U.S. patent application number 11/525782 was filed with the patent office on 2007-01-18 for acellular matrix implants for treatment of articular cartilage, bone or osteochondral defects and injuries and a method for use thereof.
This patent application is currently assigned to HISTOGENICS CORP. Invention is credited to Akihiko Kusanagi, Mary Beth Schmidt, Laurence J. B. Tarrant.
Application Number | 20070014867 11/525782 |
Document ID | / |
Family ID | 34198211 |
Filed Date | 2007-01-18 |
United States Patent
Application |
20070014867 |
Kind Code |
A1 |
Kusanagi; Akihiko ; et
al. |
January 18, 2007 |
Acellular matrix implants for treatment of articular cartilage,
bone or osteochondral defects and injuries and a method for use
thereof
Abstract
An acellular matrix implant for treatment of defects and
injuries of articular cartilage, bone or osteochondral bone and a
method for treatment of injured, damaged, diseased or aged
articular cartilage or bone, using the acellular matrix implant
implanted into a joint cartilage lesion in situ and a bone-inducing
composition implanted into an osteochondral or bone defect. A
method for repair and restoration of the injured, damaged, diseased
or aged cartilage or bone into its full functionality by implanting
the acellular matrix implant between two layers of biologically
acceptable sealants and/or the bone-inducing composition into the
osteochondral bone or skeletal bone defect. A method for
fabrication of the acellular matrix implant of the invention. A
method for preparation of bone-inducing composition.
Inventors: |
Kusanagi; Akihiko;
(Brookline, MA) ; Tarrant; Laurence J. B.;
(Northampton, MA) ; Schmidt; Mary Beth; (Pomfret
Center, CT) |
Correspondence
Address: |
PETERS VERNY JONES & SCHMITT, L.L.P.
425 SHERMAN AVENUE
SUITE 230
PALO ALTO
CA
94306
US
|
Assignee: |
HISTOGENICS CORP
|
Family ID: |
34198211 |
Appl. No.: |
11/525782 |
Filed: |
September 22, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10882581 |
Jun 30, 2004 |
|
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11525782 |
Sep 22, 2006 |
|
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60496971 |
Aug 20, 2003 |
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Current U.S.
Class: |
424/548 |
Current CPC
Class: |
A61F 2002/30535
20130101; A61L 27/58 20130101; A61L 27/52 20130101; A61F 2/30756
20130101; A61L 27/24 20130101; A61L 27/3608 20130101; A61L 27/34
20130101; A61L 27/3633 20130101; A61L 27/3654 20130101; A61F
2250/0058 20130101; A61L 2430/06 20130101 |
Class at
Publication: |
424/548 |
International
Class: |
A61K 35/34 20060101
A61K035/34 |
Claims
1. A method for treatment of injury of an articular cartilage and
for repair and restoration of damaged, injured, diseased or aged
cartilage to a functional hyaline cartilage, said method comprising
steps: a) preparing an acellular matrix implant; and b) implanting
said implant into an articular cartilage lesion.
2. The method of claim 1 wherein said acellular matrix implant is a
sponge, porous or honeycomb scaffold, sol-gel, gel, a polymer of an
aromatic organic acid or thermo-reversible hydrogel.
3. The method of claim 2 additionally comprising a step of
depositing a layer of a biologically acceptable top sealant over
said implant implanted into said lesion.
4. The method of claim 3 further comprising a step of depositing a
layer of a biologically acceptable bottom sealant over a bottom of
said lesion.
5. The method of claim 4 wherein said top and said bottom sealants
are the same or different.
6. The method of claim 5 wherein a combination of said implant and
said top sealant results in formation and growth of a superficial
cartilage layer sealing the cartilage lesion in situ.
7. The method of claim 6 additionally comprising a step of
debridement of the articular cartilage lesion and during the
debridement, preparing the lesion for implantation of the acellular
matrix implant by depositing the bottom sealant at the bottom of
the lesion thereby insulating said cavity from the surrounding
tissue.
8. The method of claim 7 further comprising a step of optionally
introducing enzymes, hormones, growth factors, proteins, peptides
and mediators, or drugs promoting an endogenous production of these
factors or mediators, into said sealed cavity or generating
conditions for their transport or transfer through the bottom
sealant.
9. The method of claim 7 further comprising a step of subjecting an
individual undergoing a surgery for repair of said lesion to a
normal physical activity thereby naturally providing an
intermittent hydrostatic pressure which promotes a formation of the
functional hyaline cartilage and its integration into a surrounding
native intact cartilage.
10. The method of claim 1 wherein the acellular matrix implant is
prepared from a material selected from the group consisting of a
Type I collagen, a Type II collagen, a Type IV collagen, a
cell-contracted collagen containing proteoglycan, a cell-contracted
collagen containing glycosaminoglycan, a cell-contracted collagen
containing glycoprotein, a polymer of an aromatic organic acid,
gelatin, agarose, hyaluronin, fibronectin, laminin, a bioactive
peptide growth factor, a cytokine, elastin, fibrin, a polymer of an
aromatic organic acid or a copolymer thereof, a synthetic polymeric
fiber made of polylactic acid, a synthetic polymeric fiber made of
polyglycolic acid, polycaprolactones, a polyamino acid, a
polypeptide gel, a polymeric thermo-reversible gelling hydrogel
(TRGH), a copolymer thereof and a combination thereof.
11. The method of claim 10 wherein said material for preparation of
the acellular matrix implant is the thermo-reversible gelation
hydrogel.
12. The method of claim 11 suitable for treatment of the articular
cartilage injury comprising steps: a) preparation of the acellular
matrix implant; b) debridement of said lesion during the surgery;
c) preparation of the cartilage lesion for implantation of said
implant, including a step of depositing the bottom sealant at the
bottom of the cartilage lesion for sealing of said lesion and
protecting the implant from migration of blood-borne agents; d)
implanting the implant into the lesion; e) depositing the top
sealant over the acellular matrix implant; and f) following the
surgery, subjecting an individual undergoing a surgery for repair
of said lesion to a normal physical activity thereby naturally
providing an intermittent hydrostatic pressure.
13. The method of claim 12 wherein said acellular matrix implant is
a biodegradable collagenous sponge, honeycomb sponge, collagenous
porous scaffold, a polymer of an aromatic organic acid or
thermo-reversible gelation hydrogel (TRGH) matrix.
14. The method of claim 13 wherein said matrix additionally
comprises matrix remodeling enzymes, matrix metalloproteinases,
aggrecanases and cathepsins.
15. The method of claim 14 wherein the acellular matrix implant is
the thermo-reversible gelation hydrogel (TRGH) deposited into a
lesion cavity formed above the bottom sealant layer, or into the
cavity between the top and bottom sealant, said TRGH deposited into
said cavity either incorporated into a collagenous sponge or
scaffold or as a sol at temperatures between about 5 to about
30.degree. C., wherein within said cavity and at the body
temperature said TRGH converts from the fluidic sol into a solid
gel.
16. A method for treatment of osteochondral defects, said method
comprising steps: a) preparing a bone-inducing composition or a
carrier comprising said composition, said composition comprising
one or several bone-inducing agents, for implantation into a bone
lesion; b) preparing an acellular matrix implant for implantation
into a cartilage lesion as a collagenous sponge, collagenous porous
scaffold, a polymer of an aromatic organic acid or
thermo-reversible gelation hydrogel (TRGH) matrix support wherein
said sponge, scaffold, polymer or TRGH are biodegradable, will
disintegrate with time, be removed and replaced with a hyaline
cartilage; c) introducing said bone-inducing composition or a
carrier comprising said composition into a bone lesion; d) covering
said bone-inducing composition or the carrier comprising said
composition with a bottom sealant; e) implanting said acellular
matrix implant into said cartilage lesion over the bottom sealant;
and f) introducing a layer of a top sealant over said implant
wherein said top and bottom sealants may or may not be the
same.
17. The method of claim 16 wherein said inducing agent is selected
from the group consisting of a demineralized bone powder,
hydroxyapatite, organoapatite, calcium phosphate, titanium oxide,
poly-L-lactic acid, polyglycolic acid, a copolymer thereof and a
bone morphogenic protein.
18. The method of claim 17 wherein said bone-inducing agent is
hydroxyapatite.
19. The method of claim 17 wherein said bone-inducing agent is the
demineralized bone powder.
20. The method of claim 19 wherein said demineralized bone powder
is dissolved in collagen.
21. The method of claims 16 wherein said carrier comprising the
bone-inducing agent is a polymer of an aromatic organic acid.
22. A method for treatment of bone defects and fractures, said
method comprising steps: a) preparing a bone-inducing composition
or an implant carrier comprising said composition, said composition
comprising one or several bone-inducing agents for implantation
into a bone lesion; b) introducing said bone-inducing composition
or said carrier comprising said composition into the bone lesion;
c) covering said bone-inducing composition or said carrier
comprising said composition with a layer of a sealant.
23. The method of claim 22 wherein said bone-inducing agent is
selected from the group consisting of a demineralized bone powder,
hydroxyapatite, organoapatite, calcium phosphate, titanium oxide,
poly-L-lactic acid, polyglycolic acid, a copolymer thereof and a
bone morphogenic protein.
24. The method of claim 23 wherein said bone-inducing agent is the
demineralized bone powder.
25. The method of claim 24 wherein said demineralized bone powder
is mixed with collagen into a stable paste.
26. The method of claims 23 wherein said carrier comprising said
bone-inducing agent is a polymer of an organic aromatic acid.
27. The method of claim 23 wherein said bone-inducing agent is the
hydroxyapatite.
28. The method of claim 23 wherein said bone-inducing composition
is the bone morphogenic protein.
29. The method of claims 23 wherein said bone-inducing agent is
titanium oxide.
30. An acellular matrix implant for implantation into a cartilage
lesion, said implant comprising a collagenous, sol-gel, polymer of
an aromatic organic acid or thermo-reversible hydrogel material
fabricated as a sponge or porous or honeycomb scaffold.
31. The implant of claim 30 wherein said collagenous material
further comprises a compound selected from the group consisting of
a mediator, growth factor, enzyme, protein, peptide and a drug
enhancing endogenous production of the mediator, factor, enzyme,
protein or peptide.
32. A sealant for sealing of a top or bottom cartilage or bone
lesion wherein said sealant is biologically compatible with
cartilage or bone tissue, non-toxic and biodegradable.
33. The sealant of claim 32 wherein the sealant is a rapidly
gelling polymer from a flowable liquid or paste to a load-bearing
gel within 30 seconds to 5 minutes.
34. The sealant of claim 33 possessing a minimal peel strengths of
at least about 3N/m to about to 30 N/m, a cohesive strength,
measured as tensile strength in the range of from about 0.2 MPa to
about 1.0 MPa or has a bond strength of at least 0.5 N/cm.sup.2 to
about 6 N/cm.sup.2.
35. The sealant of claim 34 wherein said sealant is a gel having a
cohesive strength dependent on the number of inter-chain
linkages.
36. The sealant of claim 35 which has adhesive or peel strengths at
least 10 N/m and tensile strength at least 0.3 MPa.
37. The sealant of claim 35 which has adhesive or peel strengths of
100 N/cm and tensile strength in the range from 0.8 to 1.0 MPA.
Description
[0001] This application is based on and claims priority of the
Provisional Application Ser. No. 60/496,971, filed Aug. 20, 2003,
which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
Field of Invention
[0002] The current invention concerns acellular matrix implants and
compositions for treatment of articular cartilage, bone or
osteochondral defects and injuries and a method for treatment of
such osteochondral defects and/or injured, damaged, diseased or
aged articular cartilage or bone using an acellular matrix implant
implanted into a joint cartilage lesion and/or into the
osteochondral defect in situ wherein the osteochondral or bone
defect is further implanted with a bone inducing composition or a
carrier comprising said composition. The acellular matrix implant
of the invention comprises a two or three dimensional biodegradable
scaffold structure implanted into the joint cartilage lesion
typically below or over one, two or several layers, or between two
layers of biologically acceptable sealants. The implant and the
method are particularly useful for repair and restoration of
function of the injured or traumatized articular cartilage, bone or
osteochondral defects of younger individuals. In particular, the
invention concerns a method where the implantation of the acellular
matrix implant of the invention initiates and achieves natural
healing of the cartilage by activation and migration of
chondrocytes from a native, surrounding cartilage into the
cartilage defect and/or by inducing bone formation by depositing a
bone inducing composition into the osteochondral and/or bone defect
in conjunction with the acellular matrix implant or without the
implant.
[0003] The method further concerns a formation of a new superficial
cartilage layer overgrowing and sealing the lesion in the joint
cartilage by applying a top sealant over the cartilage lesion as
well as insulation of the lesion from the cell and blood debris, by
applying a bottom sealant. Such formation of the superficial
cartilage layer is also applicable to osteochondral cartilage and
bone lesions where the bottom sealant is used for sealing and
separating the cartilage and bone lesions and the top sealant is
used to form the superficial cartilage layer.
[0004] The method for treatment of articular cartilage comprises
preparation of the acellular implant, preparation of the lesion for
implantation of said implant including a step of depositing a
bottom sealant at the bottom of the cartilage lesion for sealing
the joint cartilage lesion and protecting the implant from effects
of blood-borne agents, implanting the implant of the invention into
the lesion and depositing the top sealant over the implant. The
method for treatment of osteochondral defects additionally
typically comprises depositing a bone inducing composition or a
carrier comprising said composition into the bone lesion wherein
said bone lesion is covered by the bottom sealant thereby
separating said bone and cartilage lesions. The method for
treatment of bone defects comprises depositing the bone inducing
composition or a carrier comprising said composition in a bone
lesion which may optionally be lined with or covered with a bottom
or top sealant.
[0005] The invention further concerns a method for repair and
restoration of the injured, damaged, diseased or aged cartilage or
bone into its full functionality and for treatment of injured
cartilage by implanting the acellular matrix implant into the
cartilage lesion between two or more layers of biologically
acceptable sealants and/or depositing the bone inducing composition
or a carrier comprising said composition into the bone lesion,
covering said bone inducing composition or a carrier comprising
said composition with the bottom sealant, depositing the acellular
matrix implant into the cartilage lesion and covering said implant
with the top sealant.
[0006] Additionally, the invention concerns a method for
fabrication of an acellular implant of the invention for use in
treatment of cartilage defects and for preparation of a bone
inducing composition or a carrier comprising said composition for
use in treatment of bone or osteochondral defects.
BACKGROUND AND RELATED DISCLOSURES
[0007] Damage to the articular cartilage which occurs in active
individuals and older generation adults as a result of either acute
or repetitive traumatic injury or aging is quite common. Such
damaged cartilage leads to pain, affects mobility and results in
debilitating disability.
[0008] Typical treatment choices, depending on lesion and symptom
severity, are the rest and other conservative treatments, minor
arthroscopic surgery to clean up and smooth the surface of the
damaged cartilage area, and other surgical procedures such as
microfracture, drilling, and abrasion. All of these may provide
symptomatic relief, but the benefit is usually only temporary,
especially if the person's pre-injury activity level is maintained.
For example, severe and chronic forms of knee joint cartilage
damage can lead to greater deterioration of the joint cartilage and
may eventually lead to a total knee joint replacement. Nowadays,
approximately 200,000 total knee replacement operations are
performed annually. The artificial joint generally lasts only 10 to
15 years and the operation is, therefore, typically not recommended
for people under the age of fifty.
[0009] Osteochondral diseases or injuries, which are a combination
lesions of bone and cartilage, present yet another challenge for a
treatment of which need is not being met by the currently available
procedures and methods. For example, treatment of osteochondritis
dissecans with autologous chondrocyte transplantation, described in
J. Bone and Joint Surgery, 85A-Supplement 2: 17-24 (2003), requires
multiple surgeries and at least three weeks for cell cultivation
and growth.
[0010] It would, therefore, be extremely advantageous to have
available a method for in situ treatment of these injuries which
would effectively restore the cartilage or bone to its pre-injury
state during one surgery and with minimal time needed for recovery,
which treatment would be especially suitable for younger
individuals who are more active and have better recovery
capabilities.
[0011] Attempts to provide means and methods for repair of
articular cartilage are disclosed, for example, in U.S. Pat. Nos.
5,723,331; 5,786,217; 6,150,163; 6,294,202; 6,322,563 and in the
U.S. patent application Ser. No. 09/896,912, filed on Jun. 29,
2001.
[0012] U.S. Pat. No. 5,723,331 describes methods and compositions
for preparation of synthetic cartilage for the repair of articular
cartilage using ex vivo proliferated denuded chondrogenic cells
seeded ex vivo, in the wells containing adhesive surface. These
cells redifferentiate and begin to secrete cartilage-specific
extracellular matrix thereby providing an unlimited amount of
synthetic cartilage for surgical delivery to a site of the
articular defect.
[0013] U.S. Pat. No. 5,786,217 describes methods for preparing a
multi-cell layered synthetic cartilage patch prepared essentially
by the same method as described in '331 patent except that the
denuded cells are non-differentiated, and culturing these cells for
a time necessary for these cells to differentiate and form a
multicell layered synthetic cartilage.
[0014] U.S. application Ser. No. 09/896,912, filed on Jun. 29, 2001
concerns a method for repairing cartilage, meniscus, ligament,
tendon, bone, skin, cornea, periodontal tissues, abscesses,
resected tumors and ulcers by introducing into tissue a temperature
dependent polymer gel in conjunction with at least one blood
component which adheres to the tissue and promotes support for cell
proliferation for repairing the tissue.
[0015] U.S. patent application Ser. Nos. 10/104,677; 10/625,822;
10/625,245 and 10/626,459 filed on Jul. 22, 2003, by inventors,
hereby incorporated by reference, disclose neo-cartilage constructs
subjected to an algorithm of certain specific conditions suitable
for repair of injured or damaged articular cartilage.
[0016] None of the above cited references, however, results in
repair and regeneration of cartilage or bone in situ without a need
for several surgeries.
[0017] It is thus a primary objective of this invention to provide
a method and a means for treatment of injured or traumatized
cartilage, bone or cartilage-bone defects by depositing at least
two separate layers of biologically acceptable adhesive sealants
thereby forming a cavity in the injured lesion of the cartilage and
implanting an acellular implant into said cavity between these two
layers and, additionally, by providing a bone inducing composition
or a carrier comprising said composition containing bone inducing
agents and implanting said composition into the bone lesion of the
osteochondral defects followed by the implantation of the acellular
matrix implant into the cartilage defect. The method according to
the invention results in induction of chondrocyte activation and
migration from the surrounding native cartilage into the acellular
implant's matrix and in the growth of the superficial cartilage
layer over the implant thereby sealing the lesion and, when used
for treatment of osteochondral defects, in migration of osteoblast
into the bone lesion and in healing of the bone defect as well as
defect of the articular cartilage.
[0018] All patents, patent applications and publications cited
herein are hereby incorporated by reference.
SUMMARY
[0019] One aspect of the current invention is an acellular matrix
implant for treatment of defects and injuries of articular
cartilage.
