U.S. patent application number 12/070503 was filed with the patent office on 2009-02-12 for composition for the delivery of live cells and methods of use thereof.
Invention is credited to Lawrence J. Bonassar, Henry R. Costantino, Mark A. Tracy.
Application Number | 20090041851 12/070503 |
Document ID | / |
Family ID | 24454483 |
Filed Date | 2009-02-12 |
United States Patent
Application |
20090041851 |
Kind Code |
A1 |
Costantino; Henry R. ; et
al. |
February 12, 2009 |
Composition for the delivery of live cells and methods of use
thereof
Abstract
The invention relates to an improved method for administering
live cells to a patient and compositions useful in the method. The
composition comprises live cells and biocompatible, biodegradable
polymer microparticles. The cells and microparticles of the
cell/microparticle composition can be contacted immediately prior
to administration, or can be contacted in culture for a specified
period of time prior to administration. In the method of the
invention, an effective amount of the cell/microparticle
composition is administered to a patient in need thereof by
injection to a treatment site of the patient to provide a
therapeutic effect in the patient. The therapeutic effect can be,
for example, the formation of new tissue at the treatment site, or
the production and secretion of a biologically active secretory
molecule at the treatment site. The therapeutic effect resulting
from injection of the cell/microparticle composition into a
treatment site, is determined by the type of cell present in the
composition. The composition comprising lives cells and
biocompatible, biodegradable polymer microparticles can further
comprise a biologically active agent. In a preferred embodiment,
the biologically active agent is incorporated into the
microparticle. The biologically active agent can be, for example,
factors which modulate cell growth.
Inventors: |
Costantino; Henry R.;
(Grantham, NH) ; Bonassar; Lawrence J.; (Acton,
MA) ; Tracy; Mark A.; (Arlington, MA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD, P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Family ID: |
24454483 |
Appl. No.: |
12/070503 |
Filed: |
February 19, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10784908 |
Feb 23, 2004 |
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12070503 |
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09612744 |
Jul 10, 2000 |
6719970 |
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10784908 |
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Current U.S.
Class: |
424/501 ;
424/93.1; 424/93.7 |
Current CPC
Class: |
C12N 5/0655 20130101;
A61K 35/39 20130101; C12N 2531/00 20130101; A61K 35/32 20130101;
A61P 19/04 20180101; C12N 2533/40 20130101; A61K 35/30 20130101;
A61P 3/10 20180101 |
Class at
Publication: |
424/501 ;
424/93.1; 424/93.7 |
International
Class: |
A61K 35/12 20060101
A61K035/12; A61K 9/14 20060101 A61K009/14; A61K 35/32 20060101
A61K035/32; A61K 35/39 20060101 A61K035/39; A61P 3/10 20060101
A61P003/10; A61P 19/04 20060101 A61P019/04 |
Claims
1. A method of administering live cells to a patient in need
thereof comprising injecting into a treatment site of the patient
an effective amount of a composition comprising biocompatible,
biodegradable polymer microparticles and live cells, wherein said
cells provide a therapeutic effect in the patient.
2. The method of claim 1 wherein the therapeutic effect comprises
the generation of new tissue at the treatment site.
3. The method of claim 2 wherein the live cells are selected from
cartilage producing cells, organ cells, fibroblasts, osteoblasts,
nerve cells, smooth muscle cells, skeletal muscle cells, and
Schwann cells.
4. The method of claim 2 wherein the cells are chondrocytes.
5. The method of claim 4 wherein the new tissue is cartilage
tissue.
6. The method of claim 5 wherein the treatment site is into the
articular space of a joint of the patient.
7. The method of claim 1 wherein the therapeutic effect is the
secretion of a biologically active secretory molecule.
8. The method of claim 7 wherein the biologically active secretory
molecule is selected from hormones, cytokines, growth factors,
trophic factors, angiogenesis factors, antibodies, blood
coagulation factors, lymphokines, enzymes and agonists, precursors,
active analogs or active fragments thereof.
9. The method of claim 8 wherein the biologically active secretory
molecule is the hormone insulin.
10. The method of claim 9 wherein the live cells are pancreatic
islet cells.
11. The method of claim 8 wherein the biologically active secretory
molecule is dopamine.
12. The method of claim 11 wherein the live cells are selected from
PC-12 cells, adrenal chromaffin cells and fetal nigral primordia
cells.
13. The method of claim 1 wherein the the biocompatible,
biodegradable polymer of the microparticle is selected from
poly(lactides), poly(glycolides), poly(lactide-co-glycolides),
poly(lactic acid)s, poly(glycolic acid)s, polycarbonates,
polyesteramides, polyanydrides, poly(amino acids), polyorthoesters,
poly(dioxanone)s, poly(alkylene alkylate)s, copolymers of
polyethylene glycol and polyorthoester, polyurethanes, blends
thereof, and copolymers thereof.
