Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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METHOD FOR IN SITU REPAIR OF INJURED, DAMAGED, DISEASED
OR AGED ARTICULAR CARTILAGE USING NEO-CARTILAGE
CONSTRUCTS AND A METHOD FOR PREPARATION THEREOF
BACKGROUND OF THE INVENTION
Field of Invention
The current invention concerns a method for
treatment of injured, damaged, diseased or aged articular
cartilage using neo-cartilage constructs implanted into
a joint cartilage lesion in situ. The method is
particularly useful for repair and restoration of
function of the injured, traumatized, aged or diseased
cartilage. In particular, the invention concerns a
method where the implantation of the construct of the
invention initiates and achieves incorporation of neo-
cartilage into a native surrounding cartilage including
a formation of a new superficial cartilage layer
overgrowing and eventually completely sealing the lesion
in the joint cartilage. The neo-cartilage construct of
the invention comprises at least chondrocytes
incorporated into a support matrix processed according to
the algorithm of the invention. The construct is
implanted into the joint cartilage lesion typically below
one layer or between two layers of biologically
acceptable sealants. The method for the treatment of
articular cartilage comprises preparation of the neo-
cartilage construct from the autologous or heterologous
chondrocytes ex vivo, preparing the lesion for
implantation of said construct including an optional step
of depositing a first sealant compound at the bottom of
the lesion for sealing the joint cartilage lesion and
protecting the construct from effects of blood-borne
agents, implanting the construct of the invention on the
top of the first sealant and depositing a second sealant
compound over the construct.
The invention further concerns a method for repair
and restoration of the injured, damaged, diseased or aged
cartilage into its full functionality and for treatment
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of injured or diseased cartilage by implanting the neo-
cartilage construct between two layers of biologically
acceptable sealants.
Additionally, the invention concerns a method for
generation of the neo-cartilage and the neo-cartilage
construct of the invention.
BACKGROUND AND RELATED DISCLOSURES
Damage to the articular cartilage which occurs in
active individuals and older generation adults as a
result of either acute or repetitive traumatic injury or
aging is quite common. Such damaged cartilage leads to
pain, affects mobility and results in debilitating
disability.
Typical treatment choices, depending on lesion and
symptom severity, are rest and other conservative
treatments, minor arthroscooic surgery to clean up and
smooth the surface of the damaged cartilage area, and
other surgical procedures such as microfracture,
drilling, and abrasion. All of these may provide
symptomatic relief, but the benefit is usually only
temporary, especially if the person's pre-injury activity
level is maintained. For example, severe and chronic
forms of knee joint cartilage damage can lead to greater
deterioration of the joint cartilage and may eventually
lead to a total knee joint replacement. Approximately
200,000 total knee replacement operations are performed
annually. The artificial joint generally lasts only 10
to 15 years and the operation is, therefore, typically
not recommended for people under the age of fifty.
It would, therefore, be extremely advantageous to
have available a method for in situ treatment of these
injuries which would effectively restore the cartilage to
its pre-injury state.
Attempts to provide means and methods for repair of
articular cartilage are disclosed, for example, in U.S.
patents 5,723,331; 5,786,217; 6,150,163; 6,294,202;
6,322,563 and in the U.S. patent application No.
2002/0082220 filed on June 29, 2001.
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U.S. patent 5,723,331 describes methods and
compositions for preparation of synthetic cartilage for
the repair of articular cartilage using ex vivo
proliferated denuded chondrogenic cells seeded ex vivo,
in the wells containing adhesive surface. These cells
redifferentiate and begin to secrete cartilage-specific
extracellular matrix thereby providing an unlimited
amount of synthetic cartilage for surgical delivery to a
site of the articular defect.
U.S. patent 5,786,217 describes methods for
preparing a multi-cell layered synthetic cartilage patch
prepared essentially by the same method as described in
'331 patent except that the denuded cells are non-
differentiated, and culturing these cells for a time
necessary for these cells to differentiate and form a
multi cell-layered synthetic cartilage.
U.S. application Ser. No. 09/896,912, filed on June
29, 2001 concerns a method for repairing cartilage,
meniscus, ligament, tendon, bone, skin, cornea,
periodontal tissues, abscesses, resected tumors and
ulcers by introducing into tissue a temperature dependent
polymer gel in conjunction with at least one blood
component which adheres to the tissue and promotes
support for cell proliferation for repairing the tissue.
None of the above cited references results in repair
and regeneration of cartilage in situ including de novo
formation of the superficial cartilage layer sealing a
joint cartilage lesion in situ.
It is thus a primary objective of this invention to
provide a method and means for regeneration of injured or
traumatized cartilage by forming, in the injured lesion
of the cartilage, a cavity, by administering at least one
but typically two separate layers of a biologically
acceptable glue sealant and implanting a neo-cartilage
containing construct under the one layer or into said
cavity. The method according to the invention results in
the growth of the superficial cartilage layer over the
lesion and sealing the lesion.
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SUMMARY
One aspect of the current invention is a method for
repair and restoration of damaged, injured, diseased or
aced cartilage to a functional cartilage, said method
comprising steps:
a) preparing a neo-cartilage construct comprising
autologous or heterologous chondrocytes incorporated into
a sponge, Porous scaffold or thermo-reversible gelation
hydrogel (TRGH) matrix support and subjected to the
algorithm of the invention;
b) optionally introducing a first layer of a first
biologically acceptable sealant into a cartilage lesion;
c) implanting said construct into said lesion or
into said cavity over the first layer of said first
sealant;
d) introducing a second layer of a second
biologically acceptable sealant over said construct
wherein said second sealant may or may not be the same as
the first sealant and wherein a combination of said
construct and said second sealant results in formation
and growth of a superficial cartilage layer sealing the
cartilage lesion in situ.
Another aspect of the current invention is a method
for repair and restoration of damaged, injured, diseased
or aged cartilage to a functional cartilage, said method
comprising steps:
a) obtaining autologous or heterologous
chondrocvtes;
b) culturing said chondrocvtes ex vivo into a neo- =
cartilage, said neo-cartilage comprising autologous or
heterologous chondrocytes incorporated into a sponge or
TRGH matrix support subjected to the algorithm of the
invention;
c) optionally introducing a first layer of a first
biologically acceptable sealant into a cartilage lesion;
d) depositing a space-holding thermo-reversible
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gel (SHTG) or TRGH into a lesion or into a cavity formed
above the first sealant layer thereby permitting
sufficient time for growth and differentiation of ex vivo
cultured neo-cartilage, said space holding thermo-
reversible gel (SHTG) deposited into said cavity as a sol
at temperatures between about 5 to about 25 C, wherein
within said cavity and at the body temperature said SHTG
converts from the fluidic sol into a solid gel and in
this form SHTG holds the space for subsequent
introduction of the neo-cartilage cultured ex vivo, and
provides protection against cell and blood-borne agents
migration into the cavity from the subchondral space and
from the synovial capsule and wherein its presence
further provides a substrate for and promotes in situ
formation of a de novo superficial cartilage layer
covering the cartilage lesion;
e) depositing a second layer of a second
biologically acceptable sealant over the cartilage
lesion;
f) removing said SHTG by cooling said lesion to
change SHTG into sol;
f) depositing said neo-cartilage cultured ex vivo
into the cavity formed between two layers of sealants and
under the de novo formed superficial cartilage layer;
g) removing said SHTG or TRGH from the cavity after
the neo-cartilage integration into a native cartilage
under the formed superficial cartilage layer by cooling
said lesion to from about 5 to about 15 C to convert the
solid gel into fluidic sol and removing said sol or, in
alternative, leaving said SHTG or TRGH to disintegrate
and be removed naturally.
Another aspect of the current invention is a method
for repair and restoration of damaged, injured, diseased
or aged cartilage to a functional cartilage, said method
comprising steps:
a) preparing an intact and discreet piece of neo-
cartilage by culturing autologous or heterologous
chondrocytes ex vivo, suspending said cultured
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chondrocytes in a thermo-reversible gelation hydrogel
(TRGH) and warming said suspension of chondrocytes to
temperature above 30 C in order to convert TRGH into a
solid gel and subjecting the solid gel to the algorithm
of the invention;
b) introducing a first and a second layer of a
first and a second biologically acceptable sealant into
a cartilage lesion;
c) cooling said TRGH/neo-cartilage to 5-15 C to
sol state;
d) depositing said neo-cartilage suspended in the
TRGH into a cavity formed between two layers of sealants
as a sol at temperatures between about 5 to about 25 C
wherein, within said cavity and at the body temperature,
said TRGH converts from the sol state into the solid gel
and in this state provides protection for and enables
integration of deposited neo-cartilage into a native
surrounding cartilage and wherein the presence of TRGH
further provides a substrate and promotes in situ
formation of de novo superficial cartilage layer covering
the cartilage lesion;
e) leaving said TRGH in the lesion until its
disintegration or, in alternative, removing said TRGH
from the cavity as a sol by cooling said lesion to
temperature between 5 and 15 C after the neo-cartilage
integration and formation of superficial cartilage layer;
and
d) evaluating incorporation of said neo-cartilage
into a surrounding native cartilage.
Still another aspect of the current invention is a
method for repair and restoration of damaged, injured,
diseased or aged cartilage to a functional cartilage,
said method comprising steps:
a) preparing neo-cartilage or a neo-cartilage
containing construct comprising autologous cultured
chondrocytes incorporated into a gel or thermo-reversible
gel matrix support ex vivo and subjected to the algorithm
of the invention;
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b) introducing a first layer of a first
biologically acceptable sealant into a cartilage lesion;
C) depositing said constructor said neo-cartilage
over the first layer of the first sealant; and
d) depositing a layer of a second biologically
acceptable sealant either over the neo-cartilage
construct or the neo-cartilage deposited into a cartilage
lesion and covering the lesion with said second sealant,
wherein in time said neo-cartilage is integrated into
the native cartilage and wherein the presence of the neo-
cartilage construct and the second sealant promotes in
situ formation and growth of de novo superficial
cartilage layer covering the cartilage lesion.
Another aspect of the current invention is a neo-
cartilage construct suitable for implantation into a
cartilage lesion in situ.
Yet another aspect of the current invention is a
neo-cartilage construct implanted under one or between
two layers of biologically acceptable sealants within a
cartilage lesion.
Still another aspect of the current invention is a
neo-cartilage construct implanted in situ into a
cartilage lesion between two layers of sealants wherein
a first sealant is deposited at the bottom of a cartilage
lesion and the second sealant is deposited over the
implanted construct on the top of the cartilage lesion
and wherein the second sealant leads to formation and
growth of superficial cartilage layer which seals said
cartilage lesion.Another aspect of the current invention is a method
for fabrication of a three-dimensional neo-cartilage
construct of the invention comprising steps of:
a) preparing a support matrix structure;
b) harvesting a piece of cartilage from a donor for
isolation of chondrocytes;
c) culturing and expanding the chondrocytes;
d) suspending the expanded chondrocytes in a
suspension fluid;
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e) incorporating said suspended chondrocytes into
said matrix; and
f) propagating said chondrocytes into two or three-
dimensional neo-cartilage construct using the algorithm
of the invention.
Still another aspect of the current invention is a
method for generation of an autologous type of neo-
cartilage construct by generating a carrier support for
autologous chondrocytes cultured into neo-cartilage
wherein said support is a biologically acceptable cell-
carrier thermo-reversible polymer gel or a thermo-
reversible gelation hydrogel (CCTG, TRGH or VITROGENe),
wherein said neo-cartilage is suspended within the CCTG
and wherein a resulting CCTG/neo-cartilage or TRGH/neo-
cartilage or a suspension thereof is injected into the
cartilage lesion.
Still another aspect of the current invention is a
method for generation and maintaining integrity of the
lesion cavity for the introduction of neo-cartilage, a
neo-cartilage gel, a flea-cartilage suspension or neo-
cartilage construct from a synovial capsule and for
blocking the migration of subchondral and synovial cells
and cell and blood products into said cavity and for
providing a substrate for a formation of superficial
cartilage layer overgrowing the lesion by introducing a
biologically acceptable space-holding thermo-reversible
gel (SHTG) into a cleaned lesion for a duration of
culturing autologous chondrocytes into neo-cartilage
before introducing said neo-cartilage or neo-cartilage
construct or suspension into the lesion.
Still another aspect of the current invention is a
method for treatment of damaged, injured, diseased or
aged cartilage by utilizing any of the methods listed
above to implant the neo-cartilage construct into the
lesion.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 shows a construct comprising neo-cartilage.
Figure 1A is a schematic drawing of the sponge made of
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so/gel showing the distribution of chondrocytes within
the collagen sponge. Figure 1S is a micrograph of the
actual neo-cartilage construct held in the forceps having
4 mm in diameter and thickness of 1.5 mm. Seeding density
of the construct is 300,000 chondrocytes per 25 1 of
collagen solution (12,000,000 cells/m1).
Figure 2A shows a diagram of hydrostatic pressure
culture system. Figure 23 shows a =SS culture processor
unit.
Figure 3A is a graph representing S-GAG accumulation
in cell constructs subjected to static atmospheric
(control) or cyclic hydrostatic pressure (test). Figure
313 is a photomicrograph of Safranin-O staining for S-GAG
on paraffin sections in 18 days subjected to static
pressure. Figure 3C is a phootom'crog-r.ach of Safranin-O
staining for S-GAG on paraffin sections in cell
constructs subjected to cyclic hydrostatic pressure for
6 days followed by 12 days of static pressure.
Figure 4 illustrates effect of cyclic and constant
hydrostatic pressure on production of S-GAG (Figure 4A)
and DNA (Figure 43).
Figure 5A shows S-GAG accumulation in cell
constructs under continued culture conditions of static
culture (control), medium perfusion (COMPa), cyclic
hydrostatic pressure (Cy-HP) combined with medium
= perfusion (control) and constant hydrostatic pressure
combined with medium perfusion (constant-HP). Figure 52
illustrates DNA content at day 6 and day 18 in cells
constructs submitted to static conditions (control),
medium perfusion only (COMPa), cyclic hydrostatic
pressure (Cy-HP) and constant hydrostatic pressure
(constant-HP).
Figure 6A is a photomicrograph of Safranin-C
staining for S-GAG on paraffin sections in 18 days cell
constructs subjected to static atmospheric pressure.
Figure 62 is a photomicrograph of Safranin-O staining for
S-GAG on paraffin sections in cell constructs subjected
to cyclic hydrostatic pressure for 6 days followed by 12
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days of static pressure. Figure 6C is a photomicrograph
of type II collagen immunohistochemistry on paraffin
sections in 6 days cell constructs subjected to static
atmospheric pressure. Figure 6D is a photomicrograph of
type II collagen immunohistochemistry on paraffin
sections in cell constructs subjected to cyclic
hydrostatic pressure for 6 days.
Figure 7A is a graph illustrating effect of the
medium perfusion flow rate on cell proliferation (DNA
content) by cell constructs subjected to a medium flow
rate of either 0.005 or 0.05 ml/min. Figure 78
illustrates effect of flow rate on production of S-GAG.
Figure 8 shows accumulation detected histologically
by toluidine S-GAG blue staining after 15 days culture
submitted to perfusion (Figure 8A), cyclic hydrostatic
pressure (Figure 88) and constant hydrostatic pressure
(Figure 8C).
Figure 9 illustrates effect of low oxygen tension on
S-GAG production (Figure 9A) and cell proliferation
(Figure 9B).
Figure 10A shows an arthroscopic observation of the
control empty defect site 2 weeks after creating empty
defect. Figure 10B shows an arthroscopic observation of
the porcine neo-culture (Porcine-NeoCartTM) implant site
2 weeks after the implantation.
Figure 11 shows the control lesion without treatment
with porcine neo-cartilage where the proliferation of
fibrocartilage within the defect site is clearly visible
after 4 months. Figure 11A shows a defect site vis-a-vis
subchondral bone with a site of formation of fibro
cartilage. Figure 11B shows a defect site synovium and
synovial migration. Figure 11C shows the defect site and
formation of fibrocartilage.
Figure 12A and 12B shows integration of porcine neo-
cartilage into the lesion within the host's cartilage
after 3 months. Figure 12C shows the regenerated
hyaline-like cartilage in the porcine neo-cartilage
implanted site. Figure 12D shows the integration between
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the porcine neo-cartilage and the host cartilage
laterally and at the subchondral bone.
Figure 13A shows S-GAG production in cell constructs
subjected to cyclic hydrostatic pressure and to
atomospheric pressure (control) with medium perfusion.
Figure 13B shows DNA content in cell constructs
subjected to cyclic and constant hydrostatic pressure
with medium perfusion.
