U.S. patent application number 09/873975 was filed with the patent office on 2001-11-08 for proteoglycan-reduced soft tissue xenografts.
Invention is credited to Stone, Kevin R..
Application Number | 20010039459 09/873975 |
Document ID | / |
Family ID | 22938672 |
Filed Date | 2001-11-08 |
United States Patent
Application |
20010039459 |
Kind Code |
A1 |
Stone, Kevin R. |
November 8, 2001 |
Proteoglycan-reduced soft tissue xenografts
Abstract
The invention provides an article of manufacture comprising a
substantially non-immunogenic soft tissue xenograft for
implantation into humans. The invention further provides methods
for preparing a soft tissue xenograft by removing at least a
portion of a soft tissue from a non-human animal to provide a
xenograft; washing the xenograft in saline and alcohol; subjecting
the xenograft to cellular disruption treatment; and digesting the
xenograft with a proteoglycan-depleting factor and/or glycosidase
and optionally following with a capping treatment. The invention
also provides an article of manufacture produced by the
above-identified method of the invention. The invention further
provides a soft tissue xenograft for implantation into a human
including a portion of a soft tissue from a non-human animal,
wherein the portion has extracellular components and substantially
only dead cells. The extracellular components have reduced
proteoglycan molecules. Each of the xenografts of the invention are
substantially non-immunogenic and have substantially the same
mechanical properties as a corresponding native soft tissue.
Inventors: |
Stone, Kevin R.; (Mill
Valley, CA) |
Correspondence
Address: |
McDERMOTT, WILL & EMERY
28 State Street
Boston
MA
02109
US
|
Family ID: |
22938672 |
Appl. No.: |
09/873975 |
Filed: |
June 4, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09873975 |
Jun 4, 2001 |
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09248336 |
Feb 11, 1999 |
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6267786 |
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Current U.S.
Class: |
623/23.72 ;
623/915 |
Current CPC
Class: |
A61L 27/3687 20130101;
A61L 27/3691 20130101; A61L 27/3604 20130101; A61L 2430/34
20130101; A61L 2430/40 20130101; Y10S 623/915 20130101 |
Class at
Publication: |
623/23.72 ;
623/915 |
International
Class: |
A61F 002/02; A61L
017/00 |
Claims
What is claimed is:
1. A method of preparing a soft tissue xenograft for implantation
into a human, which comprises: a. removing at least a portion of a
soft tissue from a non-human animal to provide a xenograft; b.
washing the xenograft in water and alcohol; c. subjecting the
xenograft to a cellular disruption treatment; and d. depleting
substantially a plurality of proteoglycans from the xenograft,
whereby the xenograft is substantially non-immunogenic and has
substantially the same mechanical properties as the native soft
tissue.
2. A method according to claim 1, wherein the depleting step
comprises digesting the xenograft with at least one
proteoglycan-depleting factor selected from the group consisting of
chondroitinase ABC, hyaluronidase, chondroitin AC II lyase,
keratanase, trypsin and fibronectin fragment.
3. A method according to claim 1 further comprising the step of
after step c, piercing the xenograft.
4. A method according to claim 1 further comprising the step of
after step c, treating the xenograft with at least one enzyme.
5. A method according to claim 4, wherein the enzyme is selected
from the group consisting of ficin and trypsin.
6. A method according to claim 1 further comprising the step of
after step c, treating the xenograft with one or more agents
selected from the group consisting of anticalcification agents,
antithrombotic agents, antibiotics, and growth factors.
7. A method according to claim 1 further comprising the step of
after step c, sterilizing the xenograft.
8. A method according to claim 7, wherein the sterilizing step
comprises sterilizing the xenograft with one or more agents
selected from the group consisting of ethylene oxide and propylene
oxide.
9. A method according to claim 1 further comprising the step of:
after step c, treating the xenograft with polyethylene glycol.
10. A method according to claim 1 further comprising the step of
after step c, exposing the xenograft to a crosslinking agent in a
vapor form.
11. A method according to claim 1, wherein the cellular disruption
treatment comprises freeze/thaw cycling.
12. A method according to claim 1, wherein the cellular disruption
treatment comprises exposure to gamma radiation.
13. A method according to claim 1 further comprising the step of
after step d, removing substantially a plurality of first surface
carbohydrate moieties from the xenograft.
14. A method according to claim 1, wherein the removing step
comprises digesting the xenograft with a glycosidase.
15. A method according to claim 14, wherein the glycosidase is a
galactosidase.
16. A method according to claim 15, wherein the galactosidase is an
.alpha.-galactosidase.
17. A method according to claim 13 further comprising the step of
following the removing step, treating a plurality of second surface
carbohydrate moieties on the xenograft with a plurality of capping
molecules to cap at least a portion of the second surface
carbohydrate moieties.
18. A method according to claim 16, wherein at least a portion of
the capping molecules are a plurality of fucosyl molecules.
19. A method according to claim 16, wherein at least a portion of
the capping molecules are a plurality of n-acetyl glucosamine
molecules.
20. A method according to claim 1, wherein the removing step
comprises removing the portion of a medial or lateral meniscus
having a superior principal surface and an inferior principal
surface, each of the principal surfaces having an outer portion
being joined by an outer lateral surface, and each of the principal
surfaces having an inner portion being joined by an inner lateral
surface.
21. A method according to claim 1, wherein the removing step
comprises removing the portion of a ligament.
22. A method according to claim 21, wherein the removing step
comprises removing with the portion of the ligament a first block
of bone attached to a first end of the portion.
23. A method according to claim 22, wherein the removing step
comprises removing with the portion of the ligament a second block
of bone affixed to a second end of the portion opposite the first
end.
24. A method according to claim 1, wherein the removing step
comprises removing the portion of an articular cartilage.
25. A method according to claim 24, wherein the removing step
comprises removing with the portion of the articular cartilage a
layer of subchondral bone.
26. An article of manufacture comprising a substantially
non-immunogenic, proteoglycan-reduced soft tissue xenograft for
implantation into a human, produced by: a. removing at least a
portion of a soft tissue from a non-human animal to provide a
xenograft; b. washing the xenograft in water and alcohol; c.
subjecting the xenograft to a cellular disruption treatment; and d.
digesting the xenograft with a proteoglycan-depleting factor to
remove substantially a plurality of proteoglycans from the
xenograft, whereby the xenograft is substantially non-immunogenic
and has substantially the same mechanical properties as the native
soft tissue.
27. An article of manufacture according to claim 26, wherein the
proteoglycan-depleting factor is selected from the group consisting
of chondroitinase ABC, hyaluronidase, chondroitin AC II lyase,
keratanase, trypsin and fibronectin fragment.
28. An article of manufacture according to claim 26, wherein the
xenograft has a plurality of punctures for increasing permeability
to agents and enzymes.
29. An article of manufacture according to claim 26 further
comprising one or more agents selected from the group consisting of
anticalcification agents, antithrombotic agents, antibiotics, and
growth factors.
30. An article of manufacture according to claim 26, wherein the
xenograft is sterilized.
31. An article of manufacture according to claim 26, wherein the
xenograft is a polyethylene glycol-treated xenograft.
32. An article of manufacture according to claim 26 further
comprising a crosslinking agent.
33. An article of manufacture according to claim 26, wherein the
xenograft has a plurality of first surface carbohydrate moieties
substantially removed.
34. An article of manufacture according to claim 33, wherein the
xenograft is a glycosidase-treated xenograft.
35. An article of manufacture according to claim 34, wherein the
glycosidase-treated xenograft is a galactosidase-treated
xenograft.
36. An article of manufacture according to claim 35, wherein the
galactosidase-treated xenograft is an .alpha.-galactosidase-treated
xenograft.
37. An article of manufacture according to claim 33 further
comprising a plurality of capping molecules capped on a plurality
of second surface carbohydrate moieties on the xenograft.
38. An article of manufacture according to claim 37, wherein the
capping molecules are selected from one or more of the groups of
fucosyl molecules and n-acetyl glucosamine molecules.
39. An article of manufacture according to claim 26, wherein the
xenograft is a thawed, frozen xenograft.
40. An article of manufacture according to claim 26, wherein the
xenograft is a gamma-irradiated xenograft.
41. An article of manufacture according to claim 26, wherein the
portion is of a medial or lateral meniscus having a superior
principal surface and an inferior principal surface, each of the
principal surfaces having an outer portion being joined by an outer
lateral surface, and each of the principal surfaces having an inner
portion being joined by an inner lateral surface.
42. An article of manufacture according to claim 26, wherein the
portion is of a ligament.
43. An article of manufacture according to claim 42, wherein the
portion of the ligament comprises a first block of bone attached to
a first end of the portion.
44. An article of manufacture according to claim 43, wherein the
portion of the ligament comprises a second block of bone affixed to
a second end of the portion opposite the first end.
45. An article of manufacture according to claim 26, wherein the
portion is of an articular cartilage.
46. An article of manufacture according to claim 45, wherein the
portion of the articular cartilage comprises a layer of subchondral
bone.
47. A soft tissue xenograft for implantation into a human
comprising a portion of a soft tissue from a non-human animal,
wherein the portion includes a plurality of substantially only dead
cells and a plurality of extracellular components, the
extracellular components having reduced proteoglycans, whereby the
portion of the soft tissue is substantially non-immunogenic and has
substantially the same mechanical properties as the native soft
tissue.
48. A soft tissue xenograft according to claim 47, wherein the
extracellular components and the substantially only dead cells have
substantially no surface .alpha.-galactosyl moieties.
49. A soft tissue xenograft according to claim 47, wherein the
portion has capping molecules linked to at least a portion of a
plurality of surface carbohydrate moieties on the xenograft.
50. A soft tissue xenograft according to claim 49, wherein at least
a portion of the capping molecules are a plurality of fucosyl
molecules.
51. A soft tissue xenograft according to claim 49, wherein at least
a portion of the capping molecules are a plurality of n-acetyl
glucosamine molecules.
51. A soft tissue xenograft according to claim 47, wherein the
portion is of a medial or lateral meniscus having a superior
principal surface and an inferior principal surface, each of the
principal surfaces having an outer portion being joined by an outer
lateral surface, and each of the principal surfaces having an inner
portion being joined by an inner lateral surface.
