U.S. patent application number 11/383309 was filed with the patent office on 2006-11-02 for composition and process for bone growth and repair.
Invention is credited to Kevin J. Thorne.
Application Number | 20060246150 11/383309 |
Document ID | / |
Family ID | 25002919 |
Filed Date | 2006-11-02 |
United States Patent
Application |
20060246150 |
Kind Code |
A1 |
Thorne; Kevin J. |
November 2, 2006 |
Composition and Process for Bone Growth and Repair
Abstract
A composition for the induction of bone growth is disclosed. The
composition includes a substrate, bone growth protein, and sources
of calcium and phosphate. The composition is acidic which promotes
high activity of the bone growth protein. The calcium and phosphate
sources can be provided as an acidic calcium phosphate salt. Also
disclosed are methods of the making the composition and methods of
using it.
Inventors: |
Thorne; Kevin J.; (Austin,
TX) |
Correspondence
Address: |
WILLIAMS, MORGAN & AMERSON
10333 RICHMOND, SUITE 1100
HOUSTON
TX
77042
US
|
Family ID: |
25002919 |
Appl. No.: |
11/383309 |
Filed: |
May 15, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09746921 |
Dec 22, 2000 |
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11383309 |
May 15, 2006 |
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Current U.S.
Class: |
424/603 ;
514/16.9; 514/8.8; 514/8.9 |
Current CPC
Class: |
A61K 38/1875 20130101;
A61L 27/3608 20130101; A61L 27/46 20130101; A61L 27/22 20130101;
A61L 27/3616 20130101; A61L 2430/02 20130101; A61P 19/00 20180101;
A61L 27/12 20130101; A61P 19/08 20180101; A61L 27/227 20130101;
A61L 27/24 20130101; A61L 27/46 20130101; C08L 89/06 20130101 |
Class at
Publication: |
424/603 ;
514/012 |
International
Class: |
A61K 38/18 20060101
A61K038/18; A61K 33/42 20060101 A61K033/42 |
Claims
1. A bone growth composition, comprising: a bone growth protein
having a first bioactivity; and an acidic substrate; wherein the
bone growth protein has a second bioactivity greater than the first
bioactivity when combined with the acidic substrate.
2. The bone growth composition of claim 1, wherein the bone growth
protein is selected from transforming growth factor .beta.
(TGF.beta.)1, TGF.beta.2, TGF.beta.3, bone morphogenic protein
(BMP)-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9,
cartilage-derived morphogenic protein (CDMP)-1, CDMP-2, or
CDMP-3.
3. The bone growth composition of claim 1, wherein the acidic
substrate comprises collagen, fibrin, alginate, or a mixture of two
or more thereof.
4. The bone growth composition of claim 1, wherein the acidic
substrate comprises a calcium source and a phosphate source.
5. The bone growth composition of claim 4, wherein the calcium
source and the phosphate source are calcium hydrogen phosphate
dihydrate, monocalcium phosphate, calcium pyrophosphate, or a
mixture of two or more thereof.
6. The bone growth composition of claim 1, further comprising a
fluid selected from the group consisting of marrow, serum, whole
blood, saline, and water.
7. A bone growth composition, comprising a bone growth protein; and
an acidic substrate comprising an acidic calcium phosphate
compound; wherein the bone growth composition comprises between
about 0.5 .mu.mol and about 6 .mu.mol of the acidic calcium
phosphate compound per 1 .mu.g of total bone growth protein.
8. The bone growth composition of claim 7, wherein the bone growth
composition comprises between about 1 .mu.mol and about 5 .mu.mol
of the acidic calcium phosphate compound per 1 .mu.g of total bone
growth protein.
9. The bone growth composition of claim 8, wherein the bone growth
composition comprises between about 2 .mu.mol and about 4 .mu.mol
of the acidic calcium phosphate compound per 1 .mu.g of total bone
growth protein.
10. The bone growth composition of claim 7, wherein the bone growth
protein is selected from transforming growth factor .beta.
(TGF.beta.)1, TGF.beta.2, TGF.beta.3, bone morphogenic protein
(BMP)-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9,
cartilage-derived morphogenic protein (CDMP)-1, CDMP-2, or
CDMP-3.
11. The bone growth composition of claim 7, wherein the acidic
substrate comprises collagen, fibrin, alginate, or a mixture of two
or more thereof.
12. The bone growth composition of claim 7, wherein the acidic
calcium phosphate compound is calcium hydrogen phosphate dihydrate,
monocalcium phosphate, calcium pyrophosphate, or a mixture of two
or more thereof.
13. A method of promoting bone growth at a bone defect in a
vertebrate, comprising: delivering a bone growth protein having a
first bioactivity to the bone defect; and delivering an acidic
substrate to the bone defect, wherein the bone growth protein has a
second bioactivity greater than the first bioactivity when combined
with the acidic substrate.
14. The method of claim 13, wherein the bone growth protein is
selected from transforming growth factor .beta. (TGF.beta.)1,
TGF.beta.2, TGF.beta.3, bone morphogenic protein (BMP)-2, BMP-3,
BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, cartilage-derived
morphogenic protein (CDMP)-1, CDMP-2, or CDMP-3.
15. The method of claim 13, wherein the acidic substrate comprises
collagen, fibrin, alginate, or a mixture of two or more
thereof.
16. The method of claim 13, wherein the acidic substrate comprises
a calcium source and a phosphate source.
17. The method of claim 16, wherein the calcium source and the
phosphate source are calcium hydrogen phosphate dihydrate,
monocalcium phosphate, calcium pyrophosphate, or a mixture of two
or more thereof.
18. The method of claim 13, wherein the bone growth protein and the
acidic substrate are delivered simultaneously to the bone
defect.
19. A method of forming a bone growth composition, comprising:
combining a bone growth protein having a first bioactivity with an
acidic substrate, wherein the bone growth protein has a second
bioactivity greater than the first bioactivity when combined with
the acidic substrate.
20. The method of claim 19, wherein the bone growth protein is
selected from transforming growth factor .beta. (TGF.beta.)1,
TGF.beta.2, TGF.beta.3, bone morphogenic protein (BMP)-2, BMP-3,
BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, cartilage-derived
morphogenic protein (CDMP)-1, CDMP-2, or CDMP-3.
21. The method of claim 19, wherein the acidic substrate comprises
collagen, fibrin, alginate, or a mixture of two or more
thereof.
22. The method of claim 19, wherein the acidic substrate comprises
a calcium source and a phosphate source.
23. The method of claim 22, wherein the calcium source and the
phosphate source are calcium hydrogen phosphate dihydrate,
monocalcium phosphate, calcium pyrophosphate, or a mixture of two
or more thereof.
24. The method of claim 19, wherein the bone growth protein and the
acidic substrate are combined prior to delivery of the composition
to a patient.
25. A kit for promoting bone growth at a bone defect in a mammal,
comprising: a bone growth protein having a first bioactivity and an
acidic substrate, wherein the bone growth protein has a second
bioactivity greater than the first bioactivity when combined with
the acidic substrate.
26. The kit of claim 25, wherein the bone growth protein is
selected from transforming growth factor .beta. (TGF.beta.)1,
TGF.beta.2, TGF.beta.3, bone morphogenic protein (BMP)-2, BMP-3,
BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, cartilage-derived
morphogenic protein (CDMP)-1, CDMP-2, or CDMP-3.
27. The kit of claim 25, wherein the acidic substrate comprises
collagen, fibrin, alginate, or a mixture of two or more
thereof.
28. The kit of claim 25, wherein the acidic substrate comprises a
calcium source and a phosphate source.
29. The kit of claim 28, wherein the calcium source and the
phosphate source are calcium hydrogen phosphate dihydrate,
monocalcium phosphate, calcium pyrophosphate, or a mixture of two
or more thereof.
30. The kit of claim 25, wherein the bone growth protein and the
acidic substrate are included in a common package.
31. The bone growth composition of claim 1, produced by the process
comprising: combining the bone growth protein with an acidic
substrate, wherein the bone growth protein has a second bioactivity
greater than the first bioactivity when combined with the acidic
substrate.
32. The bone growth composition of claim 31, wherein the bone
growth protein is selected from transforming growth factor .beta.
(TGF.beta.)1, TGF.beta.2, TGF.beta.3, bone morphogenic protein
(BMP)-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9,
cartilage-derived morphogenic protein (CDMP)-1, CDMP-2, or
CDMP-3.
33. The bone growth composition of claim 31, wherein the acidic
substrate comprises collagen, fibrin, alginate, or a mixture of two
or more thereof.
34. The bone growth composition of claim 31, wherein the acidic
substrate comprises a calcium source and a phosphate source.
35. The bone growth composition of claim 31, wherein the bone
growth protein and the acidic substrate are combined ex vivo.
36. The bone growth composition of claim 34, wherein the calcium
source and the phosphate source are calcium hydrogen phosphate
dihydrate, monocalcium phosphate, calcium pyrophosphate, or a
mixture of two or more thereof.
37. A composition, comprising: about 3 parts by weight to about 10
parts by weight of a collagen:acidic calcium phosphate mineral
material having an average particle size of about 125 microns to
about 5000 microns, wherein the material comprises about 25 wt % to
about 75 wt % of the acidic calcium phosphate mineral, the material
has a porosity of about 85% to about 98%, and the collagen and the
acidic calcium phosphate mineral are dehydrothermally crosslinked;
about 1 part by weight to about 20 parts by weight of collagen
other than that crosslinked with the acidic calcium phosphate
mineral material in the collagen:acidic calcium phosphate mineral
material; and about 2 parts by weight to about 15 parts by weight
of a highly acidic calcium phosphate mineral other than that
crosslinked with collagen in the collagen:acidic calcium phosphate
mineral material.
38. The composition of claim 37, wherein the acidic calcium
phosphate mineral has a Ca:PO.sub.4 ratio from about 0.5 to about
1, and the highly acidic calcium phosphate mineral has a
Ca:PO.sub.4 ratio from about 0.25 to about 0.5.
39. The composition of claim 38, wherein the acidic calcium
phosphate mineral comprises one or more of calcium hydrogen
phosphate dihydrate (CaHPO.sub.4.2H.sub.2O), monocalcium phosphate
(Ca(H.sub.2PO.sub.4).sub.2), calcium pyrophosphate
(2CaO.P.sub.2O.sub.5), tricalcium phosphate (3CaO.P.sub.2O.sub.5),
hydroxyapatite (3.33CaO.P.sub.2O.sub.5(OH).sub.2), tetracalcium
phosphate (4CaO.P.sub.2O.sub.5), or calcium carbonate
(CaCO.sub.3).
40. The composition of claim 37, wherein the highly acidic calcium
phosphate mineral comprises monocalcium phosphate.
41. The composition of claim 37, wherein the average particle size
is about 125 microns to about 300 microns.
42. The composition of claim 37, wherein the material comprises
about 60 wt % to about 75 wt % of the acidic calcium phosphate
mineral.
43. The composition of claim 37, wherein the material has a
porosity of about 94% to about 98%.
44. The composition of claim 37, comprising about 85 parts by
volume to about 95 parts by volume of the collagen:acidic calcium
phosphate mineral material and about 5 parts by volume to about 15
parts by volume collagen.
45. The composition of claim 37, further comprising about 2 parts
by weight to about 150 parts by weight of a fluid.
46. The composition of claim 45, wherein the fluid is bone marrow
aspirate, whole blood, serum, saline, water, or two or more
thereof.
47. The composition of claim 37, further comprising one or more
bone growth proteins.
48. The composition of claim 47, wherein the one or more bone
growth proteins are selected from TGF.beta.1, TGF.beta.2,
TGF.beta.3, bone morphogenic protein (BMP)-2, BMP-3, BMP-4, BMP-5,
BMP-6, BMP-7, BMP-8, BMP-9, cartilage-derived morphogenic protein
(CDMP)-1, CDMP-2, or CDMP-3.
49. A bone growth composition, comprising: at least one bone growth
protein; and an acidic substrate comprising a substrate and at
least one calcium phosphate salt; wherein the at least one calcium
phosphate salt is more than 95 wt % insoluble when 200 mg of the
calcium phosphate are introduced at room temperature to 1 cc of an
aqueous solution at pH 7 prior to addition of the calcium phosphate
and the calcium phosphate does not excessively bind to the at least
one bone growth protein.
50. A bone growth composition, comprising: at least one bone growth
protein; and an acidic substrate comprising a substrate and at
least one calcium phosphate salt, wherein each calcium phosphate
salt has a pKa from about 4 to about 6.5.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of prior
copending U.S. patent application Ser. No. 09/746,921, filed Dec.
22, 2000.
FIELD OF THE INVENTION
[0002] The present invention relates to methods and compositions
for the induction of bone growth in mammals and to methods for the
production of such compositions.
