U.S. patent application number 11/974420 was filed with the patent office on 2008-02-14 for compositions and methods for the treatment and repair of defects or lesions in articular cartilage using synovial-derived tissue or cells.
Invention is credited to Ernst B. Hunziker.
Application Number | 20080039955 11/974420 |
Document ID | / |
Family ID | 26950934 |
Filed Date | 2008-02-14 |
United States Patent
Application |
20080039955 |
Kind Code |
A1 |
Hunziker; Ernst B. |
February 14, 2008 |
Compositions and methods for the treatment and repair of defects or
lesions in articular cartilage using synovial-derived tissue or
cells
Abstract
Compositions and methods are provided for treatment of cartilage
defects in animals and humans. The compositions of the invention
include synovial tissue, synovial cells and matrices containing
synovial (or cambium) tissue or cells for use in filling a
cartilage defect. The matrix and synovial tissue or cell
preparations may also contain a proliferation agent, transforming
factor or other active agents to promote healing. A
controlled-release delivery system may be used to administer the
transforming factor. The compositions of the invention also include
a synovial covering membrane or devitalized fascial sheet for
covering the cartilage defect. The methods of this invention are
those in which a minimally invasive surgical intervention is
performed to remove a small portion of synovial membrane from a
joint. Portions of the synovial membrane, or cells expanded in
vitro, are implanted alone or within a matrix, into the defect
site, where they produce new cartilage tissue and repair the
defect. Alternatively, partially transformed synovial-derived
tissue may be formed in situ and implanted into the defect
site.
Inventors: |
Hunziker; Ernst B.; (Boll,
CH) |
Correspondence
Address: |
ROPES & GRAY LLP;PATENT DOCKETING 39/41
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Family ID: |
26950934 |
Appl. No.: |
11/974420 |
Filed: |
October 12, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10060009 |
Jan 30, 2002 |
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11974420 |
Oct 12, 2007 |
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60265053 |
Jan 30, 2001 |
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60265064 |
Jan 30, 2001 |
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Current U.S.
Class: |
623/23.76 ;
424/93.7 |
Current CPC
Class: |
A61K 38/1841 20130101;
A61L 27/3804 20130101; A61L 27/3683 20130101; C12N 2533/56
20130101; A61F 2002/30062 20130101; A61K 38/1875 20130101; A61K
38/1875 20130101; A61L 27/3654 20130101; A61P 19/04 20180101; A61K
35/32 20130101; A61L 27/3612 20130101; A61K 2300/00 20130101; A61F
2002/30677 20130101; A61K 2300/00 20130101; A61L 27/3852 20130101;
A61P 19/00 20180101; C12N 5/0655 20130101; A61F 2210/0004 20130101;
A61K 2300/00 20130101; A61L 27/3817 20130101; A61F 2002/4635
20130101; A61K 38/1841 20130101; A61K 38/51 20130101; A61L 27/3604
20130101; A61K 38/51 20130101; A61P 19/02 20180101; A61F 2002/2817
20130101; A61F 2002/30761 20130101; A61K 35/12 20130101 |
Class at
Publication: |
623/023.76 ;
424/093.7 |
International
Class: |
A61F 2/28 20060101
A61F002/28; A61K 35/00 20060101 A61K035/00 |
Claims
1. A method for anchoring cells or tissues placed within an
articular cartilage defect, comprising: perforating the subchondral
bone plate at the base of the defect at multiple points; covering
the base of the defect with a membrane; depositing an osteogenic
factor and/or an angiogenic factor between the base of the defect
and the membrane; depositing superior to the membrane and between
the membrane and the plane of the top of the cartilage defect a
chondrogenic preparation selected from the group consisting of a
cell suspension, cells within a matrix, synovial membrane tissue
and synovial membrane tissue within a matrix; and covering the
defect with a covering membrane.
2. The method according to claim 1, further comprising placing a
transforming factor between the membrane and the covering
membrane.
3. The method according to claim 1, further comprising placing an
anti-angiogenic factor between the membrane and the covering
membrane.
4. The method according to any one of claims 1-3, wherein the
membrane and the covering membrane comprise synovial membranes.
5. The method according to any one of claims 1-3, wherein the
transforming factor is selected from the group consisting of
TGF-.beta.'s, BMP's, CDMP, IHH, SHH and SOX-9.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/060,009, filed on Jan. 30, 2002, which claims the benefit of
U.S. Provisional Application Ser. Nos. 60/265,053 and 60/265,064,
both filed on Jan. 30, 2001, the contents of each of which are
incorporated herein by reference in their entirety.
TECHNICAL FIELD OF THE INVENTION
[0002] This invention relates to the treatment and repair of
defects or lesions (used interchangeably herein) in cartilage. The
compositions of the invention include matrices and synovial tissue
or cells for use in filling a cartilage defect. Cambium cells may
also be used. The matrices and synovial tissue or cell preparations
may also contain a proliferation agent and/or transforming factor
to facilitate, respectively, expansion of synovial cells and
differentiation of synovial cells into chondrocytes, leading to the
formation of cartilage tissue. The compositions of the invention
also include synovial or cambium cells that have been transfected
with chondrogenic genes such that the cells express chondrogenic
factors that promote cartilage formation. The methods of the
invention comprise obtaining synovial membrane tissue or cells from
a minimally invasive biopsy of a joint capsule's synovium, adding
appropriate proliferation and/or transforming factors to the
synovial membrane tissue or cells and placing the tissue (as a
membrane sheet or minced) or cells in the defect with or without a
structural matrix. Alternatively, the defect may be filled with
layered synovial membrane sheets. The synovial membrane tissue may
be partially devitalized prior to filling the defect.
Alternatively, the defect may be filled with a matrix containing
chondrocytes that have been prepared from synovial cells or cambium
cells in vitro or in situ. Alternatively, partially transformed
synovial-derived tissue formed in vitro or in situ may be used to
fill the defect. The filled defect may then be covered with a
covering membrane, preferably comprising a partially devitalized
synovial membrane sheet. The methods of this invention are
particularly useful in the treatment of articular cartilage defects
found in osteoarthritis, and in other diseases and traumas that
produce cartilage injury.
[0003] U.S. Pat. Nos. 5,206,023, 5,270,300 and 5,853,746 are hereby
incorporated by reference.
BACKGROUND ART
[0004] Joints are one of the common ways bones in the skeleton are
connected. The ends of normal articulated bones are covered by
articular cartilage tissue, which permits practically frictionless
movement of the bones with respect to one another [L. Weiss, ed.,
Cell and Tissue Biology (Munchen: Urban and Schwarzenburg, 1988) p.
247].
[0005] Articular cartilage is characterized by a particular
structural organization. It consists of specialized cells
(chondrocytes) embedded in an intercellular material (often
referred to in the literature as the "cartilage matrix") that is
rich in proteoglycans, collagen fibrils of predominantly type II,
other proteins, and water [Buckwalter et al., "Articular Cartilage:
Injury and Repair," in Injury and Repair of the Musculoskeletal
Soft Tissues (Park Ridge, Ill.: American Academy of Orthopaedic
Surgeons Symposium, 1987) p. 465]. The cartilage matrix is produced
and maintained by the chondrocytes embedded within it. Cartilage
tissue is neither innervated nor penetrated by the vascular or
lymphatic systems. However, in the mature joints of adults, the
underlying subchondral bone tissue forms a thin, continuous plate
between the bone tissue and the cartilage. This subchondral bone
tissue or bone plate is innervated and vascularized. Beneath this
bone plate, the bone tissue forms trabeculae, containing the
marrow. In immature joints, articular cartilage is underlined by
only primary bone trabeculae. A portion of the meniscal tissue in
joints also consists of cartilage whose make-up is similar to
articular cartilage [Beaupre, A. et al., Clin. Orthop. Rel. Res.,
pp. 72-76 (1986)].
[0006] Two types of defects are recognized in articular surfaces.
These are full-thickness defects and superficial defects.
Full-thickness defects are those that penetrate into or through the
subchondral bone plate; superficial defects are those that do not.
These defects differ not only in the extent of physical damage to
the cartilage, but also in the nature of the repair response each
type of lesion can elicit.
[0007] Full-thickness defects of an articular surface include
damage to the hyaline cartilage, the calcified cartilage layer and
the subchondral bone tissue with its blood vessels and bone marrow.
Full-thickness defects can cause severe pain because the bone plate
contains sensory nerve endings. Such defects generally arise from
severe trauma or during the late stages of degenerative joint
disease, such as osteoarthritis. Full-thickness defects may, on
occasion, lead to bleeding and the induction of a repair reaction
from the subchondral bone [Buckwalter et al., "Articular Cartilage:
Composition, Structure, Response to Injury, and Methods of
Facilitating Repair," in Articular Cartilage and Knee Joint
Function Basic Science and Arthroscopy (New York: Raven Press,
1990) pp. 19-56]. The repair tissue formed is a vascularized,
fibrous type of cartilage that has poor biomechanical properties,
and that does not persist on a long-term basis [Buckwalter et al.
(1990), supra].
[0008] Superficial defects in articular cartilage tissue are
restricted to the cartilage tissue itself. Such defects are
notorious because they generally do not heal and show no propensity
for repair reactions.