52. A soft tissue xenograft according to claim 47, wherein the
portion is of a ligament.
53. A soft tissue xenograft according to claim 52, wherein the
portion of the ligament comprises a first block of bone attached to
a first end of the portion.
54. A soft tissue xenograft according to claim 53, wherein the
portion of the ligament comprises a second block of bone affixed to
a second end of the portion opposite the first end.
55. A soft tissue xenograft according to claim 47, wherein the
portion is of an articular cartilage.
56. A soft tissue xenograft according to claim 55, wherein the
portion of the articular cartilage comprises a layer of subchondral
bone.
Description
CLAIM OF PRIORITY
[0001] This application is a divisional application of U.S. Ser.
No. 09/248,336, filed Feb. 11, 1999.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of treatment of
defective human knee joints, and in particular, to replacement and
repair of defective or damaged human knee joint soft tissue using a
substantially immunologically compatible soft tissue from a
non-human animal.
BACKGROUND OF THE INVENTION
[0003] The term "soft tissue", as used herein, refers to
cartilaginous structures, such as meniscus and articular cartilage;
ligaments, such as anterior cruciate ligaments; tendons; and heart
valves.
[0004] Meniscus Cartilage
[0005] Specifically, the femoral condyles articulate with the
surface plateaus of the tibia, through the cartilaginous medial and
lateral menisci soft tissue, and all of these structures are held
in place by various ligaments. The medial and lateral menisci are
structures comprised of cells called fibrochondrocytes and an
extracellular matrix of collagen and elastic fibers as well as a
variety of proteoglycans. Undamaged menisci provide shock
absorption for the knee by ensuring proper force distribution,
stabilization, and lubrication for the interacting bone surfaces
within the knee joint, which are routinely exposed to repeated
compression loading during normal activity. Much of the shock
absorbing function of the medial and lateral menisci is derived
from the elastic properties inherent to cartilage. When menisci are
damaged through injury, disease, or inflammation, arthritic changes
occur in the knee joint, with consequent loss of function.
[0006] Since joint cartilage in adults does not naturally
regenerate to a significant degree once it is destroyed, damaged
adult menisci have historically been treated by a variety of
surgical interventions. Damaged menisci have been removed and
replaced with prosthetic devices. An artificial knee joint having a
rigid plastic femoral member and a metal tibial member is disclosed
in U.S. Pat. No. 4,034,418. A number of meniscus prostheses have
been devised which employ resilient materials such as silicone
rubber or natural rubber, as in U.S. Pat. No. 4,344,193 and U.S.
Pat. No. 4,502,161. Additional deformable, flexible resilient
materials for a meniscus prosthesis such as collagen, tendon, or
fibrocartilage are disclosed in U.S. Pat. No. 5,092,894 and U.S.
Pat. No. 5,171,322. A cartilage replacement apparatus constructed
of polyethylene plastic filled with small ball bearings or
gelatinous fluid is described in U.S. Pat. No. 5,358,525. However,
the known artificial prostheses have been unsatisfactory for
treatment of damaged menisci, since they are deficient in the
elastic, and therefore in the shock-absorbing, properties
characteristic of natural menisci. Moreover, the known artificial
devices have not proven able to withstand the forces inherent to
routine knee joint function.
[0007] One of the present inventors provided improved prosthetic
menisci in several of his earlier patents (U.S. Pat. No. 4,880,429;
U.S. Pat. No. 5,007,934; U.S. Pat. No. 5,116,374; and U.S. Pat. No.
5,158,574). These patents generally disclose prosthetic menisci
formulated from dry, porous matrices of processed natural fibers
such as reconstituted cross-linked collagen, which optionally
include glycosaminoglycan molecules. Generally, the source of
collagen for these prosthetic menisci has been animal Achilles
tendons or skin. The reconstitution process removes non-collagenous
materials such as glycoproteins, proteoglycans, lipids, native
glycosaminoglycans, and the like, which may confer additional
elastic properties to the original tissue.
[0008] Articular Cartilage
[0009] Articular cartilage soft tissue covers the ends of all bones
that form articulating joints in humans and animals. Articular
cartilage is made of fibrochondrocytes and an extracellular matrix
of collagen fibers as well as a variety of proteoglycans. The
cartilage acts in the joint as a mechanism for force distribution
and as a lubricant in the area of contact between the bones.
Without articular cartilage, stress concentration and friction
would occur to the degree that the joint would not permit ease of
motion. Loss of the articular cartilage usually leads to painful
arthritis and decreased joint motion.
[0010] Damaged adult articular cartilage has historically been
treated by a variety of surgical interventions including repair,
replacement, or by excision. With repair or excision, regeneration
of tissue may occur, although the tissue is usually temporary and
inadequate to withstand the normal joint forces.
[0011] Replacement of articular cartilage usually has been by
allografting (Sengupta et al. (1974) J. Bone Suro. 56B(l):167-177;
Rodrigo et al. (1978) Clin Orth. 134:342-349) by periosteal grafts
(see, e.g., Engkvist (1979) Scan J. Plast. Reconstr. Suro.
13:361-369; Rubak (1982) Acta Orthop. Scan. 53:181-186) or with
metal and/or plastic components (Rubash et al., eds. (1991) Clin.
Orth. Rel. Res. 271:2-96). Allografting dead cartilage tissue has
been tried for years with minimal success. This approach has been
only partially successful over the long term due to the host's
immunologic response to the graft, failures in the cryopreservation
process, and failures of the attachment sites. Replacement of an
entire joint surface with metal and plastic components has met
excellent success for the older, more sedentary patients, but is
generally considered insufficient for tolerating the impact of
athletic activities, and has not been shown to restore normal joint
mechanics.
[0012] In alternative prior art approaches, articular cartilage has
been replaced with prostheses composed of bone and/or artificial
materials. For example, U.S. Pat. No. 4,627,853 describes the use
of demineralized allogenic or xenogeneic bone segments as
replacements. The proper functioning of these replacements depends
on the differential demineralization of the bone segments. U.S.
Pat. No. 4,846,835 describes a grafting technique for
transplantation of fibrochondrocytes to promote healing lesions in
articular cartilage. U.S. Pat. No. 4,642,120 describes the use of
gel-like compositions containing embryonal fibrochondrocytes. U.S.
Pat. No. 5,306,311 describes a prosthetic articular cartilage which
includes a dry, porous volume matrix adapted to have in vivo an
outer contour substantially the same as that of natural articular
cartilage.
[0013] Despite these developments, the replacement of articular
cartilage soft tissue with structures consisting of permanent
artificial materials generally has been less than satisfactory, and
a structure suitable as articular cartilage and constructed from
natural resorbable materials, or analogs thereof, has not been
developed. Because the opposing articular cartilage of mammalian
joints is so fragile, it will not withstand abrasive interfaces nor
compliance variances from normal which eventually result from the
implantation of prior art artificial cartilage. Additionally, joint
forces are multiples of body weight which, in the case of the knee
and hip, are typically encountered over a million cycles per year.
Thus far, prior art permanent artificial cartilages have not been
composed of materials having natural articular cartilage
properties, nor have they been able to be positioned securely
enough to withstand such routine forces.
[0014] Ligaments
[0015] Anterior cruciate ligament soft tissue of the knee
(hereinafter the ACL) functions to resist anterior displacement of
the tibia from the femur at all flexion positions. The ACL also
resists hyperextension and contributes to rotational stability of
the fully extended knee during internal and external tibial
rotation. The ACL may play a role in proprioception. The ACL is
made up of connective tissue structures composed of cells, water,
collagen, proteoglycans, fibronectin, elastin, and other
glycoproteins. Cyril Frank, M.D. et al., Normal Ligament:
Structure, Function, and Composition. Injury and Repair of the
Musculoskeletal Soft Tissues, 2:45-101. Structurally, the ACL
attaches to a depression in the front of the intercondyloid
eminence of the tibia extending postero-superiorly to the medial
wall of the lateral femoral condyle.
[0016] The preferred treatment of damaged ACL is ligament
reconstruction, using a bone-ligament-bone autograft. Cruciate
ligament reconstruction has the advantage of immediate stability
and a potential for immediate vigorous rehabilitation. However, the
disadvantages to ACL reconstruction are significant: for example,
normal anatomy is disrupted when the patellar tendon or hamstring
tendons are used for the reconstruction; placement of
intraarticular hardware is required for ligament fixation; and
anterior knee pain frequently occurs. Moreover, recent reviews of
cruciate ligament reconstruction indicate an increased risk of
degenerative arthritis with intraarticular ACL reconstruction in
large groups of patients.
[0017] A second method of treating ACL injuries, referred to as
"primary repair", involves suturing the torn structure back into
place. Primary ACL repair has the potential advantages of a limited
arthroscopic approach, minimal disruption of normal anatomy, and an
out-patient procedure under a local anesthetic. The potential
disadvantage of primary cruciate ligament repair is the perception
that over the long term ACL repairs do not provide stability in a
sufficient number of patients, and that subsequent reconstruction
may be required at a later date. The success rate of anterior
cruciate ligament repair has generally hovered in the 60% to 70%
range.
[0018] Heart Valves
[0019] Heart valves are composed of fibrochondrocytes and an
extracellular matrix of collagen and elastic fibers, as well as a
variety of proteoglycans. Various synthetic and tissue based
materials (the latter either from the recipient organism or from a
different organism within the same species) have been used for
forming heart valve replacements. Each have their advantages and
disadvantages.
[0020] In the case of synthetic heart valves, it may be possible to
modify advantageously the properties of the heart valves by
altering the monomers and/or the reaction conditions of the
synthetic polymers. Synthetic heart valves may be associated with
thromboembolism and mechanical failure, however. See U.S. Pat. No.
4,755,593.
[0021] Tissue based heart valves may demonstrate superior blood
contacting properties relative to their synthetic counterparts.
Tissue based heart valves also may be associated with inferior in
vivo stability, however. See U.S. Pat. No. 4,755,593.
[0022] Pericardial xenograft tissue valves have been introduced as
alternatives to the synthetic and the tissue based valves described
above. See Ionescu, M. I. et al., Heart Valve Replacement With The
Ionescu-Shiley Pericardial Xenograft, J. Thorac. Cardiovas. Surg.