BACKGROUND
[0003] A number of diseases or injuries involving bones are known
for which repair, regeneration, or augmentation of bone is a
desired treatment. Formation of bone in vivo involves an
interaction of various inductive proteins which act by causing a
differentiation of mesenchymal cells into cartilage and then
bone-forming cell lines. This mechanism is not completely
understood. However, in efforts to improve orthopedic procedures,
purified protein mixtures and recombinantly produced proteins have
been developed which stimulate osteoinductive activity.
[0004] In general, autogeneous bone grafts have been viewed as the
standard for restoring skeletal defects. However, autogeneous
sources of bone in human beings are limited, expensive and painful
to obtain. Accordingly, materials such as demineralized bone matrix
have been developed to augment or replace autogeneous bone grafts.
However, an alternative to demineralized bone matrix is desired to
improve the ease of use, economy of product manufacture and to
eliminate the potential of disease transfer or immune system
incompatibilities. To date however, an acceptable substitute has
not been identified.
[0005] Currently the clinical potential of composite implants
containing a mixture of bovine tendon collagen and a proprietary
bone morphogenic protein mixture, with demineralized bone matrix
powders and simulated body fluid is being evaluated. While a number
of advances have improved the activity of osteoinductive factors
such as those present in bone morphogenic protein mixtures, their
clinical application has been limited, in part, by the requirement
for a superior delivery vehicle. Resistance to the use of
demineralized bone matrix in certain cultures, as well as the
desire to enhance the activity of the bone morphogenic protein
mixture to reduce cost, speaks to the desire to develop substitutes
for demineralized bone matrix.
[0006] The present invention provides compositions that can be used
as bone graft substitutes to obtain a product with an improved
osteoinductive response for growth factors in degradable implants
for skeletal regeneration. The compositions of the present
invention are easier to use and more economical to manufacture than
demineralized bone matrix, and they eliminate or significantly
reduce the potential of both disease and pathogen transfer and
immune system incompatibilities.
[0007] Numerous materials have been experimentally evaluated as
alternative delivery vehicles for osteoinductive growth factors.
The materials previously assessed by reconstructive surgeons and
scientists include, without limitation, hydroxyapatites, tricalcium
phosphates, aliphatic polyesters, cancellous bone allografts, human
fibrin, plaster of paris, apatite wollastonite glass ceramics,
titanium, devitalized bone matrix, non-collagenous proteins,
collagen and autolyzed antigen extracted allogenic bone. None of
these materials have been found to be entirely satisfactory.
[0008] Other growth factor carriers containing calcium phosphate
additives have been developed. For example, a macroporous collagen
sponge containing a mixture of a-tricalcium phosphate
(.alpha.-3CaO.P.sub.2O.sub.5) and hydroxyapatite
(3.33CaO.P.sub.2O.sub.5(OH).sub.2) has been developed.
Alternatively, a macroporous collagen sponge that contains
precipitated hydroxyapatite has also been disclosed (U.S. Pat. No.
5,776,193). The composition of such products are consistent with
the prevailing view that hydroxyapatite is the preferred calcium
phosphate source for bone graft substitutes or extenders due to its
compositional similarity with the mineral component of natural
bone.
[0009] There remains a desire for improved compositions for the
induction of bone growth in animals that address the problems of
existing compositions and products.
SUMMARY
[0010] As noted above, hydroxyapatite has long been considered a
preferred source of calcium phosphate in bone graft substitutes.
Indeed, evidence suggests that the inclusion of hydroxyapatite in
bone graft substitutes does provide benefits related to osteoblast
adherence. Thus, hydroxyapatite shares a compositional similarity
to naturally occurring bone mineral and stimulates certain elements
of the bone formation cascade. Certain hydroxyapatite bone graft
substitutes have also been preferred as they are generally
characterized by having a neutral or basic pH when implanted under
normal physiological conditions. Despite these long-believed
potential benefits and the resultant preference for hydroxyapatite
containing bone growth substitutes, the inventor has now shown that
the addition of calcium phosphate materials having a neutral or
basic pH, such as hydroxyapatite, to collagen actually hinders the
osteoinductive activity of bone growth proteins in vivo.
[0011] The present invention is directed to a bone growth
composition which includes an acidic substrate and a bone growth
protein, wherein the bone growth protein is characterized as having
a first activity at neutral or basic pH and a second, higher
activity at acidic pH. The acidic substrate preferably comprises a
source of calcium and a source of phosphate. The composition also
may include an acidic buffering potential in physiological
solution. In another embodiment, the composition further includes a
biocompatible buffering agent to maintain the acidity of the
composition. In further alternative embodiments, the sources of
calcium and/or phosphate can be salts such as monocalcium
phosphate, calcium hydrogen phosphate dihydrate, or calcium
pyrophosphate. The substrate can comprise collagen, fibrin,
alginate, one or more synthetic polymers (such as polyethylene
glycol (PEG), functionalized PEG, aliphatic polyesters, polylactic
acid (PLA), or polyglycolic acid (PGA)), or mixtures thereof. The
bone growth protein can be one or more purified bone growth
proteins (each protein can be purified independently or
collectively from allograft, xenograft, or autograft bone),
recombinantly produced bone growth proteins, or mixtures thereof.
In a preferred embodiment, the bone growth protein includes a
purified bone growth protein composition known as Bone Protein
Mixture or BPM.
[0012] An embodiment of the present invention also includes a
process for producing an implantable bone growth composition. The
process includes producing a dispersion of collagen fibrils
containing a solubilized sodium phosphate salt. The process may
further include adding a calcium chloride salt to the dispersion of
collagen fibrils to precipitate a calcium phosphate salt onto the
surface of the collagen fibrils to produce an implantable bone
growth composition. Alternatively, the process can include making
the dispersion with a calcium salt and adding a phosphate salt.
[0013] The present invention also includes a process for the
induction of bone formation in a mammal, which includes implanting
a bone growth composition of the present invention in a mammal.
Such a process can include the use of the bone growth composition
in a joint replacement operation (e.g., hip, knee, shoulder, elbow,
or ankle, among others), a spinal fusion, repair of periodontal
defects, treatment of osteoporosis, skeletal augmentation, repair
of bone defects, or repair of bone fractures.
[0014] The composition of the present invention and products made
therewith are superior materials for use as a demineralized bone
matrix replacement. It has been found that the calcium source, the
phosphate source, their collective pH, and the acidic buffering
potential each have independent beneficial effects for bone growth
induced by the present composition. In addition, the novel
processing technology for producing such materials produces
collagen sponges with dramatically superior physical properties.
The products are collagen dispersions containing a calcium
phosphate salt on the surface of the collagen fibrils, resulting in
the formation of water stable, collagen sponges with superior
physical properties. Composite products provide both improved
physical properties and superior osteoinductive performance. The
products can be rapidly and cost-effectively manufactured, and can
reduce the required doses of osteoinductive proteins. These
composites provide significant economic savings and eliminate
potential disease transfer due to the elimination of the use of
demineralized bone matrix. Additionally, the composites provide
more reproducible clinical results, allow simpler surgical
application, and better maintain physical dimensions during
use.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1: Indicates location of the subcutaneous implant sites
in the upper quadrants of a rat's abdomen and dorsal thorax.
[0016] FIG. 2: A. Explant mass of disks composed of osteoinductive
compounds at time of harvest, normalized to average value measured
against controls containing only collagen and bone proteins.
[0017] B. Average and normalized values of explant mass at time of
harvest.
[0018] FIG. 3: A. Histology scores of disks composed of
osteoinductive compounds at time of harvest normalized to average
value measured against controls containing only collagen and bone
proteins.
[0019] B. Average and normalized histology scores at time of
harvest.
[0020] FIG. 4: A. Mineral concentration of disks composed of
osteoinductive compounds at time of harvest normalized to average
value measured against controls containing only collagen and bone
proteins.
[0021] B. Average and normalized mineral concentration at time of
harvest.
[0022] FIG. 5: A. Mineral mass of disks composed of osteoinductive
compounds at time of harvest normalized to average value measured
against controls containing only collagen and bone proteins.
[0023] B. Average and normalized mineral mass at time of
harvest.
[0024] FIG. 6: A. Explant mass of disks composed of osteoinductive
compounds at time of harvest normalized to average value measured
against controls containing collagen, bone proteins and devitalized
bone matrix.
[0025] B. Average and normalized values of explant mass at time of
harvest.
[0026] FIG. 7: A. Histology scores of disks composed of
osteoinductive compounds at time of harvest normalized to average
value measured against controls containing collagen, bone proteins
and devitalized bone matrix.
[0027] B. Average and normalized histology scores at time of
harvest.
[0028] FIG. 8: A. Mineral concentration of disks composed of
osteoinductive compounds at time of harvest normalized to average
value measured against controls containing collagen, bone proteins
and devitalized bone matrix.
[0029] B. Average and normalized mineral concentration at time of
harvest.
[0030] FIG. 9: A. Mineral mass of disks composed of osteoinductive
compounds at time of harvest normalized to average value measured
against controls containing collagen, bone proteins and devitalized
bone matrix.
[0031] B. Average and normalized mineral mass at time of
harvest.
[0032] FIG. 10: shows the influence of adding a calcium source, a
phosphate source, or both a calcium and a phosphate source to the
implanted composition at different acidic buffering capacity on the
histology score.
[0033] FIG. 11: shows the influence of adding a calcium source, a
phosphate source, or both a calcium and a phosphate source to the
implanted composition at different acidic buffering capacity on the
relative mineral mass.
[0034] FIG. 12: Box plot representations of average (inter-animal)
and relative (intra-animal) explant mass when supplemented with
various calcium phosphates.
[0035] FIG. 13: Box plot representations of average (inter-animal)
and relative (intra-animal) mineral mass when supplemented with
various calcium phosphates.
[0036] FIG. 14: Box plot representations of average (inter-animal)
and relative (intra-animal) histological scores when supplemented
with various calcium phosphates.
[0037] FIG. 15. Photomicrographs representing Average Histological
differences between samples containing various calcium phosphate
additives (7 mg, 50 wt. %) [2.times. magnification, processed with
H&E/Von Kossa (left)and toluidine blue tissue stains
(right)]
[0038] FIG. 16: Influence of temporary pH of the paste of Example 4
on induced explant and mineral mass values.
[0039] FIG. 17: Influence of temporary pH of the paste of Example 4
on induced bone quality.
[0040] FIG. 18: Overview of natural metabolic regulation of bone
remodeling and healing.
DETAILED DESCRIPTION
[0041]
[0042] The natural process of bone resorption and subsequent bone
reformation occurs throughout a person's life. The process is
initiated by biomechanical stimuli and localized micro-damage to
skeletal tissues. Osteoclast bone resorption locally releases
essential components required for bone repair and bone reformation,
including: soluble calcium [Ca.sup.3+] and phosphate
[PO.sub.4.sup.3-] ions, collagen and osteoinductive bone
morphogenetic proteins (BMPs). The released molecular proteins
(BMPs) sequentially result in increased cellular differentiation of
active osteoblasts. Active osteoblasts deposit organic scaffolds,
referred to as osteoid matrices. In the presence of soluble
[Ca.sup.2+] and [PO.sub.4.sup.3-] ions, osteoid matrices are
gradually converted into bone through the serial precipitation of
calcium phosphate compositions. The first calcium phosphate phase
to precipitate during natural bone formation is calcium hydrogen
phosphate [CaHPO.sub.4 (DICAL)] (Brown. P; Constantz, B.
Hydroxylapatite and Related Materials. Boca Raton, Fla.; CRC Press,
1994, 9; Francis, M.; Webb, N. "Hydroxylapatite formation from a
hydrated calcium monohydrogen phosphate precursors", Calcif Tissue
Res., 6, pps. 335-342, 1971; Johnson, M. S.; Nacollas, G. "The role
of brushite and octacalcium phosphate in apatite formation".
Critical Reviews in Oral Biology and Medicine, 3 [1/2], pps. 61-82
(1992); Brown, W.; Chow, L. C. "Chemical Properties of bone
mineral". Ann. Res. Mater. Sci., 6, pps. 212-226, 1976; Brown, W.;
"Crystal chemistry of Octacalcium Phosphate", Prog Cyrstal Growth
Charact., 4, pps. 59-87, 1981). The calcium hydrogen phosphate
product is then quickly and sequentially transformed into
biomedical calcium phosphate compositions, including tricalcium
phosphate [Ca.sub.3(PO.sub.4).sub.2 (TCP)], octacalcium phosphate
[Ca.sub.4(PO.sub.4).sub.3(OH) (OCP)] and hydroxylapatite
[Ca.sub.5(PO.sub.4).sub.3(OH) (HA)]. FIG. 18 shows an overview of
these processes.
