U.S. patent application number 10/625245 was filed with the patent office on 2004-08-05 for neo-cartilage constructs and a method for preparation thereof.
Invention is credited to Kusanagi, Akihiko, Mizuno, Shuichi, Smith, Robert Lane, Tarrant, Laurence J. B., Tokuno, Toshimasa.
Application Number | 20040151705 10/625245 |
Document ID | / |
Family ID | 37035713 |
Filed Date | 2004-08-05 |
United States Patent
Application |
20040151705 |
Kind Code |
A1 |
Mizuno, Shuichi ; et
al. |
August 5, 2004 |
Neo-cartilage constructs and a method for preparation thereof
Abstract
Neo-cartilage constructs suitable for implantation into a joint
cartilage lesion in situ and a method for repair and restoration of
function of injured, traumatized, aged or diseased cartilage. The
construct comprises at least chondrocytes incorporated into a
support matrix processed according to the algorithm comprising
variable hydrostatic or atmospheric pressure or non-pressure
conditions, variable rate of perfusion, variable medium
composition, variable temperature, variable cell density and
variable time to which the chondrocytes are subjected.
Inventors: |
Mizuno, Shuichi; (Brookline,
MA) ; Kusanagi, Akihiko; (Brookline, MA) ;
Tarrant, Laurence J. B.; (Easthampton, MA) ; Tokuno,
Toshimasa; (Tokyo, JP) ; Smith, Robert Lane;
(Palo Alto, CA) |
Correspondence
Address: |
Hana Verny
Peters, Verny, Jones & Schmitt LLP
Suite 6
385 Sherman Avenue
Palo Alto
CA
94306
US
|
Family ID: |
37035713 |
Appl. No.: |
10/625245 |
Filed: |
July 22, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10625245 |
Jul 22, 2003 |
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10104677 |
Mar 22, 2002 |
|
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60425696 |
Nov 12, 2002 |
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60427627 |
Nov 18, 2002 |
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Current U.S.
Class: |
424/93.7 |
Current CPC
Class: |
A61L 27/3843 20130101;
A61F 2310/00365 20130101; A61L 27/24 20130101; A61L 27/3817
20130101; A61L 2300/64 20130101; A61L 27/3895 20130101; A61L 27/24
20130101; C12N 2521/00 20130101; C12N 5/0655 20130101; A61F 2/28
20130101; C12N 2533/54 20130101; A61L 27/56 20130101; A61L 27/38
20130101; A61L 2430/06 20130101; A61L 27/3633 20130101; A61L 27/54
20130101; A61L 27/38 20130101; A61F 2/28 20130101; A61L 27/3852
20130101; A61L 27/56 20130101; A61K 35/32 20130101; A61K 9/0024
20130101; A61K 35/12 20130101 |
Class at
Publication: |
424/093.7 |
International
Class: |
A61K 045/00 |
Claims
What is claimed
1. A neo-cartilage construct for in situ implanting into a
cartilage lesion, said construct comprising a cultured
differentiated autologous or heterologous chondrocytes or cells
which could be differentiated into chondrocytes incorporated into a
support matrix and subjected to an algorithm of the invention
wherein said algorithm comprises variable hydrostatic or
atmospheric pressure or non-pressure conditions, variable rate of
perfusion, variable medium composition, variable temperature,
variable concentration of oxygen or carbon dioxide, variable cell
density and variable time to which the chondrocytes are
subjected.
2. The construct of claim 1 wherein the support matrix is a sponge,
porous scaffold or a hydrogel 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 a
glycoprotein, gelatin, agarose, hyaluronin, fibronectin, laminin, a
bioactive peptide growth factor, cytokine, elastin, fibrin, a
synthetic polymeric fiber made of a polylactic acid, a synthetic
polymeric fiber made of a polyglycolic acid, a synthetic polymeric
fiber made of a polyamino acid, polycaprolactone, a polyamino acid,
a polypeptide gel, a polymeric thermo-reversible gelation hydrogel
(TRGH), a copolymer thereof and a combination thereof.
3. The construct of claim 2 wherein the support matrix is the
TRGH.
4. The construct of claim 3 wherein the hydrostatic pressure is a
cyclic or constant pressure.
5. The construct of claim 4 wherein the hydrostatic pressure is
from about zero MPa to about 10 MPa above atmospheric pressure at
about 0.01 to about 1 Hz, wherein the time for applying the
hydrostatic pressure is from zero to about 24 hours per day for
from about one day to about ninety days, wherein said hydrostatic
pressure is preceded or followed by a period of zero to about 24
hours per day of a static atmospheric pressure for from about one
day to about ninety days, wherein the flow rate is from about 1
.mu.L/min to about 500 .mu.L/min, wherein the cell density is from
about 3 to 60 millions and wherein the oxygen concentration is from
about 1 to about 20%.
6. The construct of claim 5 wherein the hydrostatic cyclic pressure
is from about 0.05 MPa to about 3 MPa at 0.1 to about 0.5 Hz or
constant pressure is from about zero to about 3 MPa above
atmospheric pressure and wherein such pressure is applied for about
7 to about 28 days.
7. The construct of claim 6 wherein said hydrostatic pressure is
preceded or followed by a period of about zero to about 28 days of
atmospheric pressure.
8. The construct of claim 7 wherein said chondrocytes are
autologous.
9. The construct of claim 7 wherein said chondrocytes are
heterologous.
10. A method for fabrication of a three-dimensional neo-cartilage
construct for in situ implantation into a cartilage lesion, said
method comprising steps: a) preparing a support matrix structure;
b) harvesting a piece of cartilage from a donor for isolation of
chondrocytes; c) culturing and expanding the chondrocytes; d)
suspending the expanded chondrocytes in a suspension fluid; e)
incorporating said suspended chondrocytes into said matrix; and f)
propagating said chondrocytes into two or three-dimensional
neo-cartilage construct using an algorithm of the invention.
11. The method of claim 10 wherein the support matrix structure is
prepared from a material selected from the group consisting of
collagen, a Type I collagen, a Type II collagen, a Type IV
collagen, a cell-contracted collagen containing a proteoglycan, a
cell-contracted collagen containing a glycosaminoglycan, a
cell-contracted collagen containing a glycoprotein, gelatin,
agarose, hyaluronin, fibronectin, laminin, a bioactive peptide
growth factor, a cytokine, elastin, fibrin, a synthetic polymeric
fiber made of a polylactic acid, a synthetic polymeric fiber made
of a polyglycolic acid, a synthetic polymeric fiber made of a
polyamino acid, polycaprolactone, a polyamino acid, a polypeptide
gel, a hydrogel, a copolymer thereof and a combination thereof.
12. The method of claim 11 wherein the support matrix is prepared
from collagen, the Type I collagen, the Type II collagen or the
Type IV collagen.
13. The method of claim 12 wherein said collagen, the Type I
collagen, the Type II collagen or the Type IV collagen is
freeze-dried or lyophilized into a sponge or a sponge-like
structure.
14. The method of claim 11 wherein the support matrix is the
polymeric thermo-reversible gelling hydrogel (TRGH) or a polymeric
sol-gel hydrogel.
15. The method of claim 11 wherein said cultured and expanded
chondrocytes are incorporated into said support matrix suspended in
a gel solution.
16. The method of claim 15 wherein the support matrix is the
collagen sponge or a sponge-like structure and wherein the
chondrocytes are incorporated into said sponge in the TRGH or
sol-gel hydrogel.
17. The method of claim 16 wherein the chondrocytes are
incorporated into said sponge at a density of from about 3 to about
60 millions cells/ml.
18. The method of claim 17 wherein the hydrostatic pressure is from
about zero MPa to about 10 MPa above atmospheric pressure at about
0.01 to about 1 Hz, wherein the time for applying the hydrostatic
pressure is from zero to about 24 hours per day for from about one
day to about ninety days, wherein said hydrostatic pressure is
preceded or followed by a period of zero to about 24 hours per day
of a static atmospheric pressure for from about one day to about
ninety days, wherein the flow rate is from about 1 .mu.L/min to
about 500 .mu.L/min, wherein the cell density is from about 12 to
15 millions and wherein the oxygen concentration is from about 1%
to about 20%.
19. The method of claim 18 wherein the hydrostatic cyclic pressure
is from about 0.05 MPa to about 3 MPa at 0.1 to about 0.5 Hz or
constant pressure is from about zero to about 3 MPa above
atmospheric pressure and wherein such pressure is applied for about
7 to about 28 days.
20. The method of claim 19 wherein said hydrostatic pressure is
preceded or followed by a period of about zero to about 28 days of
atmospheric pressure.
Description
[0001] This application is a continuation-in-part application of
Ser. No. 10/104,677 filed on Mar. 22, 2002 and is based on and
claims priority of the Provisional application Ser. No. 60/425,696
filed on Nov. 12, 2002 and Provisional application Ser. No.
60/427,627 filed on Nov. 18, 2002.
BACKGROUND OF THE INVENTION
[0002] 1. Field of Invention
[0003] The current invention concerns neo-cartilage constructs
suitable for implantation into a joint cartilage lesion in situ and
a method for repair and restoration of function of injured,
traumatized, aged or diseased cartilage. The construct of the
invention comprises at least chondrocytes incorporated into a
support matrix processed according to the algorithm of the
invention. In particular, the invention concerns a neo-cartilage
construct for in situ implanting into a cartilage lesion wherein
said construct comprises a cultured differentiated autologous or
heterologous chondrocytes or cells which could be differentiated
into chondrocytes incorporated into a support matrix and subjected
to an algorithm of the invention wherein said algorithm comprises
variable hydrostatic or atmospheric pressure or non-pressure
conditions, variable rate of perfusion, variable medium
composition, variable temperature, variable concentration of oxygen
or other gases, variable cell density and variable time to which
the chondrocytes are subjected.
[0004] Additionally, the invention concerns a method for generation
of the neo-cartilage and the neo-cartilage construct of the
invention.
[0005] The invention further concerns a method where the
implantation of the construct of the invention initiates and
achieves incorporation of the neo-cartilage into a native
surrounding cartilage. The construct is implanted into the joint
cartilage lesion typically below one layer or between two layers of
biologically acceptable sealants.
[0006] The invention further concerns a method for repair and
restoration of the injured, damaged, diseased or aged cartilage
into its full functionality and for treatment of injured or
diseased cartilage by implanting the neo-cartilage construct
between two layers of biologically acceptable sealants.
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 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.
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] It would, therefore, be extremely advantageous to have
available a method for in situ treatment of these injuries which
would effectively restore the cartilage to its pre-injury
state.
[0010] 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.
[0011] 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.
[0012] 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 multi
cell-layered synthetic cartilage.
[0013] 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.
[0014] None of the above cited references results in repair and
regeneration of cartilage in situ including de novo formation of
the superficial cartilage layer sealing a joint cartilage lesion in
situ.
[0015] It is thus a primary objective of this invention to provide
a method and means for regeneration of injured or traumatized
cartilage by forming, in the injured lesion of the cartilage, a
cavity, by administering at least one but typically two separate
layers of a biologically acceptable glue sealant and implanting a
neo-cartilage containing construct under the one layer or into said
cavity. The method according to the invention results in the growth
of the superficial cartilage layer over the lesion and sealing the
lesion.
[0016] All patents, patent applications and publications cited
herein are hereby incorporated by reference.
SUMMARY
[0017] One aspect of the current invention is a neo-cartilage
construct suitable for implantation into a cartilage lesion in
situ.
[0018] Another aspect of the current invention is a neo-cartilage
construct implanted under one or between two layers of biologically
acceptable sealants within a cartilage lesion.
[0019] Another aspect of the current invention is a method for
fabrication of a three-dimensional neo-cartilage construct of the
invention comprising steps of:
[0020] a) preparing a support matrix structure;
[0021] b) harvesting a piece of cartilage from a donor for
isolation of chondrocytes;
[0022] c) culturing and expanding the chondrocytes;
[0023] d) suspending the expanded chondrocytes in a suspension
fluid;
[0024] e) incorporating said suspended chondrocytes into said
matrix; and
[0025] f) propagating said chondrocytes into two or
three-dimensional neo-cartilage construct using the algorithm of
the invention.
[0026] Still another aspect of the current invention is a method
for generation of an autologous type of neo-cartilage construct by
generating a carrier support for autologous chondrocytes cultured
into neo-cartilage wherein said support is a biologically
acceptable cell-carrier thermo-reversible polymer gel or a
thermo-reversible gelation hydrogel (CCTG, TRGH or VITROGEN.RTM.),
wherein said neo-cartilage is suspended within the CCTG and wherein
a resulting CCTG/neo-cartilage or TRGH/neo-cartilage or a
suspension thereof is injected into the cartilage lesion.
[0027] Still another aspect of the current invention is a
neo-cartilage construct implanted in situ into a cartilage lesion
between two layers of sealants wherein a first sealant is deposited
at the bottom of a cartilage lesion and the second sealant is
deposited over the implanted construct on the top of the cartilage
lesion and wherein the second sealant leads to formation and growth
of superficial cartilage layer which seals said cartilage
lesion.
[0028] 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:
[0029] a) preparing a neo-cartilage construct comprising autologous
or heterologous chondrocytes incorporated into a sponge, porous
scaffold or thermo-reversible gelation hydrogel (TRGH) matrix
support and subjected to the algorithm of the invention;
[0030] b) optionally introducing a first layer of a first
biologically acceptable sealant into a cartilage lesion;
[0031] c) implanting said construct into said lesion or into said
cavity over the first layer of said first sealant;
[0032] d) introducing a second layer of a second biologically
acceptable sealant over said construct wherein said second sealant
may or may not be the same as the first sealant and wherein a
combination of said construct and said second sealant results in
formation and growth of a superficial cartilage layer sealing the
cartilage lesion in situ.
[0033] 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:
[0034] a) obtaining autologous or heterologous chondrocytes;
[0035] b) culturing said chondrocytes ex vivo into a neo-cartilage,
said neo-cartilage comprising autologous or heterologous
chondrocytes incorporated into a sponge or TRGH matrix support
subjected to the algorithm of the invention;
[0036] c) optionally introducing a first layer of a first
biologically acceptable sealant into a cartilage lesion;
[0037] d) depositing a space-holding thermo-reversible gel (SHTG)
or TRGH into a lesion or into a cavity formed above the first
sealant layer thereby permitting sufficient time for growth and
differentiation of ex vivo cultured neo-cartilage, said space
holding thermo-reversible gel (SHTG) deposited into said cavity as
a sol at temperatures between about 5 to about 25.degree. C.,
wherein within said cavity and at the body temperature said SHTG
converts from the fluidic sol into a solid gel and in this form
SHTG holds the space for subsequent introduction of the
neo-cartilage cultured ex vivo, and provides protection against
cell and blood-borne agents migration into the cavity from the
subchondral space and from the synovial capsule and wherein its
presence further provides a substrate for and promotes in situ
formation of a de novo superficial cartilage layer covering the
cartilage lesion;
[0038] e) depositing a second layer of a second biologically
acceptable sealant over the cartilage lesion;
[0039] f) removing said SHTG by cooling said lesion to change SHTG
into sol;
[0040] f) depositing said neo-cartilage cultured ex vivo into the
cavity formed between two layers of sealants and under the de novo
formed superficial cartilage layer;
[0041] g) removing said SHTG or TRGH from the cavity after the
neo-cartilage integration into a native cartilage under the formed
superficial cartilage layer by cooling said lesion to from about 5
to about 15.degree. C. to convert the solid gel into fluidic sol
and removing said sol or, in alternative, leaving said SHTG or TRGH
to disintegrate and be removed naturally.
