U.S. patent application number 10/066992 was filed with the patent office on 2002-09-19 for cell-culture and polymer constructs.
Invention is credited to Domb, Abraham J., Frondoza, Carmelita G., Hungerford, David S., Shikani, Alan H., Sohrobi, Afshin.
Application Number | 20020133235 10/066992 |
Document ID | / |
Family ID | 23051776 |
Filed Date | 2002-09-19 |
United States Patent
Application |
20020133235 |
Kind Code |
A1 |
Hungerford, David S. ; et
al. |
September 19, 2002 |
Cell-culture and polymer constructs
Abstract
Cells grown on a microcarrier are separated from the
microcarrier by enzymatically digesting the microcarrier. More
specifically, chondrocytes may be grown on dextran microcarrier
beadlets and then the beadlets digested using dextranase to
separate the chondrocytes from the carrier. Cells can also be grown
on chitosan microcarriers to be used for implantation. In addition,
cells can be grown on polysaccharide polymers to be used as implant
devices. Various polymers serve as scaffolds for cells to be used
for implantation. The polymers can be used for cell culture as well
as for preparing scaffolds useful for tissue replacement such as
cartilage tissue.
Inventors: |
Hungerford, David S.;
(Cockeysville, MD) ; Frondoza, Carmelita G.;
(Woodstock, MD) ; Sohrobi, Afshin; (McLean,
VA) ; Shikani, Alan H.; (Ruxton, MD) ; Domb,
Abraham J.; (Efrat, IL) |
Correspondence
Address: |
ARMSTRONG, WESTERMAN & HATTORI, LLP
Leonard Bloom, Senior Counsel
Suite 220
502 Washington Avenue
Towson
MD
21204
US
|
Family ID: |
23051776 |
Appl. No.: |
10/066992 |
Filed: |
February 4, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10066992 |
Feb 4, 2002 |
|
|
|
09275319 |
Mar 24, 1999 |
|
|
|
6378527 |
|
|
|
|
60081016 |
Apr 8, 1998 |
|
|
|
60104842 |
Oct 20, 1998 |
|
|
|
Current U.S.
Class: |
623/23.63 ;
435/396; 623/908; 623/919 |
Current CPC
Class: |
A61K 35/12 20130101;
A61K 47/36 20130101; C12N 5/0655 20130101; C12N 2531/00 20130101;
C08B 37/006 20130101; C12N 2533/70 20130101; C08H 1/00 20130101;
A61K 9/1652 20130101; A61L 2430/06 20130101; C08B 37/00 20130101;
C12N 5/0654 20130101; C08B 15/005 20130101; C08B 37/0021 20130101;
A61L 27/3852 20130101; C08J 3/12 20130101; C12N 2533/72 20130101;
A61F 2/08 20130101; A61L 27/3895 20130101; A61F 2/30756 20130101;
A61L 27/3817 20130101; C12N 2500/02 20130101; C08B 33/00 20130101;
C08J 2305/08 20130101; A61K 9/5036 20130101; C12N 5/0075
20130101 |
Class at
Publication: |
623/23.63 ;
623/908; 623/919; 435/396 |
International
Class: |
A61F 002/28 |
Claims
1. In a method of cell culture wherein cells are cultured on
microcarrier beadlets the improvement comprising growing the cells
on dextran microcarrier beadlets and thereafter separating the
cells from the microcarrier beadlets by digestion with
dextranase.
2. The method of claim 1 wherein the cells are a member of the
group consisting of chondrocytes, oesteocytes, oesteoblasts,
monocytes, chondrogenic cells, pluripotential cells, mucosal cells
and fibroblasts.
3. The method of claim 2 wherein the cells are chondrocytes or
chondrogenic cells.
4. The method of claim 3 wherein after separating the chondrocytes
or chondrogenic cells from the microcarrier beadlets the cells are
plated on a scaffold or template for growth thereon.
5. The method of claim 4 wherein after the cells are plated on a
scaffold or template for growth thereon, the scaffold or template
is surgically implanted into the cartilage tissue.
6. The method of claim 1 wherein after separating the cells from
the microcarrier beadlets the cells are injected into a patient in
need of said cells.
7. A crosslinked polymer prepared by crosslinking a polysaccharide
with a polyamine said polymer to be used for cell culture and
preparing scaffolds or templates for tissue replacement.
8. The crosslinked polymer of claim 7 wherein the polysaccharide is
selected from the group consisting of arabinogalactan and oxidized
arabinogalactan; and the crosslinking agent is glutamic acid and
lysine, albumin, gelatin or lysine.
9. The crosslinked polymer of claim 7 wherein the polysaccharide is
selected from the group consisting of dextran, cellulose,
arabinogalactan, pollulan and amylose.
10. In a method of cell culture wherein cells are cultured on
microcarrier beadlets the improvement comprising growing the cells
on a polyamine cross-linked polysaccharide polymer of claim 7 and
thereafter separating the cells from the microcarrier beadlets by
digestion with an enzyme compatible with the polysaccharide.
11. The method of claim 10 wherein the cells are a member of the
group consisting of chondrocytes, chondrogenic cells, oesteoblasts,
monocytes, pluripotential cells, mucosal cells and fibroblasts.
12. The method of claim 11 wherein the cells are chondrocytes or
chondrogenic cells.
13. The method of claim 12 wherein after separating the
chondrocytes or chondrogenic cells from the microcarrier beadlets
the cells are plated on a scaffold or template for growth
thereon.
14. The method of claim 13 wherein after the cells are plated on
the scaffold or template for growth thereon, the scaffold or
template is surgically implanted into the cartilage tissue.
15. A beadlet for cell culture cell culture or a scaffold used for
cartilage repair comprising a crosslinked polysaccharide derivative
of the formula 7n is an integer of the 2 and 10,000; and R.dbd.H,
alkyl or alkylene, and the polysaccharide derivative being
crosslinked by a polyamine compound.
16. The beadlet for cell culture or the scaffold used for cartilage
repair of claim 15 wherein the polysaccharide used to prepare the
crosslinked polysaccharide derivative is a member selected from the
group consisting of dextran, arabinogalactan, pollulan, cellulose
and amylose; with the polysaccharide derivative being crosslinked
by a polyamine compound selected from the group consisting of
lysine, ethylenediamine, alkylenediamine, phenylenediamine,
xylenediamine, polyethylenimine, gelatin, albumin, fibrinogen and
8
17. A beadlet for cell culture or a scaffold used for cartilage
repair comprising a crosslinked polysaccharide derivative of the
formula 9n is an integer of between 2 and 10,000 and R.dbd.H, alkyl
or alkylene, and the polysaccharide derivative being crosslinked by
a polyamine compound.
18. The beadlet for cell culture, or the scaffold used for
cartilage repair of claim 17 wherein the polysaccharide used to
prepare the crosslinked polysaccharide derivative is a member
selected from the group consisting of dextran, arabinogalactan,
pollulan, cellulose and amylose; with the polysaccharide derivative
being crosslinked by a polyamine compound selected from the group
consisting of lysine, ethylenediamine, alkylenediamine,
phenylenediamine, xylenediamine, polyethylenimine, gelatin,
albumin, fibrinogen and 10
19. The method for the surgical of cartilage wherein the
crosslinked polysaccharide derivative of claim 15 in the form of a
scaffold containing chondrocytes thereon is surgically implanted
into the cartilage tissue of a patient.
20. The method for the surgical repair of cartilage wherein the
crosslinked polysaccharide derivative of claim 17 in the form of a
scaffold containing chondrocytes thereon is surgically implanted
into the cartilage tissue of a patient.
21. A scaffold or template for cartilage repair comprising a
chitosan scaffold or template with chondrocytes incorporated
thereon to produce a scaffold or template for cartilage repair
having viable chondrocytes.
22. A method for the surgical repair of cartilage tissue wherein
the chitosan scaffold or template with chondrocytes of claim 21 is
surgically incorporated into the cartilage tissue of a patient.
23. A scaffold or template for cartilage repair comprising a member
of the group consisting of chitosan and a crosslinked
polysaccharide prepared by crosslinking said polysaccharide with a
polyamine, said member being shaped in the form of a scaffold or
template and having plated and growing thereon chondrocytes.
24. In a surgical procedure for surgically implanting chondrocytes
into cartilage tissue wherein the scaffold or template having
plated and growing thereon the chondroctyes of claim 23 is
surgically implanted into the cartilage for cartilage repair.
25. In the surgical procedure of claim 24 wherein the scaffold or
template having plated and growing thereon chondroctyes is
surgically implanted into cartilage of the knee.
26. In the surgical procedure of claim 24 wherein the scaffold or
template having plated and growing thereon chondrocytes is
surgically implanted into cartilage of the head or neck area.
27. In the surgical procedure of claim 24 wherein the scaffold or
template having plated and growing thereon chondrocytes is
surgically implanted into the knee of a horse.
28. In a surgical method for surgically implanting chondrocytes
into cartilage tissue comprising the steps of: 1) culturing
chondrocytes on dextran microcarrier beadlets and thereafter
separating the chondrocytes form the microcarrier beadlets by
digestion with dextranase, 2) plating and growing said separated
chondrocytes on a scaffold or template manufactured from a
crosslinked polysaccharide prepared by crosslinking said
polysaccharide with a polyamine or alternatively plating and
growing said separated chondrocytes on a scaffold or template
comprising chitosan and then 3) surgically implanting into
cartilage tissue said scaffold or template having chondrocytes
plated and grown thereon, to thereby repair cartilage tissue in a
patient requiring cartilage repair.
29. In combination chitosan and a member of the group consisting of
osteoblasts and chondrocytes wherein the chitosan serves as a
substrate for the growth of said osteoblasts or chondrocytes.
30. The combination of claim 29 wherein the chitosan is in the form
of a scaffold to be used in transplantation surgery.
31. A method of culturing and obtaining increased levels of
collagen type II chondrocytes comprising culturing the chondrocytes
at a low oxygen tension.
32. The method of claim 31 wherein the oxygen tension is about
5%.
33. In a surgical procedure wherein chondrocytes are taken from a
patient rapidly multiplied and transplanted into said patient
comprising the steps of: 1) obtaining cartilage tissue from a
patient, 2) treating the cartilage to obtain chondrocytes
therefrom, 3) culturing the chondrocytes to obtain an adequate
sample of chondrocytes, 4) plating and growing the chondrocytes
onto a chitosan scaffold, and 5) surgically implanting the
chondrocytes bearing chitosan scaffold into said patient, wherein
the entire surgical procedure is carried out expeditiously with
good quality chondrocytes being returned to the patient and the
scaffold being reabsorbed leaving in place viable chondrocytes.
34. A method for preparing a scaffold for tissue repair comprising
culturing chondrocytes onto a chitosan scaffold wherein the
culturing takes place in the presence of integrins.
35. The method of preparing a scaffold for tissue repair comprising
culturing chondrocytes onto a chitosan scaffold wherein the
culturing takes place at a low oxygen tension under controlled
conditions in an antomated spin culture.
Description
RELATED APPLICATIONS
[0001] This application is related to provisional applications
Serial No. 60/081,016 filed Apr. 8, 1998 and Serial No. 60/104,842
filed Oct. 20, 1998.
FIELD OF THE INVENTION
[0002] The herein disclosed invention finds applicability in the
field of cell culture, as well as in the field of tissue substitute
for tissue replacement.
BACKGROUND
[0003] Attempts at replacing or rebuilding diseased or damaged
structures in the human body go back to 3000 B.C. It was not until
the middle of the 1900's, however, that the use of synthetic
materials for rebuilding body structures met with widespread and
reproducible success. Advances in material science and biomaterials
and science have afforded much of the success. The need for new and
better implants exists in every field of medicine in which disease
or trauma can be treated surgically.
[0004] As technology advances continue to improve the state of the
art, the standards for successful implants continue to improve
including strength, biocompatibility and elasticity. The new
research being conducted today on growth factors and controlled
drug release tell of the day when implant material will be expected
to promote healing, dissipate disease and stimulate tissue
regeneration. New horizons for implants and implantable materials
are being developed through the coordinated efforts of biomedical
engineers and surgeons who are best positioned to appreciate the
problems that patients are facing and that need to be
addressed.