[0043] In one embodiment, the present invention is directed toward
a bone growth composition comprising a bone growth protein having a
first bioactivity; and an acidic substrate; wherein the bone growth
protein has a second bioactivity greater than the first bioactivity
when combined with the acidic substrate. In this embodiment, the
ability of the bone growth protein to induce bone growth is greater
at an acidic pH than it is at a neutral and/or basic pH. The bone
growth composition is particularly useful in processes of the
present invention which include implanting the product in the body
for the purpose of inducing bone formation in vivo. This embodiment
provides an acidic substrate which can be substituted for
demineralized bone matrix as a delivery vehicle that not only
avoids the risks of disease/pathogen transmission associated with
demineralized bone matrix use, but which also enhances the
osteoinductive activity of bone growth proteins. Compositions of
the present invention have been shown to improve both the quantity
(e.g., mass) and quality (e.g., histological score) of bone
produced with bone morphogenic protein at reduced doses.
[0044] 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 non-human
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 a bone growth factor can be
demonstrated by a test using a substrate onto which material to be
tested is deposited. For example, osteoinductive activity can be
graded or scored as disclosed in U.S. Pat. No. 5,290,763 or as is
described below in the Examples.
[0045] The bone growth composition comprises an acidic substrate
and includes a bone growth protein. In certain embodiments, the
substrate can provide a structure for the growth of bone and an
acidic environment to enhance the activity of the bone growth
protein. In such embodiments, the bone growth protein can be highly
active in the acidic environment of the composition and induces the
production of bone.
[0046] The acidic substrate of the bone growth composition provides
a structure for the various other components of the composition and
also allows for the ingrowth of bone induced by the composition.
More particularly, the substrate can be a matrix forming material,
such as collagen, fibrin or alginate. A preferred substrate is
collagen and a preferred collagen is Type I bovine tendon
atelocollagen or Type I bovine dermal atelocollagen.
[0047] The acidic substrate can also comprise a compound which
renders the substrate acidic. In one embodiment, the acidic
substrate comprises a calcium source and a phosphate source. In a
preferred embodiment, the acidic substrate comprises an acidic
calcium phosphate compound. The calcium and phosphate of the
compound provide an available supply of these ions for the
production of bone.
[0048] In embodiments wherein the composition includes sources of
calcium and phosphate, these sources can be used to locally enhance
the soluble concentration of dissolved calcium [Ca.sup.2+] and
phosphate [PO.sub.4.sup.-] within and around the site of
implantation of the composition. Natural bone acts as a reservoir
to, inter alia, maintain constant serum concentrations of these
components. It has been theorized that the rate of bone formation
may be limited by the diffusion of these critical ions to the site
of bone induction. In brief, bone mineralization may exhaust the
local serum concentration of calcium and phosphate, after which
bone mineralization is limited by the rates of both osteoclast
resorption of local bone (to provide soluble calcium and phosphate
ions) and diffusion of these ions to the site of bone induction. By
specifically enhancing the local concentration of these critical
components, such as by the use of sparingly soluble calcium
phosphate additives, the amount (mass) and quality of induced bone
formation can be enhanced.
[0049] Preferred sources of calcium for the composition of the
present invention include essentially any acidic calcium salt,
including calcium phosphate or calcium citrate. Particularly
preferred sources of calcium include acidic calcium phosphate
salts. Preferred acidic calcium phosphate salts include monocalcium
phosphate, calcium phosphate dihydrate (also known as dical), and
calcium pyrophosphate. Typically, the calcium source is present in
the composition in an amount of between about 1% by weight and
about 85% by weight. In one embodiment, the calcium source is
present from about 45 wt % to about 85 wt %. In a further
embodiment, the calcium source is present from about 50 wt % to
about 80 wt % . In yet a further embodiment, the calcium source is
present from about 55 wt % to about 75 wt %. In still a further
embodiment, the calcium source is present from about 60 wt % to
about 70 wt %.
[0050] Preferred sources of phosphate for the composition of the
present invention include essentially any phosphate salt, including
calcium phosphate or sodium phosphate. Particularly preferred
sources of phosphate include calcium phosphate salts, and more
particularly preferred sources of phosphate include acidic calcium
phosphate salts. Preferred acidic calcium phosphate salts include
calcium hydrogen phosphate dihydrate, monocalcium phosphate,
calcium pyrophosphate, or a mixture of two or more thereof.
[0051] Typically, the phosphate source is present in the
composition in an amount of between about 1% by weight and about
85% by weight. In one embodiment, the phosphate source is present
from about 45 wt % to about 85 wt %. In a further embodiment, the
phosphate source is present from about 50 wt % to about 80 wt %. In
yet a further embodiment, the phosphate source is present from
about 55 wt % to about 75 wt %. In still a further embodiment, the
phosphate source is present from about 60 wt % to about 70 wt
%.
[0052] The calcium source and the phosphate source can be the same
material.
[0053] As noted above, preferred sources of calcium and of
phosphate include acidic calcium phosphate salts. Calcium
phosphates can be represented by the general chemical formula of
xCaO.P.sub.2O.sub.5. The sparingly soluble calcium phosphate salts
act as solution buffers. Thus, as the salts increase in calcia
concentration (CaO), the pH increases from approximately 2 (x=1) to
11 (x=4). It is believed that the alkaline buffering nature of
hydroxyapatite (x=3.33, about pH 9) reduces the performance of bone
growth proteins. It has been found that acidic calcium phosphate
salts (i.e., monocalcium phosphate (Ca(H.sub.2PO.sub.4).sub.2),
calcium hydrogen phosphate dihydrate (CaHPO.sub.4.2H.sub.2O) and
calcium pyrophosphate (2CaO.P.sub.2O.sub.5)) stimulate the
osteoinductive performance of bone growth proteins. In comparison
to collagen composites containing devitalized bone matrix
additives, collagen dispersions containing calcium hydrogen
phosphate (CaHPO.sub.4.2H.sub.2O) have resulted in superior bone
quality.
[0054] Alternatively, in one embodiment, the acidic calcium
phosphate compound has a Ca:PO.sub.4 ratio from about 0.5 to about
1. In another embodiment, the acidic calcium phosphate compound is
a highly acidic calcium phosphate mineral, by which is meant an
acidic calcium phosphate mineral having a Ca:PO.sub.4 ratio from
about 0.25 to about 0.5. In a further embodiment, the acidic
calcium phosphate has a Ca:PO.sub.4 ratio from about 0.3 to about
0.4. The acidic calcium phosphate compound tends to lower the pH of
the composition, and in the absence of other compounds that are
basic, the composition will have an acidic pH.
[0055] Thus, in preferred embodiments, the composition of the
present invention uses calcium phosphate salts to (1) control local
pH (to enhance/control bone growth factor release activity by
providing protons to biological tissues within about 0.1 mm to
about 5 cm of the site of implantation), (2) locally enhance
soluble calcium concentration (which increases bone production
within about 0.1 mm to about 5 cm of the site of implantation), and
(3) locally enhance soluble phosphate concentration (which also
increases bone production within about 0.1 mm to about 5 cm of the
site of implantation). In comparison to collagen composites
containing devitalized bone matrix additives, collagen dispersions
containing acidic calcium phosphates have been developed that
stimulate the formation of larger explants containing bone of
superior quality. As noted above, control of each of the foregoing
three factors independently can be used to enhance bone production
by the bone growth composition. Accordingly, acidic mineral salts
other than calcium phosphate salts can be used to control pH,
thereby increasing the bone morphogenic activity of bone growth
proteins without providing additional calcium or phosphate.
Additionally, other buffering agents (e.g., a sulfate-based buffer)
or acidifying agents (e.g., lactic acid) can be used to control the
local pH surrounding the composition in the absence of a calcium
source, in the absence of a phosphate source, or in the absence of
a calcium phosphate source. Similarly, the use of specific calcium
salts (e.g., calcium citrate) which do not incorporate phosphorus
can be used without regard to control of local pH or phosphate
concentration. Likewise, the use of non-calcium phosphate salts
(i.e., sodium phosphate salts) can be used to enhance local
concentrations of phosphate ions to enhance bone morphogenic
activity without specifically controlling local pH or calcium
concentration. Each of these three factors (the addition of a
calcium source, the addition of a phosphate source, and the control
of the local pH) leads to increased bone production or growth
independently of one another (see, e.g., FIGS. 10 and 11). The bone
growth and production enhanced by these three factors may be
increased in quantity (e.g., as evidenced by increased relative
mineral mass) or quality (e.g., as evidenced by increased relative
histology score) or may be increased in both quantity and quality.
Furthermore, the effects on bone growth enhanced by these three
factors are separately additive. Thus, the combination of any two
of the three factors in the final composition will increase the
production of bone above the bone growth seen with anyone of the
factors independently.
[0056] A further aspect of the composition of the present invention
is the acidic buffering potential in physiological solutions. More
particularly, when the composition of the present invention is put
into a solution, such as a bodily fluid at physiological pH (e.g.,
in an in vivo application) or another weakly basic solution, the
composition acts to buffer the solution at an acidic pH (i.e., the
pH of the composition is less than 7). Additionally, if the
composition is implanted into a mammal, the composition can buffer
the environment within, on, or surrounding the implanted
composition to an acidic pH. More particularly, the present
compositions can buffer such solutions in the surrounding
environment to a pH between about 2 and about 7, preferably between
about 2 and about 5, more preferably between about 2 and about 4.9,
such as about 2 and 4.9, and most preferably between about 3.5 and
about 4.7. Control of the pH of the compositions can be achieved by
those skilled in the art using routine techniques. For example, the
use of buffering agents to maintain a desired pH range is
well-known. Because compositions of the present invention can be
used for in vivo applications, such buffering agents desire to be
biocompatible. Particularly preferred buffers are discussed in more
detail below.
[0057] The pKa of calcium monophosphate is known to be about 4.2,
and thus its approximate buffering range is about pH 3.2 to 5.2. A
composition composed of primarily calcium monophosphate is strongly
acidic (e.g., pH<4). For reference, standard pKa values of
various calcium phosphate salts are approximately as follows:
TABLE-US-00001 Ca(H.sub.2PO.sub.4).sub.2 (monocalcium phosphate;
MCP) pKa = 4.2 CaHPO.sub.4.2H.sub.2O (dicalcium hydrogen phosphate;
DCP) pKa = 6.5 3CaO.P.sub.2O.sub.5.2H.sub.2O (tricalcium phosphate;
TCP) pKa = 26.0 3.33CaO.P.sub.2O.sub.5.2H.sub.2O (hydroxyapatite;
HA) pKa = 57.8 4CaO.P.sub.2O.sub.5 (tetracalcium phosphate; TTCP)
pKa = 30.6
[0058] In one embodiment of the composition, each calcium phosphate
salt has a pKa from about 4 to about 6.5.
[0059] In one embodiment, the calcium phosphate is selected from
sparingly soluble calcium phosphates that do not excessively bind
to osteoinductive proteins. A mixture of such calcium phosphates
can be used. Such a calcium phosphate provides a reserve of calcium
and phosphate ions which can be slowly resorbed during the healing
process and does not interfere significantly with the activity of
osteoinductive proteins.
[0060] Biologically compatible, sparingly soluble calcium
phosphates are suitable supplements to locally increase the supply
of soluble calcium [Ca.sup.2+] and phosphate [PO.sub.4.sup.3-]
ions. As shown in Table 1, calcium phosphate salts solubilize at
different equilibrium ionic concentrations. Despite the fact that
the local supplemented concentrations of calcium [Ca.sup.2+] and
phosphate [PO.sub.4.sup.3-] ions can vary by more than four orders
of magnitude, the limited solubility of calcium phosphates ensures
that only a minor fraction of the mineral is solubilized. This
allows the calcium phosphate to continue to supplement the soluble
mineral pool during months of expected healing.
[0061] In summary, like TCP and HA, calcium hydrogen phosphate
[CaHPO.sub.4 (DICAL)] provides local concentrations of [Ca.sup.2+]
and [PO.sub.4.sup.3-] for bone healing. At the same time, DICAL's
resorption rate is essentially equivalent to conventional
tricalcium phosphate and hydroxylapatite BVF supplements.