Figure 14 shows histological evaluation of cell
constructs by Safranin-O. Figure I4A shows S-GAG
accumulation at day 0 (initial). Figure 14B shows
accumulation of S-GAG on day 21 in cell constructs
subjected to atmospheric pressure (control). Figure 14C
shows accumulation of S-GAG on day 21 in cell constructs
subjected to 7 days of cyclic hydrostatic pressure (Cy-
HP#1) followed by 14 days of to atmospheric pressure.
Figure 14D shows accumulation of S-GAG on day 21 in cell
constructs subjected to 14 days of cyclic hydrostatic
pressure (Cy-HP#2) followed by 7 days of to atmospheric
pressure. Figure 14E shows accumulation of S-GAG on day
21 in cell constructs subjected to 7 days of constant
hydrostatic pressure (Constant-HP) followed by 14 days of
atmospheric pressure.
As used herein: DEFINITIONS
"Chondrocyte" means a nondividing cartilage cell
which occupies a lacuna within the cartilage matrix.
"Isogenous chondrocytes" means clones of cartilage
cell derived from one cell of division. Isogenous
chondrocytes occur in clusters called isogenous nests.
"Autologous chondrocytes" means chondrocytes
isolated from a donor's own healthy articular cartilage.
"Heterologous chondrocytes" means chondrocytes
derived from a donor of a different species or from a
donor of the same species but not the recipient
individual or a donor tissue that is derived from the
recipient individual but is non-articular cartilage
isolated from a cartilage of the different species.
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"Support matrix" means biologically acceptable sal-
gel or sponge scaffold suitable for seeding expanded
chondrocytes that provides a structural support for
growth and three-dimensional propagation of chondrocytes.
The support matrix is prepared from such materials as
Type I collagen, Type II collagen, Type IV collagen,
gelatin, agarose, cell-contracted collagen containing
proteoglycans, glycosaminoglycans or glycoproteins,
fibronectin, laminin, bioactive peptide growth factors,
cytokines, elastin, fibrin, synthetic polymeric fibers
made of poly-acids such as polylactic, polyglycolic or
polyamino acids, polycaprolactones, polyamino acids,
polypeptide gel, copolymers thereof and combinations
thereof. The gel solution matrix may be a polymeric
thermo-reversible gelling hydrogel. The support matrix
is preferably biocompatible, biodegradable, hydrophilic,
non-reactive, has a neutral charge and be able to have or
has a defined structure.
"Neo-cartilage" means an immature hyaline cartilage
wherein the ratio of extracellular matrix to chondrocytes
is lower than in mature hyaline cartilage.
"Mature hyaline cartilage" means cartilage
consisting of groups of isogenous chondrocytes located
within lacunae cavities which are scattered throughout an
extracellular collagen matrix.
"Autologous Cultured Neo-Cartilage" means a hyaline
neo-cartilage tissue grown ex vivo from chondrocytes
isolated from a donor's own healthy articular cartilage.
"Neo-cartilage construct", "NEOCART'" or "NeoCart'TM"
means a 3-dimensional structural composition comprising
chondrocytes incorporated into a matrix support treated
by or subjected to the algorithm of the invention. Neo-
cartilage construct thus means a discrete piece of
hyaline neo-cartilage formed from cultured chondrocytes
for implantation into lesion of a damaged, aged or
diseased cartilage wherein, after implantation, the neo-
cartilage is integrated into a native cartilage within
the lesion. NeoCart cartilage is manufactured by and is
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proprietary of Histogenics Corporation, Easthampton, MA.
"TESS' means Tissue Engineering Supoort System
which is available as TESS culture processor unit for
culturing of chondrocytes prepared from arthroscopic
biopsy samples. The unit permits changes in hydrostatic
pressure, including cyclic hydrostatic pressure changes
and controls other physical parameters such as
temperature, gas concentration, medium perfusion rate and
such other parameters as may be needed. Relevant
detailed information is found in US patents 6,432,713 B2,
patent 6,607,917, 6,599,734,
PCT JP01/01516, Japanese patent applications
2001-126543 and 2001-261556.
"Sealant" means a biologically acceptable typically
rapid-gelling formulation having a specified range of
adhesive and cohesive properties.
Sealant is thus a biologically accettable rapidly
gelling synthetic compound having adhesive and/or gluing
properties, and is typically a hydrogen such as
derivatized polyethylene glycol (PEG) which is preferably
cross-linked with a collagen compound, typically
alkylated collagen. Examples of suitable sealants are
tetra-hydrosuccinimidyl or tetra-thiol derivatized PEG,
or a combination thereof, commercially available from
Cohesion Technologies, Palo Alto, CA under the trade name
CoSeal'TM, described in J. Biomed. Mater. Res opl.
Biomater., 58:545-555 (2001), or two-part polymer
compositions that rapidly form a matrix where at least
one of the compounds is polymeric, such as, polyamino
acid, polysaccharide, polyalkylene oxide or polyethylene
glycol and two parts are linked through a covalent bond,
as described in US patent 6,312,725S1, and cross-linked PEG with
methyl collagen, such as a cross-linked polyethylene
glycol hydroael with methyl-collagen. The sealant of the
invention typically gels and/or bonds rapidly upon
contact with tissue, particularly with tissue containing
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collagen.
"First sealant" means a biologically acceptable
tissue sealant which is deposited at the bottom of the
lesion.
"Second sealant" means a biologically acceptable
sealant which is deposited above and over the neo-
cartilage construct implanted into a lesion. The second
sealant may or may not be the same as the first sealant
and is preferably a cross-linked polyethylene glycol
hydrogel with methyl-collagen.
"Hydrostatic pressure" means pressure measured above
the atmospheric pressure.
"Cyclic hydrostatic pressure" or "Cy-HP" means the
application of repeated, two or multiplicity periods of
applied hydrostatic pressure within a defined loading
interval which creates a sine wave form of measured
pressure.
"Constant hydrostatic pressure", "constant-HP" or
"CHP" means the application of a non-fluctuating or non-
cyclic pressure load over a period of time.
"Loading" or "loading interval" means a period of
applied cyclic hydrostatic pressure load followed by a
return to atmospheric pressure where no external pressure
is applied.
"Resting phase" means a variable length of time
wherein cells are maintained in culture at atmospheric
pressure after exposure to or culturing under cyclic
hydrostatic pressure.
"De nova" or "de nova formation" means the new
production of cells, such as chondrocytes, fibroblasts,
fibrochondrocytes, tenocytes, osteoblasts and stem cells
capable of differentiation, or tissues such as cartilage
connective tissue, fibrocartilage, tendon, and bone
within a support structure, such as multi-layered system,
scaffold or collagen matrix or formation of superficial
cartilage layer.
"Superficial cartilage layer" means an outermost
layer of cartilage that forms the layer of squamous-like
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flattened superficial zone chondrocytes covering the
layer of the second sealant and overgrowing the lesion.
"Thermo-reversible" means a compound or composition
changing its physical properties such as viscosity and
consistency, from sol to gel, depending on the
temperature. The thermo-reversible composition is
typically completely in a sol (liquid) state at between
about 5 and 15 C and in a gel (solid) state at about 30 C
and above. The gel/sol state in between shows a lesser
or higher degree of viscosity and depends on the
temperature. When the temperature is higher than 15 C,
the sol begins to change into gel and with the
temperature closer to 30-37 the sol becomes more and
more solidified as gel. At lower temperatures, typically
lower than 15 C, the sol has more liquid consistency.
"TRGH" means thermo-reversible gelation hydrogel
material in which the sol-gel transition occurs on the
opposite temperature cycle of agar and gelatin gels.
Consequently, the viscous fluidic phase is in a sol stage
and the solid phase is in a gel stage. TRGH has very
quick sol-gel transformation which requires no cure time
and occurs simply as a function of temperature without
hysteresis. The sol-gel transition temperature can be set
at any temperature in the range from 5 C to 70 C by
molecular design of thermo-reversible gelation polymer
(TGP), a high molecular weight polymer of which less than
5 wt% is enough for hydrogel formation.
"SHTG" means space holding thermo-reversible gel.
"Sol-gel solution" means a colloidal suspension
which, under certain conditions, transitions from a
liquid (sol) to a solid material (gel). The "sol" is a
suspension of aqueous collagen that is transitioned, by
heat treatment, into a gel. " G A G " means
glycosaminoglycan.
"S-GAG" means sulfated glycosaminoglycan.
"MMP" means matrix metalloproteinase, an enzyme
associated with cartilage degeneration in an injured or
diseased joint.
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"DME" means dimethylene blue used for staining of
chondrocytes.
"MPa" means MegaPascal. One MPa is eaual to 145
psi.
"Superficial zone cartilage" means the flattened
outermost laver of chondrocytes covering the
extracellular matrix intermediate zone and deeper zone of
mature articular cartilage in which non-dividing cells
are dispersed.
"Connective tissue" means tissue that protect and
support the body organs, and also tissues that hold
organs together. Examples of such tissues include
mesenchyme, mucous, connective, reticular, elastic,
collagenous, bone, blood, or cartilage tissue such as
hyaline cartilage, fibrocartilage, and elastic cartilage.
"The algorithm" means variable defined conditions,
such as variable pressure or non-pressure conditions,
variable perfusion rate, different medium, different cell
density, different temperature, variable time, different
oxygen and carbon dioxide conditions, etc., to which a
cellular construct of neo-cartilage is subjected in
order to convert it to a mature neo-cartilage construct.
"Adhesive strength" means a peel bond strength
measurement, which can be accomplished by bonding two
plastic tabs with an adhesive formulation. The tabs can
be formed by cutting 1 x 5 cm strips from polystyrene
weighing boats. To the surface of the boat are bonded
(using commercial cyanoacrylate Superglue), sheets of
sausage casing (collagen sheeting, available from. butcher
supply houses). The sausage casing is hydrated in water
or physiological saline for 20 min to one hour and the -
adhesive is applied to a 1 x 1 cm area at one end of the
tab; the adhesive is cured. Then, the free ends of the
tab are each bent and attached to the upper and lower
grips, respectively, of a tensile testing apparatus and
pulled at 10 mm/min strain rate, recording the force in
Newtons to peel. A constant force trace allows estimation
of N/m, or force per width of the strip. A minimum force -
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Per width of 10 N/m is desired; 100N/m or higher is more
desirable.
Alternatively, the same tab can be bonded (a single
tab) over a 1 x 1 cm area to tissue, either dissected or
exposed tissue in a living animal, during surgery. The
free end of the tab is then aripced or attached throuch
a perforation to a hook affixed to a hand-held tensile
test device (Omeaa* DFC451-2 digital force gauge; Omega
Engineering, Stamford, CT) and pulled upward at
acproximately 1 cm/sec. The maximum force required to
detach the tab from the tissue is recorded. The minimum
force desired in such measurements would be 0.1 N to
detach the tab. Forces or 0.2 to 1 N are more desirable.
"Cohesive strength" means the force required to
achieve tensile failure and is (culling in extension);
measured using a tensile test apparatus. The clue or
adhesive can be cured in a "dog-bone"-shaced mold. The
wc1e ends of the formed solid adhesive can then be
affixed, using cyanoacrylate (Superglue) to plastic tabs,
and gripped in the test apparatus. Force at extensional
failure should be at least 0.2 MPa (2 N/cm2) but
preferably 0.8 to 1 MPa or higher.
"Lap shear measurements" means a test of bonding
strength, in which the sealant formulation is applied to
overlapping tabs of tissue, cured, and then the force to
pull the tabs apart is measured. The test reflects
adhesive and cohesive bonding; strong adhesives will
exhibit values of 0.5 up to 4-6 N/cm: of overlap area. DETAILED DESCRIPTION OF
THE INVENTION
This invention is based on finding that when neo-
cartilage or a neo-cartilage construct is deposited into
a lesion of injured, traumatized, aged or diseased
cartilage or under the top sealant or between layers of
a first (bottom) and a second (top) sealant, within time
the neo-cartilage is incorporated into the surrounding
native cartilage and that under these circumstances, the
second top sealant promotes in situ formation of de novo
superficial cartilage layer over the cartilage lesion
* Trade-mark
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wherein the neo-cartilage is implanted.
The invention thus, in its broadest scope, concerns
a method for repair and restoration of damaged, injured,
traumatized or aged cartilage to its full functionality,
and a method for treatment of injuries or diseases caused
by damaged cartilage due to the trauma, injury, disease
or age by implanting a neo-cartilage construct and
further includes a method for preparation of neo-
cartilage from chondrocytes harvested from a donor's
tissue, a method for formation of a support matrix, a
method for fabrication of a neo-cartilage construct and
additionally a method for de novo formation of a
superficial cartilage layer in situ.
Briefly, the invention comprises preparation of neo-
cartilage from harvested autologous or heterologous
chondrocytes, culturing and expansion of chondrocytes,
seeding the chondrocytes within a collagenous or thermo-
reversible gel support matrix and propagating said
chondrocytes in two or three-dimensions. To achieve the
chondrocyte propagation, the seeded support matrix is
subjected to the algorithm of variable conditions, such
as static atmospheric pressure, constant or cyclic
hydrostatic pressure, temperature changes, oxygen and/or
carbon dioxide level changes and changes in perfusion
flow rate of the culture medium in the presence of
various supplements, such as, growth factors, donor's
serum, ascorbic acid, ITS, etc. The chondrocyte-seeded
support matrix treated as above becomes a neo-cartilage
construct (neo-cartilage) suitable for implanting into a
joint cartilage lesion.
The neo-cartilage construct is implanted into the
lesion under a top sealant, or into a cavity formed by
two layers of adhesive sealants. The first layer of the
sealant is deposited at and covers the bottom of the
lesion and its function is to protect the integrity of
said lesion from cell migration and from effects of
various blood and tissue metabolites and also to form a
bottom of the cavity into which the neo-cartilage
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construct is deposited.
In one embodiment, after the neo-cartilage construct
is emplaced into the lesion cavity, the second adhesive
layer is deposited on the top of the neo-cartilage
construct and within several months results in formation
of the superficial cartilage layer completely sealing the
lesion.
In the alternative embodiment, two adhesive layers
may be deposited concurrently with or before the
construct is implanted into the cavity between them. In
such an instance, in the interim, said cavity may be
filed with a space holding thermo-reversible gel (SHTG).
Both sealant layers and the construct or space holding
gel are left within the lesion cavity for a certain
predetermined period of time, typically from one week to
several months, or in case of the space holding gel,
until the neo-cartilage construct is prepared ex vivo and
ready to be implanted. The second layer deposited on the
top and over the lesion promotes formation of a
superficial cartilage layer which covers the lesion on
the outside and eventually overgrows the lesion
completely thereby resulting in complete or almost
complete sealing of the lesion and of the neo-cartilage
construct deposited within said lesion leading to
incorporation of neo-cartilage into a native cartilage
and resulting in healing of the injured or damaged
cartilage. In alternative, the thermo-reversible gel may
serve as an initiator for promotion of formation of the
superficial cartilage layer.
Both the support matrix of the neo-cartilage
construct or the space holding thermo-reversible gel
deposited into the lesion are materials which are
biodegradable and permit and promote formation of the
superficial cartilage layer and integration of the
chondrocytes from the neo-cartilage construct into the
native cartilage within the lesion cavity. Such
integration begins within several weeks or months
following the implanting and may continue for several
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months and involves a growth and maturing of neo-
cartilage into normal cartilage integrated into the
healthy cartilage. The top sealant layer promotes an
overgrowth of the lesion with the superficial cartilage
layer typically in about two-three months when the
sealant is itself degraded.
In the alternative embodiment, the lesion cavity is
filled with a space-holding gel until the outer
superficial cartilage layer is formed at which time the
neo-cartilage construct comprising ex vivo propagated
chondrocytes suspended in a thermo-reversible sol is
introduced at a temperature between 50 and 15 C. After
it is introduced into the lesion as a liquid sol, the
introduced thermo-reversible sol-gel is converted into a
solid gel at body temperatures of 37 C or at the same or
similar temperature as the temperature of the synovial
cavity. The neo-cartilage construct introduced into the
lesion is integrated into the native cartilage
surrounding the cavity and is completely covered with the
superficial cartilage layer.
In the alternative, the neo-cartilage construct is
deposited into a lesion of injured, traumatized, aged or
diseased cartilage over the first (bottom) sealant layer
and the thermo-reversible gel of the neo-cartilage
construct promotes in situ formation of the superficial
membrane without a need to add the second sealant.
The method for treatment of injured, traumatized,
diseased or aged cartilage comprises treating the
injured, traumatized, diseased or aged cartilage with an
implanted neo-cartilage construct prepared by methods
described above and/or by any combination of steps or
components as described.