[0005] Tissue engineering has opened a new frontier in the
development of implant materials for surgery. The surgical
specialties which are more apt to deal with cartilage grafts
substitutes and bone graft substitutes are the fields of
orthopedics, otolaryngology-head and neck surgery and facial
plastic reconstructive surgery. Cartilage and bone graft
substitutes can be used as temporary scaffolds in which new
cartilage and/or new bone may grow, encouraging tissue regeneration
rather than inert tissue replacement. The regeneration of tissue in
a controlled and predictable manner may be achieved with
biodegradable implants, where the scaffold or template will degrade
and be absorbed by the body, and the living cartilage or bone cells
will remain. Implants such as these will eliminate the concerns
about the long-term stability and safety of implant material
currently used for tissue augmentation, and obviate the concern
about long-term implant failure due to mismatch at the
implant-tissue interface.
[0006] Articular cartilage is frequently damaged in the course of
common activities. In addition, osteoarthritis leads to the initial
focal wear of-articular cartilage in many joints, particularly the
hip and the knee. There are approximately 500,000 total hip and
knee prostheses implanted in the United States every year and more
than 1,000,000 arthroscopies of the knee. In the case of the
replacements, the condition usually started with focal loss of
articular cartilage. Under normal circumstances, articular
cartilage, once damaged, does not heal. Current treatment methods
consist mostly of alleviating symptoms through the use of activity
modification (restrictions), weight-bearing protection (canes,
crutches, walkers), analgesics and anti-inflammatory drugs.
Recently, the FDA has approved autologous cell therapy. In this
method, Chondrocytes are harvested arthroscopically, multiplied in
tissue culture and implanted in the articular cartilage defect in
the knee under a periosteal flap. The procedure costs $30,000.
Alternative concepts are in development, but none are approved.
[0007] The inventors have developed a unique way of multiplying
human chondrocytes so that they rapidly multiply and don't lose
their phenotypic expression. They invest these cells in a scaffold
that allows the interaction of the scaffold and the cells to
reproduce the physical attributes of the cartilage the implant is
intended to replace. No current therapy other than cadaver
cartilage transplant offers this potential. Cadaver cartilage
transplant is ineffective over the long term, is expensive and
transplant material is not readily available. It must be emphasized
that neither the cells nor the scaffold alone will work. In both
instances, success is dependent on an uncontrolled response from
the body in an uncontrolled environment. In the past, such response
has been unpredictable and the development of such a response has
required long periods of inactivity on the part of the patient. The
approach provides a mature mechanically functional implant to the
patient at the time of implantation, requiring only sufficient time
for the implant to unite to the host (4-6 weeks).
[0008] Most of the patients undergoing arthroplasty for the knee
started with a focal lesion of the knee that would have been
amenable to treatment with chondrocyte-implant. At least 50% of the
patients undergoing arthroscopy of the knee have a focal articular
cartilage lesion that is amenable to treatment with the chondroctye
implant.
[0009] The ideal implant material for the future will be capable of
forming a living bond to tissue, providing a scaffold or template
to allow cells to grow and multiply and lay a matrix that will fill
defects in the body and promote tissue healing. The field of this
invention involves the use of polymer constructs as scaffolds or
templates to allow the growth and multiplication of autologous
cells in culture, including chondrocytes (cartilage cells),
oesteocytes (bone cells) oesteoblasts, chondrogenic cells,
pluripotential cells and mucosal cells for tissue replacement
and/or coverage.
[0010] Chondrocytes are the sole cellular component of articular
cartilage. They are sparsely distributed in the cartilaginous
matrix and occupy less than 5% of the tissue volume. Chondrocytes
produce and break down macromolecules that make up cartilage. These
macromolecules consist primarily of a collagen type II network
embedded with high-molecular-weight proteoglycans. By maintaining
the integrity of articular cartilage, chondrocytes play a key role
in the load-bearing function of the joint.
[0011] Several strategies have been explored to expand the number
of chondrocytes ex vivo. Most of these attempts involve propagating
cells in monolayer culture, which allows them to proliferate.
However, chondrocytes propagated in monolayer culture lose their
original characteristics by assuming a fibroblastoid morphology and
shift from production of collagen type II to type I. They also
change synthesis of high- to lower-molecular-weight
proteoglycans.
[0012] Attempts to maintain the original phenotype in monolayer
culture have had some success by seeding chondrocytes in high
density. Despite these modifications in culture conditions, not all
chondrocytes reverted to the spherical, collagen type II producing
cells. Under culture conditions in which cells are restrained from
spreading out as in monolayer culture, chondrocytes were inhibited
from proliferating. These methods are not able to provide
sufficient numbers of chondrocytes with unaltered phenotype needed
for analysis. There is therefore a need for a culture system that
would allow chondrocytes to proliferate, as well as maintain their
original characteristics.
[0013] One culture system that has been shown to facilitate cell
proliferation, as well as maintain synthesis of cellular products,
is the microcarrier suspension culture. This culture system was
based on the original `bioreactor` design to grow bacteria in large
quantities.
[0014] Microcarriers have been utilized in the prior art to grow
anchorage-dependent mammalian cells in large quantities. Cells
attached onto microcarriers are maintained in suspension spinner
culture under controlled levels of pH, oxygen, nutrient supply and
mechanical agitation. At an appropriate time the cells are
harvested. Existing methods provide fairly simple procedures for
obtaining secreted products from the spent culture. However, the
ease of recovering high yields of viable and functional cells from
microcarriers has been a problem. Several factors contribute to the
problem of harvesting viable cells. These factors include the
chemical composition of the microcarriers, the charge on the
microcarrier surface and the method of cell harvesting. The most
common method of cell harvesting used is by trypsinizaion, which
may not completely retrieve cells from the microcarriers.
[0015] With all of this in mind, applicants have perfected means
for cell culture, retrieval and methods for using the cells for
tissue implants when used along with polymer constructs.
CELL-CULTURE PRIOR ART
[0016] U.S. Pat. No. 4,994,388 to Hillegas et al discloses a method
for culturing and harvesting anchorage-dependent cells employing
insoluble micro carrier beads coated with collagen. The cells
attach and grow on the collagen coated microcarrier. Once growth is
complete, the collagen is digested off of the microcarrier, and the
cultured cells are separated from the insoluble microcarrier. This
patent also discloses that the patentees have, in contrast to using
collagen coated beads, used beads made entirely of collagen and
found these latter beads to support the growth of some cells very
well, but that other cells did not proliferate well on
collagen-only beads. The patentees also found in contrast to
collagen coated beads that collagen-only beads present a slower
attachment of cells to collagen. In addition, the patentees found
that harvesting cells from collagen-only beads presented
difficulties in separation of the cells from the media in that
there was contamination of the cells by soluble fragments of
collagen. While the patent discloses applicability of their
invention to anchorage-dependant cells, the patent does not
disclose any specific cell, nor are chondrocytes specifically
disclosed. The disclosure of the patent to Hillegas et al has no
specific embodiment of a process for carrying out separation of a
specific cell from a specific substrate with a specific enzyme.
[0017] Mundt in U.S. Pat. No. 5,100,799 discloses a method for
releasing cells from microcarriers using trypsin to release the
cells. The composition of the microcarrier is not disclosed in this
patent.
[0018] Kandel in U.S. Pat. No. 5,326,357 discloses cartilage tissue
reconstituted on a substrate. The method of this patent involves
reconstituting cartilage tissue in vitro from chondrocytes cultured
on a substrate.
[0019] Green in U.S. Pat. No. 4,304,866 teaches a method for
producing a transplantable sheet of living keratinous tissue by
culturing keratinocytes in a culture vessel and subsequently
enzymatically detaching a sheet of keratinous tissue employing an
enzyme. These sheets of keratinous tissue are to be used for
producing skin grafts.
[0020] Civin in U.S. Pat. No. 5,081,030 teaches the release of
cells from an affinity matrix. The enzyme used by Civin can be an
enzyme selected from the group consisting of carbohydrases,
proteases and lipases The carbohydrase is to be used for cell
surface glycolipids and glycoproteins. Cell culture on a
microcarrier beadlet is not disclosed in this patent.
OBJECTS OF THE INVENTION
[0021] An object of this invention is to efficiently propagate
cells in vitro on the surface of microcarrier spheres or
microcarrier beadlets.
[0022] A further object of this invention is to propagate cells on
microcarriers to promote proliferation of the cells and also to
continue the synthesis of cell by-products characteristic of the
cells.
[0023] A most important object of this invention is to produce
cells free of microcarriers without the need to physically separate
the carrier from the cell.
[0024] A still further object is to be able to retrieve cells for
testing or analysis.
[0025] A further important object of this invention is to produce
viable cells by culture for further propagation in tissue
culture.
[0026] An important object of this invention is to efficiently
culture cells in vitro to be subsequently used in vivo.
[0027] A significant object of this invention is to prepare cells
in culture to be used in animal or human transplantation.
[0028] A major object of this invention is to produce cells, which
are useful for forming an implant device.
[0029] A main object of the invention is to prepare a polymer
having medical applicability.
[0030] Another object of this invention is to prepare polymers
useful for supporting the growth of cells or tissues.
[0031] An important object of this invention is to develop a
polymer to be used for preparing scaffolds for cells.
[0032] An important object of this invention is to form polymer
beads used as a substrate for culturing attached cells, as well as
a substrate for preparing cell seeded scaffold implants.
FIGURES
[0033] FIG. 1 is a schematic representation of the method of
obtaining chondrocytes by culture on microcarriers, followed by
enzyme isolation and then using the chondrocytes for cartilage
repair.
[0034] FIG. 2 is a graph describing the results of culture using
arabinogalactan polysaccharide polymer particles on human
osteoblast-like cells.
[0035] FIG. 3 is a schematic representation for the automatic
control of conditions for cell culture.
[0036] FIG. 4 is a flow chart describing rapid retrieval of
chondrocytes and repair of cartilage.
CELL CULTURE AND ENZYME SEPARATION
[0037] The inventors have discovered a relationship between cell,
substrate, enzyme and culture method.
[0038] Chondrocytes require special culture techniques in order to
produce optimum growth, viability and maintenance of the cells in a
non-altered form. Applicants' process involves primarily culturing
chondrocytes along with dextran microcarrier beads and separating
the cells from the microcarrier using dextranase.
[0039] The inventors have developed a procedure for successfully
retrieving cells adhering to the surface of microcarriers. They
employ enzymes, which completely digest the microspheres without
damaging the cells. The paragraphs, which follow, describe (1)
examples of cells that can be used, (2) types of microcarriers, (3)
method of preparing the construct or microcarrier, (4) methods of
culture, and (5) methods of harvesting the cells from the
constructs.
[0040] The invention is directed primarily to the culture,
harvesting and medical application of chondrocytes, oesteoblasts,
oesteocytes, chondrogenic cells, pluripotential cells and mucosal
cells.
[0041] The herein disclosed invention involves a rather defined
process. The process involves allowing the cells to attach to
microcarrier beadlets. The microcarrier beadlets are critical. The
operative microcarrier beadlets require a special type of surface,
as well as specific surface charge, to allow optimum cell adhesion
and cell growth. In the herein disclosed invention, the terms
microcarrier beadlets, beadlets or microcarrier are used
interchangeably.
[0042] A most significant feature of the herein disclosed invention
is the criticality between the microcarrier beadlet and the enzyme
cleavage thereof. It is most important that the enzyme cleave the
microcarrier beadlet in such a way as to not leave over sensible
microfragments and debris. These microfragments and debris can have
a deleterious affect on the cells, as well as causing cell-death.
In addition, it is important that the enzyme used to digest the
carrier not adversely affect the cell. The inventors have found
that the combination of dextran microcarrier and dextranase as the
000 enzyme are ideal for growth and retrieval of chondrocytes.
[0043] For seeding, 1 ml of 1 to 2.times.10.sup.5 cells are added
into 50 ml of media containing microcarriers with a surface area
equivalent to 400 cm.sup.2 in a silliconized spinner flask with a
250 ml volume capacity. The mixture is intermittently stirred for 2
minutes every 30 minutes during the first 4 hours at 25 to 30 rpm.
The cell microcarrier suspension is subsequently stirred at 45 rpm
for another 4 hours. The speed is gradually increased to 60 rpm.