TABLE-US-00002 TABLE 1 Equilibrium solubility of calcium and
phosphate ions from several different biologically compatible
calcium phosphate salts Equilibrium Equilibrium Insoluble fraction
[Ca.sup.2+] [PO.sub.4.sup.3-] [200 mg/cc] Plasma 2,200.0 .mu.M
1,100.0 .mu.M -- Ca(H.sub.2PO.sub.4).sub.2 14,300.0 .mu.M 28,600.0
.mu.M 97.0000 wt. % (Monocal) CaHPO.sub.4 (DICAL) 480.0 .mu.M 480.0
.mu.M 99.9700 wt. % Ca.sub.3(PO.sub.4).sub.2 (TCP) 1.4 .mu.M 0.9
.mu.M 99.9999 wt. % Ca.sub.5(PO.sub.4).sub.3(OH) (HA) 2.2 .mu.M 1.3
.mu.M 99.9999 wt. % Ca.sub.4(PO.sub.4).sub.2(OH).sub.2 (TTCP) 28.2
.mu.M 14.1 .mu.M 99.9994 wt. %
[0062] In one embodiment, the calcium phosphate can be any calcium
phosphate which is more than 95 wt % insoluble when 200 mg of the
calcium phosphate are introduced at room temperature to 1 cc of an
aqueous solution at pH 7 prior to addition of the calcium
phosphate. In a further embodiment, the calcium phosphate can be
any calcium phosphate which is more than 99 wt % insoluble when 200
mg of the calcium phosphate are introduced at room temperature to 1
cc of an aqueous solution at pH 7 prior to addition of the calcium
phosphate. In yet a further embodiment, the calcium phosphate can
be any calcium phosphate which is more than 99.9 wt % insoluble
when 200 mg of the calcium phosphate are introduced at room
temperature to 1 cc of an aqueous solution at pH 7 prior to
addition of the calcium phosphate. Any such calcium phosphate,
however, must further not excessively bind osteoinductive
proteins.
[0063] Various forms of calcium phosphates are known to have
different chemical affinities for endogenous osteoinductive
proteins (such as BMPs). A study was performed to assess the
influence of variable composition calcium phosphate salts on the
soluble concentration of osteoinductive proteins. This study
measured the residual concentration of soluble recombinant BMP-2
after exposing a controlled concentration aliquot to an equimolar
quantity of calcium phosphate salt. According to the results in
Table 2, moderately acidic calcium phosphates salts, like DICAL,
preserve greater than a 50 wt % soluble concentration of rhBMP-2,
i.e., do not excessively bind to osteoinductive proteins. Though
not to be bound by theory, we submit the enhanced local
concentration and cellular availability of bone morphogenetic
proteins (BMPs) would better stimulate bone formation.
TABLE-US-00003 TABLE 2 Equilibrium solubility of osteoinductive
recombinant human BMP-2 protein in the presence of equimolar
concentrations of various calcium phosphates. [rhBMP-2] mg/ml
[rhBMP-2] % Control 15.0 100% Ca(H.sub.2PO.sub.4).sub.2 15.0 100%
(MONO) CaHPO.sub.4 11.4 76% (DICAL) Ca.sub.3(PO.sub.4).sub.2 (TCP)
3.5 23% Ca.sub.5(PO.sub.4).sub.3(OH) (HA) 2.3 15%
[0064] As used herein, the term "osteoinductive material" refers to
one or more proteins capable of inducing bone formation when
implanted in a body. Suitable bone growth proteins of the present
invention can be produced by purification of naturally occurring
proteins (from xenograft, allograft, or autograft) or by
recombinant DNA techniques. As used herein, the term recombinantly
produced bone growth protein refers to the production of bone
growth protein using recombinant DNA technology.
[0065] A number of naturally occurring proteins from bone or
recombinant bone growth proteins have been described in the
literature and are suitable. Recombinantly produced bone growth
proteins have been produced by several entities. Creative
Biomolecules of Hopkinton, Mass., USA produces a bone growth
protein referred to as Osteoinductive Protein 1 or OP 1. Genetics
Institute of Cambridge, Mass., USA produces a series of bone growth
proteins referred to as Bone Morphogenic Proteins 1-8 which are
described in U.S. Pat. No. 5,106,748. Purified bone growth proteins
have been developed by several entities. Collagen Corporation of
Palo Alto, Calif., USA developed a purified protein mixture which
is believed to have osteoinductive 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 osteoinductive 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 osteoinductive and which is described in
U.S. Pat. No. 4,804,744. All of the foregoing patents are
incorporated herein by reference.
[0066] A preferred bone growth protein of the present invention and
process for making the same is described in detail in U.S. Pat. No.
5,290,763, which is incorporated herein by reference. Protein
mixtures prepared in accordance with the disclosure of U.S. Pat.
No. 5,290,763 are referred to herein as "Bone Protein Mixture" or
"BPM." This bone growth protein is preferred because of its high
osteoinductive activity and because it is a purified bone growth
protein. The Bone Protein of U.S. Pat. No. 5,290,763 exhibits
osteoinductive activity at about 3 micrograms when deposited onto a
suitable carrier and implanted subcutaneously.
[0067] Yet another embodiment of the preferred bone growth protein
of the invention as described in U.S. Pat. No. 5,290,763 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 bone growth protein has an
amino acid composition of about 20.7 to about 26.1 (preferably
about 23.4) mole percent of ASP (+ASN) and GLU(+OLN); 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 bone growth protein is a protein
mixture having the approximate amino acid composition shown in
Table 3. TABLE-US-00004 TABLE 3 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
[0068] In a further embodiment, the bone growth protein of the
present invention is a "TGF.beta. superfamily protein" which can be
any protein of the art-recognized superfamily of extracellular
signal transduction proteins that are structurally related to
TGF.beta.1-5. Preferably, a TGF.beta. superfamily protein suitable
for use in the present invention is selected from the following
proteins: TGF.beta.1, TGF.beta.2, TGF.beta.3, bone morphogenic
protein (BMP)-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9,
cartilage-derived morphogenic protein (CDMP)-1, CDMP-2, and/or
CDMP-3.
[0069] Other bone growth proteins that can be used in the bone
growth protein mixture include fibroblast growth factor (FGF)-1,
BMP-1, BMP-2.alpha., BMP-2.beta., BMP-3b, BMP-8b, BMP-10, BMP-11,
BMP-12, BMP-13, BMP-14, BMP-15, TGFP.beta.4, TGF.beta.5.
[0070] The amount or dose of bone growth protein used depends on
the activity of the bone growth protein and the particular
application. In the case of the bone growth protein identified in
U.S. Pat. No. 5,290,763, the bone growth protein is used in amounts
between about 10 micrograms/gram substrate and about 10,000
micrograms/g substrate, more preferably between about 100
micrograms/g substrate and about 350 micrograms/g substrate, and
more preferably between about 150 micrograms/g substrate and about
250 micrograms/g substrate.
[0071] In another embodiment, the bone growth composition can
comprise between about 0.5 micromol of the acidic calcium phosphate
compound per 1 microgram of total bone growth protein to about 6
micromol of the acidic calcium phosphate compound per 1 microgram
of total bone growth protein.
[0072] It has been determined by the present inventor that solution
pH plays a strong role in the osteoinductive performance of bone
growth proteins, with acidic environments providing dramatically
superior results. In other words, the bone growth protein has a
second bioactivity in an acidic environment, such as that provided
in its environment by the acidic substrate, greater than a first
bioactivity in a neutral or basic environment.
[0073] In one embodiment, the composition includes:
[0074] about 3 parts by weight to about 10 parts by weight of a
collagen:acidic calcium phosphate mineral material having an
average particle size of about 125 microns to about 5000 microns,
wherein the material comprises about 25 wt % to about 75 wt % of
the acidic calcium phosphate mineral, the material has a porosity
of about 85% to about 98%, and the collagen and the acidic calcium
phosphate mineral are dehydrothermally crosslinked;
[0075] about 1 part by weight to about 20 parts by weight of
collagen other than that crosslinked with the acidic calcium
phosphate mineral material in the collagen:acidic calcium phosphate
mineral material; and
[0076] about 2 parts by weight to about 15 parts by weight of a
highly acidic calcium phosphate mineral other than that crosslinked
with collagen in the collagen:acidic calcium phosphate mineral
material.
[0077] The collagen:acidic calcium phosphate mineral material can
have from about 33 mg acidic calcium phosphate mineral per 100 mg
collagen to about 300 mg acidic calcium phosphate mineral per 100
mg collagen.
[0078] In one embodiment, the acidic calcium phosphate mineral has
a Ca:PO.sub.4 ratio from about 0.5 to about 1, and the highly
acidic calcium phosphate mineral has a Ca:PO.sub.4 ratio from about
0.25 to about 0.5. The acidic calcium phosphate mineral can
comprise one or more of calcium hydrogen phosphate dihydrate
(CaHPO.sub.4.2H.sub.2O), monocalcium phosphate
(Ca(H.sub.2PO.sub.4).sub.2), calcium pyrophosphate
(2CaO.P.sub.2O.sub.5), tricalcium phosphate (3CaO.P.sub.2O.sub.5),
hydroxyapatite (3.33CaO.P.sub.2O.sub.5(OH).sub.2), tetracalcium
phosphate (4CaO.P.sub.2O.sub.5), or calcium carbonate (CaCO.sub.3),
and the highly acidic calcium phosphate mineral can comprise
monocalcium phosphate. The pH of the composition can be tuned by
the amount of the highly acidic calcium phosphate mineral used.
[0079] In further embodiments, the collagen:acidic calcium
phosphate mineral material can have an average particle size of
about 125 microns to about 300 microns, the material can comprise
about 60 wt % to about 75 wt % of the acidic calcium phosphate
mineral, and the material can have a porosity of about 94% to about
98%. In other words, the material can comprise about 150 mg acidic
calcium phosphate mineral per 100 mg collagen to 300 mg acidic
calcium phosphate mineral per 100 mg collagen.
[0080] In a further embodiment, the composition can comprise about
85 parts by volume to about 95 parts by volume of the
collagen:acidic calcium phosphate mineral material and about 5
parts by volume to about 15 parts by volume collagen.
[0081] The composition can be further combined with a fluid,
especially suitably for injection at a bone defect site. In one
embodiment, the composition further comprises about 2 parts by
weight to about 150 parts by weight of a fluid comprising water or
an organic solvent. The fluid can include bone marrow aspirate,
whole blood, plasma, platelet-rich plasma (PRP), serum, saline,
water, PBS, cell culture media, or two or more thereof.
[0082] The composition can also further comprise one or more bone
growth proteins as described above.
[0083] In another embodiment, the present invention relates to a
method of forming a bone growth composition including combining a
bone growth protein having a first bioactivity with an acidic
substrate, wherein the bone growth protein has a second bioactivity
when combined with the acidic substrate that is greater than the
first bioactivity.
[0084] The composition of the present invention can be in a variety
of different forms, such as a sponge, a paste, a fleece, or
particles, among others, comprising natural materials such as
collagen or chitin, among others, or synthetic materials such as
PLA or PGA, among others. In a preferred embodiment, a collagen
sponge is provided which contains bone growth proteins as well as
calcium phosphate salts for controlling pH and providing calcium
and phosphate to the local environment. An example of how to
prepare such a sponge is provided below.
[0085] Another embodiment of the present invention is a novel
process to produce collagen sponges for implantation which
incorporate the replacement materials generally described above. In
one embodiment, the products are prepared by producing a dispersion
of collagen fibrils that contains either solubilized calcium salts
or solubilized phosphate salts. Suitable collagen can include type
I collagen, type II collagen, type III collagen, or type N
collagen. In one embodiment, the collagen is from bovine tendon.
The collagen dispersion is typically between about 0.5% by weight
and about 20% by weight collagen, more preferably between about 1%
by weight and about 10% by weight collagen, and most preferably
between about 3% by weight and about 5% by weight collagen.
[0086] If the dispersion was made with a calcium salt, a phosphate
salt is then added to the dispersion to heterogeneously precipitate
a calcium phosphate salt directly onto the surface of the collagen
fibrils. If the dispersion was made with a phosphate salt, a
calcium salt is then added to the dispersion to heterogeneously
precipitate a calcium phosphate salt directly onto the surface of
the collagen fibrils. The interfacial adherence of the precipitate
improves the mechanical rigidity and wetability of the composite
sponges. The composition can be cross-linked. In one embodiment,
the application of dehydrothermal collagen cross-linking techniques
(e.g., 110.degree. C., 24-72 hrs, vacuum) are well known in the
art. Such cross-linking techniques result in the formation of water
stable, collagen sponges of superior physical properties. Such
sponges can then be loaded with bone growth protein and used for
induction of bone growth in vivo. In a preferred embodiment, the
products are prepared by producing a 4% (by weight) collagen
dispersion that contains solubilized calcium dichloride dihydrate
(CaCl.sub.2.2H.sub.2O). A solution of disodium phosphate
(Na.sub.2HPO.sub.4) is added to the heterogeneously precipitate
calcium hydrogen phosphate dihydrate (CaHPO.sub.4.2H.sub.2O)
directly onto the surface of collagen fibrils.
