U.S. patent application number 09/799284 was filed with the patent office on 2001-08-09 for in vitro production of transplantable cartilage tissue.
This patent application is currently assigned to Rush-Presbyterian-St. Luke's Medical Center. Invention is credited to Hejna, Michael, Masuda, Koichi, Thomar, Eugene J-M. A..
Application Number | 20010012965 09/799284 |
Document ID | / |
Family ID | 22990419 |
Filed Date | 2001-08-09 |
United States Patent
Application |
20010012965 |
Kind Code |
A1 |
Masuda, Koichi ; et
al. |
August 9, 2001 |
In vitro production of transplantable cartilage tissue
Abstract
The present invention is directed to a transplantable cartilage
matrix and a method for its in vitro production.
Inventors: |
Masuda, Koichi; (Glenview,
IL) ; Thomar, Eugene J-M. A.; (Lockport, IL) ;
Hejna, Michael; (Riverside, IL) |
Correspondence
Address: |
FITCH EVEN TABIN AND FLANNERY
120 SOUTH LA SALLE STREET
SUITE 1600
CHICAGO
IL
606033406
|
Assignee: |
Rush-Presbyterian-St. Luke's
Medical Center
|
Family ID: |
22990419 |
Appl. No.: |
09/799284 |
Filed: |
March 5, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09799284 |
Mar 5, 2001 |
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09260741 |
Mar 1, 1999 |
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6197061 |
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Current U.S.
Class: |
623/11.11 ;
623/23.72 |
Current CPC
Class: |
A61L 27/3852 20130101;
A61F 2/3094 20130101; A61L 27/24 20130101; C12N 2500/38 20130101;
A61L 27/3654 20130101; A61L 27/3817 20130101; A61K 35/12 20130101;
A61L 27/54 20130101; C12N 5/0655 20130101; A61F 2310/00365
20130101; Y10S 623/915 20130101; A61L 27/3895 20130101; C12N
2531/00 20130101; A61L 27/227 20130101; C12N 2533/74 20130101; A61L
2430/06 20130101; A61F 2/30756 20130101; A61L 27/3633 20130101;
C12N 2533/90 20130101; A61L 27/38 20130101; A61L 27/54 20130101;
A61L 27/24 20130101; A61L 27/227 20130101 |
Class at
Publication: |
623/11.11 ;
623/23.72 |
International
Class: |
A61F 002/02 |
Claims
What is claimed is:
1. A method for the production of transplantable cartilage matrix,
the method comprising: isolating chondrogenic cells; culturing
chondrogenic cells for an amount of time effective for allowing
formation of a chondrogenic cell-associated matrix; recovering the
chondrogenic cells with the cell-associated matrix; and culturing
the chondrogenic cells with the cell-associated matrix on a
semipermeable membrane in the presence of growth factor for a time
effective for allowing formation of a cartilage matrix.
2. A method for the production of transplantable cartilage matrix
according to claim 1 wherein the chondrogenic cells are cultured in
an alginate medium.
3. A method for the production of transplantable cartilage matrix
according to claim 1 wherein the chondrogenic cell-associated
matrix includes aggrecan, collagen types II, IX and XI, and
hyaluronan.
4. A method for the production of transplantable cartilage matrix
according to claim 3 wherein the ratio aggregan to hyaluronan in
the chondrogenic cell-associated matrix is at least above about
10:1.
5. A method for the production of transplantable cartilage matrix
according to claim 1 wherein the semipermeable membrane has a pore
size of less than about 5 microns and a pore density of at least
about 8.times.10.sup.5 pores per cm.sup.2.
6. A method for the production of transplantable cartilage matrix
according to claim 1 wherein the growth factor is selected from the
group consisting of osteogenic protein-1, bone morphogenetic
proteins, transforming growth factor beta, insulin-like growth
factor, and mixtures thereof.
7. A method for the production of transplantable cartilage matrix
according to claim 1 wherein the cartilage matrix includes
aggrecan, collagen types II, IX and XI, and hyaluronan.
8. A method for the production of transplantable cartilage matrix
according to claim 7 wherein the cartilage matrix has a ratio
aggregan to hyaluronan of at least above about 10:1.
9. A cohesive carilage matrix produced by a method comprising:
isolating chondrogenic cells; culturing chondrogenic cells for an
amount of time effective for allowing formation of a chondrogenic
cell-associated matrix; recovering the chondrogenic cells with the
cell-associated matrix; and culturing the chondrogenic cells with
the cell-associated matrix on a semipermeable membrane in the
presence of growth factor for a time effective for allowing
formation of a cohesive cartilage matrix.
10. A cohesive cartilage matrix according to claim 9 wherein the
chondrogenic cells are cultured in an alginate medium.
11. A cohesive cartilage matrix according to claim 9 wherein the
chondrogenic cell-associated matrix includes aggrecan, collagen
types II, IX and XI, and hyaluronan.
12. A cohesive cartilage matrix according to claim 11 wherein the
ratio aggregan to hyaluronan in the chondrogenic cell-associated
matrix is at least above about 10:1.
13. A cohesive cartilage matrix according to claim 9 wherein the
semipermeable membrane has a pore size of less than about 5 microns
and a pore density of at least about 8.times.10.sup.5 pores per
cm.sup.2.
14. A cohesive cartilage matrix according to claim 9 wherein the
growth factor is selected from the group consisting of osteogenic
protein-1, bone morphogenetic proteins, transforming growth factor
beta, insulin-like growth factor, and mixtures thereof.
15. A cohesive cartilage matrix according to claim 9 wherein the
cohesive cartilage matrix includes aggrecan, collagen types II, IX
and XI, and hyaluronan.
16. A cohesive cartilage matrix according to claim 15 wherein the
cohesive cartilage matrix has a ratio aggregan to hyaluronan of at
least above about 10:1.
17. A cohesive cartilage matrix comprising: at least about 5
mg/cc.sup.3 aggrecan; collagen types II, IX and XI, and hyaluronan;
wherein the ratio of aggrecan to hyaluronan is about 10:1 to about
200:1, and the ratio of aggrecan to collagen is about 1:1 to about
10:1.
18. A cohesive cartilage matrix according to claim 17 wherein the
matrix has a thickness of less than about 2 mm.
