U.S. patent application number 10/083825 was filed with the patent office on 2003-04-03 for manufacture of autogenous replacement body parts.
Invention is credited to Khouri, Roger K., Rueger, David C., Sampath, Kuber T..
Application Number | 20030064090 10/083825 |
Document ID | / |
Family ID | 22960109 |
Filed Date | 2003-04-03 |
United States Patent
Application |
20030064090 |
Kind Code |
A1 |
Khouri, Roger K. ; et
al. |
April 3, 2003 |
Manufacture of autogenous replacement body parts
Abstract
Disclosed are matrix materials, methods, and devices for
manufacture in vivo of autogenous replacement body parts comprising
plural distinct tissues. In one embodiment, the replacement body
part is a skeletal joint and the new plural distinct tissues
include bone and articular cartilage.
Inventors: |
Khouri, Roger K.; (St.
Louis, MO) ; Sampath, Kuber T.; (Medway, MA) ;
Rueger, David C.; (Hopkinton, MA) |
Correspondence
Address: |
TESTA, HURWITZ & THIBEAULT, LLP
HIGH STREET TOWER
125 HIGH STREET
BOSTON
MA
02110
US
|
Family ID: |
22960109 |
Appl. No.: |
10/083825 |
Filed: |
February 27, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10083825 |
Feb 27, 2002 |
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09547601 |
Apr 13, 2000 |
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09547601 |
Apr 13, 2000 |
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08459129 |
Jun 2, 1995 |
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6110482 |
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08459129 |
Jun 2, 1995 |
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08253398 |
Jun 3, 1994 |
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5906827 |
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Current U.S.
Class: |
424/426 ;
435/174 |
Current CPC
Class: |
A61L 27/3683 20130101;
A61L 2430/06 20130101; A61L 27/227 20130101; A61L 27/3612 20130101;
A61L 27/3641 20130101; A61L 27/3604 20130101; A61L 27/3608
20130101; A61L 27/3645 20130101; A61L 27/3654 20130101 |
Class at
Publication: |
424/426 ;
435/174 |
International
Class: |
A61F 002/00; C12N
011/00 |
Claims
What is claimed is:
1. A device for implantation in a mammal which serves as a template
to form in vivo a functional replacement body part comprising
plural distinct tissues, the device comprising: a) a biocompatible,
biodegradable matrix defining a structure which allows the
attachment of infiltrating cells, comprising residues having
specificity for, or derived from, said plural distinct tissues;
and, having dimensions and shape which mimic that of the body part
to be replaced; and disposed on the surface of said matrix, b) an
osteogenic protein in an amount sufficient to induce formation of
new said plural distinct tissues thereby to permit regeneration of
tissues corresponding in shape and kind to said residues within a
mammal.
2. The device of claim 1 wherein one of said plural distinct
replacement tissues is a non-mineralized tissue.
3. The device of claim 1 wherein said matrix comprises devitalized
tissue from a mammalian donor.
4. The device of claim 1 wherein said matrix comprises residues of
articular cartilage and of bone.
5. The device of claim 1 wherein said matrix comprises at least a
portion of a skeletal joint
6. The device of claim 1 or 5 wherein the matrix comprises
dehydrated mammalian tissue.
7. The device of claim 2 wherein said replacement non-mineralized
tissue is selected from the group consisting of articular
cartilage, ligament, tendon, synovial capsule and synovial membrane
tissue.
8. The device of claim 5 wherein said skeletal joint defines a
synovial or articulating joint
9. The device of claim 5 or 8 wherein said device defines a
devitalized intact skeletal joint structure.
10. A device for implantation in a mammal forming in vivo articular
cartilage replacement tissue in a skeletal joint, the device
comprising: osteogenic protein disposed on the surface of a
biocompatible, bioresorbable matrix said matrix defining a
structure which allows the attachment of infiltrating cells and
which comprises residues specific for, or derived from, articular
cartilage tissue.
11. A device for implantation in a mammal for forming in vivo
replacement non-mineralized tissue in a skeletal joint, the device
comprising: osteogenic protein disposed on the surface of a
biocompatible, bioresorbable matrix, said matrix defining a
structure which allows the attachment of infiltrating cells and
which comprises residues specific for, or derived from,
non-mineralized skeletal joint tissue corresponding in kind to said
tissue to be replaced.
12. The device of claim 11 wherein said non-mineralized tissue is
an avascular tissue.
13. The device of claim 10 wherein said non-mineralized tissue is
selected from the group consisting of articular cartilage,
ligament, synovial membrane and synovial capsule tissue.
14. The device of claim 10 or 11 wherein said matrix comprises
devitalized allogenic or xenogenic tissue.
15. The device of claim 10 or 11 wherein said matrix comprises a
material selected from the group consisting of: collagen, polymers
comprising monomers of lactic acid, glycolic acid, butyric acid and
combinations thereof, hydroxyapatite, tricalcium phosphate, and
mixtures thereof.
16. The device of claim 10 or 11 further comprising a material
suitable for binding particulate matter to form a moldable
solid.
17. The device of claim 1, 10 or 11 wherein said osteogenic protein
comprises homodimers or heterodimers of OP-1, OP-2, BMP2, BMP3,
BMP4, BMP5, BMP6, OPX, or functional equivalents thereof.
18. A method for inducing in a mammal the formation of an
autologous replacement body part comprising plural distinct
tissues, said method comprising the steps of: a) providing a device
comprising osteogenic protein disposed on the surface of a
bioresorbable, biocompatible matrix, said matrix defining a
structure which allows the attachment of infiltrating cells,
comprising residues having specificity for, or derived from, said
plural distinct tissues, and having dimensions and shape which
mimic that of the body part to be replaced; and b) implanting said
device at a locus in a mammal, thereby to induce formation of
tissues corresponding in shape and kind to said residues.
19. The method of claim 18 wherein said locus in said mammal
defines an endogenous body part to be replaced.
20. The method of claim 18 wherein said matrix further comprises
residues which are dimensioned to correspond in shape and
structural relation to said plural distinct tissues to be
replaced.
21. The method of claim 18 wherein the plural distinct tissues
comprise bone and cartilage.
22. The method of claim 18 wherein said matrix comprises
devitalized allogenic or xenogenic tissue.
23. The method of claim 18 wherein one of said plural distinct
tissues is an avascular tissue.
24. A method for repairing in vivo articular cartilage on the
surface of a bone, the method comprising the step of: providing to
said bone surface at a locus in a mammal a device comprising an
osteogenic protein disposed on the surface of a biocompatible,
bioresorbable matrix, said matrix comprising residues specific for,
or derived from, cartilage, and defining a structure which allows
the attachment of infiltrating cells.
25. The method of claim 24 wherein said locus occurs in a synovial
cavity.
26. A method for restoring in a mammal a non-mineralized tissue in
a skeletal joint, the method comprising the step of: providing to
said skeletal joint in a mammal a device comprising an osteogenic
protein disposed on the surface of a biocompatible, bioresorbable
matrix, said matrix comprising residues specific for, or derived
from, tissue corresponding in kind to said non-mineralized tissue
to be replaced, and defining a structure which allows the
attachment of infiltrating cells.
27. The method of claim 26 wherein said non-mineralized tissue to
be restored comprises avascular tissue.
28. The method of claim 26 wherein said non-mineralized tissue to
be restored is selected from the group consisting of articular
cartilage, tendon, ligament, synovial capsule and synovial membrane
tissue.
29. The method of claim 24 or 26 wherein said matrix is derived
from allogenic or xenogenic articular cartilage.
30. The method of claim 24 or 26 wherein said device comprises a
moldable solid.
31. The method of claim 24 or 26 wherein said device comprises a
flexible sheet
32. The method of claim 24 or 26 wherein said device comprises
collagen, polymers comprising lactic acid, butyric glycolic acid or
mixtures thereof; hydroxyapatite and combinations thereof.
33. The method of claim 18, 24 or 26 wherein said osteogenic
protein comprises homodimers or heterodimers of OP-1, OP-2, BMP2,
BMP3, BMP4, BMP5, BMP6, OPX, or functional equivalents thereof.
Description
RELATION TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
08/253,398, filed Jun. 3, 1994, the disclosure of which is
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to materials and methods for the
repair and regeneration of plural distinct tissues at a single
defect site in a mammal. More particularly, the invention is
concerned with materials and methods for the manufacture in vivo of
autogenous replacement body parts, including mammalian skeletal
joints, comprising plural different tissues, such as ligament,
articular cartilage and bone tissues.
BACKGROUND OF THE INVENTION
[0003] Skeletal joints provide a movable union of two or more
bones. Synovial joints are highly evolved articulating joints that
permit free movement Because mammalian lower limbs are concerned
with locomotion and upper limbs provide versatility of movement,
most of the joints in the extremities are of the synovial type
There are various types of synovial joints. Their classification is
based upon the types of active motion that they permit (uniaxial,
biaxial, and polyaxial). They are differentiated further according
to their principal morphological features (hinge, pivot,
condyloid). In contrast to fibrous and cartilaginous joints where
the ends of the bones are found in continuity with intervening
tissue, the ends of the bones in a synovial joint are in contact,
but separate. Because the bones are not bound internally, the
integrity of a synovial joint results from its ligaments and
capsule (which bind the articulation externally) and to some extent
from the surrounding muscles. In synovial joints, the contiguous
bony surfaces are covered with articular or, hyaline cartilage, and
the joint cavity is surrounded by a fibrous capsule which
segregates the joint from the surrounding vascularized environment.