[0087] In one embodiment, the composition can be formed starting
with dicalcium hydrogen phosphate [CaHPO.sub.4xH.sub.2O, (DICAL)]
(Sigma, St. Louis, Mo.), monocalcium phosphate
[Ca(H.sub.2PO.sub.4).sub.2 (MONOCAL)] (Sigma, St. Louis, Mo.),
bovine dermal collagen, (Kensey Nash Corporation, Exton, Pa.),
hydrochloric acid, and distilled, de-ionized water. After
sterilization of the calcium phosphates and preparation of a 30 mM
hydrochloric acid solution, a cross-linked, collagen-calcium
phosphate sponge particle can be prepared.
[0088] First, a composite collagen-calcium phosphate gel dispersion
(5 vol % collagen gel) can be prepared by introducing collagen and
dicalcium hydrogen phosphate to one syringe, the 30 mM HCl solution
to another, and mixing between the two. The dispersion can then be
cast in a mold and frozen (-80.degree. C. for at least 1 hr),
followed by lyophilization/freeze-drying. The samples can then be
dehydrothermally cross-linked in a vacuum oven (110.degree. C., 48
hr) and thereafter milled and collected with -20 mesh (typically,
about 0.5-1.2 micron).
[0089] Second, a high surface area (HSA), soluble collagen particle
preparation can be prepared by dual-syringe mixing of collagen and
sufficient 30 mM HCl to yield a 2 vol % solid dispersion. The
dispersion can then be cast, frozen, and lyophilized/freeze-dried
as described above. The samples can then be milled and collected
with -60 mesh.
[0090] Third, the soluble HSA collagen particles (about 60 weight
parts), collagen-DICAL DHT cross-linked particles (about 530 weight
parts), and monocalcium phosphate powder (about 100 weight parts)
can be combined to form a final dry powder.
[0091] The final dry powder can then be combined with various
fluids to yield a putty or a paste, according to Table 4:
TABLE-US-00005 TABLE 4 Per 1 cc dry powder volume, mix the
approximate volume of the specific fluids to obtain cohesive
putties. Putty consistency Saline 0.4 ml Phosphate buffered saline
0.4 ml Whole blood 0.6 ml Paste Consistency Saline 1.0 ml Phosphate
buffered saline 1.0 ml Whole blood 1.3 ml
[0092] Alternative processes for producing the composition of the
present invention are possible.
[0093] Another process of the present invention includes implanting
a composition as broadly described above into a body for induction
of bone growth. The composition can be combined with a fluid, which
can include bone marrow aspirate, whole blood, plasma,
platelet-rich plasma (PRP), serum, saline, water, PBS, cell culture
media, or two or more thereof. As noted above, most uses of the
present invention are concerned with human applications. The
process, however, is suitable for a wide variety of animals, such
as vertebrates, such as mammals, of which humans are one example.
As used herein, the term implanting refers to placing the
composition of the present invention in any bone defect or other
area in which it is desired to have bone grow or survive. By
implanting composition, bone formation is induced by the bone
growth protein. Over time, preferred calcium and phosphate
materials are resorbed allowing for uniform bone formation
throughout a defect area.
[0094] In another embodiment, the present invention relates to a
method of promoting bone growth at a bone defect in a mammal
including combining a bone growth protein having a first
bioactivity with an acidic substrate, wherein the bone growth
protein has a second bioactivity greater than the first bioactivity
when combined with the acidic substrate; and delivering the bone
growth protein to the bone defect.
[0095] The combining and delivering steps can be performed
sequentially or simultaneously. The delivering step can also be
considered an implanting step.
[0096] Compositions of the present invention can be used in a
variety of applications whenever there is a desire to generate bone
or retard bone loss. 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
and repair of bone fractures and defects.
[0097] 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 stem which is inserted into the proximal
end of the femur from which the natural ball portion was removed.
The stem can have a porous surface so that bone growth around the
stem can anchor the stem in the femur. The product of the present
invention, in particulate form, is layered, packed, or injected
between the stem and the cavity in the femur in which stem 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. The product of the present
invention, in particulate form, is placed in the natural socket
cavity so that upon placement of the artificial socket, the product
is between the natural and artificial socket. In this manner, as
bone is formed, the artificial socket is anchored in the natural
socket.
[0098] Products 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.
The product of the present invention is layered, packed, or
injected 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.
[0099] Products 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 product
can be applied, for example, between adjacent spinous and
transverse processes so that upon bone formation throughout the
composite material, two adjacent vertebrae are joined by fusion
between the respective spinous processes and transverse
processes.
[0100] In the case of periodontal defects, the product of the
present invention is conformed to the defect shape. As bone growth
is induced, bone fills in the defect.
[0101] In the treatment of osteoporosis, the present product is
injected in existing bone to offset the effects of osteoporosis in
which bone density is lost. For example, if it is determined that
bone density is low in a localized area, such an injection can be
made in that area.
[0102] In the treatment of bone fractures, traumatic osseous
defects, or surgically-created osseous defects, the product of the
present invention is layered, packed, or injected into the fracture
or defect. In this manner, as bone formation is induced, the
fracture or defect is treated.
[0103] In performing the method, it may be convenient to provide to
the surgeon performing the delivering step to be provided with a
precursor to the composition in kit form. Therefore, in another
embodiment, the present invention relates to a kit for promoting
bone growth at a bone defect in a mammal, comprising a bone growth
protein having a first bioactivity and an acidic substrate, wherein
the bone growth protein has a second bioactivity greater than the
first bioactivity when combined with the acidic substrate.
[0104] The following examples are provided for the purposes of
illustration and are not intended to limit the scope of the present
invention.
EXAMPLES
Example 1
[0105] This example illustrates the production and use of bone
growth protein containing devices that provide equivalent or
superior osteoinductive performance without the addition of
demineralized bone matrix additives as biologic supplements.
[0106] This example shows the influence of the carrier vehicle on
the in vitro and in vivo osteoinductivity attributable to
osteoinductive growth factors. An accepted protocol to assess the
osteoinductive activity of composite materials is through
implantation of samples in rats. The advantages of the rat model
for product evaluation include its moderate cost and an accelerated
rate of bone induction. Visible evidence of mineralization appears
in the implant within several days (10), with typical experiments
lasting between 14 and 21 days. Osteoinductive activity is commonly
evaluated using four standard test protocols: histological tissue
analysis, mineral concentration via x-ray and ash weight evaluation
and bone cell activity via alkaline phosphatase analysis.
[0107] This example specifically compared the osteoinductive
differences between implant samples containing Bone Protein Mixture
(BPM), collagen (bovine tendon type 1) and a powder of either
devitalized bone matrix (DVBM) or the calcium phosphate ceramic
(Ostite) [Millennium Biologix, Kingston, Canada]. Ostite is a
material containing variable concentrations of calcium
hydroxyapatite and silica stabilized tricalcium phosphate.
Similarly to alternative calcium phosphate sources, Ostite supports
the required interfacial activity of osteoblasts for bone
regeneration. A unique feature of Ostite is that it has been shown
to degrade only by osteoclastic resorption. Sample disks were
prepared with variable composition: a) collagen (100 wt. % )/BPM
and b) collagen/particle (50/50 wt. %)/BPM. The sample disks were
prepared using two distinct processing techniques. In the first
technique, the components were mixed in phosphate buffered saline
(PBS) at a collagen ratio of 4 wt. %. The mixtures were molded into
disks (h.about.3 mm, d.about.8 mm) and freeze dried. In the second
technique, the components were mixed with dilute acetic acid (1
vol. %) to form a gel with a collagen ratio of 4 wt. %. The gels
were molded into disks and freeze-dried. All disks were loaded with
BPM and freeze dried according to standard protocols.
[0108] The testing protocol involved sample implantation in
subcutaneous (to assess endochondral bone formation) and calvaria
sites (to assess membranous bone formation). The osteoinductive
responses were evaluated after 4 weeks implantation using accepted
protocols for explant mass, ash weight, x-ray mineral density and
histology.
[0109] Clinically, the application of bone morphogenic proteins
(BMPs) and other osteoinductive growth factors are desired to
assist in the surgical reconstruction of skeletal defects. BMPs are
advantageous because they induce bone formation by targeting and
activating undifferentiated perivascular connective tissue cells.
In contrast, mitogens target and accelerate the osteoinductive
activity of previously differentiated cells. Numerous advances have
improved the activity of osteoinductive factors, however, their
clinical application has been limited by the requirement for a
superior delivery vehicle.
Procedures:
Collagen Implants:
[0110] Collagen sponge disks were prepared according to standard
procedures as follows; Mix 12.0 g of 1 vol. % glacial acidic acid
and 500 mg of Bovine tendon Type 1 Collagen in an inert screw cap
container. Mix with a spatula as the gel begins to form, minimizing
the number of trapped air bubbles. Stop mixing when the gel becomes
thick. Tap gel container on bench-top to remove trapped air bubbles
and cap tightly. Allow mixture to sit for at least 1 hour at room
temperature.
[0111] To make disks from the collagen dispersion, place a Delrin
disk mold sheet on a glass plate and press the dispersion into the
holes. Remove excess dispersion with a knife or spatula. Place the
molding sheet and glass plate in a freezer at -80.degree. C. for
approximately 1 hour. Remove from the freezer and allow warming for
approximately 1 minute. Remove the glass plate and place the Delrin
plate into a freeze drying flask. Freeze dry for a minimum of 42
hours. After drying, remove the samples from the plate, trim the
edges and weigh each disk. Each disk must weigh between 6.5 to 7.3
mg to be acceptable for use.
Collagen/Powder Implants:
[0112] In an inert screw cap container, mix 600 mg of Bovine tendon
Type 1 collagen with 600 mg of either Ostite powder (NP) or
devitalized rat bone matrix powder (DVM). Add 14.4 g of acetic acid
(1 vol. %) to prepare gel dispersions containing 4 wt. % collagen.
Stir with a spatula to homogenize the mixtures and to adequately
wet the components. Vibrate the mixtures on a high intensity
orbital shaker to remove trapped air bubbles. Allow mixtures to sit
for at least 1 hour at room temperature.
[0113] To make disks from the collagen dispersions, place a Delrin
disk mold sheet on a glass plate and press the mixtures into the
holes. Remove excess mixture with a knife or spatula. Place the
molding sheet and glass plate in a freezer at -80.degree. C. for
approximately 1 hour. Remove from the freezer and allow warming for
approximately 1 minute. Remove the glass plate and place the Delrin
plate into a freeze-drying flask. Freeze dry for a minimum of 12
hours. After drying, remove the samples from the plate, trim the
edges and weigh each disk. The disks must weigh between 13.0-14.6
mg to be acceptable for use.
BPM Loading:
[0114] Dilute a volume of BPM (produced as described in U.S. Pat.
No. 5,290,763) with a volume of 10 mM HCl to prepare solutions of
10 mg BPM/100 ml (15 ml) and 35 mg BPM/100 ml (4.0 ml). In the
Delrin loading plate, pipet 50 .mu.l of a solution on each of the
top and bottom half of a collagen sponge (n=240 (10 mg), n=48 (35
mg)). Allow disks to stand in a chamber containing a moist paper
towel (to prevent drying and sponge shrinkage) at ambient
temperatures for 40-60 minutes. Cover the disk holding plate with
Saran Wrap and place in a -80.degree. C. freezer for 40-60 min.
Unwrap and carefully place in a freeze dryer flask. Freeze dry for
a minimum of 12 hours then remove. The implant samples will
respectively contain total BPM doses of 10 ng and 35 ng.
[0115] Surgical controls were used to determine the osteoinductive
response in the calvaria implants due to irritation of the
periosteum. A solution of 10 mM HCl was prepared and sterilized by
filtration through a 0.2 mm sterile syringe filter. The solution
was applied to the collagen disks in an identical manner as the BPM
loaded samples and served as negative controls.
Sample Disk Implantation
[0116] The weight of each Long-Evans rat was recorded. Acceptable
rats for bioassays weigh between 100 and 130 g. The animals was
anesthetized with 400 .mu.l of pentobarbital dosing solution
injected i.p.
[0117] Subcutaneous sample implantation was made as follows: small
(6 mm) incisions were made in the skin of the ventral or dorsal
thorax. Ventral incisions were made at the base of the rib cage. A
template, to be aligned with the base of the rib cage, was provided
to identify constant dorsal implant locations. After incision, a
pocket beneath the skin and above the incision was prepared by
blunt dissection. The loaded collagen sponges were placed in the
pocket, approximately 5 mm above the incision. Additional incisions
and implant insertions were made and then the incisions were closed
with Tedvek II 5-0 (or equivalent) sutures.
[0118] The animals were housed in compliance with the guidelines
described in QC-008. The animals were checked for lesions 3-5 days
post implantation. If lesions were detected or if animal death
occurred before sacrifice, these results were documented.
