U.S. patent application number 14/877392 was filed with the patent office on 2016-01-28 for minced cartilage systems and methods.
This patent application is currently assigned to AlloSource. The applicant listed for this patent is AlloSource. Invention is credited to Carolyn Barrett, Yaling Shi, Peter Stevens.
Application Number | 20160022740 14/877392 |
Document ID | / |
Family ID | 50681910 |
Filed Date | 2016-01-28 |
United States Patent
Application |
20160022740 |
Kind Code |
A1 |
Shi; Yaling ; et
al. |
January 28, 2016 |
Minced Cartilage Systems and Methods
Abstract
Compositions comprising a plurality of cartilage particles from
a human adult cadaveric donor, wherein the cartilage particles
comprise viable chondrocytes, and a biocompatible carrier are
provided. Methods of manufacturing cartilage compositions
comprising a plurality of cartilage particles from a human adult
cadaveric donor are also provided.
Inventors: |
Shi; Yaling; (Larkspur,
CO) ; Barrett; Carolyn; (Denver, CO) ;
Stevens; Peter; (North Richland Hills, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AlloSource |
Centennial |
CO |
US |
|
|
Assignee: |
AlloSource
Centennial
CO
|
Family ID: |
50681910 |
Appl. No.: |
14/877392 |
Filed: |
October 7, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14081913 |
Nov 15, 2013 |
9186380 |
|
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14877392 |
|
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|
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61727016 |
Nov 15, 2012 |
|
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Current U.S.
Class: |
424/489 ;
424/548; 424/549; 424/93.7 |
Current CPC
Class: |
A61K 47/20 20130101;
A61K 47/42 20130101; A61K 47/36 20130101; A61K 35/32 20130101; A61L
27/3645 20130101; A61L 24/0005 20130101; A61L 2430/06 20130101;
A61L 27/3683 20130101; A61K 35/32 20130101; A61L 27/3608
20130101 |
International
Class: |
A61K 35/32 20060101
A61K035/32; A61L 24/00 20060101 A61L024/00 |
Claims
1. A composition comprising: a plurality of cartilage particles
from a human adult cadaveric donor age 15 years or older, wherein
the cartilage particles comprise viable chondrocytes; and a
biocompatible carrier.
2. The composition of claim 1, wherein the cartilage is articular
cartilage.
3. The composition of claim 1, wherein the cartilage is
non-decellularized cartilage.
4. The composition of claim 1, wherein the cartilage particles have
an average thickness from about 0.25 mm to about 5 mm.
5. The composition of claim 4, wherein the cartilage particles have
an average thickness from about 0.5 mm to about 2 mm.
6. The composition of claim 1, wherein the cartilage particles have
an average width from about 1 mm to about 5 mm.
7. The composition of claim 1, wherein the cartilage particles have
an average diameter from about 1 mm to about 5 mm.
8. The composition of claim 1, wherein the cartilage particles have
an average volume of about 0.5 mm.sup.3 to about 100 mm.sup.3.
9. The composition of claim 8, wherein the cartilage particles have
an average volume of about 0.5 mm.sup.3 to about 30 mm.sup.3.
10. The composition of claim 1, wherein the cartilage particles are
from a human donor that is 18 years of age or older at the time of
donation.
11. The composition of claim 1, wherein the biocompatible carrier
comprises a cryopreservation medium.
12. The composition of claim 11, wherein the cryopreservation
medium comprises dimethyl sulfoxide (DMSO) and serum.
13. The composition of claim 1, further comprising a biological
adhesive.
14. The composition of claim 13, wherein the biological adhesive is
fibrin, fibrinogen, thrombin, fibrin glue, polysaccharide gel,
cyanoacrylate glue, gelatin-resorcin-formalin adhesive, collagen
gel, synthetic acrylate-based adhesive, cellulose-based adhesive,
basement membrane matrix, laminin, elastin, proteoglycans,
autologous glue, or a combination thereof.
15. The composition of claim 1, further comprising demineralized
bone.
16. The composition of claim 1, further comprising a bone or
cartilage substrate seeded with stem cells.
17-30. (canceled)
31. A method of treating a cartilage or bone defect in a subject,
the method comprising administering to the subject the composition
of claim 1.
32. A method of repairing cartilage in a subject, the method
comprising administering to the subject the composition of claim
1.
33. (canceled)
34. The composition of claim 1, wherein the plurality of cartilage
particles comprise at least 50,000 viable cells per cm.sup.3.
35. The composition of claim 1, wherein at least 50% of the
chondrocytes in the cartilage particles are viable.
36. The composition of claim 1, wherein the cartilage particles are
laser cut cartilage particles or mechanically cut cartilage
particles.
37. The composition of claim 1, wherein the composition is made by
a process comprising: obtaining cartilage tissue from a human adult
cadaveric donor age 15 years or older; mincing the cartilage tissue
into a plurality of cartilage particles, wherein the cartilage
particles comprise chondrocytes native to the cartilage tissue,
wherein the cartilage particles comprise viable chondrocytes native
to the cartilage tissue; suspending the plurality of cartilage
particles in a biocompatible medium; and packaging the suspended
plurality of cartilage particles into a container for storage and
shipping.
38. The composition of claim 37, wherein mincing step comprises
cutting the cartilage tissue with a laser cutter, with a mechanical
blade, or with a mechanical press.
39. The composition of claim 37, wherein mincing step comprises
cutting the cartilage tissue with a laser cutter.
40. The composition of claim 37, wherein the mincing comprises
cutting the cartilage tissue with a laser cutter at a power of
about 3 Watts to about 13.5 Watts and a frequency of about 400 Hz
to about 2400 Hz.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/081,913, filed Nov. 15, 2013, which claims benefit of
priority of U.S. Provisional Application No. 61/727,016, filed Nov.
15, 2012, the entire content of which is incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] Cartilage tissue can be found throughout the human anatomy.
The cells within cartilage tissue are called chondrocytes. These
cells generate proteins, such as collagen, proteoglycan, and
elastin, that are involved in the formation and maintenance of the
cartilage. Hyaline cartilage is present on certain bone surfaces,
where it is commonly referred to as articular cartilage. Articular
cartilage contains significant amounts of collagen (about
two-thirds of the dry weight of articular cartilage), and
cross-linking of the collagen imparts a high material strength and
firmness to the tissue. These mechanical properties are important
to the proper performance of the articular cartilage within the
body.
[0003] Articular cartilage is not vascularized, and when damaged as
a result of trauma or degenerative causes, this tissue has little
or no capacity for in vivo self-repair. A variety of therapeutic
solutions have been proposed for the treatment and repair of
damaged or degenerated cartilage. Although such techniques may
provide real benefits to patients in need thereof, still further
advancements in the field of cartilage repair are desirable.
BRIEF SUMMARY OF THE INVENTION
[0004] In one aspect, cartilage compositions are provided. In some
embodiments, the composition comprises: a plurality of cartilage
particles from a human adult cadaveric donor age 15 years or older,
wherein the cartilage particles comprise viable chondrocytes; and a
biocompatible carrier.
[0005] In some embodiments, on average at least 50% of the
chondrocytes in the cartilage particles are viable.
[0006] In some embodiments, the cartilage is articular cartilage.
In some embodiments, the cartilage is non-decellularized cartilage.
In some embodiments, the cartilage particles are from a human donor
that is 18 years of age or older at the time of donation.
[0007] In some embodiments, the cartilage particles have an average
thickness from about 0.25 mm to about 5 mm. In some embodiments,
the cartilage particles have an average thickness from about 0.5 mm
to about 2 mm. In some embodiments, the cartilage particles have an
average length and/or an average width from about 0.1 mm to about
25 mm. In some embodiments, the cartilage particles have an average
diameter from about 0.1 mm to about 25 mm. In some embodiments, the
cartilage particles have an average volume of about 0.5 mm.sup.3 to
about 100 mm.sup.3. In some embodiments, the cartilage particles
have an average volume of about 0.5 mm.sup.3 to about 30
mm.sup.3.
[0008] In some embodiments, the biocompatible carrier comprises a
cryopreservation medium. In some embodiments, the cryopreservation
medium comprises dimethyl sulfoxide (DMSO) and serum.
[0009] In some embodiments, the composition further comprises a
biological adhesive. In some embodiments, the biological adhesive
is fibrin, fibrinogen, thrombin, fibrin glue, polysaccharide gel,
cyanoacrylate glue, gelatin-resorcin-formalin adhesive, collagen
gel, synthetic acrylate-based adhesive, cellulose-based adhesive,
basement membrane matrix, laminin, elastin, proteoglycans,
autologous glue, or a combination thereof.