14. The method of claim 13 wherein the biocompatible, biodegradable
polymer is a poly(lactide-co-glycolide).
15. The method of claim 1 wherein the composition further comprises
a pharmaceutically acceptable carrier.
16. The method of claim 1 wherein the composition further comprises
a biologically active agent.
17. The method of claim 16 wherein the biologically active agent
has tissue regeneration inductive properties.
18. The method of claim 17 wherein the biologically active agent is
a growth factor or differentiating factor.
19. The method of claim 18 wherein the growth factor is selected
from basic fibroblast growth factor (bFGF), platelet-derived growth
factors (PDGF), transforming growth factors (TGF- , TGF- ),
cementum growth factors, epidermal growth factor (EGF), hepatocyte
growth factor, heparin binding factor, insulin-like growth factors
I or II (IGF-I, IGF-II), erythropoietin, and nerve growth factor
(NGF).
20. The method of claim 18 wherein the differentiating factor is a
morphogenic protein.
21-60. (canceled)
Description
RELATED APPLICATION(S)
[0001] This application is a continuation of U.S. application Ser.
No. 10/784,908, filed Feb. 23, 2004, which is a continuation of
U.S. application Ser. No. 09/612,744, filed Jul. 10, 2000, now U.S.
Pat. No. 6,719,970, issued Apr. 13, 2004. The entire teachings of
the above application(s) are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Implantable polymeric materials capable of being degraded
and absorbed by the body have been used in medicine for many years.
For example, implantable devices can be pre-seeded with a desired
cell type, and used as structural supports or scaffolds for guiding
tissue regeneration. One example is the regeneration of cartilage
tissue using a degradable fiber mesh pre-seeded with chondrocytes
as described in U.S. Pat. No. 5,041,138 to Vacanti et al. However,
implantation of the scaffolds requires surgical intervention which
presents disadvantages, such as the risk of infection and the need
for invasive and painful procedures.
[0003] Suspensions of liquid hydrogel-cell compositions, which
solidify in vivo following administration, have also been described
as useful for the delivery of cells to a tissue surface in need of
repair. See, for example, U.S. Pat. No. 5,944,754 to Vacanti.
However, controlled delivery and containment of a liquid system
within a particular area is difficult, and the liquid can spread to
areas other than the implant site prior to solidification.
[0004] As such, a need exists for improved compositions and methods
for administering live cells to a host to provide a therapeutic
effect in the host, such as tissue regeneration.
SUMMARY OF THE INVENTION
[0005] The invention relates to an improved method for
administering live cells to a patient and compositions useful in
the method. The composition comprises live cells and biocompatible,
biodegradable polymer microparticles. The cells and microparticles
of the cell/microparticle composition can be contacted immediately
prior to administration, or can be contacted in culture for a
specified period of time prior to administration. In the method of
the invention, an effective amount of the cell/microparticle
composition is administered to a patient in need thereof by
injection to a treatment site to provide a therapeutic effect in
the patient. The therapeutic effect can be, for example, the
formation of new tissue at the treatment site, or the production
and secretion of a biologically active secretory molecule at the
treatment site. The therapeutic effect resulting from injection of
the cell/microparticle composition into a treatment site, is
determined by the type of cell present in the composition.
[0006] Alternatively, the cell/microparticle composition of the
invention can be used to generate in vitro tissue having a specific
shape which can then be implanted in the patient at an implantation
site to replace damaged tissue. The cell/microparticle composition
is placed in a cell culture chamber having a desired shape. As the
cells proliferate and adhere to the surfaces of the individual
microparticles, a coherent mass of tissue having the shape of the
culture chamber is formed. The formation of this coherent mass is
referred to herein as "sintering". Sintering differs from other
methods of preparing tissue in specific shapes, since it is the
combination of cells and polymer microparticles which provide the
matrix responsible for the shape of the resulting tissue rather
than the polymer alone.
[0007] In a specific embodiment, the composition and method of the
invention can be used for generating new tissue growth at a
treatment site in a patient in need of tissue regeneration. The
method for generating new tissue growth in a patient in need
thereof comprises administering to the patient by injection into a
treatment site an effective amount of a composition comprising
biocompatible, biodegradable polymer microparticles and live cells
capable of generating new tissue. The cells can be, for example,
hepatocytes for the generation of liver tissue or chondrocytes for
the generation of cartilage tissue. In a preferred embodiment, the
cells are chondrocytes which generate cartilage tissue. In a more
preferred embodiment, administration of the
chondrocyte/microparticle composition is into the articular space
of a joint of the patient.
