U.S. patent application number 08/816079 was filed with the patent office on 2002-07-25 for bone paste.
Invention is credited to GROOMS, JAMIE M., WIRONEN, JOHN F..
Application Number | 20020098222 08/816079 |
Document ID | / |
Family ID | 25219628 |
Filed Date | 2002-07-25 |
United States Patent
Application |
20020098222 |
Kind Code |
A1 |
WIRONEN, JOHN F. ; et
al. |
July 25, 2002 |
BONE PASTE
Abstract
A bone paste useful in the orthopedic arts, for example in the
repair of non-union fractures, periodontal ridge augmentation,
craniofacial surgery, implant fixation, impaction grafting, or any
other procedure in which generation of new bone is deemed
necessary, is provided by a composition comprising a substantially
bioabsorbable osteogenic compound in a gelatin matrix. In various
embodiments, the osteogenic compound is selected from (i)
demineralized bone matrix (DBM); (ii) bioactive glass ceramic,
BIOGLASS.RTM., bioactive ceramic, calcium phosphate ceramic,
hydroxyapatite, hydroxyapatite carbonate, corraline hydroxyapatite,
calcined bone, tricalcium phosphate, or like material; (iii) bone
morphogenetic protein, TGF-.beta., PDGF, or mixtures thereof,
natural or recombinant; and (iv) mixtures of (i)-(iii).
Inventors: |
WIRONEN, JOHN F.;
(GAINESVILLE, FL) ; GROOMS, JAMIE M.;
(GAINESVILLE, FL) |
Correspondence
Address: |
VAN DYKE & ASSOCIATES, P.A.
1630 HILLCREST STREET
ORLANDO
FL
32803
US
|
Family ID: |
25219628 |
Appl. No.: |
08/816079 |
Filed: |
March 13, 1997 |
Current U.S.
Class: |
424/423 ;
424/424 |
Current CPC
Class: |
A61L 27/26 20130101;
A61L 27/46 20130101; A61L 24/0005 20130101; A61L 24/0094 20130101;
A61L 27/26 20130101; A61F 2310/00383 20130101; A61F 2002/30062
20130101; A61L 24/0084 20130101; A61L 27/48 20130101; A61L 27/26
20130101; A61F 2310/00293 20130101; A61P 19/00 20180101; A61L
24/0094 20130101; A61L 24/043 20130101; A61F 2/28 20130101; A61L
27/48 20130101; A61L 27/48 20130101; A61L 24/0084 20130101; A61L
27/46 20130101; A61F 2/4455 20130101; A61L 27/46 20130101; A61F
2002/2835 20130101; A61F 2310/00179 20130101; A61L 24/043 20130101;
A61L 27/3645 20130101; A61L 2430/02 20130101; A61F 2002/30622
20130101; A61F 2310/00329 20130101; A61F 2210/0004 20130101; A61L
27/36 20130101; A61L 24/0094 20130101; A61L 27/3608 20130101; A61L
24/043 20130101; A61F 2/2875 20130101; A61L 24/0084 20130101; A61L
27/222 20130101; A61L 27/227 20130101; A61L 24/104 20130101; A61F
2002/2817 20130101; A61L 27/58 20130101; A61L 27/54 20130101; C08L
89/00 20130101; C08L 89/00 20130101; A61L 24/0015 20130101; A61L
24/0015 20130101; C08L 89/00 20130101; A61L 27/54 20130101; C08L
89/00 20130101; A61F 2/28 20130101; C08L 89/00 20130101; A61L
24/0042 20130101; C08L 89/00 20130101; A61L 24/0042 20130101; A61L
24/0042 20130101; A61F 2/28 20130101; C08L 89/00 20130101; C08L
89/00 20130101; A61L 27/58 20130101; A61F 2/28 20130101; C08L 89/00
20130101; A61L 27/58 20130101; C08L 89/00 20130101; C08L 89/00
20130101; C08L 89/00 20130101; A61L 27/54 20130101; A61L 24/0015
20130101 |
Class at
Publication: |
424/423 ;
424/424 |
International
Class: |
A61K 009/00 |
Claims
1. An implantable bone paste composition comprising gelatin as a
carrier for substantially bioabsorbable osteogenic components for
use in a recipient in need thereof.
2. The bone paste of claim 1 for use in the repair of non-union
fractures, periodontal ridge augmentation, craniofacial surgery,
arthrodesis of spinal or other joints, spinal fusion procedures,
and implant fixation.
3. The composition of claim 1 wherein the gelatin is thermally
cross-linkable at or slightly above the temperature of the organism
into which it is to be implanted.
4. The composition of claim 3 wherein said composition gels at
about 38.degree. C.
5. The composition of claim 3 wherein said gelatin is present at a
concentration of between about 20-45% (w/w) gelatin as a fraction
of the weight of the composition.
6. The composition of claim 5 wherein the osteogenic component is
selected from the group consisting of: (i) demineralized bone
matrix (DBM); (ii) bioactive glass ceramic, BIOGLASS.RTM.,
bioactive ceramic, calcium phosphate ceramic, hydroxyapatite,
hydroxyapatite carbonate, corraline hydroxyapatite, calcined bone,
tricalcium phosphate, or mixtures thereof; (iii) bone morphogenetic
protein, TGF-beta, PDGF, or mixtures thereof, natural or
recombinant; and (iv) mixtures of (i)-(iii).
7. The composition of claim 6 wherein the gelatin, the
demineralized bone matrix, or both are derived from the species
into which the bone paste is to be implanted.
8. The composition of claim 7 wherein DBM is present at between
about 0-40% (w/w) of the total composite weight.
9. The composition of claim 8 wherein DBM is present at between
about 15-33% (w/w) of the total composite weight.
10. The composition of claim 6 wherein the bioactive glass is
BIOGLASS.RTM..
11. The composition of claim 6 wherein component (ii) is present at
between about 0-40% (w/w) of the total composition mass.
12. The composition of claim 6 comprising antibiotics, bone
morphogenetic or other proteins, whether derived from natural or
recombinant sources, wetting agents, glycerol, carboxymethyl
cellulose (CMC), growth factors, steroids, non-steroidal
anti-inflammatory compounds, or combinations thereof.
13. The composition of claim 6 comprising between about 0.0001 to
0.1 mg/ml bone morphogenetic protein.
14. The composition of claim 1 which is a frozen solution or is
freeze-dried.
15. The composition of claim 1 wherein the gelatin is human,
bovine, ovine, equine, canine or mixtures thereof.
16. The composition of claim 1 wherein the gelatin is derived from
human collagen sources via enzymatic, acid or alkaline
extraction.
17. The composition of claim 16 wherein said human collagen sources
are human skin, bone, cartilage, tendon, connective tissue, or
mixtures thereof.
18. The composition of claim 17 produced by treating the collagen
source with pepsin at about 33.degree. C., heat denaturing the thus
treated collagen under controlled conditions to produce gelatin,
and mixing the thus produced gelatin with an osteogenic compound
such that the gelatin is present at a final concentration of about
20-45% (w/w).
19. The composition of claim 18 wherein the denaturation is
achieved by heating to at least 50.degree. C.