[0010] In some embodiments, the composition further comprises
demineralized bone. In some embodiments, the composition further
comprises a bone or cartilage substrate seeded with stem cells.
[0011] In another aspect, methods of manufacturing a cartilage
composition are provided. In some embodiments, the method
comprises: [0012] obtaining cartilage tissue from a human adult
cadaveric donor; [0013] mincing the cartilage tissue into a
plurality of cartilage particles, wherein the cartilage particles
comprise viable chondrocytes; and [0014] suspending the plurality
of cartilage particles in a biocompatible medium.
[0015] In some embodiments, on average at least 50% of the
chondrocytes in the cartilage particles are viable.
[0016] In some embodiments, the cartilage tissue is articular
cartilage. In some embodiments, the cartilage tissue is
non-decellularized cartilage. In some embodiments, the cartilage
tissue is from a human donor that is 18 years of age or older at
the time of donation.
[0017] In some embodiments, prior to the mincing step, the
cartilage tissue is sliced to a thickness of about 0.25 mm to about
5 mm. In some embodiments, prior to the mincing step, the cartilage
tissue is sliced to a thickness of about 0.25 mm to about 2 mm.
[0018] In some embodiments, the mincing step comprises cutting the
cartilage tissue with a laser cutter, with a mechanical blade, or
with a mechanical press. In some embodiments, the mincing step
comprises cutting the cartilage tissue with a laser cutter. In some
embodiments, the mincing step comprising cutting the cartilage
tissue with the laser cutter at a speed from about 10% to about
50%, a power from about 0% to about 45%, and a frequency from about
10 Hz to about 2400 Hz.
[0019] In some embodiments, the cartilage tissue is minced into a
plurality of cartilage particles having an average length and/or an
average width from about 0.1 mm to about 25 mm. In some
embodiments, the cartilage particles have an average diameter from
about 0.1 mm to about 25 mm. In some embodiments, the cartilage
tissue is minced into a plurality of cartilage particles having an
average volume of about 0.5 mm.sup.3 to about 100 mm.sup.3. In some
embodiments, the cartilage tissue is minced into a plurality of
cartilage particles having an average volume of about 0.5 mm.sup.3
to about 30 mm.sup.3.
[0020] In some embodiments, following the mincing step, the
plurality of cartilage particles are washed with a saline
solution.
[0021] In some embodiments, the biocompatible carrier comprises a
cryopreservation medium. In some embodiments, the cryopreservation
medium comprises dimethyl sulfoxide (DMSO) and serum.
[0022] In some embodiments, prior to the suspending step, the
method further comprises combining the plurality of cartilage
particles with a biological adhesive. In some embodiments, the
biological adhesive is fibrin, fibrinogen, thrombin, fibrin glue,
polysaccharide gel, cyanoacrylate glue, gelatin-resorcin-formalin
adhesive, collagen gel, synthetic acrylate-based adhesive,
cellulose-based adhesive, basement membrane matrix, laminin,
elastin, proteoglycans, autologous glue, or a combination
thereof.
[0023] In some embodiments, prior to the suspending step, the
method further comprises combining the plurality of cartilage
particles with demineralized bone. In some embodiments, prior to
the suspending step, the method further comprises combining the
plurality of cartilage particles with a bone or cartilage substrate
seeded with stem cells.
[0024] In another aspect, methods of repairing cartilage in a
subject are provided. In some embodiments, the method comprises
administering to the subject a composition as described herein
(e.g., a composition comprising a plurality of cartilage particles
from a human adult cadaveric donor age 15 years or older, wherein
the cartilage particles comprise viable chondrocytes; and a
biocompatible carrier).
[0025] In yet another aspect, methods of treating a defect in
cartilage, bone, ligament, tendon, meniscus, joint, or muscle in a
subject are provided. In some embodiments, the method comprises
administering to the subject a composition as described herein
(e.g., a composition comprising a plurality of cartilage particles
from a human adult cadaveric donor age 15 years or older, wherein
the cartilage particles comprise viable chondrocytes; and a
biocompatible carrier).
[0026] In still another aspect, compositions for use in treating a
defect in cartilage, bone, ligament, tendon, meniscus, joint, or
muscle in a subject are provided. In some embodiments, the
composition for use is a composition as described herein (e.g., a
composition comprising a plurality of cartilage particles from a
human adult cadaveric donor age 15 years or older, wherein the
cartilage particles comprise viable chondrocytes; and a
biocompatible carrier).
[0027] In still another aspect, kits comprising a composition as
described herein (e.g., a composition comprising a plurality of
cartilage particles from a human adult cadaveric donor age 15 years
or older, wherein the cartilage particles comprise viable
chondrocytes; and a biocompatible carrier) are provided. In some
embodiments, the kits are used for treating a subject having a
defect in cartilage, bone, ligament, tendon, meniscus, joint, or
muscle. In some embodiments, the kits are used for treating a
subject having a degenerative defect or injury cartilage, bone,
ligament, tendon, meniscus, joint, or muscle; a subject having a
traumatic defect or injury cartilage, bone, ligament, tendon,
meniscus, joint, or muscle; or a subject having osteoarthritis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIGS. 1A-1D. Examples of cartilage particle shapes processed
from cartilage tissue. FIG. 1A shows cartilage particles can have a
rectangular columnar shape. FIG. 1B shows cartilage particles can
have a cylindrical or elliptical columnar shape. FIG. 1C shows
cartilage particles can be cut into tiled or mosaic configurations.
For example, the cartilage particle can have a width A, length B,
and height C. Cuts, etches, or channels in the construct have a
depth F and width H. Individual columns have a height F, width E,
and length D. Subsequent to cutting, the construct has a minimum
thickness G. FIG. 1D shows cartilage tissue can be cut, for example
using a laser, on two or more sides (e.g., top and bottom).
[0029] FIG. 2. A standard curve for samples having known
concentrations of chondrocytes. The y-axis represents fluorescence
readings from a Countess.RTM. automated cell counter, and the
x-axis represents the chondrocyte concentration (cells/.mu.l).
[0030] FIGS. 3A-3B. Mean fluorescence readings for chondrocyte
samples from adult donor A, shown in FIG. 3A, and chondrocyte
samples from juvenile donor B, shown in FIG. 3B, placed in six-well
tissue culture plates.
[0031] FIG. 4. Mean fluorescence readings for chondrocyte samples
from an adult donor and from a juvenile donor, measured at day 1
and after culturing for 6 weeks.
[0032] FIG. 5. Trypan Blue cell viability assay for Donors C, D, E,
F, and G (also referred to as donors 1, 2, 3, 4, and 5,
respectively). Cell viability was determined for laser cut and hand
cut cartilage particles. The average cell viability is presented as
a percentage. The term "Denovo" refers to a juvenile cartilage
product that is hand cut into 1 mm squares.
[0033] FIG. 6. Graph depicting the live cell count data for Trypan
Blue and Presto Blue assays shown in the lower panel of FIG. 5.
[0034] FIG. 7. Trypan Blue cell viability assay at 6 weeks for
laser cut and hand cut cartilage particles.
[0035] FIGS. 8A-8B. Confocal microscope images depicting tissue
edges (white arrow) of hand cut and laser cut cartilage pieces are
shown in FIG. 8A and FIG. 8B, respectively. Invitrogen
LIVE/DEAD.RTM. stain was used on undigested cartilage sample for
visualizing cells.
[0036] FIGS. 9A-9B. Photographic images at 4.times. magnification
of chondrocyte cells growing out of hand cut and laser cut adult
cartilage particles are shown in FIG. 9A and FIG. 9B, respectively.
Cartilage particles were placed in 12-well culture plates with
chondrocyte growth medium containing 10% FBS and 2% antibiotic. The
medium was changed twice a week. The plates were cultured under
standard cell culture conditions (37.degree. C. incubator with 5%
CO.sub.2) and the images were obtained at 18 days.
[0037] FIG. 10. Schematic of an exemplary manufacturing method for
cartilage compositions.
[0038] FIG. 11. Alcian Blue staining of cartilage samples from
adult (upper panels) and juvenile (lower panels) donors after a 6
week explant study.
[0039] FIG. 12. Collagen type II staining of cartilage samples from
adult cartilage (left panel) and juvenile cartilage (right panel)
after a 12 week explant study. Brown staining in both the adult
cartilage and juvenile cartilage indicates collagen type II
produced by cells that grew out of the cartilage.
DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
[0040] It is known in the art that juvenile cartilage contains more
cells than adult cartilage. Furthermore, it has previously been
suggested that the cells of adult cartilage do not grow out or have
the ability to repair cartilage defects. Thus, it has been
suggested that adult cartilage is not well suited for use in
allogeneic grafts.