I. Preparation of Neo-Cartilage Constructs
Preparation of neo-cartilage constructs for
implanting into the cartilage lesion involves harvesting
and culturing chondrocytes, seeding them in the support
matrix and preparation thereof, and propagating the
chondrocytes either ex vivo, in vitro, or in vivo.
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A. Cartilage and Neo-Cartilage
Cartilage is a connective tissue covering joints and
bones. Neo-cartilage is immature cartilage which
eventually, upon deposition into the lesion according to
this invention, is integrated into and acquires
properties of mature cartilage. Differences between the
two types of cartilage is in their maturity. Cartilage
is a mature tissue comprising metabolically active but
non-dividing chondrocytes; neo-cartilage is an immature
cartilage comprising metabolically and genetically
activated chondrocytes which are able to divide and
multiply. This invention utilizes properties of neo-
cartilage in achieving repair and restoration of damaged
cartilage into the full functionality of the healthy
cartilage by enabling the neo-cartilage to be integrated
into the mature cartilage surrounding the lesion and in
this way repair the defect.
a) Cartilage
Cartilage is a connective tissue characterized by
its poor vascularity and a firm consistency. Cartilage
consist of mature non-dividing chondrocytes (cells),
collagen (interstitial matrix of fibers) and a ground
proteoglycan substance (glycoaminoglycans or
mucopolysaccharides). Later two are cumulatively known
as extracellular matrix.
There are three kinds of cartilage, namely hyaline
cartilage, elastic cartilage and fibrocartilage. Hyaline
cartilage found primarily in joints has a frosted glass
appearance with interstitial substance containing fine
type II collagen fibers obscured by proteoglycan.
Elastic cartilage is a cartilage in which, in addition to
the collagen fibers and proteoglycan, the cells are
surrounded by a capsular matrix surrounded by an
interstitial matrix containing elastic fiber network.
The elastic cartilage is found, for example, in the
central portion of the epiglottis. Fibrocartilage
contains Type I collagen fibers and is typically found in
transitional tissues between tendons, ligaments or bones.
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The articular cartilage of the joints, such as the
knee cartilage, is the hyaline cartilage which consists
of approximately 5% of chondrocytes (total volume)
embedded in approximately 95% extracellular matrix (total
volume). The extracellular matrix contains a variety of
macromolecules, including collagen and proteoglycan. The
structure of the hyaline cartilage matrix allows it to
reasonably well absorb shock and withstand shearing and
compression forces. Normal hyaline cartilage has also an
extremely low coefficient of friction at the articular
surface.
Healthy hyaline cartilage has a contiguous
consistency without any lesions, tears, cracks, ruptures,
holes or shredded surface. Due to trauma, injury,
disease such as osteoarthritis, or aging, however, the
contiguous surface of the cartilage is disturbed and the
cartilage surface shows cracks, tears, ruptures, holes or
shredded surface resulting in cartilage lesions. Partly
because hyaline cartilage is avascular, the spontaneous
healing of large defects is not believed to occur in
humans and other mammals and the articular cartilage has
thus only a limited, if any, capacity for repair.
A variety of surgical procedures have been developed
and used in attempts to repair damaged cartilage. These
procedures are performed with the intent of allowing bone
marrow cells to infiltrate the defect and promote its
healing. Generally, these procedures are only partly
successful. More often than not, these procedures result
in formation of a fibrous cartilage tissue
(fibrocartilage) which does fill and repair the cartilage
lesion but, because it is qualitatively different being
made of Type I collagen fibers, it is less durable and
less resilient than the normal articular (hyaline)
cartilage and thus has only a limited ability to
withstand shock and shearing forces than does healthy
hyaline cartilage. Since all diarthroid joints,
particularly knees joints, are constantly subjected to
relatively large loads and shearing forces, replacement
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of the healthy hyaline cartilage with fibrocartilage does
not result in complete tissue repair and functional
recovery.
b) Neo-Cartilage
Neo-cartilage is an immature hyaline cartilage where
the ratio of extracellular matrix to chondrocytes is
lower than in mature hyaline cartilage. Mature hyaline
cartilage has the ratio of the extracelluar matrix to
chondrocytes approximately 95:5. The neo-cartilage has
a lower ratio of the extracelluar matrix to chondrocytes
than mature cartilage and thus comprises more than 5% of
chondrocytes.
In the process of development of this invention, it
was discovered that under the conditions described below,
the older inactive chondrocytes could be activated from
static non-dividing stage to an active stage where they
divide, multiply, promote growth of the extracellular
matrix and develop into new cartilage (neo-cartilage).
The neo-cartilage thus contains chondrocytes which were
rejuvenated and are surrounded by a newly synthesized
extracellular-matrix macromolecules. A process for
activation was found to require certain period of time,
typically from about 1 week to about 3 months and it is
thus preferred that the neo-cartilage be prepared ex vivo
where nutrients needs and mechanical loading are well
defined.
B. Preparation of Neo-Cartilage
Neo-cartilage prepared according to the current
invention is grown ex vivo from chondrocytes isolated
from the mammalian donor's source. In the alternative,
neo-cartilage may also be grown in situ or in vivo under
conditions described below.
Typical donor sources of mammalian chondrocytes are
swine or humans. Neo-cartilage of the invention for human
use is preferably grown from autologous chondrocytes
obtained from the patient during arthroscopy. While it
is preferred that for human use chondrocytes are
autologous, it is to be understood that chondrocytes
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obtained from other mammalian sources are equally
suitable for preparation of neo-cartilage for treatment
of damaged, diseased or aged cartilage. The use of both
autologous and heterologous chondrocytes is intended to
be within the scope of the invention.
a) Isolation of Chondrocvtes
Specific procedures used for isolation of mammalian
chondrocytes generally using swine cartilage as an
example are described in Example 1. The isolation of
human chondrocytes and preparation of autologous human
neo-cartilage is according to procedures described in
Example 2.
Briefly, the donor cartilage is obtained either by
arthroscopic biopsy from the human donor or from a joint
or bone, such as, for example, the femur of the
slaughtered animal and processed according to Example 1
or 2. The cartilage is preferably digested by
collagenase, a strong protease, most preferably Type I
collagenase, in a solution containing preferably about
0.15% of collagenase. The digestion is run for several
hours to several days, preferably for about 18 hours.
In alternative, the extracellular matter can be
digested with proteases or sugar lyases including but not
limited to heparitinase, heparinase, chondroitinase ABC,
chondroitinase B and chondroitinase AC. The lyases are
added in admixture with collagenase or in a sequential
enzyme digestion steps. These lyases promote further
isolation of the chondrocytes from the extracellular
matrix ECM including disruption the glycosaminoglycans of
the pericellular environment such that the chondrocytes
do not receive inhibitory signals that prevent them from
dividing or producing healthy new extracellular matrix.
This finding is especially important for osteoarthritic
chondrocytes which have very slow division rates and
reduced ability to produce extracellular matrix.
This is especially important for osteoarthritic
chondrocytes which have very slow division rates and
reduced ability to produce ECM. US patent 5,916,557
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shows that application of chon.droit inase ABC to
chondrocytes in vitro resulted couterintuitively in the
promotion of new cartilage production.
The ability to free the chondrocytes from all extra-
and pericellular inhibitory material and thereby to
promote cell expansion and differentiation is especially
important in autologous osteoarthritic tissue where the
growth is otherwise slow because these chondrocytes have
reduced ability to produce ECM where neo-cartilage
formation in the TESS processor under pressure is greatly
improved by this early step of the process. Furthermore,
this method of stimulating chondrocyte growth and
differentiation is relatively benign compared to the
application of growth factors or other chemical stimuli
at a later stage of the formation of neo-cartilage, since
the cells are washed free of the enzymes before
culturing.
Expansion of Chondrocytes
The isolated chondrocytes are then expanded by any
method suitable for such purposes such as, for example,
by incubation in a suitable growth medium, for a period
of several days, typically from about 3 to about 30 days,
preferably for 14 days, at about 37 C. Any kind of
culture or incubation apparatus or chamber may be used
for expanding chondrocytes. The expansion of the cells is
preferably associated with the removal of dead
chondrocytes, residual native extracellular matrix and
other cellular debris before the chondrocytes are
selected for culturing and multiplying. Selected
chondrocytes are collected and isolated using
trypsinization process or any other suitable method.
Expanded chondrocytes are then suspended in a
suitable solution and seeded into a support matrix to
form a seeded matrix. The seeded matrix is typically
processed in a tissue processor.
c) Suspension and Seeding of Chondrocytes in the
Support Matrix
Following the expansion, chondrocytes are suspended
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in any suitable solution, preferably collagen containing
solution. For the purposes of this invention such
solution is typically a gel, preferably sol-gel
transitional solution which changes the state of the
solution from liquid sol to solid gel above room
temperature. The most preferred such solution is the
thermo-reversible gelation hydrogel or a thermo-
reversible polymer gel. The thermo-reversible property is
important both for immobilization of the chondrocytes
within the support matrix and for implanting of the neo-
cartilage construct within the cartilage lesion.
One characteristic of the sol-gel is its ability to
be cured or transitioned from a liquid into a solid form.
This property may be advantageously used for solidifying
the suspension of chondrocytes withing the support matrix
for delivery, storing or preservation purposes.
Additionally, these properties of sol-gel also permit
its use as a support matrix by changing its sol-gel
transition by increasing or decreasing temperature, as
described in greater detail below for thermo-reversible
gelation hydrogel, or exposing the sol-gel to various
chemical or physical conditions or ultraviolet radiation.
In one embodiment the expanded chondrocytes are
suspended in a collagenous sol-gel solution before
incorporation (seeding) into the support matrix. The
sol-gel viscosity permits easy mixing of chondrocytes
avoiding need to use shear forces. One example of the
suitable sol-gel solution is the solution substantially
composed of Type I collagen, commercially available under
trade name VITROGEN from Cohesion Corporation, Palo
Alto, CA. VITROGEN is a purified pepsin-solubilized
bovine collagen dissolved in 0.012N HC1. Sterile collagen
for tissue culture may be additionally obtained from
other sources, such as, for example, Collaborative
Biomedical, Bedford, MA, and Gattefosse, SA, St Priest,
France.
When using a VITROGEN solution, the cell density is
approximately 5-10 x 10' cells/ml. However, both the
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density of the cells, the volume for their seeding and
strength of the solution are variables within the
algorithm , and the higher or lower number of
chondrocytes may be suspended in a larger or lower volume
of the suspension solution, depending on the size of the
support matrix and the size of the cartilage lesion.
Seeding of the suspended chondrocytes into the
support matrix is by any means which permit even
distribution of the chondrocytes within said support
matrix. Seeding may be achieved by bringing the
suspension and the support matrix into close contact and
seeding the cells by wicking or suction of the suspension
into the matrix by capillary action, by inserting the
support matrix into the suspension, by using suction,
positive or negative pressure, injection or any other
means which will result in even distribution of the
chondrocytes within said support matrix.
In alternative embodiment, the chondrocytes are
suspended in the thermo-reversible gelation hydrogel or
gel polymer at temperature between 5 and 15 C. At that
temperature, the hydrogel is at a liquid sol stage and
easily permits the chondrocytes to be suspended in the
sol. Once the chondrocytes are evenly distributed within
the sol, the sol is subjected to higher temperature of
about 30-37 C at which temperature, the liquid sol
solidifies into solid gel having evenly distributed
chondrocytes within. The gelling time is from about
several minutes to several hours, typically about 1 hour.
In such an instance, the solidified gel may itself become
and be used as a support matrix or the suspension in sol
state may be loaded into a separate support matrix, such
as a sponge or honeycomb support matrix.
Other means of generating suspending gels, not
necessarily thermo-reversible, are also available and
suitable for use. Polyethylene glycol (PEG) derivatives,
in which one PEG chain contains vinyl sulfone or acrylate
end groups, and the other PEG chain contains free thiol
groups will covalently bond to form thio-ether linkages.
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If one or both partner PEG molecules are branched (three-
or four-armed), the coupling results in a network, or
gel. If the molecular weight of the PEG chains is several
thousand Daltons (500 to 10,000 Daltons along any linear
chain segment), the network will be open, swellable by
water, and compatible with living cells. The coupling
reaction can be accomplished by preparing 5 to 20% (w/v)
solutions of each PEG separately in aqueous buffers or
cell culture media. Chondrocytes can be added to the
thiol-PEG solution. Just prior to incorporation into the
support matrix, the cells plus thiol PEG and the acrylate
or vinyl sulf one PEG are mixed and infused into the
matrix. Gelation will begin spontaneously in 1 to 5
minutes; the rate of gelation can be modulated somewhat
by the concentration of PEG reagent and by pH. The rate
of coupling is faster at pH 7.8 than at pH 6.9. Such gels
are not degradable unless additional ester or labile
linkages are incorporated into the chain. Such PEG
reagents may be purchased from Shearwater Polymers,
Huntsville, AL, USA; or from SunBio, Korea.
In a second alternative, alginate solutions can be
gelled in the presence of calcium ions. This reaction has
been employed for many years to suspend cells in gels or
micro-capsules. Cells can be mixed with a 1-2% (w/v)
solution of alginate in culture media devoid of calcium
or other divalent ions, and infused into the support
matrix. The matrix can then be immersed in a solution
containing calcium chloride, which will diffuse into the
matrix and gel the alginate, trapping and supporting the
cells. Analogous reactions can be accomplished with other
polymers which bear negatively charged carboxyl groups,
such as hyaluronic acid. Viscous solutions of hyaluronic
acid can be used to suspend cells and gelled by diffusion
of ferric ions.Suspension loaded into the support matrix or gelled
into the solid support is processed using the algorithm
of the invention. Such processing is performed in a
processing apparatus, such as a TESS processor.
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C. Preparation of Suipport Matrix
The support matrix for seeding expanded chondrocytes
provides a structural support for growth and three-
dimensional propagation of chondrocytes. Generally, the
support matrix is biologically biocompatible, hydrophilic
and has preferably a neutral charge.
Typically, the support matrix is a two or three-
dimensional structural composition, or a composition able
to be converted into such structure, containing a
plurality of pores dividing the space into a fluidically
connected interstitial network. In some embodiments the
support matrix is a sponge-like structure or honeycomb-
like lattice.
In general, any polymeric material can serve as the
support matrix, provided it is biocompatible with tissue
and possesses the required geometry. Polymers, natural or
synthetic, which can be induced to undergo formation of
fibers or coacervates, can then be freeze-dried as
aqueous dispersions to form sponges. Typically, such
sponges must be stabilized by crosslinking, such as, for
example, ionizing radiation. Practical exmaple includes
preparation of freeze-dried sponges of poly-hydroxyethyl-
methacrylate (pHEMA), optionally having additional
molecules, such as gelatin, entrapped within
advantageously. Such types of sponges can advantageously
function as support matrices for the present invention.
Incorporation of agarose, hyaluronic acid, or other bio-
active polymers can be used to modulate cellular
responses. A wide range of polymers may be suitable for
the fabrication of support matrix sponges, including
agarose, hyaluronic acid, alginic acid, dextrans,
polyHEMA, and poly-vinyl alcohol above or in combination.
Typically, the support matrix is prepared from a
collagenous gel or gel solution containing Type I
collagen, Type II collagen, Type Iv collagen, gelatin,
agarose, cell-contracted collagens containing
proteoglycans, glycosaminoglycans or glycoproteins,
fibronectin, laminin, bioactive peptide growth factors,
ak 02541827 2010-05-12
30
cytokines, elastin, fibrin, synthetic polymeric fibers
made of poly-acids such as polylactic, polyglycotic or
polyamino acids, Polycaprolactones, polyamino acids,
polypeptide gel, copolymers thereof and combinations
thereof. Preferably, the support matrix is a gel
solution, most preferably containing aqueous Type I
collagen or a polymeric, preferably thermo-reversible,
gel matrix.
The gel or gel solution used for preparation of the
support matrix is typically washed with water and
subsequently freeze-dried or lyophilized to yield a
sponge like matrix able to incorporate or wick the
chondrocytes suspension withing the matrix. The cellular
support matrix of the current invention acts like a
sponge when infiltrated with the chondrocyte suspension
wherein the cells are evenly distributed.
One important aspect of the support matrix is the
pore size of the support matrix. Support matrices having
different pore sizes permit faster or slower infiltration
of the chondrocytes into said matrix, faster or slower
growth and propagation of the cells and, ultimately, the
higher or lower density of the cells in the neo-cartilage
construct. Such pore size may be adjusted by varying the
pH of the gel solution, collagen concentration,
lyophilization conditions, etc. Typically, the pore size
of the support matrix is from about 50 to about 500 p,
preferably the pore size is between 100 and 300 p and
most preferably about 200 p.