The inner surface of the capsule is lined by a synovial layer or
"membrane" containing cells involved in secreting the viscous
lubricating synovial fluid. Gray, Anatomy of the Human Body, pp.
312; 333-336 (13th ed.; C. C. Clemente, ed., (1985)).
[0004] In certain synovial joints, the joint or synovial cavity may
be divided by a meniscus of fibrocartilage. Synovial joints
involving two bones and containing a single joint cavity are
referred to as simple joints. Joints that contain a meniscus
forming two joint cavities are called composite joints. The term
compound joint is used for those articulations in which more than a
single pair of articulating surfaces are present
[0005] Joint replacement, particularly articulating joint
replacement, is a commonly performed procedure in orthopedic
surgery. However, the ideal material for replacement joints remains
elusive. Typically, joint reconstruction requires repair of the
bony defect, the articular cartilage and, in addition, one or more
of the joining ligaments. To date, there are no satisfactory
clinical means for readily repairing both articular cartilage and
bony defects within a joint which reliably results in viable,
fully-functional weight-bearing joints. Prosthetic joints which
replace all the endogenous joint tissues circumvent some of these
problems. However, prosthetic joints have numerous, well documented
limitations, particularly in younger and highly active patients. In
addition, in some cigars prosthetic joint replacement is not
possible and repair options are limited to osteochondroallograft
materials.
[0006] The articular, or hyaline cartilage, found at the end of
articulating bones is a specialized, histologically distinct tissue
and is responsible for the distribution of load resistance to
compressive forces, and the smooth gliding that is part of joint
function. Articular cartilage has little or no self-regenerative
properties. Thus, if the articular cartilage is torn or worn down
in thickness or is otherwise damaged as a function of time, disease
or trauma, its ability to protect the underlying bone surface is
compromised.
[0007] Other types of cartilage in skeletal joints include rib cage
and elastic cartilage. Secondary cartilaginous joints are formed by
discs of fibrocartilage which join vertebrae in the vertebral
column. In rib cartilage, the mucopoly-saccharide network is
interlaced with prominent collagen bundles and the chondrocytes are
more widely scattered than in hyaline cartage. Elastic cartilage
contains collagen fibers which are histologically similar to
elastin fibers. As with other connective tissues the formation of
cartilaginous tissue is a complex biological process, involving the
interaction of cells and collagen fibers in a unique biochemical
milieu.
[0008] Cartilage tissue, including articular cartilage, unlike
other connective tissues, lacks blood vessels, nerves, lymphatics
and basement membrane. Cartilage is composed of chondrocytes which
synthesize an abundant extracellular milieu composed of water,
collagens, proteoglycans and noncollagenous proteins and lipids.
Collagen serves to trap proteoglycans and to provide tensile
strength to the tissue. Type II collagen is the predominant
collagen in cartilage tissue. The proteoglycans are composed of a
variable number of glycosaminoglycan chains, keratin sulphate,
chondroitin sulphate and/or dermatan sulphate, and N-linked and
O-linked oligosaccharides covalently bound to a protein core. The
sulfated glycosaminoglycans are negatively charged resulting in an
osmotic swelling pressure that draws in water.
[0009] In contrast, certain collagens such as the fibrotic
cartilaginous tissues which occur in scar tissue for example, are
keloid and typical of scar-type tissue, i.e., composed of
capillaries and abundant, irregular, disorganized bundles of Type I
and Type II collagen.
[0010] Histologically, articular or hyaline cartilage can be
distinguished from other forms of cartilage, both by its morphology
and by its biochemistry. Morphologically, articular cartilage is
characterized by superficial versus mid versus deep "zones" which
show a characteristic gradation of feats from the surface of the
tissue to the base of the tissue adjacent to the bone. In the
superficial zone, for example, chondrocytes are flattened and lie
parallel to the surface embedded in an extracellular network that
contains tangentially arranged collagen and few proteoglycans. In
the mid zone, chondrocytes are spherical and surrounded by an
extracellular network rich in proteoglycans and obliquely organized
collagen fibers. In the deep zone, close to the bone, the collage
fibers are vertically oriented The keratin sulphate rich
proteoglycans increase in concentration with increasing distance
from the cartilage surface. For a detailed description of articular
cartilage micro-structure, see, for example, (Aydelotte and
Kuettner, (1988), Conn. Tiss. Res. 18:205; Zanetti et al., (1985),
J. Cell Biol. 101:53; and Poole et al., (1984), J. Anat.
138:13.
[0011] Biochemically, articular collagen can be identified by the
presence of Type II and Type IX collagen, as well as by the
presence of well-characterized proteoglycans, and by the absence of
Type X collagen, which is associated with bone formation.
[0012] In normal articular cartilage, a balance exists between
synthesis and destruction of the above-described extracellular
network. However, in tissue subjected to trauma, for example due to
friction between misaligned bones in contact with one another, or
in joint diseases characterized by net loss of articular cartilage,
e.g., osteoarthritis, an imbalance occurs between synthesis and
degradation.
[0013] Two types of defects are recognized in articular surfaces,
i.e., full-thickness defects and superficial defects. These defects
differ not only in the extent of physical damage to the cartilage,
but also in the nature of the repair response each type of lesion
can elicit.
[0014] Full-thickness defects of an articulating surface include
damage to the hyaline cartilage, the calcified cartilage layer and
the subchondral bone tissue with its blood vessels and bone marrow.
Full-thickness defects can cause severe pain since the bone plate
contains sensory nerve endings. Such defects generally arise from
severe trauma and/or during the late stages of degenerative joint
disease, such as osteoarthritis. Full-thickness defects may, on
occasion, lead to bleeding and the induction of a repair reaction
from the subchondral bone. In such instances, however, the repair
tissue formed is a vascularized fibrous type of cartilage with
insufficient biomechanical properties, and does not persist on a
long-term basis.
[0015] In contrast, superficial defects in the articular cartilage
tissue are restricted to the cartilage tissue itself. Such defects
are notorious because they do not heal and show no propensity for
repair reactions. Superficial defects may appear as fissures,
divots, or clefts in the surface of the cartilage, or they may have
a "crab-meat" appearance in the affected tissue. They contain no
bleeding vessels (blood spots) such as are seen in full-thickness
defects. Superficial defects may have no known cause, however, they
are often the result of mechanical derangements which lead to a
wearing down of the cartilaginous tissue. Such mechanical
derangements may be caused by trauma to the joint, e.g., a
displacement of torn meniscus tissue into the joint, meniscectomy,
a laxation of the joint by a torn ligament, malalignment of joints,
or bone fracture, or by hereditary diseases. Superficial defects
are also characteristic of early stages of degenerative joint
diseases, such as osteoarthritis. Since the cartilage tissue is not
innervated or vascularized, superficial defects do not heal and
often degenerate into full-thickness defects.
[0016] Replacement with prosthetic joints is currently the
preferred option for serious degeneration of joint function
involving loss of articular cartilage. It is anticipated that a
means for functional reconstruction of joint complexes, including
regeneration and repair of articular cartilage, will have a
profound effect on alloplastic joint replacement surgery and the
management of degenerative joint disease.
[0017] Like articular cartilage, joint ligaments which serve to
connect interacting bones in the joint, have little or no
self-regenerative properties. Ligaments typically are composed of
substantially parallel bundles of white fibrous tissue. They are
pliant and flexible to allow substantially complete freedom of
movement, but are inextensile to prevent over-extension of the
interacting bones in the joint. Like cartilage, ligament tissue is
substantially devoid of blood vessels and has little or no
self-regenerative properties. Surgical repair of torn or damaged
ligament tissue to date is limited to use of autogenous grafts or
synthetic materials that are surgically attached to the articular
extremities of the bones. Allogenic ligaments typically fail
mechanically, presumably due to the treatments required to render
these materials biocompatible. Similarly, tendons are rope-like
structures which connect muscle fibers to bone or cartilage and
which are formed from substantially parallel fibroids of white
connective tissue. The synovial capsule is composed of a thin layer
of ligamentous tissue which encloses the joint and allows the joint
to be bathed in the lubricating synovial fluid. The interior of the
joint capsule is lined with a thin membrane of connective tissue
having branched connective-tissue corpuscles defining the synovial
membrane, and which is primarily responsible for secreting synovial
fluid into the cavity. The integrity of this membrane therefore, is
important to maintaining a source for the lubricating synovial
fluid. Repair of these tissues in orthopedic contexts typically is
limited to resuturing of existing tissue.
[0018] Bone tissue differs significantly from the other tissues
described hereinabove, including cartilage tissue. Specifically,
bone tissue is vascularized tissue composed both of cells and a
biphasic medium which is composed of a mineralized, inorganic
component (primarily hydroxyapatite crystals) and an organic
component comprised primarily of Type I collagen.
Glycosaminoglycans constitute less than 2% of this organic
component and less than 1% of the biphasic medium itself or of bone
tissue per se. Moreover, relative to cartilage tissue, the collagen
present in bone tissue exists in a highly-organized parallel
arrangement.
[0019] Bony defects, whether from degenerative, traumatic or
cancerous etiologies, pose a formidable challenge to the
reconstructive surgeon. Particularly difficult is reconstruction or
repair of skeletal parts that comprise part of a multi-tissue
complex, such as occurs in mammalian joints.
[0020] Mammalian bone tissue is known to contain one or more
proteinaceous materials presumably active during growth and natural
bone healing which can induce a developmental cascade of cellular
events resulting in endochondral bone formation. The developmental
cascade involved in endochondral bone differentiation consists of
chemotaxis of mesenchymal cells, proliferation of progenitor cells
into chondrocytes and osteoblasts, differentiation of cartilage,
vascular invasion, bone formation, remodeling, and finally marrow
differentiation.