[0041] However, as described herein, it has been surprisingly
discovered that adult cartilage, for example as prepared according
to the methods described herein, retains properties that are useful
in an allogeneic graft. As shown herein, it has been found that
adult cartilage particles, when cultured for a period of time,
exhibit comparable chondrocyte outgrowth and matrix production as
juvenile cartilage particles. Thus, cartilage particles derived
from human adult donors can be useful for repairing cartilage
defects in subjects in need thereof.
II. Cartilage Compositions
[0042] In one aspect, cartilage compositions comprising viable
chondrocytes are provided. In some embodiments, the composition
comprises: a plurality of cartilage particles from a human adult
cadaveric donor age 15 years or older, wherein the cartilage
particles comprise viable chondrocytes; and a biocompatible
carrier. In some embodiments, on average at least 50% of the
chondrocytes in the cartilage particles are viable.
[0043] As used herein, the term "human adult donor" refers to a
human donor that is fifteen years of age or older. The term "human
juvenile donor" refers to a human donor that is twelve years of age
or younger. In some embodiments, the donor is an adult cadaveric
donor that is between the ages of 15 and 36 at the time of the
donation. In some embodiments, the donor is an adult cadaveric
donor that is 18 years of age or older at the time of the
donation.
[0044] In some embodiments, the cartilage is articular cartilage.
In some embodiments, the articular cartilage is obtained from an
articular surface of a joint (e.g., a knee joint or an elbow joint)
or from a long bone (e.g., femur or tibia).
[0045] Cartilage particles can be shaped as circles, spheres,
squares, rectangles, cubes, cylinders, strips, tiles (e.g.
particles that are partially attached to other particles), or other
desired shapes. In some embodiments, the cartilage particles have
an average thickness of about 0.25 mm to about 5 mm (e.g., about
0.25 mm, about 0.5 mm, about 0.75 mm, about 1 mm, about 1.5 mm,
about 2 mm, about 2.5 mm, about 3 mm, about 3.5 mm, about 4 mm,
about 4.5 mm, or about 5 mm). In some embodiments, the cartilage
particles have an average thickness of about 0.5 mm to about 2 mm.
In some embodiments, the cartilage particles have an average length
and/or an average width of about 0.1 mm to about 25 mm (e.g., about
0.1 mm, about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm,
about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, about 1 mm,
about 1.5 mm, about 2 mm, about 2.5 mm, about 3 mm, about 3.5 mm,
about 4 mm, about 4.5 mm, about 5 mm, about 6 mm, about 7 mm, about
8 mm, about 9 mm, about 10 mm, about 11 mm, about 12 mm, about 13
mm, about 14 mm, about 15 mm, about 16 mm, about 17 mm, about 18
mm, about 19 mm, about 20 mm, about 21 mm, about 22 mm, about 23
mm, about 24 mm, or about 25 mm). In some embodiments, the
cartilage particles have an average length and/or an average width
of about 0.5 mm to about 10 mm, of about 0.5 mm to about 5 mm, of
about 0.5 mm to about 3 mm, of about 1 mm to about 5 mm, or of
about 1 mm to about 3 mm.
[0046] In some embodiments, the cartilage particles have an average
diameter of about 0.1 mm to about 25 mm (e.g., about 0.1 mm, about
0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm,
about 0.7 mm, about 0.8 mm, about 0.9 mm, about 1 mm, about 1.5 mm,
about 2 mm, about 2.5 mm, about 3 mm, about 3.5 mm, about 4 mm,
about 4.5 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about
9 mm, about 10 mm, about 11 mm, about 12 mm, about 13 mm, about 14
mm, about 15 mm, about 16 mm, about 17 mm, about 18 mm, about 19
mm, about 20 mm, about 21 mm, about 22 mm, about 23 mm, about 24
mm, or about 25 mm). In some embodiments, the cartilage particles
have an average diameter of about 0.5 mm to about 10 mm, of about
0.5 mm to about 5 mm, of about 0.5 mm to about 3 mm, of about 1 mm
to about 5 mm, or of about 1 mm to about 3 mm.
[0047] In some embodiments, the cartilage particles have an average
volume of from about 0.5 mm.sup.3 to about 100 mm.sup.3 (e.g.,
about 0.5 mm.sup.3, about 1 mm.sup.3, about 2 mm.sup.3, about 3
mm.sup.3, about 4 mm.sup.3, about 5 mm.sup.3, about 6 mm.sup.3,
about 7 mm.sup.3, about 8 mm.sup.3, about 9 mm.sup.3, about 10
mm.sup.3, about 15 mm.sup.3, about 20 mm.sup.3, about 25 mm.sup.3,
about 30 mm.sup.3, about 35 mm.sup.3, about 40 mm.sup.3, about 45
mm.sup.3, about 50 mm.sup.3, about 60 mm.sup.3, about 70 mm.sup.3,
about 80 mm.sup.3, about 90 mm.sup.3, or about 100 mm.sup.3). In
some embodiments, the cartilage particles have an average volume
from about 0.5 mm.sup.3 to about 30 mm.sup.3. In some embodiments,
the cartilage particles have an average volume from about 1
mm.sup.3 to about 30 mm.sup.3. In some embodiments, the cartilage
particles have an average volume from about 1 mm.sup.3 to about 25
mm.sup.3.
[0048] As one non-limiting example, as shown in FIG. 1A, cartilage
particles can have a rectangular columnar shape (e.g., with a
thickness of 0.25 to 5 mm, a width of 1 to 5 mm, and a depth of 1-5
mm). As another non-limiting example, as shown in FIG. 1B, minced
particles can have a cylindrical or elliptical columnar shape (e.g.
with a thickness of 0.25 to 5 mm and a diameter of 1 to 5 mm).
Perforated Cartilage
[0049] In some embodiments, cartilage tissue can be cut into tiled
or mosaic configurations to yield cartilage particles or constructs
comprising channels or microperforations that separate the
cartilage particles into a plurality of smaller portions. Thus, in
some embodiments, the composition comprises one or more cartilage
particles, each cartilage particle comprising one or more channels
or microperforations that separates the cartilage particle into a
plurality of smaller cartilage portions.
[0050] In some embodiments, the cartilage particle comprises one or
more channels or microperforations that separates the cartilage
particle into a plurality of smaller cartilage portions, wherein
each cartilage portion has an average length and/or an average
width of about 1 mm to about 5 mm (e.g., about 1 mm, about 1.5 mm,
about 2 mm, about 2.5 mm, about 3 mm, about 3.5 mm, about 4 mm,
about 4.5 mm, or about 5 mm). In some embodiments, the cartilage
particle comprises one or more channels or microperforations that
separates the cartilage particle into a plurality of smaller
cartilage portions, wherein each cartilage portion has an average
diameter of about 1 mm to about 5 mm (e.g., about 1 mm, about 1.5
mm, about 2 mm, about 2.5 mm, about 3 mm, about 3.5 mm, about 4 mm,
about 4.5 mm, or about 5 mm).). In some embodiments, the cartilage
particle comprises one or more channels or microperforations that
separates the cartilage particle into a plurality of smaller
cartilage portions, wherein each cartilage portion has an average
volume of from about 0.5 mm.sup.3 to about 100 mm.sup.3 (e.g.,
about 0.5 mm.sup.3, about 1 mm.sup.3, about 2 mm.sup.3, about 3
mm.sup.3, about 4 mm.sup.3, about 5 mm.sup.3, about 6 mm.sup.3,
about 7 mm.sup.3, about 8 mm.sup.3, about 9 mm.sup.3, about 10
mm.sup.3, about 15 mm.sup.3, about 20 mm.sup.3, about 25 mm.sup.3,
about 30 mm.sup.3, about 35 mm.sup.3, about 40 mm.sup.3, about 45
mm.sup.3, about 50 mm.sup.3, about 60 mm.sup.3, about 70 mm.sup.3,
about 80 mm.sup.3, about 90 mm.sup.3, or about 100 mm.sup.3).
[0051] As a non-limiting example, in FIG. 1C, the cartilage
construct or particle has a width A, length B, and height C. Cuts,
etches, or channels in the construct have a depth F and width H.
Individual columns have a height F, width E, and length D.
Subsequent to cutting, the construct has a minimum thickness G. As
shown in FIG. 1D, laser cutting can be performed on two or more
sides of a cartilage tissue (e.g. top and bottom). Thus, it is
possible to create cartilage constructs having multiple connected
pieces of small columns or blocks, with square profiles, circular
profiles, triangular profiles, irregular profiles, and the like. In
some embodiments, cartilage constructs are prepared in strips,
sheets, ribbons, zig-zag or accordion shapes, or the like. In some
embodiments, the composition comprises one or more cartilage
particles formed as a sheet, wherein the cartilage particle sheet
comprises one or more channels or microperforations that separates
the cartilage particle into a plurality of smaller cartilage
portions.