[0008] As such, in a preferred embodiment, the invention relates to
a method of generating new cartilage tissue in a patient in need
thereof comprising administering by injection to a treatment site
of the patient a composition comprising live chondrocytes and
biocompatible, biodegradable polymer microparticles. The method and
composition can be used for the treatment of cartilage
deficiencies, defects, voids and conformational discontinuities in
a patient.
[0009] In a further embodiment, the composition of the invention
can be used in a method for secreting a biologically active
secretory molecule in a patient in need of said molecule. The
patient can be a mammal, such as a human. The method for secreting
a biologically active secretory molecule in a patient in need of
said molecule comprises administering to the patient by injection
into a treatment site of the patient an effective amount of a
composition comprising biocompatible, biodegradable polymer
microparticles and live cells, wherein said cells are capable of
secreting the biologically active secretory molecule.
[0010] In a particular embodiment, the cells are live pancreatic
islet cells which secrete insulin. The composition comprising
pancreatic islet cells and biocompatible, biodegradable polymer
microparticles can be administered to the pancreas or other
suitable treatment site of the patient. As such, the invention
relates to a treatment for diabetes.
[0011] In another preferred embodiment, the cells are dopaminergic
cells capable of secreting dopamine, such as PC-12 cells, adrenal
chromaffin cells and fetal nigral primordia cells. The composition
comprising live dopaminergic cells and biocompatible, biodegradable
polymer microparticles can be administered to the striatum or other
suitable treatment site of the patient in need thereof. Therefore,
the invention relates to a treatment for Parkinson's disease.
[0012] In a particular embodiment, the composition comprising lives
cells and biocompatible, biodegradable polymer microparticles
further comprises a biologically active agent. In a preferred
embodiment, the biologically active agent is incorporated into the
microparticle. The biologically active agent can be, for example,
factors which modulate cell growth, such as factors having tissue
regeneration inductive properties, for example, growth factors and
differentiating factors, for example, morphogenic proteins; a
cytokine; an extracellular matrix molecule; an antimicrobial agent;
an anti-inflammatory agent; and immunosuppressive agent, cel which
support the therapeutic effect of the administered cells or
combinations thereof. Incorporation of the biologically active
agent into the microparticles of the cell/microparticle composition
provides a sustained delivery of the biologically active agent at
the treatment site. It is preferred that the biologically active
agent enhances the primary therapeutic effect resulting from
administration of the live cell/microparticle composition.
[0013] The composition and methods of the present invention provide
a means for eliciting a therapeutic effect in a patient in need
thereof by administering a composition comprising lives cells and a
biocompatible, biodegradable polymer microparticle. Advantageously,
the composition permits the administration to be by injection which
obviates the need for an open surgical intervention to permit
exposure of the treatment area and the disadvantages associated
with open surgery (e.g., pain, infection, recovery time and cost).
In addition, the microparticles of the composition can have
incorporated therein a biologically active agent thereby providing
at the treatement site a sustained release of an agent which can be
complementary to or enhance the primary treatment.
[0014] Advantages of the cell/microparticle composition are also
realized when the composition is used to generate tissue in vitro
having a specific shape. That is, sintering offers advantages over
known methods of shaped tissue formation since a greater number of
cells can be initially loaded into the culture chamber of a
specified shape, thereby providing the needed tissue in a shorter
period of time.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The foregoing will be apparent from the following more
particular description of example embodiments of the invention, as
illustrated in the accompanying drawings in which like reference
characters refer to the same parts throughout the different views.
The drawings are not necessarily to scale, emphasis instead being
placed upon illustrating embodiments of the present invention.
[0016] FIG. 1 is a graph showing the percentage of attached cells,
in a cell/microparticle suspension, following a 16 hour incubation.
The results of control mixtures of cells alone and microparticles
alone are also shown.
[0017] FIG. 2 is a graph showing the percentage of attached cells
in a cell/microparticle suspension as a function of time.
[0018] FIG. 3 is a Scanning Electron Micrograph (SEM), at the
indicated magnifications, of a suspension of chondrocytes and
microparticles following a 2 hour incubation.
[0019] FIG. 4 is a Scanning Electron Micrograph (SEM), at the
indicated magnifications, of a suspension of chondrocytes and
microparticles following an 8 hour incubation.
[0020] FIG. 5 is a section of tissue removed from the injection
site of an animal receiving a mixture of PLG microparticles and
chondrocytes following a 4 week treatment period. The section was
stained with hematoxylin and eosin. The presence of a mature
cartilage cluster is noted.
DETAILED DESCRIPTION OF THE INVENTION
[0021] A description of example embodiments of the invention
follows. The foregoing and other objects, features and advantages
of the invention will now be more particularly described with
reference to the accompanying drawings and pointed out in the
claims. It is understood that the particular embodiments of the
invention are shown by way of illustration and not as limitations
of the invention. The principles of the invention can be employed
in various embodiments without departing from the scope of the
invention. A description of the preferred embodiments of the
invention follows.
