U.S. patent application number 09/887901 was filed with the patent office on 2002-07-11 for matrix-free osteogenic devices, implants and methods of use thereof.
This patent application is currently assigned to STRYKER CORPORATION. Invention is credited to Rueger, David C., Tucker, Marjorie M..
Application Number | 20020091077 09/887901 |
Document ID | / |
Family ID | 26714038 |
Filed Date | 2002-07-11 |
United States Patent
Application |
20020091077 |
Kind Code |
A1 |
Rueger, David C. ; et
al. |
July 11, 2002 |
Matrix-free osteogenic devices, implants and methods of use
thereof
Abstract
Provided herein are methods for inducing bone formation in a
mammal sufficient to fill a defect defining a void, wherein
osteogenic protein is provided alone or dispersed in a
biocompatible non-rigid, amorphous carrier having no defined
surfaces. The methods and devices provide injectable formulations
for filling critical size defects, as well as for accelerating the
rate and enhancing the quality of bone formation in non-critical
size defects.
Inventors: |
Rueger, David C.;
(Southborough, MA) ; Tucker, Marjorie M.;
(Holliston, MA) |
Correspondence
Address: |
FISH & NEAVE
1251 AVENUE OF THE AMERICAS
50TH FLOOR
NEW YORK
NY
10020-1105
US
|
Assignee: |
STRYKER CORPORATION
|
Family ID: |
26714038 |
Appl. No.: |
09/887901 |
Filed: |
June 22, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09887901 |
Jun 22, 2001 |
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09019339 |
Feb 5, 1998 |
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Current U.S.
Class: |
514/8.8 |
Current CPC
Class: |
A61K 38/1875 20130101;
A61L 27/227 20130101; A61K 38/18 20130101; A61F 2/28 20130101; A61P
19/08 20180101; A61F 2/28 20130101; A61L 2430/02 20130101; A61L
27/227 20130101; A61K 38/18 20130101; A61L 27/227 20130101 |
Class at
Publication: |
514/2 |
International
Class: |
A61K 038/43 |
Claims
What is claimed is:
1. A method for inducing bone formation in a mammal sufficient to
fill a defect locus defining a void, the method comprising the step
of providing to said locus an osteogenic device comprising
osteogenic protein dispersed in a biocompatible, non-rigid
amorphous carrier having no defined surfaces.
2. The method of claim 1 wherein the volume of said device provided
to said defect locus is insufficient to fill said void.
3. The method of claim 1 wherein said device lacks scaffolding
structure.
4. The method of claim 1 wherein said defect locus defines a volume
incapable of endogenous repair.
5. The method of claim 1 wherein said bone formation is
endochondral bone formation.
6. The method of claim 1 wherein said bone formation is
intramembranous bone formation.
7. The method of claim 1 wherein said carrier comprises a gel.
8. The method of claim 1 wherein said carrier comprises an aqueous
solution.
9. The method of claim 1 wherein said carrier is selected from the
group consisting of: alkylcelluloses; pluronics; gelatins;
polyethylene glycols (PEG); dextrins; and vegetable oils.
10. The method of claim 1 wherein said carrier is selected from the
group consisting of: carboxymethylcellulose; mannitol; PEG 3350;
pluronic F127; and sesame oil.
11. The method of claim 1 wherein said osteogenic protein is
selected from the group consisting of: OP1; OP2, OP3, BMP2; BMP3;
BMP4; BMP5; BMP6; BMP9; BMP-10, BMP-11, BMP-12, BMP-15, BMP-3b,
DPP; Vg1; Vgr; 60A protein; GDF-1; GDF-3, GDF-5, GDF-6, GDF-7,
GDF-8, GDF-9, GDF-10, GDF-11; and amino acid sequence variants
thereof.
12. The method of claim 1 wherein said osteogenic protein is
selected from the group consisting of OP1; OP2, BMP2; BMP4; BMP5;
BMP6; and amino acid sequence variants thereof.
13. The method of claim 1 wherein said osteogenic protein is a
morphogen, said morphogen comprising an amino acid sequence having
at least 70% homology within the C-terminal 102-106 amino acids,
including the conserved seven cysteine domain, of human OP1.
14. The method of claim 1 wherein said osteogenic protein is
OP1.
15. The method of claim 1 wherein said osteogenic protein is mature
OP1 solubilized in a saline solution.
16. A device for inducing bone formation sufficient to fill a
defect locus defining a void, the device comprising: osteogenic
protein dispersed in a biocompatible, nonrigid amorphous carrier
having no defined surfaces.
17. The device of claim 16 wherein said carrier comprises a
gel.
18. The device of claim 16 wherein said carrier comprises an
aqueous solution.
19. The device of claim 16 wherein said carrier is selected from
the group consisting of: alkylcelluloses; pluronics; gelatins;
polyethylene glycols (PEG); dextrins; and vegetable oils.
20. The device of claim 16 wherein said carrier is selected from
the group consisting of: carboxymethylcellulose; mannitol; PEG
3350; pluronic F127; and sesame oil.
21. The device of claim 16 wherein said osteogenic protein is
selected from the group consisting of: OP1; OP2, OP3, BMP2; BMP3;
BMP4; BMP5; BMP6; BMP-10, BMP-11, BMP-12, BMP-15, BMP-3b, BMP9;
DPP; Vg1; Vgr; 60A protein; GDF-1; GDF-3, GDF-5, GDF-6, GDF-7,
GDF-8, GDF-9, GDF-10, GDF-11; and amino acid sequence variants
thereof.
22. The device of claim 16 wherein said osteogenic protein is
selected from the group consisting of: OP1; OP2, BMP2; BMP4; BMP5;
BMP6; and amino acid sequence variants thereof.
23. The device of claim 16 wherein said osteogenic protein is a
morphogen, said morphogen comprising an amino acid sequence having
at least 70% homology within the C-terminal 102-106 amino acids,
including the conserved seven cysteine domain, of human OP1.
24. The device of claim 16 wherein said osteogenic protein is
OP1.
25. The device of claim 16 wherein said osteogenic protein is
mature OP1 solubilized in a saline solution.
26. A method for inducing bone formation in a mammal sufficient to
fill a defect locus defining a void, the method comprising the step
of providing to said locus substantially pure osteogenic protein
free of a carrier or scaffolding structure.
27. A method for enhancing the quantity or quality of callus
formation at an osteogenic defect locus in a mammal, the method
comprising the step of administering the device of claim 16.
28. The method of claim 27 wherein said osteogenic protein is a
morphogen, said morphogen comprising an amino acid sequence having
at least 70% homology within the C-terminal 102-106 amino acids,
including the conserved seven cysteine domain, of human OP1.
29. The method of claim 27 wherein said osteogenic protein is
OP1.
30. The method of claim 27 wherein said osteogenic protein
comprises an amino acid sequence defined by OPX (Seq. ID No. 3);
Generic Sequence 6 (Seq. ID No. 4, Generic Sequence 7 (Seq. ID No.
5); Generic Sequence 8 (Seq. ID No. 6); or Generic Sequence 9 (Seq.
ID No. 7).
31. A method of accelerating bone formation, the method comprising
the step of providing to a defect locus the device of claim 16.
32. A method of inducing endogenous matrix formation, the method
comprising the step of providing to a defect locus the device of
claim 16.
33. A method of repairing a bone defect, chondral defect or
osteochondral defect, said method comprising the step of
administering to a defect a matrix-free osteogenic device, wherein
administering said device post-injury is delayed.
34. The method of claim 33 wherein said administering step is
delayed at least 6 hours post-injury.
35. A matrix-free device for repairing a chondral defect, said
device comprising osteogenic protein and a glycosaminoglycan
carrier.
36. The device of claim 35 wherein said carrier is hyaluronic
acid.
37. A method of repairing a chondral defect, said method comprising
the step of administering to a chondral defect the device of claim
35.
Description
CONTINUING APPLICATION DATA
[0001] This application is based on prior application U.S. Ser. No.
60/037327 (Atty. Dock. No. CRP-111PR), filed Feb. 7, 1997, the
entire contents of which is incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The invention disclosed herein relates to materials and
methods for repairing bone defects using osteogenic proteins.
BACKGROUND OF THE INVENTION
[0003] A class of proteins now have been identified that are
competent to act as true chondrogenic tissue morphogens, able, on
their own, to induce the proliferation and differentiation of
progenitor cells into functional bone, cartilage, tendon, and/or
ligamentous tissue. These proteins, referred to herein as
"osteogenic proteins" or "morphogenic proteins" or "morphogens,"
includes members of the family of bone morphogenetic proteins
(BMPs) which were initially identified by their ability to induce
ectopic, endochondral bone morphogenesis. The osteogenic proteins
generally are classified in the art as a subgroup of the TGF-.beta.
superfamily of growth factors (Hogan (1996) Genes & Development
10:1580-1594). Members of the morphogen family of proteins include
the mammalian osteogenic protein-1 (OP-1, also known as BMP-7, and
the Drosophila homolog 60A), osteogenic protein-2 (OP-2, also known
as BMP-8), osteogenic protein-3 (OP-3), BMP-2 (also known as BMP-2A
or CBMP-2A, and the Drosophila homolog DPP), BMP-3, BMP-4 (also
known as BMP-2B or CBMP-2B), BMP-5, BMP-6 and its murine homolog
Vgr-1, BMP-9, BMP-10, BMP-11, BMP-12, GDF3 (also known as Vgr2),
GDF8, GDF9, GDF10, GDF11, GDF12, BMP-13, BMP-14, BMP-15, GDF-5
(also known as CDMP-1 or MP52), GDF-6 (also known as CDMP-2), GDF-7
(also known as CDMP-3), the Xenopus homolog Vg1 and NODAL, UNIVIN,
SCREW, ADMP, and NEURAL. Members of this family encode secreted
polypeptide chains sharing common structural features, including
processing from a precursor "pro-form" to yield a mature
polypeptide chain competent to dimerize, and containing a carboxy
terminal active domain of approximately 97-106 amino acids. All
members share a conserved pattern of cysteines in this domain and
the active form of these proteins can be either a disulfide-bonded
homodimer of a single family member, or a heterodimer of two
different members (see, e.g., Massague (1990) Annu. Rev. Cell Biol.
6:597; Sampath, et al. (1990) J. Biol. Chem. 265:13198). See also,
U.S. Pat. No. 5,011,691; U.S. Pat. No. 5,266,683, Ozkaynak et al.
(1990) EMBO J. 9: 2085-2093, Wharton et al. (1991) PNAS
88:9214-9218), (Ozkaynak (1992) J. Biol. Chem. 267:25220-25227 and
U.S. Pat. No. 5,266,683); (Celeste et al. (1991) PNAS
87:9843-9847); (Lyons et al. (1989 ) PNAS 86:4554-4558). These
disclosures describe the amino acid and DNA sequences, as well as
the chemical and physical characteristics of these osteogenic
proteins. See also Wozney et al. (1988) Science 242:1528-1534); BMP
9 (WO93/00432, published Jan. 7, 1993); DPP (Padgett et al. (1987)
Nature 325:81-84; and Vg-1 (Weeks (1987) Cell 51:861-867).
[0004] Thus, true osteogenic proteins capable of inducing the
above-described cascade of morphogenic events that result in
endochondral bone formation have now been identified, isolated, and
cloned. Whether naturally-occurring or synthetically prepared,
these osteogenic factors, when implanted in a mammal in association
with a conventional matrix or substrate that allows the attachment,
proliferation and differentiation of migratory progenitor cells,
have been shown to induce recruitment of accessible progenitor
cells and stimulate their proliferation, thereby inducing
differentiation into chondrocytes and osteoblasts, and further
inducing differentiation of intermediate cartilage,
vascularization, bone formation, remodeling, and finally marrow
differentiation. Furthermore, numerous practitioners have
demonstrated the ability of these osteogenic proteins, when admixed
with either naturally-sourced matrix materials such as collagen or
synthetically-prepared polymeric matrix materials, to induce bone
formation, including endochondral bone formation under conditions
where true replacement bone otherwise would not occur. For example,
when combined with a matrix material, these osteogenic proteins
induce formation of new bone in: large segmental bone defects,
spinal fusions, and fractures. Without exception, each of the
above-referenced disclosures describes implantation or delivery of
the osteogenic protein at the defect site by packing, filling,
and/or wrapping the defect site with an admixture of osteogenic
protein and matrix, with the relative volume and surface area of
matrix being significant. In the case of non-union defects which do
not heal spontaneously, it has heretofore been conventional
practice to implant volumes of matrix-osteogenic factor admixtures
at the defect site, the volumes being sufficient to fill the defect
in order to provide a 3-dimensional scaffold for subsequent new
bone formation. While standard bone fractures, can heal
spontaneously and without treatment, to the extent the art has
contemplated treating fractures with osteogenic proteins, it has
been the practice in the art to provide the osteogenic protein
together with a matrix locally to a defect site to promote
healing.
[0005] While implanting a volume of matrix may be conventional
wisdom, particularly in the case of non-healing non-union defects,
clinical consequences may develop in certain patients as a result
of this practice. For example, patients undergoing repeated
constructions or defect repairs, or wherein the matrix volume is
large, can develop adverse immunologic reactions to matrices
derived from collagen. Collagen matrices can be purified, but
residual levels of contaminants can remain which is strongly
allergenic for certain patients. Alternatively, demineralized
autogenic, allogenic or xenogenic bone matrix can be used in place
of collagen. Such a matrix is mechanically superior to collagen and
can obviate adverse immune reactions in some cases, but proper
preparation is expensive, time consuming and availability of
reliable sources for bone may be limited. Such naturally-sourced
matrices can be replaced with inert materials such as plastic, but
plastic is not a suitable substitute since it does not resorb and
is limited to applications requiring simple geometric
configurations. To date, biodegradable polymers and copolymers have
also been used as matrices admixed with osteogenic proteins for
repair of non-union defects. While such matrices may overcome some
of the above-described insufficiencies, use of these matrices still
necessitates determination and control of features such as polymer
chemistry, particle size, biocompatability and other particulars
critical for operability.
[0006] In addition, individuals who, due to an acquired or
congenital condition, have a reduced ability to heal bone fractures
or other defects that normally undergo spontaneous repair would
benefit from methods and injectable compositions that can enhance
bone and/or cartilage repair without requiring a surgical
procedure. Finally, an injectable formulation also provides means
for repairing osteochondral or chondral defects without requiring a
surgical procedure.
[0007] Needs remain for devices, implants and methods of repairing
bone defects which do not rely on a matrix component. Particular
needs remain for devices, implants and methods which permit
delivery of bone-inducing amounts of osteogenic proteins without
concomitant delivery of space-filling matrix materials which can
compromise the recipient and/or fail to be biomechanically and
torsionally ideal. Needs also remain for providing methods and
devices, particularly injectable devices that can accelerate the
rate and enhance the quality of new bone formation.
[0008] Accordingly, it is an object of the instant invention to
provide devices, implants and methods of use thereof for repairing
bone defects, cartilage defects and/or osteochondral defects which
obviate the need for an admixture of osteogenic protein with
matrix. The instant invention provides matrix-free osteogenic
devices, implants and methods of use thereof for repairing
non-healing non-union defects, as well as for promoting enhanced
bone formation for spinal fusions and bone fractures, and for
promoting articular cartilage repair in chondral or osteochondral
defects. These and other objects, along with advantages and
features of the invention disclosed herein, will be apparent from
the description, drawings and claims that follow.
SUMMARY OF THE INVENTION
[0009] The present invention is based on the discovery that an
osteogenic or bone morphogenic protein such as OP-1, alone or when
admixed with a suitable carrier and not with a conventional matrix
material, can induce endochondral bone formation sufficient to
repair critical-sized, segmental bone defects. Thus this discovery
overcomes the above-described problems associated with conventional
materials and methods for repairing bone defects because it permits
elimination of matrix material. Furthermore, in view of existing
orthopedic and reconstructive practices, this discovery is
unexpected and contravenes the art's current understanding of the
bone repair/formation processes.
[0010] As disclosed herein, it is now appreciated that an
osteogenic protein can be admixed with a carrier as defined herein
to form a matrix-free device which, when provided to a mammal, is
effective to promote repair of non-union bone defects, fractures
and fusions. As disclosed herein, methods and devices are provided
for inducing new bone formation at a local defect site without the
need for also providing a three-dimensional structural component at
the defect site. As contemplated herein, a "matrix-free" osteogenic
device is a device devoid of matrix at the time it is provided to a
recipient. It is understood that the term "matrix" means a
structural component or substrate having a three-dimensional form
and upon which certain cellular events involved in endochondral
bone morphogenesis will occur; a matrix acts as a temporary
scaffolding structure for infiltrating cells having interstices for
attachment, proliferation and differentiation of such cells.
[0011] The invention provides, in one aspect therefore, a novel
method for inducing bone formation in a mammal sufficient to repair
a defect. One embodiment comprises the step of providing a
matrix-free osteogenic device to a defect locus defining a void.
The matrix-free device may be composed of osteogenic protein alone,
or it may be composed of osteogenic protein in admixture with a
biocompatible, amorphous non-rigid carrier having no defined
surfaces. This method induces new bone formation which fills the
defect locus, thereby repairing the defect. As contemplated herein,
the method comprises providing a matrix-free osteogenic device to a
defect locus, wherein the device is provided in a volume
insufficient to fill the void at the defect locus. In certain
embodiments, the void comprises a volume incapable of endogenous or
spontaneous repair. Examples of defects suitable for repair by the
instant method include, but are not limited to, critical-sized
segmental defects and non-union fractures.