19. A method for the surgical repair of cartilage damage
comprising: producing a transplantable cartilage matrix; and
surgically implanting the cartilage matrix.
20. A method for the surgical repair of cartilage damage according
to claim 19 wherein the transplantable cartilage matrix is produced
by a method comprising: isolating chondrogenic cells; culturing
chondrogenic cells for an amount of time effective for allowing
formation of a chondrogenic cell-associated matrix; recovering the
chondrogenic cells with the cell-associated matrix; culturing the
chondrogenic cells with the cell-associated matrix on a
semipermeable membrane in the presence of growth factor for a time
effective for allowing formation of a cartilage matrix.
21. A method for the surgical repair of cartilage damage according
to claim 20 wherein the chondrogenic cells are cultured in an
alginate medium.
22. A method for the surgical repair of cartilage damage according
to claim 20 wherein the chondrogenic cell-associated matrix
includes aggrecan, collagen types II, IX and XI, and
hyaluronan.
23. A method for the surgical repair of cartilage damage according
to claim 22 wherein the ratio aggregan to hyaluronan in the
chondrogenic cell-associated matrix is at least above about
10:1.
24. A method for the surgical repair of cartilage damage according
to claim 20 wherein wherein the semipermeable membrane has a pore
size of less than about 5 microns and a pore density of at least
about 8.times.10.sup.5 pores per cm.sup.2.
25. A method for the surgical repair of cartilage damage according
to claim 20 wherein wherein the growth factor is selected from the
group consisting of osteogenic protein-1, bone morphogenetic
proteins, transforming growth factor beta, insulin-like growth
factor, and mixtures thereof.
26. A method for the surgical repair of cartilage damage according
to claim 19 wherein the cartilage matrix includes aggrecan,
collagen types II, IX and XI, and hyaluronan.
27. A method for the production of transplantable cartilage matrix
according to claim 26 wherein the cartilage matrix has a ratio
aggregan to hyaluronan of at least above about 10:1.
Description
[0001] The present invention relates to the method of production of
cartilage tissue for surgical implantation into human joints for
the purpose of filling defects of the articular cartilage or
replacing damaged or degenerated cartilage.
BACKGROUND OF THE INVENTION
[0002] Cartilage Injury and Repair:
[0003] Human joint surfaces are covered by articular cartilage, a
low friction, durable material that distributes mechanical forces
and protects the underlying bone. Injuries to articular cartilage
are common, especially in the knee. Data from the Center for
Disease Control (CDC) and clinical studies have suggested that
approximately 100,000 articular cartilage injuries occur per year
in the United States. Such injuries occur most commonly in young
active people and result in pain, swelling, and loss of joint
motion. Damaged articular cartilage does not heal. Typically,
degeneration of the surrounding uninjured cartilage occurs over
time resulting in chronic pain and disability. Cartilage injuries
therefore Frequently lead to significant loss of productive work
years and have enormous impact on patients' recreation and
lifestyle.
[0004] Joint surface injuries may be limited to the cartilage layer
or may extend into the subchondral bone. The natural histories of
these types of injuries differ. Cartilage injuries which do not
penetrate the subchondral bone have limited capacity for healing
(1). This is due to properties inherent to the tissue. Nearly 95
percent of articular cartilage is extracellular matrix (ECM) that
is produced and maintained by the chondrocytes dispersed throughout
it. The ECM provides the mechanical integrity of the tissue. The
limited number of chondrocytes in the surrounding tissue are unable
to replace ECM lost to trauma. A brief overproduction of matrix
components by local chondrocytes has been observed (2); however,
the response is inadequate for the repair of clinically relevant
defects. Cellular migration from the vascular system does not occur
with pure chondral injury and extrinsic repair is clinically
insignificant.
[0005] Osteochondral injuries, in which the subchondral bone plate
is penetrated, can undergo healing due to the influx of reparative
cells from the bone marrow (1). Numerous studies have shown,
however, that the complex molecular arrangement of the ECM
necessary for normal cartilage function is not recapitulated. The
repair response is characterized by formation of fibrocartilage, a
mixture of hyaline cartilage and fibrous tissue. Fibrocartilage
lacks the durability of articular cartilage and eventually
undergoes degradation during normal joint use Many osteochondral
injuries become clinically asymptomatic for a period of a few to
several years before secondary degeneration occurs. However, like
isolated chondral injuries, these injuries ultimately result in
poor joint function, pain, and disability.
[0006] Molecular Organization of the ECM:
[0007] The physical properties of articular cartilage are tightly
tied to the molecular structures of type II collagen and aggrecan.
Other molecules such as hyaluronan and type IX collagen play
important roles in matrix organization. Type II collagen forms a
3-dimensional network or mesh that provides the tissue with high
tensile and shear strength (3). Aggrecan is a large, hydrophilic
molecule, which is able to aggregate into complexes of up to 200 to
300.times.10.sup.6 Daltons (4)]. Aggrecan molecules contain
glycosaminoglycan chains that contain large numbers of sulfate and
carboxylate groups. At physiological pH, the glycosaminoglycan
chains are thus highly negatively charged (5). In cartilage,
aggrecan complexes are entrapped within the collagen network. A
Donnan equilibrium is established in which small cations are
retained by electrical forces created by the sulfate and
carboxylate groups (6). Water is in turn retained by the osmotic
force produced by large numbers of small cations in the tissue.
[0008] When the joint is mechanically loaded, movement of water
results in perturbation of the electrochemical equilibrium. When
the load is removed, the Donnan equilibrium is reestablished and
the tissue returns to its pre-loaded state (7). The physical
properties of articular cartilage are tightly tied to the molecular
structures of type II collagen and aggrecan. Other matrix
molecules, such as hyaluronan (8) and type IX collagen (9), play
important roles in matrix organization. Failure to restore the
normal molecular arrangement of the ECM leads to failure of the
repair tissue over time, as demonstrated by the poor long-term
performance of fibrocartilage as a repair tissue (10).
[0009] Distinct compartments have been demonstrated within the ECM.
These differ with respect to the composition and turnover of matrix
macromolecules. Immediately surrounding each chondrocyte is a thin
shell of ECM characterized by a relatively rapid turnover of matrix
components (11). This region is termed the pericellular matrix
(11). Surrounding the pericellular matrix is the territorial
matrix. Further from the cells is the interterritorial matrix (11).