[0009] Superficial defects may appear as fissures, divots, or
clefts in the surface of the cartilage, or they may have a
"crab-meat" appearance in the affected tissue. They contain no
bleeding vessels (blood spots) such as are seen in full-thickness
defects. Superficial defects may have no known cause, but often
they are the result of mechanical derangements that lead to a
wearing down of the cartilaginous tissue. Mechanical derangements
may be caused by trauma to the joint, e.g., a displacement of torn
meniscus tissue into the joint, meniscectomy, a laxation of the
joint by a torn ligament, malalignment of joints, bone fracture or
by hereditary diseases. Superficial defects are also characteristic
of early stages of degenerative joint diseases, such as
osteoarthritis. Because cartilage tissue is not innervated [Ham's
Histology (9th ed.) (Philadelphia: J. B. Lippincott Co. 1987), pp.
266-272] or vascularized, superficial defects are typically not
painful. However, although painless, superficial defects generally
do not heal, and often degenerate into full-thickness defects.
[0010] It is generally believed that because articular cartilage
lacks a vasculature, damaged cartilage tissue does not receive
sufficient or proper stimuli to elicit a repair response [Webber et
al., "Intrinsic Repair Capabilities of Rabbit Meniscal
Fibrocartilage: A Cell Culture Model", (30th Ann. Orthop. Res.
Soc., Atlanta, February 1984); Webber et al., J. Orthop. Res., 3,
pp. 36-42 (1985)]. It is theorized that chondrocytes in
cartilaginous tissue are normally not exposed to sufficient amounts
of repair-stimulating agents such as growth factors and fibrin
clots typically present in damaged vascularized tissue.
[0011] One approach that has been used to expose damaged cartilage
tissue to repair stimuli involves drilling or scraping through the
cartilage into the subchondral bone to cause bleeding [Buckwalter
et al. (1990), supra]. Unfortunately, the repair response of the
tissue to such surgical trauma is usually comparable to that
observed to take place naturally in full-thickness defects that
cause bleeding, viz., formation of a fibrous type of cartilage that
exhibits insufficient biomechanical properties and that does not
persist on a long-term basis [Buckwalter et al. (1990), supra].
[0012] A variety of growth factors have been isolated and are now
available for research and biomedical applications [see e.g.,
Rizzino, A., Dev. Biol., 130, pp. 411-422 (1988)]. Some of these
growth factors, such as transforming growth factor beta
(TGF-.beta.), have been reported to promote formation of
cartilage-specific molecules, such as type II collagen and
cartilage-specific proteoglycans, in embryonic rat mesenchymal
cells in vitro [e.g., Seyedin et al., Proc. Natl. Acad. Sci. USA,
82, pp. 2267-71 (1985); Seyedin et al., J. Biol. Chem., 261, pp.
5693-95 (1986); Seyedin et al., J. Biol. Chem., 262, pp. 1946-1949
(1987)].
[0013] Millions of patients have been diagnosed as having
osteoarthritis, i.e., as having degenerating defects or lesions in
their articular cartilage. Nevertheless, despite claims of various
methods to elicit a repair response in damaged cartilage, none of
these treatments has received substantial application [Buckwalter
et al. (1990), supra; Knutson et al., J. Bone and Joint Surg.,
68-B, p. 795 (1986); Knutson et al., J. Bone and Joint Surg., 67-B,
p. 47 (1985); Knutson et al., Clin. Orthop., 191, p. 202 (1984);
Marquet, Clin. Orthop., 146, p. 102 (1980)]. And such treatments
have generally provided only temporary relief. Systemic use of
"chondroprotective agents" has also been purported to arrest the
progression of osteoarthritis and to induce relief of pain
[Lohmander, L. S. et al., Ann. Rheum. Dis., 55(7), pp. 424-31
(1996)]. However, such agents have not been shown to promote repair
of lesions or defects in cartilage tissue.
[0014] One approach that has been considered is illustrated in U.S.
Pat. No. 4,846,835. There, chondrocytes are harvested from mature
cartilage tissue that has been removed by biopsy, subsequently
grown or expanded in number in tissue culture, and then grafted
into the defect site in a collagen gel matrix used to fix the
chondrocytes in situ. A periosteal sheet is used to secure the
transplanted cells (i.e., the graft) in the defect. This approach
suffers from the disadvantages of being more invasive than the
instant invention, and creating additional cartilage defects by
removing mature cartilage. The use of articular chondrocytes to
repair defects is also disadvantaged because articular chondrocytes
have a more limited potential for proliferation as compared to
synovial cells.
[0015] Another approach has been to transform bone marrow-derived
mesenchymal stem cells to create chondrocytes in vitro for
transplantation into a cartilage defect [Caplan and Haynesworth,
U.S. Pat. No. 5,811,094]. These cells can only be obtained through
a bone-marrow biopsy, which may be associated with long-term local
pathology at the donor site of the patient (usually the iliac crest
of the pelvis). Biopsied bone marrow cells then must be purified
using sophisticated techniques, expanded in vitro and seeded at the
site of the defect [See Caplan et al., Clin. Orthop., 342, pp.
254-269(1997)].
[0016] A further approach found in the literature is applying an
electric potential through the tissue surrounding the defect in
order to stimulate the growth of new tissue. [U.S. Pat. No.
4,506,673].
[0017] Another approach to repairing such defects is found in U.S.
Pat. Nos. 5,270,300, 5,206,023, and 5,368,858, in which the present
inventor described inventions wherein repair cells are attracted
from the synovial compartment to the defect site, where they are
induced to proliferate and to differentiate into chondrocytes that
synthesize new cartilage matrix.
[0018] It has been reported that one means to retain cells in
suspension or a matrix within an articular cartilage defect is to
suture an appropriate thin covering membrane on the top of the
defect space. The covering membrane material used so far has been
periosteum-derived, perichondrium-derived or muscle fascia. These
covering membranes or other artificial covering membranes have the
significant disadvantage that the covering membrane itself does not
transform into cartilage tissue, or does so to only a limited
extent. Thus the defect space will never fill completely with
repair cartilage. Moreover, the degradation of fibrous types of
covering membranes is extremely slow. In addition, these covering
membranes do not integrate with the native tissue along the defect
lesion borders. Such covering membrane tissue may contain
fibroblasts, which will not transform into chondrocytes but instead
result in undesirable scar-like tissue. Thus, in certain prior art
covering membranes, fibroblasts may migrate out into the repair
space, contaminate it and lead to unwanted scar tissue
formation.
[0019] There is, therefore, a need for a simple, fast, and reliable
treatment superficial and full-thickness cartilage defects, e.g.,
as found in cases of severe mechanical injury and
osteoarthritis.
SUMMARY OF THE INVENTION
[0020] The present invention provides effective therapeutic
compositions and methods to induce the repair of lesions in
cartilage of humans and other animals. Use of the compositions and
methods of this invention also promotes the healing of traumatic
lesions and forms of osteoarthritis that would otherwise lead to
loss of effective joint function leading to probable resection and
replacement of the joint with a metal and/or plastic artificial
joint.
[0021] In the present invention, the patient suffering from a
cartilage defect in a joint is administered a local anesthetic in
the joint area, or a general anesthetic may be administered.
Appropriate surgical tools are used to extract a portion of
synovial membrane tissue. It may be advantageous to take the
synovial tissue from the same joint as the defect, as it has been
shown that cartilage and synovial cells mature into a form that
fulfills the mechanical and structural properties typical of the
particular joint from which they are derived. [Kuettner, K. E.,
Clin. Biochem., 25, pp. 155-63 (1992) (cartilage of different
joints differ biochemically); Archer, C. W., Ann. Rheum. Dis., 53,
pp. 624-30 (1994) (fetal cartilage differs joint to joint)]. In
some cases, however, it may be desirable to take synovial tissue
from a large joint for use in the repair of a small joint
containing an insufficient quantity of donor synovial tissue. The
synovial membrane tissue or cells thus procured is used in the
repair procedure as described below. Alternatively, the cambium
layer from the periosteum or perichondrium can be used as the
source of cells or repair tissue.
[0022] It is preferred that, prior to filling the defect with the
repair materials, the edges of the articular cartilage at the
defect site are treated with a proteoglycan-degrading enzyme to
remove superficial proteoglycans. This treatment exposes the
underlying collagen network, permitting greater tissue integration
and adhesion of the repair materials.
[0023] In one embodiment of the present invention, synovial
membrane tissue is used in an immediate replantation procedure. It
is preferred that the synovial membranes or minced synovial
membranes are replanted in the presence of a transforming factor
that induces differentiation of the synovial cells into
chondrocytes that in turn form new cartilage and thereby repair the
defect. Preferably, the synovial membranes or minced synovial
membranes are distributed in a biodegradable matrix that is then
implanted into the defect site. The transforming factor may be
administered via a controlled-release delivery system.
[0024] In another embodiment of the present invention, minced
synovial membranes are subjected to brief collagenase digestion and
trypsin digestion (30-45 minutes) followed by a rapid purification
in the operating room. These synovial bits are then used for
immediate transplantation into the site of the defect, suspended in
a biodegradable matrix that includes a transforming factor that
will cause them to differentiate into chondrocytes after
implantation. Alternatively synovial membrane sheets arranged in
stacks can be used to fill the defect. The addition of appropriate
transforming factors will promote synovial cell transformation into
chondrocytes in situ. Such factors are preferably placed between
the synovial membrane sheets. Appropriate anti-angiogenic agents
may also be used to prevent vascularization of new cartilage
tissue, and appropriate mineralization/calcification inhibitors may
be used to prevent ossification of new cartilage tissue.