[0020] Another aspect of the current invention is an acellular
matrix implant in combination with a bone inducing composition or a
carrier comprising said composition for treatment of osteochondral
defects and injuries.
[0021] Still another aspect of the current invention is an
acellular bone implant comprising a bone inducing composition or a
carrier comprising said composition for implantation into a bone
lesion for treatment of bone defects and injuries.
[0022] Yet another aspect of the current invention is a method for
fabrication of an acellular matrix implant of the invention.
[0023] Still another aspect of the current invention is a method
for preparation of an acellular matrix implant wherein said matrix
is a sponge, honeycomb, scaffold, thermo-reversible gelation
hydrogel (TRGH) or a polymer of an aromatic organic acid.
[0024] Yet another aspect of the current invention is a method for
treatment of injured, damaged, diseased or aged articular cartilage
using the acellular matrix implant implanted into a joint cartilage
lesion in situ.
[0025] Still yet another aspect of the current invention is an
acellular matrix implant used in a method where the implantation of
the acellular matrix implant of the invention initiates and
achieves activation and induction of migration of chondrocytes from
a native surrounding cartilage into the acellular matrix implant
deposited within a cartilage defect.
[0026] Still yet another aspect of the current invention is a
method for treatment of osteochondral defects by implanting an
acellular matrix implant into the cartilage lesion in conjunction
with depositing a bone inducing composition or a carrier comprising
said composition into an osteochondral lesion in situ.
[0027] Still another aspect of the current invention is a bone
inducing composition or a carrier comprising said composition
containing bone inducing agents such as a demineralized bone
powder, calcium phosphate, hydroxyapatite, organoapatite, titanium
oxide, poly-L-lactic or polyglycolic acid or a copolymer thereof or
a bone morphogenic protein used in a method where the deposition of
said composition into the bone lesion initiates migration of
osteoblast and achieves natural healing of the underlying bone.
[0028] Still yet another aspect of the current invention is a bone
inducing composition or a carrier comprising said composition
deposited into a bone lesion of the osteochondral defect in
conjunction with implantation of an acellular matrix implant into
the cartilage lesion useful for treatment of osteochondral
defects.
[0029] Still yet another aspect of the current invention is a
method for treatment of bone lesions caused by bone injuries or
defects said treatment accomplished by implanting a bone inducing
composition or a carrier comprising said composition into the bone
lesion in situ.
[0030] Still another aspect of the current invention is a bone
inducing composition or a carrier comprising said composition
containing bone inducing agents such as a demineralized bone
powder, calcium phosphate, hydroxyapatite, organoapatite, titanium
oxide, poly-L-lactic or polyglycolic acid or a copolymer thereof or
a bone morphogenic protein alone, in combination, or incorporated
into a carrier, such as a matrix, hydrogel, sponge, honeycomb,
scaffold or a polymer of an aromatic organic acid, used in a method
where the deposition of said composition into the bone lesion
initiates migration of osteoblast and achieves natural healing of
the underlying bone.
[0031] Still yet another aspect of the current invention is a bone
inducing composition or a carrier comprising said composition
deposited into a bone lesion for treatment of a bone defect alone
or, where appropriate, in conjunction with implantation of an
acellular matrix implant into the cartilage lesion or osteochondral
implant useful for treatment of osteochondral defects.
[0032] Yet another aspect of the current invention is a method for
treatment of injured, damaged, diseased or aged articular cartilage
using an acellular matrix implant implanted into a joint cartilage
lesion in situ, said method further comprising a formation of a new
superficial cartilage layer overgrowing and sealing the lesion in
the joint articular cartilage by applying a top sealant over the
lesion and further applying a bottom sealant over the bottom of the
lesion, said bottom sealant providing protection of the lesion
against a cell and blood debris migration.
[0033] Another aspect of the current invention is a method for
treatment of osteochondral defects by depositing a bone inducing
composition or a carrier comprising said composition comprising
bone inducing agents into a bone lesion, depositing a bottom
sealant over the bone inducing composition or a carrier comprising
said composition, implanting an acellular matrix implant into the
articular lesion and depositing a top sealant over the acellular
matrix implant.
[0034] Still another aspect of the current invention is an
acellular matrix implant for use in treatments of the cartilage or
bone lesions comprising a two or three dimensional biodegradable
sponge, honeycomb, hydrogel, scaffold or a polymer of an aromatic
organic acid matrix implanted into the joint cartilage lesion
between two layers, top and bottom, of biologically acceptable
sealants.
[0035] Still yet another aspect of the current invention is a
method for treatment of articular cartilage injury comprising
steps:
[0036] a) preparation of an acellular matrix implant;
[0037] b) preparation of a cartilage lesion for implantation of
said implant, including a step of depositing a bottom sealant at
the bottom of the cartilage lesion for sealing of said lesion and
protecting the implant from migration of blood-borne agents;
[0038] c) implanting the implant into the lesion; and
[0039] d) depositing a top sealant over the acellular matrix
implant.
[0040] Still yet another aspect of the current invention is a
method for repair and restoration of damaged, injured, diseased or
aged cartilage to a functional cartilage, said method comprising
steps:
[0041] a) preparing an acellular matrix implant as a collagenous
sponge, collagenous porous scaffold or honeycomb, thermo-reversible
gelation hydrogel (TRGH) or a polymer of an aromatic organic acid
matrix, wherein said sponge, scaffold, polymer of the aromatic
organic acid or TRGH are biodegradable, will disintegrate with time
and be metabolically removed from the healed lesion and replaced
with a hyaline cartilage, said matrix optionally comprising matrix
remodeling enzymes, such as matrix metalloproteinases,
aggrecanases, cathepsins and/or other biologically active
components;
[0042] b) introducing a layer of a biologically acceptable bottom
sealant into a cartilage lesion;
[0043] c) implanting said implant into said lesion into a cavity
formed by the bottom layer of said bottom sealant; and
[0044] d) introducing a top layer of a second biologically
acceptable top sealant over said implant wherein said top sealant
may or may not be the same as the bottom sealant and wherein a
combination of said implant and said top sealant results in
formation and growth of a superficial cartilage layer sealing the
cartilage lesion in situ.
[0045] Still another aspect of the current invention is an
acellular matrix implant comprising a thermo-reversible gelation
hydrogel (TRGH) deposited into a lesion cavity formed above the
bottom sealant layer, or into the cavity between the top and bottom
sealant, said TRGH deposited into said cavity either incorporated
into a collagenous sponge or scaffold or as a sol at temperatures
between about 5 to about 30.degree. C., wherein within said cavity
and at the body temperature said TRGH converts from the fluidic sol
into a solid gel and in this form, its presence provides a
structural support for migration of chondrocytes from a surrounding
native cartilage and formation of extracellular matrix, wherein
said TRGH is biodegradable, will disintegrate with time and be
metabolically removed from the lesion and replaced with a hyaline
cartilage.
[0046] Still yet another aspect of the current invention is a
method for treatment of osteochondral defects, said method
comprising steps:
[0047] a) preparing a bone inducing composition or a carrier
comprising said composition comprising one or several bone inducing
agents for implantation into a bone lesion;
[0048] b) preparing an acellular matrix implant for implantation
into a cartilage lesion as a collagenous sponge, collagenous porous
scaffold or honeycomb or thermo-reversible gelation hydrogel (TRGH)
matrix support wherein said sponge, scaffold or TRGH are
biodegradable, will disintegrate with time and be metabolically
removed from the lesion and replaced with a hyaline cartilage, said
matrix optionally comprising matrix remodeling enzymes, matrix
metalloproteinases, aggrecanases and cathepsins;
[0049] c) introducing said bone inducing composition or a carrier
comprising said composition into a bone lesion;
[0050] d) covering said bone inducing composition or a carrier
comprising said composition with a bottom sealant;
[0051] e) implanting said acellular matrix implant into said
cartilage lesion over the bottom sealant; and
[0052] f) introducing a layer of a top sealant over said implant
wherein said top and bottom sealants may or may not be the same and
wherein a combination of said acellular matrix implant and said top
sealant results in formation and growth of a superficial cartilage
layer sealing the cartilage lesion in situ.
[0053] Still yet another aspect of the current invention is a bone
inducing composition or a carrier comprising said composition
comprising bone inducing agents for treatment of osteochondral
defects further in combination with an acellular matrix implant
comprising a thermo-reversible gelation hydrogel (TRGH) each
deposited separately into a bone or cartilage lesion, wherein said
composition provides a means for rebuilding the bone and migration
of osteoblast into the bone lesion and wherein said implant
provides a structural support for migration of chondrocytes from a
surrounding native cartilage and formation of extracellular
matrix.
BRIEF DESCRIPTION OF DRAWINGS
[0054] FIG. 1A is an enlarged schematic representation of the
cartilage lesion within the host cartilage with underlaying
uninjured bone, showing a bottom sealant deposited at the bottom of
the lesion, an acellular matrix implant deposited over the bottom
sealant and covered with a top sealant. FIG. 1B is an enlarged
schematic representation of the osteochondral defect showing the
articular lesion, bone lesion, emplacement of the bone inducing
composition (bone material) or a carrier comprising said
composition into the bone lesion, emplacement of top and bottom
sealants and emplacement of the acellular matrix implant. FIG. 1C
is an enlarged schematic representation of the bone defect showing
the articular lesion, and combined osteochondral and skeletal bone
lesion, emplacement of the bone inducing composition or a carrier
comprising said composition into the bone and osteochondral lesion,
emplacement of top and bottom sealants and emplacement of the
acellular matrix implant. FIG. 1D is a schematic depiction of
creation of defects A and B at weight bearing site for implantation
of an acellular matrix implant or serving as an empty control
defect.
[0055] FIG. 2A is an image of an acellular matrix implant held in
the forceps. The actual size of the sponge is 5 mm in diameter and
1.5 mm of thickness. FIG. 2B is a longitudinal scheme of a
honeycomb structure of an acellular matrix implant showing a
relative localization of collagen sponge and porous collagen gel
wherein the pore size is between 200 and 400 .mu.m.
[0056] FIG. 3 shows a micrograph of the two control empty defect
sites A and B (4 mm in diameter and 1-1.5 mm in depth) created on
the weight-bearing site of the swine medial femoral condyle.
[0057] FIG. 4 is a micrograph of the two defect sites A and B
generated on the weight-bearing site of the swine medial femoral
condyle, implanted with acellular matrix implants. The defect has 4
mm in diameter and 1-1.5 mm in depth. The implants have 5 mm
diameter and 1.5 mm thickness. Each implant is sutured using 4
absorbable sutures and two non-absorbable sutures. The defect was
lined up with the bottom sealant and the implant was covered with
the top sealant.
[0058] FIG. 5 shows arthroscopic evaluation of a magnified empty
defect 2 weeks after defect creation showing the defect to be fully
exposed and empty.
[0059] FIG. 6 shows arthroscopic evaluation of a magnified defect
treated with the acellular matrix implant 2 weeks after the defect
creation. The superficial cartilage layer overgrowing the implant
site forms a smooth flat surface over the defect.
[0060] FIG. 7 is a graph illustrating a histological grading of the
repair tissue.
[0061] FIG. 8A shows a histological evaluation (29.times.
magnification) of the empty defect (D) at a control site (A). FIG.
8B shows a higher (72.times.) magnification of the defect site (D).
The defect is surrounded by the host cartilage (H) with underlying
subchondral bone (SB) area. Fibrous tissue (F) formation is seen in
both figures at the empty defect site. Fibrovascular pannus (F) is
formed at empty defect site as indicated by the absence of the
S-GAG accumulation.
[0062] FIG. 9A shows a histological evaluation (29.times.
magnification) of the empty defect (D) at a control site (B). FIG.
9B shows a higher (72.times.) magnification of the defect site (D).
The defect is surrounded by the host cartilage (H) with underlying
subchondral bone (SB) area. Fibrous tissue (F) formation is seen in
both FIGS. 9A and 9B at the empty defect site with slight
accumulation of S-GAG accumulation.
[0063] FIG. 10A shows a histological evaluation (29.times.
magnification) of the acellular implantation (I) at the implant
site (A). FIG. 10B shows acellular implantation at higher
(72.times.) magnification of the implant site (I). The implant site
is surrounded by the host cartilage (H) with underlying subchondral
bone (SB) area. Superficial cartilage layer is shown to cover the
implant site. In both FIGS. 10A and 10B normal S-GAG accumulation
and formation of hyaline-like cartilage was observed at the implant
site.
[0064] FIG. 11A shows a histological evaluation (29.times.
magnification) of the acellular implantation (I) at the implant
site (B). FIG. 11B shows acellular implantation at higher
(72.times.) magnification of the implant site (I). The implant site
is surrounded by the host cartilage (H) with underlying subchondral
bone (SB) area. Superficial cartilage layer is shown to cover the
implant site. In both FIGS. 11A and 11B normal S-GAG (*)
accumulation and formation of hyaline-like cartilage was observed
at the implant site.
[0065] FIG. 12 illustrates a degradation pattern in vivo of the top
sealant 3 months after the acellular matrix implantation. The
formed superficial cartilage layer was formed over the implant and
the sealant was partially degraded at three months after the
implantation. FIG. 12A shows a surface view of the Safranin-O
stained implantation site. FIG. 12B shows a side view of the
Safranin-O stained implantation site. FIG. 12C shows the bottom
view of the Safranin-O stained implantation site. Safranin-O
staining, seen as reddish color, indicates S-GAG accumulation.
[0066] FIG. 13 shows an example image of a full thickness defect
(D) after harvest created at femoral condyle of mini-pig at
72.times. magnification. Surrounding host cartilage (H),
subchondral bone area (SB) and remaining calcified cartilage area
are also indicated.
DEFINITIONS
[0067] As used herein:
[0068] "Acellular" means an implant lacking any biologically active
cells.
[0069] "Acellular matrix implant" or "acellular implant" means a
biologically acceptable collagenous implant whether in the form of
collagenous sponge, collagenous honeycomb, collagenous scaffold or
thermo-reversible gelation hydrogel without any biologically active
cells, forming a matrix into which the chondrocytes may
migrate.
[0070] "Articular cartilage" means a hyaline cartilage of the
joints, such as the knee joint.
[0071] "Subchondral" means a structure underlying a joint
cartilage.
[0072] "Subchondral bone" means a bone of specific composition,
typically very dense, but thin layer of bone just below the zone of
calcified cartilage and above the cancellous or trabecular bone
that forms the bulk of the bone structure of the limb.
[0073] "Osteochondral" means combined area of the cartilage and
bone where a lesion or lesions occur.
[0074] "Osteochondral defect" means a lesion which is a composite
lesion of cartilage and underlying bone.
[0075] "Bone defect" or "bone lesion" means the defect which is
localized under the subchondral bone region and is thus a
defect/lesion in a skeletal bone.
[0076] "Osteoblast" means a bone forming cell.
[0077] "Chondrocyte" means a nondividing cartilage cell which
occupies a lacuna within the cartilage matrix.
[0078] "Support matrix" means biologically acceptable sol-gel or
collagenous sponge, scaffold, honeycomb, hydrogel or a polymer of
an aromatic organic acid suitable for receiving activated migrating
chondrocytes or osteocytes that provides a structural support for
growth and three-dimensional propagation of chondrocytes and for
formulating of new hyaline cartilage or for migration of
osteochondrocytes into the bone lesions. The support matrix is
prepared from such materials as Type I collagen, Type II collagen,
Type IV collagen, gelatin, agarose, cell-contracted collagen
containing proteoglycans, glycosaminoglycans or glycoproteins,
polymers of aromatic organic acids, fibronectin, laminin, bioactive
peptide growth factors, cytokines, elastin, fibrin, synthetic
polymeric fibers made of poly-acids such as polylactic,
polyglycolic or polyamino acids, polycaprolactones, polyamino
acids, polypeptide gel, copolymers thereof and combinations
thereof. The gel solution matrix may be a polymeric
thermo-reversible gelling hydrogel. The support matrix is
preferably biocompatible, biodegradable, hydrophilic, non-reactive,
has a neutral charge and is able to have or has a defined
structure.
[0079] "Mature hyaline cartilage" means cartilage consisting of
groups of isogenous chondrocytes located within lacunae cavities
which are scattered throughout an extracellular collagen
matrix.
[0080] "Sealant" means a biologically acceptable typically rapidly
gelling formulation having a specified range of adhesive and
cohesive properties. Sealant is thus a biologically acceptable
rapidly gelling synthetic compound having adhesive and/or gluing
properties, and is typically a hydrogel, such as derivatized
polyethylene glycol (PEG), or a protein, such as albumin, which is
preferably cross-linked with a collagen compound. The sealant of
the invention typically gels and/or bonds rapidly upon contact with
tissue, particularly with tissue containing collagen.
[0081] "Modified sealant" means any suitable sealant for use in the
invention which has a polymerization time of at least 30
seconds.
[0082] "Bone-inducing composition" or "a carrier comprising said
composition" means a composition comprising at least one
bone-inducing agent or, preferably, a combination of several
agents, typically dissolved in a carrier or incorporated into a
matrix similar to the acellular matrix implant.
[0083] "Bone-inducing carrier", "carrier comprising bone-inducing
composition" or "bone acellular implant" means any carrier which
contains bone-inducing agents and which by itself promotes bone
formation or is suitable for depositing said bone-inducing
composition comprising at least one bone-inducing agent or,
preferably, a combination of several agents. Typically, the carrier
will be an acellular biodegradable porous matrix, hydrogel, sponge,
honeycomb, scaffold or a polymer of an aromatic organic acid
structure having large pores from about 50 to about 150 .mu.m,
which pores encourage migration of osteoblast and interconnecting
small pores of about 0.1 to about 10 .mu.m which promote support
and encourage formation of bone. The surface of such carrier might
be negatively charged encouraging pseudopod attachment of
osteoblasts and subsequent bone formation. One example of the
suitable carrier promoting bone formation is a polymer of an
aromatic organic acid with controllable degree of degradation which
is sufficiently hard but has a spongiform structure.
[0084] "Bone-inducing agents" means agents which induce, support or
promote bone growth and repair of bone defects. Exemplary
bone-inducing agents are calcium phosphate, hydroxyapatite,
organoapatite, titanium oxide, demineralized bone powder,
poly-L-lactic and polyglycolic acid or a copolymer thereof or a
bone morphogenic protein, among others.
[0085] "Bottom sealant" or "first sealant" means a biologically
acceptable tissue sealant which is deposited at the bottom of the
lesion. In case of the osteochondral defect, the first sealant is
deposited over the bone-inducing composition or a carrier
comprising said composition deposited into the bone lesion
effectively sealing, separating and protecting the bone lesion from
chondrocyte migration as well as protecting the cartilage lesion
from migration of osteocytes.
[0086] "Top sealant" or "second sealant" means a biologically
acceptable sealant which is deposited above and over the acellular
matrix implant implanted into a lesion and may promote formation of
the superficial cartilage layer. The second (top) sealant may or
may not be the same as the first (bottom) sealant and is preferably
a cross-linked polyethylene glycol hydrogel with
methyl-collagen.