The support matrix may be Prepared according to
procedures described in Example 3, or by any other
procedure, such as, for example, procedures described in
the U.S. Patent 6,022,744; 5,206,023; 5,656,492;
4,522,753 and 6,080,194.
One preferred type of support matrix is Type-I
collagen support matrix fabricated into a sponge,
commercially available from Koken Company, Ltd., Tokyo,
Japan, under the trade name Honeycomb Sponge.
An exemplary neo-cartilage support matrix made of
* Trade-mark
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collagen and seeded with chondrocytes is seen in Figure
1, wherein Figure lA is a schematic drawing of the sponge
made of sol/gel showing the distribution of chondrocytes
within the collagen sponge. Figure 13 shows a
microphotograph of the actual neo-cartilage construct
(Neo-Cart"") having 4 mm in diameter and thickness of 1.5
mm. The seeding density of this construct is 300,000 -
375,000 chondrocytes per 25 pl of collagen solution
corresponding to about 12,000,000 - 15,000,000 cells/ml.
a) Honeycomb Cellular Support Matrix
In one embodiment of the invention, the support
matrix is a honeycomb-like lattice matrix providing a
cellular support for activated chondrocytes, herein
described as neo-cartilage. The honeycomb-like matrix
supports a growth platform for the neo-cartilage and
permits three-dimensional propagation of the neo-
cartilage.
The honeycomb-like matrix is fabricated from a
polymerous compound, such as collagen, gelatin, Type I
collagen, Type II collagen or any other polymer having a
desirable properties. In the preferred embodiment, the
honeycomb-like matrix is prepared from a solution
comprising Type I collagen.
The pores of the honeycomb-like matrix are evenly
distributed within said matrix to form a sponge-like
structure able to taking in and evenly distributing the
neo-cartilage suspended in a viscous solution.
b) Sol-Gel Cellular Support Matrix
In another embodiment, the support matrix is
fabricated from sol-gel materials wherein said sol-gel
materials can be converted from sol to gel and vice versa
by changing temperature. For these materials the sol-gel
transition occurs on the opposite temperature cycle of
agar and gelatin gels. Thus, in these materials the sol
is converted to a solid gel at a higher temperature. Sol-
gel material is a material which is a viscous sol at
temperatures of below 15 and a solid gel at temperatures
around and above 37 . Typically, these materials change
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their form from sol to gel by transition at temperatures
between about 15 and 37 and are in transitional state
at temperatures between 15 C and 37 . The most preferred
materials are Type I collagen containing gels and a
thermo-reversible gelation hydrogel (TRGH) which has a
rapid gelation point.
In one embodiment, the sol-gel material is
substantially composed of Type I collagen and, in the
form of 99.9% pure pepsin-solubilized bovine dermal
collagen dissolved in 0.012N HC1, is commercially
available under the tradename VITROGEN from Cohesion
Corporation, Palo Alto, CA. One important characteristic
of this sol-gel is its ability to be cured by transition
into a solid gel form wherein said gel cannot be mixed or
poured or otherwise disturbed thereby forming a solid
structure containing immobilized chondrocytes.
Type I collagen sol-gel is generally suitable for
suspending the chondrocytes and for seeding them into a
separately prepared support matrix in the sol form and
gel the sol into the solid gel by heating the support
matrix to a proper temperature, usually around 30-37
and, in this form, processing the seeded support matrix.
This type of sol-gel can also be used as a support matrix
for purposes of processing the gel containing
chondrocytes in the processor of the invention into a
neo-cartilage construct.
In another embodiment, the sol-gel is thermo-
reversible gelation hydrogel (TRGH). Sol-gel thermo-
reversible material for preparation of sol-gel support
matrix is a material which is a viscous sol at
temperatures of below 15 and solid gel at temperatures
above 37 . The primary characteristic of the thermo-
reversible gelation hydrogel (TRGH) is that it gels at
body temperature and sols at lower than 15-25 C
temperature that upon its degradation within the body it
does not leave biologically deleterious material and that
it does not absorb water at gel temperatures. TRGH has
a very quick sol-gel transformation which requires no
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cure time and occurs simply as a function of temperature
without hysteresis. The sol-gel transition temperature
can be set at any temperature in the range from 5 C to
70 C by the molecular design of the thermo-reversible
gelation polymer (TGP), a high molecular weight polymer
of which less than 5 wt% is enough for hydrogel
formation.
The typical TRGH is generally made of blocks of high
molecular weight polymer comprising numerous hydrophobic
domains cross-linked with hydrophilic polymer blocks.
TRGH has low osmotic pressure and is very stable as it is
not dissolved in water when the temperature is maintained
above the sol-gel transition temperature. Hydrophilic
polymer blocks in the hydrogel prevent macroscopic phase
separation and separation of water from hydrogel during
gelation. These properties make it especially suitable
for safe storing and extended shelf-life.
The thermo-reversible gel, particularly a space-
holding thermo-reversible gel (SHTG), should be a
compressively strong and stable at 37 C and below till
about 32 C, that is to about temperature of the synovial
capsule of the joint which is typically below 37 C, but
should easily solubilize below 30-31 C to be able to be
conveniently removed from the cavity as sol. The
compressive strength of the SHTG or TRGH must be able to
resist compression by the normal activity of the joint.
In this regard, the thermo-reversible hydrogel is an
aqueous solution of thermo-reversible gelation polymer
(TGP) which turns into hydrogel upon heating and
liquefies upon cooling. TGP is a block copolymer
composed of temperature responsive polymer (TRP) block,
such as poly(N-isopropylacrylamide) or polypropylene
oxide and of hydrophilic polymer blocks such as
polyethylene oxide.
Thermally reversible hydrogels consisting of co-
polymers of polyethylene oxide and polypropylene oxide
are available from BASF Wyandotte Chemical Corporation
ak 02541827 2010-05-12
34
under the trade name of Pluronics*.
In general, thermo-reversibility is due to the
presence of hydrophobic and hydrophilic groups on the
same polymer chain, such as in the case of collagen and
for copolymers of polyethylene oxide and polypropylene
oxide. When the polymer solution is warmed, hydrophobic
interactions cause chain association and gelation; when
the polymer solution is cooled, the hydrophobic
interaction disappears and the polymer chains are dis-
associated, leading to dissolution of the gel. Any
suitably biocompatible polymer, natural or synthetic,
with such characteristics will exhibit the same
reversible gelling behavior.
This type of thermo-reversible gelation hydrogel is
particularly preferred for preparation of neo-cartilage
constructs for implantation of the construct into the
lesion. In such an instance, the harvested chondrocytes
are suspended in the TRGH sol, then warmed to about 37 C
into the solid gel which thus itself becomes a seeded
support matrix, then submitting said seeded matrix to the
processing in the tissue processor using the algorithm of
the invention, including resting period as described
below, thereby resulting in a formation of neo-cartilage
construct, then submitting said construct to cooling to
change its form into a sol and in this form injecting the
neo-cartilage into the lesion wherein upon warming to
body temperature the sol is immediately converted into
the gel containing neo-cartilage. In time, the delivered
neo-cartilage is integrated into the existing cartilage
and the TRGH is subsequently degraded leaving no
undesirable debris behind.
D. Processing Neo-Cartilage and Tissue Processors
In order to promote three-dimensional growth and
Propagation of chondrocytes and/or neo-cartilage, it is
advantageous and/or necessary in certain instances to
facilitates such growth and propagation by changing
conditions of their growth. Such facilitation may be
initiated either ex vivo, in vitro or in vivo.
* Trade-mark
ak 02541827 2011-07-07
35
This process is, in the current invention, achieved
by subjecting either the suspended expanded chondrocytes
or the support matrix incorPorated with suspended
chondrocytes to certain protocol (the algorithm) of
conditions which were found to promote such propagation.
Such conditions are, for example, application of constant
or cyclic hydrostatic pressure, resting periods,
recirculation and changing flow rate of media, regulation
of oxygen or carbon dioxide concentrations, cell density,
control pH, availability of nutrients and co-factors,
etc. Typically, this process is performed in the
apparatus, preferably in the TESS' tissue processor,
permitting changing of the conditions, as stated above.
a) Neo-Cartilage Tissue Processor
The general design of the tissue processor is the
apparatus for culturing chondrocytes comprising a culture
unit having a culture chamber containing culture medium
and a supply unit for the continuous and intermittent
delivery of the culture medium, a pressure generator for
at:plying atmospheric or constant or cyclic hydrostatic
pressure above the atmospheric pressure to chondrocytes
in the tissue chamber, said generator having means for
changing the pressure, timing, or applying the
atmospheric, constant or cyclic hydrostatic pressure at
predetermined periods and, optionally, a means capable of
delivering and/or absorbing gases such as nitrogen,
carbon dioxide and oxygen. Additionally, the processor
typically comprises a hermetically sealed space including
a heating, cooling and humidifying means.
An exemplary scheme of the tissue processor suitable
for applying hydrostatic pressure, changing flow rate of
the medium and regulating gas concentration delivered to
the seeded support system suitable for purposes of this
invention is seen in Figure 2A. The tissue processor,
seen in Figure 213, known as Tissue Engineering Support
System (TESS) is described in the U.S. patent 6,432,713
issued on August 13, 2002, and also in the U.S.
patent no. 6,607,917.
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b) Biochemical and Histoloaical Testing of
Neo-Cartilaae Constructs
The neo-cartilage constructs are tested for their
metabolic activity, genetic activation and histological
appearance.
Typically, the constructs are harvested at days 6
and 18. For histological evaluation of the immature and
mature cartilage matrix, 4% paraformaldehyde-fixed
paraffin sections are stained with Safranin-O and Type II
collagen antibody. For biochemical analysis, neo-
cartilage constructs are digested in papain at 60 C for
18 hours and DNA is measured using, for example, Hoechst*
3 2 2 5 8 dye method as described in Kun et al., "Fluorometric assay of DNA
in cartilage explants using Hoechst 33258", Anal. Biochem._, 174:168-
176 (1988). The production of alycoaminoalycan (GAG) or
sulfated-glycosaminoglycan (S-GAG) indicating a metabolic
activity of the chondrocyte culture is tested using, for
example, modified dimethylene blue (DMS) microassay
according to Farndale et al., Connective Tissue Research,
9247-248 (1982).
c) Conditions for Prooaaation of Chondroovtes,
Preparation of Neo-Cartilage and Neo-Cartilaae
Constructs
Neo-cartilage construct, as used herein, means a
matrix seeded with chondrocytes and processed according
to the invention.
Neo-cartilage constructs may be produced as 3-
dimensional patches comprising neo-cartilage having an
approximate size of the lesion into which they are
deposited or they may be produced as 3-dimensional sheet
for use in repairs of extensive cartilage injuries. Their .
size and shape is determined by the shape and size of the
support matrix. Their functionality is determined by the
conditions (the algorithm) under which they were
processed.
Conditions for three-dimensional propagation of
chondrocytes in the support matrix into neo-cartilage
construct are variable and are adjusted according to the
* Trade-mark
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intended use and/or function of the neo-cartilage and
depend on the type of used thermo-reversible hydrogel and
on the density of the seeded cells. Thus for production
of small neo-cartilage constructs, the conditions will be
different from those needed for production of large
constructs or for production of extensive neo-cartilage
sheets for partial or total replacement of extensively
damaged or diseased, for example osteoarthritic,
cartilage.
i) Processing Neo-Cartilage under Variable Flow
One aspect of this invention is the discovery that
if the support matrix seeded with chondrocytes is
perfused under varying medium flow rates, the cell
proliferation, measured by increased accumulation of the
extracellular matrix, can be advantageously increased or
decreased. Generally, the lower medium flow rate results
in the higher extracellular matrix accumulation.
Perfusion is an important variable condition for
culturing chondrocytes incorporated into support
matrices. Using a faster perfusion flow rate may slow
down extracellular matrix accumulation affecting growth
and propagation of chondrocytes, as measured by
production of sulfated glycosaminoglycan (S-GAG). A
slower perfusion rate, on the other hand, results in
higher production of S-GAG. These results are important
for controlling the neo-cartilage growth and for, for
example, storage, preservation, transport and shelf-life
of neo-cartilage constructs.
The perfusion flow rate suitable for purposes of
this invention is from about 0.001 to about 0.5 ml/min,
preferably from about of 0.005 to about 0.05 ml/min. At
the medium perfusion rate 0.005 ml/min the accumulation
of extracellular matrix is significantly (p-<0.05)
increased compared to accumulation of extracellular
matrix observed following perfusion at rate 0.05 ml/min.
The optimum flow rate depends upon the total number of
cells in the culture chamber.
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ii) Processing Meo-Cartilage Under Different Types
of Pressure
Subjecting the seeded support matrix to hydrostatic
pressure, in conjunction with a decreased perfusion flow,
is an integral part of the culture processing system
according to this invention. Different types of
hydrostatic pressure have a significant effect on
glycosaminoglycan production and thus on extracellular
matrix accumulation compared to atmospheric pressure
alone. The hydrostatic pressure, particularly cyclic
hydrostatic pressure applied according to this invention
has been found to stimulate chondrocyte proliferation and
metabolism which contributes to extracellular matrix
accumulation.
Hydrostatic pressure suitable for processing
chondrocytes seeded within the support matrix is either
a constant or cyclic hydrostatic pressure, such pressure
being the pressure above the atmospheric pressure. The
cyclic hydrostatic pressure suitable for use in
processing of the seeded support matrix is from about
0.01 to about 10.0 MPa, preferably from about 0.5 to
about 5.0 MPa and most preferably at about 3.0 MPa at
0.01 Hz to about 2.0 Hz, preferably at about 0.5 Hz,
applied for about 1 hour to about 30 days, preferably
about 7 days, with or without resting period. Typically,
the period of hydrostatic pressure is followed by the
resting period, typically from about 1 day to about 30
days, preferably about 16-18 days.
Studies performed in support of this invention
indicate that cell viability is not affected by the
hydrostatic pressure and is maintained with chondrocytes
distributed uniformly within the support matrix.
Following the treatment with hydrostatic pressure,
accumulations of both DNA and S-GAG are significantly
increased compared to cultures not experiencing applied
load, indicating that chondrocyte activation and
metabolic and genetic activity can be controlled by the
culture environment.
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iii) Processin= Neo-Cartila.e Under Reduced Ox .en
Concentration
Another variable in the processing of seeded support
matrices is the concentration of oxygen, carbon dioxide
and nitrogen.
The chondrocytes-seeded support matrix described
above may be further cultured under reduced 02
concentration (i.e. less than 20% saturation) during
formation of neo-cartilage in the TESS processor. The
reduced oxygen concentration of cartilage has been
observed in vivo, and such reduction may be due to its
normal lack of vascularization which produces a lower
oxygen partial pressure, as compared to the adjacent
tissues. In this set of studies, chondrocytes seeded in
support matrix or neo-cartilage were cultured under
oxygen concentration between about 0% and about 20%
saturation or under dioxide concentration about 5%.
E) Determination of Conditions for Optimization of
The Algorithm
The ultimate aim of this invention was to find and
confirm conditions (the algorithm) for preparation of
neo-cartilage constructs for implantation into cartilage
lesions, which in conjunction with deposition of one or
two sealant layers, would lead to healing of the damaged,
injured, diseased or aged cartilage by a) growth of
superficial cartilage layer completely overgrowing and
covering the lesion and protecting implanted neo-
cartilage construct; b) integration of neo-cartilage
implanted into the lesion as the neo-cartilage construct;
and c) subsequent degradation of the construct and
sealant materials.
The underlying studies, described below, show that
a properly designed and optimized culture conditions
utilizing hydrostatic pressure with medium perfusion
followed by constant culture result in fabrication of
neo-cartilage constructs which are integrated into the
native cartilage when implanted under the one layer or in
between two layers of sealants according to the
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invention.
General design for a method for preparation of neo-
cartilage constructs comprises steps:
a) isolation of chondrocytes from a donor tissue;
b) expanding the chondrocytes for about 3-28 days;
c) seeding chondrocytes in a thermo-reversible or
collagen gel or collagen sponge support matrix;
d) subjecting the seeded gel or sponge to constant
or cyclic hydrostatic pressure above
atmospheric pressure (about 0.5-3.0 MPa at 0.5
Hz) with medium perfusion rate of 5 1/min for
several (5-10) days; and
e) subjecting the seeded gel or sponge to resting
period for ten to fourteen days at constant
(atmospheric) pressure.