[0022] The present invention relates to a method of administering
live cells to a patient in need thereof comprising injecting into a
treatment site of the patient an effective amount of a composition
comprising biocompatible, biodegradable polymer microparticles and
live cells, wherein said cells provide a therapeutic effect in the
patient.
[0023] The invention also relates to a composition comprising
biocompatible, biodegradable polymer microparticles and live cells.
It is understood that the live cells are responsible for the
primary therapeutic effect and that the microparticles contribute
to the retention of the cells at the treatment site and/or can
provide a surface for cell growth. The microparticles and cells can
be contacted immediately prior to administration, or can be
contacted in culture for a desired period of time prior to
administration. Preincubation can result in at least a portion of
the cells to become attached to the microparticles prior to
injection. However, as described herein, preincubation of the cells
and microparticles is not needed in order to achieve a therapeutic
effect following administration of the live cell/microparticle
composition. When desired, the composition can further comprise a
pharmaceutically acceptable carrier.
[0024] "Therapeutic effect", as that term is used herein, refers to
the generation of new tissue at the treatment site, the secretion
of a biologically active secretory molecule at the treatment site
or a combination thereof.
[0025] Cells suitable for use in the invention include cells
capable of generating tissue, cells which secrete biologically
active secretory molecules, and cells which metabolize materials.
Cell types that can be used for example, include but are not
limited to, cartilage producing cells, for example, chondrocytes;
fibroblasts; osteoblasts; exocrine cells; cells of intestinal
origin; bile duct cells; parathyroid cells; thyroid cells; cells of
the adrenal-hypothalamic-pituitary axis; organ cells, such as,
heart cells, for example, heart muscle cells, kidney cells, for
example, kidney epithelial cells, kidney tubular cells, and kidney
basement membrane cells, liver cells, for example, hepatocytes,
pancreatic cells, for example, pancreatic islet cells, lung cells,
and brain cells; endothelial cells; mucosal cells; nerve cells;
blood vessel cells; smooth muscle cells; skeletal muscle cells;
pleural cells; Schwann cells; ear canal cells; tympanic membrane
cells; peritoneal cells; tracheal epithelial cells macrophages; and
dopaminergic cells capable of secreting dopamine, such as PC-12
cells, adrenal chromaffin cells and fetal nigral primordia cells,
precursor cells of any of the above and stem cells.
[0026] Cells can be obtained directly from a donor, e.g., a
patient's own cells, from a culture of cells from a donor of the
same or a different species or from established cell culture lines.
Preferably cells are of the same species and more preferably of the
same immunological profile. Such cells can be obtained, for
example, by biopsy either from the patient or a close relative. The
cells are then grown in culture until confluent using standard cell
culture techniques and conditions, and used when needed. Typically,
cells are cultured only until a sufficient number of cells have
been obtained for a particular application. For example, autologous
cultured chondrocytes can be prepared according to the commercial
process CARTICEL.RTM.
[0027] The cells can be genetically altered or manipulated using
standard techniques prior to contacting them with the
microparticles. For example, the cells can be genetically
engineered to encode and secrete a desired biologically active
secretory molecule and/or produce a desired tissue or enhance
production of a desired tissue at a treatment site. For example,
pancreatic islet cells can be genetically engineered to secrete
enhanced amounts of insulin. Suitable method of genetically
engineering cells can be found in U.S. Pat. No. 5,399,346 to
Anderson et al., the entire content of which is incorporated herein
by reference.
[0028] Cells can be cultured using any of the numerous well known
cell culture techniques. Standard cell culture techniques are
described in Freshney, "Cell Culture, A Manual of Basic Technique",
Third Edition (Wiley-Liss, New York, 1994) the entire content of
which is incorporated herein by reference.
[0029] In a particular embodiment, the composition comprising live
cells and biocompatible, biodegradable polymer microparticles
further comprises a biologically active agent. In a preferred
embodiment, the biologically active agent can be incorporated into
the microparticle. The biologically active agent can be, for
example, factors which modulate cell growth, such as factors having
tissue regerneration inductive properties, for example, a growth
factor, a morphogenic protein, a cytokine, an immunosuppressive
agent, an extracellular matrix molecule, an antimicrobial agent, an
anti-inflammatory agent, cells which support the therapeutic effect
of the administered cell or combinations thereof. Incorporation of
the biologically active agent into the microparticles of the
cell/microparticle composition provides a sustained delivery of the
biologically active agent at the treatment site. It is preferred
that the biologically active agent enhances the primary therapeutic
effect resulting from administration of the cell microparticle
composition.