[0087] "De novo" or "de novo formation" means the new production of
cells, such as chondrocytes, fibroblasts, fibrochondrocytes,
tenocytes, osteoblasts and stem cells capable of differentiation,
or tissues such as cartilage connective tissue, hyaline cartilage,
fibrocartilage, tendon, and bone within a support structure, such
as multi-layered system, scaffold or collagen matrix or formation
of superficial cartilage layer.
[0088] "Superficial cartilage layer" means an outermost layer of
cartilage that forms the layer of squamous-like flattened
superficial zone chondrocytes covering the layer of the second
sealant and overgrowing the lesion.
[0089] "Thermo-reversible" means a compound or composition changing
its physical properties such as viscosity and consistency, from sol
to gel, depending on the temperature. The thermo-reversible
composition is typically completely in a sol (liquid) state at
between about 5 and 15.degree. C. and in a gel (solid) state at
about 25-30.degree. C. and above. The gel/sol state in between
shows a lesser or higher degree of viscosity and depends on the
temperature. When the temperature is higher than 15.degree. C., the
sol begins to change into gel and with the temperature closer to
30-37.degree. the sol becomes more and more solidified as gel. At
lower temperatures, typically lower than 15.degree. C., the sol has
more liquid consistency.
[0090] "TRGH" means thermo-reversible gelation hydrogel material in
which the sol-gel transition occurs on the opposite temperature
cycle of agar and gelatin gels. Consequently, the viscous fluidic
phase is in a sol stage and the solid phase is in a gel stage. TRGH
has very quick sol-gel transformation which requires no cure time
and occurs simply as a function of temperature without hysteresis.
The sol-gel transition temperature can be set at any temperature in
the range from 5.degree. C. to 70.degree. C. by molecular design of
thermo-reversible gelation polymer (TGP), a high molecular weight
polymer of which less than 5 wt % is enough for hydrogel
formation.
[0091] "Sol-gel solution" means a colloidal suspension which, under
certain conditions, transitions from a liquid (sol) to a solid
material (gel). The "sol" is a suspension of aqueous collagen that
is transitioned, by heat treatment, into a gel.
[0092] "GAG" means glycosaminoglycan.
[0093] "S-GAG" means sulfated glycosaminoglycan.
[0094] "Aggrecanase" means aggrecanase enzyme.
[0095] "Cathepsin" means a proteinase or peptidase enzyme.
[0096] "MMP" means matrix metalloproteinase, an enzyme associated
with cartilage degeneration in an injured or diseased joint.
[0097] "DMB" means dimethylene blue used for staining of
chondrocytes.
[0098] "Superficial zone cartilage" means the flattened outermost
layer of chondrocytes covering the extracellular matrix
intermediate zone and deeper zone of mature articular cartilage in
which non-dividing cells are dispersed.
[0099] "Connective tissue" means tissue that protect and support
the body organs, and also tissues that hold organs together.
Examples of such tissues include mesenchyme, mucous, connective,
reticular, elastic, collagenous, bone, blood, or cartilage tissue
such as hyaline cartilage, fibrocartilage, and elastic
cartilage.
[0100] "Adhesive strength" means a peel bond strength measurement,
which can be accomplished by bonding two plastic tabs with an
adhesive formulation. The tabs can be formed by cutting 1.times.5
cm strips from polystyrene weighing boats. To the surface of the
boat are bonded (using commercial cyanoacrylate Superglue), sheets
of sausage casing (collagen sheeting, available from butcher supply
houses). The sausage casing is hydrated in water or physiological
saline for 20 min to one hour and the adhesive is applied to a
1.times.1 cm area at one end of the tab; the adhesive is cured.
Then, the free ends of the tab are each bent and attached to the
upper and lower grips, respectively, of a tensile testing apparatus
and pulled at 10 mm/min strain rate, recording the force in Newtons
to peel. A constant force trace allows estimation of N/m, or force
per width of the strip. A minimum force per width of 10 N/m is
desired; 100N/m or higher is more desirable. Alternatively, the
same tab can be bonded (a single tab) over a 1.times.1 cm area to
tissue, either dissected or exposed tissue in a living animal,
during surgery. The free end of the tab is then gripped or attached
through a perforation to a hook affixed to a hand-held tensile test
device (Omega DFG51-2 digital force gauge; Omega Engineering,
Stamford, Conn.) and pulled upward at approximately 1 cm/sec. The
maximum force required to detach the tab from the tissue is
recorded. The minimum force desired in such measurements would be
0.1 N to detach the tab. Forces or 0.2 to 1 N are more
desirable.
[0101] "Cohesive strength" means the force required to achieve
tensile failure and is measured using a tensile test apparatus. The
glue or adhesive can be cured in a "dog-bone"-shaped mold. The wide
ends of the formed solid adhesive can then be affixed, using
cyanoacrylate (Superglue) to plastic tabs, and gripped in the test
apparatus. Force at extensional failure should be at least 0.2 MPa
(2 N/cm.sup.2) but preferably 0.8 to 1 MPa or higher.
[0102] "Lap shear measurements" means a test of bonding strength,
in which the sealant formulation is applied to overlapping tabs of
tissue, cured, and then the force to pull the tabs apart is
measured. The test reflects adhesive and cohesive bonding; strong
adhesives will exhibit values of 0.5 up to 4-6 N/cm.sup.2 of
overlap area.
DETAILED DESCRIPTION OF THE INVENTION
[0103] This invention is based on findings that when a
biodegradable acellular matrix implant, such as a collagenous
sponge matrix, collagenous scaffold matrix or thermo-reversible
gelation hydrogel matrix implant, is deposited into a lesion of
injured, traumatized, aged or diseased cartilage or, in conjunction
with a bone-inducing composition or a carrier comprising said
composition comprising bone activating agents, into an
osteochondral or bone defect, within time, this acellular matrix
implant activates mature but non-dividing chondrocytes present in
the surrounding native cartilage, induces them to migrate to a site
of the articular cartilage defect and generates a new extracellular
matrix ultimately resulting in formation of a healthy hyaline
cartilage and/or, in case of the bone or osteochondral defect, it
induces migration of osteoblast cells from surrounding healthy bone
or subchondral bone. Under these circumstances, the second, top
sealant deposited over the acellular matrix implant will promote in
situ formation of superficial cartilage layer over the cartilage
lesion containing the implant. Such superficial cartilage layer
will be also generated when the top sealant is deposited over the
osteochondral defect, which, additionally, will comprise depositing
of the bone-inducing composition or a carrier comprising said
composition into the bone lesion and covering said composition with
a first, bottom sealant.
[0104] The invention thus, in its broadest scope, concerns a method
for repair and restoration of damaged, injured, traumatized or aged
cartilage or for repair of bone or osteochondral defects and
restoration of both the cartilage and/or bone into their full
functionality by implanting, during arthroscopic surgery, an
acellular matrix implant and/or depositing a bone-inducing
composition or a carrier comprising said composition into the bone
lesion before implanting the acellular matrix implant into the
cartilage lesion. The invention further includes a method for
fabrication of said acellular matrix implant, preparation of said
bone-inducing composition or a carrier comprising said composition
and a method for de novo formation of a superficial cartilage layer
in situ.
[0105] Briefly, for treatment of the articular lesions, the
invention comprises preparation of the acellular matrix implant for
implanting into a joint cartilage lesion, said implant comprising a
collagenous, thermo-reversible gel or an aromatic organic acid
polymer support matrix in two or three-dimensions. The acellular
matrix implant may contain various supplements, such as matrix
remodeling enzymes, metalloproteinases (MMP-9, MMP-2, MMP-3),
aggrecanases, cathepsins, growth factors, donor's serum, ascorbic
acid, insulin-transferrin-sodium (ITS), etc., in concentrations
which are known in the art to induce growth, differentiation and
phenotype stability.
[0106] For treatment of osteochondral defects, the invention
comprises preparation of a bone-inducing composition or a carrier
comprising said composition comprised of bone-inducing agents, such
as demineralized bone powder, calcium phosphate, hydroxyapatite,
organoapatite, titanium oxide, poly-L-lactic and polyglycolic acid
or a copolymer thereof, alone or in combination, or a bone
morphogenic protein, depositing said composition into the bone
lesion and covering said bone-inducing composition or a carrier
comprising said composition with the first bottom sealant followed
by depositing said acellular matrix implant into the cartilage
lesion and covering said implant with the second, top sealant.
[0107] For treatment of bone defects, the invention comprises
preparation of a bone-inducing composition or a carrier comprising
said composition comprised of bone-inducing agents, such as
demineralized bone powder, calcium phosphate, hydroxyapatite,
organoapatite, titanium oxide, poly-L-lactic and polyglycolic acid
or a copolymer thereof, alone or in combination, or a bone
morphogenic protein in amounts needed to fill the bone lesion, and
depositing said composition into the bone lesion. Said lesion may
optionally be covered with the bottom or top sealant. Typically,
the bottom sealant is not deposited at the bottom of the bone
lesion but if needed, it can be.
[0108] The acellular matrix implant is implanted into a cartilage
lesion cavity formed by at least two layers of adhesive sealants.
However, in certain circumstances, the acellular matrix implant may
be also deposited into the cartilage lesion without either the
bottom or top sealant or without both sealants.
[0109] When the sealants are used in the method for repair of
cartilage, the first (bottom) layer of the sealant is deposited at
and covers the bottom of the cartilage lesion. Its function is to
protect the integrity of said lesion from cell migration and from
effects of various blood and tissue debris and metabolites and also
to form a bottom of the cavity into which the acellular matrix
implant is deposited. The first layer of the sealant may also
become a covering layer deposited over the bone-inducing
composition or a carrier comprising said composition placed into
the bone lesion within the subchondral bone or bone area.
[0110] Studies of induced defects of the pig's femoral condyle
confirmed that implantation of a biodegradable acellular matrix
implant combined with a implantation procedure disclosed herein and
performed under defined conditions induces activation and promotes
chondrocyte migration from surrounding native host cartilage
resulting in formation of extracellular matrix (ECM) of a
regenerated hyaline-like cartilage within the lesion at the injured
site. Similarly, a deposition of a bone-inducing composition or a
carrier comprising said composition comprising bone-inducing agents
into the bone defect promotes natural healing of bone by inducing
migration of osteoblast into said bone lesion and, combined with
the acellular matrix implant as described above, leads to healing
and reconstruction of both the bone and cartilage.
[0111] The method for using the acellular matrix implant for
generation of the hyaline cartilage is particularly suitable for
treatment of lesions in younger patients with focused lesions where
the cartilage has not developed an incipient osteoarthritic
conditions, that is in patients who would typically be treated with
microfracture or with cleaning the articular cartilage in the
joint, such as in, for example, arthroscopic surgery following a
sports injury. Such patients stand a high probability of restoring
a fully functional hyaline cartilage, or in case of osteochondral
defects, a fully functional cartilage and bone, without need of and
aggravation associated with undergoing additional one or multiple
surgeries.
[0112] One advantage of using the above-described method is that
the acellular matrix implant and/or the bone-inducing composition
or a carrier comprising such composition is non-immunogenic, can be
pre-manufactured well before the operation and can be introduced
during the first arthroscopy, when the diagnosis, cleaning and
debridement of the lesion takes place without a need for further
biopsy, cell culturing, additional surgeries or treatments to
prevent immune reactions.
[0113] I. Cartilage, Bone and Properties Thereof
[0114] Cartilage and bone, both, are connective tissues providing
support in the body for other soft tissues.
[0115] Bone is a hard connective tissue forming a skeleton,
consisting of osteoblast cells embedded in a matrix of mineralized
ground substance and collagen fibers. The collagen fibers are
impregnated with a form of calcium phosphate similar to
hydroxyapatite as well as with substantial quantities of carbonate,
citrate, sodium and magnesium. Bone is composed of approximately
75% of inorganic material and 25% of organic material. Bone
consists of a dense outer layer of compact substance covered by
periosteum and an inner, loose spongy substance, i.e. bone marrow.
Bone emplaced immediately below the cartilage is called subchondral
bone and it is a bone of specific composition and structure that is
itself underlain by the cancellous bone of the limb.
[0116] Cartilage is a mature connective tissue covering joints and
bones which is comprised of metabolically active but non-dividing
chondrocytes. This results in essential non-existence of
spontaneous ability of the cartilage to self-repair following the
injury or damage caused by age or disease.
[0117] Cartilage is characterized by its poor vascularity and a
firm consistency, and consists of mature non-dividing chondrocytes
(cells), collagen (interstitial matrix of fibers) and a ground
proteoglycan substance (glycoaminoglycans or mucopolysaccharides).
Later two are cumulatively known as extracellular matrix.
[0118] There are three kinds of cartilage, namely hyaline
cartilage, elastic cartilage and fibrocartilage. Hyaline cartilage,
found primarily in joints, has a frosted glass appearance with
interstitial substance containing fine type II collagen fibers
obscured by proteoglycan. Elastic cartilage is a cartilage in
which, in addition to the collagen fibers and proteoglycan, the
cells are surrounded by a capsular matrix further surrounded by an
interstitial matrix containing elastic fiber network. The elastic
cartilage is found, for example, in the central portion of the
epiglottis. Fibrocartilage contains Type I collagen fibers and is
typically found in transitional tissues between tendons, ligaments
or bones and also as a low quality replacement of injured hyaline
cartilage. This invention utilizes properties of acellular matrix
implant combined with certain conditions existing naturally in the
surrounding native cartilage further combined with certain steps
according to the method of the invention, to achieve the healing
and replacement of injured cartilage with the healthy and
functional hyaline cartilage.
[0119] A. Articular Cartilage and Articular Cartilage Defects
[0120] The articular cartilage of the joints, such as the knee
cartilage, is hyaline cartilage which consists of approximately 5%
of chondrocytes (total volume) seeded in approximately 95%
extracellular matrix (total volume). The extracellular matrix
contains a variety of macromolecules, including collagen and
glycosaminoglycan (GAG). The structure of the hyaline cartilage
matrix allows it to reasonably well absorb shock and withstand
shearing and compression forces. Normal hyaline cartilage has also
an extremely low coefficient of friction at the articular
surface.
[0121] Healthy hyaline cartilage has a contiguous consistency
without any lesions, tears, cracks, ruptures, holes or shredded
surface. Due to trauma, injury, disease such as osteoarthritis, or
aging, however, the contiguous surface of the cartilage is
disturbed and the cartilage surface shows cracks, tears, ruptures,
holes or shredded-surface resulting in cartilage lesions.
[0122] The articular cartilage is an unique tissue with no
vascular, nerve, or lymphatic supply. The lack of vascular and
lymphatic circulation may be one of the reasons why articular
cartilage has such a poor, almost non-existent intrinsic capacity
to heal. The mature metabolically active but non-dividing
chondrocytes in their lacunae surrounded by extracellular matrix do
not respond to damage signals by generating high-quality hyaline
cartilage. After a significant injury, unique mechanical functions
of articular cartilage are never reestablished spontaneously and
never completely because the water-absorption capacity of the type
II collagen/proteoglycan network is disturbed. The usual
replacement material for hyaline cartilage, which might develop
spontaneously in response to the injury of hyaline cartilage and
which replaces the injured cartilage, is the much weaker and
functionally inferior fibrocartilage.
[0123] Defects occurring due to cartilage trauma, injury, disease
or aging are tears, cracks, ruptures or holes which are solely
located in the joint cartilage. According to the method of the
invention, when such defect is treated, the implant is deposited
within the lesion, as illustrated in FIG. 1A.
[0124] FIG. 1A is a schematic representation of an acellular matrix
implant implantation into the cartilage defect. The scheme shows
the lesion implantation site with acellular matrix implanted
therein surrounded by host cartilage with underlaying undisturbed
subchondral bone. Emplacement of the top and bottom sealants are
also illustrated.
[0125] B. Currently Available Procedures for Repair of
Cartilage
[0126] A variety of surgical procedures have been developed and
used in attempts to repair damaged cartilage. These procedures are
performed with the intent of allowing bone marrow cells to
infiltrate the defect and promote its healing. Generally, these
procedures are only partly, if at all, successful. More often than
not, these procedures result in formation of a fibrous cartilage
tissue (fibrocartilage) which does fill and repair the cartilage
lesion but, because it is qualitatively different being made of
Type I collagen fibers, it is less durable, less resilient and
generally inferior than the normal articular hyaline cartilage and
thus has only a limited ability to withstand shock and shearing
forces than does healthy hyaline cartilage. Since all diarthroid
joints, particularly knees joints, are constantly subjected to
relatively large loads and shearing forces, replacement of the
healthy hyaline cartilage with fibrocartilage does not result in
complete tissue repair and functional recovery.
[0127] Among the currently available procedures for repair of the
articular cartilage injuries are the microfracture technique, the
mosaicplasty technique and autologous chondrocyte implantation
(ACI). However, in one way or another, all these techniques are
problematic. The mosaicplasty technique and ACI, for example, need
a biopsy of cartilage from a non-damaged articular cartilage area
and subsequent cell culture to grow the number of cells. As a
consequence, these techniques require at least two separate
surgeries. One system, the Carticel.RTM. system additionally
requires a second surgery site to harvest portion of and,
therefore, disrupt the tibial periosteum. While the microfracture
technique does not require a biopsy of articular cartilage, the
resulting tissue which develops is always fibrocartilage.
[0128] The method for treatment of injured, traumatized, diseased
or aged cartilage according to the current invention obviates the
above problems as it comprises treating the injured, traumatized,
diseased or aged cartilage with an acellular matrix implant without
need to remove tissue or cells for culturing, said implant prepared
by methods described below and implanted into the cartilage lesion
during the debriding surgery, as described below.
[0129] C. Osteochondral Area and Osteochondral Defects
[0130] Osteochondral area, in this context, means an area where the
bone and cartilage connect to each other and where the
osteochondral defects often develop following the injury.
[0131] FIG. 1B is a schematic representation of implantation of an
acellular matrix implant in the osteochondral defect. The scheme
shows the cartilage lesion implantation site with the acellular
matrix implanted therein surrounded by host cartilage with
underlaying bone lesion in the subchondral bone. A bone-inducing
composition or an acellular implant carrier comprising said
composition is deposited into the bone lesion separated from the
cartilage lesion by the bottom sealant. Emplacement of the top and
bottom sealants illustrates separation of the bone lesion from the
cartilage lesion by the bottom sealant such that each the cartilage
lesion and the bone lesion are treated separately using different
means, namely the acellular matrix implant for treatment of the
cartilage lesion and the bone-inducing composition or the acellular
carrier comprising said composition for treatment of the bone
defect.
[0132] Osteochondral defects are thus defects that are composites
of cartilage and underlying bone. Up-to-date, commonly used
treatments for osteochondral defects are surgical excisions,
mosaicplasty, osteochondral autogenous grafting, allogenic
grafting, bone cementing, deposition of metal or ceramic solid
composite materials, porous biomaterials and, lately, a
transplantation of autologous chondrocytes. Regretfully, none of
these procedures was found to be successful in treating these
defects and safe or comfortable for a patient. Typically, these
procedures involve two or more surgical procedures and long period,
generally at least two to three weeks, of time to culture the
transplantable cells. For example, mosaicplasty requires removal of
circular pieces of healthy subchondral bone and cartilage to be
used as transplantable plugs at a defect site. One obvious problem
with mosaicplasty is that the surgeon, in an open surgery, is
disrupting healthy tissue in order to repair the subchondral
defect. Clearly, the multiple surgeries and long period of time
between them necessarily extend a time of recovery to fully
functional joint and often result only in partial functional
restoration as both the bone and cartilage defects are filled with
the fibrocartilage instead of the bone and hyaline cartilage.