[0021] True osteogenic factors capable of inducing the
above-described cascade of events that result in endochondral bone
formation have now been identified, isolated, and cloned. These
proteins, which occur in nature as disulfide-bonded dimeric
proteins, are referred to in the art as "osteogenic" proteins,
"osteoinductive" proteins, and "bone morphogenetic" proteins.
Whether naturally-occurring or synthetically prepared, these
osteogenic proteins, when implanted in a mammal typically in
association with a substrate that allows the attachment,
proliferation and differentiation of migratory progenitor cells,
are capable of inducing recruitment of accessible progenitor cells
and stimulating their proliferation, inducing differentiation into
chondrocytes and osteoblasts, and further inducing differentiation
of intermediate cartilage, vascularization, bone formation,
remodeling, and finally marrow differentiation. Those proteins are
referred to as members of the Vgr-1/OP1 protein subfamily of the
TGP.beta. super gene family of structurally related proteins.
Members include the proteins described in the art as OP1 (BMP-7),
OP2 (BMP-8), BMP2, BMP3, BMP4, BMP5, BMP6, 60A, DPP, Vgr-1 and Vgl.
See., e.g., 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 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).
[0022] It is an object of the instant invention to provide a
bioresorbable matrix and device, suitable for regenerating body
parts which comprise two or more functionally- and
structurally-associated yet distinct replacement tissues in a
mammal. Another object is to provide compositions and methods for
the repair or complete reconstruction of a mechanically and
functionally viable skeletal joint in a mammal, particularly an
articulating or synovial joint, as well as other body parts
comprising bone and bona fide hyaline cartilage, without relying on
prosthetic devices. Another object is to provide materials and
methods for the repair of tissue defects in an articulating
mammalian joint, so as to form a mechanically and functionally
viable joint comprising bone and articular cartilage, ligament,
tendon, synovial membrane and synovial capsule tissue. Another
object of the invention is to provide means for restoring
functional non-mineralized tissue in a skeletal joint including the
avascular tissue therein.
SUMMARY OF THE INVENTION
[0023] In accordance with the present invention, methods and
devices are provided for the manufacture of a live autogenous
replacement part comprising plural distinct tissues. In one aspect
the replacement body part includes part or all of a mammalian
skeletal joint, including an articulating or synovial joint. As
described herein below, the methods and compositions of the
invention are sufficient to restore mechanical and functional
viable of the tissues associated with a skeletal joint, including
bone (and bone marrow), articular cartilage, ligament, tendon,
synovial capsule and synovial membrane tissues. Thus the invention
provides methods and compositions for replacement of one or more of
the plural distinct tissues that define a mammalian skeletal
joint.
[0024] The invention provides, in one aspect therefore, a novel
matrix for forming a mechanically and structurally functional,
mammalian, replacement body part comprising plural distinct
tissues. The matrix comprises intact residues specific for or
characteristic of, and/or derived from at least two distinct
tissues of the replacement body part. As will be appreciated from
the description provided herein below, the matrix can include
residues specific for four or more distinct tissues. The matrix is
biocompatible and bioresorbable. Specifically, it is sufficiently
free of pathogens and antigenic stimuli that can result in graft
rejection. Preferably the matrix is derived from an allogenic or
xenogenic body part. Preferably, it is derived from a mammalian
donor, such as a cadaver. The body part may be rendered inert or
"devitalized" by dehydration, such as by ethanol extraction and
lyophilization, so that no residual cellular metabolism remains,
but the function of endogenous growth factors and the like can be
restored upon in situ reconstitution by endogenous body fluids. The
treated body part which now is substantially depleted in antigenic
and pathogenic components and now is biocompatible, maintains the
residues specific for the plural distinct tissues constituting the
body part sought to be replaced. These residues include those of
plural distinct tissues with dimensions and structural
relationships to each other which mimic those of the body part to
be replaced.
[0025] The thus treated matrix having utility in the methods and
devices of the invention lacks significant mechanical integrity as
compared with native tissue and, on its own, is not sufficient to
induce regeneration of a replacement body part or tissue when
implanted. However, by impregnating or otherwise infusing the
interstices of the matrix with osteogenic protein so that the
protein is disposed on or adsorbed to, the surfaces of the matrix,
the device of the instant invention is formed and is sufficient to
induce formation of new tissue in vivo such that regeneration of a
mechanically and functionally viable replacement body part occurs
in situ.
[0026] In one preferred embodiment, the device comprises part or
all of a skeletal joint excised from a mammalian donor allogenic or
xenogenic to the donee. Treated as described herein the device
comprising the allogenic or xenogenic skeletal joint (1) is
biocompatible, namely, it is non-pathogenic and sufficiently
non-antigenic to prevent graft rejection in vivo, and (2) is
sufficient to induce formation of a functionally viable autogenous
replacement joint in vivo, including generating functional bone,
articular cartilage, ligament and capsule tissue in correct
relation to one another such that a structurally and mechanically
functional replacement joint results.
[0027] In another embodiment, the invention provides a device which
serves as a template for forming in vivo part or all of a skeletal
synovial joint comprising plural distinct tissues and which, in
response to morphogenic signals, induces new tissue formation,
including new articular cartilage tissue from responding cells
present in the synovial environment. The newly formed tissues
assume the shape and function of the original tissue in the
skeletal joint.
[0028] In another aspect, the invention provides methods for
replacing a defective body part comprising the steps of: excising
the defective body part and implanting the device of the instant
invention. In one embodiment, the method also comprises the
additional step of providing a supply of mesenchymal cells to the
implanted device, as by threading or otherwise providing a muscle
flap prefused with a blood supply into a hollow portion of the
device. In another embodiment, the device is implanted at a locus
in the body of the individual distinct from the defect site but
which allows generation of the replacement body part. The
autogenous body part thus formed then can be implanted at the
defect site.
[0029] As will be appreciated from the description provided herein,
in another aspect, the invention provides devices and methods for
the functional and mechanical restoration of one or more individual
tissues in a mammalian skeletal joint, including the
non-mineralized and avascular tissue therein. Thus, in one
embodiment, the invention provides methods and devices competent
for restoring, without limitation, functional articular cartilage,
ligament, synovial membrane and synovial capsule tissue. The
methods and devices described herein can be used for example, to
correct superficial articular cartilage defects in a joint, to
replace torn or compromised ligaments and/or tendons, and to repair
defects in synovial capsule or membrane tissue.
[0030] The devices for repairing individual skeletal joint tissue
comprise osteogenic protein disposed on a matrix containing
residues specific for, or derived from skeletal joint tissue of the
type to be restored, including, without limitation, cartilage,
ligament, tendon, synovial capsule, or synovial membrane tissue.
The device can take the form of a solid, or it can have the
physical properties of a paste or gel. Preferably, the matrix is
derived from allogenic or xenogenic tissue, and is treated as
described herein to form a biocompatible devitalized matrix.
[0031] In another embodiment the matrix can be formulated de novo
from synthetic and/or naturally-derived components. The matrix
includes both (a) residues specific for, or characteristic of, the
given tissue and, (b) materials sufficient to create a temporary
scaffold for infiltrating cells and defining a three dimensional
structure which mimics the dimensions of the desired replacement
tissue. Useful such materials are described hereinbelow. Suitable
tissue-specific residues can be obtained from devitalized allogenic
or xenogenic tissue and combined with the structural materials as
described herein to create the synthetic matrix. In another
embodiment, the matrix comprises devitalized non-mineralized
tissue. In some circumstances, as in the formation of articular
cartilage on subchondral bone, a non-mineralized matrix material
defining a three-dimensional structure which allows the attachment
of infiltrating cells, can be sufficient, in combination with
osteogenic protein, to induce new tissue formation.
[0032] While, as described above, in a preferred embodiment the
invention contemplates a device suitable as a template for forming
in vivo a replacement skeletal joint, as will be appreciated by the
practioner in the art, the invention contemplates, and the
disclosure enables, a device suitable as a template for forming in
vivo functional replacement body parts other than skeletal joints
and which comprise plural distinct tissues.
[0033] When used in accordance with the methods of the instant
invention, the devices of the invention and/or the tissues which
result from their application, essentially satisfy the following
criteria of a preferred grafting material:
[0034] 1. They result in formation of mechanically and functionally
viable tissues normally present at the site. These tissues are of
an appropriate size and have correct structural relationships so as
to result in a functional body part. In particular, the
multi-tissue replacement part, whether produced in situ at the site
of intended use or remotely, becomes incorporated, integrating with
adjacent tissues, essentially maintaining its shape, and avoiding
abnormal resorption, regardless of the conditions present at the
recipient site. Weiland et al. (1983) Clin. Orthop. 174:87
(1983).
[0035] 2. The devices are capable of being precisely contoured and
shaped to exactly match any defect, whichever complex skeletal or
organ shape it is meant to replace.
[0036] 3. The devices virtually have unlimited supply and are
relatively easy to obtain.
[0037] 4. The devices have minimal donor site morbidity.