Turnover of matrix macromolecules is slower in the interterritorial
matrix than in the pericellular and territorial matrices (11). The
role that these various compartments play in the function of the
tissue as a whole is unclear. From the perspective of articular
cartilage repair, however, they represent a higher level of matrix
organization that must be considered in the restoration of injured
tissue.
[0010] Surgical Treatment of Articular Cartilage Injury:
[0011] Current methods of surgical restoration of articular
cartilage fall into three categories: (1) stimulation of
fibrocartilaginous repair; (2) osteochondral grafting; and (3)
autologous chondrocyte implantation. Fibrocartilage, despite its
relatively poor mechanical properties, can provide temporary
symptomatic relief in articular injuries. Several surgical
techniques have been developed to promote the formation of
fibrocartilage in areas of cartilage damage. These include
subchondral drilling, abrasion, and microfracture. The concept of
these procedures is that penetration of the subchondral bone allows
chondroprogenitor cells from the marrow to migrate into the defect
and effect repair. The clinical success rate of this type of
treatment is difficult to assess. In published series, success
rates as high as 70% are reported at 2 years; however, the results
deteriorate with time. At five years post-treatment, the majority
of patients are symptomatic.
[0012] In osteochondral grafting, articular cartilage is harvested
with a layer of subchondral bone and implanted into the articular
defect. Fixation of the graft to the host is accomplished through
healing of the graft bone to the host bone. The major advantage of
this technique is that the transplanted cartilage has the
mechanical properties of normal articular cartilage and therefore
can withstand cyclical loading. The major disadvantages are
donor-site morbidity (in the case of autograft) and risk of disease
transmission (in the case of allograft). Additionally, tissue
rejection can occur with allografts which compromises the surgical
result. Osteochondral autografting (mosaicplasty) has demonstrated
short-term clinical success. The long-term effectiveness is
unknown. Osteochondral allografts are successful in approximately
65% of cases when assessed at 10 years post-implantation, but are
generally reserved for larger areas of damage extending deep into
the subchondral bone.
[0013] Autologous chondrocyte implantation is a method of cartilage
repair that uses isolated chondrocytes. Clinically, this is a
two-step treatment in which a cartilage biopsy is first obtained
and then, after a period of ex vivo processing, cultured
chondrocytes are introduced into the defect (12). During the ex
vivo processing, the ECM is removed and the chondrocytes are
cultured under conditions that promote cell division. Once a
suitable number of cells are produced, they are implanted into the
articular defect. Containment is provided by a patch of periosteum
which is sutured to the surrounding host cartilage. The cells
attach to the defect walls and produce the extracellular matrix in
situ. The major advantages of this method are the use of autologous
tissue and the ability to expand the cell population. Difficulties
with restoration of articular cartilage by this technique fall into
three categories: cell adherence, phenotypic transformation, and
ECM production.
[0014] Cell Adherence.
[0015] The success of implantation of individual cells (without
ECM) is critically dependent upon the cells attaching to the defect
bed. Cartilage ECM has been shown to have anti-adhesive properties,
which are believed to be conferred by small proteoglycans, dermatan
sulfate, and heparan sulfate. Normal chondrocytes possess
cell-surface receptors for type II collagen (13) and hyaluronan
(11), but it is not clear to what extent ex-vivo manipulated cells
possess receptors for these matrix molecules that are functional.
An in vitro study of chondrocyte binding to ECMs suggests that the
interaction is weak. An in vivo study in rabbits suggests that only
8% of implanted chondrocytes remain in the defect bed after
implantation.
[0016] Phenotypic Transformation.
[0017] During the process of expanding the cell population in
vitro, chondrocytes usually undergo phenotypic transformation or
dedifferentiation (14). Morphologically, the cells resemble
fibroblasts. Synthesis of type II collagen and aggrecan is
diminished and synthesis of type I collagen, typical of
fibrocartilage, is increased. Limited data exist to support the
contention that the cells redifferentiate in situ following
implantation. Reestablishment of the chondrocytic phenotype is
critical to the success of the repair process, as tissue produced
by cells which are phenotypically fibroblastic functions poorly as
a replacement for articular cartilage.
[0018] ECM Production.
[0019] Prior to implantation, the cultured chondrocytes are
enzymatically denuded of ECM. The cells are injected into the
defect bed as a suspension. The graft construct is incapable of
bearing load and must be protected from weight bearing for several
weeks to months. Limited data exist on the quality of the ECM that
is ultimately produced. It has been characterized as hyaline-like
tissue at second-look arthroscopy two years post-implantation
[Petersen, L., personal communication]. The overall recovery period
from this form of treatment is 9-12 months. Good or excellent
clinical results are achieved in approximately 85% of femoral
condyle lesions 2 years post-implantation. However, it is not clear
whether the clinical results will be maintained over longer
follow-up periods.
[0020] Tissue Engineering:
[0021] Each of the current methods of cartilage repair has
substantial limitations. As a result, several laboratory approaches
to production of cartilage tissue in vitro have been proposed.
These generally involve seeding of cultured cells (either
chondrocytes or pluripotential stem cells) into a biological or
synthetic scaffold. The major drawbacks of this type of approach
are: (1) difficulty in attaining or maintaining the chondrocyte
phenotype; (2) unknown biological effects of the scaffold material
on the implanted and native chondrocytes and other joint tissues;
and (3) limited attachment of the engineered tissue construct to
the defect bed.
[0022] The present invention involves the production of an
implantable cartilage tissue. Its method of preparation and
composition address the major problems encountered with current
techniques of cartilage repair. The major advantages, features and
characteristics of the present invention will become more apparent
upon consideration of the following description and the appended
claims.
SUMMARY OF THE INVENTION
[0023] The present invention relates to a transplantable cartilage
matrix and a method for its production. Cartilage tissue produced
by this method has properties that, with time in culture, become
similar to those of a naturally occurring cell-associated ECM. At
the time of reimplantation, the matrix in the cartilage tissue has
a high rate of turnover (i.e. it is metabolically active) It is
rich in cartilage-specific aggrecan proteoglycans and contains
enough long hyaluronan chains to allow all these aggrecan molecules
to form large aggregates of very large size, but it is relatively
poor in collagen pyridinium crosslinks. These properties enhance
the implantability of the tissue and subsequent maturation of the
tissue in situ following implantation, which leads to integration
with the host.