[0133] One example of the osteochondral defect which is common and
very difficult to treat is osteochodritis dissecans. Osteochodritis
dissecans is a focal bone-cartilage lesion characterized by
separation of an osteochondral fragment from the articular surface.
Attempts to treat this injury with allograph transplants faces the
same problem of second surgery and disruption of the healthy
tissue, as described above. Thus it would be advantageous to have
available a method which would remove a need for second surgery and
yet provide a means for a cartilage and bone repair.
[0134] The current method provides a solution to the above-outlined
problems by implanting, during the first arthroscopic surgery, a
bone-inducing composition or a carrier comprising said composition
comprising a bone-inducing agents into the bone lesion and an
acellular matrix implant into the cartilage lesion thereby
providing, in one surgery, treatments for both the bone and
cartilage defects.
[0135] D. Bone and Bone Defects
[0136] The restorative method according to this invention is
additionally also suitable for repair of the skeletal bone
lesions.
[0137] The skeletal bone lesions are lesions which are either
solely or at least partially located in the skeletal part of the
bone, that is the bone placed immediately below the subchondral
bone region, as seen in FIG. 1C.
[0138] FIG. 1C is a schematic representation of the deep
osteochondro-skeletal bone injury extending into the skeletal bone.
The figure shows the positioning of the host cartilage, subchondral
bone and the skeletal bone as well as emplacement of the acellular
matrix implant into the osteochondral defect and the bone-inducing
composition into the subchondral and skeletal bone defect. The
scheme shows the cartilage lesion implantation site with the
acellular matrix implanted therein surrounded by host cartilage
with underlaying bone lesion in the subchondral bone. The
bone-inducing composition or a carrier comprising said composition
is deposited into the bone lesion. The carrier for this purpose may
be any matrix described above but is preferably collagenous,
hydrogel or a polymer of an aromatic organic acid containing
structure. Emplacement of the top and bottom sealant are also shown
wherein the bottom sealant separates the bone portion of the defect
from the cartilage lesion such that each is treated separately
using different means.
[0139] In an alternative, the bone-inducing composition and/or the
acellular implant carrier comprising such composition can be used
for treatment of simple skeletal bone defects, lesions or fractures
without a need for cartilage implant.
[0140] If and when the method of the invention is used for
treatment of skeletal bone lesions, the bone-inducing composition
alone or incorporated into a carrier, preferably dissolved in
collagen or another binding agent, is deposited directly into the
skeletal bone lesion. The bone-inducing agent is selected from the
group consisting of calcium phosphate, hydroxyapatite,
organoapatite, titanium oxide, demineralized bone powder,
poly-L-lactic, polyglycolic acid or a copolymer thereof and a bone
morphogenic protein.
[0141] A preferred bone-inducing agent is the demineralized bone
powder (DMB). DMB is derived from bone by, for example, acid
extraction of the calcium phosphate. Following such extraction, the
DMB retains, in addition to the bone collagen other chemical
elements found in the bone, including the naturally present members
of TGF-.beta. superfamily of bone development factors. These
factors may also be extracted by further treatment of bone with
such materials as quanidine hydrochloride. When these naturally
occurring TGF-.beta.s are present in the DMB, no further
bone-inducing agents are needed to be present because DMB has a
porous microstructure suitable for bone formation.
[0142] It is to be understood that the DMB itself is very light
powder and therefore, it is preferably formulated in an agent
having a binding capabilities. The most preferred binding agent is
collagen or collagen-like agents, hydrogels, alginates, etc.
[0143] II. An Acellular Matrix Implant for Treatment of Cartilage
Lesions
[0144] The current invention provides a method for treatment of
injured, damaged, diseased or aged cartilage. To this end, the
method involves implantation of the acellular matrix implant into
the injured, damaged, diseased or aged cartilage at a site of
injury or at a site of a defect caused by disease or age, in a
single surgery. The acellular matrix implant is a collagenous
construct or a polymer of an aromatic organic acid comprising
various components as described below.
[0145] A. Preparation of an Acellular Matrix Implant
[0146] Preparation of the acellular matrix implant for implanting
into the cartilage lesion involves preparation of acellular support
matrix, typically a collagenous scaffold or sponge,
thermo-reversible gelation hydrogel or a polymer of an aromatic
organic acid and implanting said matrix into the cartilage defect
in situ.
[0147] The acellular matrix implant, such as the one seen in FIG.
2A, is prepared according to the method of the invention and
implanted into artificially generated lesions in a swine's knee
weight bearing region. FIG. 2A is an image of an actual acellular
matrix sponge implant used for implantation, here held in the
forceps. The sponge has a size of 5 mm in diameter and 1.5 mm in
thickness and comprises a composition of collagen sponge and
collagen gel having pores of sizes from about 200-400 .mu.m (FIG.
2B). When the sponge is implanted into the lesion, chondrocytes are
activated and migrate into the porous structure of the sponge where
they begin to secrete a new extracellular matrix ultimately
replacing the collagen sponge and gel with the new hyaline
cartilage. The sponge and gel naturally biodegrade and are
metabolically removed from the lesion.
[0148] FIG. 2B is a cross-side view scheme of a honeycomb structure
of the acellular matrix sponge seen in FIG. 2A illustrating a
relative positioning of the collagen sponge, collagen gel and pores
within the acellular matrix sponge.
[0149] The matrices of the acellular matrix implant deposited into
the lesion are comprised of biodegradable materials which permit
said implant to function for certain period of time needed for
formation of the hyaline cartilage. Such biodegradable materials
are subsequently biodegraded and metabolically removed from the
site of implantation leaving, if any, only non-toxic residues.
These materials were additionally found to promote formation of the
superficial cartilage layer which covers the lesion containing the
implant thereby protecting a newly formed hyaline cartilage. The
biodegradable materials may additionally include enzymes, such as
metalloproteinases, paracrine or autocrine growth hormones,
GAG-lyases and such like enzymes, soluble protein mediators and
other supplements. Presence or addition of these materials may
enhance activation of mature, metabolically active but non-dividing
chondrocytes present in the surrounding native host cartilage and
migration of these chondrocytes from the native host cartilage
surrounding the lesion cavity into said acellular matrix implant
emplaced within said lesion.
[0150] The present invention thus concerns a discovery that when
the acellular matrix implant according to the invention is
implanted into a cartilage defect, under conditions described
below, the older inactive chondrocytes residing within the
surrounding native cartilage are induced to migrate into the defect
where these chondrocytes are activated from static non-dividing
stage to an active stage where they divide, multiply, promote
growth of the extracellular matrix and generate a new hyaline
cartilage in situ. Following the implantation of the acellular
matrix implant, the cartilage defect is quickly repaired,
particularly in the young individuals, by chondrocyte migration and
by formation of the extracellular matrix supported by the
metalloproteinases naturally present in sufficient amounts in
tissues of the young individuals. For the repair of lesions in
older subjects, the GAG-lyases and metalloproteinases, growth
factors and other components are added or incorporated into said
matrix before implantation or they may be conveniently used to coat
said matrix to promote degradation of the injured cell.
[0151] A process for activation of chondrocytes was found to
require certain period of time, typically from about 1 hour to
about 3 weeks, typically only about 6 hours to about 3 days. The
process for complete replacement of the implant matrix with the
hyaline cartilage typically takes from one week to several months
provided that the treated individual becomes normally physically
active subjecting said new cartilage to the intermittent
hydrostatic pressure by, for example, walking, running or
biking.
[0152] B. Induction of Chondrocyte Migration
[0153] Induction of chondrocyte migration from the surrounding
native cartilage involves biological actions of various agents
either naturally present within the cartilage, cartilage
surrounding tissue, blood or plasma or they are added either
before, during or after the surgery to promote release, activation
and migration of chondrocytes from the native surrounding host
cartilage into the implant.
[0154] One of the steps in achieving the activation of the
chondrocytes is the use of sealants at the top and bottom of the
articular cartilage lesion. This step results in creation of a
cavity into which the acellular matrix implant is deposited. A
container-like porous property of the acellular collagenous matrix
implant permits infusion and concentration of soluble protein
mediators, enzymes, growth or other factors, etc., naturally
present in the host's surrounding healthy cartilage.
[0155] Sealing of the top and bottom of the defect before and after
insertion of the acellular matrix implant results in accumulation
of autocrine and paracrine growth factors that are released by
chondrocytes in the adjacent extracellular matrix, enabling these
factors to induce cell migration into the implant. Suitable growth
factors include, among others, certain transforming growth factors,
platelet-derived growth factors, fibroblast growth factors and
insulin-like growth factor-I. Additionally, these and other
supplements, such as the GAG-lyases (matrix remodeling enzymes),
may be used to coat the implant before its insertion into the
lesion or the lesion itself may be coated.
[0156] The acellular matrix implant sequestered within the lesion
cavity by the top and bottom sealant, however, remains in flowable
communication with the adjacent cartilage. This arrangement creates
conditions resulting in decrease of levels of inhibitors of the
matrix remodeling enzymes, such as tissue inhibitors of
metalloproteinase-1 (TIMP-1), metalloproteinase-2 (TIMP-2) and
metalloproteinase-3 (TIMP-3), at the defect site. As a consequence,
the matrix metalloproteinases (MMP-1, MMP-2, MMP-3) become
accessible to enzymatic activation and degrade the adjacent
extracellular matrix thereby releasing chondrocytes localized
therein resulting in chondrocytes migration from the surrounding
host cartilage into the acellular matrix implant or coat the walls
of the lesion itself with the sugar lyases.
[0157] The acellular matrix implant sealed within the lesion also
becomes a repository of exogenous growth factors that pass through
the bottom sealant layer in response to joint loading and
hydrostatic pressure to which the joint is subjected when
undergoing a normal physical activity such as walking, running or
biking. Consequently, in response to the hydrostatic pressure load,
these factors become more concentrated within the defect site and
chondrocytes released from adjacent areas of the surrounding
extracellular matrix migrate into the lesion with ensuing
chondrocyte proliferation and initiation of the de novo
extracellular matrix synthesis within the lesion.
[0158] Moreover, the acellular matrix of the implant fills the
defect with a material that has a reduced stiffness relative to
normal articular cartilage and permits deformation of the adjacent
native cartilage matrix edges thereby increasing level of shear
stress further resulting in increased release of soluble mediators
that indicate matrix remodeling and chondrocyte migration into the
acellular matrix implant.
[0159] The presence of the acellular matrix implant sealed to the
adjacent cartilage boundaries thus creates conditions by which
matrix remodeling enzymes, namely matrix metalloproteinases,
aggrecanases and cathepsins, become concentrated at the defect site
and initiate enzymatic opening of the adjacent extracellular matrix
so that chondrocytes may migrate into the acellular matrix implant,
be deposited within its matrix, begin to divide and proliferate and
secrete the new extracellular matrix, ultimately leading to
formation of normal healthy hyaline cartilage.
[0160] C. Types of Acellular Matrix Implant
[0161] The acellular matrix implant provides a structural support
for migration, growth and two or three-dimensional propagation of
chondrocytes in situ. Generally, the acellular matrix is
biologically biocompatible, biodegradable, hydrophilic and
preferably has a neutral charge.
[0162] Typically, the implant is a two or three-dimensional
structural composition, or a composition able to be converted into
such structure, containing a plurality of pores dividing the space
into a fluidically connected interstitial network. In some
embodiments the implant is a sponge-like structure, honeycomb-like
lattice, sol-gel, gel or thermo-reversible gelation hydrogel.
[0163] Typically, the implant is prepared from a collagenous gel or
gel solution containing Type I collagen, Type II collagen, Type IV
collagen, gelatin, agarose, hyaluronin, cell-contracted collagens
containing proteoglycans, polymers of organic aromatic acids,
glycosaminoglycans or glycoproteins, fibronectins, laminins,
bioactive peptide growth factors, cytokines, elastins, fibrins,
synthetic polymeric fibers made of poly-acids such as polylactic,
polyglycotic or polyamino acids, polycaprolactones, polypeptide
gels, copolymers thereof and combinations thereof. Preferably, the
implant matrix is a gel, sol-gel, a polymer of an aromatic organic
acid or a polymeric thermo-reversible gel. Most preferably the
implant matrix contains aqueous Type I collagen.
[0164] The acellular matrix implant may be of a type of sponge,
scaffold or honeycomb sponge, scaffold or honeycomb-like lattice or
it may be a gel, sol-gel or thermo-reversible gel composition or it
may be a polymer of an aromatic organic acid.
[0165] The acellular matrix implant may be produced as two or
three-dimensional entities having an approximate size of the lesion
into which they are deposited. Their size and shape is determined
by the shape and size of the defect.
[0166] a. Acellular Sponges or Sponge-Like Implants
[0167] In general, any polymeric material can serve as the support
matrix, provided it is biocompatible with tissue and possesses the
required geometry. Polymers, natural or synthetic, which can be
induced to undergo formation of fibers or coacervates, can be
freeze-dried as aqueous dispersions to form sponges.
[0168] In addition to collagen, a wide range of polymers may be
suitable for the fabrication of sponges, including agarose,
hyaluronic acid, alginic acid, dextrans, polyHEMA, and poly-vinyl
alcohol alone or in combination.
[0169] Typically, such sponges must be stabilized by cross-linking,
such as, for example, ionizing radiation. Practical example
includes preparation of freeze-dried sponges of
poly-hydroxyethyl-methacrylate (pHEMA), optionally containing
additional molecules, such as gelatin, advantageously entrapped
within. Incorporation of agarose, hyaluronic acid, or other
bio-active polymers can be used to modulate cellular responses. All
these types of sponges can function advantageously as implant
matrices for the purposes of the present invention.
[0170] The gel or gel solution used for preparation of the sponge
or sponge-like implant is typically washed with water and
subsequently freeze-dried or lyophilized to yield a sponge like
matrix able to incorporate the migrating chondrocytes within the
matrix. The acellular matrix implant of the current invention acts
like a porous sponge when infiltrated with the migrating
chondrocytes wherein the cells are distributed within the sponge
pores, providing a mesh-like support permitting the chondrocytes to
migrate and settle there, begin to divide and proliferate and
secrete materials for generation of new extracellular matrix and
eventually for generation of hyaline cartilage contiguous with the
existing healthy surrounding cartilage.
[0171] One important aspect of the sponge implant is the pore size
of the sponge matrix. Sponges having different pore sizes permit
faster or slower infiltration of the chondrocytes into said sponge,
faster or slower growth and propagation of the cells and,
ultimately, the higher or lower density of the cells in the
implant. Such pore size may be adjusted by varying the pH of the
gel solution, collagen concentration, lyophilization conditions,
etc., during implant fabrication. Typically, the pore size of the
sponge is from about 50 to about 500 .mu.m, preferably the pore
size is between 100 and 300 .mu.m and most preferably about 200
.mu.m.
[0172] The pore size of the acellular matrix implant will be
selected depending on the recipient. In the young recipient where
the metalloproteinases are present naturally and active, the pore
size will be smaller as the activated chondrocytes will rapidly
proliferate through the pores and secrete extracellular matrix. In
older recipients, the pores will be bigger as the migrating
chondrocytes will be sluggish and will need more time to settle in
the pores and proliferate.
[0173] An exemplary acellular matrix implant made of collagen is
seen in FIG. 2. FIG. 2A is an example image of acellular
collagenous matrix implant of size 4 mm in diameter and of 1.5 mm
in thickness. The seeding density of this implant is between
300,000 chondrocytes per 25 .mu.l volume corresponding to about
12-15 millions cells/ml. The cell density following the
implantation of the acellular matrix implant is, of course,
dependent on the rapidity of the migration of chondrocytes from the
surrounding native cartilage and on their ability to divide and
rapidity of their multiplication, however, the collagenous matrix
of the implant has a capacity to accommodate this range of
migrating cells.
[0174] The acellular sponge may be prepared according to procedures
described in Example 1, or by any other procedure, such as, for
example, procedures described in the U.S. Pat. Nos. 6,022,744;
5,206,028; 5,656,492; 4,522,753 and 6,080,194 or in co-pending
application Ser. Nos. 10/625,822, 10/625,245 and 10/626,459, herein
incorporated by reference.
[0175] b. Acellular Scaffold or Honeycomb Implants
[0176] One type of the implant of the invention is an acellular
scaffold, honeycomb scaffold, honeycomb sponge or honeycomb-like
lattice. All these implants contain a honeycomb-like lattice matrix
providing a support structure for migrating and dividing
chondrocytes. The honeycomb-like matrix is similar to that of the
sponge described above but has that typical pattern of the
honeycomb. Such honeycomb matrix provides a growth platform for the
migrating chondrocytes and permits three-dimensional propagation of
the migrated and divided chondrocytes thereby providing a
structural support for formation of new hyaline cartilage.
[0177] FIG. 2B is a side view scheme of honeycomb structure of
acellular matrix showing a collagen sponge and collagen gel with
pore (*) size of each column of about 200-400 .mu.m.
[0178] The honeycomb-like matrix is fabricated from a polymerous
compound, such as collagen, gelatin, Type I collagen, Type II
collagen or any other polymer, as described above for the sponge,
having a desirable properties. In the preferred embodiment, the
honeycomb-like acellular matrix implant is prepared from a solution
comprising Type I collagen.
[0179] The pores of the honeycomb-like implant are evenly
distributed within said honeycomb matrix to form a structure able
of taking in and evenly distributing the migrated chondrocytes.
[0180] One preferred type of acellular matrix implant is Type-I
collagen support matrix fabricated into a honeycomb-lattice,
commercially available from Koken Company, Ltd., Tokyo, Japan,
under the trade name Honeycomb Sponge.
[0181] Acellular matrix implant of the invention thus may be any
suitable biodegradable structure, gel or solution, preferably
containing collagen. For the purposes of convenience in implanting,
such implant is typically a gel, preferably sol-gel transitional
solution which changes the state of the solution from liquid sol to
solid gel above room temperature. The most preferred such solution
is the thermo-reversible gelation hydrogel or a thermo-reversible
polymer gel as described below.
[0182] c. Sol-Gel Acellular Matrix Implant
[0183] Another type of acellular matrix implant is the implant
matrix fabricated from sol-gel materials wherein said sol-gel
materials can be converted from sol to gel and vice versa by
changing temperature. For these materials the sol-gel transition
occurs on the opposite temperature cycle of agar and gelatin gels.
Thus, in these materials the sol is converted to a solid gel at a
higher temperature.