[0025] Alternatively, whole periosteum, perichondrium or,
preferably, sheets of the cambium layer of the periosteum or
perichondrium are used to fill the defect. Any of several
configurations of tissue is possible: bits, sheets, sheets
interspersed with bits, tissue suspended in a biodegradable matrix,
or combinations of these elements. Also, different tissues may be
used in combination. For example, a periosteum cambium layer sheet
may be placed on the floor of the defect, a biodegradable matrix
containing synovial or periosteal bits may be used to fill the
defect and a synovial membrane sheet may be used to cover the
defect. Alternatively, mixed layers of synovial membrane sheets and
periosteal or perichondrial membrane sheets may be used to fill the
defect.
[0026] Synovial cells may also be used in an implantation
procedure. Synovial cells are obtained from the synovial membrane
or fluid, or alternatively, cambium cells are obtained from the
cambium layer of the periosteum or perichondrium. If the cells are
not of sufficient numbers (as described infra), the cells are then
cultured and expanded in vitro. When the cultured cells are
expanded to sufficient numbers, they are implanted into the
cartilage defect site. Preferably, the cells are implanted in a
biodegradable matrix that is implanted into the defect site. The
cells are implanted in the presence of a transforming factor that
induces differentiation of the synovial cells into chondrocytes
that in turn form new cartilage and thereby repair the defect. The
transforming factor may be administered via a controlled-release
delivery system.
[0027] Alternatively, chondrocytes may be isolated from transformed
synovial membrane sheets and then implanted into a defect space by
inserting them into a matrix or in suspension as described above.
Chondrocytes induced this way may also be expanded in vitro before
implantation, in order to increase their numbers, and to select for
a chondrogenic subpopulation.
[0028] In another embodiment of the present invention, synovial
cells are induced to differentiate in the presence of a
transforming factor in vitro into chondrocytes that begin to form
cartilage tissue. Partial enzymatic digestion of the pericellular
matrix may be performed to release the individual chondrocytes,
which are then implanted in the defect site as a mass.
Alternatively, the cartilage tissue formed in vitro may be
implanted into a biodegradable matrix that is then in turn
implanted into the defect site.
[0029] In another embodiment of the present invention, synovial
cells may be transformed into chondrocytes in situ. In this aspect
of the invention, synovial cells are induced to differentiate in
situ through the use of a differentiation-factor-impregnated matrix
pad sutured to the synovial membrane in one of the synovial recessi
or in proximity to a fat pad. Alternatively, this may be
accomplished by attaching the differentiation-factor-impregnated
matrix pad to synovial tissue immediately outside of the joint
space, eliminating the need to open the joint. The transformed
tissue is excised and cells rapidly separated using collagenase and
trypsin digestion for about 30-45 minutes. The newly transformed
chondrocytes may be further expanded and/or differentiated in vitro
before implantation in situ. Chondrocytes may then be implanted at
the site of the defect either in suspension (covered by a covering
membrane to prevent cell loss into the joint cavity) or in a matrix
to retain cells in the defect space. The covering membrane may be
sutured to the defect edges near the top perimeter of the
lesion.
[0030] In each of the foregoing embodiments, it is preferred that
the tissue be implanted within a biodegradable matrix to prevent
loss of tissue or cells from the defect site, to effectively fill
the defect space and as a carrier for any agents or factors
employed to treat the defect. Alternatively, or in addition, it is
preferred that the surface of the defect site following implanting
be covered by a thin biodegradable membrane ("covering membrane")
to prevent loss of tissue or cells from the defect site.
[0031] In a preferred embodiment of the present invention, a
synovial covering membrane, preferably partially devitalized (i.e.,
partially depleted in macrophages) or fully native, may be used to
cover the filled defect. Alternatively, fascial tissue sheets,
which may be completely devitalized through three freeze-thaw
cycles (to prevent contaminating cell outgrowth following exposure
to growth factors in the underlying repair matrix), may be used to
cover the defect. Alternatively, the biodegradable membrane may be
artificial (e.g., polylactic acid sheets, hyaluronic acid sheets
and thin (fine) collagen meshes) or natural (e.g., periosteum,
perichondrium). Natural periosteum or perichondrium covering
membranes are not preferred when growth factors are used according
to this invention, because such factors may stimulate cells in
those covering membranes that would contaminate the defect area.
However, this is generally not a concern when the cambium layer
alone is used.
[0032] The use of synovial tissue membranes as a covering membrane
to cover an articular cartilage defect has advantages over prior
art covering membranes comprising periosteal tissue or fascia.
Synovial tissue can be more completely transformed into
chondrocytes and cartilage and better integrated into adjacent
cartilage tissue. Both periosteal tissue and fascia typically
contain many fibroblasts, which cannot be transformed into
chondrocytes and cartilage, and which persist as scar tissue.
Synovial tissue membranes, without additional therapeutic agents in
the defects beneath them, through transformation into chondrocytes
and cartilage can be adequate to repair shallow defects.
[0033] When using synovial tissue as a covering membrane, it is
preferred that the tissue be treated with a transforming factor. It
is also preferable that the transforming factor is used in
association with a controlled-release delivery system. The
transforming factor, with or without the controlled-release
delivery system, can be added to the synovial covering membrane, or
the synovial covering membrane can be soaked in a solution of
transforming factor, with or without a controlled-release delivery
system.
[0034] The present invention is a simpler and more rapid procedure
than those in the prior art. For example, the present invention
does not require the presence of attractive factors to induce
migration of repair cells into the defect site, and it does not
require the complicated extraction and purification procedures
necessary to utilize bone marrow-derived mesenchymal cells.
[0035] In the knee, synovial tissue can be excised from recessi
near the retropatellar fat pad or in the suprapatellar recessus
where large reserve folds of synovial tissue are present. Such
removal of synovial tissue involves virtually no impairment of
joint function. Removal of pads of synovial membrane is not
associated with any joint pathology, and any lesions in these
tissues tend to heal spontaneously.
[0036] The use of the synovial tissue covering membrane of the
present invention to cover the filled defect also has a significant
advantage over the methods of the prior art in that the whole
covering membrane itself is capable of transforming into cartilage
tissue and integrating into the newly forming repair cartilage.
Moreover, it can integrate well with the adjoining native tissue,
such as bone. In addition, it is advantageous when transforming
growth factors are used because the synovial tissue covering
membrane contains primarily chondrogenic precursor cells.
[0037] The use of partially devitalized tissue covering membranes
according to the present invention is also advantageous.
Alternatively, a fascial covering membrane may be used. Preferably,
such a fascial covering membrane will be fully devitalized prior to
use (i.e., frozen and thawed three times prior to use) in order to
prevent fibroblasts, macrophages and other cell types from
contaminating the newly formed cartilage tissue and subsequently
forming undesirable scar tissue.
[0038] For synovial tissue covering membranes, partial
devitalization is preferred for selectively removing macrophages,
but not other cell types. This is accomplished through a single
freeze-thaw cycle, or other means known in the art for the
selective removal of macrophages and other inflammatory cells
(e.g., macrophages may be removed from synovial or other tissue by
using anti-macrophage antibodies or other substances to selectively
neutralize macrophages).
[0039] In each of the above embodiments, anti-inflammatory agents
may be used locally to prevent macrophage migration, activation and
proliferation, as well as pannus formation. Anti-inflammatory
agents include steroidal drugs such as prednisone, and
non-steroidal anti-inflammatory drugs ("NSAIDs") such as ibuprofen,
ketoprofen, piroxicam, naproxen, sulindac, aspirin, choline
subsalicylate, diflunisal, fenoprofen, indomethacin, meclofenamate,
salsalate, tolmetin and magnesium salicylate. Such
anti-inflammatory agents may be administered to the defect in a
controlled-release delivery system in a matrix containing synovial
tissue or cells, or placed between layered synovial membrane
sheets. Examples of delivery systems include poly(lactic) acid
(PLA), poly(D,L-lactic-co-glycolic) acid (PLGA) or
poly(epsilon-caprolactone) (PCL) microspheres, liposomes,
bioerodible polymers, collagen fibers chemically linked to heparin
sulfate proteoglycans, and carbohydrate-based corpuscles.
[0040] In each of the above embodiments, cartilage repair may be
carried out through autologous transplantation of synovial tissue
or cells, i.e., from the same individual, or alternatively,
heterologous transplantation of synovial tissue or cells may be
carried out. Heterologous transplantation may require simultaneous
administration of immunosuppressant therapies known in the art.
[0041] The synovial tissue covering membranes of the present
invention may also be used with other cartilage repair techniques
known in the art, including but not limited to those described in
U.S. Pat. Nos. 5,206,023, 5,270,300 and 5,853,746.
DETAILED DESCRIPTION OF INVENTION
[0042] In order that the invention may be more fully understood,
the following detailed description is provided. In the description
the following terms are used.
[0043] Anti-Angiogenic Agent--as used herein, refers to any
compound or composition with biological activity that prevents
ingrowth of blood vessels from the underlying bone tissue into the
cartilage tissue, such as anti-invasive factors, cartilage-derived
angiogenesis inhibitors, angiostatin, metalloprotease inhibitors,
antibodies against angiogenesis-inducing factors (including bFGF
and endothelial cell stimulating angiogenic factor (ESAF)), Suramin
(Germanin.RTM., Bayer AG, Germany), fumagillin, fumagillin
analogues and AGM-1470 [Peacock, D. J. et al., Cellular Immunology,
160, pp. 178-84 (1995)]. In vivo and in vitro assays to determine
anti-angiogenic agents are well-known in the art [e.g., Moses, M.