[0024] In accordance with the method of the invention, chondrocytes
are isolated from tissues containing chondrogenic cells. The
isolated chondrogenic cells are cultured in alginate culture for an
amount of time effective for allowing formation of a chondrogenic
cell-associated matrix. In an important aspect of the invention,
the cell-associated matrix has at least about 5 mg/cc.sup.3 of
aggrecan, a ratio of aggrecan to hyaluronan (mg/mg) between about
10:1 and about 200:1, and a ratio of aggrecan to collagen (mg/mg)
between about 1:1 to about 10:1.
[0025] Chondrogenic cells, each with a pericellular matrix, are
recovered and cultured on a semipermeable membrane system in the
presence of serum or serum containing exogenously added growth
factor(s). The chondrogenic cells with cell-associated matrix are
cultured for a time effective for formation of a cohesive cartilage
matrix.
[0026] In an important aspect, the invention relates to the use of
such in vitro-produced articular tissue in the surgical repair of
cartilage damage. Such damage would include acute partial and full
thickness chondral injuries, osteochondral injuries, and
degenerative processes. Surgical treatment includes open surgical
techniques (arthrotomy) and arthroscopic application/insertion of
the in vitro-produced cartilaginous tissue.
DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 generally illustrates the overall process for the
production of transplantable cartilage matrix in accordance with
the present invention.
[0028] FIG. 2 describes a method for the separation of cells and
their cell-associated matrix from the further removed matrix and
alginate gel.
[0029] FIG. 3 shows a culture method on a semipermeable
membrane.
[0030] FIG. 4 shows the histological appearance of in vitro
regenerated cartilage matrix produced in accordance with the
present invention. Bovine articular chondrocytes were cultured with
DMEM/F12 containing 20% FBS, an effective amount of growth factor,
10 .mu.g/ml gentamicin and 25 .mu.g/ml ascorbic acid in 1.2%
alginate. After 7 days of culture, the beads were dissolved with 55
mM sodium citrate, 0.15M sodium choloride, pH 6.8. The resulting
suspension of the cell with their associated matrix is centrifuged
at 100 g for 10 minutes. The pellet was resuspended in the same
medium described above. After 7 additional days in culture, each
insert was removed from the tissue culture plate and the tissue was
processed for histology by Toluidine Blue Staining.
[0031] FIG. 5 shows repair of a cartilage defect one month after
reimplantation of the cartilage tissue formed in vitro.
DETAILED DESCRIPTION
[0032] A generalized process of the present invention is set forth
in FIG. 1. In accordance with the invention, chondrocytes are
isolated and cultured in alginate. The resulting chondrocytes, each
with a cell-associated matrix, are recovered and then further
cultured on a semipermeable membrane. The resulting cartilage
tissue then is utilized for transplantation.
[0033] Isolation of Chondrocytes/Chondrogenic Cells
[0034] Chondrogenic cells useful in the practice of the invention
may be isolated from essentially any tissue containing chondrogenic
cells. As used herein, the term "chondrogenic cell" is understood
to mean any cell which, when exposed to appropriate stimuli, may
differentiate into a cell capable of producing and secreting
components characteristic of cartilage tissue. The chondrogenic
cells may be isolated directly from pre-existing cartilage tissue,
for example, hyaline cartilage, elastic cartilage, or
fibrocartilage. Specifically, chondrogenic cells may be isolated
from articular cartilage (from either weight-bearing or
non-weight-bearing joints), costal cartilage, nasal cartilage,
auricular cartilage, tracheal cartilage, epiglottic cartilage,
thyroid cartilage, arytenoid cartilage and cricoid cartilage.
Alternatively, chondrogenic cells may be isolated from bone marrow.
See for example, U.S. Pat. Nos. 5,197,985 and 4,642,120, and
Wakitani et al. (1994) J. bone Joint Surg. 76:579-591, the
disclosures of which are incorporated by reference herein.
[0035] Culture in Alginate for the Production of Chondrocyte
Cell-Associated Matrix
[0036] In accordance with the present invention,
chondrocytes/chondrogenic cells isolated from the tissue are
resuspended at a density of at least about 10.sup.4 cells/ml in a
solution of sodium alginate. The cells cultured under conditions
effective for maintaining their spherical conformation conducive to
the production, upon the chondrocyte membrane, of a cell-associated
matrix similar to that found in vivo. In an important aspect,
chondrocytes are cultured in alginate for at least about five days
to allow for formation of the cell-associated matrix. Culture media
used may contain a stimulatory agent, such as fetal bovine serum,
to enhance the production of the cell-associated matrix.
[0037] In an alternative aspect of the invention, the culture
medium for the chondrocytes may further include exogenously added
specific growth factors. The addition of specific growth factors,
for example those not already present in fetal bovine serum, such
as osteogenic protein-1, may act as an effective stimulator of
matrix formation. Growth factors (other than those present in fetal
bovine serum) may be advantageous as they are becoming available as
human recombinant proteins. The use of human growth factors is
advantageous in as far as they are less likely to cause an immune
response in the joint (the use of fetal bovine serum requires
extensive rinsing of the newly-formed tissue prior to its
implantation). In this aspect of the invention, growth factor is
added to the medium in an amount to near-maximally stimulate
formation of the cell-associated matrix.
[0038] In an other important aspect of the invention, amplification
of chondrocytes or chondrogenic cells in alginate does not induce
loss of the chondrocyte phenotype as occurs when amplification is
performed in monolayer culture. As used herein, "chondrocyte
phenotype" refers to a cell that has (i) a spherical shape and the
ability to synthesize and accumulate within the matrix significant
amounts of (ii) aggrecan and (iii) type II collagen without (iv)
accumulating within the matrix an effective amount of type I
collagen. As used herein, a minimal amount of collagen type I means
less than about 10% of all collagen molecules that become
incorporated within the matrix. Chondrocytes cultured in alginate
retain their spherical shape (typical of chondrocytes) and maintain
a large amount of matrix. The matrix resembles hyaline cartilage
histologically and is rich in aggrecan and collagen type II.
[0039] In addition to the three parameters already mentioned, a
phenotypically stable chondrocyte must retain the ability to
effectively incorporate the major macromolecules into a
cartilage-like matrix. Normal chondrocytes may express small
amounts of mRNA for collagen type I that they do not translate.
