table
7.
7TABLE 7 Acid time (min) Distance (mm) Peak Force (g) 50 mM
Ascorbic Acid 2 -11.75 .+-. 0.43 54.51 .+-. 4.82 " 5 -12.87 .+-.
2.30 56.90 .+-. 6.25 " 10 -10.53 .+-. 0.52 74.21 .+-. 7.24 " 20
-8.40 .+-. 0.80 58.11 .+-. 7.82 50 mM Citric Acid 2 -17.56 .+-.
1.82 35.30 .+-. 2.34 " 5 -18.56 .+-. 3.17 49.49 .+-. 5.12 " 10
-16.66 .+-. 1.50 54.74 .+-. 6.42 " 20 -18.32 .+-. 1.24 55.13 .+-.
8.53 100 mM Citric Acid 2 -11.32 .+-. 0.62 21.61 .+-. 4.13 " 5
-10.54 .+-. 0.32 19.32 .+-. 1.58 " 10 -10.79 .+-. 0.66 25.87 .+-.
3.90 " 20 -14.42 .+-. 1.10 31.84 .+-. 4.26
[0073] Distance to peak force are similar for putty materials
having a stand time of 2, 5 and 10 minutes. More importantly, the
pH of the putty materials changed slightly with time as shown on
Table 8.
8TABLE 8 pH of Putty Materials over Time Acid (amount of acid) 2
min 5 min 10 min 20 min 50 mM Ascorbic Acid (65.2 mg) 4.4 4.3 4.5
4.6 50 mM Citric Acid (71.1 mg) 3.8 3.9 4.0 4.0 100 mM Citric Acid
(142.2 mg) 3.4 3.3 3.4 3.5
Example 8
[0074] This example illustrates the effect of ethylene oxide
sterilization on the physical properties of putty materials.
[0075] Samples using 20 mM and 50 mM ascorbic acid, and 50 mM and
100 mM citric acid solutions were prepared using the procedure of
Example 5 and sterilized with ethylene oxide by MMC (Erie, Pa.). A
comparative distance to peak force and peak force are shown on
Table 9.
9TABLE 9 Effect of Ethylene Oxide Sterilization Acid Sterilized? pH
Distance (mm) Peak Force (g) 20 mM Ascorbic Acid Y 5.0 -4.30 .+-.
0.34 8.22 .+-. 1.53 " N 4.9 -5.11 .+-. 1.26 13.57 .+-. 4.13 50 mM
Ascorbic Acid Y 4.4 -9.30 .+-. 1.13 18.49 .+-. 1.68 " N 4.3 -12.54
.+-. 1.32 56.17 .+-. 7.91 50 mM Citric Acid Y 4.1 0 <3 " N 3.8
-16.24 .+-. 1.47 50.98 .+-. 4.12 100 mM Citric Acid Y -- 0 <3 "
N 3.4 -11.99 .+-. 0.67 25.17 .+-. 1.01
Example 9
[0076] This example illustrates the changes in physical properties
of putty materials due to sterilization by exposure to
g-radiation.
[0077] Samples using 20 mM, 50 mM and 100 mM ascorbic acid
solutions were prepared using the procedure of Example 5 and
irradiated with 1.0 MRad of g-radiation by Sterigenics (Charlotte,
N.C.). A comparative distance to peak force and peak force are
shown on Table 10.
10TABLE 10 Effect of g-radiation Acid Sterilized? pH Distance (mm)
Peak Force (g) 20 mM Ascorbic Y 5.2 0 <3 Acid " N 5.0 -2.94 .+-.
1.22 5.83 .+-. 1.2 50 mM Ascorbic Y 4.6 -6.53 .+-. 0.71 27.52 .+-.
3.03 Acid N 4.4 -7.48 .+-. 1.05 38.81 .+-. 5.80 100 mM Ascorbic Y
4.1 -9.02 .+-. 0.16 18.80 .+-. 1.70 Acid N 4.1 -11.79 .+-. 0.26
38.92 .+-. 1.13
[0078] Samples prepared with 50 mM or 100 mM ascorbic acid retained
its putty texture even after being exposed to g-radiation.
[0079] Those skilled in the art will appreciate that numerous
changes and modifications may be made to the preferred embodiments
of the invention and that such changes and modifications maybe made
without departing from the spirit of the invention. It is therefore
intended that the appended claims cover all such equivalent
variations as fall within the true spirit and scope of the
invention.
* * * * *