[0012] In another embodiment, the invention provides methods and
compositions for enhancing fracture repair by providing the
matrix-free osteogenic devices described herein to a fracture
defect site. The ability of the devices described herein to
substantially enhance fracture repair, including accelerating the
rate and enhancing the quality of newly formed bone, has
implications for improving bone healing in compromised individuals
such as diabetics, smokers, obese individuals and others who, due
to an acquired or congenital condition have a reduced capacity to
heal bone fractures, including individuals with impaired blood flow
to their extremities.
[0013] In another aspect, the invention provides an implant for
inducing bone formation in a mammal sufficient to repair a defect.
One preferred implant comprises a matrix-free osteogenic device
disposed at a defect locus defining a void. Practice of the
above-described method, i.e., providing an osteogenic device devoid
of scaffolding structure to a mammal at a defect locus, results in
an implant competent to induce new bone formation sufficient to
promote repair of non-union bone defects, fractures and fusions.
Upon disposition of the osteogenic device at the defect locus, the
implant so formed has insufficient volume to fill the defect
void.
[0014] In yet another aspect, the present invention provides a
matrix-free osteogenic device for inducing bone formation in a
mammal. As contemplated herein, a preferred osteogenic device
comprises an osteogenically-active protein dispersed in a suitable
carrier. Preferred osteogenic proteins, include but are not limited
to, OP-1, OP-2, BMP-2, BMP-4, BMP-5, and BMP-6 (see below). As
disclosed herein, preferred carriers are biocompatible, nonrigid
and amorphous, having no defined surfaces or three-dimensional
structural features. Thus, the devices of the instant invention
lack scaffolding structure and are substantially free of matrix
when administered to a mammal. Examples of preferred carriers
include, but are not limited to, pluronics and alkylcelluloses. As
discussed above, the method of the instant invention involves
providing such a device to a defect locus such that the volume of
the device is insufficient to fill the void volume at the defect
locus.
[0015] The methods, implants and devices of the invention also are
competent to induce and promote or enhance repair of chondral or
osteochondral defects. As a result of this discovery means now are
available for promoting bone and/or cartilage repair without
requiring a surgical procedure. Particularly as a method for
enhancing bone fracture repair, it is contemplated that a suitable
formulation can be injected to a fracture site at the time the
fracture is set so as to accelerate the rate and enhance the
quality of new bone formation.
[0016] The device of the instant invention can have a variety of
configurations. The nature of the device will be dependent upon the
type of carrier in which the osteogenic protein is dispersed. For
example, one preferred embodiment can have a paste-like or
putty-like configuration; such a device can result from dispersing
osteogenic protein in a gel-like carrier such as a Pluronic.TM.
carrier or an alkylcellulose such as carboxymethyl cellulose which
is then wetted with a suitable wetting agent such as, for example,
a saline solution. Another preferred embodiment can have a dry
powder configuration; such a device results from first dispersing
osteogenic protein in a liquid carrier such as water with or
without excipient, followed by lyophilization. A third formulation
is a solution, such as by combining the protein together with an
acidic buffered solution, e.g., pH 4.0-4.5, for example an acetate
or citrate buffer. Still another formulation is a suspension formed
by disbursing osteogenic protein in a physiologically buffered
solution, such as phosphate buffered saline (PBS). Depending upon
the configuration of the device, providing it to a defect locus can
be accomplished by a variety of delivery processes. For example, a
paste can be extruded as a bead which lays along one surface of the
defect locus. Alternatively, a viscous liquid can be brushed or
painted along one or more surfaces of the defect locus or injected
through a wide gauge needle. Less viscous fluids can be injected
through a fine gauge needle. Other configurations and modes of
delivery are contemplated and discussed below in more detail.
[0017] Generally, the proteins of the invention are dimeric
proteins that induce endochondral bone morphogenesis. Osteogenic
proteins comprise a pair of polypeptides that, when folded, adopt a
configuration sufficient for the resulting dimeric protein to
elicit a morphogenetic response. That is, osteogenic proteins
generally induce all of the following biological functions in a
morphogenically permissive environment: stimulating proliferation
of progenitor cells; stimulating the differentiation of progenitor
cells; stimulating the proliferation of differentiated cells; and
supporting the growth and maintenance of differentiated cells.
Progenitor cells are uncommitted cells that are competent to
differentiate into one or more specific types of differentiated
cells, depending on their genomic repertoire and the tissue
specificity of the permissive environment in which morphogenesis is
induced. In the instant invention, osteogenic proteins can induce
the morphogenic cascade which typifies endochondral bone
formation.
[0018] As used herein, the term "morphogen", "bone morphogen",
"bone morphogenic protein", "BMP", "osteogenic protein" and
"osteogenic factor" embraces the class of proteins typified by
human osteogenic protein 1 (hOP-1). Nucleotide and amino acid
sequences for hOP-1 are provided in Seq. ID Nos. 1 and 2,
respectively. For ease of description, hOP-1 is recited herein
below as a representative osteogenic protein. It will be
appreciated by the artisan of ordinary skill in the art, however,
that OP-1 merely is representative of the TGF-.beta. subclass of
true tissue morphogenes competent to act as osteogenic proteins,
and is not intended to limit the description. Other known, and
useful proteins include, BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6,
BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-15, GDF-1, GDF-2,
GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, GDF-12,
NODAL, UNIVIN, SCREW, ADMP, NURAL and osteogenically active amino
acid variants thereof. In one preferred embodiment, the proteins
useful in the invention include biologically active species
variants of any of these proteins, including conservative amino
acid sequence variants, proteins encoded by degenerate nucleotide
sequence variants, and osteogenically active proteins sharing the
conserved seven cysteine skeleton as defined herein and encoded by
a DNA sequence competent to hybridize to a DNA sequence encoding an
osteogenic protein disclosed herein. In still another embodiment,
useful osteogenic proteins include those sharing the conserved
seven cysteine domain and sharing at least 70% amino acid sequence
homology (similarity) within the C-terminal active domain, as
defined herein.
[0019] In still another embodiment, the osteogenic proteins of the
invention can be defined as osteogenically active proteins having
any one of the generic sequences defined herein, including OPX and
Generic Sequences 7 and 8 or Generic Sequences 9 and 10. OPX
accommodates the homologies between the various species of the
osteogenic OP1 and OP2 proteins, and is described by the amino acid
sequence presented herein below and in Seq. ID No. 3. Generic
sequence 9 is a 96 amino acid sequence containing the six cysteine
skeleton defined by hOP1 (residues 330-431 of Seq. ID No. 2) and
wherein the remaining residues accommodate the homologies of OP1,
OP2, OP3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, BMP-10,
BMP-11, BMP-15, GDF-1, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9,
GDF-10, GDF-11, UNIVIN, NODAL, DORSALIN, NURAL, SCREW and ADMP.
That is, each of the non-cysteine residues is independently
selected from the corresponding residue in this recited group of
proteins. Generic sequence 10 is a 102 amino acid sequence
containing the seven cysteine skeleton defined by hOP1 (335-431
Seq. ID No. 2) and wherein the remaining residues accommodate the
homologies of the above-recited protein group.
[0020] As contemplated herein, this family of osteogenic proteins
includes longer forms of a given protein, as well as phylogenetic,
e.g., species and allelic variants and biosynthetic mutants,
including C-terminal addition and deletion mutants and variants,
such as those which may alter the conserved C-terminal cysteine
skeleton, provided that the alteration still allows the protein to
form a dimeric species having a conformation capable of inducing
bone formation in a mammal when implanted in the mammal. In
addition, the osteogenic proteins useful in this invention may
include forms having varying glycosylation patterns and varying
N-termini, may be naturally occurring or biosynthetically derived,
and may be produced by expression of recombinant DNA in procaryotic
or eucaryotic host cells. The proteins are active as a single
species (e.g., as homodimers), or combined as a mixed species,
including heterodimers.
[0021] The methods and implants of the invention do not require a
carrier for the osteogenic protein to induce bone formation
sufficient to fill a critical size bone defect or to enhance
fracture repair in an animal. When the protein is provided in
association with a carrier in the practice of the invention, the
carrier must lack a scaffolding structure, as stated above. When a
preferred carrier is admixed with an osteogenic protein, a device
is formed which is substantially free of matrix as defined herein.
"Substantially free of matrix" is understood to mean that, the
carrier-containing device as formulated prior to administration,
does not contain a substrate competent to act as a scaffold per se.
That is, the device contains no substrate which has been introduced
from an exogenous source and is competent to act as a scaffold.
Stated another way, prior to delivery, the carrier is recognized,
by virtue of its chemical nature, to be unable to contribute a
scaffolding structure to the device. By definition, preferred
carriers are biocompatible, non-rigid and amorphous, having no
defined surfaces. As used herein, "non-rigid" means a carrier
formulation that is lax or plaint or otherwise is substantially
incapable of providing or forming a three-dimensional structure
having one or more defined surfaces. As used herein, "amorphous"
means lacking a definite three-dimensional form, or specific shape,
that is, having no particular shape or form, or having an
indeterminate shape or form. Preferred carriers are also
biocompatible, non-particulate, adherent to bone, cartilage and/or
muscle, and inert. In certain embodiments, water-soluble carriers
are preferable. Additionally, preferred carriers do not contribute
significant volume to a device of the instant invention. That is, a
preferred carrier permits dispersal of an osteogenic protein such
that the final volume of the resulting device is less than the
volume of the void at the defect locus. As discussed below, a
preferred carrier can be a gel, an aqueous solution, a suspension
or a viscous liquid. For example, particularly preferred carriers
can include, without limitation, Pluronics, alkylcelluloses,
acetate buffers, physiological saline solutions, lactose, mannitol
and/or other sugars. Alternatively, osteogenic proteins can be
provided alone to a defect site.
[0022] In summary, the methods, implants and devices of the present
invention can be used to induce endochondral and intramembranous
bone formation sufficient to repair bone defects which do not heal
spontaneously, as well as to promote and enhance the rate and/or
quality of new bone formation, particularly in the repair of
fractures and fusions, including spinal fusions. The methods,
implants and devices also are competent to induce repair of
osteochondral and/or subchondral defects. That is, the methods,
implants and devices are competent to induce formation of new bone
and the overlying surface cartilage. The present invention is
particularly suitable for use in collagen- or matrix-allergenic
recipients. It is also particularly suitable for use in patients
requiring repetitive reconstructive surgeries, as well as cancer
patients as an alternative to reconstructive procedures using metal
joints. The present invention also is useful for individuals whose
ability to undergo spontaneous bone repair is compromised, such as
diabetics, smokers, obese individuals, immune-compromised
individuals, and any individuals have reduced blood flow to their
extremities. Other applications include, but are not limited to,
prosthetic repair, spinal fusion, scoliosis, cranial/facial repair,
and massive allograft repair.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0023] In order to more clearly and concisely describe the subject
matter of the claimed invention, the following definitions are
intended to provide guidance as to the meaning of specific terms
used in the following written description and appended claims.
[0024] "Bone formation" means formation of endochondral bone or
formation of intramembranous bone. In humans, bone formation begins
during the first 6-8 weeks of fetal development. Progenitor stem
cells of mesenchymal origin migrate to predetermined sites, where
they either: (a) condense, proliferate, and differentiate into
bone-forming cells (osteoblasts), a process observed in the skull
and referred to as "intramembranous bone formation;" or, (b)
condense, proliferate and differentiate into cartilage-forming
cells (chondroblasts) as intermediates, which are subsequently
replaced with bone-forming cells. More specifically, mesenchymal
stem cells differentiate into chondrocytes. The chondrocytes then
become calcified, undergo hypertrophy and are replaced by newly
formed bone made by differentiated osteoblasts which now are
present at the locus. Subsequently, the mineralized bone is
extensively remodeled, thereafter becoming occupied by an ossicle
filled with functional bone-marrow elements. This process is
observed in long bones and referred to as "endochondral bone
formation." In postfetal life, bone has the capacity repairs itself
upon injury by mimicking the cellular process of embryonic
endochondral bone development. That is, mesenchymal progenitor stem
cells from the bone-marrow, periosteum, and muscle can be induced
to migrate to the defect site and begin the cascade of events
described above. There, they accumulate, proliferate, and
differentiate into cartilage which is subsequently replaced with
newly formed bone.
[0025] "Defect" or "defect locus" as contemplated herein defines a
void which is a bony structural disruption requiring repair. The
defect further can define an osteochondral defect, including both a
structural disruption of the bone and overlying cartilage. "Void"
is understood to mean a three-dimensional defect such as, for
example, a gap, cavity, hole or other substantial disruption in the
structural integrity of a bone or joint. A defect can be the result
of accident, disease, surgical manipulation and/or prosthetic
failure. In certain embodiments, the defect locus is a void having
a volume incapable of endogenous or spontaneous repair. Such
defects are also called critical-sized segmental defects. The art
recognizes such defects to be approximately 3-4 cm, at least
greater than 2.5 cm, gap incapable of spontaneous repair. In other
embodiments, the defect locus is a non-critical segmental defect
approximately at least 0.5 cm but not more than approximately 2.5
cm. Generally, these are capable of some spontaneous repair, albeit
biomechanically inferior to that made possible by practice of the
instant innovation. In certain other embodiments, the defect is an
osteochondral defect such as an osteochondral plug. Other defects
susceptible to repair using the instant invention include, but are
not limited to, non-union fractures; bone cavities; tumor
resection; fresh fractures; cranial/facial abnormalities; spinal
fusions, as well as those resulting from diseases such as cancer,
arthritis, including osteoarthritis, and other bone degenerative
disorders. "Repair" is intended to mean induction of new bone
formation which is sufficient to fill the void at the defect locus,
but "repair" does not mean or otherwise necessitate a process of
complete healing or a treatment which is 100% effective at
restoring a defect to its pre-defect physiological/structural
state.
[0026] "Matrix" is understood in the art to mean an osteoconductive
substrate having a scaffolding structure on which infiltrating
cells can attach, proliferate and participate in the morphogenic
process culminating in bone formation. In certain embodiments,
matrix can be particulate and porous, with porosity being a feature
critical to its effectiveness in inducing bone formation,
particularly endochondral bone formation. As described earlier, a
matrix is understood to provide certain structural components to
the conventional osteogenic device (i.e., heretofore comprising a
porous, particulate matrix component such as collagen,
demineralized bone or synthetic polymers), thereby acting as a
temporary and resorbable scaffolding structure for infiltrating
cells having interstices for attachment, proliferation and
differentiation of such cells. Accordingly, the term "matrix-free
osteogenic device" or an osteogenic device which is "substantially
free of matrix" contemplates a device which is devoid of an
art-recognized matrix at the time it is provided to a recipient.
Moreover, substantially free of matrix is understood to mean that,
when a device is provided to a defect locus, no substrate competent
to act as a scaffold per se is introduced from an exogenous source.
Matrix-free or substantially free of matrix is not intended to
exclude endogenous matrix which is induced or formed following
delivery of the devices and/or implants disclosed herein to a
defect locus. Thus the present invention further contemplates a
method of inducing endogenous matrix formation by providing to a
defect locus the matrix-free devices or implants disclosed
herein.
[0027] "Osteogenic device" is understood to mean a composition
comprising osteogenic protein dispersed in a biocompatible,
non-rigid amorphous carrier having no defined surfaces. Osteogenic
devices of the present invention are competent to induce bone
formation sufficient to fill a defect locus defining a void.
Osteogenic devices are matrix-free when provided to the defect
locus and are delivered to the defect locus in a volume
insufficient to fill the void defined by the defect locus. A device
can have any suitable configuration, such as liquid, powder, paste,
or gel, to name but a few. Preferred properties of osteogenic
devices suitable for use with the method of the instant invention
include, but are not limited to: adherent to bone, cartilage and/or
muscle; and, effective to provide at least a local source of
osteogenic protein at the defect locus, even if transient. As
contemplated herein, providing a local source of protein includes
both retaining protein at the defect locus as well as controlled
release of protein at the defect locus. All that is required by the
present invention is that the osteogenic device be effective to
deliver osteogenic protein at a concentration sufficient to induce
bone formation that fills the three-dimensional defect defining the
void requiring repair. In addition to osteogenic proteins, various
growth factors, hormones, enzymes therapeutic compositions,
antibiotics, or other bioactive agents can also be contained within
an osteogenic device. Thus, various known growth factors such as
EGF, PDGF, IGF, FGF, TGF-.alpha., and TGF-.beta. can be combined
with an osteogenic device and be delivered to the defect locus. An
osteogenic device can also be used to deliver chemotherapeutic
agents, insulin, enzymes, enzyme inhibitors and/or
chemoattractant/chemotactic factors.