Further, articular chondrocytes cultured in alginate beads for
several months may synthesize some collagen type I molecules, but
the latter never become incorporated into the forming matrix.
Consequently, the appearance of small amounts of newly-synthesized
collagen type I molecules in the medium does not necessarily denote
the onset of dedifferentiation. Further, hyaluronan is not a marker
of the chondrocytic phenotype since it is synthesized in large
amounts by many other cell types. However, it is an essential
constituent of the cartilage matrix.
[0040] Cells that are phenotypically stable should synthesize at
least about 10 times more aggrecan than collagen (on a mass basis).
Further, the ratio of aggrecan to hyaluronan in the matrix should
always remain above about 10.
[0041] Chondrocyte with Cell-Associated Matrix
[0042] Culture of chondrocytes in alginate results in the
production of an ECM which is organized into two compartments: a
cell-associated matrix compartment which metabolically resembles
the pericellular and territorial matrices of native tissues, and
(ii) a further removed matrix compartment which metabolically
resembles the interterritorial matrix of native tissue.
[0043] The formation of a highly structured cell-associated matrix
around each chondrocyte is important for several reasons. First,
the cell-associated matrix is anchored to the cell via receptors
such as anchorin CII (which binds to collagen)and CD44 (which binds
to hyaluronan in proteoglycan aggregates). Once this matrix has
been reestablished, the cells are much less likely to become
dedifferentiated. Second, the chondrocyte turns over proteoglycan
aggrecan and thus remodels this matrix relatively rapidly. The
chondrocyte is much less effective in remodeling the further
removed matrix.
[0044] In an important aspect of the invention, the cell-associated
matrix compartment of the ECM produced during culture in alginate
includes aggrecan (the major cartilage proteoglycan), collagen
types II, IX and XI, and hyaluronan. Aggrecan molecules are formed
principally in aggregates bound to receptors (including CD44) on
the chondrocyte cell membrane via hyaluronan molecules.
[0045] The relative proportions of each component in the
cell-associated matrix vary depending on the length of time in
culture. In an important aspect of the invention, the
cell-associated matrix has at least about 5 mg/cc.sup.3 of
aggrecan, a ratio of aggrecan to hyaluronan (mg/mg)between 10:1 and
200:1, and a ratio of aggrecan to collagen (mg/mg) from 1:1 to
about 10:1. Further, the molecular composition of the
cell-associated matrix (around each cell) and further removed
matrix (between the cells) can be altered by specific modifications
of the culture conditions. These modifications involve the physical
arrangement of the culture system and application of various growth
factors. Manipulation of matrix production and organization are
central to the engineering of articular cartilage in vitro for
surgical treatment of cartilage injury.
[0046] In an important aspect of the invention, the contents of
collagen and of the pyridinoline crosslinks of collagen increase
with time of culture. The crosslinks in particular show a dramatic
increase in concentration after two weeks of culture. By keeping
the length of the culture period relatively short, the collagen
fibrils in the cell-associated matrix do not become overly
crosslinked. A tissue that has good functional properties but is
relatively deficient in crosslinks is easier to mold and more
likely to become integrated within the host cartilage than a
harder, crosslink-rich tissue.
[0047] Recovery of Chondrocytes with their Cell-Associated
Matrix
[0048] Recovery of chondrocytes with their cell-associated matrix
is accomplished by solubilizing alginate beads after a sufficient
culture period. One approach is set forth in FIG. 2. Alginate beads
20 are first solubilized using known techniques. The resulting cell
suspension then is centrifuged, separating the cells with their
cell-associated matrix 40 (in the pellet) from the components of
the further removed matrix 30 (in the supernatant).
[0049] Culturing the Chondrocyte with their Cell-Associated Matrix
on a Semipermeable Membrane.
[0050] In this aspect of the invention, the chondrocytes with their
cell-associated matrix isolated as described above, are further
cultured on a semipermeable membrane. The semipermeable culture
system of the invention is shown in FIG. 3.
[0051] In accordance with the present invention, a cell culture
insert 50 is placed into a plastic support frame 60. Culture medium
70 flows around the cell culture insert 50. In an important aspect
of the invention, cell culture insert 50 includes a semipermeable
membrane 80. The semipermeable membrane 80 allows medium to flow
into the cell culture insert in an amount effective for completely
immersing the chondrocytes and their cell-associated matrix 90.
[0052] In an important aspect of the invention, the semipermeable
membrane 80 allows the chondrocytes to have continuous access to
nutrients while allowing the diffusion of waste products from the
vicinity of the cells. In this aspect, the membrane should have a
pore size effective to prevent migration of chondrocytes through
the pores and subsequent anchoring to the membrane. In this aspect
of the invention, the pore size should not be more than about 5
microns. Further, the membrane utilized should have a pore density
effective for providing the membrane with sufficient strength so
that it can be removed from its culture frame without curling, and
with sufficient strength such that the tissue on the membrane can
be manipulated and cut to its desired size. In this aspect of the
invention, the membrane has a pore density of at least about
8.times.10.sup.5 pores/cm.sup.2. The membrane may be made of any
material suitable for use in culture. Examples of suitable membrane
systems include but are not restricted to: (i) Falcon Cell Culture
Insert [Polyethylene terephthalate (PET) membrane, pore size 0.4 or
3.0 microns, diameter 12 or 25 mm]; (ii) Coaster Transwell Plate
[Polycarbonate membranes, pore size, 0.1, 0.4, 3.0 or 5.0 microns,
diameter 12 or 24.5 mm]; (iii) Nunc Tissue Culture Insert
[Polycarbonate Membrane Insert: pore size, 0.4 or 3.0 microns,
diameter 10 mm or 25 mm); Millicell Culture Plate Insert (PTFE
(polytetrafluoroethylene) membrane, polycarbonate, pore size 0.4 or
3.0 microns, diameter 27 mm]).