The speed is maintained at 60 rpm for the desired duration of two
weeks or longer. The final volume of the suspension culture is
adjusted to 100 ml with warm medium. The final cell number is
2-3.times.10.sup.2/cm.sup.2. The spinner culture is replenished by
sedimentation for 5 minutes. Approximately 50% of the volume is
replaced every three days. The spinner cultures are incubated at
37.degree. C., 5% CO.sub.2.
[0044] To harvest the cells, the spinner microcarrier culture is
sedimented. The supernatant is removed and the microcarriers are
washed with Hank's buffered salt solution. The cells are retrieved
by incubation with the dextranase enzyme at 7000 units 50/ml at
37.degree. C. as shown in Table 3, page 6. To stop the enzyme
reaction, 5 ml of medium is added and the supernatant fluid is
discarded. The cell pellet is resuspended in 5 ml of tissue culture
media for analysis.
[0045] The cells can be used for (1) direct implantation, (2)
seeding on a scaffold, (3) reculture or (4) culturing to form a
matrix.
[0046] The microcarriers of this invention must be generally smooth
spheres. Particles with jagged edges are generally inoperative.
[0047] As used herein, the terms beads, microbeadlets,
microcarriers and micropellets are used interchangeably, and they
range in size from 1 to 300 microns.
[0048] Cells will be retrieved by non-toxic enzymatic digestion of
microcarrier spheres while leaving cells viable and metabolically
functional.
[0049] Chondrocytes are the only cellular constituent of articular
cartilage. These cells are critically important in that they
produce, as well as degrade, matrix components that make up
cartilage. Understanding how these cells function has been
facilitated by success in isolating viable chondrocytes and
propagating them in vitro. Chondroctyes proliferate on a variety of
tissue culture surfaces such as polystyrene and in resorbable
biomaterials. Although chondrocytes cultured on these material
divide, they shift to fibroblastoid cells and decrease their
production of collagen type II and high molecular weight
proteoglycans. We have previously demonstrated that chondroctyes,
propagated on microcarriers made of solid collagen type I spheres,
proliferate and maintain certain features of their chondrocyte
phenotype. In the present study, we investigated whether dextran
coated microcarriers coated with collagen type I can also be used
to support the growth of chondrocytes and promote their synthesis
of collagen type II. This collagen type serves as the matrix
scaffold of articular cartilage. Identification of appropriate
culture surface material will facilitate the propagation and
analysis of chondroctyes.
[0050] Chondroctyes propaged on the surface of Cytodex 3.RTM.
dextran microcarriers adhered tightly and could not be retrieved by
trypsinization without damage. In contrast, cells recovered from
Cellagen.RTM. microcarriers and monolayer cultures were viable.
Cells on Cytodex 3.RTM. and Cellagen.RTM. microcarriers stained
intensely for collagen type II. There was insignificant staining
for type I. To further characterize de novo collagen synthesis
radiolabeled collagens were analyzed. Chondrocytes on Cytodex
3.RTM. microcarriers synthesized and retained collagen type II in
the cellular fraction (CF) more profoundly than cells propagated in
Cellagen.RTM. microcarriers. In comparison, cells cultured as
monolayer produced collagen type II which was more readily secreted
out into the media fraction (MF).
[0051] It is important that the microcarrier beadlets not be toxic
to the cells being cultured and equally important is the ability of
the cells to adhere to the beadlets. The inventors have found that
dextran microcarrier beadlets are particularly efficacious for
culturing chondrocytes. The inventors have found that dextran
microcarrier beadlets facilitate adhesion and growth of
chondrocytes. The inventors have found that using dextranase the
cells are readily separated from the dextran beadlets. Using
dextranase to digest the dextran microcarrier beadlets, the
inventors have found that dextranase is able to digest the dextran
without producing fragments in the solution, thereby facilitating
the separation of chondrocytes from the culture media.
[0052] The inventors have now found that the culture of
chondrocytes on dextran microcarriers and digestion with dextranase
to remove the carrier from the cell offers several unexpected and
unobvious results. Applicants have found that chondrocytes grown on
dextran microcarriers are able to adhere, be viable and produce
extracellular matrix components such as collagen type II. In
addition, separation of the cells from the dextran microcarriers
with dextranase is efficient in that fragments of dextran do not
interfere with efficient separation of the chondrocytes from the
microbeadlets and do not adversely affect the viability of the
chondrocytes.
[0053] The herein disclosed invention seeks to propagate cells in
vitro on the surface of microcarrier spheres. The invention
envisions the propagation of cells on microcarrier spheres that
promote proliferation and continued synthesis of cell products
characteristic of the cells being cultured. In the methods of this
invention, cells can be easily retrieved for testing and analysis,
as well as for further propagation and transplantation in animals
and humans. The cells can also be plated onto a scaffold or matrix
to be used for tissue replacement. In the processes of this
invention, the cells after removal from the microcarrier beadlets
will be viable and metabolically functional.
[0054] A variety of cells are amenable to the uses of this
invention; however, chondrocytes, oesteoblasts, oesteocytes,
monocytes, chondrogenic cells, pluripotential cells, mucosal cells
and fibroblasts are most preferred.
[0055] Cartilage is composed of chondrocytes, which synthesize an
abundant extracellular matrix, which is composed of water,
collagens, proteoglycans and noncollagenous proteins and lipids.
Collagen serves to trap proteoglycans and to provide tensile
strength to the tissue. Type II collagen is the predominant
collagen in cartilage tissue. The proteoglycans are composed of a
variable number of glycosaminoglycan chains. The sulfated
glycosaminoglycans are negatively charged resulting in an osmotic
swelling pressure that draws in water.
[0056] It is most desirable that chondrocytes be maintained in a
spherical shape which has been shown to slow down or prevent
differentiation of the chondrocyte phenotype. Accordingly, it is
important that culture systems be developed which maintain the
spherical shape of the chondrocytes.
[0057] A wide variety of cells can be propagated on the surface of
microcarrier spheres with the only requirement being that the cells
adhere to the surface of the microcarrier sphere. Table I is a list
of some of the cell types that can be successfully propagated on
microcarriers.
1TABLE I LIST OF CELLS 1. Normal cells: a) Undifferentiated bone
marrow cells i] progenitor/mesenchymal periosteal perichondral from
the cells cambium ii] precursor cells, e.g. chondrogenic b)
differentiated cells from: i] tissues chondrocytes osteoblasts
fibroblasts ii] peripheral blood monocytes macrophage cell line
osteoblast cell line 2. Tumor cells
[0058] Cells originating from several species have been used for
microcarrier culture. These include cells from rabbits, bovine,
chickens and man.
[0059] Several types of microcarriers have tested and characterized
in our laboratory as shown in Table II. These microcarriers were
purchased form sources listed in Table II.
2TABLE II Com- Com- Size mercial mercial Diameter Name Source
Description (.mu.) Cellagen ICN Collagen beads are prepared from
100-400 bovine corium insoluble collagen by pepsin treatment. The
collagen is then purified and formed into microsphere beads.
Cytodex 1 Sigma Cytodex 1 microcarriers are based on a
131-220.sup.++ cross-linked dextran matrix which is substituted
with positively charged N,N- diethylaminoethyl (DEAE) groups to a
degree which is optimal for cell growth. Cytodex 2 Sigma Cytodex 2
microcarriers are formed by 114-198.sup.++ substituting a
cross-linked dextran matrix with only a surface layer of positively
charged, N,N,N-trimethyl- 2-hydroxy-aminopropyl groups. Cytodex 3
Sigma Cytodex 3 microcarriers consist of a 133-215.sup.++ surface
layer of denatured collagen covalently bound to a matrix of cross-
linked dextran. ++Diameter at 5% and 95% of the volume of a sample
of microcarriers.
[0060] The method for preparing the cell microcarrier constructs
are described as follows:
[0061] Preparation of Cellagen microcarriers
[0062] Cellagen microcarriers are purchased ready to use in sterile
preparation.
[0063] Preparation of Cytodex microcarriers
[0064] for a surface area of 1000 cm.sup.2, the following
measurements are required for different Cytodex microcarriers:
[0065] 1) Cytodex 1-6000 cm.sup.2/g @ 1000 cm.sup.2-0.1667 g
[0066] 2) Cytodex 2-5500 cm.sup.2/g @ 1000 cm.sup.2-0.1818 g
[0067] 3) Cytodex 3-4600 cm.sup.2/g @ 1000 cm.sup.20.2174 g
[0068] To a clean siliconized 250 ml spinner 50 ml of sterile
1.times.PBS is added followed by the addition of the desired
Cytodex microcarrier. The cytodex carriers are allowed to rehydrate
at room temperature overnight. The next day, the microcarriers are
washed two times with sterile 1.times.PBS and are then ready to be
autoclaved.
[0069] The microcarriers are seeded with cells according to the
following procedures:
[0070] 1. Cellagen microcarriers
[0071] 1. A 250 ml capacity spinner is cleaned, siliconized and
autoclaved in advance.
[0072] To the spinner is added 40 ml Hy(13) media: (Dulbecco's
Modified Eagle Medium [DMEM], GIBCO-BRL); NCTC-109(GIBCO-BRL);
oxaloacetate; pyruvate; insulin; L-gluatamine (GIBCO-BRL);
gentamycin (BIOWHITAKER); fetal calf serum (Gemini Bioproducts,
Inc.). Chondrocytes (4.times.10.sup.6) are next added to 3.95
ml=1000 cm.sup.2 cellagen bead suspension.
[0073] 2. The spinner flask is placed on the stirring plate on the
incubator @ 37 degrees, 5% CO.sub.2, and the plate is set at
interval mode (2 minutes on, 30 minutes off, 35 rpm). The spinner
is incubated at these settings overnight.
[0074] 3. The stirring plate is switched to continuous mode after
the cells have attached to beads. The stirring plate is set to 45
rpm in the morning. At the end of the day, the stirring plate is
switched to 60 rpm continuous mode where it will stay for the
remainder of the experiment, e.g. for about two weeks.
[0075] 2. Cytodex I, II, III microcarriers
[0076] 1. A 250 ml spinner containing rehydrated cytodex
microcarriers is autoclaved for 15 minutes on liquid mode. After
autoclaving, the microcarriers are allowed to settle and cool off
in a sterile hood. The 1.times.PBS is replaced with 40 ml of fresh
HY media to spinner cells are finally added 4.times.10.sup.6.
[0077] The seeded microcarriers are subsequently cultured at
37.degree. C., 5% CO.sub.2 for the duration required, e.g. for
about two weeks. The schematic diagram of the procedure is shown in
FIG. 1.
[0078] Cells are retrieved from microcarriers using enzymes
selectively directed to digest the microcarrier without damaging
cells. Table III shows the enzymes to be used for each specific
microcarrier.
3TABLE III ENZYMATIC RETRIEVAL OF MICROCARRIERS Micro- Enzyme
Commercial Digestion carrier Enzyme Source Procedure Cel-
Collagenase Collagenase A from Incubate culture with lagen
Clostridium hystolyticum .1% Collagenase for Supplier: Boehringer
1.5 hours Mannheim Cytodex Dextranase Dextranase Source: Incubate
culture with 1 Penicillium sp. 7000 units/50 ml for Code: DEXC 1.5
hours Supplier: Sigma Cytodex Dextranase Dextranase Source:
Incubate culture with 2 Penicillium sp. 7000 units/50 ml for Code:
DEXC 1.5 hours Supplier: Sigma Cytodex Dextranase Dextranase
Source: Incubate culture with 3 Penicillium sp. 7000 units/50 ml
for Code: DEXC 1.5 hours Supplier: Sigma
[0079] The cell suspension isolated from the microcarriers are
sieved through a 150 (100 .mu.m) mesh collector (A. H. Thomas, PA.,
USA). The cells are pelleted by centrifugation at 200 g for 10
minutes. The supernatant fluid is discarded and the cells are
resuspended in 5 ml of tissue culture fluid for analysis or for use
in implantation.
[0080] The inventive process also involves separating the
chondrocytes from the microcarrier beadlets, plating the
chondroctyes on a scaffold or template for growth thereon and then
surgically implanting the scaffold for cartilage repair.
[0081] In another embodiment of this invention after separating the
cells from the microcarrier beadlets the cells can be injected into
a patient in need of said cells.