[0042] 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:
[0043] a) preparing an intact and discreet piece of neo-cartilage
by culturing autologous or heterologous chondrocytes ex vivo,
suspending said cultured chondrocytes in a thermo-reversible
gelation hydrogel (TRGH) and warming said suspension of
chondrocytes to temperature above 30.degree. C. in order to convert
TRGH into a solid gel and subjecting the solid gel to the algorithm
of the invention;
[0044] b) introducing a first and a second layer of a first and a
second biologically acceptable sealant into a cartilage lesion;
[0045] c) cooling said TRGH/neo-cartilage to 5-15.degree. C. to sol
state;
[0046] d) depositing said neo-cartilage suspended in the TRGH into
a cavity formed between two layers of sealants as a sol at
temperatures between about 5 to about 25.degree. C. wherein, within
said cavity and at the body temperature, said TRGH converts from
the sol state into the solid gel and in this state provides
protection for and enables integration of deposited neo-cartilage
into a native surrounding cartilage and wherein the presence of
TRGH further provides a substrate and promotes in situ formation of
de nova superficial cartilage layer covering the cartilage lesion;
and
[0047] e) leaving said TRGH in the lesion until its disintegration
or, in alternative, removing said TRGH from the cavity as a sol by
cooling said lesion to temperature between 5 and 15.degree. C.
after the neo-cartilage integration and formation of superficial
cartilage layer.
[0048] 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:
[0049] a) preparing neo-cartilage or a neo-cartilage containing
construct comprising autologous cultured chondrocytes incorporated
into a gel or thermo-reversible gel matrix support ex vivo and
subjected to the algorithm of the invention;
[0050] b) introducing a first layer of a first biologically
acceptable sealant into a cartilage lesion;
[0051] c) depositing said construct or said neo-cartilage over the
first layer of the first sealant; and
[0052] d) depositing a layer of a second biologically acceptable
sealant either over the neo-cartilage construct or the
neo-cartilage deposited into a cartilage lesion and covering the
lesion with said second sealant, wherein in time said neo-cartilage
is integrated into the native cartilage and wherein the presence of
the neo-cartilage construct and the second sealant promotes in situ
formation and growth of de novo superficial cartilage layer
covering the cartilage lesion.
[0053] Still another aspect of the current invention is a method
for generation and maintaining integrity of the lesion cavity for
the introduction of neo-cartilage, a neo-cartilage gel, a
neo-cartilage suspension or neo-cartilage construct from a synovial
capsule and for blocking the migration of subchondral and synovial
cells and cell and blood products into said cavity and for
providing a substrate for a formation of superficial cartilage
layer overgrowing the lesion by introducing a biologically
acceptable space-holding thermo-reversible gel (SHTG) into a
cleaned lesion for a duration of culturing autologous chondrocytes
into neo-cartilage before introducing said neo-cartilage or
neo-cartilage construct or suspension into the lesion.
[0054] Still another aspect of the current invention is a method
for treatment of damaged, injured, diseased or aged cartilage by
utilizing any of the methods listed above to implant the
neo-cartilage construct into the lesion.
BRIEF DESCRIPTION OF DRAWINGS
[0055] FIG. 1 shows a construct comprising neo-cartilage. FIG. 1A
is a schematic drawing of the sponge made of sol/gel showing the
distribution of chondrocytes within the collagen sponge. FIG. 1B is
a micrograph of the actual neo-cartilage construct held in the
forceps having 4 mm in diameter and thickness of 1.5 mm. Seeding
density of the construct is 300,000 chondrocytes per 25 .mu.l of
collagen solution (12,000,000 cells/ml).
[0056] FIG. 2A shows a diagram of hydrostatic pressure culture
system. FIG. 2B shows a TESS culture processor unit.
[0057] FIG. 3A is a graph representing S-GAG accumulation in cell
constructs subjected to static atmospheric (control) or cyclic
hydrostatic pressure (test). FIG. 3B is a photomicrograph of
Safranin-O staining for S-GAG on paraffin sections in 18 days
subjected to static pressure. FIG. 3C is a photomicrograph of
Safranin-O staining for S-GAG on paraffin sections in cell
constructs subjected to cyclic hydrostatic pressure for 6 days
followed by 12 days of static pressure.
[0058] FIG. 4 illustrates effect of cyclic and constant hydrostatic
pressure on production of S-GAG (FIG. 4A) and DNA (FIG. 4B).
[0059] FIG. 5A shows S-GAG accumulation in cell constructs under
continued culture conditions of static culture (control), medium
perfusion (COMPa), cyclic hydrostatic pressure (Cy-HP) combined
with medium perfusion (control) and constant hydrostatic pressure
combined with medium perfusion (constant-HP). FIG. 5B illustrates
DNA content at day 6 and day 18 in cells constructs submitted to
static conditions (control), medium perfusion only (COMPa), cyclic
hydrostatic pressure (Cy-HP) and constant hydrostatic pressure
(constant-HP).
[0060] FIG. 6A is a photomicrograph of Safranin-O staining for
S-GAG on paraffin sections in 18 days cell constructs subjected to
static atmospheric pressure. FIG. 6B is a photomicrograph of
Safranin-O staining for S-GAG on paraffin sections in cell
constructs subjected to cyclic hydrostatic pressure for 6 days
followed by 12 days of static pressure. FIG. 6C is a
photomicrograph of type II collagen immunohistochemistry on
paraffin sections in 6 days cell constructs subjected to static
atmospheric pressure. FIG. 6D is a photomicrograph of type II
collagen immunohistochemistry on paraffin sections in cell
constructs subjected to cyclic hydrostatic pressure for 6 days.
[0061] FIG. 7A is a graph illustrating effect of the medium
perfusion flow rate on cell proliferation (DNA content) by cell
constructs subjected to a medium flow rate of either 0.005 or 0.05
ml/min. FIG. 7B illustrates effect of flow rate on production of
S-GAG.
[0062] FIG. 8 shows accumulation detected histologically by
toluidine S-GAG blue staining after 15 days culture submitted to
perfusion (FIG. 8A), cyclic hydrostatic pressure (FIG. 8B) and
constant hydrostatic pressure (FIG. 8C).
[0063] FIG. 9 illustrates effect of low oxygen tension on S-GAG
production (FIG. 9A) and cell proliferation (FIG. 9B).
[0064] FIG. 10A shows an arthroscopic observation of the control
empty defect site 2 weeks after creating empty defect. FIG. 10B
shows an arthroscopic observation of the porcine neo-cartilage
(Porcine-NeoCart.TM.) implant site 2 weeks after the
implantation.
[0065] FIG. 11 shows the control lesion without treatment with
porcine neo-cartilage where the proliferation of fibrocartilage
within the defect site is clearly visible after 4 months. FIG. 11A
shows a defect site vis-a-vis subchondral bone with a site of
formation of fibrocartilage. FIG. 11B shows a defect site synovium
and synovial migration. FIG. 11C shows the defect site and
formation of fibrocartilage.
[0066] FIGS. 12A and 12B shows integration of porcine neo-cartilage
into the lesion within the host's cartilage after 3 months. FIG.
12C shows the regenerated hyaline-like cartilage in the porcine
neo-cartilage implanted site. FIG. 12D shows the integration
between the porcine neo-cartilage and the host cartilage laterally
and at the subchondral bone.
[0067] FIG. 13A shows S-GAG production in cell constructs subjected
to cyclic hydrostatic pressure and to atomospheric pressure
(control) with medium perfusion. FIG. 13B shows DNA content in cell
constructs subjected to cyclic and constant hydrostatic pressure
with medium perfusion.
[0068] FIG. 14 shows histological evaluation of cell constructs by
Safranin-O. FIG. 14A shows S-GAG accumulation at day 0 (initial).
FIG. 14B shows accumulation of S-GAG on day 21 in cell constructs
subjected to atmospheric pressure (control). FIG. 14C shows
accumulation of S-GAG on day 21 in cell constructs subjected to 7
days of cyclic hydrostatic pressure (Cy-HP#1) followed by 14 days
of to atmospheric pressure. FIG. 14D shows accumulation of S-GAG on
day 21 in cell constructs subjected to 14 days of cyclic
hydrostatic pressure (Cy-HP#2) followed by 7 days of to atmospheric
pressure. FIG. 14E shows accumulation of S-GAG on day 21 in cell
constructs subjected to 7 days of constant hydrostatic pressure
(Constant-HP) followed by 14 days of atmospheric pressure.
DEFINITIONS
[0069] As used herein:
[0070] "Chondrocyte" means a nondividing cartilage cell which
occupies a lacuna within the cartilage matrix.
[0071] "Isogenous chondrocytes" means clones of cartilage cell
derived from one cell of division. Isogenous chondrocytes occur in
clusters called isogenous nests.
[0072] "Autologous chondrocytes" means chondrocytes isolated from a
donor's own healthy articular cartilage.
[0073] "Heterologous chondrocytes" means chondrocytes derived from
a donor of a different species or from a donor of the same species
but not the recipient individual or a donor tissue that is derived
from the recipient individual but is non-articular cartilage
isolated from a cartilage of the different species.
[0074] "Support matrix" means biologically acceptable sol-gel or
sponge scaffold suitable for seeding expanded chondrocytes that
provides a structural support for growth and three-dimensional
propagation of chondrocytes. 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, 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 be able to have or has a defined
structure.
[0075] "Neo-cartilage" means an immature hyaline cartilage wherein
the ratio of extracellular matrix to chondrocytes is lower than in
mature hyaline cartilage.
[0076] "Mature hyaline cartilage" means cartilage consisting of
groups of isogenous chondrocytes located within lacunae cavities
which are scattered throughout an extracellular collagen
matrix.
[0077] "Autologous Cultured Neo-Cartilage" means a hyaline
neo-cartilage tissue grown ex vivo from chondrocytes isolated from
a donor's own healthy articular cartilage.
[0078] "Neo-cartilage construct", "NEOCART.TM." or "NeoCart.TM."
means a 3-dimensional structural composition comprising
chondrocytes incorporated into a matrix support treated by or
subjected to the algorithm of the invention. Neo-cartilage
construct thus means a discrete piece of hyaline neo-cartilage
formed from cultured chondrocytes for implantation into lesion of a
damaged, aged or diseased cartilage wherein, after implantation,
the neo-cartilage is integrated into a native cartilage within the
lesion. NeoCart.RTM. cartilage is manufactured by and is
proprietary of Histogenics Corporation, Easthampton, Mass.
[0079] "TESS.TM." means Tissue Engineering Support System which is
available as TESS culture processor unit for culturing of
chondrocytes prepared from arthroscopic biopsy samples. The unit
permits changes in hydrostatic pressure, including cyclic
hydrostatic pressure changes and controls other physical parameters
such as temperature, gas concentration, medium perfusion rate and
such other parameters as may be needed. Relevant detailed
information is found in U.S. Pat. No. 6,432,713 B2, patent
application Ser. No.: 09/895,162, Ser. No.: 09/895,161, PCT
JPO1/01516, Japanese patent applications 2001-126543 and
2001-261556, incorporated herein by reference.
[0080] "Sealant" means a biologically acceptable typically
rapid-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) which is preferably cross-linked with a
collagen compound, typically alkylated collagen. Examples of
suitable sealants are tetra-hydrosuccinimidyl or tetra-thiol
derivatized PEG, or a combination thereof, commercially available
from Cohesion Technologies, Palo Alto, Calif. under the trade name
CoSeal.TM., described in J. Biomed. Mater. Res Appl. Biomater.,
58:545-555 (2001), or 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, as described in U.S. Pat. No. 6,312,725B1, herein
incorporated by reference, and cross-linked PEG with methyl
collagen, such as a cross-linked polyethylene glycol hydrogel with
methyl-collagen. The sealant of the invention typically gels and/or
bonds rapidly upon contact with tissue, particularly with tissue
containing collagen.
[0081] "First sealant" means a biologically acceptable tissue
sealant which is deposited at the bottom of the lesion.
[0082] "Second sealant" means a biologically acceptable sealant
which is deposited above and over the neo-cartilage construct
implanted into a lesion. The second sealant may or may not be the
same as the first sealant and is preferably a cross-linked
polyethylene glycol hydrogel with methyl-collagen.
[0083] "Hydrostatic pressure" means pressure measured above the
atmospheric pressure.
[0084] "Cyclic hydrostatic pressure" or "Cy-HP" means the
application of repeated, two or multiplicity periods of applied
hydrostatic pressure within a defined loading interval which
creates a sine wave form of measured pressure.
[0085] "Constant hydrostatic pressure", "constant-HP" or "CHP"
means the application of a non-fluctuating or non-cyclic pressure
load over a period of time.
[0086] "Loading" or "loading interval" means a period of applied
cyclic hydrostatic pressure load followed by a return to
atmospheric pressure where no external pressure is applied.
[0087] "Resting phase" means a variable length of time wherein
cells are maintained in culture at atmospheric pressure after
exposure to or culturing under cyclic hydrostatic pressure.
[0088] "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, fibrocartilage,
tendon, and bone within a support structure, such as multi-layered
system, scaffold or collagen matrix or formation of superficial
cartilage layer.
[0089] "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.
[0090] "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 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.
[0091] "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.
[0092] "SHTG" means space holding thermo-reversible gel.
[0093] "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.
[0094] "GAG" means glycosaminoglycan.
[0095] "S-GAG" means sulfated glycosaminoglycan.
[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] "MPa" means MegaPascal. One MPa is equal to 145 psi.
"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. "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.
[0099] "The algorithm" means variable defined conditions, such as
variable pressure or non-pressure conditions, variable perfusion
rate, different medium, different cell density, different
temperature, variable time, different oxygen and carbon dioxide
conditions, etc., to which a cellular construct of neo-cartilage is
subjected in order to convert it to a mature neo-cartilage
construct.
[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; 100 N/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 (pulling in extension); 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/cm2) 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 finding that when metabolically
active but non-dividing chondrocytes are processed according to the
invention, they become activated and dividing. These activated and
dividing chondrocytes then may be converted into neo-cartilage and
upon incorporating this neo-cartilage into the support matrix and
submitting said neo-cartilage/support matrix to the algorithm of
the invention become a structural unit called neo-cartilage
construct. Such processed neo-cartilage construct is suitable for
implantation into a lesion of injured, traumatized, aged or
diseased cartilage or under the top sealant or between layers of a
first (bottom) and a second (top) sealant. Under these conditions
the second top sealant promotes in situ formation of de novo
superficial cartilage layer over the cartilage lesion.
[0104] The invention thus, in its broadest scope, concerns a method
for preparation of neo-cartilage from chondrocytes harvested from a
donor's tissue, a method for formation of a support matrix, a
method for fabrication of a neo-cartilage construct, a method for
de novo formation of a superficial cartilage layer in situ, a
method for repair and restoration of damaged, injured, traumatized
or aged cartilage to its full functionality, and a method for
treatment of injuries or diseases caused by damaged cartilage due
to the trauma, injury, disease or,age.