[0028] "Osteogenic protein" or bone morphogenic protein is
generally understood to mean a protein which can induce the full
cascade of morphogenic events culminating in endochondral bone
formation. As described elsewhere herein, the class of proteins is
typified by human osteogenic protein (hOP1). Other osteogenic
proteins useful in the practice of the invention include
osteogenically active forms of OP1, OP2, OP3, BMP2, BMP3, BMP4,
BMP5, BMP6, BMP9, DPP, Vg1, Vgr, 60A protein, GDF-1, GDF-3, GDF-5,
6, 7, BMP10, BMP11, BMP13, BMP15, UNIVIN, NODAL, SCREW, ADNP or
NURAL and amino acid sequence variants thereof. In one currently
preferred embodiment, osteogenic protein include any one of: OP1,
OP2, OP3, BMP2, BMP4, BMP5, BMP6, BMP9, and amino acid sequence
variants and homologs thereof, including species homologs, thereof
Particularly preferred osteogenic proteins are those comprising an
amino acid sequence having at least 70% homology with the
C-terminal 102-106 amino acids, defining the conserved seven
cystein domain, of human OP-1, BMP2, and related proteins. Certain
preferred embodiments of the instant invention comprise the
osteogenic protein, OP-1. Certain other preferred embodiments
comprise mature OP-1 solubilized in a physiological saline
solution. As further described elsewhere herein, the osteogenic
proteins suitable for use with Applicants' invention can be
identified by means of routine experimentation using the
art-recognized bioassay described by Reddi and Sampath. "Amino acid
sequence homology" is understood herein to mean amino acid sequence
similarity. Homologous sequences share identical or similar amino
acid residues, where similar residues are conservative
substitutions for, or allowed point mutations of, corresponding
amino acid residues in an aligned reference sequence. Thus, a
candidate polypeptide sequence that shares 70% amino acid homology
with a reference sequence is one in which any 70% of the aligned
residues are either identical to or are conservative substitutions
of the corresponding residues in a reference sequence. Examples of
conservative variations include the substitution of one hydrophobic
residue such as isoleucine, valine, leucine or methionine for
another, or the substitution of one polar residue for another, such
as the substitution of arginine for lysine, glutamic for aspartic
acids, or glutamine for asparagine, and the like. The term
"conservative variation" also includes the use of a substituted
amino acid in place of an unsubstituted parent amino acid provided
that antibodies raised to the substituted polypeptide also
immunoreact with the unsubstituted polypeptide.
[0029] Proteins useful in this invention include eukaryotic
proteins identified as osteogenic proteins (see U.S. Pat. No.
5,011,691, incorporated herein by reference), such as the OP-1,
OP-2, OP-3 and CBMP-2 proteins, as well as amino acid
sequence-related proteins such as DPP (from Drosophila), Vg1 (from
Xenopus), Vgr-1 (from mouse), GDF-1 (from humans, see Lee (1991),
PNAS 88:4250-4254), 60A (from Drosophila, see Wharton et al. (1991)
PNAS 88:9214-9218), dorsalin-1 (from chick, see Basler et al.
(1993) Cell 73:687-702 and GenBank accession number L12032) and
GDF-5 (from mouse, see Storm et al. (1994) Nature 368:639-643).
BMP-3 is also preferred. Additional useful proteins include
biosynthetic morphogenic constructs disclosed in U.S. Pat. No.
5,011,691, e.g., COP-1, 3-5, 7 and 16, as well as other proteins
known in the art. Still other proteins include osteogenically
active forms of BMP-3b (see Takao, et al., (1996), Biochem.
Biophys. Res. Comm. 219: 656-662. BMP-9 (see WO95/33830), BMP-15
(see WO96/35710), BMP-12 (see WO95/16035), CDMP-1 (see WO
94/12814), CDMP-2 (see WO94/12814), BMP-10 (see WO94/26893), GDF-1
(see WO92/00382), GDF-10 (see WO95/10539), GDF-3 (see WO94/15965)
and GDF-7 (WO95/01802).
[0030] Still other useful proteins include proteins encoded by DNAs
competent to bybridize to a DNA encoding an osteogenic protein as
described herein, and related analogs, homologs, muteins and the
like (see below).
[0031] "Carrier" as used herein means a biocompatible, non-rigid,
amorphous material having no defined surfaces suitable for use with
the devices, implants and methods of the present invention. As
earlier stated, "non-rigid" means a carrier formulation that is lax
or plaint or otherwise is substantially incapable of providing or
forming a three-dimensional structure having one or more defined
surfaces. As used herein, "amorphous" means lacking a definite
three-dimensional form, or specific shape, that is, having no
particular shape or form, or having an indeterminate shape or form.
Suitable carriers also are non-particulate and are non-porous,
i.e., are pore-less. Carriers suitable for use in the instant
invention lack a three-dimensional scaffolding structure and are
substantially matrix-free. Thus, "substantially free of matrix" is
also understood to mean that, when a carrier-containing device is
provided to a defect locus, no substrate competent to act as a
scaffold per se is introduced from any exogenous source, including
the carrier. Prior to delivery to and implantation in the
recipient, the carrier is recognized by virtue of its chemical
nature to be unable to contribute a three-dimensional scaffolding
structure to the device. Preferred carriers are adherent, at least
transiently, to tissues such as bone, cartilage and/or muscles.
Certain preferred carriers are water-soluble, viscous, and/or
inert. Additionally, preferred carriers do not contribute
significant volume to a device. Currently preferred carriers
include, without limitation alkylcelluloses, Pluronics, gelatins,
polyethylene glycols, dextrins, vegetable oils and sugars.
Particularly preferred carriers currently include but are not
limited to Pluronic F127, carboxymethylcelluloses, lactose,
mannitol and sesame oil. Other preferred carriers include acetate
buffer (20 mM, pH 4.5), physiological saline (PBS), and citrate
buffer. In the case of devices comprising carriers such as acetate,
Pluronics and PBS, administration by injection can result in
precipitation of certain osteogenic proteins at the administration
site.
[0032] "Implant" as contemplated herein comprises osteogenic
protein dispersed in a biocompatible, nonrigid amorphous carrier
having no defined surfaces disposed at a defect locus defining a
void. That is, the implant of the present invention is contemplated
to comprise the defect locus per se into/onto which the device of
the present invention has been delivered/deposited. It is further
contemplated that, at the time of delivery of a device, an implant
lacks scaffolding structure and is substantially matrix-free.
Implants resulting from practice of the instant method are
competent to induce bone formation in a defect locus in a mammal
sufficient to fill the defect with newly formed bone without also
requiring inclusion of a matrix or scaffolding structure, at the
time of delivery of a device, sufficient to substantially fill the
void thereby structurally defining the defect size and shape.
[0033] The means for making and using the methods, implants and
devices of the invention, as well as other material aspects
concerning their nature and utility, including how to make and how
to use the subject matter claimed, will be further understood from
the following, which constitutes the best mode currently
contemplated for practicing the invention. It will be appreciated
that the invention is not limited to such exemplary work or to the
specific details set forth in these examples.
[0034] PROTEIN CONSIDERATIONS
[0035] A. Biochemical, Structural and Functional Properties of Bone
Morphogenic Proteins
[0036] Naturally occurring proteins identified and/or appreciated
herein to be osteogenic or bone morphogenic proteins form a
distinct subgroup within the loose evolutionary grouping of
sequence-related proteins known as the TGF-.beta. superfamily or
supergene family. The naturally occurring bone morphogens share
substantial amino acid sequence homology in their C-terminal
regions (domains). Typically, the above-mentioned naturally
occurring osteogenic proteins are translated as a precursor, having
an N-terminal signal peptide sequence, typically less than about 30
residues, followed by a "pro" domain that is cleaved to yield the
mature C-terminal domain. The signal peptide is cleaved rapidly
upon translation, at a cleavage site that can be predicted in a
given sequence using the method of Von Heijne (1986) Nucleic Acids
Research 14:4683-4691. The pro domain typically is about three
times larger than the fully processed mature C-terminal domain.
Herein, the "pro" form of a morphogen refers to a morphogen
comprising a folded pair of polypeptides each comprising the pro
and mature domains of a morphogen polypeptide. Typically, the pro
form of a morphogen is more soluble than the mature form under
physiological conditions. The pro form appears to be the primary
form secreted from cultured mammalian cells.
[0037] In preferred embodiments, the pair of morphogenic
polypeptides have amino acid sequences each comprising a sequence
that shares a defined relationship with an amino acid sequence of a
reference morphogen. Herein, preferred osteogenic polypeptides
share a defined relationship with a sequence present in
osteogenically active human OP-1, SEQ ID NO: 2. However, any one or
more of the naturally occurring or biosynthetic sequences disclosed
herein similarly could be used as a reference sequence. Preferred
osteogenic polypeptides share a defined relationship with at least
the C-terminal six cysteine domain of human OP-1, residues 335-431
of SEQ ID NO: 2. Preferably, osteogenic polypeptides share a
defined relationship with at least the C-terminal seven cysteine
domain of human OP-1, residues 330-431 of SEQ ID NO: 2. That is,
preferred polypeptides in a dimeric protein with bone morphogenic
activity each comprise a sequence that corresponds to a reference
sequence or is functionally equivalent thereto.
[0038] Functionally equivalent sequences include functionally
equivalent arrangements of cysteine residues disposed within the
reference sequence, including amino acid insertions or deletions
which alter the linear arrangement of these cysteines, but do not
materially impair their relationship in the folded structure of the
dimeric morphogen protein, including their ability to form such
intra- or inter-chain disulfide bonds as may be necessary for
morphogenic activity. Functionally equivalent sequences further
include those wherein one or more amino acid residues differs from
the corresponding residue of a reference sequence, e.g., the
C-terminal seven cysteine domain (also referred to herein as the
conserved seven cysteine skeleton) of human OP-1, provided that
this difference does not destroy bone morphogenic activity.
Accordingly, conservative substitutions of corresponding amino
acids in the reference sequence are preferred. Amino acid residues
that are conservative substitutions for corresponding residues in a
reference sequence are those that are physically or functionally
similar to the corresponding reference residues, e.g., that have
similar size, shape, electric charge, chemical properties including
the ability to form covalent or hydrogen bonds, or the like.
Particularly preferred conservative substitutions are those
fulfilling the criteria defined for an accepted point mutation in
Dayhoff et al. (1978), 5 Atlas of Protein Sequence and Structure,
Suppl. 3, ch. 22 (pp. 354-352), Natl. Biomed. Res. Found.,
Washington, D.C. 20007, the teachings of which are incorporated by
reference herein.
[0039] Natural-sourced osteogenic protein in its mature, native
form is a glycosylated dimer typically having an apparent molecular
weight of about 30-36 kDa as determined by SDS-PAGE. When reduced,
the 30 kDa protein gives rise to two glycosylated peptide subunits
having apparent molecular weights of about 16 kDa and 18 kDa. In
the reduced state, the protein has no detectable osteogenic
activity. The unglycosylated protein, which also has osteogenic
activity, has an apparent molecular weight of about 27 kDa. When
reduced, the 27 kDa protein gives rise to two unglycosylated
polypeptides having molecular weights of about 14 kDa to 16 kDa
capable of inducing endochondral bone formation in a mammal. As
described above, particularly useful sequences include those
comprising the C-terminal 102 amino acid sequences of DPP (from
Drosophila), Vg1 (from Xenopus), Vgr-1 (from mouse), the OP1 and
OP2 proteins, proteins (see U.S. Pat. No. 5,011,691 and Oppermann
et al., as well as the proteins referred to as BMP2, BMP3, BMP4
(see WO88/00205, U.S. Pat. No. 5,013,649 and WO91/18098), BMP5 and
BMP6 (see WO90/11366, PCT/US90/01630) and BMP8 and 9.
[0040] In certain preferred embodiments, bone morphogenic proteins
useful herein include those in which the amino acid sequences
comprise a sequence sharing at least 70% amino acid sequence
homology or "similarity", and preferably 80% homology or similarity
with a reference morphogenic protein selected from the foregoing
naturally occurring proteins. Preferably, the reference protein is
human OP-1, and the reference sequence thereof is the C-terminal
seven cysteine domain present in osteogenically active forms of
human OP-1, residues 330-431 of SEQ ID NO: 2. Bone morphogenic
proteins useful herein accordingly include allelic, phylogenetic
counterpart and other variants of the preferred reference sequence,
whether naturally-occurring or biosynthetically produced (e.g.,
including "muteins" or "mutant proteins"), as well as novel members
of the general morphogenic family of proteins including those set
forth and identified above. Certain particularly preferred
morphogenic polypeptides share at least 60% amino acid identity
with the preferred reference sequence of human OP-1, still more
preferably at least 65% amino acid identity therewith.
[0041] In other preferred embodiments, the family of bone
morphogenic polypeptides useful in the present invention, and
members thereof, are defined by a generic amino acid sequence. For
example, Generic Sequence 7 (SEQ ID NO: 4) and Generic Sequence 8
(SEQ ID NO: 5) disclosed below, accommodate the homologies shared
among preferred protein family members identified to date,
including at least OP-1, OP-2, OP-3, CBMP-2A, CBMP-2B, BMP-3, 60A,
DPP, Vg1, BMP-5, BMP-6, Vgr-1, and GDF-1. The amino acid sequences
for these proteins are described herein and/or in the art, as
summarized above. The generic sequences include both the amino acid
identity shared by these sequences in the C-terminal domain,
defined by the six and seven cysteine skeletons (Generic Sequences
7 and 8, respectively), as well as alternative residues for the
variable positions within the sequence. The generic sequences
provide an appropriate cysteine skeleton where inter- or
intramolecular disulfide bonds can form, and contain certain
critical amino acids likely to influence the tertiary structure of
the folded proteins. In addition, the generic sequences allow for
an additional cysteine at position 41 (Generic Sequence 7) or
position 46 (Generic Sequence 4), thereby encompassing the
morphogenically active sequences of OP-2 and OP-3.
1 Generic Sequence 7 Leu Xaa Xaa Xaa Phe Xaa Xaa 1 5 Xaa Gly Trp
Xaa Xaa Xaa Xaa Xaa Xaa Pro 10 15 Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys
Xaa Gly 20 25 Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa 30 35 Xaa Xaa
Xaa Asn His Ala Xaa Xaa Xaa Xaa 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 50 55 Xaa Xaa Xaa Cys Cys Xaa Pro Xaa Xaa Xaa 60 65 Xaa
Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa 70 75 Xaa Xaa Xaa Val Xaa Leu
Xaa Xaa Xaa Xaa 80 85 Xaa Met Xaa Val Xaa Xaa Cys Xaa Cys Xaa 90
95
[0042] wherein each Xaa independently is selected from a group of
one or more specified amino acids defined as follows: "Res." means
"residue" and Xaa at res.2=(Tyr or Lys); Xaa at res.3=Val or Ile);
Xaa at res.4=(Ser, Asp or Glu); Xaa at res.6=(Arg, Gln, Ser, Lys or
Ala); Xaa at res.7=(Asp or Glu); Xaa at res.8=(Leu, Val or Ile);
Xaa at res. 11=(Gln, Leu, Asp, His, Asn or Ser); Xaa at
res.12=(Asp, Arg, Asn or Glu); Xaa at res. 13=(Trp or Ser); Xaa at
res.14=(Ile or Val); Xaa at res.15=(Ile or Val); Xaa at res.16 (Ala
or Ser); Xaa at res.18=(Glu, Gln, Leu, Lys, Pro or Arg); Xaa at
res.19=(Gly or Ser); Xaa at res.20=(Tyr or Phe); Xaa at
res.21=(Ala, Ser, Asp, Met, His, Gln, Leu or Gly); Xaa at
res.23=(Tyr, Asn or Phe); Xaa at res.26=(Glu, His, Tyr, Asp, Gln,
Ala or Ser); Xaa at res.28=(Glu, Lys, Asp, Gln or Ala); Xaa at
res.30=(Ala, Ser, Pro, Gln, Ile or Asn); Xaa at res.31=(Phe, Leu or
Tyr); Xaa at res.33=(Leu, Val or Met); Xaa at res.34=(Asn, Asp,
Ala, Thr or Pro); Xaa at res.35=(Ser, Asp, Glu, Leu, Ala or Lys);
Xaa at res.26=(Tyr, Cys, His, Ser or Ile); Xaa at res.37=(Met, Phe,
Gly or Leu); Xaa at res.38=(Asn, Ser or Lys); Xaa at res.39=(Ala,
Ser, Gly or Pro); Xaa at res.40=(Thr, Leu or Ser); Xaa at
res.44=(Ile, Val or Thr); Xaa at res.45=(Val, Leu, Met or Ile); Xaa
at res.46=(Gln or Arg); Xaa at res.47=(Thr, Ala or Ser); Xaa at
res.48=(Leu or Ile); Xaa at res.49=(Val or Met); Xaa at
res.50=(His, Asn or Arg); Xaa at res.51=(Phe, Leu, Asn, Ser, Ala or
Val); Xaa at res.52=(Ile, Met, Asn, Ala, Val, Gly or Leu); Xaa at
res.53=(Asn, Lys, Ala, Glu, Gly or Phe); Xaa at res.54=(Pro, Ser or
Val); Xaa at res.55=(Glu, Asp, Asn, Gly, Val, Pro or Lys); Xaa at
res.56=(Thr, Ala, Val, Lys, Asp, Tyr, Ser, Gly, Ile or His); Xaa at
res.57=(Val, Ala or Ile); Xaa at res.58=(Pro or Asp); Xaa at
res.59=(Lys, Leu or Glu); Xaa at res.60=(Pro, Val or Ala); Xaa at
res.63=(Ala or Val); Xaa at res.65=(Thr, Ala or Glu); Xaa at
res.66=(Gln, Lys, Arg or Glu); Xaa at res.67=(Leu, Met or Val); Xaa
at res.68=(Asn, Ser, Asp or Gly); Xaa at res.69=(Ala, Pro or Ser);
Xaa at res.70=(Ile, Thr, Val or Leu); Xaa at res.71=(Ser, Ala or
Pro); Xaa at res.72=(Val, Leu, Met or Ile); Xaa at res.74=(Tyr or
Phe); Xaa at res.75=(Phe, Tyr, Leu or His); Xaa at res.76=(Asp, Asn
or Leu); Xaa at res.77=(Asp, Glu, Asn, Arg or Ser); Xaa at
res.78=(Ser, Gln, Asn, Tyr or Asp); Xaa at res.79=(Ser, Asn, Asp,
Glu or Lys); Xaa at res.80=(Asn, Thr or Lys); Xaa at res.82=(Ile,
Val or Asn); Xaa at res.84=(Lys or Arg); Xaa at res.85=(Lys, Asn,
Gln, His, Arg or Val); Xaa at res.86=(Tyr, Glu or His); Xaa at
res.87=(Arg, Gln, Glu or Pro); Xaa at res.88=(Asn, Glu, Trp or
Asp); Xaa at res.90=(Val, Thr, Ala or Ile); Xaa at res.92=(Arg,
Lys, Val, Asp, Gln or Glu); Xaa at res.93=(Ala, Gly, Glu or Ser);
Xaa at res.95=(Gly or Ala) and Xaa at res.97=(His or Arg).