[0082] Advantages of Invention and Improvements Compared to
Existing Technology
[0083] The cell microcarrier constructs propagated in spinner
suspension culture provide a twenty fold multiplication of cell
numbers compared to monolayer cultures. The cells retrieved from
the microcarriers remain viable, metabolically active and continue
synthesis of "cell specific" products. In the case of chondrocytes,
the "cell specific" products which characterize these cells are the
cartilage matrix components-collagen type II and high molecular
weight proteoglycans. Thus the cell numbers can be scaled up for
mass production in bioreactors without compromising cellular
function.
[0084] The constructs can easily be prepared and the cells removed
for subsequent in vitro usage or for transplantation in animals or
man in conjunction with a scaffold or template. Preparation of the
cell-microcarrier construct is cost effective and requires less
involved technical time and effort, as well as lesser amounts of
tissue culture reagents as compared to preparation of monolayer
cell cultures.
[0085] Polysaccaride Polymer Constructs
[0086] There is a continuing quest for developing ways for
efficiently culturing cells and tissue. Cells are useful for
producing cell by-products used in medicine, as well as in other
fields. In addition, cells can be used for preparing products to
replace poorly functioning or inoperative tissue or body parts. The
herein disclosed invention envisions using polysaccharide polymers
in cell culture and for preparing scaffolds or templates for tissue
implants.
[0087] Chemical Pathway for Preparing Polysaccharide Polymers 1
[0088] Polysaccharide include: dextran, cellulose, arabinogalactane
Oxidizing agents include: KIO4, NaIO4, ceric ammonium nitrate
R--(NR'.sub.2).sub.x is a polyanime containing at least 2 primary
amines, R'.dbd.H,alkyl the cationic groups in the crosslinked
polymers include: 1.degree., 2.degree., 3.degree., 4.degree.
amines
[0089] In the complete structural formula of the polymer shown,
there is imine and amine crosslinking, with the polysaccharide
being linear or branched, natural or synthetic. Examples of
polysaccharides useful for practicing this invention are dextran of
various molecular weights, arabinogalactan, pollulan, cellulose and
amylose obtained from various sources. The polyamine cationic and
crosslinking agent contains at least two primary amino groups (for
crosslinking) and primary, secondary, tertiary or quaternary amino
groups. A single of mixture of polyamines can be used which include
amino acides such as lysine and omithine or peptides and
proteins.
[0090] Polysaccharide includes: dextran, cellulose, arabinogalactan
pollulan and amylose; n is an integer between 2 and 10,000 such as
in a disaccharide, oligosaccharide or polysaccharide; oxidizing
agents for oxidation include: KIO.sub.4, NaIO.sub.4, ceric ammonium
nitrate; R(NR.sub.2).sub.x is a polyamine containing at least 2
primary amines; R.dbd.H, alkyl or alkylene; the cationic groups in
the crosslinked polymers include: 1.degree., 2.degree., 3.degree.,
4.degree. amines, for example, these groups can be primary amine
1.degree. (R--NH.sub.2), secondary amine 2.degree. 2
[0091] tertiary amine 3.degree. 3
[0092] quaternary amine 4.degree. 4
[0093] x can be between 2 and 5 (x determines the number of amine
groups on the crosslinking molecule). For crosslinking it is
essential to have a molecule that has at least two amine groups
such as ethylene diamine.
[0094] The inventors envision the above chemical pathway for
preparing the polysaccharide polymers. There is imine and amine
crosslinking, with the polysaccharide being linear or branched,
natural or synthetic. Examples of polysaccharides useful for
practicing this invention are dextran of various molecular weights,
arabinogalactan, pollulan, cellulose and amylose obtained from
various sources. The polyamine cationic and crosslinking agent
contains at least two primary amino groups (for crosslinking) and
primary, secondary, tertiary or quaternary amino groups. A single
of mixture of polyamines can be used which include amino acids such
as lysine and ornithine or peptides and proteins.
[0095] The polysaccharide derived polymers with imine crosslinking,
as well as amine crosslinking, can be used as microbeadlets for
cell culture and the preparation of scaffolds for tissue
repair.
[0096] In general, the specific method for making the polymer is as
follows: The polysaccharides are first oxidized to the dialdehyde
derivatives which is then reacted with non-toxic polyamines to form
the imine crosslinking. The imine crosslinking bonds can be further
hydrogenated to the more stable amine bonds.
[0097] The disclosed polymers of this invention are to be employed
in the field of cell culture and also for providing scaffolds
useful for synthetic cartilage tissue formation. The disclosed
invention also describes the synthesis of polysaccharide beads of
different crosslinking densities, as well as the charge for
facilitating the attachment and proliferation of cartilage cells on
a polymer bead matrix. The cells (e.g., chondrocytes) will
proliferate within and on the beads and form Collagen type II which
is necessary for cartilage formation. When utilized in vivo, the
beads will degrade to non-toxic components leaving chondrocytes in
place to form cartilage tissue. The polymer beads are made of
linear or branched polysaccharides crosslinked by diamino or
polyamino molecules that are capable of forming an imine or amine
bonds with the oxidized polyscaccharide. The beads are prepared by
reacting dialdehyde polysaccharides (prepared from the reaction of
the polysaccharide with various amounts of potassium periodate at
room temperature) with various amounts diamino molecules. The beads
are designed to have a predetermined pore size formed by using
different amounts of crosslinking and cationic groups that will
enable optimum cell attachment. The polysaccharides that are useful
for this method are biocompatible linear or branched
polysaccharides and include among others: dextran, arabinogalactan,
cellulose, pollulan and hyaluronic acid. The crosslinking agents
are biocompatible diamine or polyamines exemplified by: lysine and
ornithine, as well as their oligomers and polymers, such as gelatin
or collagen. To enhance cell attachment to the beads, cationic
sites are formed by adding a polycation such as polylysine,
polyethyleneimine, gelatin, collagen, fibrin or albumin to the
crosslinking reaction mixture. The imine conjugation bonds formed
by the reaction of the amines with the aldehyde groups on the
oxidized polysaccharides can be hydrogenated to form amino
derivatives which contribute to the stability of the beads (i.e.,
amine groups are non-hydrolyzable in water solutions while the
imines do hydrolyze) and in addition these imine conjugation bonds
will also add a cationic charge to the beads. Anionic beads
containing carboxylic acid groups can be formed by the addition of
amino acids such as glutamic acid to the reaction mixture and, in
this instance, the amino group will be conjugated to the
polysaccharide and the carboxylic acid will be free.
[0098] Characteristics of the polymer: One of the main utilities of
the polymer of this invention is to serve as scaffold for cell
proliferation. The polymer should provide the proper environment
for various cells to grow within and on the polymer beads. The
characteristics and properties of the polymer should be versatile
to allow the growth of various kinds of cells. For the various
types of cells, the polymer should provide a hydrophilic micro
environment of a specific pore size with cationic sites to allow
for cell attachment. The degree of hydrophilicity, water
absorption, pore size and cationic sites are similar for the cell
types to be used in this invention but the polymer can be tuned to
optimize the micro environment to each cell type by varying the
chemistry. If the cells are seeded into the scaffold, the pore size
of the polymer should be 50-100.mu.. The polymers should be
insoluble in aqueous media but absorb water solutions to at least
30% of its weight. The polymer may swell to several times of its
original volume without dissolution. The pore size of the swollen
polymer gel should be in the range of 0.1 to 1 micron that can be
controlled by the degree of crosslinking and the reaction
conditions. The degree of crosslinking is dependent on the kind of
polysaccharide, the degree of oxidation, the kind and amount of
polyamine crosslinking agent and the method of preparation. The
polymer should contain cationic groups to allow cell attachment.
The concentration and kind of cationic groups, i.e., primary,
secondary, tertiary or quaternary amines can be controlled. The gel
may be degraded in an aqueous environment by chemical hydrolysis of
the imine crosslinking bonds or by enzymatic degradation of the
saccharide or peptide bonds.
[0099] The polymers should be biodegradable and resorb in vivo
leaving cartilage cells and a matrix in place. Most importantly,
the polymers should be compatible with cell-viability and allow
cells to proliferate without toxicity or inhibition. In actual
culture conditions, polymers of this invention have been found to
be non-toxic to chondrocytes.
[0100] The herein disclosed polymers are intended to be used as
microcarriers for cell culture; and particularly for the culture of
chondrocytes. When the cells grow on the surface of the
microcarrier, the size of the microcarrier should be 100-400.mu..
In addition, the polymers are intended to be used to form scaffolds
on which chondrocytes are cultured and, in turn, used for cartilage
replacement.
[0101] As used herein, the terms beads, microbeadlets,
microcarriers and micropellets are used interchangeably, and they
range in size from 1 to 400 microns.
[0102] Preferred Embodiments of Polysaccharide Polymers
Constructs
EXAMPLES
[0103] a: Cross linking of oxidized Arabinogalactan (AG)
[0104] Cross linking of oxidized AG was obtained from the reaction
of oxidized AG (at least 20% of the sugar units are oxidized
prepared by oxidation wit 0.1 molar KI0.sub.4 in water on
increasing mole ratio of saccharide to KIO.sub.4) with 0.5 to 15%
lysine. The gel-bead properties are dependent on the degree of
oxidation of the polysaccharide, the content of the crosslinking
agent, and the reaction conditions. The swelling capacity of the
gel was determined by incubation of the crosslined polymer (100 mg)
at room temperature over-night in 2.0 ml DDW (double-distilled
water). The water-adsorbed gel was slightly dried by tissue paper
and weighted, and then dried again by lyophilization to determine
the content of insoluble gel. The % of water adsorption
(calculated) was 210%.
[0105] b: Synthesis and characterization of poly-(carboxylic acid)
gels based on di-aldehyde Arabinogalactan (AG).
[0106] In a typical experiment, different gels were prepared by the
reaction of oxidized AG with L-glutamic acid and small quantities
of lysine to introduce the cross linked gels. These gels were first
reduced by sodium borohydride to introduce the stable amine
cross-links (FIG. 1), and swelled for 24 hours in 37.degree. C. in
different pH media to calculate their ability to absorb water as a
function of pH.
[0107] The pure oxidized AG (38% oxidation of saccharide units) was
first dissolved in 0.1 M bicarbonate buffer pH=8.5 at a 10%
.sup.w/.sub.v.
[0108] 10% (w/v) and 25% (w/w) of L-glutamic acid powder were
added. The corresponding amount of lysine (%) was added to the
reaction mixtures and stirred for 24 hours in 37.degree. C.
[0109] The different gels obtained were swelled over-night in a
solution of excess sodium borohydride (1.5 mole excess over the
aldehyde bonds) to reduce the inline conjugates to the more stable
amine bonds, and to reduce the unreacted aldehydes units. The
reduced-gels were washed several times with DDW to remove the
excess borohydride and dried for 5 hours in a speed-vac system.
[0110] Synthesis of Polycarboxylic Hydrogels
[0111] c: Albumin cross linking with oxidized Arabinogalactan
(AG)
[0112] Albumin, a natural compatible protein containing amine
groups, was reacted with increasing amounts of oxidized AG in order
to obtain a cross linked gel. The experiment was as follows: 1.0
gr. of egg-albumin (or bovine serum albumin) was dissolved in 20.0
ml of 0.1M NaHC0.sub.3 buffer and the solution was incubated for 15
minutes at 37.degree. C. to obtain a clear-yellow solution which
was then reacted with a solution of oxidized AG for 24 hours at
37.degree. C. The molecular weight (MW), as well as the gel
formation, were monitored. The results are summarized in Table 4.
No significant change in MW was observed at a 10% (w/w) AG content,
while at 0.5 to 1.0 weight ratio an insoluble gel was obtained. For
comparison, a non-oxidized AG was reacted with albumin under the
same conditions (Table 4, #6). No cross linking or interaction
between the two polymers occurred as evident by the GPC data (two
peaks for each polymer with the starting MW).