[0184] Sol-gel material is a material which is a viscous sol at
temperatures below 15.degree. C. and a solid gel at temperatures
around and above 37.degree. C. Typically, these materials change
their form from sol to gel by transition at temperatures between
about 15.degree. C. and 37.degree. C. and are in a transitional
state at temperatures between 15.degree. C. and 370. However, by
changing the hydrogel composition, the transition temperature of
the sol-gel may be predetermined to be higher or lower than those
given above. The most preferred materials are Type I collagen
containing gels and a thermo-reversible gelation hydrogel (TRGH)
which has a rapid gelation point.
[0185] In one embodiment, the sol-gel material is substantially
composed of Type I collagen and, in the form of 99.9% pure
pepsin-solubilized bovine dermal collagen dissolved in 0.012 N HCl,
commercially available under the trade name VITROGEN.RTM. from
Cohesion Corporation, Palo Alto, Calif. One important
characteristic of this sol-gel is its ability to be cured by
transition into a solid gel form wherein said gel cannot be mixed
or poured or otherwise disturbed thereby forming a solid structure
optionally containing other components supporting the chondrocytes
activation and migration. Sterile collagen for tissue culture may
be additionally obtained from other sources, such as, for example,
Collaborative Biomedical, Bedford, Mass., and Gattefosse, SA, St.
Priest, France.
[0186] Type I collagen sol-gel is generally suitable and preferred
material for fabrication of an acellular sol-gel implant.
[0187] d. Thermo-Reversible Gelation Hydrogel Implants
[0188] Additionally, the acellular matrix implant may be prepared
from thermo-reversible materials similar to sol-gel which
materials, however, have much faster point of transition, without
hysteresis, from sol to gel and vice versa.
[0189] The thermo-reversible property is important for implantation
of the acellular matrix implant into the lesion cavity as it may be
implanted into the lesion cavity in its sol state whereby filling
said cavity with the sol wherein the sol forms itself according to
the exact shape of the cavity leaving no empty space or being too
large or too small, as the case may be, for a prefabricated sponge
or a honeycomb lattice. Following the warming of the sol emplaced
within the articular lesion cavity to the natural body temperature,
the sol instantly transitions and becomes solid gel providing a
structural support for the migrating chondrocytes from the
surrounding native cartilage.
[0190] One characteristic of the sol-gel is its ability to be cured
or transitioned from a liquid into a solid form and vice versa.
This property may be advantageously used for solidifying the liquid
or liquefying the solid gel acellular matrix implant within the
cartilage lesion as well as for delivery, storing or preservation
purposes of said acellular matrix implant. Additionally, these
properties of sol-gel also permit its use as a support matrix by
changing its sol-gel transition by increasing or decreasing
temperature in the lesion, or exposing the sol-gel to various
chemical or physical conditions or ultraviolet radiation.
[0191] In one embodiment, the acellular matrix implant is a
thermo-reversible gelation hydrogel or gel polymer kept stored and
implanted at temperatures between 5.degree. C. and 15.degree. C. At
that temperature, the hydrogel is at a liquid sol stage and permits
easy emplacement into the lesion as the sol. Once the sol is
emplaced within the lesion, the sol is naturally or artificially
subjected to higher temperature of about 30.degree. C. and
37.degree. C. at which temperature the liquid sol solidifies into
solid gel. The gelling time is from about several minutes to
several hours, typically about 1 hour. In such an instance, the
solidified gel may itself become and be used as an implant or this
sol may be loaded into a separate support matrix, such as a sponge
or scaffold honeycomb implant.
[0192] The primary characteristic of the thermo-reversible gelation
hydrogel (TRGH) is that upon its degradation within the body it
does not leave biologically deleterious material and that it does
not absorb water at gel temperatures. TRGH has a very quick sol-gel
transformation which requires no cure time and occurs simply as a
function of temperature without hysteresis. The sol-gel transition
temperature can be set at any temperature in the range from
5.degree. C. to 70.degree. C. by the molecular design of the
thermo-reversible gelation polymer (TGP), a high molecular weight
polymer, of which less than 5 wt % is enough for hydrogel
formation.
[0193] The thermo-reversible gelation hydrogel (TRGH), should be
compressively strong and stable at 37.degree. C. and below till
about 32.degree. C., that is to about temperature of the synovial
capsule of the joint which is typically below 37.degree. C., but
should easily solubilize below 30-31.degree. C. to be able to be
conveniently changed to the sol within the lesion cavity. The
compressive strength of the TRGH must be able to resist compression
by the normal activity of the joint.
[0194] The typical TRGH is generally made of blocks of high
molecular weight polymer comprising numerous hydrophobic domains
cross-linked with hydrophilic polymer blocks. TRGH has a low
osmotic pressure and is very stable as it is not dissolved in water
when the temperature is maintained above the sol-gel transition
temperature. Hydrophilic polymer blocks in the hydrogel prevent
macroscopic phase separation and separation of water from hydrogel
during gelation. These properties make it especially suitable for
safe storing and extended shelf-life.
[0195] In this regard, the thermo-reversible hydrogel is an aqueous
solution of thermo-reversible gelation polymer (TGP) which turns
into hydrogel upon heating and liquefies upon cooling. TGP is a
block copolymer composed of temperature responsive polymer (TRP)
block, such as poly(N-isopropylacrylamide) or polypropylene oxide
and of hydrophilic polymer blocks such as polyethylene oxide.
[0196] Thermally reversible hydrogels consisting of co-polymers of
polyethylene oxide and polypropylene oxide are available, for
example, from BASF Wyandotte Chemical Corporation under the trade
name of Pluronics.
[0197] In general, thermo-reversibility is due to the presence of
hydrophobic and hydrophilic groups on the same polymer chain, such
as in the case of collagen and copolymers of polyethylene oxide and
polypropylene oxide. When the polymer solution is warmed,
hydrophobic interactions cause chain association and gelation; when
the polymer solution is cooled, the hydrophobic interaction
disappears and the polymer chains are dis-associated, leading to
dissolution of the gel. Any suitably biocompatible polymer, natural
or synthetic, with such characteristics will exhibit the same
reversible gelling behavior.
[0198] e) Acellular Gel Implants
[0199] The acellular matrix implants of the invention may
alternatively be prepared from various gel materials, such as
suspending gels, not necessarily thermo-reversible, which are
commercially available and may be suitable for use as acellular
matrix implants as long as they are biodegradable.
[0200] One example of such gel is polyethylene glycol (PEG) and its
derivatives, in which one PEG chain contains vinyl sulfone or
acrylate end groups and the other PEG chain contains free thiol
groups which covalently bond to form thio-ether linkages. If one or
both partner PEG molecules are branched (three- or four-armed), the
coupling results in a gel network. If the molecular weight of the
PEG chains used for the implant preparation is between 500 and
10,000 Daltons along any linear chain segment, the network will be
open and suitable for receiving migrating chondrocytes, swellable
by interstitial water, and compatible with living chondrocytes.
[0201] The coupling reaction of PEG can be accomplished, for
example, by preparing 5 to 20% (w/v) solutions of each PEG
separately in aqueous buffers or cell culture media. Just prior to
implantation, thiol, PEG and the acrylate or vinyl sulfone PEG are
mixed and infused into the lesion. Gelation will begin
spontaneously in 1 to 5 minutes. The rate of gelation can be
modulated somewhat by the concentration of PEG reagent and by pH.
The rate of coupling is faster at pH 7.8 than at pH 6.9. Thus, by
modifying the pH of the PEG containing mixture, the gelation
process may be controlled to be faster or slower, as desired by the
surgeon. Such gels are, however, typically not degradable within
the body unless the additional ester or labile linkages are
incorporated into the chain. PEG reagents may be purchased from
Shearwater Polymers, Huntsville, Ala., USA; or from SunBio,
Korea.
[0202] In a second alternative, the gelling material may be
alginate. Alginate solutions are gellable in the presence of
calcium ions. This reaction has been employed for many years to
suspend cells in gels or micro-capsules. A solution of alginate
(1-2%; w/v) in culture media devoid of calcium or other divalent
ions is mixed in a solution containing calcium chloride which will
gel the alginate. Analogous reactions can be accomplished with
other polymers which bear negatively charged carboxyl groups, such
as hyaluronic acid. Viscous solutions of hyaluronic acid can be
gelled by diffusion of ferric ions.
[0203] f. A Polymer of an Aromatic Organic Acid Matrix
[0204] The acellular implant may also conveniently be made of a
polymer of an aromatic organic acid. Polymers of this type have
typically a negative charge and are thus preferred for use as a
bone-inducing composition carriers. However, these type of
compounds may also be used and are suitable for use as cartilage
acellular implants.
[0205] G. Biodegradable Implant
[0206] The acellular matrix implant of the invention is a temporary
structure intended to provide a temporary supporting for the
migrating, dividing, proliferating and extracellular matrix
secreting chondrocytes released from the surrounding cartilage.
[0207] Consequently, the implant of the invention must be fully
biodegradable. Whether it is a sponge, honeycomb lattice, sol-gel
or TRGH, in time, the delivered implant is disintegrated or
incorporated into the existing cartilage and the TRGH is
subsequently degraded leaving no undesirable debris behind.
[0208] Overall, any of the acellular matrix implants for cartilage
defects described above is suitable for implantation into a
cartilage lesion of any size and shape and provides a support for a
structural rebuilding of the cartilage by migrating chondrocytes
therein from the surrounding healthy host cartilage. The
implantation of the implant of the invention results in the
generation of normal healthy hyaline cartilage and in complete
healing of the cartilage defect.
[0209] III. Osteochondral Defects and Treatment Thereof
[0210] Lesions of the articular cartilage are often accompanied by
lesions of the underlying bone. Such defects are thus a composite
of cartilage and underlying bone. These defects are herein
cumulatively called osteochondral defects.
[0211] A. Method for Treatment of Osteochondral Defects
[0212] The osteochondral defects are caused by injury of the
cartilage and bone. The cartilage and bone are histologically two
different connective tissues, as described above. Consequently, it
is not possible to effectively treat both using the same methods
and means and such treatment is thus complex and more difficult
than a treatment of the cartilage lesion or chipped bone alone.
Furthermore, bone development can influence cartilage development
such that it acts as a barrier to further cartilage development
during its critical developmental stages.
[0213] In one attempt to treat these complex injuries, a
mosaicplasty technique was developed. The mosaicplasty, as already
mentioned above, involves a removal of grafts from the healthy
tissue and plugging such grafts into the both bone and cartilage
lesions. An obvious defect of this technique is that in order to
treat the injured site, surgeon has to remove, during the open
surgical procedure, a healthy tissue from another site thereby
disrupting the healthy tissue in the process.
[0214] When, however, the method of the current invention is used
to treat these complex osteochondral injuries, it is possible to
treat both the bone and cartilage lesions during the same surgery
without need to remove and disturb the healthy tissue and/or
undergo multiple surgeries required, for example, for allograft
transplantation and other techniques.
[0215] The current method permits such dual treatment
simultaneously by implantation of, in combination, an acellular
matrix implant and a bone-inducing composition or a carrier
comprising said composition comprising a bone-inducing agents
further, preferably, in combination with biologically acceptable
sealants.
[0216] In practice, during the same surgery, the surgeon first
debrides both lesions and deposits the bone-inducing composition or
a carrier comprising said composition into the bone lesion and
covers said bone lesions with one or several layers of a
biologically acceptable sealant, preferably a modified highly
polymerizable sealant selected from those described below in
section IV. After the sealant polymerizes, typically within several
minutes, preferably between 0.5 and 5 minutes, the acellular matrix
implant is deposited into the cartilage lesion and covered with yet
another layer of the sealant, herein called the top sealant. In
this way, the bone-inducing composition or a carrier comprising
said composition is sequestered within the bone lesion and the bone
forming agents, such as, for example, demineralized bone powder,
calcium phosphates, calcium citrate, hydroxyapatite, organoapatite,
titanium oxide, polyacrylate, alone or in combination, and a bone
morphogenic protein and/or other known bone-inducing agents act as
inducement for osteoblast migration from the surrounding bone
without interference from the acellular matrix implant. As a
consequence of this separation of the bone and cartilage lesion,
there is no invasion of the hyaline cartilage or formation of
fibrocartilage in the bone lesion.
[0217] Conversely, when the acellular implant is separated from the
bone-inducing composition or a carrier comprising said composition,
there is no interference from any of the bone-inducing agents with
the chondrocyte migration, extracelular matrix formation and
generation of the hyaline cartilage. Each the bone and the
cartilage are treated separately and yet simultaneously during one
arthroscopic surgery.
[0218] The sealant may be deposited, preferably, as is, that is
without any additional agents being added, or it may be added to
the bone-inducing composition or a carrier comprising said
composition, if desirable.
[0219] The bone-inducing composition or a carrier comprising said
composition deposited within the bone defect covered with the
sealant is left in the lesion in order to achieve the bone
reconstruction and growth. Both the composition and the sealant are
aiding in a bone natural healing.
[0220] The acellular matrix implant implanted within the cartilage
defect separated from the bone lesion by a layer of the sealant and
covered with the top sealant is left in the cartilage lesion until
it biodegrades when the hyaline cartilage replacement is formed in
order to achieve the chondrocyte migration and formation of
extracellular matrix.
[0221] A typical process for repair of osteochondral defects is the
cleaning and debridement the osteochondral defect, depositing the
bone-inducing composition or a carrier comprising said composition
containing the bone-inducing agents, up to the upper limit of the
lesion in subchondral bone, applying a layer of the sealant over
the composition and letting the sealant to polymerize. After the
sealant polymerizes, typically in from about 0.2 to about 10
minutes, preferably about 0.5-5 minutes, the surgery proceeds with
implanting the acellular matrix implant into the cartilage lesion,
as described above. The cartilage lesion containing the implant is
then covered with yet another layer of the sealant (top sealant) to
seal and protect the wound from the exterior.
[0222] The above described procedure is particularly suitable for
treatment of osteochondral injuries as it permits dual treatment
under different conditions being implemented during the same
surgery.
[0223] One specific case of osteochondral defects is osteochodritis
dissecans, where a focal lesion of the bone and cartilage results
in a loose or totally dislocated osteochondral fragment. Currently
the only available treatment requires three independent surgeries
including biopsy harvesting of periosteum (first surgery),
culturing cells, removal of the loose fragment (second surgery),
introduction of the cultured cells into the lesion and
bone-grafting (third surgery).
[0224] The current method, as described above, or modified to
include a step of the fragment removal, during a single surgery,
eliminates a need for two or three surgeries, as all steps
necessary for repair of the osteochondritis dissecans are performed
at the same time during one surgery.
[0225] B. Bone-Inducing Agents
[0226] Bone-inducing agents are compounds or proteins having a
definite ability to promote formation of the bone.
[0227] The most suitable bone forming agents are demineralized bone
powder (DMP), calcium phosphate, calcium citrate, hydroxyapatite,
organoapatite, titanium oxide and growth factors, namely a group of
growth factors known as bone morphogenic proteins, fibroblast
growth factor (FGF), platelet derived growth factor (PDFG),
epithelial growth factor (EGF), glioma derived factor (GDF) and
transforming growth factor beta-1 (TGF-.beta.1). These growth
factors may be used individually and/or in combination with each
other or with other bone-inducing factors.
[0228] The demineralized bone powder is particularly suitable to be
used as a bone-inducing composition or as a bone-inducing carrier
and no other compounds are needed to serve as bone inducer or
supporting structure and necessary because the demineralized bone
powder mimics microporous structure of the bone. Before depositing
the DMB into the bone or subchondral bone lesion, the DMB may be
conveniently dissolved in collagen or some other adhesive fluid or
hydrogel which will permit its deposition into the lesion but
itself will have no bone-inducing function. The used amount of DMB
is such that is makes a concentrated highly viscous paste. The used
amount depends on the structure and grind of the DMB.
[0229] Bone morphogenic proteins are typically identified by the
abbreviation BMP and are further distinguished from each other by
numbering, such as BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8
and BMP-14. Some of them are further identified by a generic name,
such as, for example BMP-3 is called osteogenin, BMP-3B is GDF-10,
etc. The bone morphogenic proteins are administered generally in
concentration (per carrier volume or weight) of from about 0.01 to
about 5 mg/cm.sup.3, preferably from about 0.1 to about 1.5
mg/cm.sup.3 or from about 0.01 mg/g to about 5 mg/g, preferably
from about 0.1 mg to about 2.5 mg/g.
[0230] C. Bone-Inducing Composition
[0231] The bone-inducing composition or a carrier comprising said
composition of the invention comprises one or several bone-inducing
agents as listed above, in concentrations as disclosed. The
bone-inducing composition may be administered as a powder,
solution, gel, sol-gel, TRGH mixed in concentration given above or
incorporated into a structure similar to that of the acellular
implant, pre-prepared and implanted into the bone lesion or
fracture. The composition prepared as TRGH, for example, is
prepared as a sol solution and administered as such. The sol
subsequently changes its state into the gel filling out the whole
bone lesion. The bone-inducing agents may also be dissolved in PEG,
collagen, alginate, etc., and deposited as such. It could also be
soaked up in a second sponge system like the acellular matrix
sponge described above.
[0232] The preferred mode for the deposition of the bone-inducing
agents into the osteochondral or bone lesion is to dissolve the
agent in a gel, such as diluted collagen, alginate and such like
gels.
[0233] D. Bone-Inducing Carrier
[0234] A bone-inducing carrier or a carrier comprising
bone-inducing composition is a carrier compound which is suitable
for depositing said bone-inducing composition comprising at least
one bone-inducing agent or, preferably, a combination of several
agents into a bone lesion. Typically, the carrier will be a
biodegradable porous matrix, hydrogel, sponge, honeycomb, or
scaffold having large pores from about 50 to about 150 .mu.m, which
pores encourage migration of osteoblast. The carrier will also have
an interconnecting small pores of about 0.1 to about 10 .mu.m which
connect the large pores, permit the osteoblast to settle within the
carrier and provide a supporting matrix and connecting
microstructure for supply of nutrient and other factors thereby
permitting the bone formation. The surface of such carrier might be
negatively charged encouraging pseudopod attachment of osteoblasts
and their migration into the carrier resulting in the bone
formation.
[0235] IV. Biologically Acceptable Sealants
[0236] Generally, the implant is implanted into the cartilage or
bone lesion and between at least two layers, of top and bottom of
biologically acceptable adhesive sealants.
[0237] In practice, the first (bottom) layer of the sealant is
introduced into the lesion and deposited at the bottom of the
lesion. The first sealant's function is to prevent entry and to
block the migration of subchondral and synovial cells of the
extraneous components, such as blood-borne agents, cell and cell
debris, etc. Before the implant is deposited, such debris could
interfere with the integration of the acellular matrix implant. The
second function of the first sealant is to contain enzymes,
hormones and other components which are naturally present in the
lesion and which are needed for chondrocyte activation, migration,
secretion of other agents and proliferation of newly formed
extracellular matrix and hyaline cartilage. Then the acellular
matrix implant is implanted over the first sealant. The second
(top) sealant layer is placed over the acellular matrix implant.
The presence of both these sealants in combination with the
acellular matrix implant results in successful activation of
chondrocytes, their migration and integration into the implant
matrix and ultimately in new formation of joint hyaline
cartilage.