73; 31-42 (1977). Such valves may continue to have calcification
and durability problems, however. See Morse, D, ed. Guide To
Prosthetic Heart Valves, Springer-Verlag, New York, 225-232
(1985).
[0023] Accordingly, there is a need for mechanically durable,
flexible heart valves replacements which are capable of contacting
the blood and are stable in vivo.
[0024] Xenografts
[0025] Much of the structure and many of the properties of original
soft tissues may be retained in transplants through use of
heterograft or xenograft materials, that is, soft tissue from a
different species than the graft recipient. For example, tendons or
ligaments from cows or other animals are covered with a synthetic
mesh and transplanted into a heterologous host in U.S. Pat. No.
4,400,833. Flat tissues such as pig pericardia are also disclosed
as being suitable for heterologous transplantation in U.S. Pat. No.
4,400,833. Bovine peritoneum fabricated into a biomaterial suitable
for prosthetic heart valves, vascular grafts, burn and other wound
dressings is disclosed in U.S. Pat. No. 4,755,593. Bovine, ovine,
or porcine blood vessel xenografts are disclosed in WO 84/03036.
However, none of these disclosures describe the use of a xenograft
for soft tissue replacement.
[0026] Once implanted in an individual, a xenograft provokes
immunogenic reactions such as chronic and hyperacute rejection of
the xenograft. The term "chronic rejection", as used herein refers
to an immunological reaction in an individual against a xenograft
being implanted into the individual. Typically, chronic rejection
is mediated by the interaction of IgG natural antibodies in the
serum of the individual receiving the xenograft and carbohydrate
moieties expressed on cells, and/or cellular matrices and/or
extracellular components of the xenograft. For example,
transplantation of soft tissue cartilage xenografts from nonprimate
mammals (e.g., porcine or bovine origin) into humans is primarily
prevented by the interaction between the IgG natural anti-Gal
antibody present in the serum of humans with the carbohydrate
structure Gal.alpha.1-3Gal.beta.1-4G1cNAc-R (a-galactosyl or
.alpha.-gal epitope) expressed in the xenograft. K. R. Stone et
al., Porcine and bovine cartilage transplants in cynomolgus monkey:
I. A model for chronic xenograft rejection, 63 Transplantation
640-645 (1997); U. Galili et al., Porcine and bovine cartilage
transplants in cynomolgus monkey: II. Changes in anti-Gal response
during chronic rejection, 63 Transplantation 646-651 (1997). In
chronic rejection, the immune system typically responds within one
to two weeks of implantation of the xenograft.
[0027] In contrast with "chronic rejection", "hyper acute
rejection" as used herein, refers to the immunological reaction in
an individual against a xenograft being implanted into the
individual, where the rejection is typically mediated by the
interaction of IgM natural antibodies in the serum of the
individual receiving the xenograft and carbohydrate moieties
expressed on cells. This interaction activates the complement
system causing lysis of the vascular bed and stoppage of blood flow
in the receiving individual within minutes to two to three
hours.
[0028] The term "extracellular components", as used herein, refers
to any extracellular water, collagen and elastic fibers,
proteoglycans, fibronectin, elastin, and other glycoproteins, which
are present in soft tissue.
[0029] Xenograft materials may be chemically treated to reduce
immunogenicity prior to implantation into a recipient. For example,
glutaraldehyde is used to cross-link or "tan " xenograft tissue in
order to reduce its antigenicity, as described in detail in U.S.
Pat. No. 4,755,593. Other agents such as aliphatic and aromatic
diamine compounds may provide additional crosslinking through the
side chain carboxyl groups of aspartic and glutamic acid residues
of the collagen polypeptide. Glutaraldehyde and diamine tanning
also increases the stability of the xenograft tissue.
[0030] Xenograft tissues may also be subjected to various physical
treatments in preparation for implantation. For example, U.S. Pat.
No. 4,755,593 discloses subjecting xenograft tissue to mechanical
strain by stretching to produce a thinner and stiffer biomaterial
for grafting. Tissue for allograft transplantation is commonly
cryopreserved to optimize cell viability during storage, as
disclosed, for example, in U.S. Pat. No. 5,071,741; U.S. Pat. No.
5,131,850; U.S. Pat. No. 5,160,313; and U.S. Pat. No. 5,171,660.
U.S. Pat. No. 5,071,741 discloses that freezing tissues causes
mechanical injuries to cells therein because of extracellular or
intracellular ice crystal formation and osmotic dehydration.
SUMMARY OF THE INVENTION
[0031] The present invention provides a substantially
non-immunogenic soft tissue xenograft for implantation into a human
in need of soft tissue repair or replacement. The invention further
provides methods for processing xenogeneic soft tissue with reduced
immunogenicity but with substantially native elasticity and
load-bearing capabilities for xenografting into humans.
[0032] As used herein, the term "xenograft" is synonymous with the
term "heterograft" and refers to a graft transferred from an animal
of one species to one of another species. Stedman 's Medical
Dictionary, Williams & Wilkins, Baltimore, Md. (1995).
[0033] As used herein, the term "xenogeneic", as in, for example,
xenogeneic soft tissue, refers to soft tissue transferred from an
animal of one species to one of another species. Id.
[0034] The methods of the invention, include, alone or in
combination, treatment with radiation, one or more cycles of
freezing and thawing, treatment with a chemical cross-linking
agent, treatment with alcohol or ozonation. In addition to or in
lieu of these methods, the methods of the invention include, alone
or in combination, in any order, a cellular disruption treatment,
glycosidase digestion of carbohydrate moieties of the xenograft, or
treatment with proteoglycan-depleting factors. Optionally, the
glycosidase digestion or proteoglycan-depleting factor treatment
can be followed by further treatments, such as, for example,
treatment of carbohydrate moieties of the xenograft with capping
molecules. After one or more of the above-described processing
steps, the methods of the invention provide a xenograft having
substantially the same mechanical properties as a native soft
tissue.
[0035] As used herein, the term "cellular disruption" as in, for
example, cellular disruption treatment, refers to a treatment for
killing cells.
[0036] As used herein, the term "capping molecule(s)", refers to
molecule(s) which link with carbohydrate chains such that the
xenograft is no longer recognized as foreign by the subject's
immune system.
[0037] As used herein, the terms "to cap" or "capping", refer to
linking a capping molecule such as a carbohydrate unit to the end
of a carbohydrate chain, as in, for example, covalently linking a
carbohydrate unit to surface carbohydrate moieties on the
xenograft.
[0038] In one embodiment, the invention provides an article of
manufacture comprising a substantially non-immunogenic soft tissue
xenograft for implantation into a human.
[0039] In another embodiment, the invention provides a method of
preparing a soft tissue xenograft for implantation into a human,
which includes removing at least a portion of a soft tissue from a
non-human animal to provide a xenograft; washing the xenograft in
water and alcohol; and subjecting the xenograft to at least one
treatment selected from the group consisting of exposure to
ultraviolet radiation, immersion in alcohol, ozonation, and
freeze/thaw cycling, whereby the xenograft has substantially the
same mechanical properties as a corresponding portion of a native
soft tissue.
[0040] As used herein, the term "portion", as in, for example, a
portion of soft tissue, second surface carbohydrate moieties or
proteoglycans, refers to all or less than all of the respective
soft tissue, second surface carbohydrate moieties or proteoglycans
of the xenograft.
[0041] In another embodiment, the invention provides a method of
preparing a soft tissue xenograft for implantation into a human,
which includes removing at least a portion of a soft tissue from a
non-human animal to provide a xenograft; washing the xenograft in
water and alcohol; subjecting the xenograft to a cellular
disruption treatment; and digesting the xenograft with a
glycosidase to remove first surface carbohydrate moieties, whereby
the xenograft has substantially the same properties as a
corresponding portion of a native soft tissue.
[0042] As used herein, the term "first surface carbohydrate moiety
(moieties)" refers to a terminal .alpha.-galactosyl sugar at the
non-reducing end of a carbohydrate chain.
[0043] In still other embodiments, this method can include
additional steps such as, for example, treating second surface
carbohydrate moieties on the xenograft with capping molecules to
cap at least a portion of the second surface carbohydrate moieties,
whereby the xenograft is substantially non-immunogenic.
[0044] As used herein, the term "second surface carbohydrate moiety
(moieties)" refers to a N-acetyllactosamine residue at the
non-reducing end of a carbohydrate chain, the residue being
non-capped either naturally or as a result of prior cleavage of an
.alpha.-galactosyl epitope.
[0045] In a further embodiment, the invention provides a method of
preparing a soft tissue xenograft for implantation into a human,
which includes removing at least a portion of soft tissue from a
non-human animal to provide a xenograft; washing the xenograft in
water and alcohol; subjecting the xenograft to a cellular
disruption treatment; and digesting the xenograft with a
proteoglycan-depleting factor to remove at least a portion of the
proteoglycans from the xenograft, whereby the xenograft has
substantially the same mechanical properties as a corresponding
portion of a native soft tissue and is substantially
non-immunogenic.
[0046] In yet further embodiments, the invention provides articles
of manufacture including substantially non-immunogenic soft tissue
xenografts for implantation into humans produced by one or more of
the above-identified methods of the invention.
[0047] In another embodiment, the invention provides a soft tissue
xenograft for implantation into a human which includes a portion of
a soft tissue from a non-human animal, wherein the portion has
substantially no surface carbohydrate moieties which are
susceptible to glycosidase digestion, and whereby the portion has
substantially the same mechanical properties as a corresponding
portion of a native soft tissue.
[0048] In yet another embodiment, the invention provides a soft
tissue xenograft for implantation into a human which includes a
portion of a soft tissue from a non-human animal, wherein the
portion includes extracellular components and substantially only
dead cells, the extracellular components and dead cells having
substantially no surface .alpha.-galactosyl moieties and having
capping molecules linked to at least a portion of surface
carbohydrate moieties. The portion of the soft tissue is
substantially non-immunogenic and has substantially the same
mechanical properties as the native soft tissue.