[0105] Briefly, the invention comprises preparation of
neo-cartilage from harvested autologous or heterologous
chondrocytes, culturing and expansion of chondrocytes, seeding the
chondrocytes within a collagenous or thermo-reversible gel support
matrix and propagating said chondrocytes in two or
three-dimensions. To achieve the chondrocyte propagation, the
seeded support matrix is optionally subjected to the algorithm of
variable conditions, such as static conditions, constant or cyclic
hydrostatic pressure, temperature changes, oxygen and/or carbon
dioxide level changes and changes in perfusion flow rate of the
culture medium in the presence of various supplements, such as,
growth factors, donor's serum, ascorbic acid, ITS, etc. The
chondrocyte-seeded support matrix treated as above becomes a
neo-cartilage construct (neo-cartilage) suitable for implanting
into a joint cartilage lesion.
[0106] The neo-cartilage construct is implanted into the lesion
under a top sealant, or into a cavity formed by two layers of
adhesive sealants. The first layer of the sealant is deposited at
and covers the bottom of the lesion and its function is to protect
the integrity of said lesion from cell migration and from effects
of various blood and tissue metabolites and also to form a bottom
of the cavity into which the neo-cartilage construct is
deposited.
[0107] In one embodiment, after the neo-cartilage construct is
emplaced into the lesion cavity, the second adhesive layer is
deposited on the top of the neo-cartilage construct and within
several months results in formation of the superficial cartilage
layer completely sealing the lesion.
[0108] In the alternative embodiment, two adhesive layers may be
deposited concurrently with or before the construct is implanted
into the cavity between them. In such an instance, in the interim,
said cavity may be filed with a space holding thermo-reversible gel
(SHTG). Both sealant layers and the construct or space holding gel
are left within the lesion cavity for a certain predetermined
period of time, typically from one week to several months, or in
case of the space holding gel, until the neo-cartilage construct is
prepared ex vivo and ready to be implanted. The second layer
deposited on the top and over the lesion promotes formation of a
superficial cartilage layer which covers the lesion on the outside
and eventually overgrows the lesion completely thereby resulting in
complete or almost complete sealing of the lesion and of the
neo-cartilage construct deposited within said lesion leading to
incorporation of neo-cartilage into a native cartilage and
resulting in healing of the injured or damaged cartilage. In
alternative, the thermo-reversible gel may serve as an initiator
for promotion of formation of the superficial cartilage layer.
[0109] Both the support matrix of the neo-cartilage construct or
the space holding thermo-reversible gel deposited into the lesion
are materials which are biodegradable and permit and promote
formation of the superficial cartilage layer and integration of the
chondrocytes from the neo-cartilage construct into the native
cartilage within the lesion cavity. Such integration begins within
several weeks or months following the implanting and may continue
for several months and involves a growth and maturing of
neo-cartilage into normal cartilage integrated into the healthy
cartilage. The top sealant layer promotes an overgrowth of the
lesion with the superficial cartilage layer typically in about
two-three months when the sealant is itself degraded.
[0110] In the alternative embodiment, the lesion cavity is filled
with a space-holding gel until the outer superficial cartilage
layer is formed at which time the neo-cartilage construct
comprising ex vivo propagated chondrocytes suspended in a
thermo-reversible sol is introduced at a temperature between 50 and
15.degree. C. After it is introduced into the lesion as a liquid
sol, the introduced thermo-reversible sol-gel is converted into a
solid gel at body temperatures of 37.degree. C. or at the same or
similar temperature as the temperature of the synovial cavity. The
neo-cartilage construct introduced into the lesion is integrated
into the native cartilage surrounding the cavity and is completely
covered with the superficial cartilage layer.
[0111] In the alternative, the neo-cartilage construct is deposited
into a lesion of injured, traumatized, aged or diseased cartilage
over the first (bottom) sealant layer and the thermo-reversible gel
of the neo-cartilage construct promotes in situ formation of the
superficial membrane without a need to add the second sealant.
[0112] The method for treatment of injured, traumatized, diseased
or aged cartilage comprises treating the injured, traumatized,
diseased or aged cartilage with an implanted neo-cartilage
construct prepared by methods described above and/or by any
combination of steps or components as described.
I. Preparation of Neo-Cartilage Constructs
[0113] Preparation of neo-cartilage constructs for implanting into
the cartilage lesion involves harvesting and culturing
chondrocytes, seeding them in the support matrix and preparation
thereof, and propagating the chondrocytes either ex vivo, in vitro,
or in vivo.
[0114] A. Cartilage and Neo-Cartilage
[0115] Cartilage is a connective tissue covering joints and bones.
Neo-cartilage is immature cartilage which eventually, upon
deposition into the lesion according to this invention, is
integrated into and acquires properties of mature cartilage.
Differences between the two types of cartilage is in their
maturity. Cartilage is a mature tissue comprising metabolically
active but non-dividing chondrocytes; neo-cartilage is an immature
cartilage comprising metabolically and genetically activated
chondrocytes which are able to divide and multiply. This invention
utilizes properties of neo-cartilage in achieving repair and
restoration of damaged cartilage into the full functionality of the
healthy cartilage by enabling the neo-cartilage to be integrated
into the mature cartilage surrounding the lesion and in this way
repair the defect.
[0116] a) Cartilage
[0117] Cartilage is a connective tissue characterized by its poor
vascularity and a firm consistency. Cartilage consist 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 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.
[0119] The articular cartilage of the joints, such as the knee
cartilage, is the 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
proteoglycan. 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.
[0120] 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. Partly
because hyaline cartilage is avascular, the spontaneous healing of
large defects is not believed to occur in humans and other mammals
and the articular cartilage has thus only a limited, if any,
capacity for repair.
[0121] 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 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 and less resilient 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.
[0122] b) Neo-Cartilage
[0123] Neo-cartilage is an immature hyaline cartilage where the
ratio of extracellular matrix to chondrocytes is lower than in
mature hyaline cartilage. Mature hyaline cartilage has the ratio of
the extracelluar matrix to chondrocytes approximately 95:5. The
neo-cartilage has a lower ratio of the extracelluar matrix to
chondrocytes than mature cartilage and thus comprises more than 5%
of chondrocytes.
[0124] In the process of development of this invention, it was
discovered that under the conditions described below, the older
inactive chondrocytes could be activated from static non-dividing
stage to an active stage where they divide, multiply, promote
growth of the extracellular matrix and develop into new cartilage
(neo-cartilage). The neo-cartilage thus contains chondrocytes which
were rejuvenated and are surrounded by a newly synthesized
extracellular-matrix macromolecules. A process for activation was
found to require certain period of time, typically from about 1
week to about 3 months and it is thus preferred that the
neo-cartilage be prepared ex vivo where nutrients needs and
mechanical loading are well defined.
[0125] B. Preparation of Neo-Cartilage
[0126] Neo-cartilage prepared according to the current invention is
grown ex vivo from chondrocytes isolated from the mammalian donor's
source. In the alternative, neo-cartilage may also be grown in situ
or in vivo under conditions described below.
[0127] Typical donor sources of mammalian chondrocytes are swine or
humans. Neo-cartilage of the invention for human use is preferably
grown from autologous chondrocytes obtained from the patient during
arthroscopy. While it is preferred that for human use chondrocytes
are autologous, it is to be understood that chondrocytes obtained
from other mammalian sources are equally suitable for preparation
of neo-cartilage for treatment of damaged, diseased or aged
cartilage. The use of both autologous and heterologous chondrocytes
is intended to be within the scope of the invention.
[0128] a) Isolation of Chondrocytes
[0129] Specific procedures used for isolation of mammalian
chondrocytes generally using swine cartilage as an example are
described in Example 1. The isolation of human chondrocytes and
preparation of autologous human neo-cartilage is according to
procedures described in Example 2.
[0130] Briefly, the donor cartilage is obtained either by
arthroscopic biopsy from the human donor or from a joint or bone,
such as, for example, the femur of the slaughtered animal and
processed according to Example 1 or 2. The cartilage is preferably
digested by collagenase, a strong protease, most preferably Type I
collagenase, in a solution containing preferably about 0.15% of
collagenase. The digestion is run for several hours to several
days, preferably for about 18 hours.
[0131] In alternative, the extracellular matter can be digested
with proteases or sugar lyases including but not limited to
heparitinase, heparinase, chondroitinase ABC, chondroitinase B and
chondroitinase AC. The lyases are added in admixture with
collagenase or in a sequential enzyme digestion steps. These lyases
promote further isolation of the chondrocytes from the
extracellular matrix (ECM) including disruption the
glycosaminoglycans of the pericellular environment such that the
chondrocytes do not receive inhibitory signals that prevent them
from dividing or producing healthy new extracellular matrix. This
finding is especially important for osteoarthritic chondrocytes
which have very slow division rates and reduced ability to produce
extracellular matrix.
[0132] This is especially important for osteoarthritic chondrocytes
which have very slow division rates and reduced ability to produce
ECM. U.S. Pat. No. 5,916,557 shows that application of
chondroitinase ABC to chondrocytes in vitro resulted
couterintuitively in the promotion of new cartilage production.
[0133] The ability to free the chondrocytes from all extra- and
pericellular inhibitory material and thereby to promote cell
expansion and differentiation is especially important in autologous
osteoarthritic tissue where the growth is otherwise slow because
these chondrocytes have reduced ability to produce ECM where
neo-cartilage formation in the TESS processor under pressure is
greatly improved by this early step of the process. Furthermore,
this method of stimulating chondrocyte growth and differentiation
is relatively benign compared to the application of growth factors
or other chemical stimuli at a later stage of the formation of
neo-cartilage, since the cells are washed free of the enzymes
before culturing.
[0134] b) Expansion of Chondrocytes
[0135] The isolated chondrocytes are then expanded by any method
suitable for such purposes such as, for example, by incubation in a
suitable growth medium, for a period of several days, typically
from about 3 to about 30 days, preferably for 14 days, at about
37.degree. C. Any kind of culture or incubation apparatus or
chamber may be used for expanding chondrocytes. The expansion of
the cells is preferably associated with the removal of dead
chondrocytes, residual native extracellular matrix and other
cellular debris before the chondrocytes are selected for culturing
and multiplying. Selected chondrocytes are collected and isolated
using trypsinization process or any other suitable method.
[0136] Expanded chondrocytes are then suspended in a suitable
solution and seeded into a support matrix to form a seeded matrix.
The seeded matrix is typically processed in a tissue processor.
[0137] c) Suspension and Seeding of Chondrocytes in the Support
Matrix
[0138] Following the expansion, chondrocytes are suspended in any
suitable solution, preferably collagen containing solution. For the
purposes of this invention such solution 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. The thermo-reversible
property is important both for immobilization of the chondrocytes
within the support matrix and for implanting of the neo-cartilage
construct within the cartilage lesion.
[0139] One characteristic of the sol-gel is its ability to be cured
or transitioned from a liquid into a solid form. This property may
be advantageously used for solidifying the suspension of
chondrocytes withing the support matrix for delivery, storing or
preservation purposes. 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, as described in
greater detail below for thermo-reversible gelation hydrogel, or
exposing the sol-gel to various chemical or physical conditions or
ultraviolet radiation.
[0140] In one embodiment the expanded chondrocytes are suspended in
a collagenous sol-gel solution before incorporation (seeding) into
the support matrix. The sol-gel viscosity permits easy mixing of
chondrocytes avoiding need to use shear forces. One example of the
suitable sol-gel solution is the solution substantially composed of
Type I collagen, commercially available under trade name
VITROGEN.RTM. from Cohesion Corporation, Palo Alto, Calif. VITROGEN
is a purified pepsin-solubilized bovine collagen dissolved in
0.012N HCl. 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.
[0141] When using a VITROGEN solution, the cell density is
approximately 5-10.times.10.sup.6 cells/mL. However, both the
density of the cells, the volume for their seeding and strength of
the solution are variables within the algorithm, and the higher or
lower number of chondrocytes may be suspended in a larger or lower
volume of the suspension solution, depending on the size of the
support matrix and the size of the cartilage lesion.
[0142] Seeding of the suspended chondrocytes into the support
matrix is by any means which permit even distribution of the
chondrocytes within said support matrix. Seeding may be achieved by
bringing the suspension and the support matrix into close contact
and seeding the cells by wicking or suction of the suspension into
the matrix by capillary action, by inserting the support matrix
into the suspension, by using suction, positive or negative
pressure, injection or any other means which will result in even
distribution of the chondrocytes within said support matrix.
[0143] In alternative embodiment, the chondrocytes are suspended in
the thermo-reversible gelation hydrogel or gel polymer at
temperature between 5 and 15.degree. C. At that temperature, the
hydrogel is at a liquid sol stage and easily permits the
chondrocytes to be suspended in the sol. Once the chondrocytes are
evenly distributed within the sol, the sol is subjected to higher
temperature of about 30-37.degree. C. at which temperature, the
liquid sol solidifies into solid gel having evenly distributed
chondrocytes within. 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 a support matrix or
the suspension in sol state may be loaded into a separate support
matrix, such as a sponge or honeycomb support matrix.
[0144] Other means of generating suspending gels, not necessarily
thermo-reversible, are also available and suitable for use.
Polyethylene glycol (PEG) derivatives, in which one PEG chain
contains vinyl sulfone or acrylate end groups, and the other PEG
chain contains free thiol groups will 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 network,
or gel. If the molecular weight of the PEG chains is several
thousand Daltons (500 to 10,000 Daltons along any linear chain
segment), the network will be open, swellable by water, and
compatible with living cells. The coupling reaction can be
accomplished by preparing 5 to 20% (w/v) solutions of each PEG
separately in aqueous buffers or cell culture media. Chondrocytes
can be added to the thiol-PEG solution. Just prior to incorporation
into the support matrix, the cells plus thiol PEG and the acrylate
or vinyl sulfone PEG are mixed and infused into the matrix.
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. Such gels are not degradable unless additional ester or
labile linkages are incorporated into the chain. Such PEG reagents
may be purchased from Shearwater Polymers, Huntsville, Ala., USA;
or from SunBio, Korea.
[0145] In a second alternative, alginate solutions can be gelled in
the presence of calcium ions. This reaction has been employed for
many years to suspend cells in gels or micro-capsules. Cells can be
mixed with a 1-2% (w/v) solution of alginate in culture media
devoid of calcium or other divalent ions, and infused into the
support matrix. The matrix can then be immersed in a solution
containing calcium chloride, which will diffuse into the matrix and
gel the alginate, trapping and supporting the cells. 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 used to suspend cells
and gelled by diffusion of ferric ions.
[0146] Suspension loaded into the support matrix or gelled into the
solid support is processed using the algorithm of the invention.
Such processing is performed in a processing apparatus, such as a
TESS processor.
[0147] C. Preparation of Support Matrix
[0148] The support matrix for seeding expanded chondrocytes
provides a structural support for growth and two or
three-dimensional propagation of chondrocytes. Generally, the
support matrix is biologically biocompatible, hydrophilic and has
preferably a neutral charge.
[0149] Typically, the support matrix 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 support matrix is a sponge-like structure or
honeycomb-like lattice.
[0150] 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 then be
freeze-dried as aqueous dispersions to form sponges. Typically,
such sponges must be stabilized by crosslinking, such as, for
example, ionizing radiation. Practical example includes preparation
of freeze-dried sponges of poly-hydroxyethyl-methacrylate (pHEMA),
optionally having additional molecules, such as gelatin, entrapped
within advantageously. Such types of sponges can advantageously
function as support matrices for the present invention.
Incorporation of agarose, hyaluronic acid, or other bio-active
polymers can be used to modulate cellular responses. A wide range
of polymers may be suitable for the fabrication of support matrix
sponges, including agarose, hyaluronic acid, alginic acid,
dextrans, polyHEMA, and poly-vinyl alcohol above or in
combination.