[0043] Generic Sequence 8 (SEQ ID NO: 5) includes all of Generic
Sequence 7 and in addition includes the following sequence (SEQ ID
NO: 6) at its N-terminus:
2 Cys Xaa Xaa Xaa Xaa 1 5
[0044] Accordingly, beginning with residue 7, each "Xaa" in Generic
Sequence 8 is a specified amino acid defined as for Generic
Sequence 7, with the distinction that each residue number described
for Generic Sequence 7 is shifted by five in Generic Sequence 8.
Thus, "Xaa at res.2=(Tyr or Lys)" in Generic Sequence 7 refers to
Xaa at res. 7 in Generic Sequence 8. In Generic Sequence 8, Xaa at
res.2=(Lys, Arg, Ala or Gln); Xaa at res.3=(Lys, Arg or Met); Xaa
at res.4=(His, Arg or Gln); and Xaa at res.5=(Glu, Ser, His, Gly,
Arg, Pro, Thr, or Tyr).
[0045] In another embodiment, useful osteogenic proteins include
those defined by Generic Sequences 9 and 10, described herein
above.
[0046] As noted above, certain currently preferred bone morphogenic
polypeptide sequences useful in this invention have greater than
60% identity, preferably greater than 65% identity, with the amino
acid sequence defining the preferred reference sequence of hOP-1.
These particularly preferred sequences include allelic and
phylogenetic counterpart variants of the OP-1 and OP-2 proteins,
including the Drosophila 60A protein. Accordingly, in certain
particularly preferred embodiments, useful morphogenic proteins
include active proteins comprising pairs of polypeptide chains
within the generic amino acid sequence herein referred to as "OPX"
(SEQ ID NO: 3), which defines the seven cysteine skeleton and
accommodates the homologies between several identified variants of
OP-1 and OP-2. As described therein, each Xaa at a given position
independently is selected from the residues occurring at the
corresponding position in the C-terminal sequence of mouse or human
OP-1 or OP-2.
[0047] In still another preferred embodiment, useful osteogenically
active proteins have polypeptide chains with amino acid sequences
comprising a sequence encoded by nucleic acid that hybridizes,
under low, medium or high stringency hybridization conditions, to
DNA or RNA encoding reference morphogen sequences, e.g., C-terminal
sequences GDF3, GDF6, GDF7 and the like. As used herein, high
stringent hybridization conditions are defined as hybridization
according to known techniques in 40% formamide, 5.times.SSPE,
5.times.Denhardt's Solution, and 0.1% SDS at 37.degree. C.
overnight, and washing in 0.1.times.SSPE, 0.1% SDS at 50.degree. C.
Standard stringence conditions are well characterized in
commercially available, standard molecular cloning texts.
[0048] As noted above, proteins useful in the present invention
generally are dimeric proteins comprising a folded pair of the
above polypeptides. Such morphogenic proteins are inactive when
reduced, but are active as oxidized homodimers and when oxidized in
combination with others of this invention to produce heterodimers.
Thus, members of a folded pair of morphogenic polypeptides in a
morphogenically active protein can be selected independently from
any of the specific polypeptides mentioned above.
[0049] The bone morphogenic proteins useful in the materials and
methods of this invention include proteins comprising any of the
polypeptide chains described above, whether isolated from
naturally-occurring sources, or produced by recombinant DNA or
other synthetic techniques, and includes allelic and phylogenetic
counterpart variants of these proteins, as well as biosynthetic
variants (muteins) thereof, and various truncated and fusion
constructs. Deletion or addition mutants also are envisioned to be
active, including those which may alter the conserved C-terminal
six or seven cysteine domain, provided that the alteration does not
functionally disrupt the relationship of these cysteines in the
folded structure. Accordingly, such active forms are considered the
equivalent of the specifically described constructs disclosed
herein. The proteins may include forms having varying glycosylation
patterns, varying N-termini, a family of related proteins having
regions of amino acid sequence homology, and active truncated or
mutated forms of native or biosynthetic proteins, produced by
expression of recombinant DNA in host cells.
[0050] The bone morphogenic proteins contemplated herein can be
expressed from intact or truncated cDNA or from synthetic DNAs in
prokaryotic or eukaryotic host cells, and purified, cleaved,
refolded, and dimerized to form morphogenically active
compositions. Currently preferred host cells include E. coli or
mammalian cells, such as CHO, COS or BSC cells. Detailed
descriptions of the bone morphogenic proteins useful in the
practice of this invention, including how to make, use and test
them for osteogenic activity, are disclosed in numerous
publications, including U.S. Pat. Nos. 5,266,683 and 5,011,691, the
disclosures of which are incorporated by reference herein.
[0051] Thus, in view of this disclosure, skilled genetic engineers
can isolate genes from cDNA or genomic libraries of various
different biological species, which encode appropriate amino acid
sequences, or construct DNAs from oligonucleotides, and then can
express them in various types of host cells, including both
prokaryotes and eukaryotes, to produce large quantities of active
proteins capable of stimulating endochondial bone morphogenesis in
a mammal.
[0052] B. Preparations of Bone Morphogenic Protein, OP-1
[0053] 1. Lyophilized Protein
[0054] OP-1 can be lyophilized from 20 mM acetate buffer, pH 4.5,
with 5% mannitol, lactose, glycine or other additive or bulking
agent, using standard lyophilization protocols. OP-1 reconstituted
in this manner has been observed to be biologically active for at
least six months stored at 4.degree. C. or 30.degree. C.
[0055] OP-1 can also be lyophilized from a succinate or a citrate
buffer (or other non-volatile buffer) for re-constitution in water,
and from water for re-constitution in 20 mM acetate buffer, pH 4.5.
Generally, additives such as lactose, sucrose, glycine and mannitol
are suitable for use in lyophilized matrix-free osteogenic devices.
In certain embodiments, such devices (0.5 mg/ml OP-1 and 5%
additive) can be prepared in a wet or dry configuration prior to
lyophilization.
[0056] For example, liquid formulations of OP-1 in 10 and 20 mM
acetate buffer (pH 4, 4.5 and 5) with and without mannitol (0%, 1%
and 5%) are stable and osteogenically active for at least six
months.
[0057] II. CARRIER CONSIDERATIONS
[0058] As already explained, "carrier" as used herein means a
biocompatible, non-rigid, amorphous material having no defined
surfaces suitable for use with the devices, implants and methods of
the present invention. Suitable carriers are non-particulate and
are non-porous, i.e., are pore-less. Carriers suitable for use in
the instant invention lack a scaffolding structure and are
substantially matrix-free. Thus, "substantially free of matrix" is
also understood to mean that, when a carrier-containing device is
provided to a defect locus, no substrate competent to act as a
scaffold per se is introduced from an exogenous source, including
the carrier. Prior to delivery to and implantation in the
recipient, the carrier is recognized by virtue of its chemical
nature to be substantially unable to contribute a three-dimensional
scaffolding structure to the device. Preferred carriers are
adherent, at least transiently, to tissues such as bone, cartilage
and/or muscles. Certain preferred carriers are water-soluble,
viscous, and/or inert. Additionally, preferred carriers do not
contribute significant volume to a device. Currently preferred
carriers are selected from the group consisting of:
alkylcelluloses, Pluronics, gelatins, polyethylene glycols (PEG),
dextrins, vegetable oils and sugars. Particularly preferred
carriers currently include, but are not limited to, Pluronic F127,
carboxymethylcelluloses (CMC), (e.g., low viscosity CMC from
Aqualon), lactose, PEG, mannitol, sesame oil, and hetastarch
(Hespan, Dupont), and combinations thereof. Other preferred
carriers include, without limitation, acetate buffer (20 mM, pH
4.5), physiological saline, and citrate buffers. In the case of
devices comprising carriers such as acetate, Pluronics and PBS,
administration by injection can result in precipitation of certain
osteogenic proteins at the administration site.
[0059] III. FORMULATION AND DELIVERY CONSIDERATIONS
[0060] The devices of the invention can be formulated using routine
methods. All that is required is determining the desired final
concentration of osteogenic protein per unit volume of carrier,
keeping in mind that the delivered volume of device will be less
than the volume the void at the defect locus. The desired final
concentration of protein will depend on the specific activity of
the protein as well as the type, volume, and/or anatomical location
of the defect. Additionally, the desired final concentration of
protein can depend on the age, sex and/or overall health of the
recipient. Typically, for a critical-sized segmental defect
approximately at least 2.5 cm in length, 0.05 ml (or mg) of a
device containing 0.5-1.5 mg osteogenic protein has been observed
to induce bone formation sufficient to repair the gap. In the case
of a non-critical sized defect fresh fractures, approximately
0.1-0.5 mg protein has been observed to repair the gap or defect.
Optimization of dosages requires no more than routine
experimentation and is within the skill level of one of ordinary
skill in the art.
[0061] As exemplified below, the devices of the present invention
can assume a variety of configurations. For example, a matrix-free
osteogenic device in solution can be formulated by solubilizing
certain forms of OP-1 in solutions of acetate (20 mM, pH4.5) or
citrate buffers, or phosphate-buffered saline (PBS), pH 7.5. In
some instances, the osteogenic protein may not be entirely
solubilized and/or may precipitate upon administration into the
defect locus. Suspensions, aggregate formation and/or in vivo
precipitation does not impair the operativeness of the matrix-free
osteogenic device when practiced in accordance with the invention
disclosed herein. Matrix-free devices in solution are particularly
suitable for administration by injection, such as providing a
device to a fracture locus by injection rather than surgical
means.
[0062] Generally speaking, the configuration of matrix-free devices
suitable for delivery by injection differ from those preferred for
use at an open, surgical site. For example, lyophilized
preparations of matrix-free devices are one currently preferred
embodiment for repair of this type of defect. The above-described
matrix-free devices in solution can be used to prepare a
lyophilized configuration. For example, as described below, the
osteogenic protein OP-1 can be admixed with the above-described
buffers and then lyophilized. OP-1 can also be lyophilized from
mannitol-containing water.
[0063] As exemplified below, lyophilized configurations of
matrix-free osteogenic devices can induce bone formation in
critical-sized and non-critical sized segmental defects. For
example, providing a lyophilized device to a segmental defect locus
comprises depositing non-contiguous aliquots of the lyophilized
device along the length of exposed muscle spanning the segmental
defect, such that the total number of aliquots provides an amount
of osteogenic protein sufficient to induce bone formation which
ultimately fills the void at the defect locus. Placement is
followed by routine closure of the defect site whereby the layers
of muscles and associated tissue are sutured, layer-by-layer, to
enclose the aliquots in the void at the defect locus. This type of
delivery and surgical closure require only routine skill and
experimentation. Similar formulations and methods of delivery can
be used to induce bone formation for repair of a gap caused by a
failed prosthetic, bone tumor resection, cranial/facial
reconstruction, spinal fusions and massive allograft defects. Any
modifications of the above-described methods of delivery which may
be required for specialized applications of lyophilized matrix-free
devices are within the skill level of the artisan and require only
routine experimentation.
[0064] Yet another configuration of matrix-free devices is
exemplified below. Osteogenic protein and a carrier such as
carboxymethylcellulose (low viscosity, Aqualon, or Pluronic F127
can be admixed to form a paste. In some embodiments, approximately
saline is added to carrier to form a paste into which an osteogenic
protein such as OP-1 can be dispersed. A paste configuration can be
used to paint the surfaces of a defect such as a cavity. Pastes can
be used to paint fracture defects, chondral or osteochondral
defects, as well as bone defects at a prosthetic implant site. A
paste can also be injected or extruded into or along one of the
surfaces of a defect, in a manner similar to extruding toothpaste
or caulking from a tube, such that a bead of matrix-free device is
delivered along the length of the defect locus. Typically, the
diameter of the extruded bead is determined by the type of defect
as well as the volume of the void at the defect locus.
[0065] Carriers such as carboxymethylcellulose can also be used to
formulate a device with a configuration like putty. As will be
obvious to the skilled artisan, such a configuration results from
adjusting the proportion of carrier to wetting agent, with less
wetting agent producing a drier device and more producing a wetter
device. The precise device configuration suitable to repair a
defect will at least depend on the type of defect and the size of
the defect. The skilled artisan will appreciate the variables.
[0066] Yet another configuration which is suitable for the devices
of the instant invention is a gel. This is exemplified below by the
Pluronic-containing matrix-free devices which can induce bone
formation in vivo. Gels of this type have been used to treat
fractures as well as gap repairs. One useful feature of this
configuration is that the viscosity of the gel can be manipulated
by adjusting the amount of carrier, thereby permitting a wide-range
of applications (e.g., segmental defects, fractures, and
reconstructions) and modes of delivery (e.g., injection, painting,
extrusion, and the like). Thus this type of device can assume at
least the forms of an injectable liquid, a viscous liquid, and an
extrudable gel merely by manipulating the amount of carrier into
which the osteogenic protein is dispersed.
[0067] In yet other embodiments of the present invention,
preparation of the actual osteogenic device can occur immediately
prior to its delivery to the defect locus. For example,
CMC-containing devices can be prepared on-site, suitable for
admixing immediately prior to surgery. In one embodiment, low
viscosity CMC (Aqualon) is packaged and irradiated separately from
the osteogenic protein OP-1. The OP-1 protein then is admixed with
the CMC carrier, and tested for osteogenic activity. Devices
prepared in this manner were observed to be as biologically active
as the conventional device without CMC. Again, all that is required
is determining the effective amount of osteogenic protein to induce
bone formation sufficient to fill the defect locus and maintaining
a device volume which is less than the volume of the void at the
defect locus. The precise manner in which the device of the present
invention is formulated, and when or how formulation is
accomplished, is not critical to operativeness.
[0068] Practice of the invention will be still more fully
understood from the following examples, which are presented herein
for illustration only and should not be construed as limiting the
invention in any way.
[0069] IV. BIOASSAY
[0070] A. Bioassay of Osteogenic Activity: Endochondral Bone
Formation and Related Properties
[0071] The following sets forth protocols for identifying and
characterizing bonafide osteogenic or bone morphogenic proteins as
well as osteogenic devices within the scope of Applicants'
invention.
[0072] The art-recognized bioassay for bone induction as described
by Sampath and Reddi (Proc. Natl. Acad. Sci. USA (1983)
80:6591-6595) and U.S. Pat. No. 4,968,590, the disclosures of which
are herein incorporated by reference, is used to establish the
efficacy of the purification protocols. As is demonstrated below,
this assay consists of depositing the test samples in subcutaneous
sites in allogeneic recipient rats under ether anesthesia. A
vertical incision (1 cm) is made under sterile conditions in the
skin over the thoracic region, and a pocket is prepared by blunt
dissection. In certain circumstances, approximately 25 mg of the
test sample is implanted deep into the pocket and the incision is
closed with a metallic skin clip. The heterotropic site allows for
the study of bone induction without the possible ambiguities
resulting from the use of orthotopic sites.
[0073] The sequential cellular reactions occurring at the
heterotropic site are complex. The multistep cascade of
endochondral bone formation includes: binding of fibrin and
fibronectin to implanted matrix, chemotaxis of cells, proliferation
of fibroblasts, differentiation into chondroblasts, cartilage
formation, vascular invasion, bone formation, remodeling, and bone
marrow differentiation.
[0074] In rats, this bioassay model exhibits a controlled
progression through the stages of matrix induced endochondral bone
development including: (1) transient infiltration by
polymorphonuclear leukocytes on day one; (2) mesenchymal cell
migration and proliferation on days two and three; (3) chondrocyte
appearance on days five and six; (4) cartilage matrix formation on
day seven; (5) cartilage calcification on day eight; (9) vascular
invasion, appearance of osteoblasts, and formation of new bone on
days nine and ten; (10) appearance of osteoblastic and bone
remodeling on days twelve to eighteen; and (11) hematopoietic bone
marrow differentiation in the ossicle on day twenty-one.