4TABLE 4 Cross linking of albumin with oxidized AG Egg Albumin
mg/2.0 ml Oxidized AG (1:1) # buffer % W/W MW 1 100.0 0.0% 332,341
2 100.0 1.0% 379,332 3 100.0 10.0% 472,562 4* 100.0 50.0%
cross-linking 5** 100.0 100.0% cross-linking 6*** 100.0 100.0 mg of
the non-oxidized Arabinogalactan 372,332 (Albumin) 27,624 (Ox:AG)
(two peaks) *100.0 mg albumin reacted with 50.0 mg of oxidized
AG(1:1). Gel water with 62% insoluble gel. **100.0 mg albumin
reacted with 100.0 mg of oxidized AG(1:1). Gel water with 77%
insoluble gel. ***100.0 mg albumin reacted with 100.0 mg of
non-oxidized chromatogram show two major peaks, one peak is
relative to the non-oxidized AG.
[0113] The same procedure was employed using gelatin instead of
albumin. A cross-linked gel was obtained at a 1:1 ratio. This gel
absorb 256% of water with 87% insoluble gel. Cross-linking was
detected also by using polyethyleneimine (PEI) with Ox:AG (1:1). No
cross linking were detected by using oxidized Dextran instead of AG
with all the proteins mentioned above.
[0114] d. Synthesis of beads based on crosslinked
Arabinogalactan
[0115] 1) 5% lysine Un-red (unreduced)
[0116] 0.5 g of di-aldehyde Arabinogalactan (35% degree of
oxidation) was dissolved in 4.5 ml DDW to give a 10% (w/w/)
concentration of the polymer. 26.5 mg of lysine hydrochloride (5%
weight to polymer) was added to the polymer solution and the pH was
adjusted to 6.0 using 0.1M NaOH solution. Under these conditions
crosslinking is very slow (pH=6.0). The mixture was added dropwise
to an ice cooled decaline/buffer (0.1M NaHC03, pH=8.0) two-phase
system. While passing through the cold oil phase the droplets gel.
At the alkaline pH of the water phase, reaction of lysine with
di-aldehyde Arabinogalactan proceeds rapidly and the beads
hardened. The beads are gently stirred in the buffer for 4 hour at
0.degree. C. over-night at room temperature. Finally, the
water-phase was separated from the oil-phase and lyophilized to
dryness. The beads obtained were washed with DDW several times to
remove soluble material, and then dispersed in ethanol at 4.degree.
C. overnight, filtered and dried in vacuum. The yield was
=.about.90% by weight.
[0117] 2) 5% lysine Red (Reduced)
[0118] Reduction of the Imine-bond obtained in 1) was done by
incubating the beads in sodium borohydride solution (1.5 mol
NaBH.sub.4 for each mol sugar unit of polysaccharide) for 4 hours
at room temperature, followed by filtration and washing with
deionized water. The reduced beads were dispersed in ethanol at
4.degree. C. over-night, filtered and dried in vacuum.
[0119] 3) 20% lysine Un-red (unreduced)
[0120] These beads were synthesized exactly as 1) only using large
amounts of lysine hydrochloride (125.0 mg. 20.0% weight).
[0121] 4) 20% lysine Red (reduced)
[0122] Reduction of the Imine-bonds to the more stable amine bonds
were done using the same procedure in 2). The crosslinked
arabinogalactan beads identified as 5% lysine un-red (unreduced),
5% lysine Red (reduced), 20% lysine un-red (unreduced) and 20%
lysine Red were tested and the results are described in FIG. 3.
Bars 1-4 of the graph (FIG. 3) indicate that the particles tested
did not have any toxic affect on bone cells. The proliferative
capacity of bone cells exposed to the particles indicated by
radiolabeled thymidine uptake is not statistically different from
that of cells incubated in control medium alone (p<0.05).
[0123] 5) AG-BSA 1:1
[0124] 0.5 g di-aldehyde Arabinogalactan dissolved in 4.5 ml DDW
were mixed with an equal volume of a 0.1M Borax-buffer (pH=6.0)
containing 0.5 g Bovine-serum Albumin (BSA). The pH was maintained
at 6.0 using 0.1M sodium hydroxide solution. The beads were added
dropwise to a cold two phase system (decaline/pH=8.0 buffer) as
described above. The beads obtained after lyophilization were
dispersed in ethanol, filtered and dried in vacuum.
[0125] yield=.about.90% by weight
[0126] 6) AG-gelatin 1:1
[0127] These beads were synthesized as described in 5) only using
Gelatin instead of Bovine-serum Albumin.
[0128] A crosslinked polysaccharide prepare by: 1) oxidation of a
linear or branched polysaccharide to a dialdehyde derivative. 2)
React the oxidized polysaccharide with molecule containing 2 or
more reactive amino groups to form a crosslinked polymer with
hydrolyzable imine bonds, or the crosslinked polysaccharide is
further reduced to the amine linkages, such that the final product
has a cationic charge to allow cell attachment. The following is
the chemical reaction and polymer produced which will be useful in
carrying out the objects of this invention. For example, use in
cell culture and for scaffolds used in transplant therapy. 5
[0129] P is a polysaccharide such as: dextran, arabinogolactan,
pollulan, cellulose, amylose - - -
[0130] R is a residue of a molecule containing 2 amino groups or
more such as: lysine, ethylenediamine, alkylenediamine,
phenylenediamine, xylenediamine, polyethyleminie, gelatin, albumin
fibrinogen and 6
[0131] Preparation of Water Insoluble cationic Polymers for
Controlled Charge of Polysaccharide Beads
[0132] Three hydrophobized polyamines were prepared for use on
polysaccharide beads. Polyethyleneimine (PEI) of 30,000 molecular
weight (Polysciences, ratio of primary; secondary; tertiary amines
was 1:2:1 respectively) was alkylated with stearyl bro mide at a
degree of alkylation ranged from 1 to 5% of the amine groups of the
PEI. The reaction between dry PEI and stearyl bromide was carried
out in a dry toluene at reflux for 5 hours. The solubility of the
polymer in alcohol and in water was determined. Alkylated PEI
containing 2 to 5% stearyl groups were insoluble in water but were
soluble in alcohol. These PEI derivatives were applied on
polysaccharide beads to increase cationic charge. Polylysine was
hydrophobized to become insoluble in water by partial amidation (5
and 10% per amino groups) of the free amino side groups with
stearic acid using dicyclohexyl carbodiimide (DCC) in
dichloromethane at room temperature. The alkylated polylysine
became insoluble in water but soluble in dimethylsulfoxide (DMSO).
Preparation of acrylate based cationic polymers: To a
polymerization kettle equipped with a stirrer and a nitrogen inlet
it was added N,N-dimethylamino ethyl methacrylate (30 g), amino
ethyl methacrylate (30 g), lauryl methacrylate (60 g), benzoyl
peroxide (1.0 g) and dry toluene (300 ml). The kettle was immersed
into a water bath at 70.degree. C. and allowed to stir over night.
After cooling to room temperature, diethyl ether (500 ml) as added
and the white polymer precipitate was isolated by filtration and
washed twice with diethyl ether and dried at room air. The polymer
was soluble in dichloromethane but insoluble in water.
[0133] Coating of Polysaccharide Beads with Cationic Polymer
[0134] Crossliked arabinogalactan (AG) or dextran beads (0.5 g)
were immersed in solutions of the hydrophobized polyamines in
alcohol or dichloromethane (0.1 to 1% w/v, 5 ml) for 3 hours. The
solvent was slowly evaporated to dryness using a vacuum evaporator
at room temperature. The dry beads did not change their shape or
average particle size after the addition of the polyamine. The
amount of cationic charge was controlled by the concentration of
the polyamine solution or by the amount of solution. The amount of
amino groups on the beads was determined by nitrogen analysis.
[0135] Preparation of Micropellets
[0136] In general, an aqueous solution of the oxidized
polysaccharide and polyamine at pH6 (at this pH the crosslinking
reaction is slow) is added dropwise to a buffer solution pH 8
(where fast crosslinking occurs) through a layer of decaline with
stirring of the buffer solution. Spheres of particle size from 1 to
300 microns are formed in the high pH solution. The particle size
and pore size are affected by the polysaccharide: polyamine ratio
and concentration, the solution droplet size and stirring
conditions. The droplet size can be controlled by using an
automatic microinjunction device. Alternatively, a concentrated
solution of the oxidized polysaccharide at pH6.0 (5-20% w/v) is
added dropwise with a rapid stirring into a solution of the
polyamine at pH8. Crosslinking occurred in a few minutes of
stirring at room temperature. To add a cationic nature to the
beads, a predetermined amount of a polyamine such as arginine,
collagen, poly(ethylene imine) and its derivatives, poly(lysine),
poly(ornithine), gelatin, fibronectin and laminine. Other charged
or hydrophilic groups (carobxylates, amides and hydroxyls) can be
added to the polymer by adding the corresponding amino acids or
amino containing molecules that contain the desired functional
group.
[0137] Preparation of Scaffolding
[0138] The scaffolding. Cartilage cells or other cells are cultured
within and on the beads by common methods as described in U.S. Pat.
No. 5,326,357; Kandel et al, Biochem Biophys. Acta 1035:130, 1990.
For example isolated chondrocytes are plated at a high cell density
of between 1 and 10.times.10.sup.6/cm.sup.2 and placed in sterile
tissue culture wells and cultured in fetal bovine serum medium at
37.degree. C. in a CO.sub.2 rich cell culture incubator. The
crosslinked polysaccharide of this invention may replace the
currently used coated agarose, cellulose, collagen or gelatin
(Cultisphere) beads. Other uses of the polymer. The main use of
these polymers is as scaffold for tissue regeneration, particularly
cartilage. Beside chondrocytes, other cell types, e.g., for tissue
engineering and regeneration can be prepared using these beads of
this invention: hepatocytes, muscle cells, thyroid cells,
langerhans cells, etc. Beads with a cationic nature can be used in
chromatography for cell separation based on the bead pore size/cell
size and the difference in the cell attachment to the cationic
surface of the cell vis-a-vis, the anionic nature of the cell
surface.
[0139] The crosslinked polysaccharide polymers of this invention
are to be used for cell culture; and more specifically the
crosslinked polysaccharide polymers can be formed into beadlets and
chondroctyes, osteoblasts, monocytes and fibroblasts cultured
thereon. It is expected that the cells can be digested off of the
beadlets with an appropriate sugar digesting enzyme, e.g., amylase
for amylose and dextranase for dextran. Moreover, the cells can be
plated on a scaffold prepared from a crosslinked polysaccaride
polymer.
[0140] The chart of FIG. 2 shows the affect of the arabinogalactan
polymer particles on human osteoblast-like cells.
[0141] While this invention focuses on tissue engineering of
various cell types, it can be extended for the use of polymers in
cell separation chromatography.
[0142] Chitosan Constructs
[0143] Another elegant embodiment of this invention involves
preparing a chitosan matrix containing cells for repair of
cartilage or bone defects. Cells in the chitosan matrix are viable,
metabolically active and continue to produce tissue specific
products. The cell-chitosan constructs will be used to replace
damaged or diseased cartilage.
[0144] Chitosan, a mucopolysaccharide is the alkaline deacetylated
product of chitin, which is derived from the exoskeleton of
crustaceans (1). The structure of chitosan shown in the enclosed
figure is similar to glycosaminoglycans. It is crystallizable and
can be prepared to yield a material with mechanical properties
suitable for repairing cartilage defects (2-4). Biocompatibility
studies in vitro and in vivo have indicated that chitosan is a
non-cytotoxic, biodegradable material (5-7). With these
physico-chemical properties, chitosan has been proposed for a
variety of clinical applications. Chiosan as a biomaterial is
currently being evaluated for use as a component of hemodialysis
membranes, artificial skin, and in wound healing. The present
invention is directed to a new application of chitosan to serve as
cell scaffold for cartilage repair and particularly in articulating
joints. The cells to be used for preparing the construct of this
invention can be differentiated chondrocytes from cartilage or
undifferentiated chondrogenic cells from the bone marrow,
periosteum or perichondrium.
[0145] Articular cartilage is the resilient load bearing material
critically important for normal joint function. This aneural and
avascular tissue consists of a collagen type II flamework
interspersed with aggregated proteoglycans called aggrecan (8-9),
composed primarily of extracellular matrix and chondrocytes.