[0151] Typically, the support matrix 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, glycosaminoglycans or
glycoproteins, fibronectin, laminin, bioactive peptide growth
factors, cytokines, elastin, fibrin, synthetic polymeric fibers
made of poly-acids such as polylactic, polyglycotic or polyamino
acids, polycaprolactones, polyamino acids, polypeptide gel,
copolymers thereof and combinations thereof. Preferably, the
support matrix is a gel solution, most preferably containing
aqueous Type I collagen or a polymeric, preferably
thermo-reversible, gel matrix.
[0152] The gel or gel solution used for preparation of the support
matrix is typically washed with water and subsequently freeze-dried
or lyophilized to yield a sponge like matrix able to incorporate or
wick the chondrocytes suspension withing the matrix. The cellular
support matrix of the current invention acts like a sponge when
infiltrated with the chondrocyte suspension wherein the cells are
evenly distributed.
[0153] One important aspect of the support matrix is the pore size
of the support matrix. Support matrices having different pore sizes
permit faster or slower infiltration of the chondrocytes into said
matrix, faster or slower growth and propagation of the cells and,
ultimately, the higher or lower density of the cells in the
neo-cartilage construct. Such pore size may be adjusted by varying
the pH of the gel solution, collagen concentration, lyophilization
conditions, etc. Typically, the pore size of the support matrix is
from about 50 to about 500 .mu., preferably the pore size is
between 100 and 300 .mu. and most preferably about 200 .mu..
[0154] The support matrix may be prepared according to procedures
described in Example 3, 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 herein incorporated
by reference.
[0155] One preferred type of support matrix is Type-I collagen
support matrix fabricated into a sponge, commercially available
from Koken Company, Ltd., Tokyo, Japan, under the trade name
Honeycomb Sponge.
[0156] An exemplary neo-cartilage support matrix made of collagen
and embedded with chondrocytes is seen in FIG. 1, wherein FIG. 1A
is a schematic drawing of the sponge made of sol/gel showing the
distribution of chondrocytes within the collagen sponge. FIG. 1B
shows a microphotograph of the actual neo-cartilage construct
(Neo-Cart.TM.) having 4 mm in diameter and thickness of 1.5 mm. The
seeding density of this construct is 300,000-375,000 chondrocytes
per 25 .mu.l of collagen solution corresponding to about 12-15
millions cells/mL. The cell density range for seeding is preferably
from about 3 to about 60 millions/mL.
[0157] a) Honeycomb Cellular Support Matrix
[0158] In one embodiment of the invention, the support matrix is a
honeycomb-like lattice matrix providing a cellular support for
activated chondrocytes, herein described as neo-cartilage.
[0159] The honeycomb-like matrix supports a growth platform for the
neo-cartilage and permits three-dimensional propagation of the
neo-cartilage.
[0160] The honeycomb-like matrix is fabricated from a polymerous
compound, such as collagen, gelatin, Type I collagen, Type II
collagen or any other polymer having a desirable properties. In the
preferred embodiment, the honeycomb-like matrix is prepared from a
solution comprising Type I collagen.
[0161] The pores of the honeycomb-like matrix are evenly
distributed within said matrix to form a sponge-like structure able
to taking in and evenly distributing the neo-cartilage suspended in
a viscous solution.
[0162] b) Sol-Gel Cellular Support Matrix
[0163] In another embodiment, the support matrix is 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. Sol-gel material
is a material which is a viscous sol at temperatures of below
15.degree. and a solid gel at temperatures around and above
37.degree.. Typically, these materials change their form from sol
to gel by transition at temperatures between about 15.degree. and
37.degree. and are in transitional state at temperatures between
15.degree. C. and 37.degree.. The most preferred materials are Type
I collagen containing gels and a thermo-reversible gelation
hydrogel (TRGH) which has a rapid gelation point.
[0164] 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 0.012N HCl,
is commercially available under the tradename 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
containing immobilized chondrocytes.
[0165] Type I collagen sol-gel is generally suitable for suspending
the chondrocytes and for seeding them into a separately prepared
support matrix in the sol form and gel the sol into the solid gel
by heating the support matrix to a proper temperature, usually
around 30-37.degree. and, in this form, processing the embedded
support matrix. This type of sol-gel can also be used as a support
matrix for purposes of processing the gel containing chondrocytes
in the processor of the invention into a neo-cartilage
construct.
[0166] In another embodiment, the sol-gel is thermo-reversible
gelation hydrogel (TRGH). Sol-gel thermo-reversible material for
preparation of sol-gel support matrix is a material which is a
viscous sol at temperatures of below 15-30.degree. C. and solid gel
at temperatures above 30-37.degree. C. The primary characteristic
of the thermo-reversible gelation hydrogel (TRGH) is that it gels
at body temperature and sols at lower than 15-30.degree. C.
temperature, 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.
[0167] 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 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.
[0168] The thermo-reversible gelation hydrogel (TRGH), particularly
a space-holding thermo-reversible gel (SHTG), should be a
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 removed from the cavity as the sol. The compressive
strength of the SHTG or TRGH must be able to resist compression by
the normal activity of the joint.
[0169] 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.
[0170] Thermally reversible hydrogels consisting of co-polymers of
polyethylene oxide and polypropylene oxide are available from BASF
Wyandotte Chemical Corporation under the trade name of
Pluronics.
[0171] 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.
[0172] This type of thermo-reversible gelation hydrogel is
particularly preferred for preparation of neo-cartilage constructs
for implantation of the construct into the lesion. In such an
instance, the harvested chondrocytes are suspended in the TRGH sol,
then warmed to about 37.degree. C. into the solid gel which thus
itself becomes a seeded support matrix, then submitting said seeded
matrix to the processing in the tissue processor using the
algorithm of the invention, including resting period as described
below, thereby resulting in a formation of the neo-cartilage
construct, then submitting said construct to cooling to change its
form into a sol and in this form injecting the neo-cartilage into
the lesion wherein upon warming to body temperature the sol is
immediately converted into the gel containing neo-cartilage. In
time, the delivered neo-cartilage is integrated into the existing
cartilage and the TRGH is subsequently degraded leaving no
undesirable debris behind.
[0173] D. Processing Neo-Cartilage and Tissue Processors
[0174] In order to promote three-dimensional growth and propagation
of chondrocytes and/or neo-cartilage, it is advantageous and/or
necessary in certain instances to facilitates such growth and
propagation by changing conditions of their growth. Such
facilitation may be initiated either ex vivo, in vitro or in
vivo.
[0175] This process is, in the current invention, achieved by
subjecting either the suspended expanded chondrocytes or the
support matrix incorporated with suspended chondrocytes to certain
protocol (the algorithm) of conditions which were found to promote
such propagation. Such conditions are, for example, application of
constant or cyclic hydrostatic pressure, resting periods at static
pressure, recirculation and changing flow rate of media, regulation
of oxygen or carbon dioxide concentrations, cell density, control
pH, availability of nutrients and co-factors, etc. Typically, this
process is performed in the apparatus, preferably in the TESS.TM.
tissue processor, permitting changing of the conditions, as stated
above.
[0176] a) Neo-Cartilage Tissue Processor
[0177] The general design of the tissue processor is the apparatus
for culturing chondrocytes comprising a culture unit having a
culture chamber containing culture medium and a supply unit for the
continuous and intermittent delivery of the culture medium, a
pressure generator for applying atmospheric or constant or cyclic
hydrostatic pressure above the atmospheric pressure to chondrocytes
in the tissue chamber, said generator having means for changing the
pressure, timing, or applying the atmospheric, constant or cyclic
hydrostatic pressure at predetermined periods and, optionally, a
means capable of delivering and/or absorbing gases such as
nitrogen, carbon dioxide and oxygen. Additionally, the processor
typically comprises a hermetically sealed space including a
heating, cooling and humidifying means.
[0178] An exemplary scheme of the tissue processor suitable for
applying of static or hydrostatic pressure, changing flow rate of
the medium and regulating gas concentration delivered to the
embedded support system suitable for purposes of this invention is
seen in FIG. 2A. The tissue processor, seen in FIG. 2B, known as
Tissue Engineering Support System (TESS) is described in the U.S.
Pat. No 6,432,713 issued on Aug. 13, 2002, and also in the U.S.
application Ser. No. 09/895,162, both hereby incorporated by
reference.
[0179] b) Biochemical and Histological Testing of Neo-Cartilage
Constructs
[0180] The neo-cartilage constructs are tested for their metabolic
activity, genetic activation and histological appearance.
[0181] Typically, the constructs are harvested at days 6 and 18.
For histological evaluation of the immature and mature cartilage
matrix, 4% paraformaldehyde-fixed paraffin sections are stained
with Safranin-O and Type II collagen antibody. For biochemical
analysis, neo-cartilage constructs are digested in papain at
60.degree. C. for 18 hours and DNA is measured using, for example,
Hoechst 33258 dye method as described in Anal. Biochem.,
174:168-176 (1988). The production of glycoaminoglycan (GAG) or
sulfated-glycosaminoglycan (S-GAG) indicating a metabolic activity
of the chondrocyte culture is tested using, for example, modified
dimethylene blue (DMB) microassay according to Connective Tissue
Research, 9:247-248 (1982).
[0182] c) Conditions for Propagation of Chondrocytes, Preparation
of Neo-Cartilage and Neo-Cartilaqe Constructs
[0183] Neo-cartilage construct, as used herein, means a matrix
embedded with chondrocytes and processed according to the
invention.
[0184] Neo-cartilage constructs may be produced as 3-dimensional
patches comprising neo-cartilage having an approximate size of the
lesion into which they are deposited or they may be produced as
3-dimensional sheet for use in repairs of extensive cartilage
injuries. Their size and shape is determined by the shape and size
of the support matrix. Their functionality is determined by the
conditions (the algorithm) under which they were processed.
[0185] Conditions for three-dimensional propagation of chondrocytes
in the support matrix into neo-cartilage construct are variable and
are adjusted according to the intended use and/or function of the
neo-cartilage and depend on the type of used thermo-reversible
hydrogel and on the density of the seeded cells. Thus for
production of small neo-cartilage constructs, the conditions will
be different from those needed for production of large constructs
or for production of extensive neo-cartilage sheets for partial or
total replacement of extensively damaged or diseased, for example
osteoarthritic, cartilage.
[0186] i) Processing Neo-Cartilage under Variable Flow
[0187] One aspect of this invention is the discovery that if the
support matrix seeded with chondrocytes is perfused under varying
medium flow rates, the cell proliferation, measured by increased
accumulation of the extracellular matrix, can be advantageously
increased or decreased. Generally, the lower medium flow rate
results in the higher extracellular matrix accumulation.
[0188] Perfusion is an important variable condition for culturing
chondrocytes incorporated into support matrices. Using a faster
perfusion flow rate may slow down extracellular matrix accumulation
affecting growth and propagation of chondrocytes, as measured by
production of sulfated glycosaminoglycan (S-GAG). A slower
perfusion rate, on the other hand, results in higher production of
S-GAG. These results are important for controlling the
neo-cartilage growth and for, for example, storage, preservation,
transport and shelf-life of neo-cartilage constructs.
[0189] The perfusion flow rate suitable for purposes of this
invention is from about 1 to about 500 .mu.l/min, preferably from
about of 5 to about 50 .mu.l/min. At the medium perfusion rate 5
.mu.l/min the accumulation of extracellular matrix is significantly
(p<0.05) increased compared to accumulation of extracellular
matrix observed following perfusion at rate 5 .mu.l/min. The
optimum flow rate depends upon the total number of cells in the
culture chamber.
[0190] ii) Processing Neo-Cartilage Under Different Types of
Pressure
[0191] Subjecting the seeded support matrix to hydrostatic
pressure, in conjunction with a decreased perfusion flow, is an
integral part of the culture processing system according to this
invention. Different types of hydrostatic pressure have a
significant effect on glycosaminoglycan production and thus on
extracellular matrix accumulation compared to the effect of
atmospheric pressure alone. The hydrostatic pressure, particularly
cyclic hydrostatic pressure applied according to this invention has
been found to stimulate chondrocyte proliferation and metabolism
which contributes to extracellular matrix accumulation.
[0192] Hydrostatic pressure suitable for processing chondrocytes
embedded within the support matrix is either a constant or cyclic
hydrostatic pressure, such pressure being the pressure above the
atmospheric pressure. The cyclic hydrostatic pressure suitable for
use in processing of the seeded support matrix is from about 0.01
to about 10.0 MPa, preferably from about 0.5 to about 5.0 MPa and
most preferably at about 3.0 MPa at 0.01 Hz to about 2.0 Hz,
preferably at about 0.5 Hz, applied for about 1 hour to about 30
days, preferably about 7 to about 14 days, with or without resting
period. Typically, the period of hydrostatic pressure is followed
by the resting period, typically from about 1 day to about 60 days,
preferably for about 7 to about 28 days, most preferably for about
12 to about 18 days.
[0193] Studies performed in support of this invention indicate that
cell viability is not affected by the hydrostatic pressure and is
maintained with chondrocytes distributed uniformLy within the
support matrix. Following the treatment with hydrostatic pressure,
accumulations of both DNA and S-GAG are significantly increased
compared to cultures not experiencing applied load, indicating that
chondrocyte activation and metabolic and genetic activity can be
controlled by the culture environment.
[0194] iii) Processing Neo-Cartilage Under Reduced Oxygen
Concentration
[0195] Another variable in the processing of seeded support
matrices is the concentration of oxygen, carbon dioxide and
nitrogen.
[0196] The chondrocytes-embedded support matrix described above may
be further cultured under reduced O.sub.2 concentration (i.e. less
than 20% saturation) during formation of neo-cartilage in the TESS
processor. The reduced oxygen concentration of cartilage has been
observed in vivo, and such reduction may be due to its normal lack
of vascularization which produces a lower oxygen partial pressure,
as compared to the adjacent tissues. In this set of studies,
chondrocytes seeded in support matrix or neo-cartilage were
cultured under oxygen concentration between about 0% and about 20%
saturation or under dioxide concentration about 5%.
[0197] E) Determination of Conditions for Optimization of the
Algorithm
[0198] The ultimate aim of this invention was to find and confirm
conditions (the algorithm) for preparation of neo-cartilage
constructs for implantation into cartilage lesions, which in
conjunction with deposition of one or two sealant layers, would
lead to healing of the damaged, injured, diseased or aged cartilage
by a) growth of superficial cartilage layer completely overgrowing
and covering the lesion and protecting implanted neo-cartilage
construct; b) integration of neo-cartilage implanted into the
lesion as the neo-cartilage construct; and c) subsequent
degradation of the construct and sealant materials.
[0199] The underlying studies, described below, show that a
properly designed and optimized culture conditions utilizing
hydrostatic pressure with medium perfusion followed by constant
culture result in fabrication of neo-cartilage constructs which are
integrated into the native cartilage when implanted under the one
layer or in between two layers of sealants according to the
invention.
[0200] General design for a method for preparation of neo-cartilage
constructs comprises steps:
[0201] a) isolation of chondrocytes from a donor tissue;
[0202] b) expanding the chondrocytes for about 3-28 days;
[0203] c) seeding chondrocytes in a thermo-reversible or collagen
gel or collagen sponge support matrix;
[0204] d) subjecting the seeded gel or sponge to a static, constant
or cyclic hydrostatic pressure above atmospheric pressure (about
0.5-3.0 MPa at 0.5 Hz) with medium perfusion rate of 5.mu.l/min for
several (5-10) days; and
[0205] e) subjecting the seeded gel or sponge to resting period for
ten to fourteen days at constant (atmospheric) pressure.