Neo-cartilage constructs obtained by the above-
outlined conditions and method show that the combined
algorithm of hydrostatic pressure and constant pressure
has advantage over conventional culture methods by
resulting in higher cell proliferation and extracellular
matrix accumulation. Use of thermo-reversible or
collagen gel or collagen sponge support matrix maintains
uniform cell distribution within the support matrix and
also provides support for newly synthesized extracellular
matrix. Obtained 3-dimensional neo-cartilage construct
is easy to handle and manipulate and can be easily and
safely implanted in a surgical setting.
Combination of a period of cyclic hydrostatic
pressure under low medium, perfusion rate followed up with
a period of constant culture (resting period) results in
increased cell proliferation, increased production of
Type II collagen, increased DNA content and increased S-
GAG accumulation.
Increased cell proliferation shows that the
harvested inactive non-dividing chondrocytes have been
activated into neo-cartilage containing active, dividing
and multiplying chondrocytes. Increased level of DNA
shows genetic activation of inactive chondrocytes.
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Increased production of Type II collagen and S-GAG shows
that production of the extracellular matrix has been
activated using the algorithm described above.
Although the optimized algorithm described above is
preferred, it is to be understood that this algorithm may
be advantageously changed using variations of ranges of
cyclic hydrostatic pressure, flow rate, duration of the
pressure and resting period as disclosed above in detail
description of each condition. All variations of all
conditions and combinations thereof are intended to be
within the scope of this invention.
F. Supporting Experimental Studies
In order to test effects of different conditions on
the propagation of chondrocytes within the support matrix
for fabrication of the neo-cartilage construct, studies
combining conditions described above for process
optimization were performed during development of this
invention. Results are
shown in Figures 3-9 and in
Tables 1-3.
For all following studies, the experimental design
was as follows with changes in studies conditions.
Cartilage was harvested under sterile conditions
from the trachea of the swine hind limbs, minced and
digested, as described in Example 7. Chondrocytes were
expanded for 5 days at 37 C and suspended in VITROGEN
(300,000/30 pl). The suspension was absorbed into a
support matrix, usually a collagen sponge (4 mm in
diameter and 2 mm in thickness) as seen in Figure 1,
commercially available from Koken Co., Tokyo, Japan. The
sponges seeded with chondrocytes were pre-incubated for
1 hour at 37 C to gel the collagen, followed by
incubation in culture medium at 37 C, 5% CO, and cultured
in the Tissue Engineering Support System (TESS')
processor seen in Fig. 2.a) Evaluation of Effect of Hydrostatic Pressure
To evaluate the effect of the pressure and/or medium
perfusion rate, the cell seeded sponges were subjected to
medium perfusion at 5 pl/min (0.005 ml/min) or 50 pl/min
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(0.05 ml/min) under the cyclic (Cy-HP) or constant
hydrostatic pressure (constant-HP) of 0.5 MPa at 0.5 Hz
for 6 days in the TESS processor. Resting period under
atmospheric pressure followed for 12 days. Some seeded
sponges served as controls. These were incubated under
atmospheric pressure and without perfusion at 37 C for a
total of 18 days in culture. Sponges harvested 24 hours
after seeding with cells (day 0) served as an initial
control. More detailed conditions are to be found in
Examples and in the following text.
At the end of culture period, the support matrices
were harvested for biochemical and histological analysis.
Sulfated glycosaminoglycan production was measured using
a modified dimethylmethylene blue microassay.
Histological analysis utilized Safranin-O staining.
More detailed conditions are to be found in Examples.
The first study was directed to determination of
effect of constant (atmospheric), cyclic or constant
hydrostatic pressure on production of S-GAG.
At the end of the culture period, both control and
test matrices were harvested for biochemical and
histological analysis. For biochemical analysis,
production of sulfated glycosaminoglycan (S-GAG Agicell
construct) was measured using a modified
dimethylmethylene blue (DMB) and DNA microassays
described in Example 7. Results are seen in Tables 1 and
2 and Figures 3-6.
Results of some studies are seen in Tables 1 and 2
showing a numerical representation of observed increase
in S-GAG production in matrices treated with the
algorithm of the invention.
Table 1
Pressure Conditions
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In TESS In Incubator Total S-GAG Production
Group (3 MPa Cyclic (Atmospheric days in ( g/cell construct)
(n.6) Pressure,@0.5 Hz) Pressure) Culture (Mean+SD)
Initial 0 day 0 12.56 0.99
Control 18 days 18 57.73 6.43
Test 6 days 12 days 18 *76.32 + 4.12
(*: p<0.05, compared to Control)
Table 1 summarizes results obtained from seeded
matrices (n=6) subjected either to atmospheric pressure
in an incubator for 18 days (control) or to processing in
TESS processor under 3 MPa cyclic hydrostatic pressure at
0.5 Hz for 6 days, followed by 12 days in incubator at
atmospheric pressure (test).
As seen in Table 1, S-GAG production (mg/cell
construct) per seeded matrix was significantly increased
to 132% for test compared to 100% control (Figure 3A).
Histological results seen in Figure 3B and 3C (Safranin-O
staining for S-GAG) were consistent with the results
obtained biochemically. Figure 3B is a photomicrograph
of Safranin-O staining for S-GAG on paraffin sections in
18 days subjected to static pressure. Figure 3C is a
photomicrograph of Safranin-O staining for S-GAG on
paraffin sections in cell constructs subjected to cyclic
hydrostatic pressure for 6 days followed by 12 days of
static culture.
As seen in Figure 3B, when the cell constructs are
subjected to static atmospheric pressure (Figure 3B),
there is much lower S-GAG accumulation in the construct
than when it is subjected to a cyclic hydrostatic
pressure for 6 days, followed by 12 days of static
atmospheric pressure (Figure 3C).
To determine the effect of the hydrostatic
pressure on chondrocyte proliferation stimulation and
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matrix accumulation, cartilage was harvested under
sterile conditions as described above. Chondrocytes were
expanded for 5 days at 37 C and suspended in VITRGGEN
(300,000/30 1). The suspension was absorbed into a
honeycomb support matrix or collagen sponge as seen in
Figure 1. The cell constructs were incubated in culture
medium at 37 C, 5% CO, and 20% 02, at 0.5 MPa cyclic
hydrostatic pressure or 0.5 MPa constant hydrostatic
pressure for 6 days followed by incubation for 12 days at
atmospheric pressure in the Tissue Engineering Support
System (TESST") processor seen in Figure 2. The remaining
cell matrices comprising the control group were incubated
at atmospheric pressure for 18 days at 37 C, 5% CO, ans
20%02.
At the end of the culture period, the matrices
were harvested for biochemical analysis. Results are
seen in Table 2. Glycosaminoglycan production was
measures using a modified dimethylmethylene blue (DMB)
microassay. Cell proliferation was measured using a
modified Hoechst Dye DNA assay. Formation of neo-tissue
was evaluated by Safranin-O staining. Results are seen
in Figures, 4A, 4B, 5A and 5B and in Table 2.
Table 2
Group Pressure Conditions S-GAG DNA
(n=7)
In TEss
Days in Total GAG Production
Time Incubator days (g/cell
Type of Days (Atmospheric In construct) DNA Index
Pressure Pressure) culture (Mean t SD) (Control=1)
Control 18 18 59.85 7.69 1
Cy-HP 6
Cyclic 0.5 MPa days 12 18 *91.05t 10.88 1.49
Const-HP 0.5 MPa
Constant 6 12 18 *97.85 5.53 1.74
(*: p<0.05, compared to Control)
All cultures were incubated at 37 C, 5% CO, and 20%
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02.
In TESS culture, the medium flow rate was 0.05 ml/min. Two
cell matrices from each group were harvested for
histological analysis.
As seen in Table 2, the matrices subjected to
conditions listed in groups control, cyclic hydrostatic
pressure (Cy-HP) and constant hydrostatic pressure (const-
HP) resulted in production of 59.85, 91.05 and 97.85
pg/cell construct of S-GAG and 1, 1.49 and 1.74 (control=1)
of DNA Index, respectively. These results clearly show
that neo-cartilage cultured under hydrostatic pressure,
whether cyclic or constant, followed by static culture is
more genetically and metabolically active than one treated
under static atomospheric conditions (controls). These
results are graphically illustrated in Figure 4 which shows
effect of hydrostatic pressure on production of sulfated
glycosaminoglycan (Figure 4A) and DNA content (Figure 4B).
Figure 4A is a graphical representation of results
enumerated in Table 2 and shows the sulfated
glycosaminoglycan production in pg/cell construct wherein
control represents seeded matrices subjected to atmospheric
pressure, Cy-HP represents seeded matrices subjected to
cyclic hydrostatic pressure (0.5 MPa) and constant-HP
represent matrices subjected to constant hydrostatic
pressure (0.5 MPa).
Results seen in Table 2 are illustrated graphically
in Figure 4A, under the conditions described above. There
was significant increase in S-GAG production for both the
cyclic (Cy-HP) and constant hydrostatic pressure (constant-
HP) groups compared to atmospheric pressure (control)
group. Specifically, the production of S-GAG in group
Control was 59.85 pg/cell construct. In group Cy-HP the
production was 91.05 pg/cell construct. In group constant-
HP cell construct production was 97.85 pg/cell construct
resulting in increase of S-GAG production to 152% for group
Cy-HP and by 162% for group constant-HP compared to group
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control.
Figure 43 shows DNA production with corresponding
results presented in Table 2 for DNA, likewise showing
increased production of DNA in constructs processed under
cyclic or constant hydrostatic pressure.
Figure 5A is a graph comparing effect of constant
atmospheric pressure (Control) and zero MPa hydrostatic
pressure (OMPa) serving as pressure controls, 0.5 MPa
cyclic hydrostatic pressure (Cy-HP) and 0.5 MPa constant
hydrostatic pressure (constant-HP) at day 6 and 18 on
support matrices subjected to processing in the TESS
processor. All matrices were incubated at 37 C for 18 days.
The Cy-HP and constant-HP were applied for the first 6 days
followed by 12 days of incubation at atmospheric pressure.
Results seen in Figure 5A show that combination of
Cy-HP or constant-HP with resting period of atmospheric
pressure incubation resulted in significant (p-<0.05)
increase of S-GAG production in the processed matrices
compared to S-GAG production observed in matrices processed
at atmospheric pressure with perfusion only.
Figure 5B shows the Index of DNA content (Initial=1)
in matrices subjected to static (Control), zero hydrostatic
(OMPa), cyclic (Cy-HP) or constant (Constant-HP)
hydrostatic pressure for 6 day and 12 days of atmospheric
pressure culture. Increase in DNA content in matrices
subjected to the algorithm conditions is clearly shown in
both cyclic and constant hydrostatic pressure groups.
Comparison to initial and control DNA level to constant DNA
levels in all three groups subjected to hydrostatic
pressure reveals that the DNA level in constructs subjected
to the cyclic hydrostatic pressure at day 6 then at day 18
and the DNA level constructs subjected to constant
hydrostatic pressure is lower at day 6 than at day 18.
High levels of DNA is observed in matrices submitted to
constant hydrostatic pressure at day 18.
Figures 6A and 6B show histological evaluation of
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matrices by Safranin-0. Figure 6A shows accumulation of S-
GAG on day 18 in matrices subjected to atmospheric _
pressure. Figure 6B shows accumulation of S-GAG in
matrices subjected to 6 days of cyclic hydrostatic pressure
(Cy-HP), followed by 12 days of atmospheric pressure. The
greater S-GAG accumulation in Cy-HP culture matrices is
evident from the increased density of the photomicrograph
clearly visible in the construct. Figure 3C shows
accumulation of Type II collagen in matrices subjected to
the atmospheric pressure or to the cyclic hydrostatic
pressure (Figure 6D). Larger accumulation of Type II
collagen in Figure D is clearly seen.
These results demonstrate that chondrocytes may be
placed in culture to coalesce into a neo-cartilage
construct with accumulated extracellular matrix macro
molecules, such as sulfated glycosaminoglycan (S-GAG).
b) Evaluation of Effect of Perfusion Flow
The second type of study was performed in order to
determine the effect of perfusion flow rate on chondrocyte
proliferation (DNA content) and production of extracellular
matrix (S-GAG accumulation). Results are seen in Table 3
and Figures 7A and 7B.
Figure 7 describes determination of effect of the
perfusion flow rate on cell proliferation measured by level
of DNA (Figure 7A), S-GAG accumulation (Figure 7B) at day
0, 6 and 18.
Figure 7A shows that the lower perfusion rate (5
gl/min) results in higher DNA content index used as a
measure for determination of cell proliferation.
Specifically, the DNA content index compared to the initial
DNA content equal to 1 increased by about 50% to about 1.5
when the culture perfusion rate was 5 p1/min. The higher
perfusion rate (50 gl/min) resulted in much smaller
increase in DNA content index to about 1.2.
Table 3 shows the effect of perfusion flow rate on
the S-GAG production in matrices treated as outlined above
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where the flow rate was either 0.05 ml/min (50 pl/min) or
0.005 ml/min (5 pl/min).
Table 3
Culture duration
Medium In TESS In IncubatorTotal GAG Production
Group Perfusion (0.5 MPa days (Pg/ce11
(n=7) Flow Rate Cyclic (Atmospheric in construct)
(ml/min) Pressure Pressure) culture (Mean SD)
A 0.05 ml/min 6 days 12 days 18 days 78.75 + 6.84
0.005 ml/min 6 days 12 days 18 days 107.33 + 8.53
All cultures were incubated at 37 C, 5% CO2 and 20%
02. In the culture, 0.5 MPa cyclic pressure at 0.5 Hz was
applied to the cell matrices. Two matrices from each group
were harvested for histological analysis.
As seen in Table 3, the lower perfusion rate (5
p1/min) resulted in approximately 1.5 higher production of
S-GAG than the higher perfusion rate (50 pl/min).
These results are seen in graphical form in Figure
7B. Figure 78 is graph showing differences between S-GAG
production by seeded support matrices subjected to a medium
perfusion flow rate of 0.005 ml/min compared to matrices
subjected to a medium perfusion flow rate of 0.05 ml/min at
days 6 and 18. As seen in Figure 7B, increase in S-GAG
production up to 136% (p-<0.05) in matrices subjected to a
slower rate of 0.005 ml/min.
The results summarized in Figures 7A and 78 clearly
show a significant increase in both the DNA index and S-GAG
production in the cell construct at a flow rate of 0.005
ml/min compared to the flow rate 0.05 ml/ml. There is no
significant difference in the amount of S-GAG released into
the medium between the two flow rates. It is therefore
possible to use lower flow rate and avoid shear.
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Determination whether the combination of the
perfusion flow rate with cyclic or constant hydrostatic
pressure leads to increased formation of extracelluar
matter was also studied. Results are seen in Figure 8.
Figure 8 illustrates formation of extracellular matrix
after 15 days culture determined in matrices treated with
perfusion (0.005 ml/min) only (Figure 8A), cyclic
hydrostatic pressure 2.8 MPa at 0.015 Hz (Figure 8B) and
constant hydrostatic pressure 2.8 MPa at 0.015 Hz (Figure
8C) as determined by toluidine blue staining. This figure
clearly shows that hydrostatic pressure and medium
perfusion enhances production of extracellular matrix.
c. Evaluation of Effect of Low Oxvcren Tension
The third type of study was performed in order to
determine the effect of low oxygen tension on chondrocyte
proliferation (DNA content) and production of extracellular
matrix (S-GAG accumulation). Results are seen in Table 4
and Figure 9A and 93.
Table 4
Total UAG Vroductlon
Group Oxygen days (mg/cell
(n=8) concentration in construct)
(%) Culture duration culture (mean SD)
In TESS
(0.5 MPa In Incubator
Cyclic (Atmospheric
Pressure) Pressure)
A 20% 7 days 14 days 21 60.89 + 6.02
days
2% 7 days 14 days 21 *105.59 10.95
days
(*: p<0.05, compared to group A)
All cultures were incubated at 37 C, at 5% CO2. In
TESS culture, the medium flow rate was 0.005 ml/min. Two
cell matrices from each group were harvested for
histological analysis.
As seen in Table 4, the lower oxygen tension (2% 02
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concentration) resulted in approximately 1.7 higher
production of S-GAG than higher oxygen concentration (20%)
corresponding to atmospheric 02 concentration. These
results are seen in graphical form in Figure 9A.
Figure 9A is a graph showing differences between S-
GAG production by cell constructs subjected to 2% oxygen
concentration (Cy-HP) and to cyclic hydrostatic pressure
followed by static culture compared to cell constructs
subjected to 20% oxygen concentration and Cy-HP followed by
static culture. As already seen in Table 4, at 2% oxygen
concentration compared to 20% concentration, the production
of S-GAG rose by approximately 70%.