Articular cartilage provides the excellent friction, lubrication
and wear characteristics needed to meet the biomechical
requirements in the joint. Chondrocytes comprise about 5% of the
cartilaginous tissue but synthesize as well as degrade the
extracellular matrix. The limited capacity of chondrocytes to
proliferate and the lack of vascular supply underlie the inability
of chondrocytes to repair itself when damaged as a consequence of
mechanical trauma or disease. Superficial lesions that do not
penetrate the subchondral bone do not heal adequately. Full
thickness defects may heal but the repair tissue is made out of
fibrocartilage (10). The fibrocartilaginous repair tissue does not
have the physicochemical characteristics of the original hyaline
cartilage thus compromising the biomechanical function of the joint
(10).
[0146] Recent novel approaches for repair of cartilage include
implantation of cells alone or cells seeded onto a scaffold
(11-12). Whether cells are implanted alone or embedded in a
delivery system such as a scaffold, they need to continue
production of cartilage specific matrix components. A number of
scaffolds have been proposed for cartilage repair (13-16). Actively
metabolizing cells seeded on a scaffold have been shown to deposit
their newly synthesized matrix components into the three
dimensional framework simulating hyaline cartilage. Our proposed
use of chitosan as a scaffold is conducive to producing viable and
actively metabolizing chondrocytes or their precursors for
producing cartilage extracellular matrix components such as
collagen type II and aggrecan.
[0147] Preferred Embodiment of Chitosan Constructs
[0148] We have discovered that human chondrocytes and other cells
types such as osteoblastic cells can be successfully incorporated
into chitosan constructs. The cells remain viable and are
metabolically active indicated by their continued protein
synthesis. They do not proliferate but continue to produce tissue
specific matrix components. Chondrocytes produce collagen type II
whereas osteoblasts produce collagen type I. The section below
describes the (1) examples of cells that can be used in chitosan
constructs, (2) types of chitosan tested in our laboratory, and (3)
Method of preparing the construct.
[0149] Description and Operation
[0150] Differentiated chondrocytes or undifferentiated precursor
cells from the bone marrow, periosteum and perichondrium can be
incorporated into the chitosan scaffold described below. Several
types of chitosan have been tested and characterized in our
laboratory as shown in Table I Below. These chitosans were
purchased from different sources.
5TABLE IV Commercial Commercial Name Source Description Chitosan
Sigma (Deacetylated chitin; Poly-[1-4]- [.beta.-D-glucosamine)
Practical Grade From Crab Shells Finesse: flakes may contain
foreign matter Chitosan Seikagaku Code No. 400634; 83% Deacetylated
America Powder Crab Shell Finesse: 95% passed through 1000 .mu.m
Chitosan Seikagaku Code No. 400635 America 88% Deacetylated Powder
Crab Shell Finesse: 95% passed through 1000 .mu.m
[0151] The Method for Preparing File Encapsulation of Cells in
Chitosan are Described as Follows
[0152] 1. Preparation of Chitosan
[0153] a 2% solution of acetic acid is prepared
[0154] for 3% & 6% (W/V) chitosan, the following measurements
are required for all types of chitosan:
[0155] a) For a 3 g solution--3 g of chitosan per 100 ml of acetic
acid
[0156] b) For a 6 g solution--6 g of chitosan per 100 ml of acetic
acid
[0157] To a clean 100 ml glass bottle 50 ml of 2% acetic acid is
added followed by the addition of the desired chitosan
concentration. The chitosan is allowed to dissolve at room
temperature overnight. The next day, the chitosan is autoclaved.
Once the chitosan is ready for use, cells are encapsulated
according to the following procedures:
[0158] 1. A sterile four well plate is put in the hood on advance.
To one of the wells is added the desired amount of chitosan
solution. Next, chondrocytes, in the pellet form
(.about.4.times.10.sup.6-10.times.10.sup- .6), are added to the
chitosan solution. A spatula is used to mix the chitosan-cell
mixture. The mixture is stirred with file spatula until a ball is
formed.
[0159] 2. A 250-ml capacity beaker is cleaned and autoclaved in
advance. To the beaker is added 50 ml of sterile filtered 1 N NaOH.
Next, the chitosan-cell ball mixture is placed in the NaOH so that
it polymerizes within minutes. After polymerization, the NaOH is
quickly replaced with 50-ml of HBSS (GIBCO-BRL). The polymerized
chitosan-cell ball is cultured in AIM V media (GIBCO-BRL) at
37.degree. C., 5% CO.sub.2 for the duration required.
[0160] Chondrocytes mixed with the chitosan becomes incorporated
into a three dimensional structure as chitosan polymerizes within
minutes after addition of NaOH. The cell-chitosan polymer has a
firm consistency resembling the texture of cartilage. The
incorporated cells appear viable as they continue to produce
cartilage matrix extracellular components.
[0161] Advantages of Invention and Improvements Compared to
Existing Technology
[0162] The cell-chitosan construct provides a biocompatible
material to facilitate repair of cartilage defects. Chondrocytes or
their precursors remain viable, metabolically active and continue
synthesis of tissue-specific products. The chitosan scaffold or
template serves as an ideal three dimensional environment for
chondrocytes to maintain their production of cartilage specific
collagen type II and high molecular weight proteoglycans. The
constructs can easily be prepared for subsequent
transplantation.
[0163] Chitosan is extremely promising because it has been used in
other physiological applications and found to be compatible with
body physiology. Because chitosan has been used in physiologic
applications, FDA approval without undue delay is to be
anticipated.
[0164] The invention envisions crosslinked polysaccharides with
imine crosslinking and also amine crosslinking as exemplified in
the chemical formulas set forth above to be used for cell growth
attachment, as well as for the preparation of scaffolds for
surgically implanting cells, such as chondrocytes for cartilage
repair.
[0165] Viewed another way, the invention involves a scaffold or
template for cartilage repair comprising a member of the group
consisting of chitosan and a crosslinked polysaccharide prepared by
crosslinking said polysaccharide with a polyamine, said member
being shaped in the form of a scaffold or template and having
plated and growing on said scaffold or template chondrocytes.
[0166] Another elegant embodiment of this invention involves the
surgical implantation of the cartilage/bone polymeric scaffolds in
the head and neck area. Tissue-engineered cartilage/bone using
polymeric scaffolds and cartilage/bone cells can be used as an
implant in plastic reconstructive surgery of the head and neck
area. Studies in our laboratory have demonstrated the growth and
multiplication of cartilage cells in vitro, with different types of
polymers such as cellagen, arabinoglycan poysaccharide and
chitosan, without toxicity to the cells. This opens possibilities
for the production of viable and structurally stable cartilage in
predicatable shapes originating from an autogenous cartilage, which
in turn may be implanted for reconstructive or cosmetic purposes.
The polymer-cartilage scaffold may be shaped into specific forms to
fill defects in the head and neck area, or for the purpose of
augmentation and facial sculpting.
[0167] Surgical applications and techniques for implantation of the
polymer-cartilage scaffold include:
[0168] 1) Nasal subcutaneous implantation for dorsal
augmentation.
[0169] 2) Thyroplasty for medialization of the vocal cord and
treatment of vocal cord paralysis.
[0170] 3) Subcutaneous implantation for facial reconstruction for
congenital conditions (such as microtai, hypoplasia of mandible, of
maxilla, of nose), and for acquired conditions (such as nasolabial
fold augmentation, perioral augmentation, glabellar augmentation,
premaxilla-nasal spine, malar complex, facial reanimation, facial
defects from neoplasms, infection or trauma, defects of mandible,
of maxilla, or nose, auricular reconstruction) and for
cranioplasty.
[0171] 4) Space augmentation via implantation or injection of the
polymer-cartilage scafflod in order to improve function. For
example, injection or implantation in the larynx or the vocal cord
in order to improve vocalization, in the pharynx in order to
improve swallowing, in the middle ear in order to provide support
and improve hearing.
[0172] Besides being useful for cartilage repair in humans, the
herein disclosed invention contemplates the use of scaffold or
templates made of chitosan or a crosslinked polysaccharide prepared
by crosslinking a polysaccharide with a polyamine. These scaffolds
or templates have chondrocytes plated and growing thereon and are
useful for cartilage repair in horses and particularly the
cartilage of their knees.
[0173] In its most comprehensive form, this invention involves a
surgical method for surgically implanting chondrocytes into
cartilage tissue comprising the steps of:
[0174] 1) Culturing chondrocytes on dextran microcarrier beadlets
and thereafter separating the chondrocytes from the microcarrier
beadlets by digestion with dextranse,
[0175] 2) Plating and growing said separated chondrocytes on a
scaffold or template manufactured from a crosslinked polysaccharide
prepared by crosslinking said polysaccharide with a polyamine or
alternatively plating and growing said separated chondroctyes on a
scaffold or template comprising chitosan and then
[0176] 3) Surgically implanting into cartilage tissue said scaffold
or template having chondroctyes plated and grown thereon, to
thereby repair cartilage tissue in a patient requiring cartilage
repair.
[0177] A Surgical Technique for Cartilage Graft is Set Forth
[0178] The following methodology applies to localized cartilage
lesions suitable for laboratory grown autogenous cartilage grafts.
The applicable lesion is excised down to subchondral bone using a
#15 scalpel to create essentially vertical walls in intact
articular cartilage. The walls should actually be cut so that the
surface opening is slightly smaller than the lesion size at the
level of the subchondral plate. This will create a three
dimensional cavity which is slightly conical in nature with the top
of the cone toward the articular surface. The base of the cavity is
cleaned of all cartilage material, exposing but not penetrating the
subchondral plate. Once the cavity is created, it is filled with
cold-curing dental molding material that is allowed to set
(.about.5 minutes). The mould of the lesion cavity is trimmed to
the edges of the lesion and will serve as a template for cutting
the graft implant to the exact size of the lesion. The walls of the
graft are also trimmed to be slightly conical, with the top of the
cone toward the surface that will become the articular surface. In
this way the graft can be press fitted into the lesion and will be
mechanically held in place by virtue of the precision fit and the
conical shape of both the graft and the cavity into which it is to
be placed.
[0179] Once the graft has been positioned in the site it must be
tested for stability. If there is any question about the stability,
ancillary stabilization can be created by suturing the edge of the
graft to the lamina splendans of the periphery of the lesion with
5-0 interrupted sutures.
Comprehensive Method for Surgical Cartilage Repair
[0180] The herein disclosed invention finds applicability in the
field of cell culture to produce viable cells and then using these
cells for tissue repair in a patient. More specifically, the herein
disclosed invention finds applicability in the field of cartilage
repair. The invention contemplates the rapid and efficient
obtaining of a sample of chondrocytes, comprising the steps of
culturing the chondrocytes to obtain a sufficient sample of
chondrocytes, preparing a chondrocyte containing scaffold and then
surgically implanting the scaffold for cartilage repair.
Integrin and Collagen Expression of Human Chondrocytes
[0181] Propagated in Microcarrier Spinner Culture
[0182] Introduction
[0183] Cartilage turnover and metabolism depends on the interaction
between chondrocytes and their surrounding extracellular matrix.
Recent studies reported that the receptors on chondrocytes which
mediate these interactions belong to the family of integrins (1).
These proteins consist of heterodimeric, non-covalent complexes of
alpha and beta subunits that act as transmembrane receptors which
link the cytoskeleton and the extracellular matrix. The integrin
mediated linkage, seems to play a critical role in the elaboration
and maintenance of the extracellular matrix. We have previously
discovered that human articular chondrocytes propagated on the
surface of collagen microcarriers maintained in spinner culture
actively produce collagen type II and proteoglycans which
constitute the major extracellular matrix components of cartilage.
(2). The mechanism by which the microcarrier spinner culture
promotes expression of the chondrocytic phenotype has not been
defined. The major objective of this study was to compare the
relative expressions of integrin and collagen types I and II by
human chondrocyte: a) immediately following surgical retrieval, and
after two weeks of culture in b) monolayers or c) microcarrier
spinners. We tested the hypothesis that the expression of intern
correlates with that of collagen type II. Integrins that act as
binding sites for extracellular matrix components may help to
maintain normal chondrocyte phenotype.
[0184] Methods
[0185] Non-fibrillated articular cartilage was obtained from four
osteoarthritic patients during knee surgery. Chondrocytes isolated
by collagenase digestion were directly seeded onto Cellagen
microcarriers (100-400 .mu.m derived from bovine corium, ICN,
Cleveland, Ohio) or as monolayer culture as previously described.