[0206] Neo-cartilage constructs obtained by the above-outlined
conditions and method show that the combined algorithm of
hydrostatic pressure and static pressure has advantage over
conventional culture methods by resulting in higher cell
proliferation and extracellular matrix accumulation. Use of
thermo-reversible or collagen gel or collagen sponge support matrix
maintains uniform cell distribution within the support matrix and
also provides support for newly synthesized extracellular matrix.
Obtained 3-dimensional neo-cartilage construct is easy to handle
and manipulate and can be easily and safely implanted in a surgical
setting.
[0207] Combination of a period of cyclic hydrostatic pressure under
low medium perfusion rate followed up with a period of static
culture (resting period) results in increased cell proliferation,
increased production of Type II collagen, increased DNA content and
increased S-GAG accumulation.
[0208] Increased cell proliferation shows that the harvested
inactive non-dividing chondrocytes have been activated into
neo-cartilage containing active, dividing and multiplying
chondrocytes. Increased level of DNA shows genetic activation of
inactive chondrocytes. Increased production of Type II collagen and
S-GAG shows that production of the extracellular matrix has been
activated using the algorithm described above.
[0209] Although the optimized algorithm described above is
preferred, it is to be understood that this algorithm may be
advantageously changed using variations of ranges of cyclic
hydrostatic pressure, flow rate, duration of the pressure and
resting period as disclosed above in detail description of each
condition. All variations of all conditions and combinations
thereof are intended to be within the scope of this invention.
[0210] F. Supporting Experimental Studies
[0211] In order to test effects of different conditions on the
propagation of chondrocytes within the support matrix for
fabrication of the neo-cartilage construct, studies combining
conditions described above for process optimization were performed
during development of this invention. Results are shown in FIGS.
3-9 and in Tables 1-3.
[0212] For all following studies, the experimental design was as
follows with changes in studies conditions.
[0213] Cartilage was harvested under sterile conditions from the
trachea of the swine hind limbs, minced and digested, as described
in Example 7. Chondrocytes were expanded for 5 days at 37.degree.
C. and suspended in VITROGEN.RTM. (300,000/30 .mu.l). The
suspension was absorbed into a support matrix, usually a collagen
sponge (4 mm in diameter and 2 mm in thickness) as seen in FIG. 1,
commercially available from Koken Co., Tokyo, Japan. The sponges
seeded with chondrocytes were pre-incubated for 1 hour at
37.degree. C. to gel the collagen, followed by incubation in
culture medium at 37.degree. C., 5% CO.sub.2 and cultured in the
Tissue Engineering Support System (TESS.TM.) processor seen in FIG.
2.
[0214] a) Evaluation of Effect of Hydrostatic Pressure
[0215] To evaluate the effect of the pressure and/or medium
perfusion rate, the cell seeded sponges were subjected to medium
perfusion at 5 .mu.l/min (0.005 mL/min) or 50 .mu.l/min (0.05
mL/min) under the cyclic (Cy-HP) or constant hydrostatic pressure
(constant-HP) of 0.5 MPa at 0.5 Hz for 6 days in the TESS
processor. Resting period under atmospheric pressure followed for
12 days. Some seeded sponges served as controls. These were
incubated under the atmospheric pressure and without perfusion at
37.degree. C. for a total of 18 days in culture. Sponges harvested
24 hours after seeding with cells (day 0) served as an initial
control. More detailed conditions are to be found in Examples and
in the following text.
[0216] At the end of culture period, the support matrices were
harvested for biochemical and histological analysis. Sulfated
glycosaminoglycan production was measured using a modified
dimethylmethylene blue microassay. Histological analysis utilized
Safranin-O staining. More detailed conditions are to be found in
Examples.
[0217] The first study was directed to determination of effect of
constant (atmospheric), cyclic or constant hydrostatic pressure on
production of S-GAG.
[0218] At the end of the culture period, both control and test
matrices were harvested for biochemical and histological analysis.
For biochemical analysis, production of sulfated glycosaminoglycan
(S-GAG pg/cell construct) was measured using a modified
dimethylmethylene blue (DMB) and DNA microassays described in
Example 7. Results are seen in Tables 1 and 2 and FIGS. 3-6.
[0219] Results of some studies are seen in Tables 1 and 2 showing a
numerical representation of observed increase in S-GAG production
in matrices treated with the algorithm of the invention.
1 TABLE 1 Pressure Conditions In TESS S-GAG Production (3 MPa
Cyclic In Incubator Total (.mu.g/cell Group Pressure, (Atmospheric
days in construct) (n = 6) @0.5 Hz) Pressure) Culture (Mean .+-.
SD) Initial -- 0 day 0 12.56 .+-. 0.99 Control -- 18 days 18 57.73
.+-. 6.43 Test 6 days 12 days 18 *76.32 .+-. 4.12 (*: p < 0.05,
Compared to Control)
[0220] Table 1 summarizes results obtained from seeded matrices
(n=6) subjected either to atmospheric pressure in an incubator for
18 days (control) or to processing in TESS processor under 3 MPa
cyclic hydrostatic pressure at 0.5 Hz for 6 days, followed by 12
days in incubator at atmospheric pressure (test).
[0221] As seen in Table 1, S-GAG production (.mu.g/cell construct)
per seeded matrix was significantly increased to 132% for test
compared to 100% control (FIG. 3A). Histological results seen in
FIGS. 3B and 3C. (Safranin-O staining for S-GAG) were consistent
with the results seen in Table 1 obtained biochemically. FIG. 3B is
a photomicrograph of Safranin-O staining for S-GAG on paraffin
sections in 18 days subjected to static pressure. FIG. 3C is a
photomicrograph of Safranin-O staining for S-GAG on paraffin
sections in cell constructs subjected to cyclic hydrostatic
pressure for 6 days followed by 12 days of static culture.
[0222] As seen in FIG. 3B, when the cell constructs are subjected
to static atmospheric pressure (FIG. 3B), there is much lower S-GAG
accumulation in the constructs than when it is subjected to a
cyclic hydrostatic pressure for 6 days, followed by 12 days of
static atmospheric pressure (FIG. 3C).
[0223] To determine the effect of the hydrostatic pressure on
chondrocyte proliferation stimulation and matrix accumulation,
cartilage was harvested under sterile conditions as described
above. Chondrocytes were expanded for 5 days at 37.degree. C. and
suspended in VITROGEN.RTM. (300,000/30 .mu.l). The suspension was
absorbed into a honeycomb support matrix or collagen sponge as seen
in FIG. 1. The cell constructs were incubated in culture medium at
37.degree. C., 5% CO.sub.2 and 20% O.sub.2, at 0.5 MPa cyclic
hydrostatic pressure or 0.5 MPa constant hydrostatic pressure for 6
days followed by incubation for 12 days at atmospheric pressure in
the Tissue Engineering Support System (TESS.TM.) processor seen in
FIG. 2. The remaining cell matrices comprising the control group
were incubated at atmospheric pressure for 18 days at 37.degree.
C., 5% CO.sub.2 and 20% O.sub.2.
[0224] At the end of the culture period, the matrices were
harvested for biochemical analysis. Results are seen in Table 2.
Glycosaminoglycan production was measures using a modified
dimethylmethylene blue (DMB) microassay. Cell proliferation was
measured using a modified Hoechst Dye DNA assay. Formation of
neo-tissue was evaluated by Safranin-O staining. Results are seen
in FIGS., 4A, 4B, 5A and 5B and in Table 2.
2 TABLE 2 Pressure Conditions S-GAG Days in Total GAG Production In
TESS Incubator days (.mu.g/cell DNA Group Type of Time/
(Atmospheric In construct) DNA Index (n = 7) Pressure Days
Pressure) culture (Mean .+-. SD) (Control = 1) Control -- -- 18 18
59.85 .+-. 7.69 1 Cy-HP 0.5 MPa 6 12 18 *91.05 .+-. 10.68 1.49
Cyclic Const-HP 0.5 MPa 6 12 18 *97.85 .+-. 5.53 1.74 Constant (*:
p<0.05, compared to Control)
[0225] All cultures were incubated at 37.degree. C., 5% CO.sub.2
and 20% O.sub.2. In TESS culture, the medium flow rate was 50
.mu.l/min. Two cell matrices from each group were harvested for
histological analysis.
[0226] As seen in Table 2, the matrices subjected to conditions
listed in the control group, cyclic hydrostatic pressure (Cy-HP)
and constant hydrostatic pressure (const-HP) groups resulted in
production of 59.85, 91.05 and 97.85 .mu.g/cell construct of S-GAG
and 1, 1.49 and 1.74 (control=1) of DNA content Index,
respectively. These results clearly show that neo-cartilage
cultured under hydrostatic pressure, whether cyclic or constant,
followed by static culture is more genetically and metabolically
active than the neo-cartilage treated under static atomospheric
conditions (controls). These results are graphically illustrated in
FIG. 4 which shows effect of hydrostatic pressure on production of
sulfated glycosaminoglycan (FIG. 4A) and DNA content index(FIG.
4B).
[0227] FIG. 4A is a graphical representation of results enumerated
in Table 2 and shows the sulfated glycosaminoglycan production in
.mu.g/cell construct wherein control represents seeded matrices
subjected to atmospheric pressure, Cy-HP represents seeded matrices
subjected to cyclic hydrostatic pressure (0.5 MPa) and constant-HP
represent matrices subjected to constant hydrostatic pressure (0.5
MPa).
[0228] Results seen in Table 2 are illustrated graphically in FIG.
4A, under the conditions described above. There was significant
increase in S-GAG production for both the cyclic (Cy-HP) and
constant hydrostatic pressure (constant-HP) groups compared to
atmospheric pressure (control) group. Specifically, the production
of S-GAG in the control group was 59.85 .mu.g/cell construct. In
the group Cy-HP the production was 91.05 .mu.g/cell construct. In
the group constant-HP cell construct production was 97.85 4
.mu.g/cell construct resulting in increase of S-GAG production to
152% for group Cy-HP and to 162% for the group constant-HP compared
to the control group.
[0229] FIG. 4B shows DNA production with corresponding results
presented in Table 2 for DNA, likewise showing increased production
of DNA in constructs processed under cyclic or constant hydrostatic
pressure.
[0230] FIG. 5A is a graph comparing effect of constant atmospheric
pressure (Control) and zero MPa hydrostatic pressure (0 MPa)
serving as pressure controls, 0.5 MPa cyclic hydrostatic pressure
(Cy-HP) and 0.5 MPa constant hydrostatic pressure (constant-HP) at
day 6 and 18 on support matrices subjected to processing in the
TESS processor. All matrices were incubated at 37.degree. C. for 18
days. The Cy-HP and constant-HP were applied for the first 6 days
followed by 12 days of incubation at atmospheric pressure.
[0231] Results seen in FIG. 5A show that combination of Cy-HP or
constant-HP with resting period of atmospheric pressure incubation
resulted in significant (p<0.05) increase of S-GAG production in
the processed matrices compared to S-GAG production observed in
matrices processed at atmospheric pressure with perfusion only.
[0232] FIG. 5B shows the index of DNA content (Initial=1) in
matrices subjected to static (Control), zero hydrostatic (0 MPa),
cyclic (Cy-HP) or constant (Constant-HP) hydrostatic pressure for 6
day and 12 days of atmospheric pressure culture. Increase in DNA
content in matrices subjected to the algorithm conditions is
clearly shown in both cyclic and constant hydrostatic pressure
groups. Comparison of the initial and control DNA level to DNA
levels in all three groups subjected to hydrostatic pressure
reveals that the DNA level in constructs subjected to the cyclic
hydrostatic pressure is higher at day 6 than at day 18 and the DNA
level in constructs subjected to constant hydrostatic pressure is
lower at day 6 than at day 18. Highest levels of DNA is observed in
matrices submitted to constant hydrostatic pressure at day 18.
[0233] FIGS. 6A and 6B show histological evaluation of matrices by
Safranin-O. FIG. 6A shows accumulation of S-GAG on day 18 in
matrices subjected to atmospheric pressure. FIG. 6B shows
accumulation of S-GAG in matrices subjected to 6 days of cyclic
hydrostatic pressure (Cy-HP), followed by 12 days of atmospheric
pressure. The greater S-GAG accumulation in Cy-HP culture matrices
is evident from the increased density of the photomicrograph
clearly visible in the construct. FIG. 3C shows accumulation of
Type II collagen in matrices subjected to the atmospheric pressure
or to the cyclic hydrostatic pressure (FIG. 6D). Larger
accumulation of Type II collagen in FIG. D is clearly seen.
[0234] These results demonstrate that chondrocytes may be placed in
culture to coalesce into a neo-cartilage construct with accumulated
extracellular matrix macro molecules, such as sulfated
glycosaminoglycan (S-GAG).
[0235] b) Evaluation of Effect of Perfusion Flow
[0236] The second type of study was performed in order to determine
the effect of perfusion flow rate on chondrocyte proliferation (DNA
content) and production of extracellular matrix (S-GAG
accumulation). Results are seen in Table 3 and FIGS. 7A and 7B.
[0237] FIG. 7 describes results of studies of the effect of the
perfusion flow rate on cell proliferation measured by levels of DNA
content index (FIG. 7A) and, S-GAG accumulation (FIG. 7B) at day 0,
6 and 18.
[0238] FIG. 7A shows that the lower perfusion rate (5 .mu.l/min)
results in higher DNA content index used as a measure for
determination of cell proliferation. Specifically, the DNA content
index compared to the initial DNA content index equal to 1
increased by about 50% to about 1.5 when the culture perfusion rate
was 5 .mu.l/min. The higher perfusion rate (50 .mu.l/min) resulted
in much smaller increase in DNA content index to about 1.2.
[0239] Table 3 shows the effect of perfusion flow rate on the S-GAG
production in matrices treated as outlined above where the flow
rate was either 0.05 mL/min (50 .mu.l/min) or 0.005 mL/min (5
.mu.l/min).
3 TABLE 3 Culture duration Medium In TESS Total GAG Production
Perfusion (0.5 MPa In Incubator days (.mu.g/cell Group Flow Rate
Cyclic (Atmospheric in construct) (n = 7) (mL/min) Pressure
Pressure) culture (Mean .+-. SD) A 0.05 mL/min 6 days 12 days 18
days 78.75 .+-. 6.84 B 0.005 mL/min 6 days 12 days 18 days 107.33
.+-. 8.53
[0240] All cultures were incubated at 37.degree. C., 5% CO.sub.2
and 20% O.sub.2. In the culture, 0.5 MPa cyclic pressure at 0.5 Hz
was applied to the cell matrices. Two matrices from each group were
harvested for histological analysis.
[0241] As seen in Table 3, the lower perfusion rate (5 .mu.l/min)
resulted in approximately 1.5 higher production of S-GAG than the
higher perfusion rate (50 .mu.l/min).
[0242] These results are seen in graphical form in FIG. 7B. FIG. 7B
is graph showing differences between S-GAG production by seeded
support matrices subjected to a medium perfusion flow rate of 5
.mu.l/min compared to matrices subjected to a medium perfusion flow
rate of 50 .mu.l/min at days 6 and 18. As seen in FIG. 7B, increase
in S-GAG production up to 136% (p<0.05) in matrices subjected to
a slower rate of 5 .mu.l/min.