[0049] In still yet another embodiment, the invention provides a
soft tissue xenograft for implantation into a human which includes
a portion of a soft tissue from a non-human animal, wherein the
portion includes extracellular components and substantially only
dead cells, the extracellular components having reduced
proteoglycans. The portion of the soft tissue is substantially
non-immunogenic and has substantially the same mechanical
properties as the native soft tissue.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] The various features of the invention may be more fully
understood from the following description when read together with
the accompanying drawings.
[0051] FIG. 1 shows a simplified diagrammatic representation of a
human knee joint, with medial and lateral menisci in their native
positions.
[0052] FIG. 2 is a diagrammatic representation of a cut-away view
of a human knee joint, showing the medial and lateral menisci as
they are positioned in vivo over the medial and lateral condyles of
the tibia.
[0053] FIG. 3 is a diagrammatic representation of resection of a
torn lateral meniscus of a human knee, and preparation of the knee
for receipt of a meniscal implant.
[0054] FIG. 4 is a diagrammatic representation the preferred drill
guide placement for posterior lateral meniscal horn insertion into
a human knee.
[0055] FIG. 5 is a diagrammatic representation of a cannulated
drill overdrilling guide wire at the posterior lateral meniscal
horn insertion into a human knee.
[0056] FIG. 6 is a diagrammatic representation of the appearance of
a human knee with posterior and anterior drill holes for meniscal
horn insertion.
[0057] FIG. 7 is a diagrammatic representation of the preferred
suture passer placement for pulling a meniscal implant into a human
knee joint.
[0058] FIG. 8 is a diagrammatic representation of the appearance of
a human knee containing a meniscal implant during the insertion
stage.
[0059] FIG. 9 is a diagrammatic representation of the appearance of
a human knee containing a meniscal implant with zone-specific
meniscal repair sutures in place for final tying of the meniscal
repair sutures.
[0060] FIG. 10 is shows a simplified diagrammatic representation of
a human knee joint 3, showing the normal positioning of articular
cartilage 7 on the articulating end of femur 2 and articular
cartilage 8 on the articulating end of tibia 4.
[0061] FIG. 11 is a graphical representation of the specificity of
monoclonal anti-Gal antibodies for .alpha.-galactosyl epitopes on
bovine serum albumin (BSA), bovine thyroglobulin, mouse laminin,
Ga1.beta.1-4 G1cNAc-BSA (N-acetyllactosamine-BSA),
Gal.alpha.1-4Ga1.beta.1-4G1cNAc-BSA (P1 antigen linked to BSA), and
human thyroglobulin or human laminin.
[0062] FIG. 12 is a graphical representation of .alpha.-galactosyl
epitope elimination from .alpha.-galactosidase treated meniscal
cartilage.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0063] The present invention is directed against the chronic
rejection of xenografts for implantation into humans. Accordingly,
the soft tissue xenograft produced in accordance with the method of
the invention is substantially non-immunogenic, while generally
maintaining the mechanical properties of a native soft tissue.
[0064] While the soft tissue may undergo some shrinkage during
processing, a soft tissue xenograft prepared in accordance with the
invention will have the general appearance of a native soft tissue
xenograft. For example, a medial meniscus xenograft prepared in
accordance with the invention will have the general appearance of a
native medial meniscus, and a lateral meniscus xenograft of the
invention will have the general appearance of a native lateral
meniscus. The soft tissue xenograft may also be cut into segments,
each of which may be implanted into a joint of the recipient as set
forth below.
[0065] The invention provides, in one embodiment, a method for
preparing or processing a xenogeneic soft tissue for engraftment
into humans. The soft tissue may be harvested from any non-human
animal to prepare the xenografts of the invention. Soft tissue from
transgenic non-human animals or from genetically altered non-human
animals may also be used as xenografts in accordance with the
present invention. Preferably, bovine, ovine, or porcine knee
joints, and more preferably porcine knee joints, serve as sources
of the medial and lateral menisci and articular cartilage soft
tissue used to prepare the xenografts. Preferably, bovine and
porcine joints, and more preferably porcine joints, serve as the
source of the ligament soft tissue xenografts. Preferably, porcine
peritoneum serves as the source of the soft tissue used to prepare
the heart valve xenografts. Alternatively, porcine pericardium can
be used to form the heart valve xenografts of the present
invention. More preferably, immature animal joints are the sources
of the soft tissue, since the soft tissue of younger animals may be
inherently more elastic and engraftable than that of older animals.
Most preferably, the age of the source animal is between six and
eighteen months at time of slaughter. Additionally, the patellar
tendon, the anterior or posterior cruciate ligaments, the Achilles
tendon or the hamstring tendons may be harvested from the animal
source and used as a donor ligament.
[0066] In the first step of the method of the invention, an intact
soft tissue is removed from a non-human animal. Medial or lateral
meniscus are removed from the knee joints of the non-human animal.
Articular cartilage are removed from any joint of the non-human
animal. Ligaments and tendons, such as, for example, the Achilles
tendon, are also removed from non-human animals. Preferably soft
tissue from a corresponding joint is used to make the soft tissue
xenograft of the invention. For example, articular cartilage from a
femuro-tibial (stifle) joint is used to make an articular cartilage
xenograft for implantation into a knee. Similarly, articular
cartilage from a donor animal's hip joint is used to make an
articular cartilage xenograft for a human hip joint.
[0067] The joint which serves as the source of the soft tissue
should be collected from freshly killed animals and preferably
immediately placed in a suitable sterile isotonic or other tissue
preserving solution. Harvesting of the joints should occur as soon
as possible after slaughter of the animal and preferably should be
performed in the cold, i.e., in the approximate range of about
5.degree. C. to about 20.degree. C., to minimize enzymatic
degradation of the soft tissue.
[0068] The soft tissue is harvested in the cold, under strict
sterile technique.
[0069] With respect to meniscal soft tissue, the joint is opened by
first transecting the patellar tendon. The horns of the menisci are
dissected free of adhering tissue. A small amount of bone
representing a substantially cylindrical plug of approximately five
millimeters in diameter by five millimeters in depth may be left
attached to the horns. The meniscal synovial junction is carefully
identified and freed from the meniscus tissue itself, thereby
forming the xenograft.
[0070] With respect to articular cartilage soft tissue, a fine peel
of articular cartilage with a small layer of subchondral bone is
shaved from the donor joint to form the xenograft.
[0071] With respect to ligament soft tissue, the donor joint is
opened by standard surgical technique. Preferably, the ligament is
harvested with a block of bone attached to one or both ends,
although in some forms of the invention the ligament alone is
harvested. In one form of the invention, a block of bone
representing a substantially cylindrical plug of approximately 9-10
mm in diameter by approximately 20-40 mm in length may be left
attached to the ligament. The ligament is carefully identified and
dissected free of adhering tissue, thereby forming the
xenograft.
[0072] With respect to heart valve soft tissue, porcine peritoneum
or pericardium is harvested to form the heart valve xenografts
according to procedures known to those of ordinary skill in the
art. See, for example, the peritoneum harvesting procedure
discussed in U.S. Pat. No. 4,755,593 by Lauren.
[0073] The xenograft is then washed in about ten volumes of sterile
cold water to remove residual blood proteins and water soluble
materials. The xenograft is then immersed in alcohol at room
temperature for about five minutes, to sterilize the tissue and to
remove non-collagenous materials.
[0074] A meniscus soft tissue xenograft appears as a shiny
"C"-shaped fibrous tissue, having generally a crescent-shaped
principal surface on the top side (the "superior surface") and a
generally crescent-shaped principal surface on the bottom side (the
"inferior surface"), where the outer portions of the superior and
inferior surfaces are joined by way of an outer lateral surface and
the inner portions of the superior and inferior surfaces are joined
by way of an inner lateral surface.
[0075] The articular cartilage soft tissue xenograft appears as a
hyaline tissue supported on a bone substrate, having generally a
spherical-shaped principal surface on the tip side (the "superior
surface"), with the under surface of the bone (the "inferior
surface") being rough.
[0076] After alcohol immersion, the xenograft may be directly
implanted or may be subjected to at least one of the following
treatments: radiation treatment, treatment with alcohol, ozonation,
one or more cycles of freezing and thawing, and/or treatment with a
chemical cross-linking agent. When more than one of these
treatments is applied to the xenograft, the treatments may occur in
any order.
[0077] In one embodiment of the method of the invention, the
xenograft may be treated by exposure to ultraviolet radiation for
about fifteen minutes or gamma radiation in an amount of about 0.5
to 3 MegaRad.
[0078] In another embodiment, the xenograft may be treated by again
being placed in an alcohol solution. Any alcohol solution may be
used to perform this treatment. Preferably, the xenograft is placed
in a 70% solution of isopropanol at room temperature.
[0079] In still another embodiment, the xenograft may be subjected
to ozonation.
[0080] In a further embodiment of the method of the invention, the
xenograft may be treated by freeze/thaw cycling. For example, the
xenograft may be frozen using any method of freezing, so long as
the xenograft is completely frozen, i.e., no interior warm spots
remain which contain unfrozen soft tissue. Preferably, the
xenograft is dipped into liquid nitrogen for about five minutes to
perform this step of the method. More preferably, the xenograft is
frozen slowly by placing it in a freezer. In the next step of the
freeze/thaw cycling treatment, the xenograft is thawed by immersion
in an isotonic saline bath at room temperature (about 25.degree.
C.) for about ten minutes. No external heat or radiation source is
used, in order to minimize fiber degradation.
[0081] In yet a further embodiment, the xenograft may optionally be
exposed to a chemical agent to tan or crosslink the proteins within
the extracellular components, to further diminish or reduce the
immunogenic determinants present in the xenograft. Any tanning or
crosslinking agent may be used for this treatment, and more than
one crosslinking step may be performed or more than one
crosslinking agent may be used in order to ensure complete
crosslinking and thus optimally reduce the immunogenicity of the
xenograft. For example, aldehydes such as glutaraldehyde,
formaldehyde, adipic dialdehyde, and the like, may be used to
crosslink the extracellular collagen of the xenograft in accordance
with the method of the invention. Other suitable crosslinking
agents include aliphatic and aromatic diamines, carbodiimides,
diisocyanates, and the like.