[0238] A. A First-Bottom Sealant
[0239] In a method for treatment of cartilage lesions, the first
(bottom) sealant forms an interface between the introduced implant
and the native tissue, such as subchondral bone or cartilage. The
first sealant, deposited at the bottom of the lesion, must be able
to contain migrating chondrocytes within the lesion, to protect the
implant from influx of undesirable agents and to prevent
chondrocyte migration into the sub-chondral space. Additionally,
the first sealant prevents the infiltration of blood vessels and
undesirable cells and cell debris into the implant and it also
prevents formation of the fibrocartilage.
[0240] In a method for treatment of osteochondral defects, the
first (bottom) sealant forms a barrier between the cartilage lesion
and the bone lesion. Because these two defects are in two
qualitatively different tissues they require different treatments.
As described above, the bone lesion is treated with the
bone-inducing composition or a carrier comprising said composition
while the cartilage lesion is treated with the acellular matrix
implant. Moreover, it is not desirable that the enzymes present in
the cartilage lesion activating chondrocyte migration mix with the
bone-inducing agents and growth factors needed for bone lesion
repair. When there is no separation of one tissue from another, it
can easily end up with, for example, the fibrocartilage ingrowing
into the bone area and, in such an instance, instead of bone being
replaced with the bone, it is replaced with the inferior
fibrocartilage. Consequently, for treatment of osteochondral
defects, the bottom sealant is deposited over the bone lesion
filled with the bone-inducing composition or a carrier comprising
said composition separating the bone lesion from the cartilage
lesion implanted with the acellular implant. In this way, each the
acellular implant and the bone-inducing composition can work
independently and without interference from the other.
[0241] B. A Second-Top Sealant
[0242] The second (top) sealant acts as a protector of the
acellular matrix implant or the lesion cavity on the surface and is
typically deposited over the lesion after the implant is deposited
therein and in this way protects the integrity of the lesion cavity
from any undesirable effects of the outside environment, such as
invading cells or degradative agents and seals the acellular matrix
implant gel in place after its deposition therein.
[0243] The second sealant also acts as a protector of the acellular
implant implanted within a cavity formed between the two sealants.
In this way, the second sealant is deposited after the implant is
deposited over the first sealant and seals the implant within the
cavity or it may be deposited over the space holding gel before the
implant deposition.
[0244] The third function of the second sealant is as an initiator
or substrate for the formation of a superficial cartilage
layer.
[0245] Performed studies described below confirmed that when the
second sealant was deposited over the cartilage lesion, a growth of
the superficial cartilage layer occurred as an extension of the
native superficial cartilage layer. This superficial cartilage
layer was particularly well-developed when the lesion cavity was
filled with the thermo-reversible gel or sol gel thereby leading to
the conclusion that such gel might provide a substrate for the
formation of such superficial cartilage layer.
[0246] C. Top and Bottom Sealant Properties
[0247] The first bottom or second top sealant used according to an
embodiment of the invention must possess the following
characteristics:
[0248] Sealant must be biologically acceptable, easy to use and
possess required adhesive and cohesive properties.
[0249] The sealant must be biologically compatible with tissue, be
non-toxic, not swell excessively, not be extremely rigid or hard,
as this could cause abrasion of or extrusion of the sealant from
the tissue site, must not interfere with the formation of new
cartilage, or promote the formation of other interfering or
undesired tissue, such as bone or blood vessels and must be
bioresorbable and biodegradable by any acceptable metabolic
pathway, or be incorporated into the newly formed hyaline cartilage
tissue.
[0250] The sealant must rapidly gel from a flowable liquid or paste
to a load-bearing gel within 3 to 15 minutes, preferably within 3-5
minutes. However, the sealant must not gel or polymerize too
rapidly as it would cause problems with its even distribution over
the lesion. Gelling faster than 30 seconds is undesirable. Longer
gelation times are not compatible with surgical time constraints.
Additionally, the overall mode of use should be relatively simple
because complex and lengthy procedures will not be accepted by
surgeons.
[0251] Adhesive bonding is required to attach the sealant
formulation to tissue and to seal and support such tissue. Minimal
possessing peel strengths of the sealant should be at least 3N/m
and preferably 10 to 30 N/m. Additionally, the sealant must itself
be sufficiently strong so that it does not break or tear
internally, i.e., it must possess sufficient cohesive strength,
measured as tensile strength in the range of 0.2 MPa, but
preferably 0.8 to 1.0 MPa. Alternatively, a lap shear measurement
which define the bond strength of the formulation should have
values of at least 0.5 N/cm.sup.2 and preferably 1 to 6
N/cm.sup.2.
[0252] Sealants possessing the required characteristics are
typically polymeric. In the un-cured, or liquid state, such sealant
materials consist of freely flowable polymer chains which are not
cross-linked together, but are neat liquids or are dissolved in
physiologically compatible aqueous buffers. The polymeric chains
also possess side chains or available groups which can, upon the
appropriate triggering step, react with each other to couple or
cross-link the polymer chains together. If the polymer chains are
branched, i.e., comprising three or more arms on at least one
partner, the coupling reaction leads to the formation of a network
which is infinite in molecular weight, such as for example, a
gel.
[0253] The formed gel has cohesive strength dependent on the number
of inter-chain linkages, the length expressed as molecular weight
of the chains between links, the degree of inclusion of solvent in
the gel, the presence of reinforcing agents, and other factors.
Typically, networks in which the molecular weight of chain segments
between junction points (cross-link bonds) is between 100-500
Daltons are tough, strong, and do not swell appreciably. Networks
in which the chain segments are between 500-2500 Daltons swell
dramatically in aqueous solvents and become mechanically weak. In
some cases the latter gels can be strengthened by specific
reinforcer molecules; for example, the methylated collagen
reinforces the gels formed from 4-armed PEGs of 10,000 Daltons
(2500 Daltons per chain segment).
[0254] The gel's adhesive strength permits bonding to adjacent
biological tissue by one or more mechanisms, including
electrostatic, hydrophobic, or covalent bonding. Adhesion can also
occur through mechanical inter-lock, in which the uncured liquid
flows into tissue irregularities and fissures, then, upon
solidification, the gel is mechanically attached to the tissue
surface.
[0255] At the time of use, some type of triggering action is
required. For example, it can be the mixing of two reactive
partners, it can be the addition of a reagent to raise the pH, or
it can be the application of heat or light energy.
[0256] Once the sealant is in place, it must be non-toxic to
adjacent tissue, and it must be incorporated into the tissue and
retained permanently, degraded in situ, or be naturally removed,
usually by hydrolytic or enzymatic degradation. Degradation can
occur internally in the polymer chains, or by degradation of chain
linkages, followed by diffusion and removal of polymer fragments
dissolved in physiological fluids.
[0257] Another characteristic of the sealant is the degree of
swelling it undergoes in the tissue environment. Excessive swelling
is undesirable, both because it creates pressure and stress
locally, and because a swollen sealant gel losses tensile strength,
due to the plasticizing effect of the imbibed solvent which, in
this case, is physiological fluid. Gel swelling is modulated by the
hydrophobicity of the polymer chains. In some cases it may be
desirable to derivatize the base polymer of the sealant so that it
is less hydrophilic. For example, one function of methylated
collagen containing sealant is presumably to control swelling of
the gel. In another example, the sealant made from penta-erythritol
tetra-thiol and polyethylene glycol diacrylate can be modified to
include polypropylene glycol diacrylate, which is less hydrophilic
than polyethylene glycol. In a third example, sealants containing
gelatin and starch can also be methylated both on the gelatin and
on the starch, again to decrease hydrophilicity.
[0258] D. Suitable Sealants
[0259] Sealants suitable for purposes of this invention include the
sealants prepared from gelatin and dialdehyde starch triggered by
mixing aqueous solutions of gelatin and dialdehyde starch which
spontaneously react and gel.
[0260] In general, a sealant useful for the purposes of this
application has adhesive, or peel strengths at least 10 N/m and
preferably 100 N/cm; it needs to have tensile strength in the range
of 0.2 MPa to 3 MPa, but preferably 0.8 to 1.0 MPa. In so-called
"lap shear" bonding tests, values of 0.5 up to 4-6 N/cm.sup.2 are
characteristic of strong biological adhesives.
[0261] Such properties can be achieved by a variety of materials,
both natural and synthetic. Examples of suitable sealant include
gelatin and di-aldehyde starch described in PCT WO 97/29715,
4-armed pentaerythritol tetra-thiol and polyethylene glycol
diacrylate described in PCT WO 00/44808, photo-polymerizable
polyethylene glycol-co-poly(a-hydroxy acid) diacrylate macromers
described in U.S. Pat. No. 5,410,016, periodate-oxidized gelatin
described in U.S. Pat. No. 5,618,551, serum albumin and
di-functional polyethylene glycol derivatized with maleimidyl,
succinimidyl, phthalimidyl and related active groups described in
PCT WO 96/03159.
[0262] Another acceptable sealant is made from a copolymer of
polyethylene glycol and polylactide, polyglycolide,
polyhydroxybutyrates or polymers of aromatic organic amino acids
and sometimes further containing acrylate side chains, gelled by
light, in the presence of some activating molecules.
[0263] The acceptable sealant made from periodate-oxidized gelatin
remains liquid at acid pH, because free aldehyde and amino groups
on the gelatin cannot react. To trigger gelation, the oxidized
gelatin is mixed with a buffer that raises the pH to pH at which
the solution gels.
[0264] Still another sealant made from a 4-armed pentaerythritol
thiol and a polyethylene glycol diacrylate is formed when these two
neat liquids (not dissolvable in aqueous buffers) are mixed.
[0265] Another type of the suitable sealant is 4-armed polyethylene
glycol derivatized with succinimidyl ester and thiol plus
methylated collagen in two-part polymer compositions that rapidly
form a matrix where at least one of the compounds is polymeric,
such as polyamino acid, polysaccharide, polyalkylene oxide or
polyethylene glycol and two parts are linked through a covalent
bond, for example a cross-linked PEG with methyl collagen, such as
a cross-linked polyethylene glycol hydrogel with methyl-collagen,
as described in U.S. Pat. Nos. 6,312,725B1 and 6,624,245B2, hereby
incorporated by reference. One drawback of the type of the
bioadhesive described therein is that it gels and/or bonds
extremely fast upon contact with tissue, particularly with tissue
containing collagen. Consequently, this type of bioadhesive, which
is designed for rapid gelling or bonding during vessel or tissue
injury typically needs to be modified in order to prolong the
gelling and/or bonding time to be suitable for use as a sealant of
the invention.
[0266] One group of suitable sealants' comprises albumin. Albumin
containing sealants typically comprise at least human or bovine
serum albumin conjugated with a cross-linking agent. The
cross-linking agent may be selected from the group consisting of
glutaraldehyde, amino acids, polypeptides and proteins. Further
modification may include conjugation with a fibrous protein, such
as collagen or with a gel compound although this portion of the
sealant is, in the current invention, generally provided by the
support matrix of the invention. Sealants and bioadhesives or
portions thereof which fall within a category of this type of
suitable sealants are disclosed in U.S. Pat. Nos. 6,310,036;
6,217,894 and 6,685,726, hereby incorporated by reference.
[0267] It is worth noting that it is not the presence or absence of
particular protein or polymer chains, such as gelatin or
polyethylene glycol, which necessarily governs the mechanical
strength and degradation pattern of the sealant. The mechanical
strength and degradation pattern are controlled by the cross-link
density of the final cured gel, by the types of degradable linkages
which are present, and by the types of modifications and the
presence of reinforcing molecules, which may affect swelling or
internal gel bonding.
[0268] The first and second sealant, or the sealant used for
separation of the bone and cartilage lesions, must be a
biologically acceptable, typically rapidly gelling and
polymerizable synthetic compound having adhesive, bonding and/or
gluing properties, and is typically a hydrogel, such as derivatized
polyethylene glycol (PEG) which is preferably cross-linked with a
collagen compound, typically alkylated collagen. The sealant used
for separation of the bone and cartilage lesions should polymerize
rapidly in order to permit surgeon to continue with the surgery
without any delay. For the purposes of this invention, the sealant
should have a tensile strength of at least 0.3 MPa.
[0269] Additionally, the sealant may be two or more polymer
compositions that rapidly form a matrix where at least one of the
compounds is polymer, such as, polyamino acid, polysaccharide,
polyalkylene oxide or polyethylene glycol and two parts are linked
through a covalent bond and cross-linked PEG with collagen. The
sealant of the invention typically gels and polymerizes within
about 0.5 to about 5 minutes upon contact with tissue, particularly
with tissue containing collagen.
[0270] The second sealant or the sealant used for separation of the
bone and cartilage lesions may or may not be the same as the first
sealant and the first and second sealants may be utilized as a
barrier between the bone and cartilage lesions but the different
sealant may also be used for this purpose.
[0271] For the use in the current invention, the sealant is slowly
polymerized in situ after its deposition at the bottom of the
lesion or between the bone-inducing composition and acellular
implant. Such slow polymerization is necessary to avoid uneven
distribution of the sealant over the bottom of the lesion and also
to avoid the random and uneven accumulation of the sealant on some
parts of the surface while leaving other parts of the bottom
surface uncovered. The primary function of the sealant is to
protect the acellular implant from undesirable effects of migrating
cells, tissue debris and various factors present in the blood or
serum, as already discussed previously. Consequently, its even
distribution over the bottom of the lesion or over the
bone-inducing composition is of great importance and to achieve
such even distribution, the polymerization of the sealant must not
be too slow or too rapid in order to reach the bottom of the
lesion, cover it and then polymerize in situ and still meet
surgeon's time constraints. For arthroscopic surgery and
implantation of the acellular implant, the sealant polymerization
at the bottom of the implant site needs to occur between 30 seconds
and about 5 minutes, preferably between 30 seconds and about 3
minutes.
[0272] V. Method for Formation of Superficial Cartilage Layer Over
the Acellular Matrix Implant
[0273] An accompanying aspect of this invention is a finding that
when the acellular matrix implant produced according to procedures
described above is implanted into a cartilage lesion cavity and
covered with a biocompatible adhesive top sealant, the resulting
combination leads to a formation of a superficial cartilage layer
completely overgrowing said lesion.
[0274] In practice, the method for formation of the superficial
cartilage layer comprises several steps. First, the bottom of the
lesion is covered with a first, bottom sealant deposited as
polymerizable solution. Following the sealant polymerization, the
acellular matrix implant is implanted into said lesion and a
second, top sealant is deposited over the implant. In one
embodiment, the implant may be a thermo-reversible gel which easily
changes from sol to gel at the body temperature thereby permitting
an external preparation and delivery of the implant into the
lesion. The gel is then covered with the top sealant which promotes
formation of the superficial cartilage layer overgrowing the
cartilage lesion thereby sequestering the implant within the lesion
and protecting it from outside environment.
[0275] The superficial cartilage layer begins to form very quickly
after the implant is implanted into the cartilage lesion and
covered with the top sealant layer. As shown in FIG. 6, two weeks
after acellular matrix implantation superficial cartilage layer was
observed on the surface of acellular matrix implanted site. FIG. 6
shows arthroscopic evaluation two weeks after the defect was made
in the femoral condyle where the superficial cartilage layer is
clearly visible compared to untreated empty defect made at the same
time, seen in FIG. 5.
[0276] The top sealant gives support and promotes formation of the
superficial cartilage layer in some instances further assisted by
the gel components of the matrix. At the time when the implant
matrix is completely degraded and the new hyaline cartilage is
formed in the defect, the superficial cartilage layer completely
covers and insulates the newly formed cartilage similarly to a
synovial membrane naturally present and covering the joints. The
second top sealant is eventually also biodegraded and removed from
the site, not however, until the superficial cartilage layer, a
synovial-like membrane, has formed over.
[0277] VI. Method for Use of Acellular Matrix Implant
[0278] The method for repair and restoration of damaged, injured,
diseased or aged cartilage to a functional cartilage is based on
implantation of an acellular matrix implant into a cartilage
lesion.
[0279] The method for use of the acellular matrix implant in these
treatments comprises following steps:
[0280] a) Preparing an Acellular Matrix Implant
[0281] The first step involves preparation of the acellular matrix
implant for implanting into the cartilage lesion. Preparation of
acellular matrix implants is described in greater detail in
sections II.A.
[0282] b) Selecting and Depositing the First and Second Sealant
into the Cartilage Lesion
[0283] The second step is optional and involves selection and
depositing bottom and/or top sealant layers into a cartilage
lesion.
[0284] Specifically, this step involves deposition of the first
sealant at the bottom of the cartilage lesion and the second
sealant over the acellular matrix implant. The first and the second
sealants can be the same or different, however, both the first and
the second sealants must have certain definite properties to
fulfill their functions.
[0285] The bottom sealant, deposited into the lesion before the
acellular matrix implant is introduced, acts as a protector of the
lesion cavity integrity. It protects the lesion cavity from
contamination by extraneous substances such as blood and tissue
debris. It protects integrity of the naturally present enzymes and
other mediators needed for and involved in formation of
extracellular matrix and activation of chondrocytes and their
migration from a surrounding host cartilage into the acellular
implant implanted in the lesion. It also protects the lesion cavity
from formation of the fibrocartilage.
[0286] The top sealant deposited over the implant and effectively
sealing the lesion from external environment acts as a protector of
the lesion cavity as well as a protector of the implant deposited
within a lesion cavity formed between the two sealants as well as
an initiator of the formation of the superficial cartilage
layer.
[0287] c) Implanting the Acellular Matrix Implant
[0288] Next step in the method of the invention comprises
implanting said acellular matrix implant into a lesion cavity
formed between two layers of sealants.
[0289] The implant is preferably deposited into said lesion cavity
after the bottom sealant is deposited but before the top sealant is
deposited over it or the implant may be deposited into the lesion
cavity without the bottom sealant being deposited there and then
covered with the top sealant.
[0290] d) Generation of the Superficial Cartilage Layer
[0291] A deposition of the top sealant over the acellular matrix
implant leads to sealing of the lesion cavity and overgrowth of
said cavity with a superficial cartilage layer.
[0292] Typically, a biologically acceptable top sealant is
deposited over the acellular matrix implant implanted into the
lesion cavity. The second sealant acts as an initiator for
formation of the superficial cartilage layer which in time
completely overgrows the lesion and strongly resembles a healthy
synovial membrane. In several weeks or months, usually in about two
weeks, the superficial cartilage layer completely covers the
lesion, protects the implant, migrating, dividing and proliferating
chondrocytes and newly secreted extracellular matrix. Protecting
the implant from extraneous environment permits integration of the
newly formed cartilage tissue into the native surrounding cartilage
substantially without formation of fibrocartilage.
[0293] Formation of the superficial cartilage layer is thus a very
important aspect of the healing of the cartilage and its repair and
regeneration.
[0294] VII. Method for Treatment of Cartilage Lesions
[0295] The method for treatment of damaged, injured, diseased or
aged cartilage according to the invention is suitable for healing
of cartilage lesions due to acute injury by providing conditions
for regeneration of the healthy hyaline cartilage and for its
integration into the surrounding native cartilage.