Implantation Protocol and Analysis
[0119] The testing protocol involved subcutaneous implantation of
collagen sponges (to assess endochondral bone formation) containing
10 .mu.g BPM. The samples were placed in four subcutaneous
implantation sites: the upper quadrants of a rat's abdomen and
dorsal thorax [FIG. 1]. In addition, the testing protocol involved
calvaria implantation of collagen sponges (to assess membranous
bone formation) containing either 0 .mu.g or 35 .mu.g BPM. Samples
of variable composition and concentration can be produced.
[0120] The osteoinductive activity of the implant is evaluated
using accepted protocols for explant mass, ash weight, x-ray
mineral density and histology. A total of 20 rats/composition were
used. This population provided location-specific testing numbers of
n=10/test for the subcutaneous assays. Normalizing the samples
according to location-specific values provides a total subcutaneous
sample population of n=40/test.
[0121] Three weeks post implantation, the animals (n=20) were
sacrificed through CO.sub.2 asphyxiation. The weight of the host
rat and each implant is immediately measured post-surgical
excision. The explants are imaged with x-ray radiation to determine
mineral density as a function of composition and implant location.
Forty percent of the subcutaneous samples were analyzed using
accepted protocols for ash weight.
[0122] The remaining subcutaneous explants were analyzed for
differences in tissue quality using accepted histology protocols.
The averaged results and their standard deviations were analyzed
for statistical significance using ANOVA comparisons. The results
are shown in FIGS. 2-5.
Example 2
[0123] This example illustrates the independent effects of a
calcium source and a phosphate source in the present invention.
[0124] In vivo rat implantation assays were conducted to determine
the effects of local supplementation of calcium, phosphate, and of
both calcium and phosphate in the implanted compositions of the
present invention. The implants containing a calcium source, a
phosphate source or a source of both calcium and phosphate were
tested and evaluated in terms of relative histology score and
relative mineral mass gain. The results of these assays are shown
in FIGS. 10 and 11.
Example 3
[0125] The effect of calcium phosphate chemical composition and
microstructure (crystal structure) variations on the performance of
osteoinductive proteins was evaluated using a subcutaneous rat
implant model (Grossblatt, Guide for the Care and Use of Laboratory
Animals. Washington D.C.: National Academy Press; 1996, 1-80;
Intermedics Orthopedics/Denver, Inc. Rat Subcutaneous Bioassay:
Bone Protein Assay STM-011). The advantages of the rat model for
product evaluation include an accelerated rate of bone induction.
Visible evidence of mineralization appears in the implant within
several days (.about.10), with typical experiments lasting between
14 and 21 days. The testing protocol involved implantation of
porous collagen (bovine tendon Type 1, 7 mg, 96 vol. % porosity)
samples containing a natural mixture of bovine, osteoinductive
proteins (GFm, 10 .mu.g). A full range of calcium phosphates was
evaluated, including: monocalcium phosphate
[Ca(H.sub.2PO.sub.4).sub.2], calcium hydrogen phosphate dihydrate
[CaHPO.sub.4.2H.sub.2O], calcium pyrophosphate
[2CaO.P.sub.2O.sub.5], tricalcium phosphate
[.alpha.-3CaO.P.sub.2O.sub.5, .beta.-3CaO.P.sub.2O.sub.5],
hydroxyapatite [3.33CaO.P.sub.5(OH).sub.2 (polycrystalline and two
amorphous compositions)], tetracalcium phosphate
[4CaO.P.sub.2O.sub.5] or calcium carbonate [CaCO.sub.3 (aragonite),
CaCO.sub.3 (calcite)]. Samples were implanted in four subcutaneous
sites including the upper quadrants of the abdomen (sites 1, 2) and
the upper quadrants of the dorsal thorax (sites 6, 7). The
osteoinductive differences between controls and calcium phosphate
supplemented samples [7 mg, 50 wt. %] were assessed using three
standard test protocols: histological tissue analysis and mineral
composition via x-ray and ash weight analysis (Intermedics
Orthopedics/Denver, Inc. Histology protocol STM 009; Intermedics
Orthopedics/Denver, Inc. Alkaline phophatase protocol STM 0024,
0026).
[0126] Samples were assessed in comparison to two different control
sample populations, including: collagen sponges and collagen/GFm
sponges supplemented with 50 wt % devitalized bone matrix (DVBM)
additives. The collagen/GFm control samples were used as a
reference for a baseline osteoinductive response. A calcium
phosphate additive was considered detrimental if it either reduced
the explant or mineral mass values or if it negatively influenced
bone maturation. The composite collagen products containing
devitalized bone matrix additives serve as a reference for a
strongly positive osteoinductive response. These samples were
prepared as an alternative for demineralized bone matrix (DBM)
additives. Demineralized bone matrix provides both an ideal
osteoconductive matrix for cellular invasion and bone
mineralization and a pooled concentration of osteoinductive
proteins. Unfortunately, the osteoinductive performance of DBM
varies significantly. The composite products containing DVBM
provide ideal osteoconductive benefits and controlled, uniform
osteoinductive protein concentrations.
[0127] The experimental data are presented in box-whisker plot
format for exploratory, nonparametric data analysis. These graphs
are excellent for conveying variation information in data sets and
illustrating variability between different groups of data. This
information is represented within the three characteristic features
of a box-whisker plots: 1) the box, 2) the horizontal line within
the box and 3) the vertical lines (whiskers) that extend above and
below the box.
[0128] The box encompasses the middle 50% of the data with the
length of the box measuring data spread. The middle of the box
represents the median or `middle` value of the data set. The top of
the box represents the 75.sup.th percentile, meaning that 75% of
the data values fall below this value. It is mathematically
equivalent to the median data value plus 0.6745 of the standard
deviation. The bottom of the box represents the 25.sup.th
percentile (median minus 0.6745 of the standard deviation),
indicating the value which 75% of the data set exceeds. The
horizontal line within the box represents the mean or average value
of the data set.
[0129] The vertical lines that extend above and below the box
indicate the maximum (90.sup.th percentile) and minimum (10.sup.th
percentile) data values, respectively. The maximum and minimum
points represent the last data values within 2.7 standard
deviations of the median value. The location and length of these
lines represents the distribution of data. If the mean value is
found close to the center of the box and the length of the vertical
lines are equivalent, it can be assumed that the data follows a
normal Gaussian distribution. The data set is considered skewed if
either the mean value is not centered within the box or the length
of the maximum or minimum lines are unequal.
[0130] In a normal Gaussian distribution of data, outliers are
defined as data values that differ from the median by more than
three standard deviations. Outliers complicate statistical
interpretations of data set differences because their magnitude can
significantly influence the calculated mean and standard deviation.
In contrast, outliers have a minimal influence on the median and
quartile values used in box-whisker plots. This simplifies the
identification of outliers, as identified by circles, and
significantly improves statistical interpretations of data set
differences.
[0131] The averaged results and their standard deviations were
analyzed for statistical significance using ANOVA comparisons.
ANOVA was performed without data replications turning the analysis
into an expanded pool Student-T test. Statistical significance was
predicted using .alpha.<0.05. Calcium phosphate compositions
that had a statistically significant, negative effect on mass are
italicized in the tabulated data. Compositions that had no
significant influence on mass are indicated in plain text.
Compositions that stimulated a statistically significant
improvement in mass are indicated in bold text [Tables 5 and
6].
[0132] In comparison to the collagen controls (102.8.+-.22.9 mg
explant/11.7.+-.3.6 mg mineral), the majority of the salts had a
negligible or a detrimental effect on total explant and mineral
mass [FIGS. 12, 13]. However, a bimodal increase in explant and
mineral mass values was observed with moderately acidic calcium
phosphate salts [CaHPO.sub.4.2H.sub.2O (+40.2 wt. % mass/+153.6 wt.
% mineral), 2CaO.P.sub.2O.sub.5 (+99.6 wt. % mass, +263 wt. %
mineral)] and moderately alkaline, amorphous hydroxyapatite
[3.33CaO.P.sub.2O.sub.5(OH).sub.2 (Calcitek) (+44.1 wt. % explant
mass, +279.6 wt. % mineral)].
[0133] The quality and skeletal maturity of produced bone was
assessed through histological microscopic analysis of thin, stained
tissue sections. The subcutaneous explants were removed, fixed in
formalin and histologically processed with glycol methacrylate
according to standard protocols (Dickson, Glenn R.: Methods of
Calcified Tissue Preparation. Elsevier, 1984). Thin sections (4
.mu.m) from the explant midlines were obtained with a microtome.
One section was stained with hematoxylin & eosin (H&E) and
one was stained with toluidine blue to highlight important cellular
details. The H&E sections were counterstained with silver
nitrate (Von Kossa technique) to highlight mineralized tissue
components. The histological sections were scored for quality and
maturity using a scoring system, outlined in Table 7, previously
developed by Sulzer Biologics according to STM-021.
[0134] Using the described protocols, the influence of the various
additives on histological quality was determined. The average
histology score and the inter-animal standard deviation values are
tabulated in the first two columns of Table 8. The relative
differences in histology score, observed between experimental and
control samples within a single rat (intra-animal difference), are
included in the last four columns of Table 8. The magnitude and
range of total and relative histological scores is graphically
represented in the box plots of FIG. 14. TABLE-US-00006 TABLE 5
Average (inter-animal) and relative (intra-animal) explant mass for
implants supplemented with various calcium phosphates. Average
Explant Mass Average Explant Mass Direct Comparison Averages [mg]
.+-.SD N [mg] .+-.SD .DELTA.wt. % .+-.SD N Control 102.8 22.9 235
DVBM 142.8 46.0 290 178.9 27.8 +66.8% 39.4% 40
Ca(H.sub.2PO.sub.4).sub.2 (MCP) 104.4 29.2 90 95.3 31.6 -6.4% 26.2%
30 CaHPO4.2H.sub.2O 130.7 34.2 185 129.8 29.2 +40.2% 32.9% 40 (DCP)
2CaO.P.sub.2O.sub.5.2H.sub.2O (CP) 126.2 49.5 25 170.3 43.7 +99.6%
27.1% 10 3CaO.P.sub.2O.sub.5 (.alpha.- 39.5 31.9 35 58.7 28.3
-42.9% 26.4% 20 TCP) 3CaO.P.sub.2O.sub.5 (.beta.- 80.1 43.4 45 71.6
40 -26.3% 33.6% 20 TCP) 3.33CaO.P.sub.2O.sub.5 (HA- 113.5 32.2 20
124.7 43.1 +4.5% 37.2% 10 px) 3.33CaO.P.sub.2O.sub.5 (HA- 129.9
60.3 30 120.3 32.2 +0.3% 29.6% 15 am) 3.33CaO.P.sub.2O.sub.5 (HA-
117.9 36.3 30 140.6 30.2 +44.1% 25.0% 10 am) 4CaO.P.sub.2O.sub.5
54.8 30.9 20 70.0 38.3 -46.2% 39.2% 10 (TTCP) CaCO.sub.3 69.4 30.7
40 88.6 32.6 -27.3% 26.0% 10 (aragonite) CaCO.sub.3 67.4 27.9 40
73.1 24.0 -28.0% 20.2% 10 (calcite)
[0135] TABLE-US-00007 TABLE 6 Average (inter-animal) and relative
(intra-animal) mineral mass for implants supplemented with various
calcium phosphates. Average Mineral Mass Average Mineral Mass
Direct Comparison Averages [mg] .+-.SD n [mg] .+-.SD .DELTA.wt. %
.+-.SD n Control 11.7 3.6 74 DVBM 16.4 5.4 115 16.9 4.5 +50.4%
20.2% 8 Ca(H.sub.2PO.sub.4).sub.2 16.0 4.6 26 14.2 6.0 +50.2% 27.5%
8 (MCP) CaHPO4.2H.sub.2O 18.0 4.5 73 16.4 3.2 +53.6% 28.3% 16 (DCP)
2CaO.P.sub.2O.sub.5.2H.sub.2O (CP) 20.8 10.0 10 31.5 2.4 +163.0%
29.5% 4 3CaO.P.sub.2O.sub.5 (.alpha.- 6.5 7.7 12 9.4 8.1 -28.3%
28.6% 8 TCP) 3CaO.P.sub.2O.sub.5 (.beta.- 8.1 6.1 4 5.7 2.0 -31.6%
11.1% 4 TCP) 3.33CaO.P.sub.2O.sub.5 (HA- 19.8 8.2 8 21.9 10.1
+74.3% 25.3% 4 px) 3.33CaO.P.sub.2O.sub.5 (HA- 26.5 6.7 12 27.1 2.0
+95.6% 21.9% 4 am) 3.33CaO.P.sub.2O.sub.5 (HA- 29.4 12.1 12 30.4
5.8 +179.6% 12.7% 3 am) 4CaO.P.sub.2O.sub.5 7.7 6.1 7 10.3 8.8
-43.5% 28.3% 4 (Tetra) CaCO.sub.3 10.4 5.4 28 14.5 4.2 +3.8% 26.7%
4 (aragonite) CaCO.sub.3 7.9 5.2 27 8.1 3.0 -3.2% 28.5% 4
(calcite)
[0136] The collagen control samples produced explants with an
average histology score of approximately 2.2 (.+-.0.6). The
majority of samples resulted in histology scores of 2.0 or 3.0 with
a small fraction obtaining scores of 1.0. The collagen control
samples with DVBM produced explants with an average histology score
of 2.8 (.+-.0.8). The majority of samples resulted in histology
scores of 2.0 or 3.0 with a small fraction obtaining scores of 1.0
and 4.0.