20. The composition of claim 19 wherein the gelatin has a molecular
weight of greater than about 50,000 daltons.
21. The composition of claim 1 wherein the osteogenic component is
deminineralized bone matrix in a powdered form, and is composed of
particles in the size range between about 80-850 .mu.m in
diameter.
22. The composition of claim 21 comprising about 0-40% (w/w)
demineralized bone matrix powder, provided that if the
demineralized bone matrix is powder is absent, then a bone growth
factor is present at a concetration of at least 0.0001 mg/ml.
23. The composition of claim 22 wherein said bone growth factor is
morphogenetic protein, TGF-.beta., PDGF, or mixtures thereof,
natural or recombinant.
24. The composition of claim 6 wherein the bioactive glass is
BIOGLASS.RTM. having a diameter of between about 0.5-710 .mu.m.
25. The composition of claim 1 further comprising cortical,
cancellous or cortical and cancerous bone chips.
26. The composition of claim 25 wherein said bone chips are in the
size range of 80 .mu.m to 10 mm.
27. The composition of claim 1 which is injection molded, vacuum
molded, rotation molded, blow molded, extruded or otherwise formed
into a solid form.
28. The composition of claim 27 wherein said form is selected from
vertebral disks, acetabular hemispheres, tubes, ellipsoid, oblong,
and "U" shapes for void filling, intramedullary plug formation, and
impaction grafting.
29. A method for inducing bone formation in vivo in a recipient in
need thereof which comprises implanting an effective amount of an
implantable bone paste composition comprising gelatin as a carrier
for substantially bioabsorbable osteogenic components.
30. The method claim 29 which comprises repairing non-union
fractures, achieving periodontal ridge augmentation, conducting
craniofacial surgery, securing implants, arthrodesis of spinal or
other joints, spinal fusion procedures, or impaction grafting,
which comprises implanting said composition at the site in vivo in
need of such treatment.
31. The method according to claim 30 which comprises formation of a
series of small apertures in an interverterbral space and injection
of said composition into said space to induce artherodesis.
32. The method according to claim 30 which comprises extruding said
composition from a syringe at a temperature at a first temperature
at which it remains liquid or highly maleable, and forming a
resilient, sticky and easily formable shape from said composition
as it gels at a second temperature at or slightly above the body
temperature of the organism into which it is implanted.
33. A method for making an implantable graft which comprises
preparing a composition comprising a thermally cross-linkable
gelatin carrier and suspending therein a substantially
bioabsorbable osteogenic component.
34. The method of claim 33 wherein said osteogenic component is
selected from: (i) demineralized bone matrix (DBM); (ii) bioactive
glass ceramic, BIOGLASS.RTM., bioactive ceramic, calcium phosphate
ceramic, hydroxyapatite, hydroxyapatite carbonate, corraline
hydroxyapatite, calcined bone, tricalcium phosphate, or like
material; (iii) bone morphogenetic protein, TGF-.beta., PDGF, or
mixtures thereof, natural or recombinant; and (iv) mixtures of
(i)-(iii).
35. The method of claim 34 which further comprises injection
molding, vacuum molding, rotation molding, blow molding, extruding
or otherwise forming said composition into the desired form of a
solid graft, and allowing the composition to solidify at a
temperature at which the gelatin becomes thermally
cross-linked.
36. The method of claim 35 wherein said form is selected from
vertebral disks, acetabular hemispheres, tubes, ellipsoid, oblong,
and "U" shapes for void filling, intramedullary plug formation, and
impaction grafting.
37. The method of claim 36 which comprises raising the temperature
of the composition above its liquefaction temperature and allowing
the composition to gel in a mold of appropriate shape.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to a new osteogenic, osteoinductive
composition for use in the field of orthopedic medicine to achieve
bone fusions, fusion of implants to bone, filing of bone defects,
or any other application in which an osteoinductive, osteogenic
composition is desirable.
[0003] 2. Background
[0004] More than 100,000 bone grafting procedures are performed
every year in the United States alone. (Cornell). In the majority
of reconstruction procedures, the graft material is used as a
filler between bone particles in the belief that continuous contact
between particles of bone leads to more rapid and complete healing
at the repair site (as well as greater mechanical integrity).
(Bloebaum). In the cases of bone augmentation and spinal fusion,
these bone grafts may make up the entire structure of the graft,
since there are no bone fragments in the area. With the possible
exception of one product (whose use guidelines do not allow this),
all bone grafting materials require surgical placement with the
requisite incisions.
[0005] Osteogenic bone grafting materials may be separated into two
classes, namely those which are osteoconductive, and those which
are osteoinductive. While the exact definition of these terms
remains a matter of debate, it can be said that osteoconductive
implants "conduct" bone growth across defects when implanted into
osseous tissue. (Einhorn). Osteoinductive implants, on the other
hand, have the ability to "induce" cells in the area to generate
bone of their own accord. (Einhorn). These osteoinductive implants
will cause the generation of bone even when they are implanted into
non-osseous tissue (e.g. subcutaneous or intramuscular
implantation). (Einhorn; Benedict; Strates; Urist).
[0006] All of the artificially produced bone-grafting materials
available today fall in the osteoconductive category of grafts.
Among these are Bioglass.RTM., Norian.RTM., Collagraft.RTM.,
corraline hydroxyapatite, powdered hydroxyapatite, crystalline and
amorphous hydroxyapatite (hydroxyl apatite), and a number of other
products. All of these implants rely on their similarity to natural
bone hydroxyapatite. A likely mechanism for bone conduction lies in
the ability of these materials to enhance diffusion of trophic
factors and cells over their very large surface areas and the
mechanical support which they provide to growing tissues. FIG. 1
provides a list of relevant properties of selected bone graft
materials.
[0007] The other category of bone grafting materials currently
available is encompassed by autograft or allograft bone. If not too
harshly processed, these materials are generally osteoinductive.
(Yazdi). Since they are tissue transplants, their use imposes
certain risks. Autografts have been associated with harvest site
morbidity in excess of 20%. (Younger). Frozen or freeze-dried
allografts induce some immune response, and if not properly
screened, can be associated with disease transmission. (Hordin).
The last variety of allografts is demineralized bone matrix.
[0008] Demineralized Bone Matrix (DBM) was first described by Senn
in 1889. (Senn). It was rediscovered, largely by accident, and
thoroughly studied by Urist and Strates in the late 1960's.
(Strates; Urist). It has since become a major product of tissue
banks around the world. As the name implies, it is bone which has
been demineralized by treatment with acid. A detailed outline of
the process for producing this product is provided in FIG. 2.
[0009] DBM has the ability to induce the formation of bone even in
non-osseous tissues within 4 weeks. (Strates; Urist; Lasa). The
standard technique for determining the activity of DBM is to
implant it subcutaneously or intramuscularly. (Nathan). It is
believed that the major active factor in DBM is one or more bone
morphogenetic proteins (BMP), (see U.S. Pat. No. 4,294,753, herein
incorporated by reference). Other growth factors, including but not
limited to TGF-beta, (see U.S. Pat. No. 5,422,340, herein
incorporated by reference), platelet derived growth factor (PDGF),
and the like, may be important for this function also.