[0296] The method generally encompasses several novel features,
namely, fabrication of a biologically acceptable biodegradable
acellular matrix implant, selecting and depositing a top and bottom
adhesive sealants to the lesion and the implantation of the
acellular matrix implant within a cavity generated by two sealants,
a formation of the superficial cartilage layer covering the lesion
and protecting the integrity of the acellular matrix implant
deposited therein, and providing conditions for activation,
migration, dividing and proliferation of chondrocytes and for
secretion of extracellular matrix ultimately leading to formation
of the new hyaline cartilage and its integration into the native
cartilage.
[0297] The method generally comprises steps:
[0298] a) fabrication of the acellular matrix implant according to
the above described procedures;
[0299] b) debridement an articular cartilage lesion in surgical
procedure;
[0300] c) during the debridement, preparing the lesion for
implantation of the acellular matrix implant by depositing the
bottom sealant at the bottom of the lesion thereby insulating said
cavity from the surrounding tissue;
[0301] d) implanting the acellular matrix implant into said cavity
formed by the polymerized bottom sealant to allow the activated and
migrating chondrocytes to proliferate within said implant;
[0302] e) depositing the top sealant over the lesion, and thereby
sealing said implant within the cavity formed between the two
sealant layers;
[0303] f) optionally introducing enzymes, hormones, growth factors,
proteins, peptides and other mediators into said sealed cavity by
incorporating them into the acellular matrix, or coating said
matrix with them, introducing them separately or generating
conditions for their transport or transfer through the bottom
sealant; and
[0304] g) following the surgery, subjecting an individual
undergoing a surgery for repair of said lesion to a normal physical
activity thereby naturally providing an intermittent hydrostatic
pressure which was shown to promote formation of the healthy
hyaline cartilage and its integration into the surrounding native
intact cartilage.
[0305] There are several advantages of the current method.
[0306] The main advantage of this method is that the acellular
matrix implant is prepared beforehand and is implanted during the
first and only surgery where the cleaning and debridement is
immediately followed by implantation of the acellular matrix
implant.
[0307] Second, the acellular implant avoids immunological reactions
to develop as there is/are no foreign tissue or cells involved
because the implant is wholly synthetic and acellular.
[0308] The method using the acellular matrix implant permits a
three-dimensional expansion of chondrocytes and extracellular
matrix.
[0309] The deposition of the top sealant layer resulting in
formation of superficial cartilage layer constitutes a substitute
for synovial membrane and provides the outer surface of healthy
articular cartilage overgrowing, protecting, containing and
providing critical metabolic factors aiding in protecting the
implant and activated migrating chondrocytes in the lesion. The
superficial cartilage layer also prevent invasion of pannus as seen
in FIGS. 10A, 10B, 11A and 11B compared with FIGS. 8A, 8B, 9A and
9B, where the presence of the invading pannus is clearly visible.
In some instances, a selection of the thermo-reversible gel may be
crucial as certain TRGH may function as a promoter for growth of
the superficial cartilage layer without a need to apply the top
sealant.
[0310] Deposition of the bottom sealant layer protects the
integrity of the lesion after cleaning during surgery and prevents
migration of subchondral and synovial cells and cell products
thereby creating milieu for formation of healthy hyaline cartilage
from the activated migrating chondrocytes into the acellular matrix
implant and also preventing formation of the fibrocartilage.
[0311] The method further permits said acellular matrix implant to
be enhanced with hyaluronic acid or other components or mediators
named above, typically added in about 5 to about 50%, preferably
about 20% (v/v), wherein such hyaluronic acid or such other
components act as enhancers of the matrix-forming characteristics
of the gel and also as a hydration factor in the synovial space in
general and within the lesion cavity in particular.
[0312] Further, the method is very versatile and any of the implant
type variations may be advantageously utilized for treatment of a
specific cartilage, osteochondral or bone injury, damage, aging or
disease.
[0313] For treatment of the cartilage, a subject is treated,
according to this invention, with a prepared acellular matrix
implant implanted into the lesion, the implant is left in the
lesion covered with the top sealant for as long as needed. Usually,
during the two-three months following the surgery and implant
implantation the new hyaline cartilage is formed and integrated
into the native surrounding host cartilage. Typically also, there
is no need for any further surgical or other intervention, as
during these two-three months, at a normal physical activity, such
as walking, running or biking, etc., a sufficient hydrostatic
pressure is applied to the lesion to initiate and promote formation
of the hyaline cartilage fully integrated into the native
cartilage. Such cartilage will then become a fully functional
cartilage covered with a superficial cartilage layer which
eventually grows into or provides the same type of surface as a
synovial membrane of the intact joint.
[0314] Finally, the method also permits replacement of the age
worn-out or diseased osteoarthritic cartilage by the regenerated
hyaline-like cartilage when treated according to this
invention.
[0315] The implantation protocol may assume any variation described
above or possible within the realm of this invention. It is thus
intended that every and all variations in the treatment protocol,
the types of the implants, use of one or two sealants, implantation
process, selection of added mediators and not the least the normal
physical activity of the individual are within the scope of the
current invention.
[0316] VIII. Method for Treatment of Bone or Osteochondral
Defects
[0317] The method for treatment of osteochondral defects is
typically practiced in conjunction with treatment of cartilage. The
method for treatment of bone defects and lesions may be practiced
in conjunction with osteochondral defects or separately without
steps involving deposition of the acellular implant into the
cartilage.
[0318] A. Osteochondral Defects
[0319] Due to its anatomical arrangement where the subchondral bone
is localized directly beneath the injured cartilage and the injury
is both the injury to the cartilage and to the subchondral bone or
subchondral skeletal bone, the method for treatment of
osteochondral defects is an extension of the method for treatment
of cartilage lesions described in section VII, with exception that
during the step c) of that method, the surgeon, after debridement,
deposits into the subchondral bone lesion a bone-inducing
composition or a carrier comprising said composition typically
comprising one or several bone-inducing agent(s), as described
above, then covers said composition with a layer of the bottom
sealant, and after permitting the sealant or the composition or
both to polymerize, performs the steps a-g. The nature of this type
of defects is such that as a consequence of the thinness of the
subchondral bone layer there is high probability that the lesion
will extend into the underlying cancellous bone. In such an
instance, the bone-inducing composition or bone acellular implant
is deposited into the skeletal bone and in flowable continuation
into the osteochondral bone which is then covered with the bottom
sealant layer and the acellular implant is deposited as described
above.
[0320] B. Bone Defects
[0321] The true bone defects, lesions or fractures are stand alone
injuries in the skeletal bone. These types of injuries may also be
conveniently treated according to the invention with the
bone-inducing composition or with a carrier comprising such
bone-inducing composition.
[0322] The carrier, in this setting, corresponds to the acellular
implant utilized for treatment of bone. This bone acellular implant
comprises bone-inducing agents.
[0323] The treatment of the skeletal bone injuries comprises
depositing of the bone-inducing composition into the lesion or
fracture during the surgery. Typically, the bone-inducing
composition will be administered directly into the lesion or
fracture as a powder or a solution, such as an adhesive or
polymerizable solution, or the composition will be incorporated
into a bone-inducing carrier or porous matrix as described above.
The bone lesion may or may not then be covered with the top sealant
or any other surface to contain the composition within the
lesion.
[0324] In the preferred embodiment, the demineralized bone powder
is used as a powder or in solution wherein said powder is dissolved
in the collagen, hydrogel or some other adhesive solution which has
no bone forming effect. The bone-inducing composition is added in
amount which will completely fill the lesion or fracture.
[0325] IX. Treatment of Human Osteoarthritic Cartilage
[0326] Articular cartilage is a unique tissue with no vascular,
nerve, or lymphatic supply. The lack of vascular and lymphatic
circulation may be one of the reasons why articular cartilage has
such a poor intrinsic capacity to heal, except for formation of
fibrous or fibrocartilaginous tissue. Unique mechanical functions
of articular cartilage are never reestablished spontaneously after
a significant injury, age wear or disease, such as osteoarthritis
(OA).
[0327] Currently, the only available treatment of severe
osteoarthritis of the knee is a total knee replacement in elderly
patients. In young and middle aged patients, however, this is not
an optimal treatment.
[0328] Although the current invention is more practicable for
treatment of injuries in young individuals who naturally possess
sufficient levels of extracellular matrix building enzymes, growth
factors, and other mediators, the method may be advantageously
modified to also provide treatment for older population.
[0329] For treatment of elderly patients or for treatment of larger
lesions, the acellular matrix implant is incorporated, before
implantation, with one or more metalloproteinases, mediators,
enzymes and proteins and/or with drugs stimulating endogenous
production of these factors and mediators. These factors, as
described above, stimulate and promote chondrocytes activation,
migration and extracellular matrix secretion. The method of the
invention thus is also suitable for treatment of the cartilage
defects in older generation. It is expected, however, that such
treatment will require longer period of treatment.
[0330] In osteoarthritis, or in age worn out cartilage, disruption
of the structural integrity of the matrix by the degeneration of
individual matrix proteins leads to reduced mechanical properties
and impaired function. Consequently, the current invention reverses
this process by providing a means for rebuilding the diseased
osteoarthritic or worn cartilage with the new healthy hyaline
cartilage.
[0331] X. In Vivo Studies in Swine of the Weight-Bearing Region of
the Knee
[0332] The method according to the invention was tested and
confirmed in in vivo studies in swine.
[0333] The studies, described below, were designed to evaluate
feasibility of the porcine acellular matrix implant by detecting
chondrocyte activation and induction of chondrocyte migration on
the surrounding cartilage, generation of newly synthesized hyaline
cartilage within the lesion and formation of superficial cartilage
layer.
[0334] Studies involved the creation of defects in the
weight-bearing region of the femoral medial condyle of the knee
joint, implantation of the acellular matrix into the defect,
depositing bottom and top sealants, detection of growth of a
superficial cartilage layer after two weeks following the defect
creation, detection of chondrocyte morphology, detection of pannus
invasion and presence of fibrocartilage, detection of presence or
absence of S-GAG secretion, histochemical evaluation of presence or
absence of sealants.
[0335] Gross anatomy of the empty defect creation and acellular
matrix implantation at day zero is shown in FIGS. 3 and 4.
Formation of the healthy hyaline cartilage and generation of the
superficial cartilage layer in defects treated with the acellular
matrix implant and the fibrocartilage pannus invasion in control
defects at seven month following the defect creation are seen in
FIGS. 5-12.
[0336] FIG. 3 shows two empty defects sites A and B at a time of
the defect creation (time zero). FIG. 4 shows two defects created
at time zero implanted with the acellular matrix implants at sites
A and B.
[0337] FIGS. 5 and 6 show arthroscopic evaluation two weeks after
defect creation in the control group (FIG. 5) and in the
experimental group implanted with the acellular matrix (FIG. 6).
Histological grading is seen in FIG. 7 and histological evaluation,
in two magnifications, is seen in FIGS. 8 and 9 for the control
animals and in FIGS. 10 and 11 for the experimental group treated
with the acellular implant. Degradation of the sealant from the
cartilage lesion is seen in FIGS. 12A-12C. One example of full
thickness defect at femoral condyle of mini-pig is seen in FIG.
13.
[0338] Schematic representation of the femoral articular surface,
defect creation and implant implantation sites within said defect
is shown in FIG. 1D. FIG. 1D shows two defects A and B created in
the femoral medial condyle on the medial side of the femoral
articular surface. The defects have sizes of 4 mm in diameter and
1-1.5 mm in depth. The defects are created in the weight-bearing
region.
[0339] Table 1 is a tabulation of conditions of a study design as
schematically illustrated in FIG. 1D. TABLE-US-00001 TABLE 1 Study
Design Group Number of Number of Arthros- Number Animals Samples
Procedure copy Necropsy 1 8 16** Implantation 2 weeks 7 months
Experi- of acellular after after mental biodegradable implan-
implan- matrix* tation tation 2 8 16** Empty defect 2 weeks 7
months Control control after after defect defect creation creation
*Matrix was secured with tissue adhesive and sutures. **Each group
has two samples at weight-bearing site (site A and B, FIG. 1D).
[0340] Table 1 illustrates the study design for the seven months
study of feasability of the acellular matrix implant for treatment
of cartilage lesions. Study involved 8 castrated male Yucatan
micro-swine, 9-12 months old in each of the two groups. Two defects
(A and B) were created at time zero in the knee of each animal,
with a total number of 16 defects. The experimental group was
implanted with acellular matrix implant at a time of defect
creation. In the control group, the defect was left empty without
any treatment and was used for visual, microscopical, histological
and histochemical comparisons. Arthroscopy was performed at 2 weeks
after implantation and defect creation. Necropsy was performed 7
months after implantation and defect creation.
[0341] The acellular matrix implant was prepared from a collagen
solution VITROGEN.RTM. (35 .mu.L) obtained from Cohesion, Calif.
The collagen gel solution was absorbed into a collagen honeycomb
sponge (5 mm in diameter and 1.5 mm in thickness) obtained from
Koken Co., Japan. The combined collagen gel/sponge constructs seen
in FIG. 2A were pre-incubated for 1 hour at 37.degree. C. to gel
the collagen, followed by incubation in culture medium with 1%
penicillin and streptomycin at 37.degree. C. at 5% CO.sub.2. After
about 24 hours of polymerization, the biodegradable scaffolds were
transferred to the tissue container with pre-warmed culture medium
(37.degree. C.) for the implantation.
[0342] Arthrotomy was performed under an inhalation anesthesia.
After opening knee joint capsule, two empty full-thickness defects
(4 mm in diameter and about 1.5 mm in depth) were created in the
femoral articular cartilage on the weight-bearing site of the
medial femoral condyle of each animal. After creating defects,
tissue sealant was placed on the bottom of the defect. Then, the
pre-prepared acellular biodegradable matrix was placed over the
bottom sealant within the cartilage lesion. The acellular matrix
was secured with absorbable sutures (usually 4 to 6 sutures) and
with two non-absorbable sutures. The non-absorbable sutures were
used as a maker for arthroscopic evaluation and are visible in FIG.
6. The implanted defect was then sealed with the top sealant. For
the controls, two empty full-thickness defects were created and
left intact, that is empty, without implants, or deposition of the
bottom or top sealants.
[0343] FIG. 3 shows a photograph of the two empty full-thickness
defects A and B (4 mm in diameter and 1-1.5 mm in depth) created in
the articular cartilage on the weight-bearing site of the medial
femoral condyle. The empty defects were left intact during the
whole time of the study and were used as controls for the
experimental group.
[0344] FIG. 4 is a photograph of the two full-thickness defects
created in the same way as the empty defects seen in FIG. 3. These
two defects were treated, according to the method of the invention,
with the bottom sealant deposited on the bottom of the lesion. The
acellular matrix implant was implanted into the lesion cavity over
the bottom sealant and the top sealant deposited the over the
implanted acellular matrix implant. The implants were collagenous
sponges (FIG. 2A) and had 5 mm in diameter and 1.5 mm in thickness.
Both sites A and B were implanted. Each implant was secured with
four absorbable sutures and two non-absorbable sutures used as
markers for future arthroscopic evaluation.
[0345] Two weeks after defect creation and acellular matrix
implantation, the empty defects and implant sites were evaluated
with arthroscopy. Arthroscopic evaluation after 2 weeks is seen in
FIGS. 5 and 6.
[0346] FIG. 5 is an arthroscopic microphotograph of an empty defect
2 weeks after defect creation. Arthroscopic evaluation showed that
in the control group, if left untreated, the lesion was invaded
with synovial pannus and filled with fibrocartilage. The
arthroscopic evaluation clearly shows the defect depression
indicating that the defect is fully exposed and empty although some
synovial invasion have already occurred. Such synovial invasion is
a first step toward formation of fibrocartilage. Formation of
fibrocartilage to replace the hyaline cartilage is undesirable as
the fibrocartilage is qualitatively and functionally inferior to
hyaline cartilage.
[0347] Arthroscopic evaluation of implanted sites showed that
already at two weeks time the defects are covered with the
superficial cartilage layer. FIG. 6 is an arthroscopic
microphotograph of the defect treated with the acellular matrix
implant 2 weeks after the defect was created. The FIG. 6 shows the
superficial cartilage layer overgrowing the implant site forming a
smooth flat surface. The borders of the implant site are already
undefined compared to the empty defect which has a definite and
visible border, said implanted site indicating the beginning of
chondrocyte migration into the implant and secretion of
extracellular matrix in confluency with the host cartilage, all
this covered with the superficial cartilage layer. The arthroscopic
evaluation seen in FIG. 6 revealed that the lesion implanted with
the acellular matrix is unexposed and covered with the superficial
cartilage layer completely overgrowing the implant sites, seen as a
smooth flat surface when compared to the fully exposed and empty
defects of controls, seen in FIG. 5.
[0348] At 7 months after creating the defects and implanting the
acellular matrix implants, the animals were euthanized. The implant
and defect sites on the femoral articular condyle were harvested
for histological evaluation. The tissues were fixed with 4%
formaldehyde/PBS for 7 days at 4.degree. C. The tissues were
decalcified with 10% formic acid, processed, and embedded in
paraffin. Thin sections (5 .mu.m) were stained with Safranin-O
(Saf-O) and hematoxylin eosin (H-E) for histological
evaluation.
[0349] The stained sections were evaluated blindly by means of a
histological grading scale seen in FIG. 7, modified from J. Bone
Joint Surg. Am., 79:1452-63 (1997). Only sections from the center
of the defect were graded in order to ensure unbiased analysis and
to allow comparison among specimens studied at different
time-point. The area from the center of the defect was also chosen
because it provided the most stringent test of healing capacity,
since the least amount of cartilage healing was found consistently
in specimens taken from the middle of the defect.
[0350] Histological scoring system used for cartilage repair
evaluation is seen in Table 2. TABLE-US-00002 TABLE 2 Category 1.
Filling of defect Score Filling of Defect 0 None (or almost none) 1
<50% 2 >50% 3 All (or almost all) 2. Integration of repair
tissue with surrounding articular cartilage Score Integration 0 Gap
or lack of continuity on two sides 1 Gap or lack of continuity on
one sides 2 Non-continuous gap or lack 3 Normal continuity and
integration 3. Matrix staining with Safranin O-fast green (compared
to host cartilage) Score Matrix staining 0 None (or almost none) 1
Slight 2 Moderate 3 All (or almost all) 4. Cellular morphology
Score Chondrocytes morphology 0 Mostly spindle-shape (fibrous-like)
cells 1 <50% of round cells with morphology of chondrocytes 2
>50% of round cells with morphology of chondrocytes 3 Normal
(mostly round cells with morphology of chondrocytes) 5.