[0075] Histological sectioning and staining is preferred to
determine the extent of osteogenesis in the implants. Staining with
toluidine blue or hemotoxylin/eosin demonstrates clearly the
ultimate development of endochondrial bone. Twelve day bioassays
are usually sufficient to determine whether bone inducing activity
is associated with the test sample.
[0076] Additionally, alkaline phosphatase activity can be used as a
marker for osteogenesis. The enzyme activity can be determined
spectrophotometrically after homogentization of the excised test
material. The activity peaks at 9-10 days in vivo and thereafter
slowly declines. Samples showing no bone development by histology
should have no alkaline phosphatase activity under these assay
conditions. The assay is useful for quantitation and obtaining an
estimate of bone formation very quickly after the test samples are
removed from the rat. For example, samples containing osteogenic
protein at several levels of purity have been tested to determine
the most effective dose/purity level, in order to seek a
formulation which could be produced on an industrial scale. The
results as measured by alkaline phosphatase activity level and
histological evaluation can be represented as "bone forming units".
One bone forming unit represents the amount of protein that is need
for half maximal bone forming activity on day 12. Additionally,
dose curves can be constructed for bone inducing activity in vivo
at each step of a purification scheme by assaying various
concentrations of protein. Accordingly, the skilled artisan can
construct representative dose curves using only routine
experimentation.
[0077] B. Bone Formation Following Implantation of Matrix-Free OP-1
Osteogenic Devices
[0078] Osteogenic devices were made with 62.5 .mu.g lyophilized
OP-1, either with or without 25 mg collagen matrix. These devices
were evaluated for their ability to support bone formation using
the above-described rat ectopic bone formation assay, in both an
intramuscular and subcutaneous site. Additionally, the mass of bone
formed was assessed by measuring the calcium contents and the
weights of the removed devices. The data generated is summarized in
Tables 1 and 2 below.
[0079] As evidenced by both histology and calcium content, bone
formed in response to all of the matrix-free OP-1 samples. These
data illustrate that implanting a matrix-free OP-1 device alone is
sufficient to induce endochondral bone formation in rat ectopic
sites.
3TABLE 1 Bone Formation vs. Concentrations of OP-1 Intramuscular
Site Device 12 Day Implant ( ) Sample Size Calcium, Implant
Histology, Collagen OP-1 .mu.g/mg tissue Weight, mg % bone 25 mg
(5) 62.5 .mu.g 38.2-67.1 70-90 0 mg 51.7 .mu.g 48.6 430.7 mg 90 0
mg 61.3 .mu.g 35.8 256.1 mg 90 0 mg 18.6 .mu.g 66.3 457.8 mg 90 0
mg 59.2 .mu.g 53.9 580.0 mg 90 0 mg 59.5 .mu.g 25.9 383.1 mg 90
[0080]
4TABLE 2 Bone Formation vs. Concentration of OP-1 Subcutaneous Site
Device 12 Day Implant ( ) Sample Size Calcium, Implant Histology,
Collagen OP-1 .mu.g/mg tissue Weight, mg % bone 25 mg (5) 62.5
.mu.g 28.4-54.6 500.6-1472.6 60-80 0 mg 40.4 .mu.g 44.4 132.7 mg 90
0 mg 36.7 .mu.g 43.9 337.7 mg 80 0 mg 35.2 .mu.g 37.4 310.7 mg 90 0
mg 51.0 .mu.g 57.5 195.8 mg 90 0 mg 51.4 .mu.g 29.7 721.9 mg 90
[0081] C. Matrix-Free OP-1 Devices Containing Water-Soluble
Carriers
[0082] OP-1 admixed with water soluble carriers also support bone
formation. In this study, mannitol, carboxymethylcellulose,
dextrin, PEG3350, a Pluronic gel and collagen each were formulated
into a paste by the addition of 0.9% sterile saline. Ten .mu.g of
OP-1 dissolved in water was added to the paste to produce a
matrix-free device, and the device was immediately implanted
intramuscularly in rats. After 12 days, the implanted devices were
removed and evaluated for bone formation by both calcium content
and histology. These data confirm the above-described observation
that collagen matrix is not essential for inducing volume-filling
bone formation. Of the water-soluble carriers evaluated, mannitol
demonstrated the best results overall, with the others appearing
relatively comparable.
5 TABLE 3 Histology Calcium, .mu.g/mg implant Implant ug, mg (%)
Mannitol 100-200 28-48 >90 CMC 50-200 28-52 >80-90 Dextrin
20-80 8-12 >90 Collagen 550-800 20-45 >90 PEG 100-150 10-12
>90 Pluronic 50-150 14-28 >90
[0083] D. Matrix-Free OP-1 Devices in Solution
[0084] Matrix-free osteogenic devices in solution also were
demonstrated to induce bone formation when administered either
intramuscularly (IM), or intradermally (ID). In an exemplary
experiment, matrix-free OP-1 devices were prepared in 20 mM acetate
buffer, pH 4.5. The devices were prepared such that the desired
dose of (5-50 mg) OP-1 would be delivered in a 100 .mu.L injection
volume. Both forms of administration induced bone formation as
measured by calcium content, histology and explant weight.
[0085] E. Pluronic Gel-Containing Matrix-Free OP-1 Devices
[0086] Experiments currently preferred were initiated with a
Pluronic formulation of the matrix-free OP-1 device. In one
embodiment, this device has the unique property of being liquid at
refrigeration temperatures, but gel-like when warmed to room
temperature. This allows the device to be drawn up into syringe
when cold, allowing for easy injection after a few minutes at room
temperature. This was useful for injecting such OP-1 devices into
fracture sites, since the gel permits containment of OP-1 at the
site of injury.
[0087] Pluronic gel, prepared from 0.5 g of commercially available
Pluronic F127 to which 1.35 mL of water is added, is a viscous
liquid at refrigeration temperature and a semisolid gel at room
temperature. Bone formation was observed after 12 days in response
to IM administration of matrix-free OP-1-Pluronic gel devices. The
gel was not injected directly into the muscle, but was injected
into the muscle flap without the use of a needle. Bone also formed
in response to SC administration of these same Pluronic gel
devices. The OP-1 dose (5-25 mg) was contained in 50 .mu.l gel.
[0088] Recovery of OP-1 from the gel devices was determined by
extracting with 8M urea buffer and injecting the extract onto the
HPLC. 100% recovery of the OP-1 from the gel was obtained. In
addition, urea extracts of selected gels were as active as an OP-1
standard. For example, one of the gels was re-extracted after 10
days storage at refrigeration temperature in a syringe; 100%
recovery of OP-1 was obtained and again the extract was as active
as an OP-1 standard.
[0089] As described below, sterile filtered OP-1 can be added to
autoclaved or irradiated gel in an aspectic manner so that a
sterile device can be provided.
[0090] OP-1 has been admixed with autoclaved Pluronic gels. The gel
was prepared, autoclaved and then chilled so that it liquified
prior to admixture with the OP-1. The OP-1 gel was then filled into
syringes which were stored at 5.degree. C. The samples were
observed to be stable for at least 2 weeks at 5.degree. C.
Approximately at least 50-60% of the initial OP-1 remained after 7
weeks storage at 5.degree. C. Gels were also prepared from
irradiated Pluronic F127. Recoveries of 40-50% was observed after 7
weeks at 5.degree. C.
[0091] A second time course was carried out for the purposes of
evaluating bone formation in response to matrix-free OP-1-Pluronic
gel devices. 50 .mu.l volumes of Pluronic gel containing zero or 10
.mu.g of OP-1 were injected into a muscle flap. Implants removed at
day 7, 12 and 21 were analyzed for calcium content and alkaline
phosphatase activity. The time course of bone formation was similar
to that observed with the standard collagen-containing OP-1
device.
[0092] V. ANIMAL STUDIES: METHODS OF USE OF MATRIX-FREE OP-1
DEVICES AND IMPLANTS
[0093] A. Healing of Critical-Sized Segmental Defects in Dogs Using
Matrix-Free Osteogenic Devices
[0094] 1. Experiment 1
[0095] The following experiments demonstrate the efficacy of
injectable and freeze-dried formulations of rhOP-1 for healing both
critical and noncritical sized segmental defects in an established
canine ulna defect model. Three formulations of matrix-free
osteogenic devices, each containing OP-1, were evaluated in
critical-size defects (2.5 cm) and/or noncritical size defects (5
mm, 3 mm, 1.5 mm). As described above, critical size defects are
defects which will not heal spontaneously. The three formulations
evaluated were (1) 20 mm acetate buffered solution (pH 4.5); (2)
phosphate-buffered saline (PBS, approximately pH 7.5); and (3)
lyophilized (freeze-dried) protein alone. The amount of OP-1
provided in the critical size defects was 1.75 mg; 0.35 mg protein
was provided in the non-critical size defects. Immediately before
wound closure, OP-1 was admixed with acetate or PBS and then
injected at the defect site in a total of 1 ml. Lyophilized samples
were placed along the length of the defect in 5 separate aliquots
at discrete, non-contiguous loci along the length of the
defect.
[0096] Using standard surgical techniques, osteoperiosteal
segmental defects of the prescribed size were created bilaterally
in the mid ulna region of twenty adult bred-for-purpose mongrel
dogs. All animals were between one and two years old, weighed from
35 to 50 pounds, and were supplied by USDA licensed providers.
Special attention was paid in selecting animals of uniform size and
weight to limit the variability in bone geometry and loading. The
animals were radiographically screened preoperatively to ensure
proper size, skeletal maturity, and that no obvious osseous
abnormalities existed. The animals also were screened clinically to
exclude acute and chronic medical conditions during a two-week
quarantine period. A complete blood count with cell differential
was performed prior to surgery.
[0097] The radius was maintained for mechanical stability, but no
internal or external fixation was used. The site was irrigated with
saline to remove bone debris and spilled marrow cells and then
dried and homeostasis was achieved prior to providing the
formulation to the site. The soft-tissues were meticulously closed
in layers before injection of formulations 1 or 2 (acetate or PBS).
Formulation 3 devices (lyophilized protein alone) was placed along
the interosseous space before closing the tissue layers to contain
the implant. The procedure was then repeated on the contralateral
side, except that no OP-1 device was provided before wound
closure.
[0098] Animals were administered intramuscular antibiotics for four
days post-surgery and routine anterior-posterior radiographs were
taken immediately after surgery to insure proper placement. Animals
were kept in 3.times.4 recovery cases for 24 to 72 hours
postoperatively after which they were transferred to runs and
allowed unrestricted motion.
[0099] Biweekly radiographs were taken to study the progression of
healing. In addition, pre-operative blood (serum) was taken
biweekly until sacrifice to study antibody formation by the
sponsor. At sacrifice, all ulnae were retrieved en bloc and those
that were healed sufficiently were mechanically tested in torsion.
Segments were evaluated by histology for tissue response, bone
architecture and remodeling, and quality and amount of new bone
formation and healing.
[0100] Animals were sacrificed at 4, 6, 8 or 12 weeks post
operatively.
[0101] 1b(3). Radiographs
[0102] Radiographs of the forelimbs were obtained biweekly until
eight weeks postoperative and then again at sacrifice at twelve
postoperative weeks. Standardized exposure times and intensities
were used, and sandbags were used to position the extremities in a
consistent manner. Radiographs were evaluated and compared to
earlier radiographs to appreciate quality and speed of defect
healing.
[0103] Mechanical Testing
[0104] Immediately after sectioning, if healing was deemed
sufficient by manual manipulation, specimens were tested to failure
in torsion on an MTS closed-loop hydraulic test machine
(Minneapolis, Minn.) operated in stroke control at a constant
displacement rate of 50 mm/min in a cylindrical aluminum sleeve and
cemented with methylmethacrylate using manufacturer's protocol. One
end was rigidly fixed and the other was rotated counterclockwise.
Since the dog ulna has a slight curvature, the specimens were
mounted eccentrically to keep specimen rotation coaxial with that
of the testing device. The torsional force was applied with a lever
arm of six cm, by a servohydraulic materials testing system.
Simultaneous recordings were made of implant displacement, as
measured by the machine stroke controller, while load was recorded
from the load cell. Data was recorded via an analog-to-digital
conversion board and a personal computer and an online computer
acquisition software. Force-angular displacement curves were
generated from which the torque and angular deformation to failure
were obtained, and the energy absorption to failure computed as the
area under the load - displacement curve.
[0105] Results
[0106] The bone healing characteristics, mechanical strength, and
histology of critical size ulna defects treated with rhOP-1 without
carrier material were similar to that of defects treated with the
standard OP-1 device. In brief, the experimental observations were
as follows: New bone formation and healing patterns observed
radiographically in defects treated with rhOP-1 without a matrix
were similar to healing patterns observed previously with the
conventional collagen-containing OP-1 device. In general, new bone
formation was evident as early as two weeks postoperative. The new
bone continued to densify, consolidate and remodel until sacrifice
at twelve postoperative weeks. This study demonstrated that
functional bony union is possible with human OP-1 devices without
matrix. Additionally, the gross appearance and the twelve week
histologic characteristics were similar to that observed with the
conventional collagen-containing OP-1 device. Of the six defects
treated with a matrix-free OP-1 device, four had solid bony unions
at twelve weeks postoperative. The remaining two defects, in the
same animal, demonstrated some early new bone formation
radiographically, however at sacrifice were incompletely spanned or
filled with new bone. The mean torsional load to failure of the
healed defects was 40.05N (This represents the equivalent of about
79% of previously tested segmental defects treated with the
traditional collagen-containing OP-1 device and 61% of previously
tested intact control ulna.)
[0107] Radiographically, extensive new bone formation was observed
at two weeks postoperative with all three formulations. From two to
12 weeks, new bone increased in volume and radiodensity and filled
and spanned the defects. By 12 weeks postoperative (sacrifice),
radiodense new bone had significantly filled and bridged the
defects treated with rhOP-1 formulations. All rhOP-1 defects were
mechanically stable and bridged with new bone at sacrifice at 12
weeks. No significant differences were found among the three
formulations for radiographic appearance, although Formulations 1
and 2 scored slightly higher than Formulation 3 at all time
periods.
[0108] Histologically, proliferative new bone was present within
and surrounding the defects treated with rhOP-1. Bridging of the
defects and bony healing was significantly completed by 12 weeks
postoperative with gaps of fibrocartilage between areas of
significant new bone growth. Signs of early cortex development with
densification of the new bone borders was present in all rhOP-1
defects. There were no differences in histologic grading results
based upon formulation type, although Foundation 2 scored highly
than Formulations 3 and 1 for quality of union.
[0109] The mean load to failure of the Formulation 1 defects was
47.64N (94% of defects treated with the standard OP-1 device and
73% of previously tested intact controls). The mean load to failure
of the Formulation 2 defects was 51.96N (102% of defects treated
with the standard OP-1 device and 80% of previously tested intact
controls). The mean load to failure of the Formulation 3 defects
was 50.86N (100% of defects treated with the standard OP-1 device
and 78% of previously tested intact controls). No significant
differences were noted in the mechanical testing results among
formulation type. Formulation 2 defects scored slightly higher than
Formulations 3 and 1 in maximum load to failure.
6TABLE 4 Mechanical testing results for critical and noncritical
size defects treated with rhOP-1 versus nontreated controls. Mean
.+-. SD (sample size). Defect Size Max Load Torque % of Intact
(Treatment) (N) (Nm) Ulna 2.5 cm 46.11 .+-. 2.77 .+-. 70.64 .+-.
(rhOP-1) 17.42(10) 1.05(10) 26.69(10) 2.5 cm * * * (control) 5.0 mm
51.81 .+-. 3.11 .+-. 79.37 .+-. (rhOP-1) 17.94(6) 1.08(6) 27.48(6)
5.0 mm 9.20 .+-. 0.55 .+-. 14.09 .+-. (control) 6.62(5) 0.40(5)
10.14(5) 3.0 mm 64.10 .+-. 3.85 .+-. 98.21 .+-. (rhOP-1) 49.12(4)
2.95(4) 75.25(4) 3.0 mm 21.91 .+-. 1.31 .+-. 33.57 .+-. (control)
26.46(3) 1.59(3) 40.54(3) 1.5 mm 62.17 .+-. 3.73 .+-. 95.25 .+-.
(rhOP-1) 30.48(4) 1.83(4) 46.70(4) 1.5 mm 19.43 .+-. 1.17 .+-.
29.76 .+-. (control) 19.11(4) 1.15(4) 29.28(4) * Defects not tested
due to instability.
[0110] Formulations using 20 mm acetate buffer, pH 4.5 and 5%
mannitol also were tested. Studies using non-critical size defects
(5 mm gaps) showed a clear increase in the rate of healing when
OP-1 was present, as compared with controls. Specifically, at 8
weeks, the controls averaged 14% the strength of intact ulnas,
while the OP-1 treated defects averaged 79%. Radiographically, new
bone was evident as early as two weeks postoperative and by eight
weeks significantly began to fill and bridge the defects treated
with rhOP-1 formulations. None of the six nontreated control
defects were completely healed at the end of the study period. One
of three defects with Formulation 1 (acetate buffer) and three of
three defects treated with Formulation 2 (PBS) were completely
filled and bridged by new bone at 8 weeks. All six defects
receiving OP-1 had new bone formation and were mechanically stable.