[0010] Bioglass.RTM. is a bone grafting material which is a
SiO.sub.2, Na.sub.2O, CaO, P.sub.2O.sub.5 glass which has the
ability to produce a bio-active surface layer of hydroxylapatite
carbonate within minutes of implantation. (Hench).
[0011] Two problems are associated with the use of DBM or Bioglass.
Both of these materials are supplied as large particles, and do not
always stay in the area into which they are implanted.
(Scarborough; Frenkel). Also, due to their coarse nature, they are
hard to mold and handle in the operating room. Accordingly, there
is the need for a product which does not allow for particle
migration, while also being easier to use in the operating
environment.
[0012] As noted in table 1, in recent years, several bone-filling
surgical pastes have become commercially available. These products
range from simple mixtures of saline with a sand-like powder to a
recently released gel, known as GRAFTON.RTM., a glycerol-based,
non-cross-linkable composition. All of these products are used in
orthopedics to repair bone defects, such as voids, cavities, cracks
etc. Such defects may be the result of trauma or may be congenital,
and the known pastes may be used to patch or fill such defects, or
build upon existing bony structures. The ultimate goal of such
treatments is that the paste will induce bone formation to replace
the paste while retaining the form created by the surgeon when
applying the paste.
[0013] Desirably, a bone paste would be osteoconductive (i.e. it
conducts bone cells into a region) and osteoinductive (i.e. stem
cells are induced to differentiate into bone forming cells which
begin production of new bone). In general, bone pastes known in the
art are osteoconductive, with only weak osteoinductive effects.
Accordingly, such known pastes are inadequate for filling of large
voids and frequently do not effect proper bone formation even in
small voids. All currently available bone pastes, including those
that exhibit some osteoinductive activity, are difficult to handle,
do not adequately remain at the site of implantation, or both.
[0014] Thus, one commercially available product, GRAFTON.RTM., (see
U.S. Pat. No. 5,484,601) is a non-cross-linkable composition of
demineralized bone powder suspended in a polyhydroxy compound (e.g.
glycerol) or esters thereof, optionally including various other
ingredients, including gelatin. It is considered likely that this
material is rapidly washed away from the implant location as the
carrier matrix is glycerol, which is water soluble.
[0015] U.S. Pat. Nos. 5,236,456 and 5,405,390 (O'Leary and Prewett)
outline an "osteogenic" gel composition which is made from
demineralized bone matrix (DBM) by treating with concentrated acid
(3 M HCl) and heating to between 40 and 50.degree. C. The patent
briefly describes mixing the gel with DBM and several other
components. However, the method of manufacturing the gel
composition is such that it produces mostly collagen fibers (i.e.
the temperature elevation is insufficient to produce gelatin). As a
result, the collagen fibers are not soluble in neutral solutions.
To obtain a gel, the patent specifies that the collagen must be
dissolved in acid of low pH (e.g. HCl or 1% acetic acid, at a pH of
less than 4.0). However, compositions of low pH are not typically
very compatible with biological implantations. It is also noted
that at column 5, line 20, and column 6, line 15, it is specified
that the temperature at which the gel solidifies is 0-5.degree. C.,
which precludes gellation in vivo.
[0016] U.S. Pat. No. 4,440,750 (Glowacki and Pharris) outlines a
standard enzymatic technique for extracting collagen from tissue
using Pepsin. A highly refined collagen is obtained from animal
sources, which is then reconstituted prior to forming the working
composition. The collagen will not readily cross-link without the
addition of other chemicals (e.g. aldehydes, chondroitin sulfate),
which they do not specify in the composition. There is no mention
of a set temperature or any reference to cross-linking
behavior.
[0017] In U.S. Pat. Nos. 4,394,370 and 4,472,840, (Jefferies),
complexes of reconstituted collagen with demineralized bone or
solubilized bone morphogenetic protein, optionally cross-linked
with glutaraldehyde, were reported to be osteogenic when implanted
in vivo. The reconstituted collagen of these patents is pulverized,
lyophilized, microcrystalline collagen which has been dialyzed to
remove the hydrochloric acid used in collagen preparation.
Accordingly, the composition of those patents does not involve the
conversion of collagen to gelatin prior to formation of the
composition. Hence, the composition would not exhibit the thermal
cross-linking behaviour of the instant composition.
[0018] In U.S. Pat. No. 4,678,470 (Nashef et al.) disclosed a
non-resorbable bone-grafting material comprising demineralized bone
matrix that had been cross-linked by treatment with glutaraldehyde,
or like cross-lining agent, suspended in a gelatinous or semi-solid
carrier. Given that the demineralized bone of that patent is
chemically cross-linked, its bone inductive properties are
considered to be destroyed and the composition essentially forms a
structural filler or matrix into which recipient bone may grow.
[0019] In WO 89/04646 (Jefferies), a bone repair material having
good structural strength was disclosed. The material comprised a
demineralized bone matrix which had been surface activated by
treatment with glutaraldehyde or like cross-linking agent to
increase the binding thereof to biocompatible matrices. The
resulting material has such a rigid structure that, prior to
implantation into a biological recipient, the material may be
machined.
[0020] The bone paste of the present invention meets the needs in
the art by providing a material that is easy to handle and store,
which adheres to the site of implantation, displays both
osteoconductive and osteoinductive activities, is thermally
cross-linkable, and is substantially bioabsorbable. Preferably, the
composition is provided as a gel which contains mineral and protein
components which have been clinically shown to induce rapid bone
ingrowth. The composition may be delivered to the surgeon in a
pre-loaded syringe, ready for use. Preferably, at a first
temperature, the gel is easily formable into any shape, and is
adhesive. Once inside the biological milieu, or at a second lower
temperature, the gel desirably hardens as a rubbery solid, which
does not wash away or migrate from the site of implantation. Upon
ingrowth of bone, the implant material becomes completely
incorporated into the biological system. The mode of making and
using this composition is set forth in detail below.
BRIEF SUMMARY OF THE INVENTION
[0021] A bone paste useful in the orthopaedic arts, for example in
the repair of non-union fractures, periodontal ridge augmentation,
craniofacial surgery, implant fixation, arthrodesis of spinal or
other joints, including spinal fusion procedures, or any other
procedure in which generation of new bone is deemed necessary, is
provided by a composition comprising gelatin and additional
osteogenic components. The gelatin is preferably thermally
cross-linkable, and the osteogenic components are selected
from:
[0022] (i) demineralized bone, preferably derived from the species
into which the bone paste is to be implanted; or
[0023] (ii) bioactive glass ceramic, BIOGLASS.RTM., bioactive
ceramic, calcium phosphate ceramic, hydroxyapatite, hydroxyapatite
carbonate, corraline hydroxyapatite, calcined bone, tricalcium
phosphate, like material, or mixtures thereof; or
[0024] (iii) bone morphogenetic protein, TGF-beta, PDGF, or
mixtures thereof, natural or recombinant; or
[0025] (iv) mixtures of (i)-(iii).