Microcarrier and spinner cultures were incubated at 37.degree. C.,
5% CO.sub.2 for fourteen days. Chondrocytes were harvested and the
RNA was isolated by the TRIzol Reagent method (Life Technologies).
A total cDNA library was synthesized using the Advantage RT-for-PCR
Kit (Clonetech Laboratories, Palo Alto, Calif.) with oligo
(dT).sub.18 primer. The resulting reverse transcriptase product was
expanded using the SuperTaq Plus (Ambion, Austin, Tex.) PCR Kit and
specific primers for collagen types I and II and integrin .alpha.5.
This integrin chain sequence is of interest since it has been
detected in articular cartilage. The housekeeping gene
glyceraldehyde 3 phosphate (GPDH) was analyzed using the GPDH
Control Amplimer Kit (Clonetech Laboratories). The PCR products
were analyzed by electrophoresis in 1.5% agrose that contained
ethidium bromide and photographed with UV light excitation.
Production of integrins and collagens was verified by
immunostaining using monospecific antibodies for .alpha.5 and for
collagens with affinity purified antibodies to collagen types I and
II (Fisher Scientific)
[0186] Results
[0187] Integrin .alpha.5 and collagen type II were expressed in
chondrocytes obtained directly from articular cartilage (P-0) or
from monolayer (Mono) and spinner (Spin) cultures in all four cases
examined as shown in a representative picture below. In contrast,
collagen type I expression was not (2/2) or minimally (2/2)
detectable at P-0 and in spinner cultures but was increased in
monolayer cultures (4/4). The housekeeping gene GPDH was
upregulated from P-0 to monolayer and spinner cultures indicating
increased metabolic activity.
[0188] Discussion/Conclusion
[0189] The principal finding of the present study is that the
expression of integrin .alpha.5 parallels that of collagen type II
in chondrocytes taken directly from articular cartilage and from
monolayer or microcarrier spinner cultures. The observation that
both genes are detectable at high levels suggests the potential for
chondrocytes to readily synthesize the protein products. The
observation that collagen type I expression is more prominent in
monolayer cultures may indicate the progression into a
fibroblastoid phenotype.
[0190] A feature of this invention encompasses the use of
integrins, a glycoprotein, to serve as receptors to link
chondrocytes. The inventors intend to use integrins to help
facilitate the binding of chondrocytes to scaffold material, and
particularly to bind chondrocytes to a chitosan matrix
scaffold.
[0191] References
[0192] 1. Lapadula G., Iannone F., Zuccaro C., Grattagliano V.,
Covelli M., Patella V., Lo Bianco G., and Pipitone V. Clin and Exp
Rhemnatol. 15:247-254, 1997.
[0193] 2. Frondoza C., Sohrabi A., and Hungerford D. Biomaterials
17:879-888, 1996.
[0194] Chondrocyte Culture at Low Oxygen Levels
[0195] Introduction
[0196] Articular cartilage is an a vascular tissue and its oxygen
supply is obtained by diffusion from synovial fluid or from the
vasculature of the subchondral bone (1). It has been reported that
the oxygen tension in cartilage is lower than other tissues.
However, little is known as to the role of oxygen tension in the
regulation of chondrocyte proliferation and phenotype expression.
The present study tests the hypothesis that oxygen tension affects
chondrocyte proliferation and function. Here we compare the
response of human chondrocytes in monolayer and microcarrier
spinner cultures to different oxygen tension in vitro.
[0197] Methods
[0198] Chondrocytes were obtained from cartilage samples taken from
an osteoarthritic patient at the time of total knee replacement.
Cells were retrieved by collagenase digestion and seeded as
monolayer or microcarrier spinner cultures as previously described
(2). One set of monolayer and spinner flasks were incubated
continuously from the time of seeding at either 5% or 20% (ambient)
O.sub.2. All cells were cultured at 37.degree. C. with 5% CO.sub.2.
After 13 days of culture. samples of cells were pulsed with 8
.mu.Ci/ml of .sup.3H thymedine overnight. On day 14, the unlabeled
cells were harvested for RNA isolation. The cells pulsed with
.sup.3H thymidine were cytocentrifuged onto microscope slides using
Cytospin II (Shandon Lipshaw).
[0199] The RNA was isolated by the TRIzol Reagent method (Life
Technologies). A total cDNA library was synthesized using the
Advantage RT-for-PCR Kit (Clontech Laboratories, Palo Alto, Calif.)
with the oligo (dT) primer. The resulting reverse transcriptase
product was expanded using the SuperTaq Plus (Amabion, Austin,
Tex.) PCR Kit and specific primers for the sequences of interest.
The housekeeping gene GAPDH was analyzed using the G3PDH Control
Amplimer Kit (Clontech Laboratories). The PCR products were
analyzed by electrophoresis in 1.5% agarose that contained ethidium
bromide and photographed with UV light excitation.
[0200] The slides with .sup.3H-thymidine labeled cells were dipped
into NTB-2 emulsion (Kodak Imaging, Rochester, N.Y.) and incubated
in the dark for 2 weeks at 4.degree. C. They were developed with
Dektol Developer and Fixer (Kodak) end counterstained with
toluidine blue. The number of labeled and unlabeled cells were
enumerated blind and the proliferative capacity expressed as
percent labeling index. Statistical analysis was performed using
the Jmp In statistical software (SAIS Institute).
[0201] Results
[0202] Chondrocytes in microcarrier spinner culture incubated at 5%
oxygen tension showed a lower proliferative index (p<0.0174)
than at 20% oxygen tension. Monolayer cultures, which had a lower
proliferative index than spinner cultures, were not affected by
lower oxygen tension >0.05.
[0203] As shown in FIG. 2, RT-PCR of RNA from spinner cultures
incubated at 5% oxygen showed a decreased expression of collagen
type I RNA in the spinner cultures compared to those at 20% oxygen.
In contrast, no change in collagen type I expression was noted in
the monolayer cultures. Aggrecan RNA expression was also increased
in the spinner cultures incubated in 5% oxygen but was unaffected
in the monolayer cultures. In both spinner and monolayer cultures,
the collagen type II RNA expression was increased at 5% oxygen
cultures compared to 20% oxygen. The housekeeping gene GAPDH
indicated equal loading for all samples.
[0204] Discussion
[0205] The present study documents for the first time that low
oxygen tension (5% O.sub.2) similar to that of articular cartilage
In vivo, inhibits chondrocyte proliferation while enhancing
expression of the chondrocytic phenotype in microcarrier spinner
culture. Our finding that chondrocyte microcarrier spinner cultures
are more responsive to changes in oxygen tension than monolayer
cultures supports our previous studies (2) that the former better
simulates the physiological environment in the joint. Knowledge of
the role of oxygen tension in regulating chondrocyte proliferation
and gene function may help in propagating large numbers of
functional chondrocytes for clinic applications.
[0206] A significant feature of the herein disclosed invention is
the feature of culturing chondrocytes at a low oxygen tension when
being seeded on to a scaffold. This feature is particularly useful
when the chondrocytes are to be cultured onto a chitosan scaffold.
The low level of oxygen is about 5% oxygen, however, amounts
greater or less than 5% could be determined by those skilled in the
art. It is expected that this low level of oxygen will enhance
expression of collagen type II and aggrecan, as well as helping to
maintain the chondrocyte phenotype.
[0207] References
[0208] 1. Ysart, O E and Mason, R M. Biochemica et Biophysica Acta
1221:15-20, 1944.
[0209] 2. Frondoza, C.; Sohrabi, A.; Hungerford, D.; Biomaterials
17:879-888 1996.
[0210] An elegant embodiment of this invention involves a
servo-mechanism or mechanism for the instantaneous adjustment of
oxygen supplied to the chondrocyte cell culture in order for there
to be optimum growth conditions for the chondrocytes in culture.
This mechanism is described in FIG. 3 of the drawings. The
mechanism has a revolving culture chamber or cell grower 100 in
which the culture takes place. This at times is referred to as a
spinner culture. There is an oxygen monitor 102 which senses the
optimum oxygen concentration and sends, a message to the oxygen
supply unit 101 when the culture chamber has less than the optimum
supply of oxygen. The mechanism also has a controller
(microprocessor) 103, along with a motor 104 and rotary drive
104.
Chitosan Supports the Expression of Extracellular Matrix Proteins
in Human Osteoblasts and Chondroctyes
[0211] The need for biocompatible materials that can successfully
support the growth and phenotypic expression of osteoblasts and
chondrocytes is a major challenge in the tissue engineering of bone
and cartilage. One candidate material that may fulfill this need is
chitosan. Over the last twenty years, chitosan has been
investigated for application in skin grafting, dental implantation,
and drug delivery systems..sup.1-4 Chitosan is the deactylated
product of chitin, a ubiquitous biopolymer found in the exoskeleton
of marine invertebrates and the cell walls of fungi and yeast. Its
unique properties include biocompatibility, bioactivity, and
bioresorbability. Earlier reports described the utility of chitosan
as a substrate for the growth of keratinocytes, fibroblasts,
hepatocytes, and rabbit chondrocytes. However, little is known
about the potential usefulness of chitosan in propagating human
osteoblasts and chondrocytes. In the present study, we test the
hypothesis that chitosan can support the proliferation and function
of human osteoblasts and chondrocytes.
[0212] Methods and Materials
[0213] A 4% (w/v) solution of chitosan was sterilized by
autoclaving and 100 .mu.l was evenly pipetted into 24 well culture
plates (Becton Dickinson, Lincoln Park, N.J.) fitted with 15 mm
plastic coverslips (Nalge Nunc Intl., Naperville, Ill.). The
chitosan films were then allowed to dry over a period of 24 hours.
The acidity of the film was neutralized with a 0.5 N solution of
NaOH. Excess base was then removed with repeat washings of Hanks
Balanced Salt Solution. Plates were then sterilized under UV
radiation for 48 hours. Normal human osteoblasts (Clonetics,
Walkersville, Md.) maintained in monolayer cultures and condrocytes
retrieved from two week old microcarrier spinner cultures were used
for these experiments (5). Cells were seeded (1.times.10.sup.5/1
ml) into wells containing plastic coverslips coated with chitosan
films. The same number of cells were seeded into control wells
containing only (1.times.10.sup.5/1 ml) plastic coverslips. Cells
were incubated over a period of 7 days at 37.degree. and 5%
CO.sub.2. Cells propagated in the presence or absence of chitosan
were retrieved by typsinization, cytocentrifuged, and immunostained
for collagen type I, collagen type II, and keratan sulfate. Cell
viability in situ was assessed using Live/Dead Assay (Molecular
Probes, Eugene, Oreg.).
[0214] RNA was isolated from the remaining wells by the Trizol
Reagent method (Life Technologies). A total cDNA library was
synthesized using the Advantage RT-for-PCR Kit (Clontech
Laboratories, Palo Alto, Calif.) with the oligo (dT) primer. The
resulting reverse transcriptase product was expanded using the
SuperTaq Plus (Ambion, Austin, Tex.) PCR Kit and specific primers
for the sequences of interest. The housekeeping gene GAPDH was
analyzed using the G3PDH Control Amplimer Kit (Clontech
Laboratories). The PCR products were analyzed by electrophoresis in
a 1.5% agarose gel that contained ethidium bromide and photographed
with UV light excitation.
[0215] The search for biocompatible materials that can support the
growth and phenotypic expression of osteoblasts and chondrocytes is
a major challenge in the tissue engineering of bone and cartilage.
Chitosan, a copolymer of glucosamine and N-acetylglucosamine, may
provide a solution to this search. It is a deacetylated product of
chitin, a ubiquitous biopolymer found in the exoskeleton of marine
invertebrates and the cell walls of fungi and yeast. Little is
known about its potential utility in propagating human osteoblasts
and chondrocytes. Here, we tested the hypothesis that chitosan can
promote the survival and function of osteoblasts and chondrocytes.
Chitosan (4% w/v in 2% HAC) was prepared as: (1) film coatings or,
(2) strand meshes. These were used for seeding human osteoblasts,
MG-63 osteoblast-like cells or articular chondrocytes at
1.times.10.sup.5/cells/well with or with chitosan film; or
2.times.10.sup.6 cells/0.6 g of chitosan mesh. Cultures were
incubated at 37.degree., 5% CO.sub.2. RTPCR and immunocylochemistry
were used to analyze the phenotype expression. Cell viability was
assessed using a molecular probe vital dye.