[0137] According to statistical analysis (Student T-test), the
addition of DVBM significantly enhances osteoinductive performance
(.alpha.<0.05). The magnitude of the osteoinductive enhancement
is represented in the lower box plot of FIG. 15. This plot includes
intra-animal, relative histological score comparisons between the
collagen and collagen DVBM controls. The intra-animal comparison
indicates that the DVBM additives enhance histological scores by an
average of +33.3% wt. % (.+-.19.8 wt. %) with the majority of the
improvements ranging between +0% and +50%.
[0138] In contrast to the previous results, only acidic calcium
phosphate additives produced explants with histological scores
equivalent and superior to that observed with the addition of DVBM.
Statistically superior improvements in histological score were
observed with monocalcium phosphate [Ca(H.sub.2PO.sub.4).sub.2,
Monocal] (+9.9 wt. %), calcium hydrogen phosphate dihydrate
[CaHPO.sub.4.2H.sub.2O, DICAL] (+53.6 wt. %) and calcium
pyrophosphate [2CaO.P.sub.2O.sub.5, Pyro] (+163 wt. %) additives.
Statistically significant reductions were observed in histological
score for all other salt compositions.
[0139] The experimental data indicates that osteoinductive
performance is hindered with calcium phosphate salts of high (>2
Ca/P) calcia (CaO) content. In addition, the Ca/P ratio in the
calcium phosphate salt directly correlates with its pH buffering
potential, with high ratios being strongly alkaline. Monocalcium
phosphate [Ca(H.sub.2PO.sub.4).sub.2] is highly acidic
(pH.about.2). Dicalcium hydrogen phosphate and calcium
pyrophosphate are moderately acidic (pH.about.5.5). The neutral
transition point (pH.about.7) is located with tricalcium phosphate
compositions. Hydroxyapatites are moderately alkaline (pH.about.8).
Tetracalcium phosphate and calcium carbonates are highly alkaline
(pH.about.10-11). This information indicates that osteoinductive
performance is hindered with neutral and alkaline pH buffering
additives or calcium phosphate salts having a high calcium
content.
[0140] The effect of pH on bone quality and maturity is clearly
demonstrated in the photomicrographs included in FIG. 15. The
photomicrographs show the bone pattern on the periphery of the
explanted material (2.times. magnification). Histological sections
were selected from samples that matched both the average mass and
the average histology score for each test group. The sample and its
intra-animal collagen control are presented side-by-side.
[0141] In the Hematoxylin and eosin stained samples (FIG. 15,
left), practically all cytoplasmic structures and intercellular
substances are stained various shades of pink. The addition of
silver nitrate (Von Kossa technique) stains all mineral black,
making it simple to detect mineralized tissue. Although this
simplifies assessments of mineralization patterns, it does not aid
in distinguishing between new bone, mineralized cartilage, residual
calcium phosphate additives and calcified carrier. Induced bone is
distinguished from other mineralized tissues only by the combined
presence of osteoid matrix seams (bright pink) and layered
osteoblasts. Mature bone is represented by a continuous and thick
cortical rim, lined with a continuous seam of osteoid matrix and
active osteoblasts. Marrow quality is also easily assessed with
this staining technique since the nuclear structures are stained
dark purple or blue. Mature marrow is represented by samples that
contain high concentrations of hemopoietic granulocytes (stained
dark blue) and fat cells (adipocytes). The location and
concentration of fat cells is represented by solubilized white
voids.
[0142] The toluidine blue tissue stained samples were used to
identify cartilage tissue. Cartilage tissue is stained light to
deep purple, depending on the local concentration of proteoglycans.
Mature cartilage contains a high concentration of proteoglycans.
This stain is also useful for visualizing the number and activity
of osteoblasts (Ob) and osteocytes (Oc) which are stained dark
blue. Bone (B) appears lavender.
[0143] It is clearly observed that the acidic calcium phosphates
universally stimulated the amount of bone formation (section
diameter) and the depth of bone mineralization (bone staining
content). Collagen samples supplemented with calcium hydrogen
phosphate dihydrate [CaHPO.sub.4.2H.sub.2O] matched the cortical
rim bone quality observed in collagen/DVBM controls. The perimeters
for samples containing monocalcium phosphate and calcium
pyrophosphate were comparatively reduced in maturity but were
greater than control samples. Improvements in marrow quality were
realized with each acidic calcium phosphate additive, with the
samples supplemented with calcium hydrogen phosphate being the most
mature. These samples contained high density concentrations of
hemopoeitic granulocytes, red blood cell sinuses, and small
adipocyte concentrations characteristic of mature bone.
[0144] Despite the increase in mass values, a negative effect on
bone ossicle maturation, characterized by reduced cellular activity
and dystrophic mineralization, was observed with hydroxyapatite and
other alkaline calcium phosphate salts. In contrast, the samples
supplemented with moderately acidic calcium phosphate salts
enhanced bone maturation as well as the positive influences noted
above. In fact, the histological qualities for samples containing
calcium hydrogen phosphate dihydrate [CaHPO.sub.4.2H.sub.2O]
additives were superior to that observed with devitalized bone
matrix (DVBM) additives.
[0145] As indicated by the above described results, collagen
dispersions containing calcium hydrogen phosphate dihydrate salts
[CaHPO.sub.4.2H.sub.2O] stimulated the performance of
osteoinductive proteins resulting in bone of increased mass and
superior bone maturity. Based on this evidence, the salt and other
acidic calcium salts can be used as an alternative for
demineralized bone additives. This substitution should provide
significant economic savings, eliminate potential allograft disease
transfer and provide more reproducible and superior clinical
results as a bone void filler or autogeneous graft extender.
TABLE-US-00008 TABLE 7 Histological scores and sample requirements.
Histological Score Sample criteria 0 No residual implanted sample
found. Section shows no silver stained deposits or those deposits
are associated with acellular events. Explants are generally small,
soft and avascular. 1 Focal areas of silver stained mineralized
tissues are of cellular origin. This may include mineralized
cartilage as well as mineralized osteoid matrix. Silver stained
areas are randomly located throughout the explant, and typically
encompass less than 50% of the explant. Generally small 2 Silver
stained areas are mineralized cartilage or very early woven bone.
Osteoblasts appear in rows of only about 6 to 10 cells. If osteoid
is present, it is generally present on less than 10% of the
mineralizing tissue in the section. Small areas of hematopoietic
marrow elements may be visible (generally sinusoids containing red
blood cells). 3 Sheets of active osteoblasts, (e.g., cells are
plump and cuboidal or polygonal) generally consisting of 10 or more
cells, appear in less than 50% of the active mineralized portion.
They are generally not continuous. Bone associated with osteoblasts
is generally woven, containing some osteocytes. Woven bone appears
at outer regions of explant and may have breaks of fibrous tissue
or mineralized cartilage <10% of surface. Some hematopoietic
marrow elements may be visible. (Hemopoietic cords and sinusoids
containing red blood cells). 4 Mineralized tissue at the periphery
is generally not woven, but a mature band containing lamellar bone.
Mature bone is associated with continuous osteoblast surfaces in at
least 50% of bony area. Osteoid contains active osteoblasts and a
visible osteoid matrix. Evidence of bone marrow through presence of
granulocytes, hemopoietic cords and sinusoids is common. Evidence
of osteoclastic re-adsorption. 5 Solid rim of mature bone with few
breaks around outer edge. Mature bone contains osteocytes in
organized patterns. Mature bone contains wide dark staining (in TBO
stain) osteoid. Osteoid seams are continuous; very thick with
osteoblasts. Bone marrow contains hemopoietic cords packed with
cells, granulocytes, sinusoids and adipocytes. Trabecular bone in
marrow is reabsorbing and may appear as focal areas with little
branching. Explant center may contain mature woven bone or be
infarcted and largely acellular. Strong presence of osteoclasts
and/or lacunae.
[0146] TABLE-US-00009 TABLE 8 Average (inter-animal) and relative
(intra-animal) histological scores for implants supplemented with
various calcium phosphates. Average Histology Score Average Mineral
Mass Direct Comparison Averages [0-5] .+-.SD n [0-5] .+-.SD [%]
.+-.SD n Control 2.2 0.6 123 DVBM 2.8 0.8 156 3.0 0.3 +33.3% 19.8%
18 Ca(H.sub.2PO.sub.4).sub.2 2.2 0.8 48 1.9 0.6 +9.9% 19.8% 18
(MCP) CaHPO4.2H.sub.2O 2.9 0.8 111 2.5 0.7 +39.1% 23.2% 24 (DCP)
2CaO.P.sub.2O.sub.5.2H.sub.2O (CP) 2.3 0.6 15 2.3 0.5 +11.1% 22.8%
6 3CaO.P.sub.2O.sub.5 (.alpha.- 1.1 0.4 21 1.3 0.5 -35.1% 27.4% 12
TCP) 3CaO.P.sub.2O.sub.5 (.beta.- 1.3 0.5 9 1.3 0.5 -23.9% 22.2% 6
TCP) 3.33CaO.P.sub.2O.sub.5 (HA- 1.3 0.5 12 1.2 0.4 -46.7% 27.4% 6
am) 3.33CaO.P.sub.2O.sub.5 (HA- 1.4 0.5 18 1.7 0.5 -20.0% 27.4% 6
am) 3.33CaO.P.sub.2O.sub.5 (HA- 1.4 0.5 18 2.0 0.0 -26.7% 18.3% 6
px) 4CaO.P.sub.2O.sub.5 1.0 0.0 12 1.0 0.0 -36.1% 18.7% 6 (TTCP)
CaCO.sub.3 1.0 0.0 24 1.0 0.0 -63.9% 6.8% 6 (aragonite) CaCO.sub.3
1.0 0.0 24 1.0 0.0 -58.3% 9.1% 6 (calcite)
Example 4
[0147] A paste was made comprising (i) about 90 parts by volume
dehydrothermally-crosslinked collagen:dical particles (about 66 wt
% dical) having particle sizes of 125-300 mm and 96% porosity; (ii)
about 10 parts by volume soluble collagen; (iii) monocalcium
phosphate; and (iv) about 100 parts by volume water. The
monocalcium phosphate was included for pH control and the amount
varied, with greater amounts leading to lower paste pH. Generally,
the amount of monocalcium phosphate was about 2-10 wt % relative to
components (i)-(iii), i.e., not including the weight of water.
Generally, sufficient monocalcium phosphate was added to yield pH
values of the paste from about 4.5 to 4.9. The dehydrothermal
crosslinking was performed at about 110.degree. C. for about 48
hr.
[0148] The high porosity of component (i) was chosen to encourage
high rate and depth of cellular penetration into the paste. In
essence, the particles (i) act as extrinsic bone nucleation sites
throughout the entire mass.
[0149] The relatively small concentration of soluble collagen (ii)
was chosen to allow for the development of a cohesive, taffy-like
paste consistency. The cross-linked collagen:dical particles are
completely insoluble and lack cohesion in water or marrow. In
contrast, the addition of soluble collagen allows the formation of
a collagen gel. Collagen forms highly viscous gels near its
isoelectric point (pH 4-5.5). Simple, short-term pH control causes
the collagen to solubilize and gel, trapping the cross-linked
collagen particles in a cohesive, pliable mass.
[0150] Monocalcium phosphate (iii) was selected as a salt for
temporary acidic pH control in the paste. Its use, in comparison to
alternatives, results in dramatically superior improvements in
stimulated bone quantity and histological quality. These results
were also supported in prior sponge optimization research. The pH
effect was temporary primarily as a result of the solubility of
monocalcium phosphate in body fluids.
[0151] The pastes generated according to this example were tested
in the rat model described in Example 3.
[0152] The effect of initial pH of the paste on explant mass and
mineral mass is shown in FIG. 16. The effect of initial pH of the
paste on the histology score is shown in FIG. 17.
[0153] The results in FIG. 16 clearly demonstrate that pH has a
significant effect on total explant and mineral mass. The results
in FIG. 17 also indicate that within the tested range acidic pH
values have little influence on histological bone maturity. It
should be emphasized when reviewing these results that, in
comparison to reference controls with equivalent inductive growth
factor doses, the paste formulations yield histologically superior
explants with average bone masses around 250 mg, which corresponds
to a 400% improvement in bone mass induction.