Figure 9B shows the DNA content index (initial=1) in
cell constructs subjected to 2% or 20% oxygen concentration
with Cy-HP culture followed by static culture. There are
no significant differences in the DNA content index between
2% oxygen concentration and 20% oxygen concentration.
These results indicate that the lower oxygen tension
stimulates S-GAG production in cell constructs when
combined with the cyclic hydrostatic culture followed by
static culture. However, the cell proliferation, expressed
as DNA content index, is not affected by changes in oxygen
tension.
The algorithm of the invention thus comprises
combination of the low perfusion flow rate and low oxygen
tension with a certain predetermined period of hydrostatic
pressure followed by the period of a resting culture
period in the absence of externally applied load.
d) General Applicability of Algorithm to Various
Cell Types
The algorithm described above for chondrocytes is
similarly applicable to other types of cell and tissue,
such as fibroblasts, fibrochondrocytes, tenocytes,
osteoblasts and stem cells capable of differentiation, or
tissues such as cartilage connective tissue,
fibrocartilage, tendon and bone. The algorithm conditions
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may be the same or different but would be generally within
the above described ranges.
II. Neo-Cartilage Composition Construct
The neo-cartilage composition construct is a
multilayered three-dimensional structure comprised at least
of living chondrocytes incorporated into a cellular support
matrix. The support matrix is seeded with living
chondrocytes.
The construct is fabricated in vitro and ex vivo
prior to implanting into the cartilage lesion. The
construct is fabricated using the method and conditions,
cumulatively called the algorithm, described above, with
all conditions being variable within the given ranges and
depending on the intended use or on the method of delivery.
In one embodiment, the autologous or heterologous
chondrocytes are cultured as described, seeded into the
support matrix and processed into the neo-cartilage
construct using predetermined medium perfusion flow rate,
cyclic or constant hydrostatic pressure and reduced or
increased concentration of oxygen and/or carbon dioxide.
The neo-cartilage construct is delivered into the cartilage
lesion cavity and deposited between two layers of sealant
and left in situ to be integrated into the native
cartilage.
I. Method for Formation of Superficial Cartilage
Layer
The primary aspect of this invention is a finding
that when the flea-cartilage, neo-cartilage construct or
seeded support matrix produced according to procedures and
conditions described above is implanted into a cartilage
lesion cavity and covered with a biocompatible adhesive
sealant, the resulting combination leads to a formation of
a superficial cartilage layer completely overgrowing said
lesion.
The method is based on producing a neo-cartilage and
neo-cartilage construct comprising support matrix seeded
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with expanded chondrocytes processed according to the
algorithm of the invention. Chondrocytes are typically
suspended in a collagen sol which is thermo-reversible and
easily changes from sol to gel at the body temperature
thereby permitting external preparation of and delivery of
the neo-cartilage construct into the lesion in form of the
sol which changes its state into gel upon delivery to the
lesion and warming to the body temperature.
The neo-cartilage cohstruct is implanted into the
lesion and covered by a layer of a biologically acceptable
adhesive sealant. Optionally, the first layer of the
sealant is introduced into the lesion and deposited at the
bottom of the lesion. This first sealant's function is to
prevent entry and to block the migration of subchondral and
synovial cells of the extraneous components, such as blood-
borne agents, cell and cell' debris, etc., into the cavity
and their interference with the integration of the neo-
cartilage therein. The second sealant layer is placed over
the surface of the construct. The presence of both these
sealants in combination with the neo-cartilage construct
results in successful integration of the neo-cartilage into
the joint cartilage.
The method may be practiced in several modes and each
mode involves generic steps outlined below in variable
combinations.
General way to practice the method for repair and
restoration of damaged, injured, diseased or aged cartilage
to a functional cartilage is to follow steps:
a) Preparing Neo-Cartilage, Neo-Cartilage Construct
or Chondrocvte SupDort matrix
This step involves preparation of neo-cartilage, neo-
cartilage comprising constructs and support matrix
comprising autologous or heterologous chondrocytes
incorporated therein. Preparation of any of the three
entities named above is described in greater detail above
in sections I.B-D.
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b) Depositing the First and Second Sealant into the
Lesion
This step involves introducing a first and a second
layer of a first and a second biologically acceptable
sealant into a cartilage lesion. The first and second
sealants may be the same or different. It is to be
understood that the utilization of the first bottom layer
is optional and that the method for a formation of the
superficial cartilage layer is enabled without the first
layer.
Specifically, this step involves deposition of the
first sealant at the bottom of the lesion and of the second
sealant over the lesion. The first and the second sealants
can be the same or different, however, both the first and
the second sealants must have certain definite properties
to fulfill their functions.
The first sealant, deposited into the cavity before
the neo-cartilage is deposited, acts as a protector of the
lesion cavity integrity, that is, it protects the lesion
cavity not only from extraneous substances but it also
protect this cavity from formation of the fibrocartilage in
the interim when the cavity is filled with a space-holding
gel in expectation of implantation of the neo-cartilage
after processing. The second sealant acts as a protector
of the lesion cavity on the outside as well as a protector
of the neo-cartilage construct deposited within a cavity
formed between the two sealants and as well as an initiator
of the formation of the superficial cartilage layer.
1. First Sealant
The optionally deposited first sealant forms an
interface between the introduced neo-cartilage construct
and the native cartilage. The first sealant, deposited at
the bottom of the lesion, must be able to protect the
construct from and prevent chondrocyte migration into the
sub-chondral space. Additionally, the first sealant
prevents the infiltration of blood vessels and undesirable
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cells and cell debris into the neo-cartilage construct and
it also prevents formation of the fibrocartilage.
2. Second Sealant
The second sealant acts as a protector of the neo-
cartilage construct or the lesion cavity on the outside and
is typically deposited over the lesion either before or
after the neo-cartilage is deposited therein and in this
way protects the integrity of the lesion cavity from any
undesirable effects of the outside environment, such as
invading cells or degradative agents and seals the space
holding gel in place before the neo-cartilage is deposited
therein. The second sealant also acts as a protector of
the neo-cartilage construct implanted within a cavity
formed between the two sealants. In this way, the second
sealant may be deposited after the neo-cartilage is
deposited over the first sealant and seal the neo-cartilage
within the cavity or it may be deposited over the space
holding gel. The third function of the second sealant is
as an initiator or substrate for the formation of a
superficial cartilage layer. Studies performed during the
development of this invention discovered that when the
second sealant was deposited over the cartilage lesion, a
growth of the superficial cartilage layer occurred as an
extension of the native superficial cartilage layer. This
superficial cartilage layer is particularly well-developed
when the lesion cavity is filled with the space-holding or
thermo-reversible gel thereby leading to the conclusion
that such a gel might provide a substrate for the formation
of such superficial cartilage layer.
3. First and Second Sealant Properties
The first or second sealant of the invention must
possess the following characteristics:
Sealant must be biologically acceptable, easy to use
and possess required adhesive and cohesive properties.
The sealant is biologically compatible with tissue,
be non-toxic, not swell excessively, not be extremely rigid
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or hard, as this could cause abrasion of or extrusion of
the sealant from the tissue site, must not interfere with
the formation of new cartilage, or promote the formation of
other interfering or undesired tissue, such as bone or
5 blood vessels and must resorb and degrade by an acceptable
pathway or be incorporated into the tissue.
The sealant must rapidly gel from a flowable liquid
or paste to a load-bearing gel within 3 to 15 minutes,
preferably within 3-5 min. Longer gelation times are not
10 compatible with surgical time constraints. Additionally,
the overall mode of use should be relatively simple because
complex procedures will not be accepted by surgeons.
Adhesive bonding is required to attach the sealant
formulation to tissue and to seal and support such tissue.
15 Minimal possessing peel strengths of the sealant should be
at least 3N/m and preferably 10 to 30 N/m. Additionally,
the sealant must itself be sufficiently strong so that it
does not break or tear internally, i.e., it must possess
sufficient cohesive strength, measured as tensile strength
20 in the range of 0.2 MPa, but preferably 0.8 to 1.0 MPa.
Alternatively, a lap shear measurement may be given to
define the bond strength of the formulation should have
values of at least 0.5 N/cm2 and preferably 1 to 6 N/cm2.
Sealants possessing the required characteristics are
25 typically polymeric. In the un-cured, or liquid state, such
sealant materials consist of freely flowable polymer chains
which are not cross-linked together, but are neat liquids
or are dissolved in physiologically compatible aqueous
buffers. The polymeric chains also possess side chains or
30 available groups which can, upon the appropriate triggering
step, react with each other to couple, or cross-link the
polymer chains together. If the polymer chains are
branched, i.e., comprising three or more arms on at least
one partner, the coupling reaction leads to the formation
35 of a network which is infinite in molecular weight, i.e.,
a gel.
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The formed gel has cohesive strength dependent on the
number of inter-chain linkages, the length (molecular
weight) of the chains between links, the degree of
Inclusion of solvent in the gel, the presence of
reinforcing agents, and other factors. Typically, networks
in which the molecular weight of chain segments between
junction points (cross-link bonds) is 100-500 Daltons are
tough, strong, and do not swell appreciably. Networks in
which the chain segments are 500-2500 Daltons swell
dramatically in aqueous solvents and become mechanically
weak. In some cases the latter gels can be strengthened by
specific reinforcer molecules; for example, the methylated
collagen reinforces the gels formed from 4-armed PEGs of
10,000 Daltons (2500 Daltons per chain segment).
The gel's adhesive strength permits bonding to
adjacent biological tissue by one or more mechanisms,
including electrostatic, hydrophobic, or covalent bonding.
Adhesion can also occur through mechanical inter-lock, in
which the uncured liquid flows into tissue irregularities
and fissures, then, upon solidification, the gel is
mechanically attached to the tissue surface.
At the time of use, some type of triggering action is
required. For example, it can be the mixing of two
reactive partners, it can be the addition of a reagent to
raise the pH, or it can be the application of heat or light
energy.
Once the sealant is in place, it must be non-toxic to
adjacent tissue, and it must be incorporated into the
tissue and retained permanently, or removed, usually by
hydrolytic or enzymatic degradation. Degradation can occur
internally in the polymer chains, or by degradation of
chain linkages, followed by diffusion and removal of
polymer fragments dissolved in physiological fluids.
Another characteristic of the sealant is the degree
of swelling it undergoes in the tissue environment.
Excessive swelling is undesirable, both because it creates
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pressure and stress locally, and because a swollen sealant
gel loses tensile strength, due to the plasticizing effect
of the imbibed solvent (in this case, the solvent is
physiological fluid). Gel swelling is modulated by the
hydrophobicity of the polymer chains. In some cases it may
be desirable to derivatize the base polymer of the sealant
so that it is less hydrophilic. For example, one function
of methylated collagen containing sealant is presumably to
control swelling of the gel. In another example, the
sealant made from penta-erythritol tetra-thiol and
polyethylene glycol diacrylate can be modified to include
polypropylene glycol diacrylate, which is less hydrophilic
than polyethylene glycol. In a third example, sealants
containing gelatin and starch can also be methylated both
on the gelatin and on the starch, again to decrease
hydrophilicity.
4. Suitable and Non-suitable Sealants
Sealants suitable for purposes of this invention
include the sealants prepared from gelatin and di-aldehyde
starch triggered by mixing aqueous solutions of gelatin
and dialdehyde starch which spontaneously react and gel.
The gel bonds to tissue through a reaction of aldehyde
groups on starch molecules and amino groups on proteins of
tissue, with an adhesive bond strength to up to 100 Nim and
an elastic modulus of 8 x 106 Pa, which is a characteristic
of a relatively tough, strong material. After swelling in
physiological fluids this cohesive strength declines. The
gelled sealant is degraded by enzymes that cleave the
peptide bonds of gelatin and the glycosidic bonds of
starch.
Another acceptable sealant is made from a copolymer
of polyethylene glycol and poly-lactide or -glycolide,
further containing acrylate side chains and gelled by
light, in the presence of some activating molecules. The
linkage is formed by free-radical chemistry. The gel bonds
to tissue by mechanical interlock, having flowed into
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tissue surface irregularities prior to curing. The sealant
degrades from the tissue by hydrolytic cleavage of the
linkage between polyethylene glycol chains, which then
dissolve in physiological fluids and are excreted.
The acceptable sealant made from periodate-oxidized
gelatin remains liquid at acid pH, because free aldehyde
and amino groups on the gelatin cannot react. To trigger
gelation, the oxidized gelatin is mixed with a buffer that
raises the pH, and the solution gels. Bonding to tissue is
through aldehyde groups on the gelatin reacting with amino
groups on tissue. After gelation, the sealant can be
degraded enzymatically, due to cleavage of peptide bonds in
gelatin.
Still another sealant made from a 4-armed
pentaerythritol thiol and a polyethylene glycol diacrylate
is formed when these two neat liquids (not dissolved in
aqueous buffers) are mixed. The rate of gelation is
controlled by the amount of a catalyst, which can be a
quaternary amino compound, such as tri-ethanolamine. A
covalent linkage is formed between the thiol and acrylate,
to form a thio-ether bond. The final gel is firm and swells
very little. The tensile strength of this gel is high,
about 2 MPa, which is comparable to that of cyanoacrylate
acceptable Superglue. Degradation of such gels in vivo is
slow. Therefore, the gel may be encapsulated or
incorporated into tissue.
Another example is the composition, preferred for use
in this invention, that contains 4-armed tetra-succinimidyl
ester or tetra-thiol derivatized PEG, plus methylated
collagen. The reactive PEG reagents in powder form are
mixed with the viscous, fluid methylated collagen
(previously dissolved in water); this viscous solution is
then mixed with a high pH buffer to trigger gelation. The
tensile strength of this cured gel is about 0.3 MPa.
Degradation presumably occurs through hydrolytic cleavage
of ester bonds present in the succinimidyl ester PEG,
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releasing the soluble PEG chains which are excreted.
In general, a sealant useful for the purposes of this
application has adhesive, or peel strengths at least 10N/m
and preferably 100 N/cm; it needs to have tensile strength
in the range of 0.2 MPa to 3 MPa, but preferably 0.8 to 1.0
MPa. In so-called "lap shear" bonding tests, values of 0.5
up to 4-6 N/cm2 are characteristic of strong biological
adhesives.
Such properties can be achieved by a variety of
materials, both natural and synthetic. Examples include:
1) gelatin and di-aldehyde starch (International Patent
Publication Number WO 97/29715; 21 Aug. 1997;); 2) 4-armed
penta-erythritol tetra-thiol and polyethylene glycol
diacrylate (International Patent Application Number WO
00/44808; 3 Aug 2000; example 14); 3) photo-polymerizable
polyethylene glycol-co-poly(a-hydroxy acid) diacrylate
macromers (US Pat No. 5,410,016; Apr. 25, 1995); 4)
periodate-oxidized gelatin (US Pat No. 5,618,551, Apr. 8,
1997); 5) serum albumin and di-functional polyethylene
glycol derivatized with maleimidyl, succinimidyl,
ohthalimidvl and related active groups (International
Patent Publication Number WO 96/03159, Feb 8, 1996) and 6)
4-armed polyethylene glycols derivatized with succinimidyl
ester and thiol, plus methylated collagen, referred to as
"CT3" (US Pat No 6,312, 725B1, Nov. 6, 2001).
Various other sealant formulations are available
commercially or are described in the literature. However,
the majority of these are not suitable for practicing this
invention for a variety of reasons.
For example, fibrin sealant is unsuitable because it
interferes with the formation of cartilage.
Cyanoacrylate, or Superglue, is extremely strong but
it might exhibit toxic reactions in tissue.
Un-reinforced hvdrogels of various types typically
exhibit tensile strengths of lower than 0.02 MPa, which is
too weak to support the adhesion required for the purpose
Trade-mark
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of this application because such gels will swell too much,
Lear too easily, and break down too rapidly.
It is worth noting that it is not the presence or
absence of particular protein or polymer chains, such as
gelatin or polyethylene glycol, which necessarily govern
the mechanical strength and degradation pattern of the
sealant. The mechanical strength and degradation pattern
are controlled by the cross-link density of the final cured
gel, by the types of degradable linkages which are
present, and by the types of modifications and the presence
of reinforcing molecules, which may affect swelling or
internal gel bonding.