[0216] Osteoblasts and chondrocytes appeared spherical and
refractile on chitosan films whereas control cells (.gtoreq.90%) on
plastic coverslips were elongated and spindle shaped through 7 days
of culture. Greater than 90% of cells propagated in the presence or
absence of chitosan remained viable. Human osteoblasts propagated
on chitosan films continued to express collagen type 1, while
chondrocytes express collagen type II and aggrecan. Osteoblastic
cells cultured overnight on chitosan strands were adherent,
remained viable and retained their spherical morphology.
[0217] The present work demonstrates the biocompatibility of
chitosan as a substrate for the growth and continued function of
human osteoblasts and chondrocytes. The ability of chitosan to
promote cell survival and extracellular matrix synthesis suggests
its potential utility in tissue engineering.
[0218] Osteoblasts and chondrocytes plated on chitosan films
appeared spherical and refractile when viewed by phase contrast
microscopy after 24 hours of culture. In contrast, cells grown on
plastic coverslips were elongated and spindle shaped. This
difference was most striking in 3-day old control cultures where
the majority of cells displayed a fibroblastic appearance. In
comparison, less than 10% of cells grown on chitosan appeared
fibroblastic. These differences persisted through the 7-day time
point. Greater than 90% of human osteoblasts and chondrocytes
propagated in the presence or absence of chitosan stained green
indicating viability based on the vital dye assay. Less than 10%
stained red, indicating insignificant toxicity after 7 days of
culture. Human osteoblasts propagated in chitosan continued to
express collagen type I. Similarly, human chondrocytes grown on
chitosan film expressed matrix proteins, collagen type II and
aggrecan. The ability of the cells propagated on chitosan to
continue synthesis of extracellular matrix proteins, indicated by
mRNA expression, was further confirmed by immuno-localization of
the collagens and keratan sulfate. Osteoblasts propagated on
chitosan stained intensely for collagen type I. Chondrocytes grown
on chitosan, did not show significant staining for collagen type I,
but stained intensely for collagen type II and keratan sulfate.
[0219] Of significance is the fact that osteoblasts and
chondrocytes propaged on chitosan survive and continue expression
of phenotype markers. Once the scaffolds are seeded with
osteoblasts or chondrocytes, these seeded scaffolds can be used for
repairing bone or cartilage defects.
[0220] In experiments a 4% (w/v) solution of chitosan was
sterilized and pipetted into well culture plates and allowed to dry
to form a film. The acidity of the film was neutralized and excess
base was washed away with Hanko Balanced Salt Solution. Osteoblasts
and chondrocytes were seeded (1.times.10.sup.5/ml) onto the
chitosan film. After incubation for 7 days at 37.degree. and 5%
CO.sub.2 and cell viability was assessed. Osteoblasts and
chondrocytes plated on chitosan film appeared sperical and
refractile after 24 hours of culture, while the same cells grown on
cover slips were elongated and spindle shaped. Cells cultivated on
chitosan continued their synthesis of extracellular matrix
proteins.
[0221] In the herein disclosed invention, a preferred scaffold is
one made of sponge chitosan.
[0222] References
[0223] 1. Chandy, T. et al. Biomat, Art Cells. Art Org 18(1): 1-24
(1990)
[0224] 2. Denuziere, A el al., Biomaterials 19(1):1275-85
(1998)
[0225] 3. Yagi, K el al., Biol Pharm Bull 20(12): 1290-4 (1997)
[0226] 4. Koyano, T. J Biomed Mater Res 39:486-90 (1998)
[0227] 5. Frondoza. C el al., Biomaterials 17:879-88 (1996)
[0228] Aside from being available from the knee, chondrocytes can
be obtained from nasal septal cartilage and ear cartilage and/or
costo-chondral cartilage. These cartilage sources of chondrocyte
cells may be more readily available and easier to conveniently
obtain.
Comprehensive Method of Use
[0229] The comprehensive method of this invention contemplates an
efficient procedure for cartilage repair as shown in the flow chart
of FIG. 4. The inventors contemplate cartilage repair in a patient
wherein a small sample of cartilage is removed from the patient.
Chondrocytes are separated from the cartilage tissue. These
chondrocytes are then cultured to produce adequate numbers for
seeding on a scaffold. Once seeding is complete, the scaffold with
chondrocytes viable thereon is surgically implanted into the
patient. The procedure disclosed herein contemplates a quick
turn-around between the time of obtaining the patient-sample of
cartilage and surgically implanting the chondrocyte-containing
scaffold.
[0230] In its most comprehensive method, this invention
involves:
Retrieval and Isolation of Chondrocytes from Cartilage
[0231] 1. Cartilage will be obtained from the knee, nasal septal
cartilage, ear cartilage and/or costo-chondral cartilage.
[0232] 2. Cartilage tissue will be minced into small pieces in a
plastic petri dish under sterile condition in the laminar flow
hood.
[0233] 3. The minced tissue will be placed in a culture jar
containing lml of 0.1% collagenae in HY:DMEM (1:1) medium.
[0234] 4. The minced tissue will be incubated for 18 hours in an
incubator at 37.degree. C., 5% CO.sub.2 to digest the matrix and
separate chondrocytes.
[0235] 5. The digested tissue and cell suspension will be passed
through the Cellector screen (screen filter) to separate cells from
undigested material.
[0236] 6. The separated cell suspension will be centrifuged at 1000
rpm. The supernatant fluid will be pipetted out and then discarded.
The cell pellet will be washed by resuspending in Hanks' balanced
salt solution followed by centrifugation at 1000 rpm. The wash
fluid will be pipetted out and then discarded. The cells will
finally be resuspended in HY media and then counted for subsequent
use or cryopreserved for storage in liquid nitrogen.
Cell Culture
[0237] The cells obtained from tissue will be propaged on
microcarriers and retrieved therefrom for use.
Preparation of the Cell-Seeded Scaffold or Construct
[0238] 1. The material will be fashioned into a porous three
dimensional sponge and scaffold. And more particularly, the
fashioned material will be chitosan.
[0239] 2. The shape, thickness and size for cell seeding will be
configured to fit into the defect to be filled.
[0240] 3. The cells will be seeded into the scaffold by applying
the cell suspension on the surface of the scaffold such that the
entire volume will penetrate through the scaffold.
[0241] Surgical Repair of Articular Cartilage
[0242] 1. The cartilage defect will be prepared for transplantation
of the cell-seeded construct or scaffold.
[0243] A) The defect will be debrided and the area will be cleaned
by saline irrigation.
[0244] B) The defect may be treated with material (e.g.
transglutamase) that could help glue and keep the construct in
place.
[0245] 2. A chondrocyte scaffold having been previously prepared
will then be implanted into the prepared defect. The construct will
be fitted into the defect to the level of the surrounding normal
articular cartilage and then left as is.
[0246] Advantages of the Invention
[0247] The herein disclosed invention seeks to perfect cartilage
repair surgery. The invention envisions overcoming many of the
failings practiced by the medical profession and, particularly, the
commercial practice of cartilage repair. The invention seeks to
improve cartilage repair surgery in the following ways:
[0248] 1) The invention will make available sufficient quantities
of compatible chondrocytes required for repair (and in a relatively
short period of time) by growing or culturing the chondrocytes in a
spinning culture chamber and, preferably, at reduced oxygen
levels.
[0249] 2) The patient's own chondrocytes will be used to culture
cells for plating onto the scaffold. Cadaver cells will not have to
be used.
[0250] 3) The invention envisions rapidly growing chondrocytes in
culture.
[0251] 4) Chondrocytes cultured will be characterized as having an
approximately normal phenotype.
[0252] 5) Scaffolds prepared for transplant will be physiologically
compatible and resorbable into the body.
[0253] 6) Cell culture and surgical facilities will be at a single
location which will assure rapid and efficient surgical procedures
envisioning a high surgical success rate.
[0254] This should be contrasted with the correct medical
procedures, which may be outlined as follows:
[0255] Taking chondrocytes from the patient for subsequent growing
in a chamber (non-spinning and at ordinary oxygen levels) which
reuires a relatively long time period to grow sufficient quantities
of the chondrocytes for subsequent transplanting into the
patient.
[0256] Taking chondrocytes from a cadaver, growing the chondrocytes
and plating on a scaffold (or other plastic or inorganic support)
and thereafter transplanting into the patient.
[0257] Obviously, many modifications may be made without departing
from the basic spirit of the present invention. Accordingly, it
will be appreciated by those skilled in the art that within the
scope of the appended claims, the invention may be practiced other
than has been specifically described herein.
References
[0258] 1. Tomihata, Kenji and Ikada, Yoshito: In vitro and in vivo
degradation of films of chitin and its deacetylated derivatives.
Biomaterials 18: 567-575, 1997.
[0259] 2. Amiji, Mansoor M.: Surface modification of chitosan
membranes by complexation-interpenetration of anionic
polysaccharides for improved blood compatibility in hemodialysis.
J. Biomater. Sci. Polymer Edn, 8: 281-298, 1996.
[0260] 3. Austin, P. R.; Brine, C. J.; Castle, J. E.; and Zikakis,
J. P.: Chitin: new facets of research. Science 212: 749-753,
1981.
[0261] 4. Vasudev, Sindhu C.; Chandy, Thomas; and Sharma, Chandra
P.: Development of chitosan/polyethylene vinyl acetate co-matrix:
controlled release of aspirin-heparin for preventing cardiovascular
thrombosis. Biomaterials 18: 75-381, 1997.
[0262] 5. Taxavel, M. N. and Domard, A.: Collagen and its
interaction with chitosan. Biomaterials 16: 865-871, 1995.
[0263] 6. Maruyama, Masaaki and Ito, Michio: In vitro properties of
a chitosan-bonded self-hardening paste with hydroxyapatite
granules. Journal of Biomedical Materials Research, 32: 527-532,
1996.
[0264] 7. Wan, Andrew C. A.; Khor, Eugene; and Hastings, Garth W.:
Hydroxyapafite modified chitin as potential hard tissue substitute
material. J Biomed Mater Res 38:235-241, 1997.
[0265] 8. Kuettner, K. E.: Biochemistry of articular cartilage in
health and disease. Clin. Biochem. 25: 155, 1992.
[0266] 9. Mankin, H. J.: The reaction ofarticular cartilage to
injury and osteoarthritis. New Engl. d. Med. 291: 1285, 1974.
[0267] 10. Mankin, H. J. and Lippiello, L.: Biochemical and
metabolic abnormalities in articular cartilage from osteo-arthritic
human hips. J. Bone Joint Surg. 52(3, Suppl. A): 424, 1970.
[0268] 11. Brittberg, M.; Lindahl, A.; Nilsson, A.; Ohlsson, A.;
Isaksson, O.; and Peterson, L.: Treatment of deep cartilage defects
in the knee with autologous chondrocyte transplantation. New
England J. Med. Res. 331: 889, 1994.
[0269] 12. Vacanti, C. A.; Kim, W.; Schloo, B.; Upton, .I.; and
Vancanti, J. P.: Joint resurfacing with cartilage grown in situ
from cellpolymer structures. Amer. d. SportsMed. 22: 485, 1994.
[0270] 13. Ma, P. X.; Schloo, B.; Mooney, D.; and Langer, R.:
Development of biomechanical properties and morphogenesis of in
vitro tissue engineered cartilage. J. Biomed. Mater. Res. 29: 1587,
1995.
[0271] 14. Chu, C. R.; Coutts, R. D.; Yoshioka, M.; Harwood, F. L.;
Monosov, A. Z.; and Amiel, D.: Articular cartilage repair using
allogenic perichondrocyte seeded biodegradable porous polylactic
acid (PLA): A tissue-engineering study. J. Biomed Mater. Res.
29:1147, 1995.
[0272] 15. Grande, D. A.; Halberstadt, C.; Naughton, G.; Schwartz,
R.; and Manji, R.: Evaluation of matrix scaffolds for tissue
engineering of articular cartilage grafts. J. Biotaed. Mater. Res.
34:211,1997.
* * * * *