Formulation 2 defects scored significantly higher in radiographic
grading results, energy absorbed to failure, and histologic quality
of union that defects treated with Formulation 1. Otherwise, no
differences were fund between the two formulations for mean load to
failure, angular deformation and overall histologic appearance.
Both formulations scored significantly higher in all categories
compared to control defects. Histologically, proliferative new bone
was present within and surrounding the defects treated with rhOP-1.
Bridging of the defects and bony healing was almost completed by
eight weeks postoperative with gaps of fibrocartilage between areas
of significant new bone growth. Evidence of early cortex
development and bone remodeling was present in some of the rhOP-1
defects. All nontreated controls resulted in incomplete unions
although potential longer term healing was indicated by some new
bone formation fro the host bone ends and endosteal regions. The
mean load to failure of the Formulation 2 defects was 53.23N
(104.5% of critical size defects treated with the standard OP-1
device and 81.6% of previously tested intact controls). Comparison
of noncritical size defects treated with 0.35 mg OP-1 to previously
tested critical size defects treated with 1.5 mg OP-1 without a
carrier material (Injectable Formulations) showed no significant
difference in mean load to failure.
[0111] C. Healing of Fracture Defects Using Matrix-Free Osteogenic
Devices
[0112] 1. Rabbit Fracture Study
[0113] A rabbit fracture repair model study (ulna midshaft
fracture) also demonstrates the efficacy of the methods and devices
of the invention. This study compared the effect of administration
of matrix-free OP-1 devices in three configurations: 1) acetate
buffer pH 4.5 (soluble OP-1), 2) PBS (suspension OP-1) and 3)
pluronic gel. Four rabbits were treated in each group immediately
after fracture creation; contralateral controls were no-defect
arms. Animals were sacrificed 3 weeks post treatment. In summary,
animals injected with the acetate- or Pluronic-containing
OP-1-devices showed a significantly larger fracture callus by
radiographic, gross and histological examination. The mean
torsional load to failure for all ulna treated with OP-1 was
8.89.+-.2.99 N (mean.+-.standard deviation) (8 samples). While the
mean load to failure for non-treated control ulna was 7.9.+-.2.92 N
(9 samples).
[0114] 1a. Test Material Description
[0115] Matrix-free OP-1 devices in solution were utilized. The
three solution configurations evaluated were: (1) rhOP-1 admixed
with phosphate buffered saline, 8.71 mg OP-1/ml. The devices were
packaged in individual vials. The estimated range of device volume
delivered was between 30 .mu.l and 110 .mu.l per site; (2) rhOP-1
admixed with 20 mM acetate buffer, pH 4.5, 0.99 mg OP-1/ml. The
devices were packaged in individual vials containing 130 .mu.l. The
device was drawn up into a syringe. In all cases less than 100
.mu.l was delivered to each site. The estimated range of implant
volume delivered was between 60 .mu.l and 90 .mu.l per site; and
(3) rhOP-1 in Pluronic gel, 0.87 mg OP-1/ml. This device was
packaged in a syringe. The device was kept refrigerated until
administration to the defect site (lapse time less than one
minute). All Thick gel was delivered in all cases using a large
gauge (18) needle.
[0116] In all cases, dosages were calibrated to deliver
approximately 100 .mu.g of rhOP-1 to each fracture site.
[0117] A total of twelve adult male rabbits, adult male While New
Zealand rabbits bred for purpose, at least 9 months of age at onset
of study were utilized. All animals were skeletally mature and
weighed between 2.4 and 3.0 kg were supplied by USDA licensed
vendors. The animals were screened to exclude acute and chronic
medical conditions during a quarantine period, and were
radiographically screened to ensure proper size, skeletal maturity,
and that no obvious osseous abnormalities exist. Specific attention
was paid to selecting animals of uniform sex, size and weight to
limit the variability of healed fracture strength. Experimental
traverse fractures were created bilaterally in the center ulna of
each animal using standard surgical techniques. The left ulna
served as an untreated control in each animal. Briefly, using
standard aseptic techniques, bilateral fractures were induced by
making lateral incision approximately 2.0 cm in length and exposing
the right ulna was obtained using sharp and blunt dissection. A
transverse osteotomy was created in the mid-ulna using an
electrical surgical saw. The site then was closed with resorbable
suture. A matrix-free OP-1 device in solution was then injected
through the soft tissues into the fracture site. The procedure was
then repeated on the left side with the exception that no OP-1
device was provided to these fracture sites.
[0118] Animals were administered intramuscular antibiotics for four
days post-surgery. Animals were kept in recovery cages
postoperatively until fully conscious and weight bearing, after
which they were transferred to standard cages and allowed
unrestricted motion. The limbs were not casted.
[0119] Weekly radiographs were taken to study the progression of
healing. All animals were sacrificed at three postoperative weeks.
All ulna were retrieved en bloc and mechanically tested in torsion.
Fracture healing was further evaluated by histology for quality and
amount of new bone formation and healing.
[0120] At one week postoperative, early new bone formation was
evident in all fractures. Traces of lightly radiodense material was
present along the periosteal borders. The amount of new bone
formation was significantly greater in fractures with OP-1
matrix-free devices than the (untreated) fractures at one week
postoperative. At two weeks postoperative continuing new bone
formation was evident in all fractures treated with the matrix-free
OP-1 device. At three weeks, the bone callus was large and the
fractures were substantially or completely healed in the presence
of OP-1. On the left (non-treated) side, however, the fracture line
was still evident at three weeks and the amount of callus formed
was less.
[0121] The mean torsional load to failure for all ulna treated with
any OP-1 device was 8.89.+-.2.99N (8) (mean.+-.standard deviation
(sample size)). The mean load to failure for non-treated control
ulnas was 7.91.+-.2.92N (9).
[0122] Greater new bone volume and complete bridging across the
fracture site was observed in all right (OP-1 device treated)
fractures compared to the left. Proliferation of callus was
observed that extended into the soft tissues of the treated
fractures. The left (untreated) sides uniformly demonstrated new
bone proliferation at the periosteal and endosteal borders and
early cartilage formation at the fracture, but did not demonstrate
consistent complete bony bridging of the fracture.
[0123] Consistent with the radiographic results, greater volume of
new bone was observed in sites treated with OP-1 devices.
[0124] 2. Goat Fracture Study
[0125] Still another animal model for evaluating enhanced fracture
repair using matrix-free OP-1 devices is a goat model (tibia
midshaft acute fracture). The study compares 0.5 mg of OP-1 in
acetate buffer, 1 mg OP-1 in acetate buffer and 1 mg OP-1
precipitated in PBS, injected immediately after fracture creation
using standard surgical techniques. Animals are followed and cared
for as for the dog and rabbit studies described above and typically
are sacrificed at 2, 4 and 6 weeks post treatment.
[0126] It is anticipated that enhanced fracture repair results from
inclusion matrix-free osteogenic devices in these animals as
demonstrated for the rabbit study.
[0127] D. Repair of Osteochondral Defects Using Matrix-Free OP-1
Devices
[0128] 1. Osteochondral Defects in Rabbits
[0129] The following study demonstrates that matrix-free osteogenic
devices can enhance repair of both the articular cartilage
overlying the bone, as well as enhancing repair of the underlying
bone. In this study, a standard rabbit osteochondral defect model
was used to evaluate the various injectable forms of OP-1 to heal
this kind of defect.
[0130] Matrix-free devices containing OP-1 were prepared in two
different injectable delivery formulations and one freeze-dried
formulation. All samples contained 125 .mu.g OP-1. Formulation 1:
20 mM acetate buffer, pH 4.5 with 5% mannitol, 50 .mu.l full
volume; Formulation 2: Phosphate Buffered Saline (PBS) suspension;
and Formulation 3: Freeze-dried in 1 sample aliquots.
[0131] A total of six adult male rabbits were utilized. Full
thickness 4.0 mm in diameter osteochondral defects were created
bilaterally in the patellar sulcus of each animal, for a total of
12 defects, using standard surgical techniques. The left defect
received one of three OP-1 formulations and the right side defects
acted as an untreated control. All animals were sacrificed at
twelve postoperative weeks and the distal femurs retrieved en bloc.
The defect sites were evaluated histologically and grossly as
described herein above.
[0132] In all except one of the PBS group defects, the OP-1 side
shows significant healing with regeneration of both the bone and
cartilage. Although healing can be observed in most of the control
defects without OP-1, the repair is inferior; there is usually
incomplete healing of the underlying bone and a significant
underproduction of glycosaminoglycans (GAG) in the cartilage (as
seen by light toluidine staining).
[0133] 2. Sheep Model
[0134] Osteochondral and chondral defect repair also can be
evaluated in a standard goat or sheep model. For example, using
standard surgical techniques, each sheep in a study is operated on
both foreknee joints, and two defects per joint are created (one
each on the medial and the lateral condyle). One of the joints has
two standardized partial thickness chondral defects (5 mm in
diameter) on each condyle, while the other joint has two similar
but deeper full thickness osteochondral defects (about 1-2 mm in
the subchondral bone). One joint animal is treated with a
matrix-free osteogenic device formulation, and the other joint is
left as an untreated control. Each group has a subgroup sacrificed
early at 8 weeks and another kept for long term evaluation for 6-7
months. It is anticipated that matrix-free devices using any of the
formulations described herein will substantially enhance the speed
and quality of repair of both the articular cartilage and the
underlying bone, consistent with the results described herein
above.
[0135] E. Healing of Non-Critical Size Segmental Defects in Dogs
Using Matrix-Free Osteogenic Devices
[0136] 1. Experiment 2
[0137] As already exemplified in Experiment 1 above (see Section
V.A.1.), injectable formulations of rhOP-1 can be used to heal
non-critical size (e.g., 5 mm, 3 mm, 1.5 mm) defects. The
experiment which follows is an extension of Experiment 1 and
focuses on the 3 mm defect model. As is exemplified below in more
detail, noncritical size (3 mm) defects treated with rhOP-1
demonstrated advanced healing and more extensive new bone
formation. As demonstrated below, a 3 mm defect provided a
consistent and reproducible model to evaluate acceleration of the
fracture repair process.
[0138] This experiment evaluates the healing of noncritical size
defects treated with two OP-1 formulations, rhOP-1 in an
acetate/lactose buffer (OP/Buffer) and rhOP-1 in a
carboxymethylcellulose (CMC) (OP/CMC) gel, at four weeks
postoperative. The results summarized below demonstrate that
noncritical size defects treated with injectable rhOP-1 in CMC
solution and in an acetate buffer solution healed significantly
faster compared to CMC and buffer vehicle controls and untreated
controls: Radiographically, defects in both OP-1 treatment groups
(OP/CMC and OP/Buffer) showed early radiodense bone formation and
bridging bone by 4 weeks postoperative. The OP/CMC treated defects
were almost completely filled and spanned with nonuniform density
bone along the lateral ulna border and incorporating with the host
bone cortices. Proliferative new bone was present in the OP/Buffer
treated defects. None of the vehicle control defects (CMC and
Buffer only) showed evidence of bone defect healing at 4 weeks. The
histologic appearance of OP/CMC and OP/Buffer treated defects was
similar. In the OP treated defects, significant amounts of new bone
had formed at the defect cortices and along the ulna periosteum
extending across the defect site. Bone defect bridging was nearly
complete at the 4 week time period. Mineralizing cartilage and
fibrous tissue were present in OP treated defects. In contrast, the
vehicle control defects were filled and surrounded with fibrous
tissue and had minimal amounts of new bone formation at the defect
cortices. On average, the OP/CMC treated defects at 4 weeks had a
torsional strength that was 51% of the strength of intact ulnas
compared to 14% in the CMC vehicle controls. Defects treated with
the OP/Buffer solution had a mean torsional strength that was 44%
of intact ulna strength, while the buffer control defects achieved
only 9% of the torsional strength of intact ulnas. Both the OP/CMC
and OP/Buffer treated groups had mechanical strengths greater than
untreated controls at 4 weeks (9%) and 8 weeks (27%).
Experimental Design
[0139] The test samples consisted of recombinant human osteogenic
protein-1 (rhOP-1) in an injectable delivery matrix system. Two
rhOP-1 formulations and two vehicle only controls were evaluated
and compared to previously tested and reported nontreated control
defects. Only two of these three formulations are reported here.
One formulation (OP/CMC) consisted of 0.35 mg rhOP-1 in 100 .mu.l
carboxymethylcellulose (CMC) gel supplied in three sterile
syringes. A second formulation (OP/Buffer) consisted of 0.35 mg
rhOP-1 in 100 .mu.l acetate buffer supplied as an OP-1 solution.
The vehicle only controls consisted of 100 .mu.l CMC gel (CMC
control) supplied in three sterile syringes and 100 .mu.l acetate
buffer (Buffer control) supplied as a control solution. Samples of
known amount and content were fabricated and supplied sterile by
Creative BioMolecules, Inc. (Hopkinton, Mass.).
[0140] Bilateral ulna segmental defects, 3.0 mm in length, were
created in all animals. The right defects received one of two
rhOP-1 formulations such that three sites of each formulation were
studied. The left defects received the vehicle only control
containing no rhOP-1. Weekly radiographs were taken to study the
progression of healing. At sacrifice, all ulnae were retrieved en
bloc and if healed sufficiently, mechanically tested in torsion.
Segments were evaluated by histology for tissue response, quality
and amount of new bone formation and extent of healing. Adult male
mongrel dogs bred for purpose were utilized in this study because
of their availability, ease of handling, anatomical size, and known
bone repair and remodeling characteristics. All animals were
skeletally mature, weighed from 44 to 63 pounds (mean 54 lbs), and
were supplied by Martin Creek Kennels, USDA number 71-B-108
(Willowford, Ark.). Special attention was paid in selecting animals
of uniform size and weight to limit the variability in bone
geometry and loading.
[0141] Surgery
[0142] Anesthesia was administered by intravenous injection of
sodium pentothal at the dosage of 5.0 mg/lb body weight. Following
induction, an endotracheal tube was placed and anesthesia was
maintained by isofluorane inhalation. Both forelimbs were prepped
and draped in sterile fashion. A lateral incision approximately two
centimeters in length was made and exposure of the ulna was
obtained using blunt and sharp dissection. The 3.0 mm sized defect
was created in the mid-ulna using an oscillating saw. The radius
was maintained for mechanical study and no internal or external
fixation was used. The site was irrigated with saline to remove
bone debris and spilled marrow cells. The soft-tissues were
meticulously closed in layers around the defect. The appropriate
sample formulation was then injected into the defect site as per
the treatment schedule. The procedure was then repeated on the
contralateral side with the appropriate sample.
[0143] Radiographs
[0144] Radiographs of the forelimbs were obtained weekly until four
weeks postoperative. Standardized exposure times and intensities
were used. In order to quantify the radiographic results, each
radiograph was assigned a numerical score based on the grading
scale described in Table 5.
7TABLE 5 RADIOGRAPHIC GRADING SCALE Grade: No change from immediate
postoperative appearance 0 Trace of radiodense material in defect 1
Flocculent radiodensity with flecks of new calcification 2 Defect
bridged at least one point with material of non-uniform 3
radiodensity Defect bridged on both medial and lateral sides of
defect with 4 material of uniform radiodensity, cut end of the
cortex remain visible Same as grade 3; at least one of four
cortices is obscured by new 5 bone Defect bridged by uniform new
bone; cut ends of cortex are no 6 longer distinguishable
[0145] Animals were sacrificed using an intravenous barbituate
overdose. The ulna and radius were immediately harvested en bloc
and placed in saline soaked diapers. Both ulna were
macrophotographed and contact radiographs taken. Soft tissues were
carefully dissected away from the defect site. A watercooled saw
was used to cult the ulna to a uniform length of 9 cm with the
defect site centered in the middle of the test specimen.
[0146] Mechanical Testing
[0147] Immediately after sectioning, if healing was deemed
sufficient by manual manipulation, specimens were tested to failure
in torsion on an MTS closed-loop hydraulic test machine
(Minneapolis, Minn.) operated in stroke control at a constant
displacement rate of 50 mm/min. Each end of the bone segment was
mounted in a cylindrical aluminum sleeve and cemented with methyl
methacrylate. One end was rigidly fixed and the other was rotated
counterclockwise. Since the dog ulna has a slight curvature, the
specimens were mounted eccentrically to keep specimen rotation
coaxial with that of the testing device. The torsional force was
applied with a lever arm of 6 cm by a servohydraulic materials
testing system. Simultaneous recordings were made of implant
displacement, as measured by the machine stroke controller, while
load was recorded from the load cell. Data was recorded via an
analog-to-digital conversion voarch and a personal computer and an
online computer acquisition software. Force angular displacement
curves were generated from which the torque and angular deformation
to failure were obtained, and the energy absorption to failure
computed as the area under the load-displacement curve.
[0148] Histology
[0149] Both tested and untested specimens were prepared for
histologic evaluation. The individual specimens were fixed by
immersion in 10% buffered formalin solution immediately following
mechanical testing or after sectioning in untested specimens. On a
water cooled diamond saw the specimens were divided by bisecting
the specimen down its long axis. This procedure resulted in two
portions of each specimen for different histologic preparations
including undecalcified ground sections and undecalcified microtome
sections.