[0026] Where present (ii) or like material is included to enhance
the range of manipulable characteristics of strength and
osteoinduction exhibited by the composition. Where present, (iii)
reduces the need for demineralized bone, which otherwise provides a
source of osteoinductive factors.
[0027] Demineralized bone has been shown to be highly effective in
inducing bone formation. The gelatin provides a cross-linkable,
adhesive and easily manipulated matrix in which the osteoconductive
and osteoinductive elements of the composition are carried. Other
factors, such as antibiotics, bone morphogenetic or other proteins,
whether derived from natural or recombinant sources, wetting
agents, glycerol, dextran, carboxymethyl cellulose (CMC), growth
factors, steroids, non-steroidal anti-inflammatory compounds, or
combinations thereof or any other material found to add to the
desirable properties of the essential composition of this invention
may be included.
[0028] The composition may be freeze-dried or pre-constituted, and
may be provided in a convenient dispensing device, such as a
pre-loaded syringe. The gel is preferably in a liquid or highly
malleable state at temperatures above about 40.degree. C., but sets
up as a hard gel at or preferably slightly above the body
temperature of the organism into which it is implanted (e.g. at
38.degree. C. in humans).
BRIEF SUMMARY OF THE FIGURES
[0029] FIG. 1 is a chart of existing bone grafting materials.
[0030] FIG. 2 represents a bone demineralization process.
[0031] FIG. 3 is a graph of the kinematic viscosity (centistokes)
versus concentration (%) for human gelatin processed at various
temperatures in phosphate buffered saline solution (PBS).
[0032] FIG. 4A is a photomicrograph of a section of an implant
comprising demineralized bone matrix (DBM) without any carrier
after four weeks intramuscularly in a rat.
[0033] FIG. 4B is a photomicrograph of a section of an implant
comprising 33% DBM in gelatin (i.e. the paste of this invention)
after four weeks intramuscularly in a rat.
DETAILED DESCRIPTION OF THE INVENTION
[0034] It will be appreciated by those skilled in the art that the
specifics of the composition of this invention, its method of
preparation and use are applicable to such compositions for use in
any vertebrate species. Nonetheless, because human use is
considered likely to be the principal orthopedic application of
this new material, the following description concentrates on
exemplying this material for human applications.
[0035] The composition of this invention comprises gelatin and
additional osteogenic components. The gelatin is preferably
thermally cross-linkable, and the osteogenic components are
selected from:
[0036] (i) demineralized bone, preferably derived from the species
into which the bone paste is to be implanted; or
[0037] (ii) bioactive glass ceramic, BIOGLASS.RTM., bioactive
ceramic, calcium phosphate ceramic, hydroxyapatite, hydroxyapatite
carbonate, corraline hydroxyapatite, calcined bone, tricalcium
phosphate, like material, or mixtures thereof; or
[0038] (iii) bone morphogenetic protein, TGF-beta, PDGF, or
mixtures thereof, natural or recombinant; or
[0039] (iv) mixtures of (i)-(iii).
[0040] The composition is fluid at a first temperature (e.g., above
38.degree. C.) and becomes thermally cross-linked at or just above
a second temperature, corresponding to the normal body temperature
of the organism into which the composition is to be implanted
(e.g., at 38.degree. C. in humans).
[0041] The terms "thermally cross-linked" or "thermally
cross-linkable" are used herein to describe the property of a
composition which contains molecules which, at or below a given
temperature and concentration, associate in such a fashion as to
result in gelation of a solution containing these molecules.
[0042] The term "substantially bioabsorbable" is used herein to
describe the property of a material able to cooperate in and become
incorporated with new bone formation. Accordingly, for example,
demineralized bone matrix which has been chemically cross-linked
with an agent such as glutaraldehyde, is not considered to be
substantially bioabsorbable. However, demineralized bone matrix
itself, bioactive glass or like ceramics, gelatin, and bone
morphogenetic factors are all considered to be substantially
bioabsorbable as they cooperate in new bone formation, rather than
purely providing structural rigidity or support.
[0043] The gelatin acts as a carrier phase and has the ability to
thermally cross-link over a very small temperature range. This
thermal cross-linking reaction is largely controlled by physical
entanglement and hydrogen bonding between chains, and so is
dependant on concentration and temperature. (Sperling).
Additionally, since gelatin has been used extensively in the
medical market, its in vivo properties are thoroughly studied.
(McDonald). The gel-foam sponge is the most familiar application of
this biopolymer. Studies have indicated that gelatin is only mildly
antigenic upon implantation, and is comparable in some of its
properties to collagen, (McDonald). However, collagen does not
exhibit the thermal cross-linking property so important to the
composition of this invention.
[0044] Where present, the bioactive glass, such as BIOGLASS.RTM.,
bioactive ceramic, calcium phosphate ceramic, hydroxyapatite,
hydroxyapatite carbonate, calcined bone, tricalcium phosphate, or
like material, is included to enhance the range of manipulable
characteristics of strength and osteogenesis (osteoinduction and
osteoconduction) exhibited by the composition.
[0045] The manufacture of gelatin is based on the partial
hydrolysis of collagen. Collagen is available from skin, bone,
cartilage, tendon and other connective tissue. Skin and bone yield
Type I and Type III collagen molecules, while tendon yields nearly
pure Type I collagen, and cartilage yields a mixture of Type II and
rarer types of collagen molecules. Gelatin molecules resemble
collagen triple helices, however, they are partially hydrolyzed. As
a result, in solution they have little organization. But, as the
solution cools, the gelatin molecules begin to form helical
structures. As the solution cools further, the viscosity increases
and a phase transformation from a solution to a gel occurs. This
phase change is reversible when heat is added.
[0046] The set time and set temperature of a gelatin solution are
dependent on the concentration of gelatin in solution, the
molecular weight, or intrinsic viscosity, of the gelatin molecules,
and the pH of the solution. At the isoelectric point, or the pH at
which the gelatin molecules are electrically neutral, the set time
is the shortest.
[0047] Collagen can be partially hydrolyzed by several methods. The
Type A process is the simplest and most rapid process, in which
dilute acid (e.g. less than 1 M HCl) is used to partially hydrolyze
the collagen. Type A processing is generally used with porcine skin
and demineralized bovine bone. The Type B process uses an alkaline
solution to partially hydrolyze the collagen. Type B processing is
generally used with bovine hide and demineralized bovine bone.
Finally, enzymes, such as pepsin, may be used to partially
hydrolyze collagen. Pepsin preferentially cleaves peptide bonds
between aromatic amino acids. Pepsin also acts as an esterase, but
amides of amino acids are not hydrolyzed.
[0048] As one example of this method, the gelatin is prepared from
the bones of the species into which the compositions are to be
implanted, by crushing and defatting the bones followed by soaking
for about 24 hours in approximately 300 mg/L pepsin in a 0.5 M
acetic acid at 33.degree. C. The pH of the resulting solution is
brought to 9.0 with sodium hydroxide to denature the pepsin, then
it is returned to 7.0 with hydrochloric acid. The temperature of
the solution is raised to 60.degree. C. for about 15 to 30 minutes
and returned to 4.degree. C. to effect denaturation of remaining
collagen and complete conversion to gelatin. The resulting solution
is filtered to remove particulates and dialyzed against distilled
water for 48 hours in a 50K-100K molecular weight cut-off (50K-100K
MWCO) dialysis membrane. After lyophilization, the gelatin is
redissolved in phosphate buffered saline (PBS) or water to an
effective concentration of about 30-45 weight percent of gelatin in
solution.