Biocompatible Carrier
[0052] In some embodiments, the biocompatible carrier comprises a
buffered solution. In some embodiments, the biocompatible carrier
comprises a cryopreservation medium. In some embodiments, the
cryopreservation medium comprises dimethyl sulfoxide (DMSO) and
serum. In some embodiments, the biocompatible carrier comprises one
or more cryoprotective agents such as, but not limited to,
glycerol, DMSO, hydroxyethyl starch, polyethylene glycol,
propanediol, ethylene glycol, butanediol, polyvinylpyrrolidone, or
alginate.
[0053] In some embodiments, the biocompatible carrier comprises a
growth medium. Suitable examples of growth medium include, but are
not limited to, Dulbecco's Modified Eagle's Medium (DMEM) with 5%
Fetal Bovine Serum (FBS). In some embodiments, growth medium
includes a high glucose DMEM. In some embodiments, the
biocompatible carrier (e.g., growth medium) comprises one or more
antibiotics.
[0054] In some embodiments, the composition comprising the
cartilage particles is formed into a paste.
Quantifying Viable Chondrocytes and Characterizing Cartilage
Compositions
[0055] In some embodiments, the composition comprises cartilage
particles having an average chondrocyte viability of at least 50%,
at least 55%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 85% or higher. In some embodiments, the
composition comprises cartilage particles having at least about
50,000, at least about 60,000, at least about 70,000, at least
about 80,000, at least about 90,000, at least about 100,000, at
least about 150,000, at least about 200,000, at least about
250,000, at least about 300,000, at least about 350,000, at least
about 400,000, at least about 450,000, at least about 500,000, at
least about 550,000, at least about 600,000, at least about
650,000, at least about 700,000, at least about 750,000, at least
about 800,000, at least about 850,000, at least about 900,000, at
least about 950,000, or at least about 1 million viable
chondrocytes per cubic centimeter (cc). In some embodiments, the
average chondrocyte viability or the amount of chondrocytes per cc
is measured on day 1 following from the day of cutting.
[0056] The amount of chondrocytes in the cartilage particles can be
measured by any of a number of cell counting assays. For example,
in some embodiments, a Trypan Blue assay or a Presto Blue assay is
used to quantify the number of chondrocytes in the cartilage
particles. In some embodiments, the cartilage particles are cut
from cartilage tissue on day 0 and then the amount of chondrocytes
in the cartilage particles is measured on day 1. In some
embodiments, the amount of chondrocytes and/or cell viability is
measured on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8,
day 9, day 10, day 11, day 12, day 13, or day 14 from the day of
cutting. In some embodiments, the amount of chondrocytes and/or
cell viability is measured 1 week, 2 weeks, 3 weeks, 4 weeks, 5
weeks, or 6 weeks from the day of cutting. In some embodiments, for
determining the amount of chondrocytes in a sample, the sample is
subjected to digestion, e.g., with collagenase, in order to isolate
chondrocytes for cell count and/or viability testing.
[0057] In some embodiments, a Trypan Blue assay is used to evaluate
cell count and/or cell viability. The Trypan Blue assay is based
upon the principle that viable cells do not take up impermeable
dyes such as Trypan Blue, but dead cells are permeable and take up
the dye. Typically, Trypan Blue stain is added to a sample, then
the sample is mixed. An aliquot of the sample is placed on a cell
counter slide and the number of cells is counted. The number of
cells per cc is calculated based on the starting cartilage particle
sample size.
[0058] In some embodiments, a Presto Blue assay is used to evaluate
cell count and/or cell viability. The Presto Blue protocol involves
an indirect chondrocyte cell count, using a metabolic assay. The
cell count is performed by using a standard curve of known
concentrations of chondrocytes to determine the count in the
unknown samples. Typically, a 1:10 ratio of PrestoBlue.RTM. reagent
(Life Technologies, Carlsbad, Calif.) to cell culture medium is
added to a sample so that the sample is covered by the medium. The
metabolic activity of the cells changes the color of the medium.
After 3 hours incubation, 100 .mu.l aliquots are taken from each
sample and added to a multi-well plate for reading in a plate
reader.
[0059] In some embodiments, a cell counting technique other than
the Trypan Blue assay or Presto Blue assay is used to determine
chondrocyte cell counts in a sample comprising cartilage particles.
For example, the LIVE/DEAD.RTM. stain (Life Technologies, Carlsbad,
Calif.) or the CellTiter-Glo.RTM. Luminescent Cell Viability Assay
(Promega, Madison, Wis.) can be used to evaluate cell viability. In
some embodiments, a Quant-iT.TM. DNA Assay Kit (Life Technologies,
Carlsbad, Calif.), such as with PicoGreen, can be used to assess
DNA content, thereby determining cell count.
[0060] In some embodiments, cell viability can be calculated using
the following formula:
(number of live cells/total number of live+dead
cells)*100%=viability percentage
[0061] The cartilage particles can also be evaluated for
characteristics of or chondrocyte outgrowth. For example, the
cartilage particles can be cultured for a period of time (e.g., 1,
2, 3, 4, 5, or 6 weeks) and then assayed for one or more
characteristics of chondrocyte outgrowth, such as glycosaminoglycan
production, the presence of collagen, or the presence of one or
more cartilage-specific biomarkers. In some embodiments, the
cartilage particles exhibit one or more characteristics of
chondrocyte outgrowth, including but not limited to
glycosaminoglycan production, collagen content, or
cartilage-specific biomarker expression, that is comparable to
those obtained from cartilage particles from a juvenile donor and
cultured under the same conditions.
[0062] In some embodiments, the cartilage particles exhibit
glycosaminoglycan (GAG) production after being cultured for a
period of time (e.g., as described herein in the Examples section).
Chondrocytes function in part by producing GAGs and other
components of the cartilaginous extracellular matrix. Hence, it is
possible to evaluate the chondrocyte activity of cartilage tissue
by observing glycosaminoglycan production. The glycosaminoglycan
content can be measured, for example, using a dimethylmethylene
blue (DMMB) assay or using Alcian Blue staining. In some
embodiments, the levels of sulfated GAGs (sGAGs) are measured.
sGAGS are an important component of healthy cartilage and can
decrease with age and lead to the development of osteoarthritis.
sGAGs can be measured, for example, using a commercially available
sGAG Assay Kit (Kamiya Biomedical Company, Seattle, Wash.).
[0063] In some embodiments, the cartilage particles exhibit
collagen production after being cultured for a period of time
(e.g., as described herein in the Examples section). Collagen
production and collagen content can be measured, for example, using
a hydroxyproline assay (BioVison, Milpitas, Calif.). Collagen
production and collagen content can also be measured using an
immunoassay (e.g., immunohistochemistry or an immunosorbent assay,
e.g, ELISA assay), including but not limited to a Collagen Type II
Antibody Staining Protocol.
Additional Biological Components
[0064] In some embodiments, the cartilage particles are combined
one or more other biological components in the composition. For
example, in some embodiments, the cartilage particles are combined
with a biological adhesive. Suitable biological adhesives include,
but are not limited to, fibrin, fibrinogen, thrombin, fibrin glue
(e.g., TISSEEL), polysaccharide gel, cyanoacrylate glue,
gelatin-resorcin-formalin adhesive, collagen gel, synthetic
acrylate-based adhesive, cellulose-based adhesive, basement
membrane matrix (e.g., MATRIGEL.RTM., BD Biosciences, San Jose,
Calif.), laminin, elastin, proteoglycans, autologous glue, and
combinations thereof.
[0065] In some embodiments, the cartilage particles are combined
with demineralized bone matrix. For example, in some embodiments
the cartilage particles are combined with demineralized bone matrix
at a ratio of about 1 cubic centimeter (cc) demineralized bone
matrix: 4 cc cartilage particles to about 1 cc demineralized bone
matrix: 1 cc cartilage particles (e.g., about 4:1, about 3:1, about
2:1, or about 1:1 cc demineralized bone matrix:cartilage
particles). Demineralized bone matrix can be prepared, e.g., by
subjecting a bone substrate to acid, e.g., hydrochloric acid (HCl).
Demineralized bone matrix is also commercially available.