A., Clinical & Exptl. Rheumatology, 11 (Suppl. 8), pp. 567-69
(1993); Moses, M. A. et al., J. Cell Bio., 119(2), pp. 475-82
(1992); Moses, M. A. et al., Science, 248, pp. 1408-10 (1990);
Ingber, D. et al., Nature, 348(6), pp. 555-57 (1990)].
[0044] Arthroscopy--as used herein, refers to the use of an
arthroscope to examine or perform surgery on a joint.
[0045] Cambium Cells--as used herein, refers to cells found in the
cambium layer of the perichondrium and periosteum of joints and
bones.
[0046] Cartilage--as used herein, refers to a type of connective
tissue that contains chondrocytes embedded in an intercellular
material (often referred to as the "cartilage matrix") comprising
fibrils of collagen (predominantly type II collagen along with
other minor types, e.g., types IX and XI), various proteoglycans
(e.g., chondroitinsulfate-, keratansulfate-, and dermatansulfate
proteoglycans), other proteins, and water. Cartilage as used herein
includes articular and meniscal cartilage. Articular cartilage
covers the surfaces of the portions of bones in joints and allows
movement in joints without direct bone-to-bone contact, and thereby
prevents wearing down and damage to apposing bone surfaces. Most
normal healthy articular cartilage is also described as "hyaline,"
i.e., having a characteristic frosted glass appearance. Meniscal
cartilage is usually found in joints that are exposed to concussion
as well as movement. Such locations of meniscal cartilage include
the temporo-mandibular, stemo-clavicular, acromio-clavicular, wrist
and knee joints [Gray's Anatomy (New York: Bounty Books,
1977)].
[0047] Cell Adhesion Promoting Factor--as used herein, refers to
any compound or composition, including fibronectin and other
peptides as small as tetrapeptides, that comprises the tripeptide
Arg-Gly-Asp, which mediates the adhesion of cells to extracellular
material [Ruoslathi et al., Cell, 44, pp. 517-518 (1986)].
[0048] Chondrocytes--as used herein, refers to cells that are
capable of producing components of cartilage tissue, e.g., type II
cartilaginous fibrils and fibers and proteoglycans.
[0049] Covering membrane--as used herein, refers to any material
that can be used to cover a filled defect site following
implantation to prevent loss of cells into the adjoining space, as
well as any material that can be placed between the bone defect
portion and the cartilage defect portion of a full thickness defect
and that prevents cell migration and blood vessel infiltration from
the bone defect portion into the cartilage defect portion of the
full thickness defect. The membranes used in the methods and
compositions of this invention are preferably biodegradable, and
may include synovial membranes, polylactic acid sheets, hyaluronic
acid sheets, thin (fine) collagen meshes, periosteum, perichondrium
or fascia. Synovial membranes typically are transformed into
cartilage tissue, becoming integrated into the repair cartilage
tissue. Non-biodegradable membranes may include teflon
(Goretex.RTM.), Millipore.RTM. membrane, or Verigen.RTM.
membrane.
[0050] Fibroblast growth factor (FGF)--any member of the family of
FGF polypeptides [Gimenez-Gallego et al., Biochem. Biophys. Res.
Commun., 135, pp. 541-548 (1986); Thomas et al., Trends Biochem.
Sci., 11, pp. 81-84 (1986)] or derivatives thereof, obtained from
natural, synthetic or recombinant sources, which exhibits the
ability to stimulate DNA synthesis and cell division in vitro [for
assays see, e.g., Gimenez-Gallego et al., 1986, supra; Canalis et
al., J. Clin. Invest., 81, pp. 1572-1577 (1988)] of a variety of
cells, including primary fibroblasts, chondrocytes, vascular and
corneal endothelial cells, osteoblasts, myoblasts, smooth muscle
and glial cells [Thomas et al., 1986, supra]. FGF's may be
classified as acidic (aFGF) or basic (bFGF) FGF, depending on their
isoelectric points (pI).
[0051] Matrix--as used herein, refers to a porous composite, solid
or semi-solid substance having pores or spaces sufficiently large
to allow cells to populate the substance. The term matrix includes
matrix-forming materials, i.e., materials that can form matrices
within a defect site in cartilage or bone. Matrix-forming materials
may require addition of a polymerizing agent to form a matrix, such
as adding thrombin to a solution containing fibrinogen to form a
fibrin matrix. Other matrix materials include but are not limited
to collagen, combinations of collagen and fibrin, agarose (e.g.,
Sepharose.RTM.), gelatin, hyaluronic acid (hyaluronan), hyaluronic
acid in combination with collagen, photo-polymerizable matrices,
albumin-based matrices, polylactic acid-based matrices,
polyglycolic acid-based matrices and fibrin-based matrices. Calcium
phosphates, such as tricalcium phosphate, hydroxyapatite or other
calcium salts that form solid matrices may be used alone or in
combination with other matrix materials in treating defects in
bones.
[0052] Proliferation (mitogenic) Agent--as used herein, refers to
any compound or composition, including peptides, proteins, and
glycoproteins, that is capable of stimulating proliferation of
cells in vitro. In vitro assays to determine the proliferation
(mitogenic) activity of peptides, polypeptides and other compounds
are well-known in the art [see, e.g., Canalis et al., 1988, supra;
Gimenez-Gallego et al., Biochem. Biophys. Res. Commun., 135, pp.
541-548 (1986); Rizzino, "Soft Agar Growth Assays for Transforming
Growth Factors and Mitogenic Peptides", in Methods Enzymol., 146A
(New York: Academic Press, 1987), pp. 341-52; Dickson et al.,
"Assay of Mitogen-Induced Effects on Cellular Incorporation of
Precursors for Scavengers, de novo, and Net DNA Synthesis", in
Methods Enzymol., 146A (New York: Academic Press, 1987), pp.
329-40]. One standard method to determine the proliferation
(mitogenic) activity of a compound or composition is to assay it in
vitro for its ability to induce anchorage-independent growth of
nontransformed cells in soft agar [e.g., Rizzino, 1987, supra].
Other mitogenic activity assay systems are also known [e.g.,
Gimenez-Gallego et al., 1986, supra; Canalis et al., 1988, supra;
Dickson et al., 1987, supra]. Mitogenic effects of agents are
frequently very concentration-dependent, and their effects may be
reversed at lower or higher concentrations than the optimal
concentration range for mitogenic effectiveness.
[0053] Synovial Cell--as used herein, refers to a cell
physiologically associated with the synovial membrane or present in
the subsynovial space; a cell obtained from a joint's synovial
membrane or synovial fluid. When exposed to appropriate stimuli,
the synovial cell will differentiate and be transformed into a
chondrocyte. Synovial-derived repair cells include mesenchymal
cells, fibroblasts, fibroblast-like cells, macrophages,
de-differentiated chondrocytes, synovial lining cells, and synovial
fibroblast-like cells.
[0054] Transforming Factor--as used herein, refers to any peptide,
polypeptide, protein, or any other compound or composition that
induces differentiation of, e.g., synovial-derived cells into
chondrocytes. This also includes the products of genes introduced
(e.g., by transfection) into cells used to repair the defect. The
ability of the compound or composition to induce or stimulate
production of cartilage-specific proteoglycans and type II collagen
by cells can be determined by in vitro assays known in the art
[Seyedin et al., Proc. Natl. Acad. Sci. USA, 82, pp. 2267-71
(1985); Seyedin et al., Path. Immunol. Res., 7, pp. 38-42
(1987)].
[0055] Transforming Growth Factor Beta (TGF-.beta.)--any member of
the family of TGF-13 polypeptides [Derynck, R. et al., Nature, 316,
pp. 701-705 (1985); Roberts et al., "The transforming growth
factor-.beta.'s", In Peptide growth factors and their receptors I
(Berlin: Springer Verlag, 1990), p. 419)] or derivatives thereof,
obtained from natural, synthetic or recombinant sources, that
exhibits the characteristic TGF-.beta. ability to stimulate normal
rat kidney (NRK) cells to grow and form colonies in a soft agar
assay [Roberts et al., "Purification of Type .beta. Transforming
Growth Factors From Non-neoplastic Tissues", in Methods for
Preparation of Media, Supplements, and Substrata for Serum-Free
Animal Cell Culture (New York: Alan R. Liss, Inc., 1984)] and that
is capable of inducing transformation of synovial-derived repair
cells into chondrocytes as evidenced by the ability to induce or
stimulate production of cartilage-specific proteoglycans and type
II collagen by cells in vitro [Seyedin et al., 1985, supra].
[0056] Preparing the Defect and Obtaining Synovial Tissue
[0057] To carry out the methods of treating defects or lesions in
cartilage according to this invention, an articular cartilage
defect to be repaired is first identified. Cartilage defects in
animals are readily identifiable visually during arthroscopic
examination of the joint or during simple examination of the lesion
or defect during open surgery. Cartilage defects may also be
identified inferentially by using computer aided tomography (CAT
scanning), X-ray examination, magnetic resonance imaging (MRI),
analysis of synovial fluid or serum markers, or by any other
procedure known in the art.
[0058] Once a defect has been identified, prior to, or at the time
of repair, the surgeon may elect to surgically modify the defect to
enhance the ability of the defect to physically retain the synovial
membrane tissue, cells and/or matrix material that is added in the
treatment methods described herein. Preferably, instead of having a
flat or shallow concave geometry, the defect has, or is shaped to
have, vertical edges or is undercut in order to better retain the
synovial membrane tissue, cells and/or matrix materials added in
the treatment methods described herein. Both full-thickness and
shallow defects may be treated to create discrete areas of bleeding
in a process called "microfracture," which involves making measured
perforations in the subchondral bone plate. Released marrow
elements form a surgically induced clot that provides an enriched
environment for new tissue formation [Steadman et al. Clin.