[0049] The gelatin content of the composition is desirably between
about 20-45% (w/w). The gelatin may be derived from the same or
different species than that into which the composition is to be
implanted. For example, human, porcine, bovine, equine, or canine
gelatin is derived from collagen sources such as bone, skin,
tendons, or cartilage, and may then be mixed with DBM or other
osteogenic materials. As noted above, the collagen is converted to
gelatin via, liming, acidification or by enzymatic extraction, for
example by pepsin or like enzymatic treatment, followed by
denaturation by heat or other means. The gelatin may be derived
from tissue by mastication of the tissue, followed by an extended
treatment capable of breaking cross-links in the long collagen
chains. In one embodiment, the tissue is ground then soaked for
about 24-72 hours at between about 2-40.degree. C. in dilute acid,
such as 0.1 normal acetic acid. Preferably, an enzyme such as
pepsin at a sufficiently high concentration is added. Pepsin
concentrations of between about 10-20,000 i.u./liter, 100-2,000
i.u/liter, or like concentrations are added to the dilute acid at
the start of the treatment, with the period of treatment being
adjusted according to the enzyme concentration used. Solids are
removed from the composition, for example by centrifugation, and
the supernatant material in solution having a molecular weight of
about 50,000 daltons or higher is retained. This may be achieved by
any of a number of methods known in the art including, but not
limited to, dialyzing the supernatant in a 50,000 dalton molecular
weight cut-off membrane against several changes of solution,
ultrafiltration against a membrane having a like molecular weight
cut-off, (MWCO) or gel permeation chromatography through a medium
having a 50,000 dalton molecular mass cut-off. It will be
recognized by those skilled in the art that the higher the MWCO of
the gelatin, the lower the yield. Accordingly, lower MWCO gelatin
preparations, down to abut 1000 dalton MWCO's could be used,
recognizing that undesirable low molecular weight species might
thereby be retained.
[0050] The gelatin solution resulting from the foregoing extraction
is preferably denatured, for example by heat-treatment to above
about 50.degree. C. The denatured protein is then stored in a
frozen state or it may be freeze-dried or precipitated, for example
in a volatile organic solvent, and reconstituted in a solution,
such as an isotonic saline solution, at a concentration of between
about 30-45% (w/w) gelatin.
[0051] The demineralized bone is preferably in a powdered form, and
is preferably composed of particles in the size range between about
80-850 .mu.m in diameter. Methods for producing demineralized bone
powder are known in the art (see for example U.S. Pat. No.
5,405,390, herein incorporated by reference for this purpose), and
are not, therefore, elaborated here. Demineralized bone powder,
extracted by standard techniques, is mixed with the gelatin
solution prepared as described above, to form a composition
comprising about 0-40% (w/w) demineralized bone powder. Where
present, bone morphogenetic proteins (BMP) reduce the percentage of
DBM required in the composition. The BMP is preferably present at a
concentration of between about 0.0001 to 0.1 mg/ml, 0.001 mg/ml to
0.01 mg/ml, or like concentration, depending on the amount of DBM
present (0-40% w/w).
[0052] The final composition preferably comprises gelatin having a
viscosity of about 3600 centipoise at 44.degree. C. (when measured
in the linear range of a viscosity/sheer rate plot--0.87/s), or a
kinematic viscosity of about 0.7 centistokes at 44.degree. C. The
concentration of the gelatin in the carrier phase (i.e. absent
added osteogenic components) is preferably about 30-45% (w/w),
(approximately 50-60% w/v), to ensure that gelation at 38.degree.
C. will occur in a reasonable amount of time. Naturally, those
skilled in the art will recognize that, depending on the species of
the organism into which the composition is to be implanted,
different temperatures may be required. These needs are
accommodated by altering the gelatin concentration, increasing the
concentration if a higher gel temperature is desired, and lowering
the concentration if a lower gel temperature is desired.
[0053] The DBM content of the composition is defined herein by the
concentration required to obtain bone formation similar to that
seen with DBM alone. We have found that about 5-40% (w/w) DBM in
the composition is effective. Anything lower than about 5% seems to
do very little by way of bone formation, unless added BMPs
(component iii) are present in the composition, in which case the
DBM concentration may be substantially reduced or eliminated
altogether. Naturally, based on this disclosure, those skilled in
the art will recognize that by addition of different concentrations
and compositions of bone morphogenetic proteins or other osteogenic
or osteoinductive factors, the weight percent of DBM in the
composition may be manipulated up or down. In addition, it will be
recognized that, depending on the species into which the
composition is implanted, the DBM weight percent may need to be
adjusted up or down.
[0054] We have found in in vivo studies that the compositions with
DBM contents from 15 to 33% all produce calcified tissue. We have
found that there is a good correlation between the amount of DBM in
the composition and the level of bone induction, as long as the DBM
concentration is greater than about 19% (w/w). About 38-40% (w/w)
is the upper mass limit for DBM. Accordingly, 0-40% (w/w) DBM, and
more preferably 5-30% (w/w), 7-33%(w/w) or 15-25% (w/w) is
desirable for this component.
[0055] We have observed histologically that, subsequent to
implantation into an animal, the gelatin phase is totally absorbed
within about 2 weeks. Additionally, cartilage and mineralized bone
formed within two weeks, with mature bone being evident by about
the fourth week. The animals in these studies did not exhibit any
gross health problems or any indications of irritation, hematoma,
soreness, fever, or weight loss during the study. The composition
according to this invention, whether it comprises gelatin and
osteogenic components (i-iv) may act as a carrier for cortical,
cancellous or cortical and cancerous bone chips. Such compositions
are useful for fling larger bone voids. In addition, when these
bone chips are not demineralized, they provide an added spectrum of
biological properties not exhibited by the gelatin alone or the
gelatin plus osteogenic components (i-iv). When present, it is
preferred for such bone chips to be in the size range of about 80
.mu.m to about 10 mm.
[0056] In a further embodiment of this invention, the composition
of gelatin and osteogenic components (i-iv) is injection molded,
vacuum molded, rotation molded, blow molded, extruded or otherwise
formed into a solid form. Such forms would desirably take the form
of vertebral disks, acetabular hemispheres, tubes, ellipsoid shapes
for void filling, and intramedullary plugs, which are useful to
plug the intramedullary canal of various bones (i.e. the marrow
containing portion of the bone) to prevent bone cement from
entering healthy bone tissue. These forms are produced, for
example, by raising the temperature of the composition above its
liquefaction temperature (e.g. about 45.degree. C.), and allowing
the composition to gel in a mold of appropriate shape. For such
forms, the gelatin content is preferably made as high as possible
to ensure that the form remains solid upon grafting into a
vertebrate recipient.