[0038] Furthermore, the instant invention provides practitioners
with materials and methods for skeletal joint repair including the
repair of the bone and articular cartilage present therein, and
which solve problems that occur using the methods and devices of
the art. For example, the instant invention can induce formation of
bona fide hyaline cartilage rather than fibrocartilage at a defect
site. Using the materials and methods disclosed herein, functional
hyaline cartilage forms on the articulating surface of bone at a
defect site and does not degenerate over time to fibrocartilage. By
contrast, prior art methods of repairing cartilage defects
generally ultimately result in development of fibrous cartilage at
the defect site. Unlike hyaline cartilage, fibrocartilage lacks the
physiological ability to restore articulating joints to their full
capacity. Thus, when the instant materials are used in accordance
with the instant methods, the practitioner can substantially
functionally restore a cartilage defect in an articulating joint,
particularly a superficial articular cartilage defect and
substantially avoid the undesirable formation of fibrocartilage
typical of prior art methods, or degeneration into a
"full-thickness defect". The invention also provides means for
repairing individual tissue of a joint not readily reparable
individually using prior art methods, and which, in some cases,
previously warranted replacement of the entire joint with a
prosthetic device. The invention further allows use of allogenic
replacement materials for repairing the avascular tissue in a
skeletal joint, and which result in the formation of mechanically
and functionally viable replacement tissues at a joint locus.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] While the specification concludes with claims particularly
pointing out and specifically claiming the subject matter which is
regarded as constituting the present invention, it is believed that
the invention will be better understood from the following detailed
description of preferred embodiments taken in conjunction with the
accompanying drawings in which:
[0040] FIG. 1 is a fragmentary front elevational view of a
mammalian knee joint with sufficient tissue removed to show the
articular cartilage on the condyles of the femur, the ligaments,
synovial membrane, joint capsule, and further showing a damaged
area in the articular cartilage requiring repair;
[0041] FIGS. 2A through 2D are schematic representations of the
elements used to generate a viable, functional glenohumoral
hemi-joint in one embodiment of the invention. FIG. 2A depicts a
lyophilized allograft; FIG. 2B depicts osteogenic protein for
application to the lyophilized allograft of FIG. 2A; FIG. 2C
depicts a muscle flap of cutaneous maximus muscle to be threaded
inside the shaft of the lyophilized allograft; and, FIG. 2D depicts
a viable, functional hemi-joint resting from the combination of
elements in FIGS. 2A, 2B and 2C. FIG. 2D represents one embodiment
of the device of the instant invention;
[0042] FIGS. 3A through 3D are schematic representations of the
four allografts tested in the hemi-joint of Example 2 (5 week);
and
[0043] FIGS. 4A through 4D are schematic representations of the
four allografts tested in the hemi-joint of Example 3 (6
month).
DETAILED DESCRIPTION
[0044] In accordance with the present invention, novel materials
and methods are provided for the repair and regeneration of plural
distinct tissues, including manufacture of a live autogenous
replacement part comprising plural distinct tissues. In one
embodiment the replacement body part is a skeletal joint,
particularly an articulating joint, and includes, without
limitation, residues specific for, or derived from, bone,
cartilage, ligament, tendon, synovial capsule and synovial membrane
tissue.
[0045] More particularly, in one aspect, the invention provides a
device comprising an osteogenic protein disposed on the surfaces of
a matrix or substrate for forming a functional, mammalian
replacement body part comprising plural distinct tissues. As used
herein, the term "matrix" is understood to define a structure
having interstices for the attachment, proliferation and
differentiation of infiltrating cells. It comprises residues
specific for the tissue to be replaced and/or derived from the same
tissue type, and has a shape and dimension when implanted which
substantially mimics that of the replacement tissue desired.
[0046] As used herein, the term "residue" is intended to mean a
constituent of a given tissue, which has specificity for, or is
characteristic of, the given tissue, and which is derivable from
the non-viable constituents of the given tissue. A matrix
comprising these residue(s), when combined with osteogenic protein,
and implanted in a mammal in an environment which mimics the
tissue's local environment under physiological conditions, and is
sufficient for formation of specific, mechanically and functionally
viable replacement tissue.
[0047] The term "plural distinct tissue" is intended to mean
physiologically distinguishable tissues, such as biochemically or
ultrastructurally distinguishable tissues which reside at an
anatomically similar locus. In an articulating replacement joint
device for example, the matrix can comprise residues specific for,
or derived from, bone, cartilage, ligament, tendon and synovial
membrane tissue. Thus, a significant aspect of the matrix of the
invention is a single structure comprising residues of plural,
distinct tissues, and which, when combined with an osteogenic
protein as defined herein, is suitable for inducing repair or
regeneration of a body part that is mechanically and functionally
viable over time in vivo.
[0048] As used herein, the terms "bone" and "articular cartilage"
are intended to mean the following: Bone refers to a calcified
(mineralized) connective tissue primarily comprising a composite of
deposited calcium and phosphate in the form of hydroxyapatite,
collagen (predominately Type I collagen) and bone cells, such as
osteoblasts, osteocytes and osteoclasts, as well as to the bone
marrow tissue which forms in the interior of true endochondral
bone. Cartilage refers to a type of connective tissue that contains
chondrocytes embedded in an extracellular network comprising
fibrils of collagen (predominately Type II collagen along with
other minor types, e.g. Types IX and XI), various proteoglycans
(e.g., chondroitin sulfate, keratan sulfate, and dermatan sulfate
proteoglycans), other proteins, and water. Articular cartilage
refers to hyaline or articular cartilage, an avascular, non-mineral
tissue which covers the articulating surfaces of the portions of
bones in joints and allows movement in joints without direct
bone-to-bone contact, and thereby prevents wearing down and damage
to opposing bone surfaces. Most normal healthy articular cartilage
is referred to as "hyaline," i.e., having a characteristic frosted
glass appearance. Under physiological conditions, articular
cartilage tissue rests on the underlying, mineralized bone surface,
the subchondral bone, which contains highly vascularized ossicles.
These highly vascularized ossicles can provide diffusible nutrients
to the overlying cartilage, but not mesenchymal stem cells.
[0049] "Ligament" is intended to mean both the rope-like structures
of white fibrous connective tissue which attach anterior
extremities of intracting bones, as well as the tissue defining a
synovial capsule. "Synovial membrane" is intended to define the
connective tissue membrane lining the interior of the synovial
cavity and which is involved in synovial fluid secretion. "Tendon"
is intended to define the connective tissue structure which joins
muscle to bone.
[0050] Replacement Body Parts
[0051] As disclosed herein, the instant invention provide methods
and compositions for replacing and repairing a defective body part.
The method comprises the steps of surgically excising the defective
body part, implanting a device comprising a matrix of the type
described above at the site of excision, and, as necessary,
surgically repairing tissues adjacent the site of excision as
described herein below. For example, for synovial joint
replacement, it is desirable to repair the joint capsule, including
the synovial membrane and ligaments, so as to surgically
approximate the joint structure as it occurs physiological
conditions, thereby recreating the avascular environment which is
the synovial cavity and which is bathed in synovial fluid. It also
is preferable to suture or otherwise mechanically temporarily
connect the implanted device to surrounding tissue.
[0052] In one embodiment the device is constructed to replace part
or all of a mammalian skeletal joint structure and includes a
matrix having residues for plural, distinct tissues, including two
or more of bone, cartilage, ligament, tendon, synovial capsule
and/or synovial membrane tissue.
[0053] In another embodiment the device is constructed to replace
an individual tissue of a mammalian skeletal joint, including an
individual avascular and/or non-mineralized tissue. As demonstrated
herein, the device is competent to induce functional replacement
tissue formation, including articular cartilage, from responding
cells present in the local environment, including a synovial
environment, and without requiring cellular infiltration of
mesenchymal cells from a vascularized muscle flap. The matrix of
this embodiment comprises residues specific for, or characteristic
of, and/or derived from, tissue of the same type as the individual
tissue to be replaced. In another embodiment, the matrix comprises
devitalized non-mineralized tissue. In a preferred embodiment, the
replacement tissue can include articular cartilage, ligament, bone,
tendon or synovial capsule tissue.
[0054] In a partial or complete joint replacement, it is preferred
but not required to include in the practice of the method the
additional step of threading a muscle flap into a hollow portion of
the implanted device. For example, using the method described in
Khouri, U.S. Pat. No. 5,067,963, the disclosure of which is
incorporated herein by reference and herewith below, a muscle flap,
which can itself be pretreated with osteogenic protein, can be
surgically introduced into a cavity in the implanted matrix, such
as the marrow cavity of devitalized bone, to provide a blood supply
to expedite morphogenesis of vascularized tissue and to provide a
ready supply of mesenchymal stem cells.
[0055] The matrix of instant invention has utility as an
implantable device when osteogenic protein is disposed on the
surfaces of the matrix, present in an amount sufficient to induce
formation of each of the replacement tissues. This permits
regeneration of the body part within the mammal, including plural
tissues of appropriate size, interrelationship, and function.
Osteogenic proteins contemplated to be useful in the instant
invention are described below and have been earlier-described in,
for example, U.S. Pat. Nos. 4,968,550, 5,258,499 and 5,266,683, the
disclosures of which are incorporated by reference herein. The
osteogenic protein can be, for example, any of the known bone
morphogenetic proteins and/or equivalents thereof described herein
and/or in the art and includes naturally sourced material
recombinant material and any material otherwise produced which is
capable of inducing tissue morphogenesis.
[0056] The methods and materials of the instant invention are
especially useful for the repair and/or partial or complete
replacement of mammalian body joints, including, without
limitation, articulating joints, particularly joints enclosed by a
ligamentous capsule and bathed in synovial fluid
[0057] In some synovial joints, the movement is uniaxial, i.e., all
movements take place around one axis: Among these are the ginglymus
or hinge joint in which the axis of movement is transverse to the
axes of the bones, and the trochoid or pivot joint in which the
axis is longitudinal. In the case of biaxial synovial joints,
movements are around two axes at a right angle or any other angle
to each other. These include the condyloid, the ellipsoid, and the
saddle joints. There is a third type of synovial joint, the
spheroidal or ball-and-socket joint, in which the movements are
polyaxial, i.e., movements are permitted in an infinite number of
axes. Finally, there are the plane or gliding type synovial
joints.