[0066] In some embodiments, the cartilage particles are combined
with cells such as stem cells. In some embodiments, the cartilage
particles are combined with a bone or cartilage substrate that is
seeded with stem cells. For example, in some embodiments, the
cartilage particles are combined with a bone or cartilage substrate
(e.g., cortical and/or cancellous bone substrate, demineralized
cortical and/or cancellous bone substrate, an osteochondral
substrate, or a cartilage substrate) that is seeded with
mesenchymal stem cells. Stem cell-seeded bone and cartilage
substrates and methods of preparing such substrates are described
in U.S. 2010/0124776 and U.S. application Ser. No. 12/965,335, the
contents of each of which are incorporated by reference herein.
III. Methods of Manufacturing Cartilage Compositions
[0067] In another aspect, methods of manufacturing cartilage
compositions are provided. In some embodiments, the method
comprises: [0068] obtaining cartilage tissue from a human adult
cadaveric donor; [0069] mincing the cartilage tissue into a
plurality of cartilage particles, wherein the cartilage particles
comprise viable chondrocytes; and [0070] suspending the plurality
of cartilage particles in a biocompatible medium.
[0071] In some embodiments, on average at least 50% of the
chondrocytes in the cartilage particles are viable. In some
embodiments, an average at least 50%, at least 55%, at least 60%,
at least 65%, at least 70%, at least 75%, at least 80%, at least
85% or more of the chondrocytes in the cartilage particles are
viable. In some embodiments, the cartilage particles comprise at
least about 50,000, at least about 60,000, at least about 70,000,
at least about 80,000, at least about 90,000, at least about
100,000, at least about 150,000, at least about 200,000, at least
about 250,000, at least about 300,000, at least about 350,000, at
least about 400,000, at least about 450,000, at least about
500,000, at least about 550,000, at least about 600,000, at least
about 650,000, at least about 700,000, at least about 750,000, at
least about 800,000, at least about 850,000, at least about
900,000, at least about 950,000, or at least about 1 million viable
chondrocytes per cubic centimeter (cc). In some embodiments, the
average chondrocyte viability or the amount of chondrocytes per cc
is measured on day 1 following from the day of cutting. The amount
of chondrocytes and/or number of viable chondrocytes in a cartilage
particle sample can be measured as described herein, for example as
described in Section II above.
[0072] In some embodiments, the cartilage tissue is harvested from
an adult cadaveric donor that is 18 years of age or older at the
time of the donation. In some embodiments, the cartilage tissue is
harvested from an adult cadaveric donor that is between the ages of
15 and 36 at the time of the donation. Tissue can be harvested from
any cartilaginous region of the cadaveric donor. In some
embodiments, cartilage is harvested from the knee joint of the
donor or from a long bone. In some embodiments, articular cartilage
is harvested from the donor. In some embodiments, the cartilage
that is obtained from the donor is sliced to a thickness of about
0.25 mm to about 5 mm (e.g., about 0.25 mm, about 0.5 mm, about
0.75 mm, about 1 mm, about 1.5 mm, about 2 mm, about 2.5 mm, about
3 mm, about 3.5 mm, about 4 mm, about 4.5 mm, or about 5 mm, or
from about 0.5 mm to about 2 mm) before the mincing step.
[0073] In some embodiments, the cartilage tissue is minced by hand.
In some embodiments, the cartilage tissue is minced using a cutting
mechanism. In some embodiments, the cutting mechanism is a laser
cutting apparatus, a mechanical blade, a manual cutting apparatus,
a manual pressing apparatus, or the like. In some embodiments, the
cutting mechanism comprises a pneumatic press, such as an air press
or an oil press, or a screw press.
[0074] In some embodiments, the cartilage tissue is minced using a
laser cutting apparatus. For example, in some embodiments, the
laser cutting apparatus is a laser engraver. Non-limiting examples
of suitable engraving lasers include CO.sub.2 engraving lasers,
such as the Epilog Zing 30 Watt CO.sub.2 engraving laser. In some
embodiments, the mincing step comprises cutting the cartilage
tissue with the laser cutting apparatus at a speed from about 10%
to about 50% (e.g., about 10%, about 15%, about 20%, about 25%,
about 30%, about 35%, about 40%, about 45%, or about 50%), a power
from about 0% to about 45% (e.g., about 0%, about 1%, about 2%,
about 5%, about 10%, about 15%, about 20%, about 25%, about 30%,
about 35%, about 40%, or about 45%), and a frequency from about 10
Hz to about 2400 Hz (e.g., about 10 Hz, about 20 Hz, about 30 Hz,
about 40 Hz, about 50 Hz, about 60 Hz, about 70 Hz, about 80 Hz,
about 90 Hz, about 100 Hz, about 150 Hz, about 200 Hz, about 250
Hz, about 300 Hz, about 350 Hz, about 400 Hz, about 450 Hz, about
500 Hz, about 550 Hz, about 600 Hz, about 650 Hz, about 700 Hz,
about 750 Hz, about 800 Hz, about 850 Hz, about 900 Hz, about 950
Hz, about 1000 Hz, about 1100 Hz, about 1200 Hz, about 1300 Hz,
about 1400 Hz, about 1500 Hz, about 1600 Hz, about 1700 Hz, about
1800 Hz, about 1900 Hz, about 2000 Hz, about 2100 Hz, about 2200
Hz, about 2300 Hz, or about 2400 Hz). In some embodiments, the
mincing step comprising cutting the cartilage tissue with the laser
cutter at a speed from about 10% to about 50%, a power from about
0% to about 45%, and a frequency from about 10 Hz to about 2400 Hz.
In some embodiments, the mincing step comprising cutting the
cartilage tissue with the laser cutter at a speed from about 20% to
about 35%, a power from about 2% to about 45%, and a frequency from
about 400 Hz to about 2400 Hz. In some embodiments, the mincing
step comprises cutting the cartilage tissue with the laser cutting
apparatus at a speed from about 25% to about 35%, a power from
about 20% to about 45%, and a frequency from about 1400 Hz to about
2400 Hz. Suitable speeds, powers, and frequencies for cutting the
cartilage tissue are shown in Table 1.
[0075] According to some embodiments, small cartilage pieces can be
created by laser cutting at a certain energy level without
sacrificing cell viability. For example, as surprisingly
demonstrated herein, cartilage tissue can be cut using a laser
cutter to yield cartilage particles in which at least about at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85% or more of the
chondrocytes in the cartilage particles are viable. In some
embodiments, a laser cutting mechanism is used to produce pieces of
shaved cartilage. In some embodiments, a laser cutter is used to
mince the cartilage tissue into particles having an average length
and/or an average width of about 1 mm to about 5 mm (e.g., about 1
mm, about 1.5 mm, about 2 mm, about 2.5 mm, about 3 mm, about 3.5
mm, about 4 mm, about 4.5 mm, or about 5 mm, or from about 1 mm to
about 3 mm). In some embodiments, a laser cutter is used to mince
the cartilage tissue into particles having an average diameter of
about 1 mm to about 5 mm (e.g., about 1 mm, about 1.5 mm, about 2
mm, about 2.5 mm, about 3 mm, about 3.5 mm, about 4 mm, about 4.5
mm, or about 5 mm, or from about 1 mm to about 3 mm). In some
embodiments, a laser cutter is used to mince the cartilage tissue
into particles having an average volume of about 0.5 mm.sup.3 to
about 100 mm.sup.3 (e.g., about 0.5 mm.sup.3, about 1 mm.sup.3,
about 2 mm.sup.3, about 3 mm.sup.3, about 4 mm.sup.3, about 5
mm.sup.3, about 6 mm.sup.3, about 7 mm.sup.3, about 8 mm.sup.3,
about 9 mm.sup.3, about 10 mm.sup.3, about 15 mm.sup.3, about 20
mm.sup.3, about 25 mm.sup.3, about 30 mm.sup.3, about 35 mm.sup.3,
about 40 mm.sup.3, about 45 mm.sup.3, about 50 mm.sup.3, about 60
mm.sup.3, about 70 mm.sup.3, about 80 mm.sup.3, about 90 mm.sup.3,
or about 100 mm.sup.3, e.g., from about 0.5 mm.sup.3 to about 30
mm.sup.3, from about 1 mm.sup.3 to about 30 mm.sup.3, or from about
1 mm.sup.3 to about 25 mm.sup.3).
[0076] In some embodiments, the mincing step comprises cutting the
cartilage tissue into circles, spheres, squares, rectangles, cubes,
cylinders, strips, sheets, ribbons, zig-zag or accordion shapes,
tiles (e.g. particles that are partially attached to other
particles), or other desired shapes. In some embodiments, the
mincing step comprises cutting the cartilage tissue (e.g., using a
laser cutter) into tiled or mosaic configurations, for example as
shown in FIG. 1D.