[0243] The results summarized in FIGS. 7A and 7B clearly show a
significant increase in both the DNA content index and S-GAG
production in the cell construct at a flow rate of 5 .mu.l/min
compared to the flow rate 50 .mu.l/mL. There is no significant
difference in the amount of S-GAG released into the medium between
the two flow rates. It is therefore possible to use lower flow rate
and avoid shear.
[0244] Determination whether the combination of the perfusion flow
rate with cyclic or constant hydrostatic pressure leads to
increased formation of extracelluar matter was also studied.
Results are seen in FIG. 8.
[0245] FIG. 8 illustrates a formation of extracellular matrix after
15 days culture determined in matrices treated with perfusion (5
.mu.l/min) only (FIG. 8A), cyclic hydrostatic pressure 2.8 MPa at
0.015 Hz (FIG. 8B) and constant hydrostatic pressure 2.8 MPa at
0.015 Hz (FIG. 8C) as determined by toluidine blue staining. This
figure clearly shows that hydrostatic pressure and medium perfusion
enhances production of extracellular matrix.
[0246] C. Evaluation of Effect of Low Oxygen Tension
[0247] The third type of study was performed in order to determine
the effect of low oxygen tension on chondrocyte proliferation (DNA
content) and production of extracellular matrix (S-GAG
accumulation). Results are seen in Table 4 and FIGS. 9A and 9B.
4TABLE 4 Culture duration In TESS Total GAG Production Oxygen (0.5
MPa In Incubator days (.mu.g/cell Group concentration Cyclic
(Atmospheric in construct) (n = 8) (%) Pressure) Pressure) culture
(Mean .+-. SD) A 20% 7 days 14 days 21 60.89 .+-. 6.02 days B 2% 7
days 14 days 21 *105.59 .+-. 10.95 days (*: p<0.05, compared to
group A)
[0248] All cultures were incubated at 37.degree. C., at 5%
CO.sub.2. In TESS culture, the medium flow rate was 5 .mu.l/min.
Two cell matrices from each group were harvested for histological
analysis.
[0249] As seen in Table 4, the lower oxygen tension (2% O.sub.2
concentration) resulted in approximately 1.7 higher production of
S-GAG than higher oxygen concentration (20%) corresponding to
atmospheric O.sub.2 concentration. These results are seen in
graphical form in FIG. 9A.
[0250] FIG. 9A is a graph showing differences between S-GAG
production by cell constructs subjected to 2% oxygen concentration
(Cy-HP) and to cyclic hydrostatic pressure followed by static
pressure compared to cell constructs subjected to 20% oxygen
concentration and Cy-HP followed by static pressure. As already
seen in Table 4, at 2% oxygen concentration compared to 20%
concentration, the production of S-GAG rose by approximately
70%.
[0251] FIG. 9B shows the DNA content index (initial=1) in cell
constructs subjected to 2% or 20% oxygen concentration and Cy-HP
pressure followed by static pressure. There are no significant
differences in the DNA content index between 2% oxygen
concentration and 20% oxygen concentration. These results indicate
that the lower oxygen tension stimulates S-GAG production in cell
constructs when combined with the cyclic hydrostatic culture
followed by static culture. However, the cell proliferation,
expressed as DNA content index, is not affected by changes in
oxygen tension.
[0252] The algorithm of the invention thus comprises at least a
combination of the low perfusion flow rate from about 1 to 500
.mu.L/minute, preferably about 5 to 50 .mu.L/minute, most
preferably about 5 .mu.L/minute, low oxygen concentration from
about 1% to about 20%, preferably about 2% to about 5%, with a
certain predetermined period of cyclic or constant hydrostatic
pressure from zero to about 10 MPa at about 0.01 to about 1 Hz,
preferably about 0.1 to about 0.5 Hz, from about zero to about 10
MPa of cyclic or constant hydrostatic pressure, preferably about
0.05 MPa to about 3 MPa at about 0.1 to about 0.5 Hz, followed by
the period of a static atmospheric pressure. The algorithm
conditions are applied from about 1 hour to about 90 days wherein
the time for applying the hydrostatic pressure is from zero to
about 24 hours per day for from about one day to about ninety days,
wherein said hydrostatic pressure is preceded or followed by a
period of zero to about 24 hours of a static atmospheric pressure
for from about one day to about ninety days with preferred time for
applying the hydrostatic cyclic or constant pressure of about 7 to
28 days followed or preceded by a period of zero to about 28 days
of the atmospheric pressure.
[0253] d) General Applicability of the Algorithm of the Invention
to Various Cell Types
[0254] The algorithm described above for chondrocytes is similarly
applicable to other types of cell and tissue, such as fibroblasts,
fibrochondrocytes, tenocytes, osteoblasts and stem cells capable of
differentiation, or tissues such as cartilage connective tissue,
fibrocartilage, tendon and bone. The algorithm conditions may be
the same or different but would be generally within the above
described ranges.
II. Neo-Cartilage Composition Construct
[0255] The neo-cartilage composition construct is a multilayered
three-dimensional structure comprised at least of living
chondrocytes incorporated into a cellular support matrix. The
support matrix is embedded with living chondrocytes.
[0256] The construct is fabricated in vitro and ex vivo prior to
implanting into the cartilage lesion. The construct is fabricated
using the method and conditions, cumulatively called the algorithm,
described above, with all conditions being variable within the
given ranges and depending on the intended use or on the method of
delivery.
[0257] In one embodiment, the autologous or heterologous
chondrocytes are cultured as described, embedded into the support
matrix and processed into the neo-cartilage construct using
predetermined medium perfusion flow rate, cyclic or constant
hydrostatic pressure and reduced or increased concentration of
oxygen and/or carbon dioxide. The neo-cartilage construct is
delivered into the cartilage lesion cavity and deposited between
two layers of sealant and left in situ to be integrated into the
native cartilage.
III. Method for Formation of Superficial Cartilage Layer
[0258] The primary aspect of this invention is a finding that when
the neo-cartilage, neo-cartilage construct or seeded support matrix
produced according to procedures and conditions described above is
implanted into a cartilage lesion cavity and covered with a
biocompatible adhesive sealant, the resulting combination leads to
a formation of a superficial cartilage layer completely overgrowing
said lesion.
[0259] The method is based on producing a neo-cartilage and
neo-cartilage construct comprising support matrix seeded with
expanded chondrocytes processed according to the algorithm of the
invention. Chondrocytes are typically suspended in a collagen sol
which is thermo-reversible and easily changes from sol to gel at
the body temperature thereby permitting external preparation of and
delivery of the neo-cartilage construct into the lesion in form of
the sol which changes its state into gel upon delivery to the
lesion and warming to the body temperature.
[0260] The neo-cartilage construct is implanted into the lesion and
covered by a layer of a biologically acceptable adhesive sealant.
Optionally, the first layer of the sealant is introduced into the
lesion and deposited at the bottom of the lesion. This 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., into the
cavity and their interference with the integration of the
neo-cartilage therein. The second sealant layer is placed over the
surface of the construct. The presence of both these sealants in
combination with the neo-cartilage construct results in successful
integration of the neo-cartilage into the joint cartilage.
[0261] The method may be practiced in several modes and each mode
involves generic steps outlined below in variable combinations.
[0262] General way to practice the method for repair and
restoration of damaged, injured, diseased or aged cartilage to a
functional cartilage is to follow steps:
[0263] a) Preparing Neo-Cartilage, Neo-Cartilage Construct or
Chondrocyte Support Matrix
[0264] This step involves preparation of neo-cartilage,
neo-cartilage comprising constructs and support matrix comprising
autologous or heterologous chondrocytes incorporated therein.
Preparation of any of the three entities named above is described
in greater detail above in sections I.B-D.
[0265] b) Depositing the First and Second Sealant Into the
Lesion
[0266] This step involves introducing a first and a second layer of
a first and a second biologically acceptable sealant into a
cartilage lesion. The first and second sealants may be the same or
different. It is to be understood that the utilization of the first
bottom layer is optional and that the method for a formation of the
superficial cartilage layer is enabled without the first layer.
[0267] Specifically, this step involves deposition of the first
sealant at the bottom of the lesion and of the second sealant over
the lesion. 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.
[0268] The first sealant, deposited into the cavity before the
neo-cartilage is deposited, acts as a protector of the lesion
cavity integrity, that is, it protects the lesion cavity not only
from extraneous substances but it also protect this cavity from
formation of the fibrocartilage in the interim when the cavity is
filled with a space-holding gel in expectation of implantation of
the neo-cartilage after processing. The second sealant acts as a
protector of the lesion cavity on the outside as well as a
protector of the neo-cartilage construct deposited within a cavity
formed between the two sealants and as well as an initiator of the
formation of the superficial cartilage layer.
[0269] 1. First Sealant
[0270] The optionally deposited first sealant forms an interface
between the introduced neo-cartilage construct and the native
cartilage. The first sealant, deposited at the bottom of the
lesion, must be able to protect the construct from and 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 neo-cartilage construct
and it also prevents formation of the fibrocartilage.
[0271] 2. Second Sealant
[0272] The second sealant acts as a protector of the neo-cartilage
construct or the lesion cavity on the outside and is typically
deposited over the lesion either before or after the neo-cartilage
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 space holding gel in place before the neo-cartilage is
deposited therein. The second sealant also acts as a protector of
the neo-cartilage construct implanted within a cavity formed
between the two sealants. In this way, the second sealant may be
deposited after the neo-cartilage is implanted over the first
sealant and seal the neo-cartilage within the cavity or it may be
deposited over the space holding gel. The third function of the
second sealant is as an initiator or substrate for the formation of
a superficial cartilage layer. Studies performed during the
development of this invention discovered 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 is
particularly well-developed when the lesion cavity is filled with
the space-holding or thermo-reversible gel thereby leading to the
conclusion that such a gel might provide a substrate for the
formation of such superficial cartilage layer.
[0273] 3. First and Second Sealant Properties
[0274] The first or second sealant of the invention must possess
the following characteristics:
[0275] Sealant must be biologically acceptable, easy to use and
possess required adhesive and cohesive properties.
[0276] The sealant is 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 resorb and
degrade by an acceptable pathway or be incorporated into the
tissue.
[0277] 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
min. Longer gelation times are not compatible with surgical time
constraints. Additionally, the overall mode of use should be
relatively simple because complex procedures will not be accepted
by surgeons.
[0278] 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 3 N/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
may be given to 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.
[0279] 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, i.e., a gel.
[0280] The formed gel has cohesive strength dependent on the number
of inter-chain linkages, the length (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 100-500 Daltons are
tough, strong, and do not swell appreciably. Networks in which the
chain segments are 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).
[0281] 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.
[0282] 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.
[0283] 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, or 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.
[0284] 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 loses tensile strength,
due to the plasticizing effect of the imbibed solvent (in this
case, the solvent 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.
[0285] 4. Suitable and Non-suitable Sealants
[0286] Sealants suitable for purposes of this invention include the
sealants prepared from gelatin and di-aldehyde starch triggered by
mixing aqueous solutions of gelatin and dialdehyde starch which
spontaneously react and gel. The gel bonds to tissue through a
reaction of aldehyde groups on starch molecules and amino groups on
proteins of tissue, with an adhesive bond strength to up to 100 N/m
and an elastic modulus of 8.times.10.sup.6 Pa, which is a
characteristic of a relatively tough, strong material. After
swelling in physiological fluids this cohesive strength declines.
The gelled sealant is degraded by enzymes that cleave the peptide
bonds of gelatin and the glycosidic bonds of starch.
[0287] Another acceptable sealant is made from a copolymer of
polyethylene glycol and poly-lactide or -glycolide, further
containing acrylate side chains and gelled by light, in the
presence of some activating molecules. The linkage is formed by
free-radical chemistry. The gel bonds to tissue by mechanical
interlock, having flowed into tissue surface irregularities prior
to curing. The sealant degrades from the tissue by hydrolytic
cleavage of the linkage between polyethylene glycol chains, which
then dissolve in physiological fluids and are excreted.
[0288] 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, and the solution
gels. Bonding to tissue is through aldehyde groups on the gelatin
reacting with amino groups on tissue. After gelation, the sealant
can be degraded enzymatically, due to cleavage of peptide bonds in
gelatin.
[0289] Still another sealant made from a 4-armed pentaerythritol
thiol and a polyethylene glycol diacrylate is formed when these two
neat liquids (not dissolved in aqueous buffers) are mixed. The rate
of gelation is controlled by the amount of a catalyst, which can be
a quaternary amino compound, such as tri-ethanolamine. A covalent
linkage is formed between the thiol and acrylate, to form a
thio-ether bond. The final gel is firm and swells very little. The
tensile strength of this gel is high, about 2 MPa, which is
comparable to that of cyanoacrylate acceptable Superglue.
Degradation of such gels in vivo is slow. Therefore, the gel may be
encapsulated or incorporated into tissue.
[0290] Another example is the composition, preferred for use in
this invention, that contains 4-armed tetra-succinimidyl ester or
tetra-thiol derivatized PEG, plus methylated collagen. The reactive
PEG reagents in powder form are mixed with the viscous, fluid
methylated collagen (previously dissolved in water); this viscous
solution is then mixed with a high pH buffer to trigger gelation.
The tensile strength of this cured gel is about 0.3 MPa.
Degradation presumably occurs through hydrolytic cleavage of ester
bonds present in the succinimidyl ester PEG, releasing the soluble
PEG chains which are excreted.
[0291] 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.
[0292] Such properties can be achieved by a variety of materials,
both natural and synthetic. Examples include: 1) gelatin and
di-aldehyde starch (International Patent Publication Number WO
97/29715; 21 Aug. 1997); 2) 4-armed penta-erythritol tetra-thiol
and polyethylene glycol diacrylate (International Patent
Application Number WO 00/44808; 3 Aug. 2000; example 14); 3)
photo-polymerizable polyethylene glycol-co-poly(a-hydroxy acid)
diacrylate macromers (U.S. Pat. No. 5,410,016; Apr. 25, 1995); 4)
periodate-oxidized gelatin (U.S. Pat. No. 5,618,551, Apr. 8, 1997);
5) serum albumin and di-functional polyethylene glycol derivatized
with maleimidyl, succinimidyl, phthalimidyl and related active
groups (International Patent Publication Number WO 96/03159, Feb.
8, 1996) and 6) 4-armed polyethylene glycols derivatized with
succinimidyl ester and thiol, plus methylated collagen, referred to
as "CT3" (U.S. Pat. No. 6,312,725 B1, Nov. 6, 2001).
[0293] Various other sealant formulations are available
commercially or are described in the literature. However, the
majority of these are not suitable for practicing this invention
for a variety of reasons.
[0294] For example, fibrin sealant is unsuitable because it
interferes with the formation of cartilage.
[0295] Cyanoacrylate, or Superglue, is extremely strong but it
might exhibit toxic reactions in tissue.
[0296] Un-reinforced hydrogels of various types typically exhibit
tensile strengths of lower than 0.02 MPa, which is too weak to
support the adhesion required for the purpose of this application
because such gels will swell too much, tear too easily, and break
down too rapidly.
[0297] 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 govern 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.