[0150] Following fixation, the specimens designated for
undecalcified sections were hydrated in graduated ethyl alcohol
solutions from 70% to 100%. The specimens were then placed in
methyl methacrylate monomer and allowed to polymerize. The ground
sections were obtained by cutting the specimens on a high speed,
water cooled Mark V CS600-A (East Grandy, Conn.) sectioning saw
into sections approximately 700 to 1,000 microns thick. These
sections were mounted on acrylic slides and ground to 100 micron
thickness using a metallurgical grinding wheel, and
microradiographs were made using standardized techniques. Following
microradiography the sections were further ground to approximately
50 microns and stained with basic fuchsin and toluidine blue for
histologic grading that evaluated the quality of the union, the
appearance and quality of the cortical and cancellous bone, the
presence of bone marrow elements, bone remodeling, and inflammatory
response (Table 6).
8TABLE 6 HISTOLOGIC GRADING SCALE Grade: Quality of Union: No sign
of fibrous or other union 0 fibrous union 1 osteochondral union 2
bone union 3 bone union with reorganization of cortices 4 Cortex
Development: none present in the defect 0 densification of borders
1 recognizable formation 2 intact cortices but not complete 3
complete formation of normal cortices 4 Inflammatory Response:
severe response 0 severe/moderate response 1 moderate response 2
mild response 3 no response 4 TOTAL POINTS: 12
EXPERIMENTAL RESULTS
[0151] Radiographic Evaluation
[0152] A summary of the radiographic grades for each site is
provided in Table 7. At 4 weeks postoperative, defects treated with
OP/CMC had a mean radiographic grade of 3.0 out of 6 possible
points. Defects treated with OP/Buffer had a mean radiographic
grade of 4.0. Defects treated with CMC vehicle control and buffer
only control averaged final radiographic grades of 1.33 and 1.0,
respectively. In both OP-1 treated groups, OP/CMC and OP/Buffer,
there were signs of radiodense new bone forming in the defects and
along the lateral defect borders as early as three weeks
postoperative. At four weeks, significant amounts of new bone had
formed within the defects and in surrounding subcutaneous tissue.
The OP/CMC defects were almost completely filled and spanned with
nonuniform density bone along the lateral ulna border. New bone was
significantly incorporated with the defect cortices. In two of
three OP/Buffer treated defects, the host cortices remained visible
although proliferative new bone was present. In contrast, none of
the OP/CMC or OP/Buffer defects were completely bridged or filled
by four weeks postoperative. In the CMC control group, early new
bone obscured the host bone cortices at three weeks and continued
to increase in radiodensity. Again, in contrast, the buffer control
defects showed only a slight increase in radiodensity at the defect
cortices at four weeks. None of the control defects in either group
showed evidence of bony defect healing.
9TABLE 7 RADIOGRAPHIC GRADING RESULTS Implant Type 1 week 2 weeks 3
weeks 4 weeks OP/CMC 0 1 2 3 OP/CMC 0 0 1 3 OP/CMC 0 1 2 3 CMC
Control 0 0 1 1 CMC Control 0 1 1 2 CMC Control 0 0 0 1 OP/Buffer 0
1 2 3 OP/Buffer 0 1 2 4 OP/Buffer 0 1 2 5 Buffer control 0 0 0 1
Buffer control 0 0 0 1 Buffer control 0 0 1 1 OP/CMC 0.0 .+-. 0.0
0.67 .+-. 0.58 1.67 .+-. 0.58 3.0 .+-. 0.0 mean .+-. st dev (n) (3)
(3) (3) (3) CMC Control 0.0 .+-. 0.0 0.33 .+-. 0.58 0.67 .+-. 0.58
1.33 .+-. 0.58 mean .+-. st dev (n) (3) (3) (3) (3) OP/Buffer 0.0
.+-. 0.0 1.0 .+-. 0.0 2.0 .+-. 0.0 4.0 .+-. 1.0 mean .+-. st dev
(n) (3) (3) (3) (3) Buffer control 0.0 .+-. 0.0 0.0 .+-. 0.0 0.33
.+-. 0.58 1.0 .+-. 0.0 mean .+-. st dev (n) (3) (3) (3) (3) OP/CMC
= 0.35 mg rhOP-1 in 100 .mu.l CMC gel CMC control = 100 .mu.l CMC
vehicle only gel OP/Buffer = 0.35 mg rhOP-1 in 100 .mu.l acetate
buffer solution Buffer control = 100 .mu.l acetate buffer vehicle
only solution OP/CMC-4 weeks
[0153] At one week postoperative, there were no changes in
radiographic appearance of any OP/CMC defects. At 2 weeks, trace
radiodense areas were present at the cut bone ends. At 3 weeks,
there was an increase in radiodensity of new bone forming within
the defects and along the lateral defect borders. One defect showed
signs of early bony bridging. At 4 weeks, the OP/CMC defects had a
significant amount of radiodense new bone both within the defect.
The defects were almost completely filled and spanned with
nonuniform density bone along the lateral ulna border. New bone was
significantly incorporated with the defect cortices. None of the
OP/CMC treated defects were completely filled or solidly bridged
with new bone at 4 weeks postoperative. The final radiographic
grade for each defect was 3 out of 6 possible points (mean
3.0.+-.0.0, n=3).
[0154] CMC Control--4 weeks
[0155] At two weeks postoperative, there were no significant
changes in radiographic appearance of any CMC control defects. At 3
weeks, the host cortices were beginning to obscure with new bone in
2 of 3 defects. At 4 weeks, the CMC defects showed some evidence of
new bone activity at the defect cortices but no evidence of bony
defect healing. The final radiographic grades were 1, 2 and 1 out
of 6 possible points (mean 1.33.+-.0.58, n=3).
[0156] OP/Buffer--4 weeks
[0157] At one week postoperative, there were no changes in
radiographic appearance of any OP/Buffer treated defect. At two
weeks postoperative, trace radiodense areas were present within the
OP/buffer defects and along the defect borders. Significant new
bone formation was also seen in the subcutaneous tissues
surrounding the defects. At 3 weeks, flecks of new bone appeared in
the defects and new bone formed in the overlying soft tissues. At 4
weeks, there was a significant increase in radiodense new bone
formation filling and bridging the OP-1 treated defects. In two of
three defects the cortices remained visible although proliferative
new bone filled and spanned the defects. None of the OP/Buffer
treated defects were completely filled or solidly bridged with new
bone at the sacrifice period of 4 weeks. The final radiographic
grades were 3, 4 and 5 out of 6 possible points, respectively (mean
4.0.+-.1.0, n=3).
[0158] Buffer Control--4 weeks
[0159] At 3 weeks postoperative, there were no significant changes
in radiographic appearance of any defect treated with the buffer
only control. At 4 weeks, an increase in radiodensity at the
cortices was observed although no signs of defect healing were
evident. The final radiographic grade for each site was 1 out of 6
possible points (mean 1.0.+-.0.0, n=3).
[0160] Gross Observations
[0161] OP-1 defects: All OP/CMC and OP/Buffer treated defects were
manually stable and visibly had a mass of new bone formation at the
defect site.
[0162] Vehicle control defects: None of the CMC and Buffer only
control defects were manually stable at 4 weeks although all were
mechanically tested.
[0163] Mechanical Testing
[0164] A summary of the mechanical testing results appears in Table
8.
[0165] OP/CMC
[0166] At 4 weeks postoperative, the mean load to failure of 3 mm
defects treated with OP/CMC was 33.08.+-.16.41N (n=3). This
represented 51% of the strength of intact controls tested
previously. The mean angular deformation was 31.13.+-.15.32
degrees. The mean energy absorbed to failure was 41.64.+-.30.52
Nm-degrees.
[0167] CMC Control
[0168] At 4 weeks postoperative, the mean load to failure of 3 mm
defects treated with CMC control was 9.32.+-.16.41N (n=3). This
represented 14% of the strength of intact controls tested
previously. The mean angular deformation was 33.36.+-.25.95
degrees. The mean energy absorbed to failure was 10.53.+-.8.62
Nm-degrees.
[0169] OP/Buffer
[0170] At 4 weeks postoperative, the mean load to failure of 3 mm
defects treated with OP/Buffer was 29.03.+-.16.79N (n=3). This
represented 44% of the strength of intact controls tested
previously. The mean angular deformation was 36.14.+-.14.71
degrees. The mean energy absorbed to failure was 37.87.+-.27.73
Nm-degrees.
[0171] Buffer Control
[0172] At 4 weeks postoperative, the mean load to failure of 3 mm
defects treated with Buffer control was 5.62.+-.1.65N (n=3). This
represented 9% of the strength of intact controls tested
previously. The mean angular deformation was 24.91.+-.12.03
degrees. The mean energy absorbed to failure was 3.94.+-.4.12
Nm-degrees.
10TABLE 8 MECHANICAL TESTING RESULTS Energy Maximum Percent
absorbed Load to intact to failure Failure Torque control Angula-
(Nm- Implant (N) (Nm) (%) tion degrees) OP/CMC 49.37 2.96 75.65
35.72 63.57 OP/CMC 16.56 0.99 25.37 14.04 6.78 OP/CMC 33.32 2.00
51.05 43.62 54.56 MEAN .+-. 33.08 1.99 50.69 31.13 41.64 STANDARD
.+-. .+-. .+-. .+-. .+-. DEVIATION 16.41 0.98 25.14 15.32 30.52 CMC
Control 12.81 0.77 19.63 33.26 14.06 CMC Control 11.00 8.00 16.85
59.35 16.83 CMC Control 4.14 0.25 6.34 7.46 0.70 MEAN .+-. 9.32
3.01 14.27 33.36 10.53 STANDARD .+-. .+-. .+-. .+-. .+-. DEVIATION
4.57 4.33 7.01 25.95 8.62 OP/Buffer 32.47 1.95 49.75 50.11 55.91
OP/Buffer 43.83 2.63 67.15 37.53 51.77 OP/Buffer 10.79 0.65 16.53
20.78 5.94 MEAN .+-. 20.03 1.74 44.48 36.14 37.87 STANDARD .+-.
.+-. .+-. .+-. .+-. DEVIATION 16.79 1.01 25.72 14.71 27.73 Buffer
control 4.82 0.29 7.38 11.12 0.73 Buffer control 4.53 0.27 6.94
30.32 2.50 Buffer control 7.52 0.45 11.52 33.29 8.59 MEAN .+-. 5.62
0.34 8.62 24.91 3.94 STANDARD .+-. .+-. .+-. .+-. .+-. DEVIATION
1.65 0.10 2.53 12.03 4.12
[0173] Histology
[0174] A summary of the histologic grading results appears in Table
9. Out of 12 total points, the mean histologic grade of defects
treated with OP/CMC was 7.00.+-.0.87. The mean histologic grade of
the CMC control defects was 4.50.+-.0.87. The mean histologic
grades of the OP/Buffer defects and the Buffer controls were
6.08.+-.0.14 and 4.0.+-.1.0, respectively.
[0175] OP/CMC
[0176] Treatment resulted in early osteochondral bridging with
areas of mineralizing cartilage at four weeks. In defects treated
with OP/CMC, significant new bone formation was observed in the
periosteal and endosteal regions of the ulna and extended beyond
the defect borders. Areas of mineralizing cartilage and some
fibrous tissue were present within the defects. Bridging of the
defects was not complete by four weeks. The host cortices remained
visible although there were signs of new bone incorporation and
remodelling.
[0177] CMC control
[0178] No complete bony healing was observed in any CMC control
defects at four weeks. Control defects resulted in fibrous unions
with no signs of bony bridging. Fibrous tissue and mineralizing
cartilage was observed filling and surrounding the defects. Very
small amounts of new bone had formed along the ulna periosteum and
at the endosteal region of the ulna near the host cortices. Signs
of host cortex resorption were observed at the defect ends.
[0179] OP/Buffer
[0180] Treated defects were filled with mineralizing cartilage and
fibrous tissue. New bone formed in the endosteal and periosteal
regions of the ulna near the defect borders and early signs of
bridging with new bone was evident although none of the defects
were completely spanned. The host bone cortices showed signs of
incorporation with new bone but were not completely obscured by
four weeks. Some remodelling of the host bone cortices and early
densification along the new bone borders was observed. New bone
also formed in the subcutaneous tissue layers overlying the defect
site and extended beyond the defect borders.
[0181] Buffer control
[0182] No complete bony healing was observed in any buffer control
defects at four weeks postoperative. Untreated defects exhibited
fibrous unions with no signs of bony bridging; fibrous tissue was
observed filling and surrounding the defects. Other untreated
defects showed no sign of fibrous or other union. Very little new
bone formation was observed in the buffer control defects.
Endosteal new bone extended from the ulna marrow cavity and
periosteal new bone formed along the lateral defect borders. The
host bone ends were visible with signs of cortical resorption.
11TABLE 9 HISTOLOGIC GRADING RESULTS Inflam- Quality Cortex matory
Total Implant of Union Development Response Score OP/CMC 1.5 1 4
6.5 OP/CMC 3 1 4 8 OP/CMC 1.5 1 4 6.5 MEAN .+-. 2.0 .+-. 0.87 1.0
.+-. 0.0 4.0 .+-. 0.0 7.0 .+-. 0.87 STANDARD DEVIATION CMC Control
1 0 4 5 CMC Control 1 0 2.5 3.5 CMC Control 1 0 4 5 MEAN .+-. 1.0
.+-. 0.0 0.0 .+-. 0.0 3.50 .+-. 0.87 4.50 .+-. 0.87 STANDARD
DEVIATION OP/Buffer 1.25 1 4 6.25 OP/Buffer 1 1 4 6 OP/Buffer 1 1 4
6 MEAN .+-. 1.08 .+-. 0.14 1.0 .+-. 0.0 4.0 .+-. 0.0 6.08 .+-. 0.14
STANDARD DEVIATION Buffer control 1 0 2 3 Buffer control 1 0 4 5
Buffer control 0 0 4 4 MEAN .+-. 0.67 .+-. 0.58 0.0 .+-. 0.0 3.33
.+-. 1.15 4.0 .+-. 1.0 STANDARD DEVIATION
[0183] 2. Experiment 3
[0184] Recombinant human osteogenic protein-1 (rhOP-1), when
implanted in combination with bone collagen matrix, has been shown
to heal critical-sized diaphyseal segmental defects in animals with
the formation of new bone that is both biologically and
biomechanically functional. The purpose of this study was to
evaluate the efficacy of matrix-free injectable formulations of
rhOP-1 for accelerating bone healing in a canine non-critical-sized
defect model.
[0185] Bilateral osteoperiosteal segmental defects, 3.0 mm in
length, were created in the mid-ulna of 18 adult male mongrel dogs.
The radius was maintained for mechanical stability without
additional fixation. Soft tissues were closed prior to injection of
rhOP-1. Nine animals received rhOP-1 formulations in one defect and
vehicle controls in the contralateral defect and were sacrificed at
4 weeks postoperative. Nine untreated control defects were
evaluated at periods of 4, 8 and 12 weeks for comparison with the
rhOP-1 treatment. Radiographs were taken at regular intervals to
study the progression of healing. At sacrifice, all ulnae were
mechanically tested in torsion if healing was sufficient.
Undecalcified histologic sections were evaluated for quality and
amount of new bone formation and extent of healing.
[0186] Radiographically, new bone formation was evident as early as
two weeks postoperative in rhOP-1 treated defects and at 4 weeks,
new bone bridged the defect. In contrast, vehicle control sites
showed little or no bone formation at 4 weeks postoperative.
Moreover, torsional strengths of defects treated with rhOP-1 were
significantly greater at 4 weeks than vehicle or untreated controls
at 4 weeks. Furthermore, torsional strength of treated defects at 4
weeks virtually equaled the strength of untreated controls at 12
weeks. A clear acceleration of defect healing and bone formation
resulted from rhOP-1 treatment. Histologic findings correlated with
radiographic and mechanical testing results.
[0187] The results of this study confirm that osteogenic proteins
injected in noncritical-sized defects can accelerate defect
healing, including fracture callus formation and bridging bone
formation. Defects treated with rhOP-1 formed new bone
significantly faster and restored fracture strength and stiffness
earlier than untreated controls.
[0188] In summary, the ability of the matrix-free devices described
hereinabove to substantially enhance defect repair, including
accelerating the rate and enhancing the quality of newly formed
bone, has implications for improving bone healing in compromised
individuals such as diabetics, smokers, obese individuals, aged
individuals, individuals afflicted with osteoporosis, steroidal
users and others who, due to an acquired or congenital condition,
have a reduced capacity to heal bone fractures, including
individuals with impaired blood flow to their extremities. Such
individuals experience refractory healing, resulting from a reduced
capacity to promote progenitor cells, and are subject to gangrene
and/or sepsis.
[0189] The methods and formulations disclosed herein provide
enhanced bone repair by accelerating bone formation. Specifically,
following the methods and protocols disclosed herein, the rate of
bone formation, including bone callus formation and bridging can be
accelerated. As exemplified herein, bridge formation occurs faster,
and in a shorter time frame, allowing for more stable bone
formation, thereby enhancing biomechanical strength of the newly
forming bone.