Orthop., 391 Suppl., pp. S362-69 (October 2001)].
[0059] In one embodiment, under local anesthesia, a small
arthroscopic or surgical intervention is performed. The defect site
may optionally be treated prior to implantation with a
proteoglycan-degrading enzyme or other materials to improve
adhesion of the replanted synovial membrane tissue, cells or
matrix. The surface of the defect is dried by blotting the area
using sterile absorbent tissue, and the defect volume is filled
with a sterile enzyme solution for a period of 2-10 minutes to
degrade the proteoglycans present on the surface of the cartilage
and locally within approximately 1 to 2 .mu.m deep from the surface
of the defect. Various enzymes may be used, singly or in
combination, in sterile buffered aqueous solutions to degrade the
proteoglycans. The pH of the solution should be adjusted to
optimize enzyme activity.
[0060] Enzymes useful to degrade the proteoglycans in the methods
of this invention include chondroitinase ABC, chondroitinase AC,
hyaluronidase, pepsin, trypsin, chymotrypsin, papain, pronase,
stromelysin and Staph V8 protease [Jurgensen, K. et al., J. Bone
Joint Surg. Am., 79(2, pp. 185-93 (1997); Hunziker, E. B. et al.,
J. Bone Joint Surg. Br., 80(1), pp. 144-50 (1998)]. The appropriate
concentration of a particular enzyme or combination of enzymes will
depend on the activity of the enzyme solution.
[0061] In a preferred embodiment of this invention, the defect is
filled with a sterile solution of chondroitinase AC at a
concentration of 1 U/ml and digestion is allowed to proceed for 4
minutes. The preferred concentration of chondroitinase AC is
determined according to the procedure described in Example 1. Any
other enzyme used should be employed at a concentration and for a
time period such that only superficial proteoglycans down to a
depth of about 1-2 .mu.m are degraded.
[0062] The amount of time the enzyme solution is applied should be
kept to a minimum to effect the degradation of the proteoglycans
predominantly in the repair area. For chondroitinase ABC or AC at a
concentration of 1 U/ml, a digestion period longer than 10 minutes
may result in the unnecessary and potentially harmful degradation
of the proteoglycans outside the defect area. Furthermore,
digestion times longer than 10 minutes contribute excessively to
the overall time of the procedure. The overall time for the
procedure should be kept to a minimum especially during open
arthrotomy, because cartilage may be damaged by exposure to air
[Mitchell et al., (1989), supra]. For these reasons, in the
embodiments of the methods of this invention that include the step
of degradation of proteoglycans by enzymatic digestion, digestion
times of less than 10 minutes are preferred and digestion times of
less than 5 minutes are most preferred.
[0063] According to the methods of this invention, after the enzyme
has degraded the proteoglycans from the surface of the defect, the
enzyme solution should be removed from the defect area. Removal of
the enzyme solution may be effected by using an aspirator equipped
with a fine suction tip followed by sponging with cottonoid.
Alternatively, the enzyme solution may be removed by sponging up
with cottonoid alone.
[0064] Following removal of the enzyme solution, the defect should
be rinsed thoroughly, preferably three times, with sterile
physiologic saline (e.g., 0.15 M NaCl). The rinsed defect site
should then be dried. Sterile gauze or cottonoid may be used to dry
the defect site.
[0065] For intraoperative synovial membrane replantation or
implantation, proteoglycan degrading enzymes must be washed out
extensively in order to remove any proteases and thus prevent the
inactivation of added growth factors.
[0066] Alternatively, or in addition to the enzyme treatment step,
the defect site may be dressed with a compound, such as fibrin glue
or transglutaminase, to enhance adhesion of the matrix to the
defect site. In a preferred embodiment, fibrin glue or
transglutaminase is applied to the defect site after the defect
site has been rinsed and dried following enzyme treatment. Fibrin
glue promotes chemical bonding (cross-linking) of the fibrils of
the matrix to the cartilage collagen fibrils on the defect surface
[see Gibble et al., Transfusion, 30(8), pp. 741-47 (1990)]. The
enzyme transglutaminase may be used to the same effect [see e.g.,
Ichinose et al., J. Biol. Chem., 265(23), pp. 13411-14 (1990);
"Transglutaminase," Eds: V. A. Najjar and L. Lorand, Martinus
Nijhoff Publishers (Boston, 1984)]. Suturing, cauterization or
compounds other than fibrin glue or transglutaminase that can
promote adhesion of extracellular materials may also be used.
[0067] A patient suffering from the defect is anesthetized locally
in the joint containing the defect or given a general anesthetic,
and appropriate surgical tools (e.g., a biopsy set, scalpel or
arthroscope) are used to extract an appropriate amount of synovial
membrane and/or synovial fluid from the joint. In the knee joint,
synovial membrane material is preferably removed from the distal
recessus near or from the fat pad in the knee joint or in the
suprapatellar recessus. Synovial membrane tissue including fat pad
tissue may also be used when additional tissue bulk is useful to
completely fill the defect. Generally, synovial membrane should be
removed from areas lying in reserve folds and not directly opposing
articular cartilage surfaces.
[0068] Use of Synovial Membrane Tissue
[0069] Synovial membrane tissue obtained may be used in a rapid
intraoperative synovial membrane implantation procedure. Synovial
membrane tissue may optionally be partially devitalized with a
single freeze-thaw cycle and then placed within the defect. A
matrix containing a proliferation agent and/or transforming factor
may optionally be used together with the synovial membrane tissue
to fill the defect.
[0070] In one embodiment of the present invention, synovial
membrane tissue is directly implanted into the defect site. In
order to prevent loss of the implanted synovial membrane tissue
into the joint space, the surface of the defect site may be covered
with a thin biodegradable covering membrane. This covering membrane
may be artificial or natural (e.g., synovial membrane, as described
below). The covering membrane is sealed to the edges of the defect
site with sutures, fibrin glue, tissue transglutaminase,
cauterization or the like. If sutures are used in conjunction with
a synovial membrane, the sutures may be impregnated with a
transforming factor such as BMP-2 at an appropriate concentration
to promote differentiation of the synovial cells in the membrane
into chondrocytes.
[0071] In another embodiment of the present invention, synovial
membranes are removed from recessi and cut up into very small
pieces (i.e., minced into small bits) for transplantation. Minced
synovial membranes may be subjected to brief collagenase digestion
and trypsin digestion (30-45 minutes) followed by a rapid
purification in the operating room. Alternatively, cambium tissue
may be obtained and minced by physical scraping and/or gentle
proteolytic digestion of periosteum or perichondrium tissue. The
minced synovial membranes or cambium tissue are mixed into a matrix
and transplanted within the matrix into the defect space. It is
preferred that the matrix contains a chemotactic/proliferation
agent in order to populate the whole matrix with cells and a
transforming factor with a controlled-release system in order to
induce cartilage tissue transformation within this matrix. The
whole matrix may be covered with a synovial tissue covering
membrane or other membrane to retain the matrix within the defect
space.
[0072] In another embodiment of the present invention, one or more
layers of excised synovial membrane sheets are stacked one on top
of the other until the defect space is filled. The edges of the top
layer and, optionally, the corners of the lower layers, may be
sutured or glued to the defect edges to retain the whole stack in
situ. Between the sheets, transforming growth factors are deposited
in order to induce cartilage transformation of the synovial
membrane sheets, preferably using a controlled-release delivery
system as described above. This may be done by depositing, between
the sheets, a thin matrix layer (e.g., fibrin or collagen)
impregnated with a transforming factor to induce the tissue to
transform into cartilage. Alternatively, transforming factors
contained in microsomes, microspheres, nanospheres or liposomes
could be added between the synovial membrane sheets without a
matrix. Another embodiment is to place an emulsion containing such
factors between the sheets. Alternatively, the synovial membrane
sheets may be soaked in a transforming growth factor-containing
solution before replantation.
[0073] The minced synovial membrane, minced cambium tissue and
synovial membrane sheets of the above embodiments may also be
partially devitalized through a single freeze-thaw cycle in order
to reduce cell density and reduce the presence of unwanted cells.
Alternatively, anti-macrophage antibodies or other substances may
be used to deplete the macrophage population.
[0074] Partially transformed synovial membrane tissue may also be
used to fill a defect space. In a first surgical intervention,
matrix pads, impregnated with transforming growth factors for slow
release, are sutured and attached to recessi of synovial membrane
in a joint, or alternatively attached to synovial tissue
immediately outside the synovial capsule. Eight to fourteen days
after this intervention, the tissue beneath such a matrix pad will
have been transformed into cartilage-like tissue. This is then
removed and stacked one on top of another in the defect site to
fill the defect space with the partially transformed synovial
tissues. Additional transforming factors as well as maintenance
factors known in the art (e.g., IGF I, IGF II and IGF-BP's) may be
added between the layers of such tissue stacks.
[0075] In areas where defects extend to vascularized tissues such
as subchondral bone and subchondral bone marrow spaces (or near
synovial membranes that are heavily vascularized), there is a
possibility that ingrowing blood vessels may contaminate the repair
tissue in the defect. In order to prevent such undesirable vascular
ingrowth, anti-angiogenic factors may be added to the matrix or
deposited between the synovial tissue layers, among the synovial
bits or among the synovial-derived transplanted chondrocytes or
synovial cells.