[0082] When an aldehyde such as, for example, glutaraldehyde is
used as the crosslinking agent, the xenograft may be placed in a
buffered solution containing about 0.001% to about 5.0%
glutaraldehyde and preferably, about 0.01% to about 5.0%
glutaraldehyde, and having a pH of about 7.4. More preferably about
0.01% to about 0.10% aldehyde, and most preferably about 0.01% to
about 0.05% aldehyde is used. Any suitable buffer may be used, such
as phosphate buffered saline or trishydroxymethylaminomethane, and
the like, so long as it is possible to maintain control over the pH
of the solution for the duration of the crosslinking reaction,
which may be from one to fourteen days, and preferably from one to
five days, and most preferably from three to five days.
[0083] Alternatively, the xenograft can be exposed to a
crosslinking agent in a vapor form, including, but not limited to,
a vaporized aldehyde crosslinking agent, such as, for example,
vaporized formaldehyde. The vaporized crosslinking agent can have a
concentration and a pH and the xenograft can be exposed to the
vaporized crosslinking agent for a period of time suitable to
permit the crosslinking reaction to occur. For example, the
xenograft can be exposed to vaporized crosslinking agent having a
concentration of about 0.001% to about 5.0% and preferably, about
0.01% to about 5.0%, and a pH of about 7.4. More preferably, the
xenograft is exposed to the aldehyde in an amount ranging from
about 0.01% to about 0.10%, and most preferably to an aldehyde
ranging in an amount from about 0.01% to about 0.05%. The xenograft
is exposed to the aldehyde for a period of time which can be from
one to fourteen days, and preferably from one to five days, and
most preferably from three to five days. Exposure to vaporized
crosslinking agent can result in reduced residual chemicals in the
xenograft from the crosslinking agent exposure.
[0084] The crosslinking reaction should continue until the
immunogenic determinants are substantially eliminated from the
xenogeneic soft tissue, but the reaction should be terminated prior
to significant alterations of the mechanical properties of the
xenograft. When diamines are also used as crosslinking agents, the
glutaraldehyde crosslinking should occur after the diamine
crosslinking, so that any unreacted diamines are capped. After the
crosslinking reactions have proceeded to completion as described
above, the xenograft should be rinsed to remove residual chemicals,
and 0.01-0.10 M glycine, and preferably, 0.01-0.05 M glycine may be
added to cap any unreacted aldehyde groups which remain.
[0085] In addition to or in lieu of the above treatments, the
xenograft can be subjected to a cellular disruption treatment to
kill the xenograft's cells. Optionally, the cellular disruption
treatment precedes or follows digestion of the xenograft with
glycosidases to remove first surface carbohydrate moieties from the
xenograft. In addition or in lieu of the glycosidase treatment,
either preceding or following the glycosidase treatment, the
xenograft is treated with proteoglycan-depleting factors. Further,
the glycosidase and/or proteoglycan-depleting factor digestion in
turn is optionally followed by linkage with capping molecules such
as fucosyl or n-acetyl glucosamine molecules to cap surface
N-acetyllactosamine ends of carbohydrate chains of the
xenograft.
[0086] In embodiments of this method of the invention, the
xenograft is subjected to a cellular disruption treatment to kill
the cells of the soft tissue. Typically after surface carbohydrate
moieties have been removed from living cells and the extracellular
components, the living cells reexpress the surface carbohydrate
moieties. Reexpression of antigenic moieties of a xenograft can
provoke continued immunogenic rejection of the xenograft. In
contrast, dead cells are unable to reexpress surface carbohydrate
moieties. Removal of antigenic surface carbohydrate moieties from
dead cells and the extracellular components of a xenograft
substantially permanently eliminates antigenic surface carbohydrate
moieties as a source of immunogenic rejection of the xenograft.
[0087] Accordingly, in the above-identified embodiments, the
xenograft of the present invention is subjected to freeze/thaw
cycling as discussed above to disrupt, i.e., to kill the cells of
the soft tissue. Alternatively, the xenograft of the present
invention is treated with gamma radiation having an amount of 0.2
MegaRad up to about 3 MegaRad. Such radiation kills the soft tissue
cells and sterilizes the xenograft. Once killed, the soft tissue
cells are no longer able to reexpress antigenic surface
carbohydrate moieties such .alpha.-gal epitopes which are factors
in the immunogenic rejection of the transplanted xenografts.
[0088] Either before or after the soft tissue cells are killed, in
embodiments of the invention, the xenograft is subjected to in
vitro digestion of the xenograft with glycosidases, and
specifically galactosidases, such as .alpha.-galactosidase, to
enzymatically eliminate antigenic surface carbohydrate moieties. In
particular, .alpha.-gal epitopes are eliminated by enzymatic
treatment with .alpha.-galactosidases, as shown in the following
reaction: 1
[0089] The N-acetyllactosamine residues are epitopes that are
normally expressed on human and mammalian cells and thus are not
immunogenic. The in vitro digestion of the xenograft with
glycosidases is accomplished by various methods. For example, the
xenograft can be soaked or incubated in a buffer solution
containing glycosidase. In addition, the xenograft can be pierced
to increase permeability, as further described below.
Alternatively, a buffer solution containing the glycosidase can be
forced under pressure into the xenograft via a pulsatile lavage
process.
[0090] Elimination of the .alpha.-gal epitopes from the xenograft
diminishes the immune response against the xenograft. The
.alpha.-gal epitope is expressed in nonprimate mammals and in New
World monkeys (monkeys of South America) as
1.times.10.sup.6-35.times.10.sup.6 epitopes per cell, as well as on
macromolecules such as proteoglycans of the extracellular
components. U. Galili et al., Man, apes, and Old World monkeys
differ from other mammals in the expression of .alpha.-galactosyl
epitopes on nucleated cells, 263 J. Biol. Chem. 17755 (1988). This
epitope is absent in Old World primates (monkeys of Asia and Africa
and apes) and humans, however. Id. Anti-Gal is produced in humans
and primates as a result of an immune response to .alpha.-gal
epitope carbohydrate structures on gastrointestinal bacteria. U.
Galili et al., Interaction between human natural
anti-.alpha.-galactosyl immunoglobulin G and bacteria of the human
flora, 56 Infect. Immun. 1730 (1988); R. M. Hamadeh et al., Human
natural anti-Gal IgG regulates alternative complement pathway
activation on bacterial surfaces, 89 J. Clin. Invest. 1223 (1992).
Since nonprimate mammals produce .alpha.-gal epitopes,
xenotransplantation of xenografts from these mammals into primates
results in rejection because of primate anti-Gal binding to these
epitopes on the xenograft. The binding results in the destruction
of the xenograft by complement fixation and by antibody dependent
cell cytotoxicity. U. Galili et al., Interaction of the natural
anti-Gal antibody with .alpha.-galactosyl epitopes: A major
obstacle for xenotransplantation in humans, 14 Immunology Today 480
(1993); M. Sandrin et al., Anti-pig IgM antibodies in human serum
reactpredominantly with Gala1-3Gal epitopes, 90 Proc. Natl. Acad.
Sci. USA 11391 (1993); H. Good et al., Identification of
carbohydrate structures which bind human anti-porcine antibodies:
implications for discordant grafting in man. 24 Transplant. Proc.
559 (1992); B. H. Collins et al., Cardiac xenografts between
primate species provide evidence for the importance of the
.alpha.-galactosyl determinant in hyperacute rejection, 154 J.
Immunol. 5500 (1995). Furthermore, xenotransplantation results in
major activation of the immune system to produce increased amounts
of high affinity anti-Gal. Accordingly, the substantial elimination
of .alpha.-gal epitopes from cells and from extracellular
components of the xenograft, and the prevention of reexpression of
cellular .alpha.-gal epitopes can diminish the immune response
against the xenograft associated with anti-Gal antibody binding
with .alpha.-gal epitopes.
[0091] Further, the cartilage soft tissue xenografts of the present
invention are particularly well suited to in vitro enzymatic
elimination of the .alpha.-gal epitopes. In contrast to organs and
other tissues, the cartilage extracellular components undergo
extremely slow turnover. Moreover, once the cartilage cells, i.e.,
the fibrochondrocytes are killed, these cells are prevented from
reexpressing the .alpha.-gal epitopes, as discussed above.
[0092] In addition, the soft tissue xenografts may be treated with
polyethylene glycol (PEG) prior to or concurrently with treatment
with glycosidase. PEG acts as a carrier for the glycosidase by
covalently bonding to the enzyme and to the collagen extracellular
components. Further, PEG-treated xenografts have reduced
immunogenicity.
[0093] Either before or after the soft tissue cells are killed, in
embodiments of the invention, the xenograft is washed or digested
with one or more different types of proteoglycan-depleting factors.
The proteoglycan-depleting factor treatment can precede or follow
glycosidase treatment. Proteoglycans such as glycosaminoglycans
(GAGs) are interspersed either uniformly as individual molecules or
within varying amounts within the extracellular components of the
present invention's xenograft. The GAGs include mucopolysaccharide
molecules such as chondroitin 4-sulfate, chondroitin 6-sulfate,
keratan sulfate, dermatan sulfate, heparin sulfate, hyaluronic
acid, and mixtures thereof. The proteoglycans including such GAGs
contain attached carbohydrates such as .alpha.-gal epitopes. Such
epitopes stimulate an immune response once the xenograft is
transplanted, as discussed above. Washing or digesting the
xenograft with the proteoglycan-depleting factor removes at least a
portion of the proteoglycans and attached .alpha.-gal epitopes from
the extracellular components of the xenograft, and thereby
diminishes the immune response against the xenograft upon its
transplantation. After the proteoglycan-depleting factor treatment
and subsequent transplantation, natural tissue can repopulate the
remaining collagen shell.
[0094] Non-limiting examples of the proteoglycan-depleting factors
used in the present invention include proteoglycan-depleting
factors such as chondroitinase ABC, hyaluronidase, chondroitin AC
II lyase, keratanase, and trypsin. Other proteoglycan-depleting
factors used in the present invention include fragments of
fibronectin. Homanberg et al. suggest that fibronectin fragments,
such as the amino-terminal 29-kDa fragment, bind to the superficial
surface of articular cartilage soft tissue and penetrate the
cartilage to surround the cartilage cells. G. A. Homandberg et al.,
Fibronectin-fragment-induced cartilage chondrolysis is associated
with release of catabolic cytokines, Biochem. J. (1997) 321,
751-757; G. A. Homandberg et al., Hyaluronic acid
suppressesfibronectin fragment mediated cartilage chondrolysis: I.