[0298] 5. Preferred Sealants
[0299] The first or second sealant of the invention must be a
biologically acceptable, typically rapidly gelling 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. Sealant should have a tensile
strength of at least 0.3 MPa. Examples of suitable sealants are
tetra-hydrosuccinimidyl or tetra-thiol derivatized PEG, or a
combination thereof, commercially available from Cohesion
Technologies, Palo Alto, Calif. under the trade name CoSeal.TM.,
described in J. Biomed. Mater. Res (Appl. Biomater.), 58:545-555
(2001). Other compounds suitable to be used are the rapid gelling
biocompatible polymer compositions described in the U.S. Pat. No.
6,312,725 B1, herein incorporated by reference. Additionally, the
sealant may be two or more-part polymers 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 methyl collagen, commercially
available.
[0300] The sealant of the invention typically gels rapidly upon
contact with tissue, particularly with tissue containing collagen.
The second sealant may or may not be the same as the first sealant.
Both the first and the second is preferably a cross-linked
polyethylene glycol hydrogel with methyl-collagen, which has
adhesive properties.
[0301] c) Implanting the Neo-Cartilage Construct
[0302] Next step in the method of the invention comprises
implanting said neo-cartilage into a lesion cavity formed under the
second sealant or between two layers of sealants, said cavity
either filled with neo-cartilage construct deposited therein or,
optionally, with a space holding thermo-reversible gel (SHTG)
deposited into said cavity as a sol at temperatures between about 5
to about 30.degree. C. wherein, within said cavity and at the body
temperature, said SHTG converts the sol into gel and in this form
the SHTG holds the space for introduction of the neo-cartilage
construct and provides protection for the neo-cartilage and wherein
its presence further promotes in situ formation of de novo
superficial cartilage layer covering the cartilage lesion.
[0303] The above step is versatile in that the neo-cartilage may be
deposited into said lesion cavity after the first sealant is
deposited but before the second sealant is deposited over it or the
first and second sealants may be deposited first and the cavity is
filled with the space-holding thermo-reversible gel for the interim
period when the neo-cartilage is cultured and processed or it may
be deposited into the lesion cavity without the first sealant and
covered with the second sealant.
[0304] The neo-cartilage is either autologous or heterologous and
is prepared as described above in sections I.B. a-c.
[0305] d) Removing the Space-Holding or Thermo-Reversible Gel from
the Lesion Cavity
[0306] The neo-cartilage is deposited into the cavity either before
or after the formation of the superficial cartilage layer. In all
cases when the first sealant is used, the first sealant is
deposited first. In one embodiment, the neo-cartilage construct
containing, typically, the heterologous neo-cartilage might be
deposited on the top of the first sealant layer and immediately
covered by the second sealant layer. In such an instance, the
neo-cartilage is left in the cavity until the superficial cartilage
layer is formed and the neo-cartilage is integrated into the
surrounding cartilage. Then, depending on the material used for
neo-cartilage construct, the sponge gel or thermo-reversible
gelling hydrogel are left in the cavity to disintegrate.
[0307] In the instance when the two sealants are deposited first,
the space within the lesion cavity is optionally filled with a
polymer gel, such as the space-holding thermo-reversible gel. Such
gel is left in the cavity until the neo-cartilage construct is
cultured, processed and ready to be implanted. Since such
thermo-reversible gel might or might not be completely or partially
degraded during this time, it may be removed from the cavity by
cooling the lesion to about 50.degree. C. at which temperature the
gel becomes a sol, and by removing said sol from the cavity, for
example, by injection. Using the same process, that is by cooling
the solid gel of the neo-cartilage, the process may be reversed for
introduction of the neo-cartilage construct into said lesion cavity
wherein, after the sol is warmed into the body temperature, the sol
is converted into a solid gel.
[0308] Thus, the primary premise of this process is that the
removal and/or introduction of the space holding gel or
introduction of neo-cartilage construct proceeds at the cold
temperature where the composition is in the sol state and converts
into solid gel at warmer temperatures. In this way the gel may be
removed from the cavity as the sol after the neo-cartilage
integration and formation of superficial cartilage layer.
[0309] e) Generation of the Superficial Cartilage Layer
[0310] A combination of the neo-cartilage construct comprising the
neo-cartilage suspended in the thermo-reversible gel or support
matrix embedded with chondrocytes with the adhesive polymeric
second sealant leads to overgrowth and complete or almost complete
sealing of the lesion cavity.
[0311] In alternative, depending on the surface chemistry of the
thermo-reversible gel, the superficial layer could grow directly
over the neo-cartilage construct if such surface chemistry is
propitious to such growth.
[0312] Typically, a biologically acceptable second sealant,
preferably a cross-linked PEG hydrogel with methyl collagen
sealant, is deposited either over the neo-cartilage construct
implanted into the lesion cavity or is deposited over the lesion
before the neo-cartilage construct is deposited therein. The second
sealant acts as an initiator for formation of the superficial
cartilage layer which in time completely overgrows the lesion. The
superficial cartilage layer in several weeks or months completely
covers the lesion and permits integration of the neo-cartilage of
the neo-cartilage construct or chondrocytes embedded within the
support matrix into the native surrounding cartilage substantially
without formation of fibrocartilage.
[0313] Formation of the superficial cartilage layer is a very
important aspect of the healing of the cartilage and its repair and
regeneration.
IV. In vivo Studies in Swine of Weight-Bearing Region of the
Knee
[0314] The method according to the invention was tested and
confirmed in in vivo studies wherein the generation of the
superficial cartilage layer has been confirmed in a three month
study performed in a swine model in order to evaluate porcine
neo-cartilage construct integration into the surrounding
cartilage.
[0315] The neo-cartilage construct prepared according to the method
of the invention was implanted into an artificially generated
lesion in a pig's knee. Detailed conditions of the study are
described in Example 8. Results of this study are illustrated in
FIGS. 10, 11 and 12 depicting histological evaluation using
Safranin-O staining method of artificially created cartilage
lesions.
[0316] Briefly, the study comprised of an open arthrotomy of the
right knee joint performed on all animals. A biopsy of the
cartilage was obtained. Chondrocytes were isolated from the
cartilage biopsy and cultured within a collagen matrix in a Tissue
Engineering Support System (TESS.TM.) as described in detail above
to produce porcine neo-cartilage construct for subsequent
implantation.
[0317] A defect was created in the medial femoral condyle of the
right knee. This defect, which served as a control, was not
implanted with the neo-cartilage construct. The empty defect is
seen in FIG. 10A. Following surgery, the joint was immobilized with
an external fixation device for a period of about two weeks. Three
weeks after the arthrotomy on the right knee was performed, an open
arthrotomy was performed on the left knee and the same defects were
created in this medial femoral condyle. The porcine neo-cartilage
was implanted within defects in this knee which was similarly
immobilized. The porcine implant site is seen in FIG. 10B which
also show initiation of formation of a superficial cartilage layer
two weeks after implantation.
[0318] The operated sites were periodically viewed via arthroscopy
at monthly intervals. Subsequently, approximately 3 months after
porcine neo-cartilage implantation, animals were euthanized and
joints harvested and prepared for histological examination. The
implanted sites were prepared and examined histologically.
Comparison of FIG. 11 (control at four months after arthrotomy) and
FIG. 12 shows test knee three month following arthrotomy and
neo-cartilage implantation according to the invention. This figure
shows that in the control knee there is a visible formation of
fibrocartilage. In the test group (FIGS. 12A-12D), the implanted
porcine neo-cartilage construct resulted in production of dense
regenerating hyaline cartilage and in the same test group, there
was clearly visible cell integration (FIGS. 12C and 12D) and
formation of the superficial cartilage layer (FIGS. 12A and
12B).
[0319] FIGS. 11A-11C thus shows the control lesion at 4 months
following the surgery without a treatment with the neo-cartilage
construct. Noticeable in FIG. 11A is the proliferation of
undesirable fibrocartilage within the defect site. Also seen is
synovial tissue that has infiltrated into the subchondral
space.
[0320] FIGS. 12A-12D, on the other hand, show that after 3 months
post implantation in a weight bearing region of the knee, the
porcine neo-cartilage has produced dense hyaline-like cartilage and
has integrated with the host cartilage laterally and at the
interface of the subchondral bone.
[0321] Additionally, FIG. 12A shows a formation of regenerated
hyaline-like cartilage in the implant site; FIG. 12B shows the
beginning of integration between the porcine neo-cartilage and the
native cartilage laterally and at the subchondral bone. FIG. 12C
shows already regenerated hyaline-like cartilage and FIG. 12D shows
chondrocytes integration into the surrounding native cartilage.
[0322] The porcine neo-cartilage was delivered to the defect by
implantation of neo-cartilage construct between two layers of
sealant. The newly formed superficial cartilage layer formed over
the defect at three months following the implantation is clearly
visible.
[0323] FIG. 12 thus shows and confirms that 3 months after
implantation in a weight-bearing region of the knee, the
porcine-NeoCart.TM. has produced dense hyaline-like cartilage and
has integrated with the host cartilage laterally and at the
interface of the subchondral bone.
[0324] These results confirm that the damaged, injured, diseased or
aged cartilage may be repaired by using neo-cartilage implants
prepared according to the algorithm of the invention.
V. Human Osteoarthritic Cartilaqe
[0325] 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 or disease, such as osteoarthritis (OA).
[0326] In osteoarthritis, disruption of the structural integrity of
the matrix by the degeneration of individual matrix proteins leads
to reduced mechanical properties and impaired function.
[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, there is no
optimal treatment.
[0328] In order to evaluate suitability of the current invention
for treatment of osteoarthritis, studies using algorithm of the
invention including a TESS culture system using neo-cartilage
scaffold construct and algorithm of the invention (hydrostatic
pressure and medium perfusion) on human OA chondrocytes, cell
proliferation and extracellular matrix accumulation in OA
chondrocytes was investigated.
[0329] Results are seen in Table 5 and in FIGS. 13-15.
5TABLE 5 Pressure Conditions DNA content In TESS Total S-GAG
Production (.mu.g/cell Group Type of days (.mu.g/cell construct)
construct) (n = 7) Pressure Time In Incubator In culture (Mean .+-.
SD) (Mean .+-. SD) Initial -- -- 0 day 0 day 19.23 .+-. 0.87 1.88
.+-. 0.40 Control -- -- 21 days 21 days 23.81 .+-. 2.61 2.34 .+-.
0.32 Cy-HP#1 0.5 MPa 7 days 14 days 21 days *29.53 .+-. 1/60 2.33
.+-. 0.12 Cyclic Cy-HP#2 0.5 MPa 14 days 7 days 21 days *34.39 .+-.
0.99 2.35 .+-. 0.09 Cyclic Const-HP 0.5 MPa 7 days 14 days 21 days
26.94 .+-. 5.14 **2.65 .+-. 0.28 Constant (*: p<0.05, compared
to Control in S-GAG production) (**: p<0.05, compared to Initial
in DNA content)
[0330] In the TESS processor, the medium flow rate was 5 .mu.l/min
and the hydrostatic pressure was applied as indicated. Two cell
matrices from each group were harvested for histological
analysis.
[0331] As seen in Table 5 and FIG. 13A, S-GAG production in cell
constructs subjected to cyclic hydrostatic pressure with medium
perfusion was significantly greater than those subjected to
atmospheric pressure (control). Especially, S-GAG production
(.mu.g/cell construct) was significantly increased (144%) for
Cy-HP#2 where the cyclic hydrostatic pressure was used for 14 days
followed by 7 days of static atmospheric pressure compared to
control.
[0332] FIG. 13B shows DNA content index with corresponding results
presented in Table 5 for DNA, likewise showing increased production
of DNA. Increase in DNA content index in cell constructs using the
neo-cartilage construct subjected to constant hydrostatic pressure
was clearly shown in a comparison to initial level. DNA level in
cell constructs subjected to constant hydrostatic pressure with
medium perfusion was significantly increased to 142% compared to
initial DNA level index.
[0333] FIGS. 14A-14E show histological evaluation of cell
constructs by Safranin-O. FIG. 14A shows S-GAG accumulation at day
0 (initial). FIG. 14B shows accumulation of S-GAG on day 21 in cell
constructs subjected to atmospheric pressure (control). FIG. 14C
shows accumulation of S-GAG on day 21 in cell constructs subjected
to 7 days of cyclic hydrostatic pressure (Cy-HP#1) followed by 14
days of the atmospheric pressure. FIG. 14D shows accumulation of
S-GAG on day 21 in cell constructs subjected to 14 days of cyclic
hydrostatic pressure (Cy-HP#2) followed by 7 days of to atmospheric
pressure. FIG. 14E shows accumulation of S-GAG on day 21 in cell
constructs subjected to 7 days of constant hydrostatic pressure
(Constant-HP) followed by 14 days of atmospheric pressure. The
greater S-GAG accumulation in both cell constructs subjected to
cyclic hydrostatic pressure (7 and 14 days) is evident from the
increased density of the photomicrograph clearly visible in the
FIGS. 14C and 14D.
[0334] These results demonstrate that hydrostatic pressure combined
with a medium perfusion promotes both cell proliferation and
neo-cartilage phenotypic activity, that is, cartilage extracellular
matrix production, in the scaffold neo-cartilage constructs seeded
with human OA chondrocytes. This evidence confirms that the
algorithm of invention using TESS culture system and hydrostatic
pressure combined with medium perfusion regenerates human OA
chondrocytes and transforms the OA cartilage into the healthy
hyaline cartilage.
VI. Method for Treatment of Cartilage Lesions
[0335] The method for treatment of damaged, injured, diseased or
aged cartilage according to the invention is suitable for healing
of small lesion due to acute injury as well as healing of the large
lesions caused by osteoarthritis or other joint degenerative
diseases and/or transforming the diseased OA cartilage into the
healthy hyaline cartilage.
[0336] The method generally encompasses five novel features,
namely, employing a biologically acceptable thermo-reversible
polymer gel as a carrier support matrix for neo-cartilage generated
from autologous chondrocytes, producing the autologous
neo-cartilage by a process of the invention, employing a
biologically acceptable thermo-reversible gel as a space-holding
means for the interim period when the autologous neo-cartilage is
produced, depositing one or two adhesive sealants to the lesion
and, following depositing the sealants and implantation of the
neo-cartilage within a cavity generated thereby, a formation of the
superficial cartilage layer covering the lesion and protecting the
integrity of the neo-cartilage deposited therein.
[0337] The method generally comprises steps:
[0338] a) debriding an articular cartilage lesion and during the
debriding harvesting a small quantity (50-4000 mg) of
non-osteoarthritic hyaline cartilage;
[0339] b) fabrication and processing of the neo-cartilage construct
according to the above described procedures;
[0340] c) preparing the lesion for implantation of the
neo-cartilage construct by depositing the one or two sealant
layers, the first (optional) at the bottom of the lesion and the
second one over and on the top of the lesion, and, using all
variation already described above, depositing either the
neo-cartilage construct within the cavity formed below the top
sealant and/or between the two sealant layers or depositing the
space holding thermo-reversible polymer gel into the cavity between
the two layers to uphold the integrity of the cavity in the interim
when the neo-cartilage construct is being prepared;
[0341] d) implanting the neo-cartilage construct into said cavity
formed between the two sealant layers to allow for integration of
the neo-cartilage into the surrounding native intact cartilage and
formation of the superficial cartilage layer; and
[0342] e) optionally removing the space holding polymer gel from
the cavity before the neo-cartilage implantation.
[0343] In the alternative method for treatment, expanded and
differentiated chondrocytes may be deposited directly into a joint
lesion in a suitable typically thermo-reversible gelation hydrogel
solution.