[0058] In hinge joints, the articular surfaces are molded to each
other in such a manner as to permit motion in only one plane around
the transverse axis. Flexion at the elbow joint is an example;
other examples include the interphalangeal joints of both the
fingers and toes. In pivot joints, movement in a pivot joint also
occurs around a single axis, however, it is the longitudinal axis.
There are several pivot joints in the human body, such as the
proximal radioulnar articulation. In condylar joints include,
movement occurs principally in one plane. The tibiofemoral
articulation of the knee joint is an example. In ellipsoid joint
include, movement is around two principal axes which are at right
angles to each other. Examples of these joints include the
radiocarpal and metacarpophalangeal joints. In a saddle joint, the
articular end of the proximal bone is concave in one axis and
convex in a perpendicular axis. These surfaces fit reciprocally
into convex and concave surfaces of the distal bone. The best
example of a saddle joint is the carpometacarpal joint of the
thumb. A ball-and-socket joint is one in which the distal bone is
capable of motion around an indefinite number of axes with one
common center. Examples of this form of articulation are found in
the hip and shoulder joints. A plane or gliding-type joint allows a
slight slipping or sliding of one bone over the other. Unlike the
above-described joints, the amount of motion between the surfaces
is limited by the ligaments or osseous poses that surround the
articulation. This is the form present in the joints between the
articular processes of certain vertebrae, the carpal joints, and
the intermetatarsal joints.
[0059] Although it is contemplated that the present invention is
usable to repair defects including bone and articular cartilage
elsewhere in a mammalian body, aspects of the invention are here
illustrated in connection with the articulating surfaces on the
femur in a knee joint 10 illustrated in FIG. 1.
[0060] FIG. 1 illustrates a knee joint 10 between the bottom of a
femur 11 and the top of a tibia 12. For clarity of illustration,
only portions 13 and 14 of the medial and lateral collateral
ligaments which movably tie the femur 11 to the underlying tibia 12
and fibula 15, are shown in FIG. 1. Similarly, the joint capsule is
represented by the exterior dark lining 25, and the synovial
membrane, which lines the synovial cavity and secretes the
lubricating synovial fluid, is represented by the interior dark
lining 26. Normally interposed between the opposing surfaces of the
femur 11 and tibia 12 are lateral and medial meniscus cartilages 16
and 17 and anterior and posterior cruciate ligaments (not shown).
The convexly curved condyles 20 and 21 at the lower end of the
femur 11 are normally supported by the meniscus cartilages 16 and
17, respectively, on the upper end of the tibia 12. Normally, the
lower end of the femur 11, including the condyles 20 and 21, are
covered by a layer 22 of hyaline cartilage material, referred to as
the articular cartilage 22. The articular cartilage 22 forms a
generally resilient padding which is fixed on the surface of the
lower end of the femur 11 to protect the latter from wear and
mechanical shock Moreover, the articular cartilage 22, when
lubricated by the synovial fluid in the knee joint 10, provides a
surface which is readily slidable on the underlying surfaces of the
meniscus cartilages 16 and 17 (or on the upper surface of the tibia
12 should one or both of the meniscus cartilage 16 and 17 be partly
or totally absent) during articulation of the knee joint 10.
[0061] A portion of the articular cartilage may become damaged by
injury or disease, or become excessively worn. FIG. 1 illustrates
an example of a damaged area 23.
[0062] Matrix Considerations
[0063] As will be appreciated by the skilled artisan, provided the
matrix has a three dimensional structure sufficient to act as a
scaffold for infiltrating cells, and includes the residues specific
for, or characteristic of, and/or which are derived from, the same
tissue type as the tissue to be repaired, the precise nature of the
substrate per se used for the matrices disclosed herein is not
determinative of a matrix's ultimate ability to repair and
regenerate replacement tissue. In the instant invention, the
substrate serves as a scaffold upon which certain cellular events,
mediated by an osteogenic protein, necessarily will occur. The
specific responses to the osteogenic protein ultimately are
dictated by the endogenous microenvironment at the implant site and
the developmental potential of the responding cells. As also will
be appreciated by the skilled art the precise choice of substrate
utilized for the matrices disclosed herein will depend, in part,
upon the type of defect to be repaired, anatomical considerations
such as the extent of vascularization at the defect site, and the
like.
[0064] The matrix of the invention may be obtained as follows. A
replacement tissue or body part to be used as a replacement body
part and which comprises at least two distinct tissues in
association to form the body part, is provided, as from a cadaver,
or from a bone bank and treated, as by ethanol treatment and
dehydrated by lyophilization, so that the remaining material is
non-pathogenic and sufficient non-antigenic to prevent graft
rejection As described above, the thus treated material having
utility in the devices of the invention further comprises the
residues of the extracted tissue or tissues from which it is
derived. A replacement body part matrix thus treated further is
dimensioned such that the residues have a structural relationship
to each other which mimic that of the body part to be replaced.
[0065] Natural-sourced Matrices
[0066] Suitable allogenic or xenogenic matrices can be created as
described herein below, using methods well known in the art.
Preferably, the replacement body part or tissue is obtained fresh,
from a cadaver or from a tissue bank which freezes its tissues upon
harvest. In all cases and as will be appreciated by the
practitioner in the field, it is preferable to freeze any tissue
upon harvest, unless the tissue is to be put to immediate use.
Prior to use, the tissue is treated with a suitable agent to
extract the cellular non-structural components of the tissue so as
to devitalize the tissue. The agent also should be capable of
extracting any growth inhibiting components associated with the
tissue, as well as to extract or otherwise destroy any pathogens.
The resulting material is an acellular matrix defining interstices
that can be infiltrated by cells, and is substantially depleted in
non-structurally-associated components.
[0067] In a currently preferred procedure, the tissue is
devitalized following a methodology such as that used in the art
for fixing tissue. The tissue is exposed to a non-polar solvent,
such as 100% (200 proof) ethanol, for a time sufficient to
substantially replace the water content of the tissue with ethanol
and to destroy the cellular structure of the tissue. Typically, the
tissue is exposed to 200 proof ethanol for several days, at a
temperature in the range of about 4-40.degree. C., taking care to
replace the solution with fresh ethanol every 6-12 hours, until
such time as the liquid content of the tissue comprises 70-90%
ethanol. Typically, treatment for 3-4 days is appropriate. The
volume of liquid added should be more than enough to submerge the
tissue. The treated tissue then is lyophilized. The resulting, dry
matrix is substantially depleted in non-structural components but
retains both intracellular and extracellular matrix components
derived from the tissue.
[0068] Numerous other methods are described in the art for
extracting tissues, including mineralized tissue such as bone, and
for rendering these tissues biocompatible for allogenic or
xenogenic implants. See, for example, Sampath et al. (1983) PNAS
80:6591-6595, U.S. Pat. No. 5,011,691, and U.S. Pat. Nos. 4,975,526
and 5,171,574. These publications describe extraction with 4M
guanidine-HCl, 50 mM Tris-HCl, pH 7.0 for 16 hours at 4.degree. C.,
and various deglycosylating and collagen fibril modifying agents,
including hydrogen fluoride, trifluorocetic acid, dichloromethane,
acetonitrile, isopropanol, heated, acidic aqueous solutions, and
various combinations of these reagents. The disclosures of the
patents is incorporated herein by reference. As described therein
and below, where the matrix is treated with a fibril-modifying
agent, the treated matrix can be washed to remove any extracted
components, following a form of the procedure set forth below:
[0069] 1. Suspend matrix preparation in TBS (Tris-buffered saline)
1 g/200 ml and stir at 4.degree. C. for 2 hrs; or in 6 M urea, 50
mM Tris-HCl, 500 mM NaCl, pH 7.0 (UTBS) or water and stir at room
temperature (RT) for 30 minutes (sufficient time to neutralize
pH);
[0070] 2. Centrifuge and repeat wash step; and
[0071] 3. Centrifuge; discard supernatant water wash residue; and
then lyophilize.
[0072] Treated allogenic or xenogenic matrices are envisioned to
have particular utility for creating devices for forming
replacement body parts comprising plural distinct tissues, as well
as for creating devices for replacing individual joint tissues,
such as ligament and articular cartilage tissue. For example, a
replacement ligament device can be formulated from an allogenic
ligament matrix and osteogenic protein, and implanted at a skeletal
joint locus following standard surgical procedures for autogenous
ligament replacement. Similarly, an allogenic articular cartilage
device can be formed from devitalized cartilage tissue, or other
inert, non-mineralized matrix material and osteogenic protein, and
the device laid on the subchondral bone surface as a sheet.
Alternatively, a formulated device can be pulverized or otherwise
mechanically abraded to produce particles which can be formulated
into a paste or gel as described herein for application to the bone
surface.