[0057] Those skilled in the art will recognize the many orthopedic
applications of the bone paste of this invention. However, by way
of illustration rather than limitation, for purposes of arthrodesis
of the spine, one particularly preferred mode of using this
composition would be at an early stage of vertebral disk
degeneration or subsequent to trauma. Diagnosis of trauma or
degeneration is followed by formation of a small orifice, or a
plurality of small orifices in the intervertebral cartilage at the
site of degeneration. The bone paste is then injected into the
intervertebral space to induce arthrodesis. A similar procedure
could be used with other joints or bone damage.
[0058] Having generally described the invention, the following
examples are provided to show specific features and applications of
the invention. It should be recognized that this invention is in no
way limited to the specifics of the examples as set forth below,
and that the limits of this invention are defined by the claims
which are appended hereto.
EXAMPLE 1
Gelatin Production, Kinematic Viscosity, and Critical Concentration
for Gelation at 38.degree. C.
[0059] In this experiment, the source of collagen was from
demineralized human cortical bone powder in the size range of
250-850 .mu.m. The demineralized bone matrix powder (DBM), 0.5 M.
acetic acid solution, and pepsin were added to a centrifuge tube.
The centrifuge tube was tumbled for 24 hours at the desired
temperature: 4.degree. C., 30.degree. C., 33.degree. C. or
37.degree. C. The pH was adjusted to 9.0 then down to 7.0 with 1 N
NaOH and 1N HCl, respectively, deactivating the pepsin. The
solution was placed in a 60.degree. C. water bath for 15 minutes,
then quenched in ice water. The solution was centrifuged and the
supernatant was poured into dialysis membrane tubing with a 1000
Daltons molecular weight cut off. The supernatant was dialyzed to
obtain a 1000:1 dilution factor, frozen and lyophilized until
completely dry. This experiment was performed in quintuplicates for
each temperature.
[0060] The kinematic viscosities of dilute concentrations of
gelatin, 0.0625 w/v %, 0.125 w/v %, 0.25 w/v %, and 0.5 w/v % in
phosphate buffered saline solutions (pH 7.4 at 25.degree. C.), were
measured with an Ubbelhode viscometer at 44.degree. C. The
kinematic viscosities of human gelatin processed at 4.degree. C.,
30.degree. C., 33.degree. C., and 37.degree. C., were measured in
duplicates, except for 33.degree. C. which was only measured once.
The kinematic viscosities (centistokes) were graphed versus
concentration of human gelatin solution, FIG. 3. The linear
regression was extrapolated to zero to determine the kinematic
viscosity at zero concentration. The optimum processing temperature
was determined by the temperature that yielded the highest solution
viscosity at zero concentration, largest slope of the linear
regression, greatest yield, and lastly, the gelatin that produced a
solid bone composite at slightly above human body temperature.
[0061] As the processing temperature increased, the yield of
gelatin, normalized for the same pepsin to DBM ratio (0.03% (w/v)
pepsin/1 g DBM), increased. The kinematic viscosity at zero
concentration, or y-intercept, followed a reverse trend. As the
processing temperatures increased, the extrapolated kinematic
viscosities decreased, Table 1.
[0062] The human gelatin processed at 30.degree. C. had the highest
slope on the kinematic viscosity versus concentration plot, 0.40
(centistokes/%), followed by the human gelatin processed at
4.degree. C., 0.26 (centistokes/%), the human gelatin processed at
33.degree. C., 0.21 (centistokes/%), and lastly the human gelatin
processed at 37.degree. C., 0.17 (centistokes/%), Table 1.
[0063] In order to correlate the kinematic viscosities to molecular
weight of gelatin, the kinematic viscosities must be translated
into intrinsic viscosities. However, the intrinsic viscosities were
undefined due to the polyelectrolytic nature of gelatin. As a
result, a direct relationship between viscosity and molecular
weight of human gelatin can not be made.
1TABLE 1 Physical properties of human gelatin and human gelatin in
phosphate buffered saline solution. Human gelatin was processed at
4.degree. C., 30.degree. C., 33.degree. C., and 37.degree. C.,
resulting from 1 g of DBM and 0.03 w/v % pepsin solution in 0.5 N
acetic acid: Human Slope of Gelatin Linear Processed Average Yield
Extrapolated Regression r.sup.2 Value of at Various Percent by
y-intercept (centistokes/ Linear Temp. Weight (centistokes) %)
Regression 4.degree. C. 6% (n = 5) 0.72 (trial 0.26 (trial 0.985
(trial 1 & 2) 1 & 2) 1 & 2) 30.degree. C. 18% (n = 5)
0.71 (trial 0.40 (trial 0.993 (trial 1 & 2) 1 & 2) 1 &
2) 33.degree. C. 30% (n = 4) 0.71 (trial 1) 0.21 (trial 1) 0.994
(trial 1) 37.degree. C. 60% (n = 5) 0.70 (trial 0.17 (trial 0.996
(trial 1 & 2) 1 & 2) 1 & 2)
[0064] The set temperatures for various bone paste compositions
were determined, Table 2. Human gelatin made from DBM via pepsin at
33.degree. C., 35.degree. C., and 37.degree. C. was used in the
bone paste compositions. Gelatin concentrations were varied from 19
w/w % of total composite to 25 w/w % of total composite
(corresponding to 40 w/v % to 60 w/v % gelatin in the carrier
matrix) in a pH 7.4 phosphate buffered saline solution (PBS). All
bone paste composites tested contained DBM at a concentration of 33
w/w % of the total composite. Different ambient temperatures were
used to test whether the bone paste was solid or liquid, 45.degree.
C., 43.degree. C., 41.degree. C., 40.degree. C., 38.degree. C., and
35.5.degree. C. The set temperature was determined both by
subsequent lowering of the ambient temperature and raising of the
ambient temperature.
2TABLE 2 Ambient temperatures corresponding to solidified
(non-syringe-able) bone paste composites. Human Gelatin as a
Percent of Total 37.degree. C. Process 35.degree. C. Process
33.degree. C. Process Composite Weight Temp Temp Temp 25 w/w %
<35.5.degree. C. <35.5.degree. C. 40.degree. C. 24 w/w %
<35.5.degree. C. <35.5.degree. C. <35.5.degree. C. 22 w/w
% <35.5.degree. C. <35.5.degree. C. <35.5.degree. C. 21
w/w % <35.5.degree. C. <35.5.degree. C. <35.5.degree. C.
19 w/w % <35.5.degree. C. <35.5.degree. C. <35.5.degree.
C.
[0065] Accordingly, the critical concentration of gelatin in a bone
paste composite that was solid at slightly above human body
temperature, 38.degree. C. to 39.degree. C., was 25 w/w % of the
total composite for human gelatin, processed at 33.degree. C., and
with 33 w/w % of the composite being DBM, the remainder being PBS.
The human gelatin processed at 33.degree. C. had a zero
concentration kinematic viscosity of 0.71 centistokes. Human
gelatin solutions of lower kinematic viscosities were found to have
critical concentrations in excess of about 25 w/w %.