5. Preferred Sealants
The first or second sealant of the invention must be
a biologically acceptable, typically rapidly gelling
synthetic compound having adhesive, bonding and/or gluing
properties, and is typically a hydrogel, such as
derivatized polyethylene glycol (PEG) which is preferably
cross-linked with a collagen compound, typically alkylated
collagen. Sealant should have a tensile strength of at
least 0.3 MPa. Examples of suitable sealants are tetra-
hydrosuccinimidyl or tetra-thiol derivatized PEG, or a
combination thereof, commercially available from Cohesion
Technologies, Palo Alto, CA under the trade name CoSearm,
described in Wallace et al., "A tissue sealant based on reactive
multifunctional
polyethylene glycol", J. Biomed. Mater. Res (Aool. Biomater.),
58:545-555 (2001). Other compounds suitable to be used are
the rapid gelling biocompatible polymer compositions
described in the U.S. patent 6,312,725 51.
Additionally, the sealant may
be two or more-part polymers compositions that rapidly form
a matrix where at least one of the compounds is polymer,
such as, polyamino acid, polysaccharide, polyalkylene oxide
or polyethylene glycol and two parts are linked through a
covalent bond and cross-linked PEG with methyl collagen,
commercially available.
The sealant of the invention typically gels rapidly
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upon contact with tissue, particularly with tissue
containing collagen. The second sealant may or may not be
the same as the first sealant. Both the first and the
second is preferably a cross-linked polyethylene glycol
hydrogel with methyl-collagen, which has adhesive
properties.
c) Implanting the Neo-Cartilage Construct
Next step in the method of the invention comprises
implanting said neo-cartilage into a lesion cavity formed
under the second sealant or between two layers of sealants,
said cavity either filled with neo-cartilage construct
deposited therein or, optionally, with a space holding
thermo-reversible gel (SHTG) deposited into said cavity as
a sol at temperatures between about 5 to about 25 C
wherein, within said cavity and at the body temperature,
said SHTG converts the sol into gel and in this form the
SHTG holds the space for introduction of the neo-cartilage
construct and provides protection for the neo-cartilage and
wherein its presence further promotes in situ formation of
de novo superficial cartilage layer covering the cartilage
lesion.
The above step is versatile in that the neo-cartilage
may be deposited into said lesion cavity after the first
sealant is deposited but before the second sealant is
deposited over it or the first and second sealants may be
deposited first and the cavity is filled with the space-
holding thermo-reversible gel for the interim period when
the neo-cartilage is cultured and processed or it may be
deposited into the lesion cavity without the first sealant
and covered with the second sealant.
The neo-cartilage is either autologous or
heterologous and is prepared as described above in sections
I.B. a-c.
a) Removing the Space-Holding or Thermo-Reversible
Gel from the Lesion Cavity
The neo-cartilage is deposited into the cavity either
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before or after the formation of the superficial cartilage
layer. In all cases, when the first sealant is used, the
first sealant is deposited first. In one embodiment, the
neo-cartilage construct containing, typically, the
heterologous neo-cartilage might be deposited on the top of
the first sealant layer and immediately covered by the
second sealant layer. In such an instance, the neo-
cartilage is left in the cavity until the superficial
cartilage layer is formed and the neo-cartilage is
integrated into the surrounding cartilage. Then, depending
on the material used for neo-cartilage construct, the
sponge gel or thermo-reversible gelling hydrogel are left
in the cavity to disintegrate.
In the instance when the two sealants are deposited
first, the space within the lesion cavity is optionally
filled with a polymer gel, such as the space-holding
thermo-reversible gel. Such gel is left in the cavity
until the neo-cartilage construct is cultured, processed
and ready to be implanted. Since such thermo-reversible gel
might or might not be completely or partially degraded
during this time, it may be removed from the cavity by
cooling the lesion to about 5 C at which temperature the
gel becomes a sol, and by removing said sol from the
cavity, for example, by injection. Using the same process,
that is by cooling the solid gel of the neo-cartilage, the
process may be reversed for introduction of the neo-
cartilage construct into said lesion cavity wherein, after
the sol is warmed into the body temperature, the sol is
converted into a solid gel.
Thus, the primary premise of this process is that the
removal and/or introduction of the space holding gel or
introduction of neo-cartilage construct proceeds at the
cold temperature where the composition is in the sol state
and converts into solid gel at warmer temperatures. In
this way the gel may be removed from the cavity as the sol
after the neo-cartilage integration and formation of
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superficial cartilage layer.
e. Generation of the Superficial Cartilage Laver
A combination of the neo-cartilage construct
comprising the neo-cartilage suspended in the thermo-
reversible gel or support matrix seeded with chondrocytes
with the adhesive polymeric second sealant leads to
overgrowth and complete or almost complete sealing of the
lesion cavity.
In alternative, depending on the surface chemistry of
the thermo-reversible gel, the superficial layer could grow
directly over the neo-cartilage construct if such surface
chemistry is propitious to such growth.
Typically, a biologically acceptable second sealant,
preferably a cross-linked PEG hydrogel with methyl collagen
sealant is deposited either over the neo-cartilage
construct implanted into the lesion cavity or is deposited
over the lesion before the neo-cartilage construct is
deposited therein. The second sealant acts as an initiator
for formation of the superficial cartilage layer which in
time completely overgrows the lesion. The superficial
cartilage layer in several weeks or months completely
covers the lesion and permits integration of the neo-
cartilage of the neo-cartilage construct or chondrocytes
seeded within the support matrix into the native
surrounding cartilage substantially without formation of
fibrocartilage.
Formation of the superficial cartilage layer is a
very important aspect of the healing of the cartilage and
its repair and regeneration.
I. In vivo Studies in Swine of Weight-Bearinq
Region of the Knee
The method according to the invention was tested and
confirmed in in vivo studies wherein the generation of the
superficial cartilage layer has been confirmed in a three
month study performed in a swine model in order to evaluate
porcine neo-cartilage construct integration into the
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surrounding cartilage.
The neo-cartilage construct prepared according to the
method of the invention was implanted into an artificially
generated lesion in a pig's knee. Detailed conditions of
the study are described in Example 8. Results of this
study are illustrated in Figures 10, 11 and 12 depicting
histological evaluation using Safranin-O staining method of
artificially created cartilage lesions.
Briefly, the study comprised of an open arthrotomy of
the right knee joint performed on all animals. A biopsy of
the cartilage was obtained. Chondrocytes were isolated
from the cartilage biopsy and cultured within a collagen
matrix in a Tissue Engineering Support System (TESS') as
described in detail above to produce porcine neo-cartilage
construct for subsequent implantation.
A defect was created in the medial femoral condyle of
the right knee. This defect, which served as a control,
was not implanted with the neo-cartilage construct. The
empty defect is seen in Figure 10A. Following surgery, the
joint was immobilized with an external fixation device for
a period of about two weeks. Three weeks after the
arthrotomy on the right knee was performed, an open
arthrotomy was performed on the left knee and the same
defects were created in this medial femoral condyle. The
porcine neo-cartilage was implanted within defects in this
knee which was similarly immobilized. The porcine implant
site is seen in Figure 10B which also show initiation of
formation of a superficial cartilage layer two weeks after
implantation.
The operated sites were periodically viewed via
arthroscopy at monthly intervals. Subsequently,
approximately 3 months after porcine neo-cartilage
implantation, animals were euthanized and joints harvested
and prepared for histological examination. The implanted
sites were prepared and examined histologically. Comparison
of Figure 11 (control at four months after arthrotomy) and
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Figure 12 shows test knee three month following arthrotomy
and neo-cartilage implantation according to the invention.
This figure shows that in the control knee there is a
visible formation of fibrocartilage, in the test group
(Figures 12A-D), the implanted porcine neo-cartilage
construct resulted in production of dense regenerating
hyaline cartilage and in the same test group, there was
clearly visible cell integration (Figures 12C and 12D) and
formation of the superficial cartilage layer (Figures 12A
and 12B).
Figures 11A-11C thus shows the control lesion at 4
months following the surgery without a treatment with the
neo-cartilage construct. Noticeable in Figure 11A is the
proliferation of fibrocartilage within the defect site.
Figure 11 shows the control without porcine-NeoCart'
at 4 months. The proliferation of fibrocartilage in the
defect site is clearly noticeable. Also seen is synovial
tissue that has infiltrated into the subchondral space.
Figures 12A-12D, on the other hand, show that after
3 months post implantation in a weight bearing region of
the knee, the porcine flea-cartilage has produced dense
hyaline-like cartilage and has integrated with the host
cartilage laterally and at the interface of the subchondral
bone.
Figure 12 shows formation of regenerated hyaline-like
cartilage in the implant site. Figure 12B illustrates the
beginning of integration between the porcine flea-cartilage
and the native cartilage laterally and at the subchondral
bone.
The porcine flea-cartilage was delivered to the defect
by implantation of neo-cartilage construct between two
layers of sealant. The newly formed superficial cartilage
layer formed over the defect at three months following the
implantation is clearly visible.
Figure 12 shows and confirms that 3 months after
implantation in a weight-bearing region of the knee, the
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porcine-NeoCartTM has produced dense hyaline-like cartilage
and has integrated with the host cartilage laterally and at
the interface of the subchondral bone.
These results confirm that the damaged, injured,
diseased or aged cartilage may be repaired by using neo-
cartilage implants preparing according to the algorithm of
the invention.
V. Human Osteoarthritic Cartilage
Articular cartilage is a unique tissue with no
vascular, nerve, or lymphatic supply. The lack of vascular
and lymphatic circulation may be one of the reasons why
articular cartilage has such a poor intrinsic capacity to
heal, except for formation of fibrous or fibrocartilaginous
tissue. Unique mechanical functions of articular cartilage
are never reestablished spontaneously after a significant
injury or disease, such as osteoarthritis (OA).
In osteoarthritis, disruption of the structural
integrity of the matrix by the degeneration of individual
matrix proteins leads to reduced mechanical properties and
impaired function.
Currently, the only available treatment of severe
osteoarthritis of the knee is a total knee replacement in
elderly patients. In young and middle aged patients,
however, there is no optimal treatment.
In order to evaluate suitability of the current
invention for treatment of osteoarthritis, studies using
algorithm of the invention including a TESS culture system
using neo-cartilage scaffold construct and algorithm of the
invention (hydrostatic pressure and medium perfusion) on
human OA chondrocytes, cell proliferation and extracellular
matrix accumulation in OA chondrocytes was investigated.
Results are seen in Table 5 and in Figures 13-15.
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Table 5
Group Total S-GAG Production DNA
content
(n='7) days ( g/cell construct) (&g/cell
Pressure Conditions In culture (Mean SD) construct)
(Mean SD)
In TESS In Incubator
I ype ot
Pressure Time
Initial 0 day 0 day 19.23 0.87 1.88
0.40
Control 21 days 21 days 23.81 2.61 2.34
0.32
Cy-HP#1 0.5 MPa 7 days 14 days 21 days *29.53 1/60 2.33
0,12
Cyclic
Cy-HP#2 0.5 MPa 14 days 7 days 21 days *34.39 0.99 2.35
0.09
Cyclic
Const-HP 0.5 MPa 7 days 14 days 21 days 26.94 5.14 **/65
0.28
Constant
(*: p<0.05, compared to Control in S-GAG production)
(**: p<0.05, compared to Initial in DNA content)
In the TESS processor, the medium flow rate was 0.005
ml/min and the hydrostatic pressure as indicated. Two cell
matrices from each group were harvested for histological
analysis.
As seen in Table 5 and Figure 13A, S-GAG production
in cell constructs subjected to cyclic hydrostatic pressure
with medium perfusion was significantly greater than those
subjected to atmospheric pressure (control). Especially,
S-GAG production (g/cell construct) was significantly
increased (144%) for Cy-HP#2 where the cyclic hydrostatic
pressure was used for 14 days followed by 7 days of static
atmospheric pressure compared to control.
Figure 13B shows DNA content index with corresponding
results presented in Table 5 for DNA, likewise showing
increased production of DNA. Increase in DNA content index
in cell constructs using the neo-cartilage construct
subjected to constant hydrostatic pressure was clearly
shown in a comparison to initial level. DNA level in cell
constructs subjected to constant hydrostatic pressure with
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medium perfusion was significantly increased to 142%
compared to initial DNA level index.
Figured 14A-14E show histological evaluation of cell
constructs by Safranin-O. Figure 14A shows S-GAG
accumulation at day 0 (initial). Figure 143 shows
accumulation of S-GAG on day 21 in cell constructs
subjected to atmospheric pressure (control). Figure 14C
shows accumulation of S-GAG on day 21 in cell constructs
subjected to 7 days of cyclic hydrostatic pressure (Cy-
HP#1) followed by 14 days of to atmospheric pressure.
Figure 14D shows accumulation of S-GAG on day 21 in cell
constructs subjected to 14 days of cyclic hydrostatic
pressure (Cy-HP#2) followed by 7 days of to atmospheric
pressure. Figure 14E shows accumulation of S-GAG on day 21
in cell constructs subjected to 7 days of constant
hydrostatic pressure (Constant-HP) followed by 14 days of
atmospheric pressure. The greater S-GAG accumulation in
both cell constructs subjected to cyclic hydrostatic
pressure (7 and 14 days) is evident from the increased
density of the photomicrograph clearly visible in the
Figure 14C and 14D.
These results demonstrate that hydrostatic pressure
with medium perfusion promotes both cell proliferation and
neo-cartilage phenotypic activity, that is, cartilage
extracellular matrix production, in the scaffold neo-
cartilage constructs seeded with human OA chondrocytes.
This evidence indicates that the algorithm of invention
using TESS culture system and hydrostatic pressure combined
with medium perfusion regenerates human OA chondrocytes and
transforms the OA cartilage into the healthy hyaline
cartilage.
VI. Method for Treatment of Cartilage Lesions
The method for treatment of damaged, injured,
diseased or aged cartilage according to the invention is
suitable for healing of small lesion due to acute injury as
well as healing of the large lesions caused by
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osteoarthritis or other joint degenerative diseases and/or
transforming the diseased OA cartilage into the healthy
hyaline cartilage.
The method generally encompasses five novel features,
namely, employing a biologically acceptable thermo-
reversible polymer gel as a carrier support matrix for neo-
cartilage generated from autologous chondrocytes, producing
the autologous neo-cartilage by a process of the invention,
employing a biologically acceptable thermo-reversible gel
as a space-holding means for the interim period when the
autologous neo-cartilage is produced, depositing one or two
adhesive sealants to the lesion and, following depositing
the sealants and implantation of the flea-cartilage within
a cavity generated thereby, a formation of the superficial
cartilage layer covering the lesion and protecting the
integrity of the neo-cartilage deposited therein.
The method generally comprises steps:
a) debriding an articular cartilage lesion and during
the debriding harvesting a small quantity (50-4000 mg) of
non-osteoarthritic hyaline cartilage;
b) fabrication and processing of the neo-cartilage
construct according to the above described procedures;
c) preparing the lesion for implantation of the neo-
cartilage construct by depositing the one or two sealant
layers, the first (optional) at the bottom of the lesion
and the second one over and on the top of the lesion, and,
using all variation already described above, depositing
either the neo-cartilage construct within the cavity formed
below the top seal and/or between the two sealant layers or
depositing the space holding thermo-reversible polymer gel
into the cavity between the two layers to uphold the
integrity of the cavity in the interim when the neo-
cartilage construct is being prepared;
d) implanting the neo-cartilage construct into said
cavity formed between the two sealant layers to allow for
integration of the neo-cartilage into the surrounding
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native intact cartilage and formation of the superficial
cartilage layer; and
e) optionally removing the space holding polymer gel
from the cavity before the neo-cartilage implantation.
In the alternative method for treatment, expanded and
differentiated chondrocytes may be deposited directly into
a joint lesion in a suitable typically thermo-reversible
gelation hydrogel solution.
There are several advantages of the current method.
First, the method is very versatile and any of the
variations may be advantageously utilized for treatment of
a specific injury, damage, aging or disease.
The method permits generation of autologous neo-
cartilage by providing alternative means for maintaining a
space between two sealant layers until the autologous neo-
cartilage is prepared. The method permits generation of
more dense neo-cartilage and three-dimensional expansion of
chondrocytes and extracellular matrix.
The deposition of the second top sealant layer
resulting in formation of superficial cartilage layer
constitutes a substitute for synovial membrane and provides
the outer surface of healthy articular cartilage
overgrowing, protecting, containing and providing critical
metabolic factors aiding in growth and incorporation of
autologous neo-cartilage in the lesion.
Deposition of the first bottom sealant layer protects
the integrity of the lesion after cleaning during surgery
and prevents migration of subchondral and synovial cells
and cell products thereby creating milieu for formation of
healthy hyaline cartilage from the neo-cartilage and also
preventing formation of the fibrocartilage.