[0076] Use of Synovial Cells
[0077] The synovial membrane sample obtained as described above is
partially digested such that the individual synovial lining cells
and subsynovial fibroblast-like cells are released from the
membranous tissue. Standard trypsinization may be used or other
means of dissociating the cells from the surrounding tissue. The
individual cells are collected (e.g., via differential
sedimentation through a ficoll density gradient) and cultured in
vitro under standard cell culture conditions ensuring their rapid
proliferation and expansion (a proliferation agent may be added to
more rapidly effect this step). Synovial cell culturing techniques
are known in the art [Taguchi, K. et al., Cell Struct. Funct., 22,
pp. 443-53 (1997), Rodel, J. et al., Exp. Toxicol. Pathol., 48, pp.
243-7 (1996)]. Within a few weeks, the synovial cells will have
expanded to sufficient numbers (e.g., 10,000 to 300,000 cells per
milliliter of solution or matrix), depending on the size of the
defect, to be ready for implantation into the defect site.
[0078] In another embodiment of the present invention, cultured
synovial cells are induced to differentiate in vitro. It has been
shown that mesenchymal stem cells can be induced to differentiate
in vitro [Caplan and Haynesworth, U.S. Pat. No. 5,486,359]. It has
also been shown that synovium-derived mesenchymal-type cells
stimulated in vitro with BMP (bone morphogenetic protein)
differentiate into cartilage [Iwata et al., Clin. Orthop., 296, pp.
295-300 (1993)].
[0079] In this embodiment, synovial cells removed from a patient
are cultured in the presence of TGF-.beta. and/or BMP (preferred
concentrations are provided below) in order to induce
differentiation into chondrocytes. The resulting chondrocytes are
then implanted into the defect site as in the previously described
embodiments, that is, either as a suspension or as part of a matrix
system.
[0080] In an additional embodiment, synovial cells induced to
differentiate into chondrocytes by TGF-.beta. and/or BMP are
allowed to produce cartilage tissue in vitro. Cartilage tissue thus
produced is implanted into the defect site after molding the size
and shape of the cartilage tissue to snugly fit the defect.
Alternatively, the cartilage matrix may be subjected to partial
enzymatic digestion in order to collect cartilage-producing
chondrocytes, which are then implanted as a suspension or mass.
[0081] Use of Synovial Tissue Covering Membranes
[0082] When using synovial membranes as covering membranes, it is
preferred that a partial devitalization step be carried out because
this tissue contains macrophages, which are not ideal for cartilage
tissue formation, as they may lead to the proliferation of
inflammatory cells or produce signal substances that may attract
blood vessels and thus lead to unwanted ossification. Macrophages
may be removed by a single quick-freeze and thaw process that
reduces the number of cells in the synovial covering membrane. This
primarily removes macrophages, while mesenchymal cell numbers,
although decreased, remain adequate for chondrocyte formation and
thus eventual cartilage formation. In addition, the partial
devitalization, by reducing the cell density, will create a more
appropriate physiological cell density for cartilage formation in
the repair tissue. Other methods known in the art for removing
macrophages (e.g., anti-macrophage antibodies) may be employed in
addition or as an alternative.
[0083] Instead of the immediate synovial tissue autotransplantation
step for covering a defect as described above, a pre-transformed
synovial membrane may be used. In this approach, a two-step
surgical method is employed. The first step is an arthroscopic
intervention wherein a matrix pad that is impregnated with a
transforming factor (e.g., BMP-2, TGF-.beta.) is sutured (1) to the
synovial membrane in a recessus, (2) to synovial tissue immediately
outside the synovial space, including the fat pad or (3) to
subsynovial connective tissue, close to the synovial lining cells.
Options (2) and (3) have the advantage that the joint does not need
to be opened. Three to four weeks later, apposing this matrix pad,
the synovial membrane will have become transformed into
cartilage-like tissue. The second step is a surgical intervention
wherein this cartilage-like tissue area is excised and transplanted
into the defect site in order to fill and/or cover the defect. In a
small joint with a very thin articular cartilage layer (e.g., a
finger joint) such a partially transformed synovial membrane may
suffice to fill the whole defect space. Such a partially
transformed synovial membrane induced in the knee joint and
autotransplanted into a small finger joint (or other joint) may be
useful for reconstruction purposes.
[0084] Synovial covering membranes used according to the present
invention as a cover to retain tissue and/or matrix material may be
oriented upside down or in the physiological direction; each way
will be operative. It may be applied as fully vital or partially
devitalized material in order to eliminate specific cell types to
optimize the outcome.
[0085] In one embodiment involving the use of synovial covering
membranes according to the instant invention, a synovial covering
membrane may be used to cover the floor of a full-thickness defect,
or the bottom surface of a microfractured full-thickness or shallow
defect (that just reaches the subchondral bone plate). Such a
covering membrane may function as a structural and functional
barrier in these defects. In full-thickness or shallow defects
containing one or more bleeding points, an osteogenic factor,
optionally with a controlled-release system (e.g., microspheres,
liposomes or contained in a matrix), may be placed at the base of
the bone portion of the defect, and then covered with a first
synovial covering membrane. Optionally, an angiogenic agent may be
placed under the first synovial covering membrane to further
promote bone formation, although factors in the blood will also
promote bone formation. The first synovial covering membrane may be
held in place using sutures, cauterization, glue, other means, or
nothing at all. An anti-angiogenic agent may be used at the
perimeter of the first synovial covering membrane to inhibit
invasion of the cartilage repair space above by blood vessels.
[0086] Above the first synovial covering membrane, i.e., not
adjacent to the bone portion of the defect, chondrogenic agents may
be used with or without a matrix to promote cartilage formation. It
is preferred that stacks of synovial membranes are used to fill the
defect, interleaved with chondrogenic agent. Cell-based therapies
may also be used instead of synovial membrane stacks, with or
without a matrix, to promote cartilage formation. Such cell-based
therapies include the use of synovial cells, chondrocytes, bone
marrow stromal cells, cells transfected with chondrogenic genes or
any chondrogenic cell/matrix system.
[0087] The top of the defect void, after having been filled with
chondrogenic cell/matrix system, is preferably covered by a second
synovial covering membrane that may directly overlay chondrogenic
agents in order to promote its complete transformation into
cartilage tissue and to simultaneously function as a retention
device for the chondrogenic cell/matrix system filling the defect
void.
[0088] The first synovial covering membrane according to the above
embodiment may function as an anchoring device, performing three
repair functions in the full-thickness defect: (1) the side of the
synovial covering membrane facing the bone portion of a
full-thickness defect, or the bleeding points of a shallow defect,
will participate in the formation of bone and will eventually fuse
with newly formed bone over a period of approximately one to four
weeks; (2) the side of the synovial covering membrane facing away
from the bone portion of the defect will participate in the
formation of cartilage and will eventually fuse with newly formed
cartilage; and (3) the first synovial covering membrane itself will
function as a transient barrier to blood vessel upgrowth from the
bone portion or from the fat tissue of the defect and also as a
barrier to unwanted migration and contamination by stem cells of
various origins from bone and fat tissue into the cartilage repair
space of the defect.
[0089] Matrices and Other Repair Materials
[0090] Matrix materials useful in the methods of this invention for
filling or otherwise dressing cartilage defects include fibrinogen
(activated with thrombin to form fibrin in the defect or lesion),
collagen, agarose, gelatin and any other biodegradable material
that forms a matrix with pores sufficiently large to allow synovial
cells or chondrocytes to populate and proliferate within the matrix
and that can be degraded and replaced with cartilage during the
repair process.
[0091] The matrices useful in the methods of this invention may be
preformed or may be formed in situ, for example, by polymerizing
compounds and compositions such as fibrinogen to form a fibrin
matrix. Matrices that may be preformed include collagen (e.g.,
collagen sponges and collagen fleece), chemically modified
collagen, gelatin beads or sponges, a gel-forming substance such as
agarose, and any other gel-forming or composite substance that is
composed of a matrix material that will fill the defect and allow
cultured or native synovial cells or chondrocytes to populate the
matrix, or mixtures of the above.
[0092] In one embodiment of this invention, the matrix is formed
using a solution of fibrinogen and minced synovial tissue, to which
is added thrombin to initiate polymerization shortly before use. A
fibrinogen concentration of 0.5-5 mg/ml of an aqueous buffer
solution may be used. Preferably, a fibrinogen solution of 1 mg/ml
of an aqueous buffer solution is used. Polymerization of this
fibrinogen solution in the defect area yields a matrix with a pore
size sufficiently large (e.g., approximately 50-200 .mu.M) so that
synovial cells or chondrocytes are free to populate the matrix and
proliferate in order to fill the volume of the defect that the
matrix occupies. Preferably, a sufficient amount of thrombin is
added to the fibrinogen solution shortly before application in
order to allow enough time for the surgeon to deposit the material
in the defect area prior to completion of polymerization.
Typically, the thrombin concentration should be such that
polymerization is achieved within a few to several (2-4) minutes
since exposure of cartilage to air for lengthy periods of time has
been shown to cause damage [Mitchell et al., J. Bone Joint Surg.,
71A, pp. 89-95 (1989)]. Excessive amounts of thrombin should not be
used since thrombin has the ability to cleave growth factor
molecules and inactivate them. Thrombin solutions of 10-500 units
per ml, and preferably 100 units per ml, of an aqueous buffer
solution may be prepared for addition to the fibrinogen
solution.