[0073] Synthetic Matrices
[0074] As an alternative to a natural-sourced matrix, or as a
supplement to be used in combination with a natural-sourced matrix,
a suitable matrix also can be formulated de novo, using (1)
residues derived from and/or characteristic of, or specific for,
the same tissue type as the tissue to be repaired, and (2) one or
more materials which serve to create a three-dimensional
scaffolding structure that can be formed or molded to take on the
dimensions of the replacement tissue desired. In some
circumstances, as in the formation of articular cartilage on a
subchondral bone surface, osteogenic protein in combination with a
matrix defining a three-dimensional scaffolding structure
sufficient to allow the attachment of infiltrating cells and
composed of a non-mineralized material can be sufficient. Any one
or combination of materials can be used to advantage, including,
without limitation, collagen; homopolymers or copolymers of
glycolic acid, lactic acid, and butyric acid, including derivatives
thereof; and ceramics, such as hydroxyapatite, tricalcium phosphate
and other calcium phosphates and combinations thereof.
[0075] The tissue-specific component of a synthetic matrix ready
can be obtained by devitalizing an allogenic or xenogenic tissue as
described above and then pulerizing or otherwise mechanically
breaking down the insoluble matrix remaining. This particulate
material then can be combined with one or more structural
materials, including those described herein. Alternatively,
tissue-specific components can be further purified from the treated
matrix using standard extraction procedures well characterized in
the art and, using standard analysis procedures, the extracted
material at each purification step can be tested for its
tissue-specificity capability. See, for example, Sampath et al
(1987) PNAS 78:7599-7603 and U.S. Pat. No. 4,968,590 for exemplary
tissue extraction protocols.
[0076] A synthetic matrix may be desired where, for example,
replacement articular cartilage is desired in an existing joint to,
for example, correct a tear or limited superficial defect in the
tissue, or to increase the height of the articular cartilage
surface now worn due to age, disease or trauma. Such "resurfacing"
of the articular cartilage layer can be achieved using the methods
and compositions of the invention by, in one embodiment, treating a
sheet of allogenic or xenogenic articular cartilage tissue as
described herein, coating the resulting matrix with osteogenic
protein, rolling up the formed device so that it can be introduced
to the joint using standard orthoscopic surgical techniques and,
once provided to the site, unrolling the device as a layer onto the
articular bone surface. In another embodiment, the device is
formulated as a paste or injectable gel-like substance that can be
injected onto the articular bone surface in the joint also using
standard orthoscopic surgical techniques. In this embodiment, the
formulation may comprise a pulverized or otherwise mechanically
degraded device comprising both matrix and osteogenic protein and,
in addition, one or more components which serve to bind the
particles into a paste-like or gel-like substance. Binding
materials which characterized in the art include, for example,
carboxymethylcellulose, glycerol, polyethylene-glycol and the like.
Alternatively, the device can comprise osteogenic protein dispersed
in a synthetic matrix which provides the desired physical
properties. As an example, a synthetic matrix having tissue
specificity for cartilage and bone is described in WO91/18558,
published Dec. 21, 1991 and herein below. Briefly, the matrix
comprises a porous crosslinked structural polymer of biocompatible,
biodegradable collagen and appropriate, tissue-specific
glycosaminoglycans as tissue-specific cell attachment factors.
Collagen derived from a number of sources can be used, including
insoluble collagen, acid-soluble collagen, collagen soluble in
neutral or basic aqueous solutions, as well as those collagens
which are commercially available.
[0077] Glycosaminoglycans (GAGs) or mucopolysaccharides are
hexosamine-containig polysaccharides of animal origin that have a
tissue specific distribution, and therefore may be used to help
determine the tissue specificity of the morphogen-stimulated
differentiating cells. Reaction with the GAGs also provides
collagen with another valuable property, i.e., inability to provoke
an immune reaction (foreign body ion) from an animal host.
[0078] Chemically, GAGs are made up of residues of hexoamines
glycosidically bound and alternating in a more-or-less regular
manner with either hexouronic acid or hexose moieties (see, e.g.,
Dodgson et al. in Carbohydrate Metabolism and its Disorders
(Dickens et al., eds.) Vol. 1, Academic Press (1968)). Useful GAGs
include hyaluronic acid, heparin, heparin sulfate, chondroitin
6-sulfate, chondroitin 4-sulfate, dermatan sulfate, and keratin
sulfate. Other GAGs also can be used for forming the matrix
described herein, and those skilled in the art will either know or
be able to ascertain other suitable GAGs using no more than routine
experimentation. For a more detailed description of
mucopolysaccharides, see Aspinall, Polysaccharides, Pergamon Press,
Oxford (1970).
[0079] Collagen can be reacted with a GAG in aqueous acidic
solutions, preferably in diluted acetic acid solutions. By adding
the GAG dropwise into the aqueous collagen dispersion,
coprecipitates of tangled collagen fibrils coated with GAG results.
This tangled mass of fibers then can be homogenized to form a
homogeneous dispersion of fine fibers and then filtered and
dried.
[0080] Insolubility of the collagen-GAG products can be raised to
the desired degree by covalently cross-linking these materials,
which also serves to, raise the resistance to resorption of these
materials. In general, any covalent cross-linking method suitable
for cross-linking collagen also is suitable for cross-linking these
composite materials, although crosslinking by a dehydrothermal
process is preferred
[0081] Formulation Considerations
[0082] The devices of the invention can be formulated using any of
the methods described in the art for formulating ostegenic devices.
See, for example, U.S. Pat. No. 5,266,683, the disclosure of which
is incorporated herein by reference. Briefly, osteogenic protein
typically is dissolved in a suitable solvent and combined with the
matrix. The components are allowed to associate. Typically, the
combined material then is lyophilized, with the result that the
osteogenic protein is disposed on, or adsorbed to the surfaces of
the matrix. Useful solubilizing solvents include, without
limitation, an ethanoltrifluoroacetic acid solution, e.g., 47.5%
EtOH/0.01% TFA, and acetonitrile/TFA solution, ethanol or ethanol
in water, and physiologically buffered saline solutions.
Formulations in an acidic buffer can faciliate adsorption of OP1
onto the matrix surface. For the replacement body part devices of
the invention, the currently preferred formulation protocol is
incubation of matrix and osteogenic protein in an ethanol/TFA
solution (e.g., 30-40% EtOH/0.01-0.1% TFA) for 24 hours, followed
by lyophilization. This procedure is sufficient to adsorb or
precipitate 70-90% of the protein onto the matrix surface.
[0083] The quantity of osteogenic protein used will depend on the
size of replacement device to be used and on the specific activity
of the osteogenic protein. Typically, 0.5 mg-100 mg/10 g of matrix,
dry weight, can be used to advantage.
[0084] In addition to osteogenic proteins, various growth factors,
hormones, enzymes, therapeutic compositions, antibiotics, or other
bioactive agents also can be adsorbed onto, or impregnated within,
a substrate and released over time when implanted and the matrix
slowly is absorbed Thus, various known growth factors such as EGF,
PDGF, IGF, FGF, TGF-a, and TF-B can be released in vivo. The matrix
can also be used to release chemotherapeutic agents, insulin,
enzymes, enzyme inhibitors or chemotactic-chemoattractant
factors.
[0085] Protein Considerations
[0086] As defined herein, the osteogenic proteins useful in the
composition and methods of the invention include the family of
dimeric proteins having endochondral bone activity when implanted
in a mammal in association with a matrix and which comprise a
subclass of the "super family" of "TGF.beta.-like" proteins.
[0087] The natural-sourced osteogenic protein in its mature, native
form is a glycosylated diner 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. Useful sequences include
those comprising the C-terminal 102 amino acid sequences of DPP
(from Drosophila), Vgl (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.
[0088] The members of this family of proteins share a conserved six
or seven cysteine skeleton in the C-terminal region. See, for
example, 335-431 of Seq. ID No. 1 and whose sequence defines the
six cysteine skeleton residues referred to herein as "OPS", or
residues 330-431 of Seq. ID No. 1, comprising 102 amino acids and
whose sequence defines the seven cysteine skeleton.
[0089] This family of proteins includes longer forms of a given
protein, as well as phylogenetic, e.g., species and allelic
variants and biosynthetic mutants, including addition and deletion
mutants and variants, such as those which may alter the conserved
C-terminal cysteine skeleton, provided hat 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 association with a matrix. In addition, the
osteogenic proteins useful in devices of 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.
[0090] In one embodiment, the osteogenic protein contemplated
herein comprises OP1 or an OP1-related sequence. Useful OP1
sequences are recited in U.S. Pat. Nos. 5,011,691; 5,018,753 and
5,266,683; in Ozkaynak et al. (1990) EMBO J. 2:2085-2093; and
Sampath et al. (1993) PNAS 90: 6004-6008. OP-1 related sequences
include xenogenic homologs, e.g.; 60A, from Drosophila, Wharton et
al. (1991) PNAS 88 9214-9218; and proteins sharing greater than 60%
identity with OP1 in the C-terminal seven cysteine domain,
preferably at least 65% identity. Examples of OP-1 related
sequences includes BMP5, BMP6 (and its species homolog Vgr-1, Lyons
et al. (1989) PNAS .86:4554-4558), Celeste, et al. (1990) PNAS
87:9843-9847 and PCT international application WO93/00432; OP-2
(Ozkayna et al. (1992) J. Biol. Chem. 267:13198-13205) As will be
appreciated by those having ordinary skill in the art, chimeric
constructs readily can be created using standard molecular biology
and mutagenesis techniques combining various portions of different
morphogenic protein sequences to create a novel sequence, and these
forms of the protein also are contemplated herein
[0091] In another preferred aspect, the invention contemplates
osteogenic proteins comprising species of polypeptide chains having
the generic amino acid sequence herein referred to as "OPX" which
accommodates the homologies between the various identified species
of the osteogenic OP1 and OP2proteins, and which is described by
the amino acid sequence presented below and in Sequence ID No.
3.