In vitro, Osteoarthritis and Cartilage (1997) 5, 309-319; G. A.
Homandberg et al., High concentrations of fibronectin fragments
cause short-term catabolic effects in cartilage tissue while lower
concentrations cause continuous anabolic effects, Archives Of
Biochemistry And Biophysics, Vol. 311, No. 2, June, pp. 213-218
(1994); G. A. Homandberg et al., Agents that block fibronectin
fragment-mediated cartilage damage also promote repair,
Inflammation Research 46 (1997) 467-471. At selected
concentrations, Homanberg et al. further suggest that the addition
of such fibronectin fragments to cartilage in vitro or in vivo
results in the temporary suppression of proteoglycan synthesis and
the enhancement of extracellular metalloproteinases which in turn
cause a rapid proteoglycan loss from cartilage tissue. Id.
[0095] Other proteoglycan-depleting factors known to those of
ordinary skill in the art are also possible for use with the
present invention, however. The present invention's xenograft is
treated with proteoglycan-depleting factor in an amount effective
for removing at least a portion of the proteoglycans from the
extracellular components of the xenograft. Preferably, the
xenograft is treated with proteoglycan-depleting factor such as
hyaluronidase in an amount ranging from about 1.0 TRU/ml to about
100.0 TRU/ml or proteoglycan-depleting factor such as
chondroitinase ABC in an amount ranging from about 0.01 u/ml to
about 2.0 u/ml or most preferably, in an amount ranging from about
1.0 ul/ml to about 2.0 u/ml. The xenograft can also be treated with
proteoglycan-depleting factor such as fibronectin fragment, (e.g.,
amino terminal 29-kDa fibronectin fragment) in an amount ranging
from about 0.01 .mu.M to about 1.0 .mu.M, and preferably in an
amount ranging from about 0.1 .mu.M to about 1.0 .mu.M.
[0096] Following treatment with glycosidase or treatment with
proteoglycan-depleting factors, the remaining carbohydrate chains
(e.g., glycosaminoglycans) of the xenograft are optionally treated
with capping molecules to cap at least a portion of the remaining
carbohydrate chains. Examples of capping molecules used in the
present invention include fucosyl and N-acetyl glucosamine.
[0097] Prior to treatment, the outer surface of the xenograft
(e.g., the outer lateral surface of meniscus soft tissue
xenografts) optionally may be pierced to increase permeability to
agents used to render the xenograft substantially non-immunogenic.
A sterile surgical needle such as an 18 gauge needle may be used to
perform this piercing step, or, alternatively a comb-like apparatus
containing a plurality of needles may be used. The piercing may be
performed with various patterns, and with various pierce-to-pierce
spacings, in order to establish a desired access to the interior of
the xenograft. Piercing may also be performed with a laser. In one
form of the invention, one or more straight lines of punctures
about three millimeters apart are established circumferentially in
the outer lateral surface of the xenograft.
[0098] Prior to implantation, the soft tissue xenograft of the
invention may be treated with limited digestion by proteolytic
enzymes such as ficin or trypsin to increase tissue flexibility, or
coated with anticalcification agents, antithrombotic coatings,
antibiotics, growth factors, or other drugs which may enhance the
incorporation of the xenograft into the recipient joint. The soft
tissue xenograft of the invention may be further sterilized using
known methods, for example, with additional glutaraldehyde or
formaldehyde treatment, ethylene oxide sterilization, propylene
oxide sterilization, or the like. The xenograft may be stored
frozen until required for use.
[0099] The soft tissue xenograft of the invention, or a segment
thereof, may be implanted into damaged human joints by those of
skill in the art using known arthroscopic surgical techniques.
Specific instruments for performing arthroscopic techniques are
known to those of skill in the art, which ensure accurate and
reproducible placement of soft tissue implants.
[0100] Meniscus Cartilage Soft Tissue Xenograft Implantation
[0101] For meniscal cartilage replacement to succeed, the following
goals are preferably accomplished:
[0102] 1. The torn fragmented pieces of native meniscal cartilage
must be removed.
[0103] 2. The attachment sites for the meniscal horns must be
anatomically placed.
[0104] 3. The periphery of the meniscal implant must be attached
securely enough to permit axial and rotational loads.
[0105] 4. The surrounding capsule and ligaments of the knee joint
must be neither excessively violated nor constrained by the
fixation technique. The method of meniscal implantation described
in detail below is derived from K. R. Stone, et al., Arthroscopy:
The Journal of Arthroscopic and Related Surgery 9, 234-237 (1993);
other methods of meniscal implantation may also be employed to use
the xenogeneic meniscal xenografts of the present invention.
[0106] Initially, complete diagnostic arthroscopy of the knee joint
is accomplished using known methods. If ACL surgery is to be
performed simultaneously, the femural and tibial tunnels for the
cruciate reconstruction should be drilled first. The torn portion
of the meniscal cartilage is evaluated. If meniscal repair cannot
be accomplished due to severity of the tear or poor quality of the
tissue, then preparation of the meniscal rim is undertaken by
removing the torn portions of the cartilaginous tissue (FIG. 3).
When the entire human meniscus is to be replaced by a xenogeneic
meniscus xenograft of the invention, nearly all of the human
meniscus is removed. Additionally, for replacement of the entire
human meniscus with a xenogeneic meniscus xenograft of the
invention, resection of the human meniscal horns and preparation of
bony tunnels to accept bone plugs may be required. When only a
portion of the human meniscus is to be replaced with a segment of
the xenogeneic meniscus xenograft of the invention, only the
damaged portions are removed, preserving the peripheral rim and
horns for attachment of the xenogeneic meniscus xenograft segment.
If absolutely no human meniscal rim is present, then partial
replacement of the meniscus should not be performed. If the joint
is excessively tight, a joint distractor may be applied or the
medial collateral ligament may be partially released.
[0107] For medial or lateral meniscal replacement, the arthroscope
is placed in the mid-lateral or anterior lateral portal and the
tibial guide is placed through the anterior medial portal. The tip
of the guide is brought first to the posterior horn of the
meniscus. It should be noted that the posteromedial horn inserts on
the posterior slope of the tibial eminence. A drill pin is then
brought from the anterior medial side of the tibial tuberosity to
the posterior horn insertion (FIG. 4). The pin placement can be
confirmed by passing the arthroscope through the intercondylar
notch and viewing the exit site of the pin. Extreme care must be
undertaken to avoid penetration through the posterior capsule of
the knee, endangering the neurovascular bundle. When the pin
position is confirmed, the pin is then overdrilled with a 4.5-mm
cannulated drill bit with the option of a drill stop to prevent
posterior capsular penetration (FIG. 5). The bit is left in place
and used as a tunnel for passage of a suture passer with a suture
such as a #2 Ethibond.TM. suture available from Johnson &
Johnson. The suture is passed up the bore of the drill bit, the
drill bit removed, and the suture left in place.
[0108] The anterior medial meniscus insertion point in humans
varies considerably, most often being found anterior to the medial
tibial eminence. The anterior horn of the lateral meniscus inserts
just posterior to the anterior cruciate ligament. An anterior drill
hole is made by first identifying the insertion point of the
anterior horn of the lateral meniscus, by placing the tip of the
drill guide so that a relatively vertical hole will be made (FIG.
6). The drill pin is placed, then the cannulated drill bit is used
to overdrill the drill pin placement to form the anterior drill
hole. A suture passer is placed in the anterior drill hole.
Alternatively, the anterior horn of the medial meniscus is affixed
with a suture anchor directly to bone as opposed to a drill
hole.
[0109] Before the suture is grasped from the anterior and posterior
drill holes, the anterior portal is widened to approximately 2 cm.
The suture grasper is then passed through the widened portal, and
both the anterior and the posterior sutures brought out
simultaneously. This technique prevents the sutures from becoming
entangled in two different planes of the fat pad and capsular
tissue.
[0110] The implant is now brought onto the field. Two horizontal
mattress sutures, for example, #2-0 Ethibond.TM. sutures or the
like, are placed through each horn of the xenogeneic meniscus
xenograft with the free ends exiting the inferior surface (FIG. 7).
The two posterior sutures are then drawn through the knee and out
the posterior tibial tunnel (FIG. 8). If viewing from a mid-lateral
portal, the anterolateral portal can be used for probe insertion to
push the implant medially into place through a 1-inch incision. No
insertion cannula is required. The anterior sutures are then
similarly passed. The horn sutures are then tied over the anterior
tibial bony bridge.
[0111] Next, zone specific meniscal repair cannulae are brought
into place. For medial insertions, a posterior medial vertical
incision is made one third of the distance from the back of the
knee for protection of the saphenous nerve and for retrieval of the
inside-out meniscal repair needles. A second vertical incision is
usually required further anteriorly, next to the anterior medial
arthroscopy portal, to capture the anterior exiting needles.
Through these two incisions, the suture needles can be captured and
the knots placed directly over the capsule (FIG. 9).
[0112] When using the meniscal repair needles, the posterior
cannulae should be used first, with the sutures placed vertically
and evenly spaced. The repair should proceed from posterior to
anterior so that a buckle is not produced within the xenograft.
Each knot is tied as it is placed to avoid the chance of suture
tangling. The knots are spaced approximately 4 mm apart. The knee
is cycled through several complete ranges of motion of ensure that
the xenograft moves smoothly without impingement.
[0113] When performing a lateral meniscal replacement, the medial
portal is suitable for xenograft insertion. This may require
excision of the ligamentous mucosa and removal of a portion of the
fat pad. The drill guide for the posterior horn of the lateral
meniscus is inserted through the anteromedial portal. The posterior
slope of the lateral tibial spine must be identified for accurate
meniscal horn insertion. The anterior horn inserts on the anterior
slope of the lateral tibial spine in approximation to the lateral
aspect of the anterior cruciate ligament. The advantage of drilling
these holes from the medial side is that the tunnel divergence will
be greater, providing a larger bony bridge between the horn
insertions. The remainder of the insertion technique remains the
same, except that great care should be taken to protect the
neurovascular bundle when suturing the posterior horn. Accessing
posterolateral exposure is necessary to safeguard the common
peroneal nerve and to expose the lateral capsule. If there is any
doubt about the suture placement, open posterior horn suturing
should be performed in the standard fashion. Alternatively,
meniscus and/or stabilization devices such as arrows or staples can
be used instead of sutures. Stabilization arrows manufactured by
Bionix, Inc., Malvern, Pa., are non-limiting examples of such
stabilization arrows. Other stabilization devices known to those of
ordinary skill in the art can also be used.