[0030] "Treatment site" as that term is used herein refers to any
internal structure or organ of the patient or a subcutaneous space
needing treatment and includes, but is not limited to, joints
including the articular space of the joints; internal organs, such
as the liver, pancreas, brain, heart, lung and kidney; pleural
cavities; the tracheal region; the thoracic cavity; the
gastrointestinal tract; the genito-urinary tract; the bladder;
vessels of the cardiovascular system; the gastointestinal tract
including the stomach, colon and esophagus and subcutaeous spaces
frequently accessed in cosmetic surgery, for example, the
subcutaneous space involving the ear, cheek, nose etc. . . .
[0031] For example, the therapeutic effect can be the generation of
new tissue, often referred to as guided tissue regeneration.
"Guided tissue regeneration" as that term is used herein refers to
the restoration and/or regeneration of the morphology and function
of hard and soft tissues that have been destroyed by disease or
trauma. In tissue regeneration, the regenerating tissues repopulate
the same site and space previously occupied by the healthy tissues
that have been destroyed. As such, the composition and method of
the invention can be used in a method for treating a tissue defect
in a patient using guided tissue regeneration.
[0032] For example, the therapeutic effect can be the generation of
new cartilage tissue. As such, the invention relates to a method of
generating new cartilage tissue in a patient in need thereof
comprising administering by injection to a treatment site of the
patient a composition comprising live chondrocytes or other
cartilage producing cells and biocompatible, biodegradable polymer
microparticles. The method and composition can be used for the
treatment of cartilage deficiencies, defects, voids and
conformational discontinuities in the patient.
[0033] The need for cartilage regeneration can result from damage
produced by diseases such as arthritis, trauma or congenital
deformities. Damaged cartilage is a major cause of physical
disability and deformity. For example, spinal discs, knees and hips
are common sites of cartilage damage. The current therapy for loss
of cartilage is replacement with a prosthetic material, such as
silicon for cosmetic repairs or metal alloys for joint realignment.
The use of a prosthesis is commonly associated with the significant
loss of underlying tissue and bone without recovery of the full
function allowed by the original cartilage. The prosthesis is also
a foreign body which can become an irritating presence in the
tissues. Other long-term problems associated with the permanent
foreign body can include infection, erosion and instability. As
such, the compositions and methods of the present invention provide
a desirable alternative to current therapies for treating cartilage
deficiencies.
[0034] "Cartilage" as used herein refers to a specialized type of
dense connective tissue having cells embedded in a matrix. There
are several kinds of cartilage. Hyaline cartilage is a
bluish-white, glassy translucent cartilage having a homogeneous
matrix containing collagenous fibers. Hyaline cartilage is found in
articular cartilage, costal cartilage, the septum of the nose, the
larynx and trachea. Articular cartilage is hyaline cartilage
covering the articular surfaces of bones. Costal cartilage connects
the ribs and the sternum. Fibrous cartilage contains collagen
fibers. Yellow cartilage is a network of elastic fibers holding
cartilage cells which is primarily found in the epiglottis, the
external ear, and the auditory tube.
[0035] In another embodiment, the therapeutic effect can be the
generation of new tissue of an internal organ. For example, the
generation of tissues of the liver, brain, lung, pancreas, heart,
and kidney. As such, the invention relates to a method of
generating new internal organ tissue in a patient in need thereof
comprising administering by injection to a treatment site of the
patient a composition comprising live internal organ cells and
biocompatible, biodegradable polymer microparticles. Preferably,
the treatment site is the organ of the patient which is the same
tissue type as the administered cells. More preferably the cells
are of the same tissue type and species as the recipient organ.
Most preferably, the cells are the patient's own cells of the same
tissue type as the recipient organ. For example, the patient's
hepatocytes can be preferably injected into the patient's
liver.
[0036] In a further embodiment, the therapeutic effect can be the
secretion of a biologically active secretory molecule in a patient
in need of said molecule. As such, the invention relates to a
method for secreting a biologically active secretory molecule in a
patient in need of said molecule comprising administering to the
patient by injection into a treatment site of the patient an
effective amount of a composition comprising biocompatible,
biodegradable polymer microparticles and live cells, wherein said
cells secrete the biologically active secretory molecule.
[0037] In a particular embodiment, the cells are live pancreatic
islet cells which secrete insulin. The composition comprising
pancreatic islet cells and biocompatible, biodegradable polymer
microparticles can be administered to the pancreas or other
suitable treatment site of the patient. As such, the invention
relates to a method for treating diabetes in a patient in need of
treatment comprising administering to the patient by injection into
a treatment site of the patient an effective amount of a
composition comprising biocompatible, biodegradable polymer
microparticles and live pancreatic islet cells, wherein said cells
secrete insulin. In a preferred embodiment the treatment site is
the pancreas. In another embodiment, the pancreatic islet cells can
be genetically engineered to provide enhanced secretion of
appropriate amounts of insulin in response to varying glucose
levels as described in U.S. Pat. No. 5,534,404 to Laurance et al.,
the contents of which is incorporated herein by reference.