1 Cys Xaa Xaa His Glu Leu Tyr Val Ser Phe 1 5 10 Xaa Asp Leu Gly
Trp Xaa Asp Trp Xaa Ile 15 20 Ala Pro Xaa Gly Tyr Xaa Ala Tyr Tyr
Cys 25 30 Glu Gly Glu Cys Xaa Phe Pro Leu Xaa Ser 35 40 Xaa Met Asn
Ala Thr Asn His Ala Ile Xaa 45 50 Gln Xaa Leu Val His Xaa Xaa Xaa
Pro Xaa 55 60 Xaa Val Pro Lys Xaa Cys Cys Ala Pro Thr 65 70 Xaa Leu
Xaa Ala Xaa Ser Val Leu Tyr Xaa 75 80 Asp Xaa Ser Xaa Asn Val Ile
Leu Xaa Lys 85 90 Xaa Arg Asn Met Val Val Xaa Ala Cys Gly 95 100
Cys His,
[0092] and wherein Xaa at res. 2=(Lys or Arg); Xaa at res. 3=(Lys
or Arg); Xaa at res. 11 =(Arg or Gln); Xaa at res. 16=(Gln or Leu);
Xaa at res. 19=(Ile or Val); Xaa at res. 23=(Glu or Gln); Xaa at
res. 26=(Ala or Ser); Xaa at res. 35=(Ala or Ser); Xaa at res.
39=(Asn or Asp); Xaa at res. 57=(Phe or Leu); Xaa at res. 39=(Asn
or Asp); Xaa at res. 41=(Tyr or Cys); Xaa at res. 50=(Val or Leu);
Xaa at res. 52=(Ser or Thr); Xaa at res. 56=(Phe or Leu); Xaa at
res. 57=(Ile or Met); Xaa at res. 58 (Asn or Lys); Xaa at res.
60=(Glu, Asp or Asn); Xaa at res. 61=(Thr, Ala or Val); Xaa at res.
65=(Pro or Ala); Xaa at res. 71=(Gln or Lys); Xaa at res. 73=(Asn
or Ser); Xaa at res. 75=(Ile or The); Xaa at res. 80=(Phe or Tyr);
Xaa at res. 82=(Asp or Ser); Xaa at res. 84=(Ser or Asn); Xaa at
res. 89=(Lys or Arg); Xaa at res. 91=(Tyr or His); and Xaa at res.
97=(Arg or Lys).
[0093] In still another preferred aspect, one or both of the
polypeptide chain subunits of the osteogenerically active dimer is
encoded by nucleic acids which hybridize to DNA or RNA sequences
encoding the active region of OP1 under stringent hybridization
conditions. As used herein, stringent hybridization conditions are
defined as hybridization 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.
[0094] Given the foregoing amino acid and DNA sequence information,
the level of skill in the art, and the disclosures of numerous
publications on osteogenic proteins, including U.S. Pat. No.
5,011,691 and published PCT specification U.S. patent application
Ser. No. 89/01469, published Oct. 19, 1989, various DNAs can be
constructed which encode at least the active domain of an
osteogenic protein useful in the devices of this invention, and
various analogs thereof (including species and allelic variants and
those containing genetically engineered mutations), as well as
fusion proteins, truncated forms of the mature proteins, deletion
and addition mutants, and similar constructs which can be used in
the devices and methods of the invention. Moreover, DNA
hybridization probes can be constructed from fragments of any of
these proteins, or designed de novo from the generic sequence.
These probes then can be used to screen different genomic and cDNA
libraries to identify additional osteogenic proteins useful in the
prosthetic devices of this invention.
[0095] The DNAs can be produced by those skilled in the art using
well known DNA manipulation techniques involving genomic and cDNA
isolation, construction of synthetic DNA from synthesized
oligonucleotides, and cassette mutagenesis techniques. 15-100 mer
oligonucleotides may be synthesized on a DNA synthesizer, and
purified by polyacrylamide gel electrophoresis (PAGE) in
Tris-Borate-EDTA buffer. The DNA then may be electroeluted from the
gel. Overlapping oligomers may be phosphorylated by T4
polynucleotide kinase and ligated into larger blocks which may also
be purified by PAGE.
[0096] The DNA from appropriately identified clones then can be
isolated, subcloned (preferably into an expression vector), and
sequenced. Plasmids containing sequences of interest then can be
transfected into an appropriate host cell for protein expression
and further characterization. The host may be a procaryotic or
eucaryotic cell since the former's inability to glycosylate protein
will not destroy the protein's morphogenic activity. Useful host
cells include E. coli, Saccharomyces, the insect/baculovirus cell
system, myeloma cells, CHO cells and various other mammalian cells.
The vectors additionally may encode various sequences to promote
correct expression of the recombinant protein, including
transcription promoter and termination sequences, enhancer
sequences, preferred ribosome binding site sequences, preferred
mRNA leader sequences, preferred signal sequences for protein
secretion and the like.
[0097] The DNA sequence encoding the gene of interest also may be
manipulated to remove potentially inhibiting sequences or to
minimize unwanted secondary structure formation The recombinant
osteogenic protein also may be expressed as a fusion protein. After
being translated, the protein may be purified from the cells
themselves or recovered from the culture medium. All biologically
active protein forms comprise dimeric species joined by disulfide
bonds or otherwise associated, produced by folding and oxidizing
one or more of the various recombinant polypeptide chains within an
appropriate eucaryotic cell or in vitro after expression of
individual subunits. A detailed description of osteogenic proteins
expressed from recombinant DNA in E. coli and in numerous different
mammalian cells is disclosed in U.S. Pat. No. 5,266,963, the
disclosure of which is hereby incorporated by reference.
[0098] Alternatively, osteogenic polypeptide chains can be
synthesized chemically using conventional peptide synthesis
techniques well known to those having ordinary skill in the art For
example, the proteins may be synthesized intact or in parts on a
solid phase peptide synthesizer, using standard operating
procedures. Completed chains then are deprotected and purified by
HPLC (high pressure liquid chromatography). If the protein is
synthesized in parts, the parts may be peptide bonded using
standard methodologies to form the intact protein. In general, the
manner in which the osteogenic proteins are made can be
conventional and does not form a part of this invention.
[0099] Exemplification
[0100] The means for making and using the matrices and devices of
the invention, as well as other material aspects concerning the
nature and utility of these compositions, 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.
[0101] In the exemplification, a hemi-joint reconstruction of an
articulating synovial joint is resected into an existing joint
locus. As will be appreciated by those having ordinary skill in the
art, the methods and compositions of the invention equally can be
applied to the formation of replacement body parts other than
skeletal joints, as well as to skeletal joints other than
articulating or synovial joints. Moreover, if desired, a
replacement autogenous joint can be constructed in the recipient
first by placing the device of the invention at another convenient
locus distal to the defect site, for a time sufficient to induce
formation of the replacement body part, and the autogenous body
part thus formed then sutured into the joint locus for use.
EXAMPLE 1
Reconstruction of a Mammalian Hemi-Joint
[0102] New Zealand white rabbits were used as the experimental
model. Standard orthopedic surgical equipment and procedures were
used.
[0103] As depicted in FIG. 2A, joint defects were created in a
recipient by surgically resecting the entire gleno-humeral
hemiarticular complex with the proximal two-thirds of the humerus.
Allografts for implantation were prepared from hemi-joints excised
from a donor animal with the articular surface of the glenohumoral
joint. All allografts were extracted in ethanol and lyophilized
using standard procedures, and as described herein above, to
destroy the pathogenicity and antigenicity of the material.
Specifically, intact joint complexes were excised, demarraowed and
ethanol treated by exposure to 200ml-500ml of 200 proof ethanol for
72 hours at 40 C. Fresh ethanol was provided every 6-8 hours.
Following ethanol treatment, the matrix was lyophilized and
rehydrated in ethano/TFA, with or without osteogenic protein The
treated hemi-joints comprised devitalized bone, articular
cartilage, ligament, tendon, synovialcapsule and synovial membrane
tissue.
[0104] As illustrated in FIG. 2B, all lyophilized, osteogenic
protein-treated allografts were coated with OP-1 as described in
U.S. Pat. No. 5,011,691. Specifically, mature, dimeric recombinant
OP-1 (rhOP1) was solubilized in an acetonitrile trifluoro-acetic
acid solution, combined with the lyophilized allograft, and
implanted. 15-20 mg protein/8-10 g matrix, dry weight, was used.
The distal bone portions of all allografts were secured in place
with a four hole titanium miniplate. A meticulous surgical
reconstruction of the joint capsule was performed by suturing the
lyophilized capsule ends to the endogenous capsule using standard
surgical procedures well established in the art using standard
surgical procedures well established in the art This recreated an
intact capsule and synovial lining, thereby restoring the synovial
milieu of the grafted articular surface. Motion was permitted
almost immediately after surgery, again to restore normal joint
conditions.
[0105] In some animals, local muscle flaps (cutaneous maximus
muscle; FIG. 2C) were incorporated into the region of the defect by
threading muscle into the marrow cavity of the allograft as
depicted in FIG. 2D, using the method of Khouri as described in
U.S. Pat. No. 5,067,963 the disclosure of which is incorporated
herein by reference. Briefly, vascularized and convenient muscle
flaps were dissected using standard procedures well known to the
practitioner in reconstructive surgery, so as to maintain a
perfusing blood supply, and threaded inside the bone marrow
cavities of the allografts.
[0106] Preliminary evaluations of the reconstructed hemi-joints
were obtained by serial weekly radiographs using X-ray. and/or
magnetic resonance imaging (MRI). Histological and mechanical
confirmatory evaluations were conducted upon sacrifice at 5 weeks
and 6 months after surgery.