Forming Perforated Cartilage
[0077] In some embodiments, the cartilage tissue is incompletely
cut so as to form channels or microperforations that separate the
cartilage tissue into smaller cartilage portions. For example,
laser or other or cutting disruption means can be used to create
microperforations, channels, bores, apertures, and other passages
from one side of a cartilage construct to another side, or through
individual blocks or segments of a tiled cartilage construct. Thus,
in some embodiments, the method comprises: [0078] obtaining
cartilage tissue from a human adult cadaveric donor; [0079]
processing the cartilage tissue to form a cartilage construct
comprising one or more microperforations or channels that separates
the cartilage construct into a plurality of smaller cartilage
portions, wherein the cartilage construct comprises viable
chondrocytes; and [0080] suspending the cartilage construct in a
biocompatible medium.
[0081] In some embodiments, the processing step comprises
perforating the cartilage tissue with a laser cutter to form the
one or more microperforations or channels. In some embodiments, the
processing step comprises cutting the cartilage tissue with the
laser cutting apparatus at a speed from about 20% to about 30%
(e.g., about 20%, about 25%, or about 30%), a power from about 0%
to about 8% (e.g., about 0%, about 1%, about 2%, about 5%, about
6%, about 7%, or about 8%), and a frequency from about 10 Hz to
about 750 Hz (e.g., about 10 Hz, about 20 Hz, about 30 Hz, about 40
Hz, about 50 Hz, about 60 Hz, about 70 Hz, about 80 Hz, about 90
Hz, about 100 Hz, about 150 Hz, about 200 Hz, about 250 Hz, about
300 Hz, about 350 Hz, about 400 Hz, about 450 Hz, about 500 Hz,
about 550 Hz, about 600 Hz, about 650 Hz, about 700 Hz, or about
750 Hz). In some embodiments, the processing step comprises
separating the cartilage construct into a plurality of smaller
cartilage portions, wherein each cartilage portion has an average
length and/or an average width of about 1 mm to about 5 mm (e.g.,
about 1 mm, about 1.5 mm, about 2 mm, about 2.5 mm, about 3 mm,
about 3.5 mm, about 4 mm, about 4.5 mm, or about 5 mm). In some
embodiments, the processing step comprises separating the cartilage
construct into a plurality of smaller cartilage portions, wherein
each cartilage portion has an average diameter of about 1 mm to
about 5 mm (e.g., about 1 mm, about 1.5 mm, about 2 mm, about 2.5
mm, about 3 mm, about 3.5 mm, about 4 mm, about 4.5 mm, or about 5
mm).). In some embodiments, the processing step comprises
separating the cartilage construct into a plurality of smaller
cartilage portions, wherein each cartilage portion has an average
volume of from about 0.5 mm.sup.3 to about 100 mm.sup.3 (e.g.,
about 0.5 mm.sup.3, about 1 mm.sup.3, about 2 mm.sup.3, about 3
mm.sup.3, about 4 mm.sup.3, about 5 mm.sup.3, about 6 mm.sup.3,
about 7 mm.sup.3, about 8 mm.sup.3, about 9 mm.sup.3, about 10
mm.sup.3, about 15 mm.sup.3, about 20 mm.sup.3, about 25 mm.sup.3,
about 30 mm.sup.3, about 35 mm.sup.3, about 40 mm.sup.3, about 45
mm.sup.3, about 50 mm.sup.3, about 60 mm.sup.3, about 70 mm.sup.3,
about 80 mm.sup.3, about 90 mm.sup.3, or about 100 mm.sup.3).
[0082] In some embodiments, such microperforations or passages may
be on the order of tens of microns in dimension, or less. In some
embodiments, such microperforations or passages may be on the order
of millimeters in dimension, or less.
Further Processing Steps
[0083] In some embodiments, following the mincing step, the
cartilage particles or constructs can be subjected to one or more
additional processing steps prior to suspending the cartilage
particles in the biocompatible carrier. In some embodiments, the
cartilage particles are washed with a saline solution. In some
embodiments, the cartilage particles are treated with one or more
enzymes that promote the release of chondrocyte cells from
cartilage matrix. For example, collagenase can be applied to help
release chondrocyte cells from the cartilage matrix of the tissue
particles. In some embodiments, the cartilage particles are mixed
with collagenase and/or pronase and incubated in a growth medium
such as Dulbecco's Modified Eagle's Medium (DMEM) for a suitable
length of time for releasing the chondrocytes.
[0084] In some embodiments, the cartilage particles are combined
with demineralized bone matrix. For example, in some embodiments
the cartilage particles are combined with demineralized bone matrix
at a ratio of about 1 cc:4 cc demineralized bone matrix:cartilage
particles to about 1 cc:1 cc demineralized bone matrix:cartilage
particles (e.g., about 4:1, about 3:1, about 2:1, or about 1:1 cc
demineralized bone matrix:cartilage particles). Demineralized bone
matrix can be prepared, e.g., by subjecting a bone substrate to
acid, e.g., hydrochloric acid (HCl). Demineralized bone matrix is
also commercially available.
[0085] In some embodiments, the cartilage particles are combined
with cells such as stem cells. In some embodiments, the cartilage
particles are combined with a bone or cartilage substrate that is
seeded with stem cells. For example, in some embodiments, the
cartilage particles are combined with a bone or cartilage substrate
(e.g., cortical and/or cancellous bone substrate, demineralized
cortical and/or cancellous bone substrate, an osteochondral
substrate, or a cartilage substrate) that is seeded with
mesenchymal stem cells. Stem cell-seeded bone and cartilage
substrates and methods of preparing such substrates are described
in U.S. 2010/0124776 and U.S. application Ser. No. 12/965,335, the
contents of each of which are incorporated by reference herein.
[0086] In some embodiments, the cartilage particles are combined
with a biological adhesive. Suitable biological adhesives include,
but are not limited to, fibrin, fibrinogen, thrombin, fibrin glue
(e.g., TISSEEL), polysaccharide gel, cyanoacrylate glue,
gelatin-resorcin-formalin adhesive, collagen gel, synthetic
acrylate-based adhesive, cellulose-based adhesive, MATRIGEL.RTM.
(BD Biosciences, San Jose, Calif.), laminin, elastin,
proteoglycans, and combinations thereof.
[0087] In some embodiments, the cartilage particles are suspended
in a biocompatible carrier. In some embodiments, the biocompatible
carrier comprises a buffered solution (e.g., an aqueous buffered
solution). In some embodiments, the biocompatible carrier comprises
a cryopreservation medium. In some embodiments, the
cryopreservation medium comprises dimethyl sulfoxide (DMSO) and
serum. In some embodiments, the biocompatible carrier comprises one
or more cryoprotective agents such as, but not limited to,
glycerol, DMSO, hydroxyethyl starch, polyethylene glycol,
propanediol, ethylene glycol, butanediol, or
polyvinylpyrrolidone.
IV. Therapeutic Uses of Cartilage Compositions
[0088] The cartilage compositions described herein can be used to
treat subjects in need thereof. Without being bound to a particular
theory, it is believed that the methods of mincing cartilage
described herein can facilitate the migration of cells out of the
cartilage. When cartilage particles are administered to a subject,
chondrocytes can migrate out of the minced pieces and carry out
repair and regeneration functions. For example, the chondrocytes
can reproduce and form new cartilage via chondrogenesis. In this
way, minced cartilage which is applied to a site within a patient
can be used to treat cartilage and/or bone defects. For example,
chondrocytes from the minced cartilage pieces can reproduce and
generate new cartilage in situ. The newly established chondrocyte
population and cartilage tissue can fill defects, and integrate
with existing native cartilage and/or subchondral bone at the
treatment site.
[0089] In some embodiments, the cartilage compositions described
herein are administered to a subject having a bone or cartilage
defect. In some embodiments, the composition is administered to a
defect in cartilage, bone, ligament, tendon, meniscus, joint, or
muscle. In some embodiments, the subject has a degenerative defect
or injury. In some embodiments, the subject has a traumatic defect
or injury. In some embodiments, the subject has osteoarthritis. In
some embodiments, the subject has a muscle defect.
[0090] In some embodiments, the cartilage compositions described
herein are administered to a subject to repair cartilage or promote
cartilage growth or regeneration in the subject. In some
embodiments, the composition is administered to a joint (e.g., knee
joint), to bone (e.g., femur or humerus), or to cartilage.
[0091] In some embodiments, the cartilage compositions described
herein are administered to a subject having soft tissue defects,
for the repair and regeneration thereof. In some embodiments, the
composition is administered to a ligament, tendon, or muscle. In
some embodiments, the soft tissue defect is a sprain, strain,
contusion, or stress injury to a ligament, tendon, or muscle.