[0154] With respect to FIGS. 2-5, the osteogenic effects of
collagen disks containing various calcium phosphate salt
compositions and bone growth protein (BMP) were assessed based on
explant mass (FIGS. 2A-B), histology score (FIGS. 3A-B), mineral
concentration (FIGS. 4A-B) and mineral mass (FIGS. 5A-B). As
comparatives, some conventional, commercially available
osteoinductive compositions (ProOsteon 200R-acidic, ProOsteon
200R-neutral, Ostite C1-C3, GB9N and Bioglass) were similarly
tested. Various osteoinductive compositions (first column in FIGS.
2B-5B) were formed into disks together with collagen and BMP, and
tested ("CPB" in FIGS. 2B-5B). A control composition comprising
collagen and BMP was also tested with each of the above-described
samples, and its performance is reported under the heading "CB" in
FIGS. 2B-5B).
[0155] Similarly, with respect to FIGS. 6-9, the same
CP/collagen/BMP disks were again tested for osteogenic performance
(("CPB" in FIGS. 6B-9B) and compared to the performance of control
disks containing devitalized bone matrix instead of collagen ("CDB"
in FIGS. 6B-9B).
[0156] Acidic mineral salts other than calcium phosphate salts can
be used to control pH, e.g., sulfate-based buffer, lactic acid,
calcium citrate, sodium phosphate, and others (page 13, line
22-page 14, line 9). As shown in FIG. 10, implanting a non-calcium
phosphate/collagen/BMP composition of pH ranging from 4.5 to 6.5,
supplemented with calcium ion, improved the histology score
(.about.130-135%), compared to the histology score obtained with
the unsupplemented composition (100%). On the other hand, a
phosphate-supplemented composition of pH ranging from 4.5 to 6.5
did not alter the histology score significantly. Simultaneous
supplementation of the composition with both phosphate and calcium
ions resulted in significant improvements in bone maturity or
histology (FIG. 10) and additional bone mass improvements (FIG.
11).
[0157] The in vivo performance of osteogenic proteins is influenced
by the presence in the osteogenic composition of essential bone
components (collagen, calcium, phosphate) and solution pH. Only
acidic calcium phosphate additives, however, produced explants with
histological scores equivalent and superior to that observed with
the addition of devitalized bone matrix (CDB). Statistically
superior improvements in histological score were observed with the
monocalcium phosphate-based compositions
[Ca(H.sub.2PO.sub.4).sub.2, MCP], calcium hydrogen phosphate
dihydrate-based compositions [CaHPO.sub.4.2H.sub.2O, DCP], and
calcium pyrophosphate-based compositions [2CaO.P.sub.2O.sub.5, CP].
Statistically significant reductions were observed in histological
score for all other salt-based compositions tested. Osteogenic
performance is hindered with calcium phosphate salts of high (>2
Ca/P) calcia (CaO) content. The Ca/P ratio in the calcium phosphate
salt directly correlated with its pH buffering potential (i.e.,
pKa), with high ratios being strongly alkaline. Monocalcium
phosphate [Ca(H.sub.2PO.sub.4).sub.2] is highly acidic
(pH.about.2). Dicalcium hydrogen phosphate and calcium
pyrophosphate are moderately acidic (pH.about.5.5). The neutral
transition point (pH.about.7) is located with tricalcium phosphate
compositions. Hydroxyapatites are moderately alkaline (pH.about.8).
Tetracalcium phosphate and calcium carbonates are highly alkaline
(pH.about.10-11). Osteogenic performance is hindered with neutral
and alkaline additives.
[0158] In conclusion, regarding the influence of composition pH and
soluble ion supplementation on osteogenic bone formation, first,
the pH of the composition, and thus temporary local pH control of
the initial bone growth environment, could be exploited to
significantly increase explant and mineral mass values. Explant
mass (FIGS. 2A,B and 6A,B), mineral concentration (FIGS. 4A,B and
8A,B) and mineral mass (FIGS. 5A,B and 9A,B) values were
appreciably enhanced with either hydroxyapatite or with CP or
DCP-based compositions (i.e., moderately alkaline (pH.about.8.5) or
moderately acidic (pH.about.4.5-6.5) salt components).
[0159] Second, statistically significant improvements in bone
maturity or histology score (FIGS. 3A,B and 7A,B) were realized
with the combined use of moderately acidic compositions containing
both soluble calcium and phosphate ions and BMP, compared to
hydroxyapatite-based compositions with BMP, on either collagen
(FIGS. 3A,B) or devitalized bone matrix (FIGS. 7A,B). In contrast,
moderately alkaline pH compositions (i.e., the hydroxyapatite-based
compositions) hindered bone maturation and significantly reduced
cellular activity (FIGS. 3A,B and 7A,B).
[0160] Clearly, some of the results are contrary to conventional
thinking at the time the subject invention was conceived and
reduced to practice. The fact that the majority of the calcium
phosphate additives had no effect, or had a negative effect on
explant mass contradicts the hypothetical benefits that calcium and
phosphate ion supplementation would reasonably have been expected
to offer. Based on the available knowledge in this field at the
time of the invention, it was originally hypothesized that the
clinical performance of osteogenic proteins could be enhanced by
supplementing the local availability of essential bone components
(collagen, calcium, phosphate). However, if local supplementation
were beneficial, one of skill in this field would have expected
that improvements in explant mass would have been observed with
every calcium phosphate additive that was tested. Furthermore,
variations in explant mass improvements would have been expected
due to basic solubility, pH and compositional differences [Ca/P
ratio]. However, it is shown herein that both acidic composition
and Ca/PO.sub.4 ion supplementation can enhance and improve
osteoinductive growth factor-induced bone formation independently.
It was unexpected that the moderately acidic DCP and CP-based
compositions significantly enhanced explant and mineral mass
values, compared to the other compositions tested. Our observation
that the majority of the calcium phosphate additives had no effect,
or had a negative effect on explant mass is contrary to the
expected benefits of supplementation with calcium and phosphate
ion.
Example 5
[0161] A variety of sparingly soluble calcium phosphate salts,
[Ca.sub.x(PO.sub.4).sub.y] were used to assess the influence of
differences in local, soluble [Ca.sup.2+] and [PO.sub.4.sup.3-] ion
concentrations and in chemical BMP affinity as demonstrated by bone
formation in a small animal model.
[0162] The effect of calcium phosphate chemical composition and
microstructure (crystal structure) variations on the performance of
osteoinductive proteins was evaluated using a subcutaneous rat
implant model (FIG. 1). This model has an accelerated rate of bone
induction with visible evidence of mineralization appearing in the
implant within 1-2 weeks (.about.10 days), with typical experiments
lasting between 14 and 21 days. Osteogenic activity is commonly
evaluated using three standard test protocols: histological tissue
analysis and mineral composition via x-ray and ash weight
analysis.
[0163] The testing protocol involved implantation of porous
collagen (bovine tendon Type 1, 7 mg, 96 vol. % porosity) samples
containing a natural mixture of bovine, osteoinductive proteins
(BMPs, 10 .mu.g). A full range of calcium phosphates was evaluated,
including: monocalcium phosphate [Ca(H.sub.2PO.sub.4).sub.2],
calcium hydrogen phosphate [CaHPO.sub.4], calcium pyrophosphate
[2CaO.P.sub.2O.sub.5], tricalcium phosphate
[.alpha.,.beta.-Ca.sub.3(PO.sub.4).sub.2], hydroxylapatite
[Ca.sub.5(PO.sub.4).sub.3(OH)], tetracalcium phosphate
[Ca.sub.4(PO.sub.4).sub.2(OH).sub.2] or calcium carbonate
[CaCO.sub.3]. The experimental results were used to identify a
synthetic additive for collagen to improve its osteoinductive
performance.
[0164] The effect of variable calcium phosphate compositions on
bone quality and maturity is clearly demonstrated in the
photomicrograph shown in FIG. 15. The photomicrographs show the
bone pattern on the periphery of the explanted material at 2.times.
magnification. Histological sections were selected from samples
that matched both the average mass and the average histology score
for each test group. The sample and its intra-animal collagen
control are presented side-by-side.
[0165] In the hematoxylin and eosin stained samples, practically
all cytoplasmic structures and intercellular substances are stained
various shades of pink. The addition of silver nitrate (Von Kossa
technique) stains all mineral black making it simple to detect
mineralized tissue. Although this simplifies assessments of
mineralization patterns, it does not aid in distinguishing between
new bone, mineralized cartilage, residual calcium phosphate
additives and calcified carrier. Induced bone is distinguished from
other mineralized tissues only by the combined presence of osteoid
matrix seams (bright pink) and layered osteoblasts. Mature bone is
represented by a continuous and thick cortical rim, lined with a
continuous seam of osteoid matrix and active osteoblasts. Marrow
quality is also easily assessed with this staining technique since
the nuclear structures are stained dark purple or blue. Mature
marrow is represented by samples that contain high concentrations
of hemopoietic granulocytes (stained dark blue) and fat cells
(adipocytes). The location and concentration of fat cells is
represented by solubilized white voids.
[0166] The toluidine blue tissue stained samples were used to
identify cartilage tissue. Cartilage tissue is stained light to
deep purple, depending on the local concentration of proteoglycans.
Mature cartilage contains a high concentration of proteoglycans.
This stain is also useful for visualizing the number and activity
of osteoblasts (Ob) and osteocytes (Oc) which are stained dark
blue. Bone (B) appears lavender; however residual calcium phosphate
salts and other mineralized tissues are equally stained. Marrow
elements are difficult to distinguish with this stain.
[0167] It is clearly observed that the moderately acidic calcium
phosphate salts universally stimulated the amount of bone formation
(section diameter) and the depth of bone mineralization (bone
staining content). Collagen samples supplemented with calcium
hydrogen phosphate dihydrate [CaHPO.sub.4] were the only samples
that match the cortical rim bone quality observed in collagen/DVBM
controls. The perimeters for samples containing monocalcium
phosphate and calcium pyrophosphate were comparatively reduced in
maturity. Although improvements in marrow quality were realized
with each moderately acidic calcium phosphate additive, the samples
supplemented with calcium hydrogen phosphate were the most mature.
These samples contained high density concentrations of hemopoeitic
granulocytes, red blood cell sinuses, and small adipocyte
concentrations characteristic of mature bone.
[0168] In contrast, it is observed that the addition of neutral and
alkaline calcium phosphate salts significantly inhibits bone
formation. The histological quality of the samples supplemented
with tricalcium phosphate, hydroxyapatite, tetracalcium phosphate,
or calcium carbonate are comparatively reduced. Cellular content
and cellular activity correspondingly decreased within samples
supplemented with calcium phosphates of increased alkalinity. In
the histological sections provided, few areas of active cellular
activity are observed. Although the representative sections confirm
the experimental observations of increased explant and mineral mass
by the differences in sample size and mineral staining, the mineral
is of extremely poor quality. The mineral content predominantly
includes dystrophically mineralized carrier collagen.
[0169] The experimental evidence demonstrates that synthetic bone
void fillers supplemented with calcium hydrogen phosphate
[CaHPO.sub.4 (DICAL)] enhances both the quantity and histological
quality of bone produced. Moderately acidic microenvironments
improve protein-stimulated osteoinduction in several different
ways. First, an acidic microenvironment can enhance the rates of
protein solubilization and protein release from collagen. The
resultant increase in local concentration and cellular availability
of bone morphogenetic proteins (BMPs) could explain the observed
enhancements in bone formation and bone quality. Local pH may also
affect protein conformation or cellular activity.
[0170] Calcium phosphate compositions with alkaline buffering
potentials hinder protein stimulated osteogenic bone formation by
two mechanisms. First, bone morphogenetic proteins are insoluble
and precipitate in alkaline solutions. The alkaline calcium
phosphate additives may precipitate a significant fraction of the
inductive proteins and inhibit their osteogenic capabilities.
Second and more importantly, alkaline environments initiate the
direct precipitation of soluble calcium and phosphate ions. It is
likely the alkaline additives cause the serum calcium and phosphate
ions to immediately precipitate as apatite onto collagen. If the
BMPs simultaneously precipitate within these dystrophic crystals,
they would be unavailable to cells until after osteoclastic
resportion of the mineralized deposits. The general appearance and
lack of cellular activity and the enhanced dystrophic mineral
content supports this theory.
[0171] While various embodiments of the present invention have been
described in detail, it is apparent that modifications and
adaptations of those embodiments will occur to those skilled in the
art. It is to be expressly understood, however, that such
modifications and adaptations are within the scope of the present
invention, as set forth in the following claims.
* * * * *