[0344] There are several advantages of the current method. First,
the method is very versatile and any of the variations may be
advantageously utilized for treatment of a specific injury, damage,
aging or disease.
[0345] The method permits generation of autologous neo-cartilage by
providing alternative means for maintaining a space between two
sealant layers until the autologous neo-cartilage is prepared. The
method permits generation of more dense neo-cartilage and
three-dimensional expansion of chondrocytes and extracellular
matrix.
[0346] The deposition of the second 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 growth and
incorporation of autologous neo-cartilage in the lesion.
[0347] Deposition of the first 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 neo-cartilage and also preventing formation of the
fibrocartilage.
[0348] The method further permits deposition of the space-holding
gel or thermo-reversible polymer gel to be deposited whether alone
or with suspended processed neo-cartilage into the lesion at
temperature between 5 and 30.degree. C. as a sol. Selection of
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 second sealant.
[0349] The method further permits said thermo-reversible hydrogel
be enhanced with hyaluronic acid, typically added in about 5 to
about 50%, preferably about 20% (v/v), wherein such hyaluronic acid
acts as an enhancer of the matrix-forming characteristics of the
gel and to act as a hydration factor in the synovial space in
general and within the lesion cavity in particular.
[0350] Additionally, the gel acts as a slow-release unit for
hyaluronic acid, greatly increasing a period of hydration within
the cavity and also as a substrate for formation of the superficial
cartilage layer and it can also be conveniently removed, if needs
be, by cooling the lesion so that the solid gel formed at
37.degree. C. is converted to sol and can be removed by injection
or otherwise.
[0351] For treatment of the cartilage, a subject is treated,
according to this invention, with a prepared autologous or
heterologous neo-cartilage or neo-cartilage construct implanted
into the lesion, the neo-cartilage or the construct is left in the
lesion for two-three months and typically, it does not need any
further intervention as during these three months, the
neo-cartilage is fully integrated into the native cartilage and
becomes 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.
[0352] Finally, the diseased, osteoarthritic cartilage may be fully
replaced by the regenerated hyaline-like cartilage when processed
according to the algorithm of this invention.
[0353] The algorithm and/or 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 (algorithm of the cartilage) are within the
scope of the current invention.
EXAMPLE 1
Isolation of Chondrocytes from Source Tissue
[0354] This example describes the procedure used for isolation of
chondrocytes from swine cartilage.
[0355] Chondrocytes were enzymatically isolated from cartilage
harvested under sterile conditions from the hind limbs of 6-month
old swine. The femur was detached from the tibia and the trachea
head exposed. Strips of cartilage were removed from the trachea
using a surgical blade.
[0356] The cartilage was minced, digested in a 0.15% collagenase
type I solution in DMEM/Nutrient Mixture F-12 (DMEM/F-12) 1:1
mixture with 1% penicillin-streptomycin (P/S) and gently rotated
for 18 hours at 37.degree. C. Chondrocytes were collected and
rinsed twice by centrifugation at 1500 rpm for 5 min. Chondrocytes
were re-suspended in DMEM/F-12 containing 1%
penicillin-streptomysin and 10% FBS.
[0357] Chondrocytes were expanded for about 5 days at 37.degree.
C.
EXAMPLE 2
The Production of Human Neo-Cartilage Construct
[0358] This example describes conditions for production of
neo-cartilage for human use.
[0359] The patient undergoes arthroscopic biopsy of a small
(200-500 mg) piece of healthy cartilage from the ipsilateral knee.
The biopsy is taken from the non-weight bearing portion of the
femoral condyle or from the femoral notch as deemed most
appropriate for the patient. The biopsy sample is placed into a
sterile, non-cytotoxic, non-pyrogenic specimen container which is
packaged and shipped to the laboratory.
[0360] At the laboratory the biopsy sample is examined against
acceptance criteria and then transferred to the chondrocyte
isolation and expansion area. Samples from the biopsy specimen
transport buffer are tested for sterility and for mycoplasma. The
expanded chondrocytes are suspended in VITROGEN.RTM. gellable
collagen solution, commercially available from Cohesion Corp., Palo
Alto, Calif. A pre-formed collagen sponge (22.times.22 mm square
and 2-4 mm in thickness, wherein the thickness depends on the
thickness of patient's cartilage), commercially available from
Koken Co., Japan or honeycomb matrix produced according to this
invention is placed into the resulting chondrocyte suspension which
absorbs the chondrocyte/collagen suspension into this matrix.
[0361] The resulting chondrocyte-loaded matrix is warmed to
37.degree. C. to gel the VITROGEN in order to spatially secure the
chondrocytes within the support matrix. The loaded support matrix
is then placed into Tissue Engineering Support System (TESS.TM.)
culture unit. Typical time for cell expansion from removal of a
biopsy sample to placement of the chondrocyte loaded culture matrix
in the TESS.TM. culture unit is 10-40 days. Within the TESS.TM.
culture unit, cyclic or constant hydrostatic pressure is used to
induce the chondrocytes to begin growing and expressing their
cartilage generating program for about 1 hour to about 30 days.
[0362] The still developing new cartilage is transferred to a
constant, resting culture phase. The neo-cartilage production
process requires a minimum time of 10 days in resting culture.
After this minimum 10-day period the neo-cartilage, hereinafter
called neo-cartilage construct, undergoes final inspections and is
packaged for return to the clinic to be implanted. At the time of
release, tests for sterility, endotoxin, and mycoplasma
contamination must be negative for microbial and mycoplasma
contamination and must show .ltoreq.0.5 EU/ml of endotoxin.
EXAMPLE 3
Preparation of Support Matrices
[0363] This example illustrates preparation of the cellular support
matrix, also called the TESS matrix.
[0364] 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.
[0365] 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.
[0366] Alternatively, the support matrix may be prepared as
follows.
[0367] 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 construction of
densely packed fibers that are intertwined into a multi-layer
structure.
[0368] 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.
EXAMPLE 4
Seeding Cells in the TESS Matrix
[0369] This example describes procedures used for seeding cells in
the TESS matrix.
[0370] Isolated chondrocytes were incubated for a period of five
days at 37.degree. C. in a standard incubator. Cells were then
collected by trypsinization.
[0371] A cell suspension of 150,000 cells in 18 .mu.l of VITROGEN
solution was seeded per matrix having an approximate volume of 19
.mu.l, with nine matrices per group. The seeded matrix (collagen
sponge 4 mm in diameter and 1.5 mm in thickness) may be scaled-up
to an increased volume, where approximately 1 .mu.l of the above
described cell suspension is seeded in 1 .mu.l of matrix. The
control group matrices were incubated in a 37.degree. C. incubator
and the test group was incubated in the TESS.
[0372] In alternative set-up, isolated chondrocytes were incubated
for a period of five days at 37.degree. C. in a standard incubator.
Cells were then collected by trypsinization. A cell suspension of
300,000 cells in 18 .mu.l of VITROGEN solution was seeded per
matrix having an approximate volume of 19 .mu.l with eight matrices
per group.
EXAMPLE 5
Effect of Cyclic Hydrostatic Pressure
[0373] This example describes procedures used for determination of
effect of cyclic hydrostatic pressure in vitro formation of
chondrocyte-seeded support matrices.
[0374] Swine articular chondrocytes (sACs) were enzymatically
isolated from cartilage with type I collagenase. The cells were
suspended in collagen (VITROGEN) as described above and wicked into
the honeycombed sponge element of the cellular support matrix. The
cells seeded in the support matrix were incubated at 37.degree. C.,
5% CO.sub.2 and 20% O.sub.2 After 24 hours, some of these cells
matrices were transferred to the TESS.TM. processor and incubated
at 0.5 or 3.0 MPa cyclic or constant hydrostatic pressure with
medium perfusion (0.05 ml/min) as described above for 6 or 7 days
followed by a 12 or 14 day resting phase. The control group
comprised of chondrocytes seeded in matrices incubated for 18 or 21
days at atmospheric pressure, at 37.degree. C., 5% CO.sub.2 and 20%
or 2% O.sub.2.
[0375] At the end of the culture period (18 or 21 days), the
matrices were harvested for biochemical and histological analysis.
For biochemical analysis, sulfated glycosaminoglycan (S-GAG)
production was measured using a modified dimethylmethylene blue
(DMB) microassay.
[0376] Two matrices from each group were harvested for histological
analysis.
EXAMPLE 6
Effect of Medium Flow Rate on Extracellular Matrix Accumulation of
Chondrocytes in Collagen Sponges
[0377] This example described conditions used to determine effect
of medium flow on production and accumulation of extracellular
matrix by chondrocytes seeded into collagen sponges.
[0378] Chondrocyte Isolation
[0379] Swine legs were obtained from a local abattoir. Within 4-6
hours after slaughter, cartilage was harvested under sterile
conditions from the trochlea of the hind limbs. The cartilage was
minced and digested in 0.15% collagenase type I in DMEM/F-12
containing 1% penicillin-streptomycin (P/S) for 18 hours at
37.degree. C. Isolated swine articulate chondrocytes (sACs) were
collected, rinsed, and resuspended in DMEM/F-12 supplemented with
10% fetal bovine serum (FBS) and 1% P/S. sACs then were expanded
for 5 days at 37.degree. C.
[0380] Cell Seeding in Collagen Sponges
[0381] sACs were harvested with Trypsin EDTA and cell viability was
measured by trypan-blue exclusion. Three hundred thousand sACs were
suspended in 30 .mu.l of a neutralized 0.25% collagen solution
(VITROGEN.RTM., Cohesion Corp., Palo Alto, Calif.), and the
suspension was absorbed into a collagen sponge, 4 mm in diameter
and 2 mm in thickness, commercially available from Koken Co.,
Japan. Seeded sponges were pre-incubated for 1 hour at 37.degree.
C. to gel the collagen, followed by incubation in culture medium at
37.degree. C. in 5% CO.sub.2.
[0382] Tissue Engineering Support System (TESS.TM.) Culture
[0383] Following the incubation, the seeded sponges were
transferred to and cultured in the Tissue Engineering Support
System (TESS.TM.) processor. To evaluate the effect of medium
perfusion rate, sponges were subjected to medium perfusion at 5
.mu.l/min or 50 .mu.l/min. Cyclic hydrostatic pressure (Cy-HP)
0-0.5 MPA pressure at 0.5 Hz applied was for 6 days. Some sponges
were incubated under constant conditions at atmospheric pressure
and no perfusion at 37.degree. C. for a total of 18 days in
culture. Sponges harvested 24 hours after seeding with cells (day
0) served as an initial control.
[0384] Histological and Biochemical Analysis
[0385] Cell constructs were harvested after 6 and 18 days of
culture.
[0386] For histological evaluation, 4% paraformaldehyde-fixed,
paraffin sections were stained with Safranin-O (Saf-O) and Type II
collagen antibody.
[0387] 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 7
Biochemical and Histological Assays
[0388] This example describes assays used for biochemical and
histological studies (DMB assay).
[0389] Biochemical (DMB) Assay
[0390] At the end of the culture six matrices from each group were
used in the biochemistry assay.
[0391] The matrices were 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. GAG production in the matrices was measured
using a modified dimethylene blue (DMB) microassay with shark
chondroitin sulfate as a control Connective Tissue Research, 9:
247-248 (1982).
[0392] DNA content was determined by Hoechst 33258 dye method
according to Anal. Biochem., 174:168-176 (1988).
[0393] Histological Assay
[0394] The remaining matrices from each group were fixed in 4%
paraformaldehyde. The matrices were processed and embedded in
paraffin. 10 .mu.m sections were cut on a microtome and stained
with Safranin-O (Saf O).
EXAMPLE 8
Evaluation of Porcine Neo-Cartilage Integration in a Swine
Model
[0395] This example describe the procedure and results of study
performed for evaluation of integration of porcine neo-cartilage in
a swine model.
[0396] An open arthrotomy of the right knee joint was performed on
all animals, and a biopsy of the cartilage was obtained.
[0397] Chondrocytes were isolated from the cartilage biopsy and
cultured within a collagen matrix in a Tissue Engineering Support
System (TESS.TM.) to produce porcine-Neocart for subsequent
implantation.
[0398] A defect was created in the medial femoral condyle of the
pig's right knee. This defect (control) was not implanted with
porcine-NeoCart.TM.. Following surgery, the joint was immobilized
with an external fixation construct for a period of about two
weeks. Three 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
porcine-NeoCart.TM. 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. Animals were
euthanized and the joints harvested and prepared for histological
examination approximately 3 months after porcine-NeoCart.TM.
implantation. The implanted sites were prepared and examined
histologically.
[0399] Results are seen in FIGS. 10-12. FIG. 10 shows results of
the arthroscopic examination. The empty defect is seen in FIG. 10A.
The porcine NeoCart.TM. implant site is seen in FIG. 10B which also
shows still-evident absorbable sutures and the superficial
cartilage layer growing over the porcine NeoCart.TM..
EXAMPLE 9
Protocol for In Vivo, Ex Vivo or In Vitro Growth of Porcine
Neo-Cartilage
[0400] Autologous porcine chondrocytes are seeded into the cellular
support matrix and incubated under cyclic hydrostatic pressure at
37.degree. C. and 5% CO.sub.2. Cyclic hydrostatic pressure is
either 0.5 or 3.0 MPa at 0.5 Hz. The duration of said cyclic
pressure is approximately 6 days followed by a resting phase of 12
days in an incubator maintained at 37.degree. C. at atmospheric
pressure. At the end of this resting phase, the matrices were
harvested for biochemical and histological analysis.
[0401] In the alternative protocol, the algorithm for the growth
cells of in vivo and in vitro, the application of hydrostatic
pressure is used on isolated in situ cartilage, or application of
hydrostatic pressure for about 1-8 hours followed by about 16-23
hours of recovery period.
EXAMPLE 10
Regeneration of Human Chondrocytes
[0402] This example describes the procedure used for regeneration
of human chondrocytes.
[0403] Chondrocytes from osteoarthritic (OA) patients (40 years
old) were expanded for 18 days in monolayer culture at 37.degree.
C. and suspended in VITROGEN.RTM. (300,000 cells/30 fl). The cell
suspension was absorbed into a support matrix, usually a collagen
honeycomb sponge (4 mm in diameter and 2 mm in thickness, Koken
Co., Japan). The cell constructs were incubated in culture medium
supplemented with 10% FBS and 1% ITS (insulin-transferrin-sodium
selenite, Sigma) at 37.degree. C., 5% CO.sub.2 and 20% O.sub.2, at
0.5 MPa cyclic hydrostatic pressure (Cy-HP) or 0.5 MPa constant
hydrostatic pressure (Constant-HP) for 7 or 14 days in the TESS.TM.
processor followed by incubation for 7 or 14 days at atmospheric
pressure for 7 or 14 days in an CO.sub.2 incubator at 37.degree. C.
The remaining cell constructs compromising the control group were
incubated atmospheric pressure for 21 days at 37.degree. C., 5%
CO.sub.2 and 20% O.sub.2.
[0404] Before starting the culture, some cell constructs were
harvested for biochemical and histological analysis as an initial
condition. At the end of the culture period, the cell constructs
were harvested for biochemical and histological analysis. Sulfated
glycosaminoglycan production was measured using a modified
dimethylmethylene blue (DMB) micro assay. Cell proliferation was
measured using a modified Hoechst Dye DNA assay. Formation of
neo-tissue was analyzed by Safranin-O staining.
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