[0038] In another embodiment, the cells are dopaminergic cells
capable of secreting dopamine, such as PC-12 cells, adrenal
chromaffin cells and fetal nigral primordia cells. The composition
comprising live dopaminergic cells and biocompatible, biodegradable
polymer microparticles can be administered to the striatum or other
suitable treatment site of the patient in need thereof. Therefore,
the invention relates to a method for treating Parkinson's disease
in a patient in need thereof, comprising administering to the
patient by injection into a treatment site of the patient an
effective amount of a composition comprising biocompatible,
biodegradable polymer microparticles and live dopaminergic cells,
wherein said cells secrete dopamine. In a preferred embodiment the
treatment site is the striatum of the patient.
[0039] Other diseases such as hypoparathyroidism and anemia can be
treated using cells in the cell/microparticle composition which
secrete parathyroid hormone and erythropoietin, respectively. The
secretory cells can be cells that naturally secrete the
biologically active secretory molecule of interest or the cells can
genetically engineered to do so or enhance the secretion of the
desired biologically active secretory molecule. The cells can be
chosen based on the biologically active secretory molecule desired.
For example, the secretion of hormones, cytokines, growth factors,
trophic factors, angiogenesis factors, antibodies, blood
coagulation factors, lymphokines, enzymes and agonists, precursors,
active analogs or active fragments thereof at a desired treatment
site can be accomplished following the method of the invention
described herein.
[0040] As such, the invention relates to a method of gene therapy
comprising administering to the patient by injection into a
treatment site of the patient an effective amount of a composition
comprising biocompatible, biodegradable polymer microparticles and
live cells which have been genetically engineered to secrete a
biologically active secretory molecule. Such methods of gene
therapy are described in, for example, U.S. Pat. No. 5,399,346 to
Anderson et al.
[0041] In another embodiment, the invention relates to a method of
restoring function to an organ or structure.
[0042] "Biologically active secretory molecule" as that term is
used herein refers to a biologically active molecule which is
released or secreted in vivo from a cell of the administered
cell/microparticle composition. The biologically active secretory
molecule can exert an effect on a separate target cell or on a
target molecule in the patient. For example, hepatocytes can
produce clotting factors and pancreatic islet cells can produce
insulin and/or glycogen. The cells generally should retain normal
morphology and cell function for the secretion of the bioactive
molecules.
[0043] Examples of biologically active secretory molecules include,
but are not limited to, hormones, cytokines, growth factors,
trophic factors, angiogenesis factors, antibodies, blood
coagulation factors, lymphokines, enzymes and agonists, precursors,
active analogs or active fragments thereof at a desired treatment
site can be accomplished following the method of the invention
described herein.
[0044] An effective amount of the composition of this invention can
be administered in vivo, for example, to a human, by injection at
desired treatment site. As used herein, an "effective amount" is
the amount needed to elicit the therapeutic effect, for example,
new tissue formation and/or secretion of a biologically active
secretory molecule in the patient following administration. For
example, the composition can contain from about
0.5.times.10.sup.6cells/mL to about 50.times.10.sup.6cells/mL, such
as from about 1.times.10.sup.6cells/mL to about
25.times.10.sup.6cells/mL. Further, the composition can contain
microparticles at a concentration of about 1 mg/mL to about 500
mg/mL, such as from about 1 mg/mL to about 250 mg/mL, for example,
1 mg/mL to about 100 mg/mL.
[0045] "Injection" as that term is used herein, includes
administration through a delivery port alone or in combination with
a surgical scope such as a laparoscope, endoscope, laryngoscope,
cystoscope, protoscope or thoracoscope. The delivery port can be,
for example, a surgical tube such as a catheter with an
appropriately sized bore, or a needle or needle-like port. As such,
delivery can include a minor incision in the patient to permit
entry of a delivery port, such as a needle or catheter, or a
combination of a delivery port an a surgical scope. Advantageously,
injection of the composition avoids the need for an open surgical
procedure to expose the treatment area.
[0046] "Patient" as that term is used herein refers to the
recipient of the treatment. Mammalian and non-mammalian patients
are included. In a specific embodiment, the patient is a mammal,
such as a human, canine, murine, feline, bovine, ovine, swine or
caprine. In a preferred embodiment, the patient is a human.
[0047] In an alternative embodiment, the cell/microparticle
composition can be used to prepare tissue having a specific shape.