[0092] In some embodiments, a cartilage composition as described
herein is administered locally to the subject. In some embodiments,
the composition is surgically implanted in the subject. In some
embodiments, the composition is administered in a minimally
invasive procedure, e.g., arthroscopy.
V. Kits
[0093] In still another aspect, kits comprising a cartilage
composition as described herein are provided. In some embodiments,
the kit comprises a composition comprising a plurality of cartilage
particles from a human adult cadaveric donor, wherein the cartilage
particles comprise viable chondrocytes; and a biocompatible
carrier. In some embodiments, the kit comprises a composition
comprising cartilage particles having an average thickness from
about 0.25 mm to about 5 mm; having an average length, width, or
diameter from about 1 mm to about 5 mm; and/or having an average
volume of from about 0.5 mm.sup.3 to about 100 mm.sup.3.
[0094] In some embodiments, the kits are used for treating a
subject having a defect in cartilage, bone, ligament, tendon,
meniscus, joint, or muscle. In some embodiments, the kits are used
for treating a subject having a degenerative defect or injury
cartilage, bone, ligament, tendon, meniscus, joint, or muscle; a
subject having a traumatic defect or injury cartilage, bone,
ligament, tendon, meniscus, joint, or muscle; or a subject having
osteoarthritis.
[0095] In some embodiments, a kit comprises a cartilage composition
as described herein packaged in a container for storage and/or
shipment. In some embodiments, the kit further comprises
instructions for administering the composition.
[0096] In some embodiments, a kit comprises a composition
comprising cartilage particles as described herein, optionally
along with biological adhesive components (e.g. fibrinogen and
thrombin, for a fibrin glue). In some embodiments, cartilage
particles and biological adhesive (e.g., fibrin glue) components
are packaged separately, and a surgeon or user adds the fibrin glue
to the surgery site prior to placement of the cartilage. In some
embodiments, the biological adhesive (e.g., fibrin glue) is
combined with the cartilage particles prior to administration at
the treatment site.
[0097] In some instances, a kit comprises the packaged cartilage
particles with bone and/or stem cell components. For example, in
some embodiments, a kit comprises cartilage particles with
demineralized bone matrix. In some embodiments, a kit comprises
cartilage particles with cells (e.g., stem cells). In some
embodiments, a kit comprises cartilage particles with a bone or
cartilage substrate seeded with cells (e.g., adipose derived
mesenchymal adult stem cells combined with a bone substrate, as
described in U.S. 2010/0124776, or adipose derived mesenchymal
adult stem cells combined with an osteochondral or cartilage
substrate, as described in U.S. application Ser. No.
12/965,335).
VI. EXAMPLES
[0098] The following examples are offered to illustrate, but not to
limit, the claimed invention.
Example 1
Laser Cutting to Generate Minced Cartilage
[0099] Laser cutting techniques can provide a cost effective
approach for the preparation of minced cartilage particles, with a
decreased opportunity for tissue contamination during the mincing
process. As described below, minced cartilage particles, tiles,
mosaics, and the like as prepared by laser processing techniques
showed cell viability results that were comparable to the cell
viability results observed when using manual cutting techniques. By
using a laser to prepare minced particles, cost, contamination, and
processing time can be reduced. Further, it is possible to provide
increased amounts of donor tissue product.
[0100] Tissue cutting experiments were performed using an Epilog
Zing 30 Watt CO2 engraving laser on juvenile or adult cartilage
slices. Table 1 shows the results of the tissue cutting experiments
at varying speeds, powers, and frequencies.
TABLE-US-00001 TABLE 1 Laser Settings Laser Settings Speed (%)
Power (%) Frequency (Hz) Result/outcome: A. Low Range Settings
Test: 2 mm square pattern cut, 1 mm thick samples used 30 10 1350
Etches tissue, no burning, doesn't cut entirely through(mosaic) 30
10 1000 Etches tissue, no burning, doesn't cut entirely
through(mosaic) 30 10 750 Etches tissue, no burning, doesn't cut
entirely through(mosaic) 30 8 750 Some browning of tissue,
perforations through tissue 25 8 750 Completely cut through tissue,
some brown edges 25 8 650 Completely cut through tissue, some brown
edges 25 8 400 Completely cut through tissue, no browning 25 5 400
Etched tissue, some browning, does not cut entirely through 25 5
300 Etched tissue, no browning, does not cut entirely through 20 5
300 Etched tissue, no browning, nearly complete full thickness cut
20 2 300 Etched tissue, no browning, does not cut entirely through
20 0 300 Etched tissue, no browning, etching not very deep 20 2 200
Etched tissue, no browning, nearly complete full thickness cut 20 2
100 Etched tissue, no browning, nearly complete full thickness cut
20 2 50 Etched tissue, no browning, nearly complete full thickness
cut 20 2 25 Etched tissue, no browning, nearly complete full
thickness cut with perforations through tissue 20 2 10 Perforations
(full thickness) only through tissue no complete etched line 20 1
10 Perforations only, not a full thickness cut 20 0 10 Perforations
only, not a full thickness cut 10 0 10 Laser very slow moving,
tissue etched with perforations(full thickness), no solid line cut
B. High Range Settings Test: 2 mm square pattern cut, 1 mm thick
samples used 30 30 2000 Some browning of edges, complete cut full
thickness cut 35 30 2000 Less browning than above settings,
complete full thickness cut 35 35 2000 Some browning of edges,
complete cut full thickness cut 35 35 2200 Some browning of edges,
complete cut full thickness cut 35 40 2200 Some browning of edges,
complete cut full thickness cut 35 40 2400 Browning of edges,
complete full thickness cut 35 45 2400 dark brown edges, complete
cut through
[0101] Based at least in part upon these findings, it was
determined that laser settings at 25-35% speed, 2-45% power, and
400-2400 Hz frequency provide desirable results for mincing
cartilage.
Example 2
Characterization of Minced Articular Cartilage From Adult or
Juvenile Donors
[0102] Fresh cadaveric adult and juvenile articular cartilage
tissue samples were processed using either a laser cutting protocol
or a hand cutting protocol. The adult donors were between fifteen
and thirty six years of age, and the juvenile donors were between
the ages of three months and 12 years. For the laser cutting
method, the cartilage was shaved into thin slices (e.g., sheets
having a thickness of 1-5 mm) using a scalpel, and the sliced
sheets were minced into small particles (e.g., 1 mm, 2 mm, and/or 3
mm particles) using an Epilog Zing 30 Watt engraving laser. The
laser cutting pattern was designed with a CorelDRAW.RTM. graphics
software program. The cartilage was minced into square shaped
particles, using energy levels and other laser parameters as
described in Table 1. During the laser cutting procedure, the
cartilage was maintained in a hydrated state. The minced particles
were then washed with a phosphate buffered saline (PBS)
solution.
[0103] Cartilage particles were characterized for cell count, cell
viability, and chondrocyte growth as described below.
[0104] Using samples having known concentrations of chondrocytes, a
standard curve was prepared as shown in FIG. 2. The y-axis
represents fluorescence readings from a Countess.RTM. automated
cell counter, and the x-axis represents the chondrocyte
concentration (cells/.mu.l).
[0105] Cell Counting, Donors A (Adult) and B (Juvenile), Day
One:
[0106] Some of the harvested chondrocytes were tested for cell
count on the day of mincing (day 1) using a Trypan blue staining
protocol followed by analysis in a Countess.RTM. automated cell
counter. Cartilage particles were digested with collagenase to
isolate chondrocytes, and that mixture was then filtered through a
105 micron filter to separate any undigested matrix from the
isolated cells. For the experiments illustrated by FIGS. 3A and 3B,
equal amounts of chondrocyte samples were placed in the individual
plate wells for evaluation.
[0107] As depicted in FIG. 3A, adult donor cartilage tissue that
was minced with laser cutting provided a mean fluorescence reading
of 21,636 (Std. Dev. 578; CV % 2.67), which corresponds to a cell
count of 42,622 chondrocytes/.mu.l, using the standard curve of
FIG. 2. The adult donor cartilage tissue that was minced with hand
cutting provided a mean fluorescence reading of 24,853 (Std. Dev.
1507; CV % 6.06), which corresponds to a cell count of 52,642
chondrocytes/.mu.l. As depicted in FIG. 3B, juvenile donor
cartilage tissue that was minced with laser cutting provided a mean
fluorescence reading of 27,528 (Std. Dev. 2494; CV % 9.06), which
corresponds to a cell count of 60,974 chondrocytes/.mu.l. The
juvenile donor cartilage tissue that was minced with hand cutting
provided a mean fluorescence reading of 41,088 (Std. Dev. 3472; CV
% 8.45), which corresponds to a cell count of 103,211
chondrocytes/.mu.l. Based on these results, it was observed that in
terms of cell count, there may be no large differences between the
laser cutting and hand cutting methods.