[0053] The beads containing chondrocytic cells are first cultured
in equal parts of Dulbecco's modified Eagle medium and Ham's F12
medium containing 20% fetal bovine serum (Hyclone, Logan, Utah),
about 25 .mu.g/ml ascorbate and 50 .mu.g/ml gentamicin or another
antibiotic (Gibco). In an alternative approach, the beads are
cultured in another type of medium conducive to the maintenance of
chondrocytes in culture. In an alternative approach, the beads are
cultured in a closed chamber that allows for continuous pumping of
medium. In an important aspect, the medium contains fetal bovine
serum containing endogenous insulin-like growth factor-1 at a
concentration of at least about 10 ng/ml. In this usage, fetal
bovine serum may also be considered a growth factor. Suitable
growth factors that may be exogenously added to the medium to
maximally stimulate formation of the cell-associated matrix include
but are not limited to osteogenic protein-1 (OP-1), bone
morphogenic protein-2 and other bone morphogenetic proteins,
transforming growth factor beta, and insulin-like growth
factor.
[0054] In another aspect of the invention, cells with their
reestablished cell-associated matrix are further cultured in medium
on the semipermeable membrane for an amount of time effective for
allowing formation of a cohesive cartilage matrix. Culture times
will generally be at least about 3 days under standard culture
conditions. Partial inhibition of matrix maturation prior to
implantation is important in providing a matrix that is not as
stiff as mature cartilage, but which has enough tensile strength to
retain its shape and structure during handling. Such a tissue
should be malleable enough to be press fitted into the defect.
[0055] In an important aspect of the invention, mechanical
properties of the cartilage matrix can be controled by increasing
or decreasing the amount of time that the cartilage tissue is
cultured on the membrane. Longer culture time will result in
increased crosslink densities.
[0056] Cartilage Matrix
[0057] In an important aspect of the invention, the cartilage
matrix that forms on the semipermeable membrane has a concentration
of aggrecan of at least about 5 mg/cc.sup.3. The cartilage matrix
contains an amount of hyaluronan effective for allowing all the
newly synthesized molecules to become incorporated into
proteoglycan aggregates. The matrix of the tissue formed on the
membrane contains aggregated aggrecan molecule at a concentration
not less than 5 mg/cc.sup.3, a ratio of aggrecan to hyaluronan of
about 10:1 to about 200:1, and a ratio of aggrecan to collagen of
about 1:1 to about 10:1. In addition, the short period of culture
ensures that concentration of pyridinium crosslinks remains low
enough to permit remodeling of the tissue in vivo but high enough
to allow the orthopedic surgeon to handle it easily.
[0058] In an important aspect, cartilage matrix which forms on the
membrane should have a thickness of less than about 2 mm, as cells
in a thicker sheet are not likely to gain access to nutrients as
readily. Cartilage matrix will generally have a disk-like structure
conforming to the membrane; however, there is no requirement that
the cartilage matrix have a disk-like structure. In this aspect of
the invention, the shape of the cartilage matrix should be
effective for allowing an orthopedic surgeon to handle the tissue
(either a disk or sheet) and cut it into the size needed for a
press fit into a defect. The size of the cartilage matrix will
generally be slightly bigger than the size of the defect.
[0059] The following examples illustrate methods for carrying out
the invention and should be understood to be illustrative of, but
not limiting upon, the scope of the invention which is defined in
the appended claims.
EXAMPLES
Example I
[0060] Methods
[0061] Chondrocytes
[0062] Feasibility studies were performed using chondrocytes from
young bovine articular cartilage as described below. A similar
approach can be (and has been) used to promote cartilage matrix
formation by human adult articular chondrocytes.
[0063] Culture Conditions
[0064] Full-thickness articular cartilage is dissected from the
metacarpophalangeal joints of 14- to 18-month-old bovine
steers--special attention is given to prevent contamination by
synovial tissue. The cartilage slices are digested at 37.degree. C.
for 1 hour with 0.4% Pronase (Calbiochem, La Jolla, Calif.) and
then for 16 hours with 0.025% collagenase P from Clostridium
hystolyticum (Boehringer Mannheim, Indianapolis, Ind.) in DMEM/F12
(Gibco BRL, Grand Island, N.Y.) containing 5% fetal bovine serum.
The resulting digest is filtered through a 4-.mu.m cell strainer
(Cat #2340, Beckton Dickinson, Franklin Lakes, N.J.) and the
chondrocytes are recovered. The chondrocytes are resuspended at a
density of 4.times.10.sup.6 cells/ml in a 1.2% solution of sterile
alginate (Kelton LV, Kelco, Chicago, Ill.) in 0.15 M NaCl. The cell
suspension is slowly expressed through a 22-gauge needle and
dropped into a 102 mM calcium chloride solution. The beads are
allowed to polymerize in this solution for 10 minutes and then
washed twice in 0.15 M NaCl and then twice in DMEM/F12. The beads
then are transferred to complete culture medium (200 beads in 10
ml) consisting of DMEM/F12, 10 .mu.g/ml gentamicin, 20% fetal
bovine serum, an effective amount of growth factor and 25 .mu.g/ml
ascorbic acid (Gibco BRL). The cultures are kept at 37.degree. C.
in a humidified atmosphere of 5% CO.sub.2 in air with the medium
replaced by fresh medium daily.
[0065] After 7 days of culture, the medium is collected and the
beads dissolved at 4.degree. C. by incubation for 20 minutes in 55
mM sodium citrate, 0.15 M NaCl, pH 6.8. The resulting suspension of
cells (with their associated matrix) is centrifuged at 4.degree. C.
at 100 g for 10 minutes. The pellet, containing the cells with
their cell-associated matrix, then is resuspended in DMEM/F12
containing 20% fetal bovine serum, an effective amount of growth
factor and the supplements described above.
[0066] Three milliliters of complete medium are added to each well
of a Falcon Cell Culture Insert Companion plate (Cat. #3090) and
kept in the incubator at 37.degree. C. in the presence of 5%
CO.sub.2 for 20 minutes. A Falcon Cell Culture Insert (Cat. #3090,
0.45 um, PET membrane, transparent, diameter 23.1 mm, Beckton
Dickinson) is aseptically placed into each well of a 6-well
multiwell plate. A 2.5 ml aliquot (corresponding to the cells and
their associated matrix present in 200 beads) is plated onto each
Insert. The cultures are maintained at 37.degree. C. in a
humidified atmosphere of 5% CO.sub.2. After 7 additional days in
culture (referred to as days 8-14 of culture), each Insert is
removed from the tissue culture plate and the PET membrane is cut
using a scalpel.