Currently, there is a lack of acceptably compatible, functional
prosthesis to replace cartilage in individuals who have experienced
loss of contoured cartilage, for example, noses or ear. Loss of
contoured cartilage can result from bums or trauma. Typically, the
patient is subjected to additional surgery involving carving a
piece of cartilage out of a piece of lower rib to approximate the
necessary contours and inserting the cartilage piece into a pocket
of skin in the area where the nose or ear is missing. As such, the
present invention provides a desirable treatment alternative for
individuals needing repair of cartilage tissue wherein the tissue
is in a specific anatomical shape.
[0048] Therefore, the invention relates to a method of generating
new tissue having a specified anatomical shape comprising placing a
composition comprising live cells and biocompatible, biodegradable
polymer microparticles in a cell culture chamber having the
specified anatomical shape and sintering the composition. Briefly,
as the cells proliferate and adhere to the surfaces of the
individual microparticles in culture, a coherent mass of tissue
having the shape of the culture chamber is formed. The formation of
this coherent mass is referred to herein as "sintering". Sintering
differs from known methods of generating tissue in specific shapes,
since it is the cells which are primarily responsible for the shape
of the resulting tissue rather than the polymer matrix.
[0049] The term "biologically active agent," as used herein, is an
agent, or its pharmaceutically acceptable salt, which when released
in vivo, possesses the desired biological activity, for example
therapeutic, diagnostic and/or prophylactic properties in vivo. It
is understood that the term includes stabilized biologically active
agents as described herein. A sustained release composition of the
invention can contain from about 0.01% (w/w) to about 90% (w/w) of
active agent (dry weight of composition). The amount of agent can
vary depending upon the desired effect of the agent, the planned
release levels, and the time span over which the agent is to be
released. A preferred range of agent loading is between about 0.1%
(w/w) to about 30% (w/w). A more preferred range of agent loading
is between about 0.5% (w/w) to about 20% (w/w) agent.
[0050] When the composition comprising live cells and
biocompatible, biodegradable polymer microparticles further
comprises a biologically active, the biologically active agent can
be, for example, factors which modulate cell growth, for example,
factors having tissue regeneration inductive properties, such as
growth factors, and differentiating factors, for example,
morphogenic proteins, such as bone morphogenic proteins (BMPs) and
osteogenic proteins (OPs). In a preferred embodiment, the
biologically active agent is incorporated into the microparticle of
the cell/microparticle composition.
[0051] Growth factors suitable for use include, but are not limited
to, basic fibroblast growth factor (bFGF), platelet-derived growth
factors (PDGF), transforming growth factors (TGF- , TGF- ),
cementum growth factors, epidermal growth factor (EGF), hepatocyte
growth factor, heparin binding factor, insulin-like growth factors
I or II (IGF-I, IGF-II), erythropoietin, and nerve growth factor
(NGF).
[0052] Morphogenic proteins, which are capable of inducing bone and
other tissue formation, include, but are not limited to, OP-1,
OP-2, OP-3 (Osteogenic Protein), BMP2, BMP3, BMP4, BMP5, BMP6 and
BMP7 (Bone Morphogenic Protein). Morphogenic proteins and active
fragments and derivatives of the proteins are described in, for
example, U.S. Pat. Nos. 6,017,708 to Jones et al. issued on Jun. 6,
2000, U.S. Pat. No. 5,011,691 to Oppermann et al. issued on Apr.
30, 1991 and U.S. Pat. No. 4,968,590 to Kuberasampath et al. issued
on Nov. 6, 1990 the entire contents of all of which are hereby
incorporated by reference.
[0053] Other biologically active agents which can be present in the
cell/microparticle composition, preferably incorporated into the
microparticle of the cell/microparticle composition include
antimicrobial agents, anti-inflammatory agents, immunosuppressive
agents, extracellular matrix molecules, cytokines and cells which
support the therapeutic effect of the administered cells.
[0054] Suitable antimicrobial agents include, but are not limited
to, antibiotics such as penicillin and derivatives thereof,
cephalosporins, tetracyclines, streptomycin, gentamicin and
sulfonamide. Also included are antifungal agents such as
myconazole.
[0055] Suitable immunosuppressive agents include, but are not
limited to, cyclosporin, methotrexate or other agents which inhibit
the immune response of the patient against the administered
composition.
[0056] Examples of extracellular matrix molecules suitable for use
in the cell/microparticle composition, preferably for incorporation
into the microparticles of the cell/microparticle composition
include, but are not limited to, fibronectin, laminin, collagens,
and proteoglycans.
[0057] Cytokines suitable for use include, but are not limited to,
lymphokines, chemokines and monokines. For example, the
interleukins (IL), such as, IL-1 ( or ), IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, GM-CSF, M-CSF, LIF,
LT, -IFN, -IFN, -IFN, TNF- , BCGF, CD2, ICAM, MAdCAM, MCP-1, MCP-2,
MCP-3.