[0093] In a preferred embodiment of this invention, approximately
20 .mu.L of thrombin (100 U/ml) are mixed with each ml of a
fibrinogen solution (1 mg/ml) approximately 200 seconds before
filling the defect. Polymerization will occur more slowly if a
lower concentration of thrombin is added. It will be appreciated
that the amount of thrombin solution needed to achieve fibrin
polymerization within 2-4 minutes can be given only approximately,
since it depends upon the environmental temperature, the
temperature of the thrombin solution, the temperature of the
fibrinogen solution, etc. Alternatively, where convenient, the
thrombin may be added by placing it on top of the matrix solution
after the solution has been placed in the defect site and allowing
it to diffuse through the solution. The polymerization of the
thrombin-activated matrix solution filling the defect is easily
monitored by observing the thrombin-induced polymerization of an
external sample of the fibrinogen solution. Preferably, in the
compositions and methods of this invention, fibrin matrices are
formed from fibrinogen molecules derived from the blood of the same
mammalian species as the species to be treated. Non-immunogenic
fibrinogen from other species may also be used.
[0094] Matrices comprising fibrin and collagen or, more preferably,
fibrin and gelatin may also be used in the compositions and methods
of this invention. Collagenous matrices may also be used in
repairing cartilage defects, including full thickness defects. Note
that for repairing the bone portion of deep full-thickness defects,
the procedures disclosed in U.S. Pat. Nos. 5,270,300 and 5,853,746
may be employed.
[0095] When collagen is used as a matrix material, sufficiently
viscous solutions can be made, e.g., using
Collagen-Vliess.RTM.("fleece"), Spongostan.RTM., or
gelatine-blood-mixtures, and there is no need for a polymerizing
agent. Collagen matrices may also be used with a fibrinogen
solution activated with a polymerizing agent so that a combined
matrix results.
[0096] Polymerizing agents may also be unnecessary when other
biodegradable compounds are used to form the matrix. For example,
Sepharose.RTM. solutions may be chosen that will be liquid matrix
solutions at 39-42.degree. C. and become solid (i.e., gel-like) at
35-38.degree. C. The Sepharose should also be at concentrations
such that the gel filling the defect has a mesh size to allow the
synovial-derived repair cells or chondrocytes to freely populate
the matrix and defect area.
[0097] Use of Transforming Factors and Matrices
[0098] The replanted synovial membrane tissue or synovial cell
suspension, once in the defect site, may partially spontaneously
transform into chondrocytes that ultimately produce mature
cartilage tissue, initially adhering to the surfaces treated with
the proteoglycans-degrading enzyme [Hunziker, E. B. et al., J. Bone
Joint Sure. Br., 80(1), pp. 144-50 (1998)]. Preferably, a suitable
concentration of a transforming factor such as transforming growth
factor .beta. (TGF-.beta.), bone morphogenetic proteins (BMP's)
[Majumdar, M. K., J. Cell. Physiol., 189(3), pp. 275-84 (2001)],
cartilage-derived morphogenetic protein (CDMP) [Luyten, F. P., Acta
Orthop. Scand. Suppl., 266, pp. 51-4 (1995)], Indian hedgehog
protein (IHH protein) [St-Jacques, B. et al., Genes Dev., 13(16),
pp. 2072-86 (1999)], sonic hedgehog protein (SHH protein) [Iwamoto,
M. et al., Crit. Rev. Oral Biol. Med., 10(4), pp. 477-86 (1999)] or
SOX-9 [Kolettas, E. et al., Rheumatology, 40(10), pp. 1146-56
(2001)] may be added to the implanted synovial tissue to induce
homogeneous differentiation into chondrocytes. The transforming
factor is preferably administered in a controlled-release delivery
system. Continued proliferation and cartilage production fill in
the defect site, thereby repairing the defect. As the new cartilage
is formed and densifies, it replaces the biodegradable matrix and
the thin covering membrane dissolves or is integrated into the
repair tissue, leaving behind a repaired lesion. Maintenance
factors known in the art (e.g., IGF I, IGF II and IGF-BP's) may
also be used to stabilize the repair cell population within the
defect.
[0099] Transforming factors useful in the compositions and methods
of this invention to promote cartilage repair include any peptide,
polypeptide, protein or any other compound or composition that
induces differentiation of the synovial-derived repair cells into
chondrocytes that produce cartilage-specific proteoglycans and type
II collagen. Transforming factors may also induce cells to form
bone or other cell types. The ability of a compound or composition
to induce or stimulate production of cartilage-specific
proteoglycans and type II collagen in cells can be determined using
assays known in the art [e.g., Seyedin et al., 1985, supra; Seyedin
et al., 1987, supra]. The transforming factors useful in the
compositions and methods of this invention include, for example,
TGF-.beta.'s, TGF-.alpha.'s, FGF's (acidic or basic) and BMP's,
including BMP-2. These transforming factors may be used singly or
in combination. Dimers and multimers of these factors may also be
used. In addition, TGF-.beta. may be used in combination with
EGF.
[0100] In particular, TGF-.beta.I or TGF-.beta.II or BMP-2 may be
used as the transforming factor. Other TGF-8 forms (e.g.,
TGF-.beta.III, TGF-.beta.IV, TGF-.beta.V, or any member of the
TGF-.beta. superfamily) or polypeptides having TGF-.beta. activity
(see Roberts, 1990, supra) may also be useful for this purpose, as
well as other forms of this substance to be detected in the future,
and other growth factors.
[0101] In a preferred embodiment, a TGF-.beta. concentration is
preferably greater than 200 ng/ml of matrix and, most preferably,
is greater than or equal to 500 ng/ml of solution or matrix.
Alternatively, BMP may be used as a transforming factor at a
preferable concentration of 100-2000 ng per ml. It will be
appreciated that the preferred concentration of TGF-.beta. or BMP
to induce differentiation of synovial cells may vary with the
particular species or individual to be treated.
[0102] The transforming factors in the compositions of this
invention are applied in the defect site within the matrix. Their
presence is thus restricted to a very localized site. This is done
to avoid their free injection or infusion into a joint space. Such
free infusion may produce the adverse effect of stimulating the
cells of the surrounding synovial membrane to produce joint
effusion.
[0103] In a preferred embodiment of this invention, the tissue or
matrix to be implanted contains an anti-angiogenic agent in
addition to TGF-.beta. or BMP as the transforming factor.
[0104] In another embodiment of the present invention, the defect
site is treated with a proteoglycan-degrading enzyme and/or another
adhesion-enhancing compound as described above. The synovial
membrane tissue or cultured synovial cells are first added to a
biodegradable matrix system. The matrix system containing the
synovial membrane tissue or cells in an adequate concentration
(e.g., from 10,000 to 300,000 cells per milliliter of tissue,
solution or matrix) is then implanted into the defect site. As
described above, a thin covering membrane, artificial or natural,
may be sealed over the defect surface to prevent loss of tissue
into the joint space; however, particularly with smaller defects, a
covering membrane may not be necessary, as matrix containing
synovial membrane tissue or cells used to fill the defect will tend
to remain within the defect. Cells within synovial membrane tissue
used to fill the defect will tend to proliferate within the matrix
system.
[0105] In embodiments using the matrix system as discussed above, a
transforming factor, preferably a member of the TGF-.beta.
superfamily, is provided within the matrix system upon
implantation, such that the implanted synovial cells are induced to
differentiate into cartilage-producing chondrocytes. A delivery
system may be employed with the transforming factor to enable the
implanted cells to have continued and prolonged exposure to the
transforming factor. Examples of appropriate delivery systems
include polylactate microspheres and liposomes. A delivery schedule
may be developed based on the specific parameters of the case such
as the size and density of the defect site, concentration of target
cells at a given time and transforming factor used. Such systems
are disclosed in U.S. Pat. No. 5,206,023.
[0106] Additionally, if the defect is large relative to the
concentration of cells within the synovial membrane sample or cell
suspension used to fill the defect, a proliferation agent may be
added to the synovial membrane sample or matrix as a means of
expanding the number of synovial cells filling the defect. The
proliferation agent or agents should be present in an appropriate
concentration range to have a proliferative effect on the synovial
cells within the defect. Preferably, the same agent should also
have a chemotactic effect on the cells (as in the case of
TGF-.beta.); however, a factor having exclusively a proliferative
effect may be used, particularly when a covering membrane is
present to retain tissue and cells in the defect space.
Alternatively, to produce a chemotactic effect on the cells placed
in the defect, followed by induction of cell proliferation, two
different agents may be used, each one having just one of those
specific effects (either chemotactic or proliferative). Such agents
are described in U.S. Pat. No. 5,206,023. Fibronectin or other cell
adhesion promoting factors may also be included in the matrix, as
described in U.S. Pat. No. 5,206,023. Subsequent administration of
a transforming factor as discussed above will induce the cells in
the synovial membrane sample or cell suspension to differentiate
into cartilage-producing chondrocytes. A controlled-release
delivery system as discussed above may be employed to administer
either or both of the proliferation agent and transforming
factor.
[0107] In another embodiment of the present invention, in
particular where the cartilage defect is full-thickness, one or
more anti-angiogenic agents may be added to the synovial tissue,
cell solution or matrix in an appropriate concentration range to
prevent blood vessel growth into the cartilage tissue.