[0067] Characterization of the Chondrocytes and Cartilage Matrix
Formed after 7 Days of Culture in Alginate Beads
[0068] On day 7, both (i) whole beads and (ii) the cells recovered
with their cell-associated matrix after solubilization of the beads
were fixed, sectioned and visualized by phase contrast microscopy,
as previously described. The matrix in both matrix compartments
(cell-associated matrix and further removed matrix) was
characterized for contents of proteoglycan, hyaluronan, collagen,
and collagen crosslinks as described below.
[0069] Characterization of the Chondrocytes and Cartilage Matrix
Formed After 7 Days of Culture in Alginate Beads Followed by 7 More
Days of Culture on the Membrane
[0070] On day 14, the morphological appearance of the tissue on the
membrane was assessed by histology, its composition determined
using a battery of biochemical assays, and the metabolism of the
chondrocytes assessed in culture.
[0071] (i) Histology.
[0072] On day 14, the tissue, still on the PET membrane, was fixed
using 4% paraformaldehyde in PBS and embedded in paraffin.
Eight-.mu.m-thick sections were cut and stained with Toluidine Blue
for sulfated glycosaminoglycans. For electron microscopy, a small
piece of tissue was cut and fixed in 2% glutaraldehyde, 0.1M sodium
cacodylate buffer, 10 .mu.M CaCl.sub.2, pH 7.4.
[0073] (ii) Biochemical Composition of the Tissue.
[0074] At the end of the culture period (day 14), the tissue was
removed from the membrane, blotted onto dry gauze and the net
weight measured. The tissue then was lyophilized and weighed again
to obtain a measure of water content. The lyophilized tissue was
digested at 56.degree. C. for 24 hours with papain (20 .mu.g/ml) in
0.1 M Sodium acetate, 50 mM EDTA, 5 mM cysteine hydrochloride, pH
5.53.
[0075] DNA content was measured using the bisbenzimidazole
fluorescent dye [Hoechst 33258 (Polyscience, Warrington, Pa.)]
method with calf thymus DNA as a standard.
[0076] Total content of sulfated glycosaminoglycan was determined
by the dimethylmethylene blue (DMMB: Polyscience) assay as
previously described.
[0077] Hydroxyproline content was measured by reverse-phase HPLC,
using the PICO tag labeling technique, after hydrolysis of the
sample for 16 hours at 110.degree. C. in 6N HCl. Collagen content
in each sample was estimated by multiplying the hydroxyproline
content by 8.2.
[0078] Hyaluronan content was measured using sandwich ELISA
technique as previously described and reported relative to the
collagen content.
[0079] (iii) Characterization of Collagen Types Synthesized on Day
14 of Culture.
[0080] On day 14 of culture (i.e. 7 days after the chondrocytes and
their cell-associated matrix were placed upon the membrane of the
tissue culture insert), the tissue on the membrane was incubated
for 16 hours in DMEM containing [.sup.3H]-proline at 50 .mu.Ci/ml,
fetal bovine serum at 200 .mu.l/ml, ascorbic acid at 25 .mu.g/ml)
and beta-aminoproprionitrile (BAPN) at 10 .mu.g/ml to prevent
crosslink formation. The tissue then was minced and extracted
overnight with 1.0 M NaCl, 50 mM Tris containing proteinase
inhibitors (1 mM N-ethylmaleimide, 1 mM phenylmethylsulfonyl
fluoride, 5 mM EDTA) at 40.degree. C. The residue was separated
from the NaCl extract by centrifugation at 3000 rpm for 15 minutes
and solubilized in 1% SDS. The labeling medium, the NaCl extract
and the SDS fraction were dialyzed against distilled water to
remove unincorporated isotopes. The samples were further dialyzed
against 0.5 M acetic acid and incubated overnight at 4.degree. C.
with pepsin (100 .mu.g/ml) in 0.2 M NaCl, 0.5 M acetic acid. The
pepsin then was inactivated by the addition of NaOH in each sample
to raise the pH to 8.6. The samples were further dialyzed against
0.4 M NaCl, 10 mM Tris, pH 7.4. Aliquots of the samples were
analyzed by SDS-PAGE in an 8% acrylamide gel under reducing
conditions. The gel was subjected to fluorography and the images
were scanned and quantified as previously described.
[0081] (iv) Characterization of Proteoglycans Synthesized on Day 14
of Culture.
[0082] On day 14 of culture, the tissue was incubated for 4 hours
in DMEM/F12 containing .sup.35S-sulfate at 20 .mu.Ci/ml, 20% fetal
bovine serum (200 .mu.l/ml) and an effective amount of growth
factor. The tissue then was extracted with 4 M guanidine chloride,
0.05 M sodium acetate, pH 6.0, in the presence of protease
inhibitors as previously described. Radiolabeled proteoglycans were
purified by DEAE column chromatography using a step-wise
concentration gradient of sodium chloride. The purified
proteoglycans were analyzed for size by sieve chromatography on
Sepharose CL2B (Pharmacia) under dissociative conditions.
[0083] (v) Quantification of Collagen-Specific Crosslinks
[0084] Collagen-specific crosslinks (pyridinoline and
deoxypyridinoline) were quantified using fluorescence detection
following reverse-phase HPLC as previously described. Briefly, the
samples were hydrolyzed in 6N HCl for 24 hours at 110.degree. C.
and the hydrolysates were applied to a CF-1 cellulose column to
separate the crosslinking amino acids. The bound fraction was
eluted with distilled water and dried. The samples were separated
by reverse-phase HPLC on a C18 ODS column (Beckman) and the
fluorescence of the eluted peaks was monitored using a
spectrofluorimeter as previously described. The concentrations of
crosslinking amino acid were reported as equivalents of external
standards of pyridinoline and deoxypyridinoline.
[0085] vi) Measurement of Mechanical Property of the Cartilage
Tissue Formed in Vitro
[0086] The compressive and tensile properties of the transplantable
construct were determined using standard methods. For compressive
tests, disks (6.4 mm diameter) were cut from constructs and tested
in a uniaxial confined compression apparatus on a mechanical
testing machine (Dynastat: IMASS, Cambridge, Mass., USA) under
computer control as described previously (15). Equilibrium
load-displacement data were acquired, and the equilibrium confined
compression modulus was calculated using the formulation of Kwan et
al (16). For tensile tests, tapered specimens (1 mm width in the
gage region) were cut from constructs (17) and subjected to
elongation at a constant strain rate until failure. The load at
failure was normalized to the initial cross-sectional area to
determine the ultimate stress. In all tests, samples were immersed
in a physiological saline buffer.