[0190] It is well-known in the art that callus formation is one
stage in the multi-staged healing process culminating in bone
formation. Specifically, the healing process involves five stages:
impact, inflammation, soft callus formation, hard callus formation,
and remodeling. Impact begins with the initiation of the fracture
and continues until energy has completely dissipated. The
inflammation stage is characterized by hematoma formation at the
fracture site, bone necrosis at the ends of the fragments, and an
inflammatory infiltrate. Granulation tissue gradually replaces the
hematoma, fibroblasts produce collagen, and osteoclasts begin to
remove necrotic bone. The subsidence of pain and swelling marks the
initiation of the third, or soft callus, stage. This stage is
characterized by increased vascularity and abundant new cartilage
formation. The end of the soft callus stage is associated with
fibrous or cartilaginous tissue uniting the fragments. During the
fourth, or hard callus, stage, the callus converts to woven bone
and appears clinically healed. The final stage of the healing
process involves slow remodeling from woven to lamellar bone and
reconstruction of the medullary canal (see "Current Diagnosis &
Treatment in Orthopedics," ed. H. B. Skinner (LANGE Medical Book
Publ.)).
[0191] F. Repair of Chondral Defects with Matrix-Free Osteogenic
Devices (Sheep)
[0192] 1. Experiment 1
[0193] Using materials and methods similar to those described above
(see relevant portions of D.2), the following study was conducted
to further demonstrate that the exemplary osteogenic protein OP-1,
when administered in an matrix-free device, can induce active
chondrogenesis and chondral defect repair in weight-bearing
joints.
[0194] As already described above, a defect is a structural
disruption of the cartilage and can assume the configuration of a
void, a three-dimensional defect such as, for example, a gap,
cavity, hole or other substantial disruption in structural
integrity. Defects in articular cartilage may extend through the
entire depth of articular cartilage and or into the subchondral
bone (osteochondral defects) or defects may be superficial and
restricted to the cartilage tissue itself (chondral or subchondral
defects).
[0195] Initially, damaged cartilage matrix undergoes degradation by
metalloproteinases that are released by nearby cellular
constituents. Proteolytic degradation clears damaged matrix
components thereby releasing anabolic cytokines entrapped in the
matrix. As currently understood, cytokines released from the matrix
stimulate proliferation of chondrocytes and, importantly, synthesis
of a new macromolecular matrix. The presence of clusters of
proliferating chondrocytes, as determined microscopically, is one
of the first indicators of a cartilage reparative response.
Presumably, this repair response counters the catabolic effect of
proteases and stabilizes the tissue by enhanced matrix
synthesis.
[0196] Articular cartilage and repair of articular cartilage is
readily studied by standard histological and histochemical means.
The techniques are well-known in the art and include microscopic
examination of sections of cartilage stained by any one of a number
of histochemical stains including, but not limited to, toluidine
blue, hematoxylin and eosin, von Kossa, safranin O, and Masson's
trichrome stain. Following the application of different stains, the
skilled artisan can assess the reparative response of cartilage by
identification of proliferating chondrocytes and determination of
the quality and quantity of matrix, such as collagen and
proteoglycans, synthesized by chondrocytes.
[0197] As used herein, articular cartilage refers specifically to
hyaline cartilage, an avascular, non-mineralized tissue which
covers the articulating surfaces of the portions of bones in
joints. Under physiological conditions, articular cartilage
overlies highly vascular mineralized bone called subchondral bone.
Articular cartilage is characterized by specialized cartilage
forming cells, called chondrocytes, embedded in an extracellular
matrix comprising fibrils of collagen (predominantly Type II
collagen as well as the minor types such as Types IX and XI),
various proteoglycans, including glycosaminoglycans, other proteins
and water.
[0198] In this study, sheep were used as a model to assess repair
of 1-2 mm total depth.times.7 mm total diameter chondral defects on
the weight-bearing condylar surface of the knee. The defects were
partial thickness chondral defects and did not involve the
subchondral bone as was evident by a lack of bleeding following
defect creation. Further confirmation was obtained by histology of
thin sections at the time of sacrifice; the defects did not extend
into subchondral bone.
[0199] The experimental protocol is provided in Table 10. Using
standardized surgical techniques, a 2 mm total depth.times.7 mm
total diameter defect was surgically made on the weight-bearing
surface of each condyle of the right and left knee. The right knee
served as the control knee. A liquid matrix-free OP-1 device (50 or
250 .mu.g OP-1) in 20 mM sodium acetate, pH 4.5, was delivered
either as a single bolus via injection into the intra-articular
joint, or intermittently delivered (0.5 .mu.L per hour for a 2 week
duration; 200 .mu.L total) via a locally implanted, subcutaneous
mini-pump (ALZET.RTM. 2002, ALZA Scientific Products, Palo Alto,
Calif.). Numerous suitable mini-pumps are readily available and
routinely used by the skilled practitioner for delivery of
pharmaceuticals and/or therapeutic agents; the skilled artisan will
appreciate the preferred mode and rate of delivery under the
circumstances. Healing of chondral defects was assessed by standard
histological and histochemical methods.
12TABLE 10 Chondral Defect Repair in Sheep Group Left Knee
(Matrix-free Device) Right Knee (Cntrl) I 50 .mu.g OP-1 No Rx II
250 .mu.g OP-1 No Rx III 50 .mu.g OP-1 via mini-pump Vehicle via
mini-pump IV 250 .mu.g OP-1 via mini-pump Vehicle via mini-pump
[0200] The results collected to date of a 3 month mini-pump study
(Group III and IV) reveal that matrix-free OP-1 devices can induce
chondrogenesis and subsequent repair of chondral defects. Little
evidence of chondral defect repair was observed at 12 weeks in the
control defects. However, by standard histological and
histochemical evaluation, new cartilage formation as well as fusion
of the old and new cartilage was found in the matrix-free OP-1
treated animal. Using art-recognized histological and histochemical
indicia as a measure of chondral repair, OP-1 stimulated the
ingrowth of synovial cells into the defect area. These cells
differentiated into full thickness, proteoglycan-rich articular
chondrocytes and repair of the chondral defect resulted
therefrom.
[0201] The healing of a partial thickness cartilage defect without
subchondral bone involvement in an adult animal is unprecedented
and demonstrates that active chondrogenesis is a feature of the
repair process that is induced by a matrix-free osteogenic device.
It is concluded from these studies that a matrix-free osteogenic
device can be used to repair chondral defects in vivo. It is
particularly significant that such repair can occur at a
weight-bearing joint in a large animal model such as the sheep.
[0202] Other studies of chondral defect repair using matrix-free
OP-1 devices (for example, experiments as outlined in Group I and
II above) are currently still in progress. Results similar to those
obtained with the mini-pump delivery experimental paradigm
described above are expected, that is, a single bolus of injectable
matrix-free device injected into the intra-articular joint is
expected to repair chondral defects in weight-bearing joints.
[0203] G. Alternative Methods of Healing Non-Critical Size
Segmental Defects Using Matrix-Free Osteogenic Devices
[0204] 1. Experiment 1: The Effects of Delayed Administration of
Matrix-Free Osteogenic Device on Repair of Non-Critical Size
Defects (Dogs)
[0205] The purpose of this study was to evaluate the healing of
non-critical size defects treated with matrix-free OP-1 devices at
various delayed administration times post-injury. The particular
device exemplified below is an injectable formulation of the
matrix-free osteogenic device. Other device embodiments are
expected to yield similar results.
[0206] Briefly, the experimental observations are as follows: In
general, non-critical size segmental defects treated with
matrix-free OP-1 devices healed to a significantly greater degree
compared to injectable carrier controls at 4 weeks post-injury. Of
particular significance are the unexpected results which indicate
that at least one indicia of defect healing, specifically, enhanced
ulnar mechanical strength, can be enhanced by manipulating the
post-injury time at which matrix-free OP-1 devices are
administered.
[0207] For purposes of this experiment and as used herein, injury
means accidental occurrence of a defect (such as an unexpected
physical mishap resulting in the occurrence of a non-critical size
defect), purposeful occurrence of a defect (such as surgical
manipulation resulting in the occurrence of a non-critical size
defect), or non-traumatically induced defects caused by one or more
of the following diseases or disorders: hypoxia; ischemia; primary
and metastatic neoplasia; infectious diseases; inflammatory
diseases; so-called collagen diseases (including defects in
collagen synthesis, degradation or normal matrix); congenital,
genetic or developmental diseases; nutritional diseases; metabolic
diseases; idiopathic diseases; and diseases associated with
abnormal mineral homeostasis, to name but a few.
[0208] Certain of the methods exemplified herein contemplate the
step of administering a matrix-free device to a defect site after
onset of the healing process; the stages of the healing process,
and the physiological events associated therewith, were earlier
described. Another of the methods of the present invention
comprises the step of administering a matrix-free device to a
defect site during maturation of the endogenous matrix at the site;
the events associated with endogenous matrix formation during
endochondral bone formation were also earlier described. In a
currently preferred embodiment, the present invention provides a
method of repairing a bone defect, chondral defect, or
osteochondral defect involving the step of administering a
matrix-free device at times post-injury which are delayed. Such
delays can be short-term, moderate or long-term as described below.
The extent to which administration is delayed depends upon the
circumstances and the skilled artisan will readily appreciate the
significance thereof.
[0209] As demonstrated below, improved healing and defect repair
results from administration of a matrix-free device to a defect
site at elapsed times post-injury. For example, delayed
administration times can include times from at least 0.5 hours to
at least 6.0 hours post-injury; alternatively, delayed
administration times can include times from at least 6 hours to 24
hours or from at least 24 hours to 48 hours post-injury. In one
currently preferred embodiment, the delay is at least 6 hours.
Other post-injury administration times are also contemplated by the
instant invention. In certain other currently preferred
embodiments, delayed administration times can range from at least
48 hours to at least 72 hours post-injury. In yet other
embodiments, administration times can range significantly beyond 72
hours, e.g., matrix-free osteogenic devices can be administered to
the defect site as late as the remodeling stage of bone healing.
Also contemplated are methods wherein matrix-free osteogenic
devices are administered to a non-critical size defect site at a
plurality of time points post-injury. For example, a currently
preferred plurality is from 0.5 to 6 hours and 7 days post-injury.
A plurality of delayed administrations can be accomplished by
manual delivery to the defect locus or by automated delivery using
a mini-pump as described earlier.
[0210] Experimental Design
[0211] A total of 12 adult mongrel dogs were utilized. As described
earlier, bilateral ulnar segmental defects, 3.0 mm in length, were
created in all animals. As exemplified in this particular study,
the matrix-free formulation of OP-1 used was 3.5 mg OP-1/ml
delivered in 100 microL lactose/acetate buffer as described
earlier. Twelve animals were administered matrix-free devices in
the right defect at various post-injury time points and control
devices were administered in the left defect at various post-injury
time points. Three animals were treated at defect creation (0
hours), three at 6 hours post-injury, and three at 48 hours
post-injury. All animals were sacrificed 4 weeks after surgery.
Weekly radiographs were taken to study the progression of healing.
At sacrifice, segments of bone were evaluated by histology for
tissue response, quality and amount of new bone formation, and
extent of healing. All ulnae were retrieved en bloc and
mechanically tested in torsion.
[0212] As described earlier, immediately after sectioning, ulna
were tested to failure in torsion on an MTS closed-loop hydraulic
test machine operated in stroke control at a constant displacement
rate of 50 mm/min. One end was rigidly fixed and the other was
rotated counterclockwise. The torsional force was applied with a
lever arm of six cm, by a servohydraulic materials testing system.
Simultaneous recordings were made of implant displacement, as
measured by the machine stroke controller, while load was recorded
from the load cell. Data was recorded via an analog-to-digital
conversion board and a personal computer and on-line computer
acquisition software. Force-angular displacement curves were
generated from which the torque and angular deformation to failure
were obtained, and the energy absorption to failure computed as the
area under the load-displacement curve.
[0213] Results
[0214] All specimens were mechanically tested at 4 weeks
post-surgery. Mechanically, defects receiving matrix-free OP-1
devices at 6 hours post-injury had the highest torsional strength;
73% of intact ulnae compared to 64% at 48 hours and 60% at 0 hours.
The control defects at 0 hours, 6 hours, and 48 hours post-injury
had strengths of 23%, 28%, and 24%, respectively.
[0215] This study demonstrates the surprising result that, in
certain embodiments of the present invention, improved healing of a
non-critical size defect can be achieved by delayed administration
of a matrix-free osteogenic device to the defect locus post-injury.
This unexpected result is related to the stage of bone healing or
endogenous matrix formation at the defect site including but not
limited to events such as clot formation, progenitor cell
infiltration, and callus formation, particularly soft callus, to
name but a few. Moreover, it is expected that other defect repair
processes involving bone, such as repair of osteochondral defects
similar to those described herein, can be improved by delayed
administration of matrix-free osteogenic devices to the defect site
post-injury.
[0216] H. Further Studies of Chondral Defect Repair Using
Matrix-Free Osteogenic Devices (Sheep)
[0217] 1. Experiment 1: Glycosaminoglycans and Other Polymers as a
Carrier for Osteogenic Protein
[0218] As described earlier, certain preferred categories of
compounds are suitable as carriers in the matrix-free devices
contemplated herein. Among the currently preferred categories are
compounds appreciated by the art as lubricating agents, especially
those which occur naturally and naturally perform physiological
functions such as protection and lubrication of cells and
maintenance of tissue integrity, to name but a few. Such compounds
are generally also wetting and moisture-preserving agents. One
sub-category of currently preferred lubricating agents includes the
biopolymers known as glycosaminoglycans. Glycosaminoglycans
contemplated by the present invention include, but are not limited
to, hyaluronic acid, chondroitin, dermatan, keratan to name but a
few. Sulfonated as well as non-sulfonated forms can be used in the
present invention. Other glycosaminoglycans are suitable for
formulating matrix-free devices, and those skilled in the art will
either know or be able to ascertain other suitable compounds using
no more than routine experimentation. For a more detailed
description of glycosaminoglycans, see Aspinall, Polysaccharides,
Pergamon Press, Oxford (1970).
[0219] A particularly preferred glycosaminoglycan is hyaluronic
acid (HA). HA is a naturally occurring anionic polysaccharide or
complex sugar. It is found in cartilage and synovial fluid. HA is
available both in cosmetic grade and medical grade; medical grade
is generally preferred for use with the present invention. HA can
range in molecular weight from low to high. In certain embodiments
of the present invention, high molecular weight material is
preferable; as an example only, HA 190 (1.9 a 10.sup.6 Da; 1% is
currently preferred, yet concentrations ranging from 0.5 -2.0% are
suitable) admixed with saline can be administered (0.1ml/kg) twice
weekly intra-articularly to repair chondral defects. In other
embodiments, low molecular weight HA (such as HA80,
0.8.times.10.sup.6 Da; 1% is preferred, yet concentrations less
than or equal to 4% are suitable) can be used. Using the teachings
provided herein, the skilled artisan can assess the circumstances
under which high molecular weight HA is preferable to low molecular
weight material for defect repair, and vice versa. Moreover, the
skilled artisan will further appreciate that HA in solution is a
viscous liquid and that the viscosity can be manipulated by
adjusting the molecular weight and the HA content. For example, in
some embodiments, it is preferred to approximate the viscosity of
synovial fluid in the joint. Using ordinary skill and routine
experimentation, together with the teachings provided herein, the
skilled artisan can formulate matrix-free osteogenic devices using
HA as a carrier for chondral defect repair; the viscosity of the
device as well as the protein content can be readily adjusted as
required by the circumstances and as taught herein. HA is available
commercially from several sources including Sigma Chemical Company
(St. Louis, Mo.), Genzyme Pharmaceuticals (Cambridge, Mass.) and
Collaborative Laboratories (East Setauket, N.Y.).
[0220] Glycosaminoglycan and other polymeric carriers, such as
hyaluronic acid, suitable for use with the instant matrix-free
osteogenic devices can be evaluated in the sheep chondral defect
model described above. For example, two 2.times.7 mm defects are
made by standard surgical procedures in the weight bearing surface
of the medial and lateral condyles of both knee joints in a sheep.
One knee joint is treated by intra-articular administration of an
OP-1/hyaluronic acid matrix-free device and the other joint is
treated with hyaluronic acid alone.
[0221] Two groups of sheep are studied: Group I is sacrificed at 8
weeks and Group II is sacrificed at 6 months. As described earlier,
healing of chondral defects can be assessed by radiology and
standard histological and histochemical methods. Radiographs of
each knee are taken at monthly intervals. Arthroscopic examination
is performed using standard techniques and equipment under
anesthesia just prior to sacrifice. Immediately after sacrifice,
specimens of the knee joint are fixed in 10% neutral buffered
formalin. Specimens are bisected longitudinally and one section
decalcified in graduated ethyl alcohol solutions from 70-100%, and
embedded in methylmethacrylate, sectioned and stained for
histologic grading.
[0222] It is expected that hyaluronic acid-containing matrix free
devices can enhance the rate of chondral defect repair and can
improve the extent of repair achieved. Repair can be assessed in
animal models by standard cartilage characterization methods,
including histologic grading of stained and fixed tissue sections,
localization of cartilage-specific macromolecules (such as type II
collagen and aggrecan), determination of the proteoglycan profile
and mechanical testing. All the foregoing can be accomplished by
the skilled artisan using routine experimentation and the knowledge
in the art.
[0223] Equivalents
[0224] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather than limiting on the invention
described herein. Scope of the invention is thus indicated by the
appended claims rather than by the foregoing description, and all
changes which come within the meaning and range of equivalency of
the claims are therefore intended to be embraced therein.
Sequence CWU 1
1
* * * * *