Anti-angiogenic agents that may be used include any agent with
biological activity that prevents ingrowth of blood vessels from
the underlying bone tissue or neighboring synovial membrane into
the cartilage tissue. Such anti-angiogenic agents are described in
U.S. Pat. No. 5,853,746. Some examples of anti-angiogenic agents
that may be useful for this invention are set forth above. The
anti-angiogenic agent should be freely available to provide
immediate activity and may also be present in a sustained-release
form, e.g., associated with a delivery system for prolonged
activity. Such systems are also disclosed in U.S. Pat. No.
5,853,746.
[0108] Alternatively, a membrane is disposed toward the bone tissue
before implantation of the synovial membrane tissue, cells or
matrix, to prevent the ingrowth of blood vessels and perivascular
cells from the underlying bone marrow spaces or other surrounding
tissue, thereby preventing the formation of bone tissue in the
defect site. See U.S. Pat. Nos. 5,270,300 and 5,853,746.
Anti-inflammatory agents may also be added in a controlled-release
delivery system to prevent contamination with inflammatory
cells.
[0109] In the above embodiments involving either in vitro cell
culture or in situ transformation of synovial tissue or cells,
synovial cells may be stably or transiently transfected with one or
more chondrogenic genes. In the case of in vitro cell culture, this
will be done before implantation into the defect, during the in
vitro cell culture process. In the case of in situ transformation,
transfection may also be accomplished in situ, by means known in
the art, such as liposomes or adenoviral vectors.
[0110] One embodiment involving transfected chondrogenic genes is
the use of stacks of synovial membrane tissue interleaved with the
selected transfection vehicle in the defect so as to effect
transfection in situ. Examples of chondrogenic genes include, but
are not limited to, BMP, BMP-2, BMP-9, TGF-.beta., CDMP, IHH, SHH
and SOX-9, and may be transfected according to methods known in the
art, such as electroporation, liposomal delivery and viral vectors,
as appropriate to the circumstance. DNA encoding chondrogenic genes
known in the art may be obtained through PCR amplification of
commercially available DNA libraries using primer sequences
obtained from published DNA sequence of the chondrogenic gene or
genes to be transfected.
[0111] In each of the above embodiments, tissue or matrices may
nonspecifically become mineralized or calcified in the repair
tissue area. Calcification or mineralization inhibitors such as
bisphosphonates may therefore be added to the tissue or matrix to
prevent such unwanted mineralization or calcification and preserve
the hyaline-like character of the repair tissue. It is also
desirable to inhibit differentiation of chondrocytes into
hypertrophic chondrocytes that act to mineralize the tissue repair
area. This may be accomplished by the use of parathyroid hormone
receptor protein or other known active agents.
[0112] In each of the foregoing embodiments, following implantation
of the synovial membrane tissue or cells, and optionally covering
the filled defect with the biodegradable covering membrane, the
joint space is surgically closed in layers.
[0113] The methods of this invention allow for a treatment of
cartilage defects in animals, including humans, that is simple to
administer and is restricted in location to an affected joint area.
The entire treatment may be carried out by arthroscopic, open
surgical or percutaneous procedures.
[0114] In order that the invention described herein may be more
fully understood, the following examples are set forth. It should
be understood that these examples are for illustrative purposes and
are not to be construed as limiting this invention in any
manner.
EXAMPLE I
Use of Synovial Membranes to Cover Articular Cartilage Defects In
Vivo
[0115] In order to test the effectiveness of using a synovial
membrane to cover articular cartilage defects, defects 5 mm wide,
10 mm long and 0.7 mm deep were created with a planing instrument
in mature goats. The new defects were filled with a fibrin matrix
containing free proliferation agent (IGF-1) at a concentration of
40 ng/ml and a liposome-encapsulated transforming growth factor
(BMP-2) at a concentration of 1.0 .mu.g/ml. The defect was then
covered with a synovial membrane that was excised from the joint
wall, of the same dimensions, and sutured to the defect borders by
vycril 7.0 suture material by using single interrupted sutures.
After closure of the joint the animals were kept with the joint
immobilized in a soft cast over 4 weeks (n=6 animals). Following
euthanasia and histological analysis, it was found that the
synovial membrane was well incorporated into the surrounding
cartilage tissue border, and it had also transformed into
cartilage-like tissue. In 3 of the animals, the synovial tissue was
oriented with the synovial lining cells towards the joint cavity
and in 3 of them the lining cells were oriented towards the defect
space. In both groups similar results were obtained (i.e., covering
membrane orientation does not appear to play a significant role in
the methods of this invention).
EXAMPLE II. A
Use of Synovial Bits to Repair Articular Cartilage Defects In
Vivo
[0116] In large articular cartilage defects, the process of
synovial cell migration from the synovium into the articular
cartilage defect to populate the defect with cells that can be
transformed into chondrocytes to repair the cartilage may be too
slow or provide insufficient numbers of cells to achieve complete
filling by cell proliferation and tissue differentiation within the
first few weeks following surgery. To provide a greater number of
sources of cells for repair, synovial membrane material was cut
into small tissue bits and mixed into a fibrin matrix and
deposited, together with a transforming factor, within a defect.
The defect was then covered by a synovial covering membrane, as
described in the Example I above. All aspects of the experiment
were as described above except for the addition of synovial bits to
the fibrin matrix. Upon sacrifice of the animal, numerous areas of
tissue transformation were present, and the number of repair cells
was sufficient for the formation of new cartilage tissue.
EXAMPLE II. B
Partial Devitalization of Synovial Tissue Prior to
Transplantation
[0117] The experiment in II. A. (above) was modified such that the
transplanted synovial covering membrane was frozen a single time
and immediately thawed. This was also done with the synovial tissue
bits in order to reduce the number of macrophages present in the
synovial tissue. By adding this step, a more homogenous
transformation of cartilage tissue was achieved.
EXAMPLE III
Use of Stacks of Synovial Membranes to Repair Articular Cartilage
Defects In Vivo
[0118] The experiment described in Example I. (above) was modified
such that the defect was filled with stacks of synovial membranes
of the approximate dimensions of the defect itself. Prior to
placement in the defect, each of the synovial membranes was soaked
in a BMP-2 solution at a concentration of 4.4 mg/ml, to induce
transformation into cartilage tissue. Additionally, between the
layers of synovial membranes, a small amount of fibrin matrix or
microspheres containing transforming factors (BMP-2, 4.4 mg/ml) was
deposited to allow for the controlled release of the transforming
factor. Macroscopic results showed transformation of the synovial
tissue into cartilage-like tissue.
EXAMPLE IV
Use of Cultured Synovial Cells to Repair Articular Cartilage
Defects In Vivo
[0119] Treatment of defects similar to those described in Example I
(above) can be conducted such that following a surgical
intervention in which an appropriate amount of synovial membrane
and/or synovial fluid is extracted, preferably from the joint to be
repaired, the synovial cells obtained are then cultured in vitro
until a concentration of from 10,000 to 300,000 cells per
milliliter of solution is reached. A proliferation agent can be
used to expedite expansion of the cultured synovial cell
population.
[0120] The cultured synovial cell population is then directly
implanted into the defect site in suspension. Under local
anesthesia, a small arthroscopic or surgical intervention is
performed. The surface of the defect is dried by blotting the area
using sterile absorbent tissue, and the defect volume is filled
with a sterile proteoglycan degrading enzyme solution for a period
of 2-10 minutes to degrade the proteoglycans present on the surface
of the cartilage and, locally, to a depth of approximately 1 to 2
.mu.m from the surface of the defect. The proteoglycan degrading
enzyme is then removed and the defect is rinsed.
[0121] In suspension or a matrix (a preferred concentration being
10,000 to 300,000 cells per milliliter of solution or matrix), the
cultured synovial cells are then implanted into the defect site,
preferably in combination with a transforming factor, such as
TGF-.beta., to promote differentiation of the synovial cells into
cartilage-producing chondrocytes. In order to prevent loss of the
implanted cells into the joint space, the surface of the defect
site is covered with a thin biodegradable covering membrane,
preferably a synovial membrane. The covering membrane is sealed to
the edges of the defect site with sutures, fibrin glue, tissue
transglutaminase, or the like. If sutures are used in conjunction
with a synovial covering membrane, the sutures may be impregnated
with a transforming factor such as BMP-2 at an appropriate
concentration to promote differentiation of the synovial cells in
the covering membrane into chondrocytes.
[0122] Following implantation of the cultured synovial cells and
covering with the biodegradable covering membrane, the joint space
is surgically closed in layers.
EXAMPLE V
Transformation of Synovial Tissue In Situ
[0123] The time course of synovial tissue transformation by BMP-2
in situ was examined in adult rabbits. The study consisted of five
groups of five rabbits in each group. A collagenous matrix
containing BMP-2-loaded liposomes at a concentration of 100 ng/ml
was sutured to synovial membrane in the knee joint of each animal.
Following implantation of the collagenous matrix, the joint space
was surgically closed in layers.
[0124] Joint histology was examined at day 0, day 6, day 8, day 12
and day 14. Whole joints were fixed and embedded in plastic, after
which 1.5 mm serial sections were made. In this way it was possible
to observe changes in tissue composition over time. Four categories
of tissue were identifiable: (1) normal synovial tissue; (2) new
connective tissue; (3) new cartilage; and (4) new bone (with bone
marrow).
[0125] On day 6, only normal synovial tissue and new connective
tissue was observed. On day 8, cartilaginous foci appeared. On day
10, the new cartilage had acquired more mass. On day 12, bone foci
appeared, and on day 14 a substantial amount of bone was seen in
addition to the cartilage. These results indicate that synovial
tissue in the knee joint contains adult stem cells that are capable
of transformation into cartilage and bone under the proper
conditions.
* * * * *