Correspondingly, gelatins with viscosities higher than about 0.71
centistokes are expected to thermally cross-link at concentrations
lower than about 25% (w/w).
EXAMPLE 2
In Vivo Bone Paste Composition and Activity
[0066] This study demonstrates that the bone paste of this
invention is osteoinductive. In addition, this study demonstrates
particle sizes for the DBM component of the composition which
operate well in promoting new bone growth in an animal into which
it is implanted.
[0067] The intra-muscular rat model is the standard model for
testing the osteoinductivity of demineralized bone and other
osteoinductive factors. Strates et al. have used this model for
many years (Strates).
[0068] As noted in Example 1 above, we determined that for gelation
at 38.degree. C., a gelatin solution concentration of 40-60% w/v
(30-45% w/w of the solution absent added osteogenic components) is
required. At this concentration, gelatin acting as a carrier matrix
thermally cross-links at 38.degree. C. within approximately 8
minutes. In this study we addressed the question of how much DBM
must be present in this fixed 40-60% gelatin carrier matrix to
induce bone formation which favorably compares with positive
controls. We compared 4 different compositions of a DBM/Gelatin
composite with both positive and negative controls in a rat
intra-muscular model.
[0069] A. Implant Preparation:
[0070] The femurs, tibiae, and fibulae were harvested from
fresh-killed (within 24 hours, refrigerated at 4.degree. C.)
Sprague-Dawley rats. The diaphyses were cut from the bones and the
marrow removed from the mid-shaft with a dissecting probe and
sterile water wash. Mid-shaft segments were then demineralized in
0.6 M. HCl for 24 hours at 4.degree. C. with the mass ratio of bone
to acid maintained at {fraction (1/10)} or lower. The bone segments
were lyophilized and then mixed with dry ice and ground in a
lab-scale bone mill. DBM powder was sieved and the fraction from
125-450 .mu.m was retained.
[0071] A carrier matrix of 50% (w/v) gelatin was made by heating
phosphate buffered saline (PBS) to 60.degree. C. and then adding
powdered porcine gelatin (Sigma, 300 bloom) and stirring
vigorously. Carrier matrix was allowed to age for 15 minutes (to
even out the distribution of gelatin in solution) and then it was
allowed to cool to 50.degree. C. DBM was added to the gelatin
solution at this point in the following amounts: 0 (negative
control), 15, 19, 24, and 33% w/w of the total composite. The
composite was blended thoroughly by hand mixing.
[0072] Implants were prepared by ejecting a thread of composite
onto a petri dish. These threads were cut into short segments
(.sup..about.4 mm.), weighed, and placed into sterile petri dishes.
Positive controls were prepared by pelletizing DBM mixed with PBS
in a centrifuge. To maintain pellet integrity during the hazards of
surgery, these pellets were frozen and implanted as such.
[0073] B. Rat Surgery:
[0074] Young Sprague-Dawley rats (200-410 g) were anesthetized with
86 mg/kg Ketamine, and 13 mg/kg Xylazine administered
intramuscularly (in the thigh). A parallel-mid-line incision was
made from the tip of the sternum to just above the groin. The
lateral aspects of the rectus abdominus were accessed by blunt
dissection to either side of the animal. Three short incisions were
made in the muscle on each side and the implants inserted, followed
by 1 to 2 stitches with Prolene.sunburst. 3-0 suture (to mark the
location and prevent the ejection of the implant mass). One
positive or one negative control as well as two experimental
compositions were inserted on each side. Implant locations were
random except that each rat had one positive control on one side
and one negative control on the contralateral side.
[0075] Animals were returned to their cages and provided food and
water ad-lib. All members of the study group were kept for 4 weeks
except one animal (R1) which was sacrificed after 2 weeks for
histology.
[0076] After 4 weeks, animals were sacrificed with an overdose of
Nembutal. The rectus abdominus was removed by sharp dissection,
removing as much tissue as possible.
[0077] C. Explant Analysis:
[0078] Each muscle was notched to mark the superior side of the
animal and placed into a labeled petri dish. The muscle was X-rayed
with mammography equipment, using mammography film (DuPont).
Roentgenograms were analyzed using a digital camera attached to an
Apple LCII equipped with NIH Image 4.1 software. Images were
thresholded to highlight the implant shadow and then the area of
the shadow was determined by pixel counting.
[0079] Two of each variety of explant were removed from the muscle
and fixed in 10% buffered formalin. Histological sections were
taken and consecutive sections were stained with H&E and
Masson's trichrome stain. These histological samples were examined
by a qualified pathologist.
[0080] Remaining explants were cut from the muscle tissue and ashed
in a muffle furnace for 4.5 hours at 700-750.degree. C. Ash weight
was determined and normalized to original implant weight. Ash was
dissolved in 1.0N HCl and analyzed for calcium content by atomic
absorption spectroscopy.
[0081] All analyses were conducted in a blinded manner with
decoding done only after processing of the data was complete.
[0082] D. Histology:
[0083] Two week histology samples of 15% and 19% DBM composites
indicated that bone formation was occurring, even at this early
date. The route of bone formation is not readily apparent, but
appears to be endochondral. Four week histology samples revealed
that mature bone was formed at the site of implantation. The
quality of bone formed was comparable to that of natural bone as
shown by the ash and percent calcium analyses. All implants
containing DBM were found to lead to the production of some bone.
Those containing greater than about 20% DBM yielded the highest
quality bone. FIGS. 4A and 4B provide photomicrographs of sections
of implants after four weeks in vivo in the rat intramuscular
model. We found that 33% (w/w) DBM in gelatin carrier (FIG. 4B)
according to this invention produced as much new bone as pure, 100%
DBM (FIG. 4A). In these figures, the following structures are
evident: 10 is mature bone, as evidenced by red stain uptake from
Masson's stain; 20 is new cartilage formation, as evidenced by
uptake of blue stain from Masson's stain and the presence of cells;
30 is residual DBM, as evidenced by uptake of blue stain and the
absence of cells, from which all cartilagenous and bone structures
in the muscle cross section arose; and 40 is immature bone, as
evidenced by light blue staining and the presence of cells. The
cells seen are osteoclasts, degrading the newly formed cartilage,
and osteoblasts, laying down new bone. In addition, vascular
infiltration in the mature bone is evident in the Masson's stained
sections, from which the black and white prints were made.
[0084] E. Compositional Analysis:
[0085] There was no statistically significant difference, using a
2.sigma. test, in ash content between the negative control, the
positive control, or compositions comprising 15% or 19% (w/w) DBM.
This does not necessarily imply that these compositions do not work
(examination of the Roentgenograms obviates this conclusion).