Architecture within entire defect (not including margins) Score
Architecture within entire defect 0 Clefts or fibrillations 1 <3
large voids 2 >3 large voids 3 Normal 6. Architecture of surface
Score Architecture of surface 0 Severe fibrillation or irregularity
1 Moderate fibrillation or irregularity 2 Slight fibrillation or
irregularity 3 Normal (or nearly normal) 7. Penetration of tissue
to subchondral bone area Score Penetration 0 Severe penetration 1
Moderate penetration 2 Slight penetration 3 Normal (or nearly
normal)
[0351] Cumulative results of the histological grading of the
repaired chondral cartilage is seen in Table 3. TABLE-US-00003
TABLE 3 Histological Grading of the Repaired Cartilage Acellular
Matrix Empty Defect Cateqory Group Group Filling of defect 3.00
2.60 Integration 2.00 1.40 Matrix staining 2.33 2.10 Chondrocyte
morphology 1.78 0.80 Architecture within entire defect 2.33 0.30
Architecture of surface 2.33 1.90 Tissue penetration into
subchondral 2.11 1.40 bone area Average total score 15.88 10.50
SD.+-. 1.90 3.60
[0352] As seen in Table 3 the average total score for histological
grading at 7 months after the defect creating and treatment with
the acellular matrix implant was much higher in the implant group,
with the score for all indicators in the implant group being higher
then in the empty defect group.
[0353] Histological grading of the repair tissue is shown in FIG.
7, which graphically illustrates results shown in Table 5. The
average total scores on the histological grading scale were
significantly better (p<0.001) for the defects treated with
acellular matrix implants than for the untreated defects.
[0354] At seven months following the defect creation, animals were
sacrificed, their joints were harvested and evaluated by Safranin-O
staining. Results are seen in FIGS. 8-11.
[0355] The non-implanted, empty defects A and B at 7 months after
defect creating are shown in FIGS. 8A, 8B, 9A and 9B.
[0356] FIG. 8A is a Safranin-O staining microphotograph (29.times.
magnification) of the empty, non-implanted defect (D) at a control
site A seven months after defect creation. In higher magnification
(FIG. 8B), the defect clearly shows a fibrous tissue (F) filling
the defect surrounded by the host cartilage (H) with underlying
subchondral bone (SB) area (FIG. 8A). None or a very small amount
of S-GAG accumulation, depicted by red color, was observed at the
defect site. S-GAG accumulation is evidence of the extracellular
matrix formation. If there is a little or none S-GAG present, there
is no extracellular matrix generated, indicating the absence of
migrating chondrocytes and absence of formation of hyaline
cartilage. It also indicates the presence and formation of
fibrocartilage within the lesion. FIG. 8B shows a 72.times.
magnification of the defect site confirming a presence of
fibroblasts, that is fibrous cells, indicating invasion of a
fibrovascular pannus (F) from synovium. Chondrocyte morphology
showed presence of mostly spindle (fibrous) cells.
[0357] FIG. 9A is a Safranin-O staining microphotograph (29.times.
magnification) of the empty, non-implanted defect (D) at a site B
of the control defect seven months after defect creation showing a
formation of fibrous tissue filling the defect surrounded by the
host cartilage (H) with underlying subchondral bone (SB) area.
Severe irregularity of the lesion surface was observed. Only very
slight S-GAG accumulation, depicted by red color, was observed at
the defect site. S-GAG accumulation is evidence of the
extracellular matrix formation.
[0358] FIG. 9B shows a 72.times. magnification of the defect site
showing a presence of fibroblasts indicating a fibrovascular pannus
(F) invasion from synovium. Cell morphology observed at this site
shows mostly spindle fibrous cells.
[0359] FIGS. 8A, 8B, 9A and 9B clearly show that non-implanted
control defects without treatment with the acellular implant of the
invention do not indicate a formation of the healthy hyaline
cartilage which would show as S-GAG accumulation, in Safranin-O
stained microphotograps seen as a red color. Rather, these
microphotographs show fibrovascular pannus synovial invasion into
the defect with an accumulation of spindly fibrous cells present in
the empty defect sites.
[0360] While the no-treatment of the lesion resulted in the filing
of the defect with the fibrocartilage, the implantation of the
acellular matrix implant into the defect induced chondrocyte
activation and migration from the surrounding native cartilage and
resulted in massive formation of cartilage extracellular matrix
(ECM accumulation) with minimal fibrovascular pannus in the implant
sites. ECM accumulation was detected by the strong red color
present at the implanted sites of experimental animals. Results are
seen in FIGS. 10A, 10B, 11A and 11B.
[0361] FIG. 10A is a micrograph of Safranin-O staining histological
evaluation (29.times. magnification) of the acellular matrix
implant (I) implanted withing the defect site A, seven month after
defect creation and implantation of the acellular matrix implant.
FIG. 10A clearly shows inducement of cell migration from the
surrounding native host cartilage (H) into the implant (I)
implanted within the defect site. After seven months following the
implantation, hyaline-like cartilage was observed at the acellular
implant site. The presence of the hyaline cartilage is indicated by
the normal S-GAG accumulation, seen as a predominant red present in
the defect site A. Superficial cartilage layer formed over the
lesion is also seen. There was minimal fibrovascular pannus in the
implant sites. Implant is surrounded by the host cartilage (H) with
underlying subchondral bone area (SB).
[0362] FIG. 10B shows a higher magnification (72.times.) of the
implant area with red color indicative of S-GAG accumulation and
chondrocyte morphology showing primarily normal mostly round cells
as compared to spindly fibrous cells observed in the non-treated
control defects.
[0363] FIG. 11A is a Safranin-O staining histological evaluation
(29.times. magnification) of the acellular matrix implant (I)
implanted withing the defect site B, seven month after
implantation. FIG. 11A confirms results seen in FIG. 10A. It
clearly shows inducement of cell migration from the surrounding
native host cartilage (H) into the implant (I) implanted within the
defect site. At seven months after implantation, hyaline-like
cartilage was observed at the acellular implant site. The presence
of the hyaline cartilage was indicated by the normal S-GAG
accumulation, seen as a predominant red color present in the defect
site B. Superficial cartilage layer formed over the lesion and
traces of non-absorbable suture are also seen. No fibrovascular
pannus synovial invasion was observed in the implant site. Implant
is surrounded by the host cartilage (H) with underlying subchondral
bone area (SB). The non-absorbable suture indicates the original
border between the host cartilage and the implant, now almost
completely obscured.
[0364] FIG. 11B shows a higher magnification (72.times.) of the
implant area with high accumulation of red color indicative of
S-GAG presence. Chondrocyte morphology again show primarily normal,
mostly round cells confirming results observed at site A.
[0365] As seen in FIGS. 10A and 10B, 11A and 11B, there was clearly
visible integration between the biodegradable acellular matrix and
the host cartilage. Such integration is not observed in FIGS. 8A
and 9A where the defect is surrounded by the normal hyaline
cartilage. These figures show different cell morphology at the
defect sites than those at the implantation sites seen in FIGS. 10A
and 10B. Cell morphology of the empty sites shows the presence of
spindly fibrous cells dissimilar to those cells of the surrounding
hyaline cartilage. Cell morphology at the implanted sites, on the
other hand, show the presence of the normal (round) cells also
observed in the surrounding healthy hyaline cartilage. The
implanted site thus, after seven months does not show difference
between the previously uninjured cartilage and the one formed
within the defect following the implantation.
[0366] Additionally, the use of a top sealant deposited over the
implant implanted at a defect site had resulted in formation of the
superficial cartilage layer and minimizing synovial tissue invasion
at the implant site.
[0367] A superficial cartilage layer is formed over the cartilage
lesion after the top sealant is deposited over the lesion implanted
with the acellular implant. As seen in FIG. 6, the presence of the
superficial cartilage layer was already observed in two weeks after
the implantation. The top sealant which causes the superficial
cartilage layer to be formed is biodegradable and biodegrades
within the time. At three months after the sealant deposition,
remaining sealant was still observed at the surface area along with
the superficial cartilage layer. At seven months after
implantation, the top sealant was completely biodegraded and
superficial cartilage layer was formed in its place, as seen in
FIGS. 10A and 11A.
[0368] In order to determine the sealant (top and bottom)
degradation in vivo, articular cartilage samples implanted with an
autologous chondrocyte construct using the scaffold matrix were
stained with Safranin-O (FIGS. 12A-12C). Reddish color in
Safranin-O stained figures indicates S-GAG accumulation. Purple
color indicates remaining tissue adhesive with amorphous
structure.
[0369] FIG. 12 thus illustrates a degradation pattern, in time, of
the top and bottom sealants three months after the acellular matrix
implantation. At that time, the superficial cartilage layer was
formed over the implant and the top sealant was partially degraded.
The bottom sealant was, at three months following its deposition at
the bottom of the lesion, completely degraded and removed from the
lesion site.
[0370] FIG. 12A shows a surface view of the Safranin-O stained
implantation site with the superficial cartilage layer clearly
visible and the small amount of the top sealant remaining under the
superficial cartilage layer. FIG. 12B shows a side view of the
Safranin-O stained implantation site. FIG. 12C shows the bottom
view of the Safranin-O stained implantation site where at time zero
the bottom sealant was deposited.
[0371] In this test, the remaining top sealant was observed only at
the surface between the top of the regenerated hyaline like
cartilage region and superficial cartilage layer (FIG. 12A). There
was no indication in side view of any remaining top or bottom
sealant between the interface of the implant site and the
surrounding host cartilage (FIG. 12B). There was no remaining
bottom sealant at the bottom of the lesion interfacing with the
subchondral bone region where the bottom sealant was deposited at
time zero (FIG. 12C).
[0372] These results indicate that the bottom sealant is completely
biodegraded and removed from the lesion site in about three month
after implantation. At that time, there are still remnants of the
top sealant visible on the surface of the lesion where the sealant
protects the acellular implant from any migration or invasion of
synovium and at the same time supports the formation of the
superficial cartilage layer. With time even these remnants of the
top sealant are biodegraded and removed from the healed lesion as
evidenced by a complete absence of any top or bottom sealant at the
defect site.
[0373] A reason why the top sealant is still present at three
months time is that, compared to the surface area, the side and
bottom of the acellular implant site are more active regions for
cell migration which is important for cell integration and
formation of hyaline cartilage. In these regions, the sealant was
completely degraded within 3 months. This phenomenon occurred and
was observed in both the cellular and acellular matrix implantation
in vivo. Cellular implant is described in copending application
Ser. No. 10/625,245 filed on Jul. 22, 2003.
[0374] In order to confirm that the surgical technique used for
creation of the cartilage defects in control and experimental
animals is distinguished from the microfracture technique which
penetrates the subchondral bone area, an image of full thickness
defect at femoral condyle of mini-pig was created and is shown,
with 72.times. magnification, in FIG. 13. FIG. 13 shows a paraffin
embedded and Safranin-O stained reference tissue of the created
full thickness defect. The defect was created of non-treated
articular cartilage and bone from the femoral condyle surrounded by
the host cartilage and underlying subchondral bone area. The
remaining calcified cartilage area is seen in the area above the
subchondral bone. This tissue was utilized in all studies as a
reference tissue used for histological evaluation.
[0375] The results described above show that implantation of the
biodegradable acellular matrix implant into the cartilage lesion
according to the invention induces chondrocyte migration from
surrounding native cartilage and formation of an extracellular
matrix and leads to synthesis of a new hyaline cartilage with
minimal synovial invasion of fibrovascular pannus at the implant
sites.
[0376] Synthesis of the new hyaline cartilage was measured by the
extracellular matrix accumulation expressed as accumulation of
S-GAG. Also observed was a cell integration between the
biodegradable acellular implant and the host cartilage. The use of
a bottom and top sealants and sutures primarily to secure the
implant within the defect suggest that these could have a secondary
effect of minimizing synovial tissue invasion at the implant site.
On the other hand, the results described above and illustrated by
the figures clearly show that the intact nontreated control defects
result in synovial invasion of the defect with fibrovascular
pannus.
[0377] The acellular matrix implant most suitable for practicing
the invention comprises a porous honeycomb sponge of Type I
atelocollagen filled with a thermoreversing hydrogel of Type I
collagen sandwiched between a bottom layer and a top layer of the
sealant. The type I collagen cell walls of the porous honeycomb add
further strength to the sealing capacity of the sealant by adding
to the collagen-PEG chemical interaction analogously to the
reaction of metal reinforcing bar to concrete.
[0378] The acellular implant itself is fully biodegradable in time.
During that time the following conditions are observed in sexually
mature but not fully epiphysealy-fused mini-swine. It is observed
that in a 2 mm lesion of the femoral condyle covered with the top
sealant, a superficial cartilage layer extending from the edge of
the healthy cartilage region peripheral to the acellular implant
proceeds to overgrow the lesion and the sealant layer.
Additionally, chondrocyte migration into the acellular implant and
production of the new hyaline cartilage matrix that eventually
fills and replaces the implant is observed. This new cartilage
matrix is or closely resembles hyaline cartilage as measured by
sulfated glycoaminoglycan content and histological appearance. The
source of these migrating chondrocytes are likely to be both the
peripheral deeper layers of healthy chondrocytes peripheral to the
acellular implant, and also the overgrown superficial cartilage
layer, since it is shown that this layer is the source of
differentiated chondrocytes capable of producing hyaline cartilage.
Eventually hyaline-like cartilage is found to fill the implant
while at the same time the implanted acellular matrix is gradually
biodegraded.
[0379] In the current methodological arrangement, the top and
bottom sealants is intended to prevent debris from subchondral
space to enter the implant (bottom sealant) and to sequester the
implant within a lesion space (top sealant). The acellular matrix
implant sequestered within the lesion permits chondrocytes from the
surrounding healthy cartilage to migrate and enter the matrix.
Naturally applied hydrostatic pressure during a normal physical
activity promotes chondrogenesis leading to a formation of true
hyaline cartilage and to a healing of the lesion.
[0380] Results of studies described above confirm that the damaged,
injured, diseased or aged cartilage may be repaired by using
acellular implants prepared according to the invention and that the
acellular matrix implant of the invention induces cell migration
from surrounding healthy host cartilage and its implantation
induces the inward growth of the superficial cartilage membrane
from the healthy tissue on the periphery. This membrane,
superficial cartilage layer, protects the implant within the lesion
from any synovial invasion. Once the implant is properly implanted
within the lesion, the natural physicochemical factors, such as
intermittent hydrostatic pressure, low oxygen tension and growth
factors induce the cartilage recovery.
[0381] The advantages of the acellular matrix implant system are
multiple. There is no need for biopsy and cell harvesting, no need
to cover periosteum over the lesion, no damage to healthy tissue,
the second and third surgery is eliminated resulting in faster
recovery and elimination of waiting periods for the next
surgery.
[0382] Advantages listed above are similarly attached to treatments
of subchondral or bone lesions.
EXAMPLE 1
Preparation of Acellular Collagenous Implants
[0383] This example illustrates preparation of the acellular matrix
implant.
[0384] 300 grams of a 1% aqueous atelocollagen solution
(VITROGEN.RTM.), maintained at pH 3.0, is poured into a 10.times.20
cm tray. This tray is then placed in a 5 liter container. A 50 ml
open container containing 30 ml of a 3% aqueous ammonia solution is
then placed next to the tray, in the 5 liter chamber, containing
300 grams of said 1% aqueous solution of atelocollagen. The 5 liter
container containing the open trays of atelocollagen and ammonia is
then sealed and left to stand at room temperature for 12 hours.
During this period the ammonia gas, released from the open
container of aqueous ammonia and confined within the sealed 5 liter
container, is reacted with the aqueous atelocollagen resulting in
gelling said aqueous solution of atelocollagen.
[0385] The collagenous gel is then washed with water overnight and,
subsequently, freeze-dried to yield a sponge like matrix. This
freeze dried matrix is then cut into squares, sterilized, and
stored under a sterile wrap.
[0386] Alternatively, the support matrix may be prepared as
follows.
[0387] A porous collagen matrix, having a thickness of about 4 mm
to 10 mm, is hydrated using a humidity-controlled chamber, with a
relative humidity of 80% at 25.degree. C., for 60 minutes. The
collagen material is compressed between two Teflon sheets to a
thickness of less than 0.2 mm. The compressed material is then
cross-linked in a solution of 0.5% formaldehyde, 1% sodium
bicarbonate at pH 8 for 60 minutes. The cross-linked membrane is
then rinsed thoroughly with water, and freeze-dried for about 48
hours. The dense collagen barrier has an inner implantation of
densely packed fibers that are intertwined into a multi-layer
structure.
[0388] In alternative, the integration layer is prepared from
collagen-based dispersions or solutions that are air dried into
sheet form. Drying is performed at temperatures ranging from
approximately 4 to 40.degree. C. for a period of time of about 7 to
48 hours.
[0389] For histological evaluation, 4% paraformaldehyde-fixed,
paraffin sections were stained with Safranin-O (Saf-O) and Type II
collagen antibody.
[0390] For biochemical analysis, seeded sponges were digested in
papain at 60.degree. C. for 18 hours and DNA content was measured
using the Hoechst 33258 dye method. Sulfated glycosaminoglycan
(S-GAG) accumulation was measured using a modified
dimethylmethylene blue (DMB) microassay.
EXAMPLE 2
Biochemical and Histological Assays
[0391] This example describes assays used for biochemical and
histological studies.
[0392] For biochemical (DMB) assay, the implant taken from the
animal after certain time following the implantation, transferred
to microcentrifuge tubes and digested in 300 .mu.l of papain (125
.mu.g/ml in 0.1 M sodium phosphate, 5 mM disodium EDTA, and 5 mM
L-cysteine-HCl) for 18 hours at 60.degree. C. S-GAG production in
the implant is measured using a modified dimethylene blue (DMB)
microassay with shark chondroitin sulfate as a control according to
Connective Tissue Research, 9: 247-248 (1982).
[0393] DNA content is determined by Hoechst 33258 dye method
according to Anal. Biochem., 174:168-176 (1988).
[0394] For histological assay, the remaining implants from each
group were fixed in 4% paraformaldehyde. The implants were
processed and embedded in paraffin. 10 .mu.m sections were cut on a
microtome and stained with Safranin-O (Saf O).
[0395] For immunohistochemistry, the samples are contacted with
diaminobenzidine (DAB). The DAB is a color substrate showing brown
color when the reaction is positive.
EXAMPLE 3
Evaluation of Integration of Acellular Matrix Implant in a Swine
Model
[0396] This example describe the procedure and results of study
performed for evaluation of integration of porcine in a swine
model.
[0397] An open arthrotomy of the right knee joint was performed on
all animals, and a biopsy of the cartilage was obtained.
[0398] A defect was created in the medial femoral condyle of the
pig's right knee. This defect (control) was not implanted with an
acellular matrix implant but was left intact. Following surgery,
the joint was immobilized with an external fixation implant for a
period of about two weeks. Two weeks after the arthrotomy on the
right knee was performed, an open arthrotomy was performed on the
left knee and defects were created in this medial femoral condyle.
The acellular matrix implant was implanted within the defect(s) in
this knee which was similarly immobilized. The operated sites were
subsequently viewed via arthroscopy two weeks after implantation or
defect creation and thereafter at monthly intervals.
[0399] Animals were euthanized and the joints harvested and
prepared for histological examination approximately 7 months after
acellular matrix implant implantation. The implanted sites were
prepared and examined histological.
* * * * *