[0108] FIG. 4 shows mean fluorescence readings as described above.
The numbers were calculated using a standard curve and the
fluorescence reading from a Presto Blue metabolic assay when
evaluated in the plate reader. Six week cell counts were also
performed using a Presto Blue assay.
[0109] Cell Counting, Donors C to G (Six Week):
[0110] To compare how chondrocytes from both adult and juvenile
cartilage grow out of the cartilage matrix, a 6-week explant study
was conducted. Three research-consented adult donors (donors C, E,
and G) and two research-consented juvenile donors (donors D and F)
were obtained. Samples were cut into sheets approximately 1 mm
thick and minced by hand or laser cut into 2 mm cubes and measured
into 0.3 ml aliquots. Cartilage particles were placed into plate
wells along with TISSEEL fibrin glue (Baxter, Deerfield, Ill.),
which provided a support from which the chondrocytes could grow out
of the cartilage samples. No collagenase was used on the cells.
Chondrocyte media (Cell Applications, San Diego, Calif.) was then
added and changed twice weekly.
[0111] Cell counting was conducted after six weeks using either (A)
a Trypan Blue staining protocol followed by analysis in a
Countess.RTM. automated cell counter, or (B) a Presto Blue staining
protocol followed by analysis in a Synergy.TM. H1 hybrid plate
reader. The Presto Blue protocol involves an indirect chondrocyte
cell count, using a metabolic assay. The cell count is performed by
using a standard curve of known concentrations of chondrocytes to
determine the count in the unknown samples. Typically, where the
chondrocytes are combined with fibrin, a metabolic assay and hybrid
reader can be used to indirectly determine the chondrocyte cell
count, by evaluating the metabolic activity. Here, it may be
assumed that a majority of the cells (e.g., 95% to 98% or more) are
viable.
[0112] FIG. 5 shows the live cell number count and viability
results for the Trypan Blue protocol, and the live cell count
number results for the Presto Blue protocol. As depicted in the
Trypan Blue live cell test results, there were 1,052,167.+-.989,536
of live cells per cc of fresh cartilage using laser cutting, and
375,333.+-.295,846 live cells per cc of fresh cartilage using hand
cutting.
[0113] FIG. 6 shows the live cell count number results for the
Trypan Blue and Presto Blue protocols, and is based on cell count
data shown in FIG. 5. With regard to the Trypan Blue and Presto
Blue cell count results shown here, a single ANOVA analysis was
performed and there was no significant difference using these two
methods regarding live cell number.
[0114] Cell Counting, Donors C to G:
[0115] FIG. 7 shows day 1 (i.e., one day after cutting) cell
viability assay for Donors C to G using the Trypan Blue protocol,
which are based on the viability % results depicted in FIG. 5. As
depicted here, the average cell viability is about 86% for both
laser cut cartilage and hand cut cartilage. Hence, it was observed
that cartilage tissue can be minced with laser cutting, without
sacrificing cell viability relative to hand cutting methods. With
regard to the Trypan Blue viability results shown in FIG. 7, a
single ANOVA analysis was performed and there was no significant
difference using these two methods regarding cell viability.
[0116] FIGS. 8A and 8B are confocal microscope images depicting
tissue edges (white arrow) of hand cut and laser cut (respectively)
cartilage pieces. These results indicate that there was not a
significant difference of cell viability when comparing laser cut
and hand cut cartilage tissue samples. For this study,
LIVE/DEAD.RTM. stain (Life Technologies, Carlsbad, Calif.) was
used. Briefly, undigested cartilage particles were placed in wells
of a 24-well plate. 1 ml PBS was added to each well and 0.5 .mu.l
of the red and green dye was then added. The plates were covered
with foil and allowed to sit for a minimum of 15 minutes. The
cartilage particles were then placed on slides and the images
captured by confocal microscopy on the laser setting.
[0117] It was also observed that laser cutting could be
accomplished more quickly than hand cutting. For example, an
equivalent amount of tissue could be minced in 8 hours via manual
cutting, versus 0.5 hours via laser cutting. Moreover, it was
observed that it was easier to obtain uniformly shaped tissue
pieces using laser cutting, as compared with hand cutting.
[0118] Microscopy Observations at Eighteen Days:
[0119] FIGS. 9A and 9B provide photographic images of chondrocyte
cells growing out of hand cut (FIG. 9A) and laser cut (FIG. 9B)
adult cartilage particles. Specifically, cartilage was obtained
from an adult donor, and minced with either laser cutting or manual
cutting protocols. The minced cartilage particles were placed in 12
well culture plates, using chondrocyte growth medium with 10% FBS
and 2% antibiotic. The media was changed twice a week. The plates
were cultured in a 37.degree. C. incubator with 5% CO.sub.2 (e.g.
standard cell culture conditions). The images (4.times.
magnification) were obtained at 18 days. As shown here,
chondrocytes were observed to grow out of the minced particles.
[0120] Alcian Blue Staining at Six Weeks:
[0121] After six week of culture, samples were fixed and stained
using Alcian Blue (IHC world, Woodstock, Md.) to show
glycosaminoglycan content. As shown in FIG. 11, both adult laser
cut cartilage particles and juvenile laser cut cartilage particles
stained positive for the presence of glycosaminoglycans after 6
weeks.
Example 3
12-Week Explant Study to Characterize Cartilage Samples
[0122] To further compare chondrocyte outgrowth and matrix
production between adult and juvenile donors, a 12-week explant
study was performed. Three research consented adult donors and two
research consented juvenile donors were obtained. Samples were
sliced by hand into 1 mm thick sheets and laser cut into 2 mm
cubes. The samples were measured into 0.3 ml aliquots (5 samples
per donor) and glued to a 12 well plate using TISSEEL (Baxter,
Deerfield, Ill.) for a 12 week explant study to be performed. A
1:10 ratio of PrestoBlue.RTM. (Life Technologies, Carlsbad, Calif.)
to media was used for weekly cell counting. Collagen type II
immunohistochemistry was performed on samples after the 12 week
time point, as well as sulfated glycosaminoglycans (sGAG) assay
(Kamiya Biomedical Company, Seattle, Wash.), hydroxyproline assay
(BioVison, Milpitas, Calif.), and DNA analysis with a Pico Green
Assay (Invitrogen, Grand Island, N.Y.). All outcome measures were
evaluated using single ANOVA analysis. Significance was considered
as p<0.05.
[0123] Results:
[0124] The 12-week study confirmed a similar trend of cell
outgrowth and matrix production as was demonstrated in the 6-week
explant study. The results of the hydroxyproline assay, Pico Green
assay, and sGAG assay are presented in Table 2 below.
TABLE-US-00002 TABLE 2 Assay results Result Standard Deviation
Statistically Assay Adult Juvenile Adult Juvenile P-value
Different? Hydroxyproline 17.1413 13.48556 0.215065 0.997325 0.9 NO
(ug/well) DNA (ng/mL) 3773.414 4168.478 677.499 365.6574 0.87 NO
sGAG(ug/mL) 268929 242163.9 9485.124 18392.75 0.985 NO
[0125] A hydroxyproline assay was used to determine the content of
collagen in the explants; since about 13% of cartilage is
hydroxyproline, the content was divided by 0.13 to obtain the
collagen content. As shown in Table 2, adult donors had a total
collagen content of 17.14.+-.1.65 mg/ml. Juvenile donors had a
total of 13.48.+-.7.67 mg/ml, resulting in no statistical
difference. sGAGS are an important component of healthy cartilage
and can decrease with age and lead to the development of
osteoarthritis. sGAG content for adult cartilage was 268929.+-.9485
.mu.g/mL, while sGAG content for juvenile donors was
242163.9.+-.18392 .mu.g/mL of sGAG, showing that sGAG content has
no statistical difference. DNA content was calculated to estimate
the total number of cells, based on the assumption that there are
approximately 6 pg DNA per cell. After the 12-week explant study,
adult donors had an average of 628902.+-.112916 cells and juvenile
donors had an average of 694746.+-.60942 cells, showing that the
total number of cells in adult and juvenile donors after 12 weeks
of outgrowth was not statistically different. Collagen Type II IHC
staining showed that both groups have type II collagen allowing for
hyaline cartilage production (FIG. 12).
[0126] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, one of skill in the art will appreciate that
certain changes and modifications may be practiced within the scope
of the appended claims. In addition, each reference provided herein
in incorporated by reference in its entirety to the same extent as
if each reference was individually incorporated by reference.
* * * * *