[0087] Results
[0088] Studies of the Matrix Formed after Seven Days of Culture in
Alginate Beads
[0089] On day 7 of culture, tissue formed by chondrocytes cultured
in the presence of growth factor contained an abundant, voluminous
ECM. Examination of the cells in the beads by phase contrast
microscopy showed evidence of only a moderate degree of cell
division. After dissolving the beads with 55 mM sodium citrate in
0.9% NaCl, the cells and their associated matrix also were
visualized. The structure of this cell-associated matrix was
well-preserved, consistent with the view that the cell-associated
matrix is tightly bound to the cell membrane via cell-surface
receptors such as CD44, integrins and anchorin CII. Biochemical
analyses showed that the accumulated matrix was primarily composed
of proteoglycan and to a lesser extent hyaluronan. It contained
relatively little collagen immediately prior to transfer upon the
membrane of the culture insert. Collagen-specific crosslinking was
barely detectable at this stage.
[0090] Studies of the Matrix Formed after Seven Additional Days of
Culture on the Membrane.
[0091] Between days 8 and 14 of culture, the individual cells and
their associated matrix progressively became incorporated into a
single mass of cartilagineous tissue. The regenerated
cartilagineous tissue had a disk-like structure with a thickness of
approximately one millimeter. The regenerated cartilage was readily
recovered from the tissue culture insert by cutting the membrane.
Histological examination of the tissue revealed it contained a
cartilage-like matrix that strongly stained with Toluidine blue and
thus was rich in proteoglycars (FIG. 4). The staining was
especially strong in the territorial (pericellular) areas of the
matrix. Most of the chondrocytes were spherical in shape as
expected from chondrocytes that are phenotypically stable). A thin
layer of flattened cells (similar in shape to the chondrocytes
found in the most superficial layer of articular cartilage) were
observed on the surface of the culture and on the interface to the
membrane of the tissue culture insert. Examination in the electron
microscope showed the presence of thin fibrils in the territorial
areas and the absence of fibrils in the interterritorial area.
[0092] Biochemical analyses of the tissue on day 14 of culture
revealed that, as native articular cartilage matrix, it was very
rich in proteoglycan and contained significant amounts of
hyaluronan. In contrast, collagen was present at a much lower
concentration than in cartilage. Further, the concentration of
pyridinoline crosslinks (18 mmol/mol collagen), which by
crosslinking the collagen fibrils make the fibrillar network more
difficult to resorb, was very much lower than in articular
cartilage. Although the stiffness of this tissue was considerably
lower than that of normal adult articular cartilage, the tissue
nevertheless was easy to handle. It should be easy for to
orthopedic surgeons to press-fit it, if needed, into a defect in
the articular cartilage surface. Preferably, this cartilaginous
tissue should be dissected into a size that is 0.5 mm larger than
the real defect to allow the surgeon to press-fit it into the
defect: such a fit would allow the implanted tissue to make close
contact with the patient's cartilage. This approach may prove
useful in maximizing its integration within the articular tissue of
the patient.
[0093] Aggrecan, the major proteoglycan of normal articular
cartilage, made up more than 90% of the .sup.35S-proteoglycans
synthesized on day 14 of culture and incorporated into the matrix.
Small nonaggregating .sup.35S-proteoglycans were recovered from the
tissue in much smaller amounts. Analysis of the newly synthesized
collagens showed that the chondrocytes produced mostly the
cartilage-specific collagen type II, although small amounts of
other cartilage collagens were detected.
[0094] Measurement of Mechanical Property of the Cartilage Tissue
Formed in vitro revealed that the equilibrium confined compression
modulus after 1 week of culture in the insert was 0.001 MPa, which
is markedly lower than normal full thickness cartilage (about 0.4
MPa). At the same time point, the peak tensile stress was 0.01 Mpa,
which was also lower than normal full cartilage. However, both
values increased markedly with time in culture.
Example II
[0095] In Vivo Animal Study
[0096] Preparation of the Tissue to be Implanted. The articular
cartilage from rabbits weighing 1-1.5 kg was dissected from each
joint and digested with pronase and collagenase sequentially. The
chondrocytes thus obtained were encapsulated in alginate beads and
cultured for 1 week. After 12 weeks, the beads were dissolved by
addition of sodium citrate solution and the cells with their
cell-associated matrix recovered by mild centrifugation. After
washing with physiological saline, the cells were placed into a
tissue culture insert (Falcon, CAT #3090) and allowed to reform a
cartilage-like tissue over seven days of culture in DMEM/Ham F-12
medium supplemented with 20% FBS, 25 ug/ml ascorbic acid, 10
.mu.g/ml gentamicin. The grafts for transplantation were then
removed from the tissue culture insert and placed into sterile
culture tubes.
[0097] Transplantation.
[0098] Twelve male rabbits (3-3.5 kg) underwent surgery. After
general anesthesia with ketamine and xylozine followed by
isoflurane inhalation, the rabbit was placed in a supine position.
Following proper sterilization and draping, the knee joints were
exposed through a medial parapatellar approach. An incision of the
capsule was performed and the patella was dislocated laterally. A
3.5 mm-full thickness cartilage defect was made (using a biopsy
punch) at the center of the patellar groove. The defects was then
treated as follows.
[0099] Group 1 (control): the defect was not treated.
[0100] Group 2 (cartilaginous graft): a cartilaginous graft
(generated as described above) was placed into the defect
[0101] In all cases, the joint was then washed several times with
sterile saline containing antibiotics and closed with layered
sutures. The animal was allowed to recover from anesthesia in the
cage. After 4 weeks, the animals were euthanatized as described
above and a photograph of cartilage surface was taken.
[0102] Results:
[0103] The defect in Group 1 showed partial spontaneous repair by a
white scar tissue. On the other hand, the defect in Group 2 was
filled with transparent cartilage whose surface resembled the
surface of normal articular cartilage (FIG. 5).
[0104] Numerous modifications and variations in the practice of the
invention are expected to occur to those skilled in the art upon
consideration of the foregoing detailed description of the
invention. Consequently, such modifications and variations are
intended to be included within the scope of the following
claims.
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