[0114] Routine skin closure and dressings are applied. Thirty
milliliters of 0.5% Marcaine (Astra) with epinephrine may be
instilled if desired. A postoperative hinged knee brace may be
applied with the range of motion limited to 30.degree. of extension
and 90.degree. of flexion.
[0115] Articular Cartilage Soft Tissue Xenograft Implantation
[0116] The underlying bone bed of the recipient joint is prepared
with a bone burr to produce a cancellous bleeding bed. Grafting can
involve either the entire articular surface or a portion of the
articular surface. The substantially non-immunogenic articular
cartilage xenograft of the invention is applied to the recipient
joint as a cover, which is held in place by one or more suture
anchors, absorbable pins, screws, staples, and the like. A fibrin
clot may also be used to hold the substantially non-immunogenic
articular cartilage xenograft in place.
[0117] Ligament Soft Tissue Xenograft Implantation
[0118] The irreparably damaged ligament is removed with a surgical
shaver. The anatomic insertion sites for the ligament are
identified and drilled to accommodate a bone plug. The size of the
bone plug can be about 9-10 mm in width by about 9-10 mm in depth
by about 20-40 mm in length. The xenogeneic ligament is brought
through the drill holes and affixed with interference screws.
Routine closure is performed.
[0119] This invention is further illustrated by the following
Examples which should not be construed as limiting. The contents of
all references and published patents and patent applications cited
throughout the application are hereby incorporated by
reference.
[0120] Heart Valve Soft Tissue Xenograft Implantation
[0121] The heart valve xenograft of the invention, or a segment
thereof, may be formed from harvested porcine peritoneum or
pericardium and may be implanted to replace and/or to repair
damaged heart valves by those of skill in the art using known
techniques. Such techniques are performed with specific instruments
which insure accurate and reproducible placement of the implants
and which are known to those of ordinary skill in the art.
EXAMPLE 1
[0122] Assay For .alpha.-gal Epitopes' Elimination from Soft Tissue
by .alpha.-galactosidase
[0123] In this example, an ELISA assay for assessing the
elimination of .alpha.-gal epitopes from soft tissue is
conducted.
[0124] A monoclonal anti-Gal antibody (designated M86) which is
highly specific for .alpha.-gal epitopes on glycoproteins is
produced by fusion of splenocytes from anti-Gal producing knock-out
mice for .alpha. 1,3 galactosyltransferase, and a mouse hybridoma
fusion partner.
[0125] The specificity of M86 for .alpha.-gal epitopes on
glycoproteins is illustrated in FIG. 11. M86 binds to synthetic
.alpha.-gal epitopes linked to .cndot.-bovine serum albumin (BSA),
to -bovine thyroglobulin which has 11 .alpha.-gal epitopes, R. G.
Spiro et al., Occurrence of .alpha.-D-galactosyl residues in the
thyroglobulin from several species. Localization in the saccharide
chains of complex carbohydrates, 259 J. Biol. Chem. 9858 (1984); or
to .box-solid.-mouse laminin which has 50 .alpha.-gal epitopes, R.
G. Arumugham et al., Structure of the asparagine-linked sugar
chains of laminin. 883 Biochem. Biophys. Acta 112 (1986); but not
to .quadrature.-human thyroglobulin or human laminin,
.largecircle.-Gal.beta.1-4 G1cNAc-BSA (N-acetyllactosamine-BSA) and
Gala1-4Gal.beta.1-4G1cNAc-BSA (P1 antigen linked to BSA), all of
which completely lack .alpha.-gal epitopes. Binding is measured at
different dilutions of the M86 tissue culture medium.
[0126] Once the M86 antibody is isolated, the monoclonal antibody
is diluted from about 1:20 to about 1:160, and preferably diluted
from about 1:50 to about 1:130. The antibody is incubated for a
predetermined period of time ranging between about 5 hr to about 24
hr, at a predetermined temperature ranging from about 3.degree. C.
to about 8.degree. C. The antibody is maintained in constant
rotation with fragments of soft tissue about 5 .mu.m to about 100
.mu.m in size, and more preferably with soft tissue fragments
ranging from about 10 .mu.m to about 50 .mu.m in size, at various
soft tissue concentrations ranging from about 200 mg/ml to about
1.5 mg/ml. Subsequently, the soft tissue fragments are removed by
centrifugation at centrifugation rate ranging from about
20,000.times.g to about 50,000.times.g. The proportion of M86 bound
to the soft tissue is assessed by measuring the remaining M86
activity in the supernatant, in ELISA with .alpha.-gal-BSA as
described in the prior art in, for example, U. Galili et al.,
Porcine and bovine cartilage transplants in cynomolgus monkey: II.
Changes in anti-Gal response during chronic rejection, 63
Transplantation 645-651 (1997). The extent of binding of M86 to the
soft tissue is defined as a percentage inhibition of subsequent
binding to .alpha.-gal-BSA. There is a direct relationship between
the amount of .alpha.-gal epitopes in the soft tissue and the
proportion of M86 complexed with the soft tissue fragments, thus
removed from the supernatant (ie., percentage inhibition).
[0127] An example of the assay is shown in FIG. 12. Fragments of
homogenized meniscus cartilage (.smallcircle.) or meniscus
cartilage (.cndot.) treated with .alpha.-galactosidase are
incubated with the M86 monoclonal antibody (diluted 1: 100) for 20
hr at 4.degree. C. Subsequently, the meniscus cartilage fragments
are removed by centrifugation at 35,000.times.g and the remaining
M86 in the supernatant is assessed in ELISA with .alpha.-gal-BSA as
solid phase antigen. FIG. 12 shows that treatment of the meniscus
cartilage with 200 U/ml .alpha.-galactosidase for 4 hour at
30.degree. C. followed by five washes with phosphate-buffered
solution (PBS) completely eliminates the .alpha.-gal epitopes.
Thus, since there is no inhibition of subsequent M86 binding to
.alpha.-gal-BSA even at a high meniscus cartilage fragment
concentration of 200 mg/ml.
EXAMPLE 2
[0128] Assessment of Primate Response to Implanted Porcine Meniscus
Cartilage, Articular Cartilage, Ligament and Heart Valve Soft
Tissue Xenografts Treated With .alpha.-galactosidase
[0129] In this example, porcine meniscus cartilage, articular
cartilage, ligament, and peritoneum heart valve soft tissue
implants are treated with .alpha.-galactosidase to eliminate
.alpha.-galactosyl epitopes, the implants are transplanted into
cynomolgus monkeys, and the primate response to the soft tissue
implants is assessed.
[0130] Porcine stifle joints are sterilely prepared and meniscus
cartilage and articular cartilage and other surrounding attached
soft tissues surgically removed. Porcine peritoneum is also
harvested for forming heart valve xenografts and adherent fatty
and/or muscular tissues surgically removed. The meniscus cartilage,
articular cartilage and heart valve soft tissue specimens are
washed for at least five minutes with an alcohol, such as ethanol
or isopropanol, to remove synovial fluid and lipid soluble
contaminants. The meniscus cartilage, articular cartilage and heart
valve soft tissue specimens are frozen at a temperature ranging
from about -35.degree. C. to about -90.degree. C., and preferably
at a temperature up to about -70.degree. C., to disrupt, that, is
to kill, the specimens' fibrochondrocytes.
[0131] Porcine stifle joints are also sterilely prepared and
ligaments, each with a block of bone attached to one or both ends,
are removed in the cold, under strict sterile technique. Each of
the blocks of bone represents a substantially cylindrical plug of
approximately 9 mm in diameter by about 40 mm in length. Each
ligament soft tissue specimen is carefully identified and dissected
free of adhering tissue, thereby forming the xenograft. The
ligament soft tissue xenograft specimens are then washed for at
least five minutes with an alcohol, such as ethanol or isopropanol,
to remove synovial fluid and lipid soluble contaminants.
Subsequently, the specimens are frozen at a temperature of about
-70.degree. C. to disrupt, that is, to kill, the ligament
specimens' cells.
[0132] Each meniscus cartilage, articular cartilage, heart valve
and ligament soft tissue xenograft specimen is cut into two
portions. Each first portion is immersed in a buffer solution
containing a-galactosidase at a predetermined concentration. The
specimens are allowed to incubate in the buffer solutions for a
predetermined time period at a predetermined temperature. Each
second portion is incubated under similar conditions as the
corresponding first portion in a buffer solution in the absence of
.alpha.-galactosidase and serves as the control.
[0133] At the end of the incubation, the soft tissue xenograft
specimens are washed under conditions which allow the enzyme to
diffuse out. Assays are performed to confirm the complete removal
of the .alpha.-gal epitopes.
[0134] Each meniscus cartilage soft tissue xenograft specimen is
implanted in the supra patellar pouch of six cynomolgus monkeys.
With the animals under general inhalation anesthesia, an incision
of about 1 cm is made directly into the supra patellar pouch at the
superior medial border of the patella extending proximally. A piece
of the porcine cartilage soft tissue of about 0.5 cm to about 1 cm
in length is placed into the pouch with a single 3-0 nylon stitch
as a marking tag.
[0135] The articular cartilage xenograft specimens are implanted in
the supra patellar pouch of six cynomolgus monkeys substantially
following the above-identified implantation procedure.
[0136] The porcine peritoneum is formed into heart valves and the
heart valve xenograft specimens are implanted in the six cynomolgus
monkeys according to heart valve implantation procedures known to
those of ordinary skill in the art.
[0137] The ligament soft tissue xenograft specimens are implanted
in six cynomolgus monkeys using the following implantation
procedure. With the animals under general inhalation anesthesia,
the anatomic insertion sites for the xenogeneic ligament are
identified and drilled to accommodate a substantially 9 mm in
diameter by 40 mm in length bone plug. The xenogeneic ligament is
brought through the drill holes and affixed with interference
screws.