[0107] Mechanical evaluations involved standard range of motion
(ROM) measurements obtained serially until sacrifice. Histological
evaluations involved staining sagital sections through the
harvested allografts using standard techniques.
[0108] Briefly, identification of bona fide articular cartilage can
be accomplished using ultrastructural and/or biochemical
parameters. For example, articular cartilage forms a continuous
layer of cartilage tissue possessing identifiable zones. The
superficial zone is characterized by chondrocytes having a
flattened morphology and an extracellular network which does not
stain, or stains poorly, with toluidine blue, indicating the
relative absence of sulphated proteoglycans. Chondrocytes in the
mid and deep zones have a spherical appearance and the matrix
contains abundant sulphated proteoglycans, as evidenced by staining
with toluidine blue. Collagen fibers are present diffusely
throughout the matrix. The chondrocytes possess abundant rough
endoplasmic reticulum and are surrounded by extracellular network.
The pericellular network contains numerous thin non-banded collagen
fibers. The collagen in the interterritorial network is less
compacted and embedded in electron translucent amorphous material,
similar to articular cartilage. Collagen fibers in the
interterritorial region of the network exhibit the periodic banding
characteristic of collagen fibers in the interterritorial zone of
cartilage tissue.
[0109] Biochemically, the presence of Type II and Type IX collagen
in the cartilage tissue is indicative of the differentiated
phenotype of chondrocytes. The presence of Type II and/or Type IX
collagen can be determined by standard gel electrophoresis, Western
blot analysis and/or immuno histo-chemical staining using, for
example, commercially available antibody. Other biochemical markers
include hematoxylin, eosin, Goldner's Thichrome and Safranin-O.
[0110] Articular cartilage regeneration was evaluated
histologically in the examples described herein using
glycosaminoglycan-specific stains and techniques well-known in the
art. For the initial histologic evaluation, the defect sites were
bisected lengthwise through the center of the defect The resulting
halves and surrounding tissue were embedded in paraffin and
sectioned across the center of the defect. One half of each defect
was utilized for histological staining with toluidine blue and/or
hematoxlin and eosin, Goldner's Trichrome and Safranin-O. The other
half was used in preparing sections for immunostaining.
Histological evaluations involved assessment of: glycosaminoglycan
content in the repair cartilage; cartilage and chondrocyte
morphology; and, structural integrity and morphology at the defect
interface. The morphology of the repair cartilage was exhibited for
the type of cartilage formed: articular vs. fibrotic by evaluating
glysaminoglycan content, degree of cartilage deposition, and the
like.
[0111] Histological evaluations using standard methodologies well
characterized in the art also allows assessment of new bone and
bone marrow formation. See, for example, U.S. Pat. No. 5,266,683,
the disclosure of which is incorporated hereinabove by reference.
Similarly, ligament and synovial capsule integrity can be monitored
by MRI, as well as by histology upon sacrifice.
EXAMPLE 2
Five Weeks Duration (Short Term)
[0112] For the 5 week study, four groups with 10 rabbits per group
were implanted with lyophilized allografts. See FIGS. 3A, 3B, 3C,
and 3D. In Group 1, control lyophilized allograft 30 free of
osteogenic protein, was implanted (FIG. 3A). In Group 2,
experimental lyophilized allograft 31 was impregnated with OP-1
prior to implantation (FIG. 3B). In Group 3, control lyophilized
allograft 30 free of osteogenic protein, was implanted, with muscle
flap 32 threaded into marrow cavity 33 (FIG. 3C). In Group 4,
experimental lyophilized allograft 31 was impregnated with OP-1
prior to implantation, and muscle flap 32 was threaded within the
marrow cavity 33 (FIG. 3D).
[0113] As stated above, graft healing was followed non-invasively
with serial X-rays and standard MRI (magnetic resonance imaging).
By X-ray assessment, allografts treated with osteongic protein had
a noticeably thickened cortex by 1 week post-operative, as compared
with control allografts (Groups 1, 3) which evidenced only a thin
egg-shell-like cortex. By four weeks the majority control
allografts had fractured and were unstable. In contrast, OP-1
treated allografts (Groups 2, 4) remained stable.
[0114] MRI also was used as a non-invasive means for following
reformation of articular cartilage in the allografts. A dark signal
produced by MRI represents absent or nonviable cartilage, while a
bright signal indicates live, viable cartilage. Control allografts
produced only a dark signal, when tested at 1, 3 and 5 weeks
post-operative. These MRI findings were confirmed by histological
analysis performed at 5 weeks post-operative. Sagital sectioning
through control allografts showed a degenerated articular surface
with no live cells.
[0115] By contrast the MRI findings of the articular caps from
OP-1-treated allografts showed a bright signal by week 3
post-operative, indicating regeneration of viable articular
cartilage. Histological analysis of the OP-1-treated allografts at
week 5 revealed a layer of newly generated articular cartilage on
top of the allograft matrix The allografts of Group 4 showed
somewhat thicker cartilage layers than those of Group 2, suggesting
that the addition of the muscle flap may further enhance the rate
of joint regeneration.
[0116] Additionally, joints regeneracy with the OP-1-treated
allografts regained near normal range of motion by the time they
were harvested at 5 weeks post-reconstruction. The near normal
range of motion also is indicative of the presence of lubricating
synovial fluid. By contrast, the harvested control allografts were
stiff and contracted at harvest Thus, hemi-joint replacement
devices of the invention succeeded in forming mechanically and
functionally viable replacement joints, with an intact capsule, and
synovium, and functioning ligament, bone and articular cartilage
tissue. In the absence of osteogenic protein, the allografts, while
not rejected by the donor, are insufficient on their own to
generate a functional, weight bearing joint.
EXAMPLE 3
Six Months Duration--(Long Term)
[0117] For the 6 month study, the variable of shaving off the old
cartilaginous cap in the lyophilized allografts was introduced.
Briefly, this was accomplished by mechanically sharing the
articular cartilage cap of the joint surface.
[0118] The following groups were used with 4 rabbits per group: in
Group 5, lyophilized allograft 34 with shaved articular surface,
and muscle flap 32 were implanted (see FIG. 4A); in Group 6,
control lyophilized allograft 30 with non-shaved articular surface,
and muscle flap 32 were implanted (see FIG. 4B); in Group 7,
lyophilized allograft 35 with shaved articular surface and OP-1,
and muscle flap 36 treated with OP-1 were implanted (see FIG. 4C);
and, in Group 8, lyophilized allograft 37 with a non-shaved
articular surface and OP-1, and muscle flap 36 treated with OP-1
were implanted (see FIG. 4D). Grafts in Groups 5-8 were harvested
at 6 months after surgery.
[0119] Based upon pre-harvest imaging studies, the results
collected by 3 months post-operative are consistent with the above
described results collected at 5 weeks. Intact allografts treated
with OP-1 (Group 8) regenerated a live cartilaginous articular
surface by 3 weeks when evaluated using MRI. This articular cap is
still present and even better developed at 3 months. Without OP-1
treatment of the allograft, (Group 6) there was negligible
cartilage regeneration relative to the OP-1 treated groups.
[0120] Similarly, Group 8 rabbits (allograft+OP1, non-shaved)
regained near normal range of motion (greater than 80%) in the
reconstructed joint Group 7 rabbits (allograft+OP1, shaved)
achieved only 50% range of motion, and Groups 5 and 6 (no OP1)
achieved less than 30%.
[0121] As determined by histology, the devices of the invention
were competent to induce and maintain both bone and articular
cartilage formation in the appropriate context to one another in a
long term study (greater than 6 months). Specifically, the rabbits
of Group 8, demonstrated articular cartilage formation on the
surface of bone, as evidenced morphologically by the presence of
resting, central and deeper zone chondrocytes. By contrast, in
groups treated only with muscle flap, (Group 5 and 6) muscle was
replaced with scar issue. In the groups treated with shaved bone
matrices, no significant cartilage regeneration was identified,
demonstrating the requirement for cartilage-specific residues in
articular cartilage formation in a non-vascularized mileiu.
[0122] In both the short term and long term study, mechanically and
functionally viable synovial joints resulted from the reconstructed
hemijoints treated with osteogenic protein, as evidenced by
morphology and biochemistry. In addition, new tissue formed,
including articular cartilage, corresponding in shape, kind and
structural relationship to the residues in the devitalized tissue
which formed the matrix of the device. Collectively, these examples
demonstrate that a device comprising osteogenic protein and an
off-the-shelf, non-viable lyophilized, devitalized matrix can be
transformed into aviable, mechanically and structurally functional
replacement body part structure comprising plural distinct newly
formed tissues which assume the shape and function of the original
tissue. The device can restore normal function to a destroyed body
part, including a destroyed skeletal joint, restoring mechanically
and functionally viable plural distinct tissues, including bone and
bone marrow, articular cartilage, ligament, tendon, synovial
capsule and synovial membrane tissue. Moreover, these tissues are
restored under substantially physiological conditions including,
for example, from responding cells present in a synovial
environment, and without exposure to avascularized muscle flap.
[0123] A device comprising osteogenic protein-treated matrices,
including lyophilized allografts or xenografts as disclosed herein
can lead to the formation of a new, mechanically, structurally and
functionally viable replacement tissue, and to replacement body
parts comprising plural distinct tissues, populated by the host
cells, and without any of the limitations of prosthetic
materials.
[0124] Those skilled in the art will know, or be able to ascertain
using no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. These and
all other equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
* * * * *