The method further permits deposition of the space-
holding gel or thermo-reversible polymer gel to be
deposited whether alone or with suspended processed neo-
cartilage into the lesion at temperature between 5 and 25 C
as a sol. Selection of thermo-reversible gel may be
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crucial as certain TRGH may function as a promoter for
growth of the superficial cartilage layer without a need to
apply the second sealant.
The method further permits said thermo-reversible
hydrogel be enhanced with hyaluronic acid, typically added
in about 5 to about 50%, preferably about 20% (v/v),
wherein such hyaluronic acid acts as an enhancer of the
matrix-forming characteristics of the gel and to act as a
hydration factor in the synovial space in general and
within the lesion cavity in particular.
Additionally, the gel acts as a slow-release unit
for hyaluronic acid, greatly increasing a period of
hydration within the cavity and also as a substrate for
formation of the superficial cartilage layer and it can
also be conveniently removed, if needs be, by cooling the
lesion so that the solid gel formed at 37 C is converted to
sol and can be removed by injection or otherwise.
For treatment of the cartilage, a subject is treated,
according to this invention, with a prepared autologous or
heterologous neo-cartilage or neo-cartilage construct
implanted into the lesion, the neo-cartilage or the
construct is left in the lesion for two-three months and
typically, it does not need any further intervention as
during these three months, the neo-cartilage is fully
integrated into the native cartilage and becomes a fully
functional cartilage covered with a superficial cartilage
layer which eventually grows into or provides the same type
of surface as a synovial membrane of the intact joint.
Finally, the diseased, osteoarthritic cartilage may
be fully replaced by the regenerated hyaline-like cartilage
when processed according to the algorithm of this
invention.
The algorithm and/or implantation protocol may assume
any variation described above or possible within the realm
of this invention. It is thus intended that every and all
variations in the treatment protocol (algorithm of the
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cartilage) are within the scope of the current invention.
EXAMPLE 1
Isolation of Chondrocytes from Source Tissue
This example describes the procedure used for
isolation of chondrocytes from swine cartilage.
Chondrocytes were enzymatically isolated from
cartilage harvested under sterile conditions from the hind
limbs of 6-month old swine. The femur was detached from
the tibia and the trachea head exposed. Strips of
cartilage were removed from the trachea using a surgical
blade.
The cartilage was minced, digested in a 0.15%
collagenase type I solution in DMEM/Nutrient Mixture F-12
(DMEM/F-12) 1:1 mixture with 1% penicillin-streptomycin
(P/S) and gently rotated for 18 hours at 37 C.
Chondrocytes were collected and rinsed twice by
centrifugation at 1500 rpm for 5 min. Chondrocytes were
re-suspended in DMEM/F-12 containing 1% penicillin-
streptomysin and 10% FBS.
Chondrocytes were expanded for about 5 days at 37 C.
EXAMPLE 2
The Production of Human Neo-Cartilage Construct
This example describes conditions for production of
neo-cartilage for human use.
The patient undergoes arthroscopic biopsy of a small
(200-500 mg) piece of healthy cartilage from the
ipsilateral knee. The biopsy is taken from the non-weight
bearing portion of the femoral condyle or from the femoral
notch as deemed most appropriate for the patient. The
biopsy sample is placed into a sterile, non-cytotoxic, non-
pyrogenic specimen container which is packaged and shipped
to the laboratory.
At the laboratory the biopsy sample is examined
against acceptance criteria and then transferred to the
chondrocyte isolation and expansion area. Samples from the
biopsy specimen transport buffer are tested for sterility
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and for mycoplasma. The expanded chondrocytes are
suspended in VITROGEN gellable collagen solution,
commercially available from Cohesion Corp., Palo Alto, CA.
A pre-formed collagen sponge (22x22 mm square and 2-4 mm in
thickness, wherein the thickness depends on the thickness
of patient's cartilage), commercially available from Koken
Co., Japan or honeycomb matrix produced according to this
invention is placed into the resulting chondrocyte
suspension which absorbs the chondrocyte/collagen
suspension into this matrix.
The resulting chondrocyte-loaded matrix is warmed to
37 C to gel the VITROGEN in order to spatially secure the
chondrocytes within the support matrix. The loaded support
matrix is then placed into Tissue Engineering Support
System (TESS') culture unit. Typical time for cell
expansion from removal of a biopsy sample to placement of
the chondrocyte loaded culture matrix in the TESS' culture
unit is 10-40 days. Within the TESS' culture unit, cyclic
or constant hydrostatic pressure is used to induce the
chondrocytes to begin growing and expressing their
cartilage generating program for about 1 hour to about 30
days.
The still developing new cartilage is transferred to
a constant, resting culture phase. The neo-cartilage
production process requires a minimum time of 10 days in
resting culture. After this minimum 10-day period the neo-
cartilage, hereinafter called neo-cartilage construct,
undergoes final inspections and is packaged for return to
the clinic to be implanted. At the time of release, tests
for sterility, endotoxin, and mycoplasma contamination must
be negative for microbial and mycoplasma contamination and
must show <0.5 EU/ml of endotoxin.
EXAMPLE 3
Preparation of Support Matrices
This example illustrates preparation of the cellular
support matrix, also called the TESS matrix.
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300 grams of a 1% aqueous atelocollagen solution
(VITROGENe), maintained at pH 3.0, is poured into a 10 x 20
cm tray. This tray is then placed in a 5 liter container.
A 50 ml open container containing 30 ml of a 3% aqueous
ammonia solution is then placed next to the tray, in the 5
liter chamber, containing 300 grams of said 1% aqueous
solution of atelocollagen. The 5 liter container
containing the open trays of atelocollagen and ammonia is
then sealed and left to stand at room temperature for 12
hours. During this period the ammonia gas, released from
the open container of aqueous ammonia and confined within
the sealed 5 liter container, is reacted with the aqueous
atelocollagen resulting in gelling said aqueous solution of
atelocollagen.
The collagenous gel is then washed with water
overnight and, subsequently, freeze-dried to yield a sponge
like matrix. This freeze dried matrix is then cut into
squares, sterilized, and stored under a sterile wrap.
Alternatively, the support matrix may be prepared as
follows.
A porous collagen matrix, having a thickness of about
4 mm to 10 mm, is hydrated using a humidity-controlled
chamber, with a relative humidity of 80% at 25 C, for 60
minutes. The collagen material is compressed between two
Teflon sheets to a thickness of less than 0.2 mm. The
compressed material is then cross-linked in a solution of
0.5% formaldehyde, 1% sodium bicarbonate at pH 8 for 60
minutes. The cross-linked membrane is then rinsed
thoroughly with water, and freeze-dried for about 48 hours.
The dense collagen barrier has an inner construction of
densely packed fibers that are intertwined into a multi-
layer structure.
In alternative, the integration layer is prepared
from collagen-based dispersions or solutions that are air
dried into sheet form. Drying is performed at temperatures
ranging from approximately 4 to 40 C for a period of time
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of about 7 to 48 hours.
EXAMPLE 4
Seeding Cells in the TESS Matrix
This example describes procedures used for seeding
cells in the TESS matrix.
Isolated chondrocytes were incubated for a period of
five days at 37 C in a standard incubator. Cells were then
collected by trypsinization.
A cell suspension of 150,000 cells in 18 pl of
VITROGEN solution was seeded per matrix having an
approximate volume of 19 pl, with nine matrices per group.
The seeded matrix (collagen sponge 4mm in diameter and 1.5
mm in thickness) may be scaled-up to an increased volume,
where approximately 1 pl of the above described cell
suspension is seeded in 1 pl of matrix. The control group
matrices were incubated in a 37 C incubator and the test
group was incubated in the TESS.
In alternative set-up, isolated chondrocytes were
incubated for a period of five days at 37 C in a standard
incubator. Cells were then collected by trypsinization.
A cell suspension of 300,000 cells in 18 p of VITROGEN
solution was seeded per matrix having an approximate volume
of 19 pl with eight matrices per group.
EXAMPLE 5
Effect of Cyclic Hydrostatic Pressure
This example describes procedures used for
determination of effect of cyclic hydrostatic pressure in
vitro formation of chondrocyte-seeded support matrices.
Swine articular chondrocytes (sACs) were
enzymatically isolated from cartilage with type I
collagenase. The cells were suspended in collagen
(VITROGEN) as described above and wicked into the
honeycombed sponge element of the cellular support matrix.
The cells seeded in the support matrix were incubated at
37 C, 5% CO2 and 20% 02. After 24 hours, some of these
cells matrices were transferred to the TESS' processor and
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incubated at 0.5 or 3.0 MPa cyclic or constant hydrostatic
pressure with medium perfusion (0.05 ml/min) as described
above for 6 or 7 days followed by a 12 or 14 day resting
phase. The control group comprised of chondrocytes seeded
in matrices incubated for 18 or 21 days at atmospheric
pressure, at 37 C, 5% CO, and 20% or 2% 02.
At the end of the culture period (18 or 21 days), the
matrices were harvested for biochemical and histological
analysis. For biochemical analysis, sulfated
glycosaminoglycan (S-GAG) production was measured using a
modified dimethylmethylene blue (DMB) microassay.
Two matrices from each group were harvested for
histological analysis.
EXAMPLE 6
Effect of Medium Flow Rate on Extracellular Matrix
Accumulation of Chondrocytes in Collagen Sponges
This example described conditions used to determine
effect of medium flow on production and accumulation of
extracellular matrix by chondrocytes seeded into collagen
sponges.
Chondrocyte Isolation
Swine legs were obtained from a local abattoir.
Within 4-6 hours after slaughter, cartilage was harvested
under sterile conditions from the trochlea of the hind
limbs. The cartilage was minced and digested in 0.15%
collagenase type I in DMEM/F-12 containing 1% penicillin-
streptomycin (P/S) for 18 hours at 37 C. Isolated swine
articulate chondrocytes (sACs) were collected, rinsed, and
resuspended in DMEM/F-12 supplemented with 10% fetal bovine
serum (FBS) and 1% P/S. sACs then were expanded for 5 days
at 37 C.
Cell Seeding in Collagen Sponges
sACs were harvested with Trypsin EDTA and cell
viability was measured by trypan-blue exclusion. Three
hundred thousand sACs were suspended in 30 Al of a
neutralized 0.25% collagen solution (VITROGEN , Cohesion
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Corp., Palo Alto, CA), and the suspension was absorbed into
a collagen sponge, 4 mm in diameter and 2 mm in thickness,
commercially available from Koken Co., Japan. Seeded
sponges were pre-incubated for 1 hour at 37 C to gel the
collagen, followed by incubation in culture medium at 37 C
in 5% CO,.
Tissue Engineering Support System (TESS') Culture
Following the incubation, the seeded sponges were
transferred to and cultured in the Tissue Engineering
Support System (TESS') processor. To evaluate the effect
of medium perfusion rate, sponges were subjected to medium
perfusion at 5 Al/min or 50 Al/min. Cyclic hydrostatic
pressure (Cy-HP) 0 - 0.5 MPA pressure at 0.5 Hz applied was
for 6 days. Some sponges were incubated under constant
conditions at atmospheric pressure and no perfusion at 37 C
for a total of 18 days in culture. Sponges harvested 24
hours after seeding with cells (day 0) served as an initial
control.
Histological and Biochemical Analysis
Cell constructs were harvested after 6 and 18 days of
culture.
For histological evaluation, 4% paraformaldehyde-
fixed, paraffin sections were stained with Safranin-O (Saf-
0) and Type II collagen antibody.
For biochemical analysis, seeded sponges were
digested in papain at 60 C for 18 hours and DNA content was
measured using the Hoechst 33258 dye method. Sulfated
glycosaminoglycan (S-GAG) accumulation was measured using
a modified dimethylmethylene blue (DMB) microassay.
EXAMPLE 7
Biochemical and Histological Assays
This example describes assays used for biochemical
and histological studies (DMB assay).
Biochemical (DMB) Assay
At the end of the culture six matrices from each
group were used in the biochemistry assay.
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The matrices were transferred to microcentrifuge
tubes and digested in 300 yl of papain (125 yg/mi in 0.1 M
sodium phosphate, 5 mM disodium EDTA, and 5 mM L-cysteine-
HC1) for 18 hours at 60 C. GAG production in the matrices
was measured using a modified dimethylene blue (OMB)
microassay with shark chondroitin sulfate as a control
Farndaleetal., Connective Tissue Research, 9: 247-248 (1982).
DNA content was determined by Hoechst 33258 dye
method according to Kim et al., "Fluorometric assay of DNA in cartilage
explants using Hoechst 33258", Anal. Biochem., 174:168-176 (1988).
Histological Assay
The remaining matrices from each group were fixed in
4% paraformaldehyde. The matrices were processed and
embedded in oaraffin. 10 ym sections were cut on a
microtome and stained with Safranin-O (Saf 0).
YA.MPLE 8
Evaluation of Porcine Neo-Cartilage integration
in a Swine Model
This example describe the procedure and results of
study performed for evaluation of integration of porcine
neo-cartilage in a swine model.
An open arthrotomy of the right knee joint was
performed on all animals, and a biopsy of the cartilage was
obtained.Chondrocytes were isolated from the cartilage biopsy
and cultured within a collagen matrix in a Tissue
Engineering Support System (TESS') to produce porcine-
Neocart for subsequent implantation.
A defect was created in the medial femoral condyle of
the pig's right knee. This defect (control) was not
implanted with porcine-NeoCart". Following surgery, the
joint was immobilized with an external fixation construct
for a period of about two weeks. Three weeks after the
arthrotomy on the right knee was performed, an open
arthrotomy was performed on the left knee and defects were
created in this medial femoral condyle. The porcine-
NeoCart" was implanted within the defect(s) in this knee
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which was similarly immobilized. The operated sites were
subsequently viewed via arthroscopy two weeks after
implantation or defect creation and thereafter at monthly
intervals. Animals were euthanized and the joints
harvested and prepared for histological examination
approximately 3 months after porcine-NeoCartim implantation.
The implanted sites were prepared and examined
histologically.
Results are seen in Figures 10-12. Figure 10 shows
results of the arthroscopic examination. The empty defect
is seen in Figure 10A. The porcine NeoCart'm implant site
is seen in Figure 10B which also shows still-evident
absorbable sutures and the superficial cartilage layer
growing over the porcine NeoCart1m.
EXAMPLE 9
Protocol for In Vivo, Ex Vivo or In Vitro Growth of
Porcine Neo-Cartilage
Autologous porcine chondrocytes are seeded into the
cellular support matrix and incubated under cyclic
hydrostatic pressure at 37 C and 5% CO2. Cyclic
hydrostatic pressure is either 0.5 or 3.0 MPa at 0.5 Hz.
The duration of said cyclic pressure is approximately 6
days followed by a resting phase of 12 days in an incubator
maintained at 37 C at atmospheric pressure. At the end of
this resting phase, the matrices were harvested for
biochemical and histological analysis.
In the alternative protocol, the algorithm for the
growth cells of in vivo and in vitro, the application of
hydrostatic pressure is used on isolated in situ cartilage,
or application of hydrostatic pressure for about 1-8 hours
followed by about 16-23 hours of recovery period.
EXAMPLE 10
Regeneration of Human Chondrocvtes
This example describes the procedure used for
regeneration of human chondrocytes.
Chondrocytes from osteoarthritic (OA) patients (40
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years old) were expanded for 18 days in monolayer culture
at 37 C and suspended in VITROGEN (300,000 cells/30 ftl).
The cell suspension was absorbed into a support matrix,
usually a collagen honeycomb sponge (4 mm in diameter and
2 mm in thickness, Koken Co., Japan). The cell constructs
were incubated in culture medium supplemented with 10% FBS
and 1% ITS (Insulin-transferrin-sodium selenite, Sigma) at
37 C, 5% CO, and 20% 02, at 0.5 MPa cyclic hydrostatic
pressure (Cy-HP) or 0.5 MPa constant hydrostatic pressure
(Constant-HP) for 7 or 14 days in the TESS' processor
followed by incubation for 7 or 14 days at atmospheric
pressure for 7 or 14 days in an CO, incubator at 37 C. The
remaining cell constructs compromising the control group
were incubated atmospheric pressure for 21 days at 37 C, 5%
CO2 and 20% 02.
Before starting the culture, some cell constructs
were harvested for biochemical and histological analysis as
an initial condition. At the end of the culture period,
the cell constructs were harvested for biochemical and
histological analysis. Sulfated glycosaminoglycan
production was measured using a modified dimethylmethylene
blue (DMB) micro assay. Cell proliferation was measured
using a modified Hoechst Dye DNA assay. Formation of neo-
tissue was analyzed by Safranin-O staining.