[0138] The implantation procedures are performed under sterile
surgical technique, and the wounds are closed with 3-0 vicryl or a
suitable equivalent known to those of ordinary skill in the art.
The animals are permitted unrestricted cage activity and monitored
for any sign of discomfort, swelling, infection, or rejection.
Blood samples (e.g., 2 ml) are drawn periodically (e.g., every two
weeks) for monitoring of antibodies.
[0139] The occurrence of an immune response against the xenograft
is assessed by determining anti-Gal and non-anti-Gal anti-soft
tissue antibodies (i.e., antibodies binding to soft tissue antigens
other than the .alpha.-gal epitopes) in serum samples from the
transplanted monkeys. At least two ml blood samples are drawn from
the transplanted monkeys on the day of implant surgery and at
periodic (e.g., two week) intervals post-transplantation. The blood
samples are centrifuged and the serum samples are frozen and
evaluated for the anti-Gal and other non-anti-Gal anti-soft tissue
antibody activity.
[0140] Anti-Gal activity is determined in the serum samples in
ELISA with .alpha.-gal-BSA as solid phase antigen, according to
methods known in the prior art, such as, for example, the methods
described in Galili et al., Porcine and bovine cartilage
transplants in cynomolgus monkey: II Changes in anti-Gal response
during chronic rejection, 63 Transplantation 645-651 (1997).
[0141] Assays are conducted to determine whether
.alpha.-galactosidase treated xenografts induce the formation of
anti-soft tissue antibodies. For measuring anti-soft tissue
antibody activity, ELISA assays are performed according to methods
known in the prior art, such as, for example, the methods described
in K. R. Stone et al., Porcine and bovine cartilage transplants in
cynomolgus monkey: I. A model for chronic xenograft rejection, 63
Transplantation 640-645 (1997).
[0142] The soft tissue xenograft specimens are optionally explanted
at one to two months post-transplantation, sectioned and stained
for histological evaluation of inflammatory infiltrates.
Post-transplantation changes in anti-Gal and other anti-cartilage
soft tissue antibody activities are correlated with the
inflammatory histologic characteristics (i.e., granulocytes or
mononuclear cell infiltrates) within the explanted soft tissue, one
to two months post-transplantation, using methods known in the art,
as, for example, the methods described in K. R. Stone et al.,
Porcine and bovine cartilage transplants in cynomolgus monkey: I. A
model for chronic xenograft rejection, 63 Transplantation 640-645
(1997).
[0143] Where the soft tissue is explanted, the soft tissue
xenografts are aseptically harvested, using anesthetic procedure,
surgical exposure of joints, removal of the implants and closure of
the soft tissue (where the animals are allowed to recover). At the
time of the xenograft removal, joint fluid, if present in amounts
sufficient to aspirate, is collected from the stifle joints for
possible immunologic testing if the gross and histopathologic
evaluation of the transplants indicate good performance of the
transplanted soft tissue.
[0144] The animals which have had meniscus cartilage or articular
cartilage xenograft implantations are allowed to recover and are
monitored closely until the incisions have healed and the gait is
normal. The xenograft samples are collected, processed, and
examined microscopically.
[0145] Portions of the meniscus cartilage, articular cartilage,
heart valve and ligament implants and surrounding tissues are
frozen in embedding mediums for frozen tissue specimens in
embedding molds for immunohistochemistry evaluation according to
the methods known in the prior art. "TISSUE-TEK.RTM." O.C.T.
compound which includes about 10% w/w polyvinyl alcohol, about 4%
w/w polyethylene glycol, and about 86% w/w nonreactive ingredients,
and is manufactured by Sakura FinTek, Torrence, Calif., is a
non-limiting example of a possible embedding medium for use with
the present invention. Other embedding mediums known to those of
ordinary skill in the art may also be used. The remaining implant
and surrounding tissue is collected in 10% neutral buffered
formalin for histopathologic examination.
EXAMPLE 3
[0146] Assessment of Primate Response to Implanted Meniscus
Cartilage, Articular Cartilage, Ligament and Heart Valve Soft
Tissue Xenografts Treated with .alpha.-galactosidase, Fucosyl and
Fucosyltransferase
[0147] In this example, porcine meniscus cartilage, articular
cartilage, ligament and peritoneum heart valve soft tissue implants
are treated with .alpha.-galactosidase to eliminate .alpha.-gal
epitopes, as described in Example 2. The soft tissue implants are
further treated with fucosyl and fucosyl transferase to cap
remaining carbohydrate chains with fucosyl. Fucosyltransferase
facilitates the transfer of fucosyl to the xenograft. The fucosyl
links to and thus caps the remaining carbohydrate chains. Capping
with fucosyl interferes with the ability of the subjects immune
system to recognize the xenograft as foreign. The soft tissue
implants are transplanted into cynomolgus monkeys, and the primate
response to the soft tissue implants is assessed.
[0148] Meniscus cartilage and articular cartilage implants from
porcine stifle joints, heart valve implants from porcine peritoneum
and ligament implants from porcine stifle joints are prepared as
the implants are prepared in Example 2 including the
.alpha.-galactosidase treatment. Prior to implantation into the
monkeys, however, the implants are further treated with a
predetermined amount of fucosyl and fucosyltransferase, at
specified concentrations for a predetermined time and at a
predetermined temperature, to cap remaining carbohydrate chains
with fucosyl. For example, the implants are immersed in buffer
solutions at predetermined concentrations of fucosyl and fucosyl
transferase. The implants are incubated for a predetermined time
period at a predetermined temperature.
[0149] Other molecules, such as N-acetyl glucosamine in combination
with the corresponding glycosyltransferase, can also be used for
capping the carbohydrate chains of the implants.
[0150] Subsequently, the implants are washed to remove the enzyme
and implanted into the monkeys, and the occurrence of an immune
response against the xenograft is assessed as described above in
Example 2.
EXAMPLE 4
[0151] Assessment of Primate Response to Implanted Meniscus
Cartilage, Articular Cartilage, Ligament and Heart Valve Soft
Tissue Xenografts Subjected to Freeze Thaw Cycling and Treatment
with Proteoglycan-depleting Factors.
[0152] In this example, porcine meniscus cartilage, articular
cartilage, ligament and peritoneum heart valve soft tissue implants
are prepared and frozen to disrupt, that is, to kill the specimens'
cells, as described above in Example 2. The soft tissue implants
are further treated with proteoglycan-depleting factors to
eliminate substantially the proteoglycans from the xenograft.
Subsequently, the xenografts are treated with glycosidase to remove
substantially remaining .alpha.-gal epitopes from the xenograft, as
described in Example 2. Substantial elimination of the
proteoglycans and the remaining .alpha.-gal epitopes interferes
with the ability of the recipient subject's immune system to
recognize the xenograft as foreign. The soft tissue implants are
transplanted into cynomologous monkeys, and the primate response to
the soft tissue implants is assessed.
[0153] Meniscus cartilage, articular cartilage and ligament
implants from porcine stifle joints and heart valve implants from
porcine peritoneum are prepared following the preparation
procedures outlined in Example 2 including the sterilization, and
freeze/thaw cycling treatments. A chondroitinase ABC solution is
then prepared by combining 0.05M Tris-HCL (7.88
gm/liter-MW=157.60), 5 mM benzamidine-HCL (0.783
.mu.m/liter-MW=156.61), 0.010 M N-ethylmaleimide (1.2513
.mu.m/liter-MW=125.13), and 0.001M phenylmethylsulfonyl fluoride
(0.17420 gm/liter-MW=174.2), dissolved in methanol. A mixture of
0.15 M NaCl (8.775 gm/liter-MW=58.5), penicillin and streptomycin
(1% (v/v) 10 ml/liter) along with enzyme in the amount of 1 unit
chondroitinase ABC (Sigma #C-3509) Enzyme Solution per 1 ml of
solution is added to bring the solution to 1 liter.
[0154] Each soft tissue xenograft specimen is incubated in the
chondroitinase ABC enzyme solution at a concentration of 1 ml of
solution per a 3 mm diameter soft tissue plug. The incubations are
performed at a pH of 8.0 and 37 degrees C in a shaker water bath
for 48 hours. After the incubation, each soft tissue specimen is
washed in appropriate buffer and the washings are added to the
chondroitinase ABC solution. Each soft tissue specimen is then
re-incubated with the chondroitinase ABC solution at a
concentration of 1 unit chondroitinase ABC (Sigma #C-3509) Enzyme
Solution per 1 ml of solution for another 48 hours as described
above. Each soft tissue specimen is again washed in appropriate
buffer solution, and the washings are added to the chondroitinase
ABC solution.
[0155] Each soft tissue specimen is then incubated in 1 ml of
trypsin solution (1 mg/ml trypsin, 0.15 M NaCl, 0.05 M Na
Phosphate) at a pH of 7.2 for 24 hours. The incubation is performed
in a shaker water bath at 37 degrees C. Each soft tissue specimen
is washed in appropriate buffer solution, and the washings are
added to the trypsin solution.
[0156] Each specimen is then placed in 1 ml of hyaluronidase
solution (0.01 mg/ml testicular hyaluronidase, 0.005 M Benzamidine
HCL, 001 M PMSF, 0.OlOM Nethylmaleimide, 0.005 M Benzamidine HCL,
1% v/v penicillin and streptomycin) at a pH 6.0 for 24 hours. The
incubation is performed in a shaker water bath at 37 degrees C.
Each soft tissue specimen is then rinsed again in an appropriate
buffer solution, and the washings are added to the hyaluronidase
solution.
[0157] Subsequently, the implants are treated with glycosidase as
described above in Example 2, implanted into the monkeys, and the
occurrence of an immune response against each of the xenografts is
assessed as described above in Example 2.
[0158] Those of skill in the art will recognize that the invention
may be embodied in other specific forms without departing from the
spirit or essential characteristics thereof. The presently
described embodiments are therefore to be considered in all
respects as illustrative and not restrictive, the scope of the
invention being indicated by the appended claims rather than by the
foregoing description, and all variations of the invention which
are encompassed within the meaning and range of equivalency of the
claims are therefor intended to be embraced therein.
* * * * *