Rather, it indicates that the sensitivity of the ash method does
not allow the detection of the difference. Examination of the data
for the 24% and 33% composites indicates that they are
significantly better than 19%, 15%, and the negative controls, and
are not significantly different from the (positive) control, see
Table 3:
3 TABLE 3 Composition % Yield Ash/g (% DBM) Implant Standard
Deviation 0 {- control} 10.1 9 (n = 6) 15 5.5 12.7 (n = 6) 19 11.9
12.2 (n = 6) 24 34.5 14.9 (n = 5) 33 30.0 8.0 (n = 4) 100 {+
control} 31.9 8.8 (n = 6)
[0086] F. Atomic Absorption Spectroscopy:
[0087] The atomic absorption spectroscopy of ashed compositions of
DBMI/gelatin composites yielded the amount of calcium in the
samples. The 15% and 19% compositions did not show a statistically
significant difference from the negative controls. However, it is
expected that with greater assay sensitivity, positive effects of
DBM at concentrations as low as about 7% (w/w) in gelatin carrier
would be measurable. The average calcium content produced by
compositions greater than or equal to 24% appeared to be
proportional to the amount of DBM, by weight, in the
composition:
4TABLE 4 Comparison between the atomic absorption spectroscopy
results of ashed samples of six different DBM/gelatin composites
explanted from rats after 4 weeks in vivo. Composition Average Ca
(% DBM w/w) Content/gram Standard Deviation (.sigma.) 0 {(-)
control} 1.2 1.2 (n = 6) 15 3.9 2.4 (n = 4) 19 7.3 7.5 (n = 4) 24
23.1 8.7 (n = 3) 33 28.0 4.4 (n = 4) 100 {(+) 81.3 30.0 (n = 5)
control}
[0088] G. X-Ray Digital Analysis:
[0089] Gross examination/comparison of the x-rays reveals that the
24% and 33% compositions are not significantly different from the
(+) controls. The 15% and 19% compositions do not appear to
generate significant bone. However, it is expected that with
greater assay sensitivity, positive effects of DBM at
concentrations as low as about 7% (w/w) in gelatin carrier would be
measurable. No bone formation was apparent on the x-rays at the
locations of the (-) controls. Accordingly, we conclude that DBM at
a concentration of between about 24% to 33% (w/w) in gelatin is
active in inducing bone formation. These same data indicate that
concentrations of DBM below about 20% are less effective in
generating significant bone in comparison to positive controls. It
is noted that Grafton.TM. contains only 8% DBM in a glycerol
carrier.
5TABLE 5 Composition Normalized Area (% of (% DBM w/w) + ve
control) Standard Deviation (.sigma.) 0 {(-) control} 0 0 (n = 10)
15 2.8 1.9 (n = 7) 19 4.1 4.2 (n = 7) 24 33.0 15.2 (n = 10) 33 36.7
14.9 (n = 10) 100 {(+) 100 43.1 (n = 10) control}
EXAMPLE 3
Procedure for the Production Bone Paste of this Invention
[0090] This example provides one procedure for the manufacture of
bone paste from gelatin and demineralized bone. As fractions of the
total mass of composition desired, the following components are
weighed (percentages given are of total composite weight):
6 Dry demineralized bone: 0-40% (w/w) Lyophilized thermally 20-45%
(w/w) cross-linkable gelatin: BIOGLASS .RTM.: 0-40% (w/w) bone
morphogenetic protein: 0.001 mg/ml
[0091] These components are thoroughly blended while dry, and the
balance of the composition mass is made up by addition of water,
phosphate buffered saline, or any other physiologically acceptable
liquid carrier. The composition may be packaged in this form or
lyophilized for later reconstruction with water. The malleable
properties of the composition are achieved by heating the
composition to a temperature sufficient to exceed the liquefaction
point of the gelatin, and then allowing the composition to cool to
the temperature at which it gels.
REFERENCES
[0092] Cornell, C. Techniques in Orthopaedics 1992, 7, 55-63.
[0093] Bloebaum, R. D. Human Bone Ingrowth and Materials; Bloebaum,
R. D., Ed.; Society for Biomaterials: Denver, Colo., 1996.
[0094] Einhorn, T. A. Enhancement of Bone Repair Using
Biomaterials; Einhorn, T. A., Ed.; Society for Biomaterials:
Denver, Colo., 1996.
[0095] Benedict, J. J. The Role of Carrier Matrices on Bone
Induction In Vivo; Benedict, J. J., Ed.; Society for Biomaterials:
Denver, Colo., 1996.
[0096] Strates, B.; Tiedeman, J. European Journal of Experimental
Musculoskeletal Research 1993, 2, 61-67.
[0097] Urist, M. R. Bone Morphogenetic Protein; Urist, M. R., Ed.;
W. B. Saunders Co.: Philadelphia, 1992, pp 70-83.
[0098] Yazdi, M.; Bernick, S.; Paule, W.; Nimni, M. Clinical
Orthopaedics and Related Research 1991, 262, 281-285.
[0099] Younger, E.; Chapman, M. Journal of Orthopaedic Trauma 1989,
3, 192-195.
[0100] Hardin, C. K. Otolaringologic Clinics of North America 1994,
27, 911-925.
[0101] Senn, N. The American Journal of the Medical Sciences 1889,
98, 219-243.
[0102] Urist, M. R.; Huo, Y. K; Brownell, A. G.; Hohl, W. M.;
Buyske, J.; Lietze, A.; Tempst, P.; Hunkapiller, M.; DeLange, R. J.
Procedures of the National Acadamy of Sciences, USA 1984, 81,
371-375.
[0103] Urist, M. R.; Chang, J. J.; Lietze, A.; Huo, Y. K.;
Brownell, A. G.; DeLang, R. J. Methods in Enzymology 1987, 146,
294-313.
[0104] Lasa, C.; Hollinger, J.; Droham, W.; MacPhee, M. Plastic and
Reconstructive Surgery 1995, 96, 1409-1417.
[0105] Nathan, R.; Bentz, H.; Armstrong, R.; Piez, K; Smestad, T.;
Ellingsworth, L.; McPherson, J.; Seyedin, S. Journal of Orthopaedic
Research 1988, 6, 324-334.
[0106] Hench, L. L.; Andersson, O. H. Bioactive Glasses; Hench, L.
L.; Andersson, O. H., Ed.; World Scientific Publishing Co. Pte.
Ltd.: Singapore, 1993, pp 41-63.
[0107] Scarborough, N. Bone Repair Using Allografts; Scarborough,
N., Ed.; Society for Biomaterials, 1996.
[0108] Frenkel, S. R.; Moskovich, R.; Spivak, J.; Zhang, Z. H.;
Prewett, A. B. Spine 1993, 18, 1634-1639.
[0109] Sperling, L. H. Introduction to Physical Polymer Science;
John Wiley and Sons, Inc.: New York, 1992.
[0110] McDonald, T. O.; Britton, B.; Borgmann, A. R.; Robb, C. A.
Toxicology 1977, 7, 37-44.
[0111] Culling, C. F. A.; Allison, R. T.; Barr, W. T. Cellular
Pathology Technique; 4 ed.; Butterworths: London, 1985.
[0112] U.S. Pat. No. 5,481,601
[0113] U.S. Pat. No. 5,236,456
[0114] U.S. Pat. No. 5,405,390
[0115] U.S. Pat. No. 4,440,750
[0116] U.S. Pat. No. 4,394,370
[0117] U.S. Pat. No. 4,472,840
[0118] U.S. Pat. No. 4,678,470
[0119] WO 89/04646
* * * * *