[0058] The biologically active agent can be incorporated into the
microparticles as is known in the art and described herein. It is
understood that more that one agent can be incorporated into the
microparticles of the composition. For example, agents can be
coincorporated into the same microparticle or separately
incorporated into separate microparticle and the microparticles
mixed prior to administration. Alternatively, a biologically active
agent can be administered without prior encapsulation to provide an
immediate availability at the treatment site.
[0059] When the biologically active agent is a macromolecule, such
as a protein, the agent can be a stabilized biologically active
agent. The biologically active agent can be stabilized against
degradation, loss of potency and/or loss of biological activity,
all of which can occur during formation of the sustained release
composition having the biologically active agent dispersed therein,
and/or prior to and during in vivo release of the biologically
active agent. In one embodiment, stabilization can result in a
decrease in the solubility of the biologically active agent, the
consequence of which is a reduction in the initial release of
biologically active agent, in particular, when release is from a
sustained release composition. In addition, the period of release
of the biologically active agent can be prolonged.
[0060] Stabilization of the biologically active agent can be
accomplished, for example, by the use of a stabilizing agent or a
specific combination of stabilizing agents. "Stabilizing agent", as
that term is used herein, is any agent which binds or interacts in
a covalent or non-covalent manner or is included with the
biologically active agent. Stabilizing agents suitable for use in
the invention are described in U.S. Pat. Nos. 5,716,644, 5,674,534,
5,654,010, 5,667,808, and 5,711,968, and co-pending U.S. patent
applications Ser. Nos. 08/934,830 to Burke et al., filed on Sep.
22, 1997 and Ser. No. 09/104,549 to Burke, filed on Jun. 25, 1998
the entire teachings of which are incorporated herein by
reference.
[0061] For example, a metal cation can be complexed with the
biologically active agent, or the biologically active agent can be
complexed with a polycationic complexing agent such as protamine,
albumin, spermidine and spermine, or associated with a
"salting-out" salt. In addition, a specific combination of
stabilizing agents and/or excipients may be needed to optimize
stabilization of the biologically active agent.
[0062] Further, excipients can be added to maintain the potency of
the biologically active agent over the duration of release and
modify polymer degradation. Suitable excipients include, for
example, carbohydrates, amino acids, fatty acids, surfactants, and
bulking agents, and are known to those skilled in the art. An
acidic or a basic excipient is also suitable. The amount of
excipient used is based on ratio to the biologically active agent,
on a weight basis. For amino acids, fatty acids and carbohydrates,
such as sucrose, trehalose, lactose, mannitol, dextran and heparin,
the ratio of carbohydrate to biologically active agent is typically
between about 1:10 and about 20:1. For surfactants the ratio of
surfactant to biologically active agent is typically between about
1:1000 and about 2:1. Bulking agents typically comprise inert
materials. Suitable bulking agents are known to those skilled in
the art.
[0063] The excipient can also be a metal cation component which
acts to modulate the release of the biologically active agent. The
metal cation component can optionally contain the same species of
metal cation, as is contained in the metal cation stabilized
biologically active agent, if present, and/or can contain one or
more different species of metal cation. The metal cation component
acts to modulate the release of the biologically active agent from
the polymer matrix of the sustained release composition and can
enhance the stability of the biologically active agent in the
composition. A metal cation component used in modulating release
typically comprises at least one type of multivalent metal cation.
Examples of metal cation components suitable to modulate release
include or contain, for example, Mg(OH).sub.2, MgCO.sub.3 (such as
4MgCO.sub.3.Mg(OH).sub.2.5H.sub.2O), MgSO.sub.4, Zn(OAc).sub.2,
Mg(OAc).sub.2, ZnCO.sub.3 (such as 3Zn(OH).sub.2
2ZnCO.sub.3)ZnSO.sub.4, ZnCl.sub.2, MgCl.sub.2, CaCO.sub.3,
Zn.sub.3(C.sub.6H.sub.5O.sub.7).sub.2 and
Mg.sub.3(C.sub.6H.sub.5O.sub.7).sub.2.
[0064] A suitable ratio of metal cation component to polymer is
between about 1:99 to about 1:2 by weight. The optimum ratio
depends upon the polymer and the metal cation component utilized. A
polymer matrix containing a dispersed metal cation component to
modulate the release of a biologically active agent from the
polymer matrix is further described in U.S. Pat. No. 5,656,297 to
Bernstein et al. and co-pending U.S. patent application Ser. No.
09/056,566 filed on Apr. 7, 1998, the teachings of both of which
are incorporated herein by reference in their entirety.
[0065] In yet another embodiment, at least one pore forming agent,
such as a water soluble salt, sugar or amino acid, can be included
in the sustained release composition to modify the microstructure.
The proportion of pore forming agent in the microparticle can be
from about 1% (w/w) to about 30% (w/w) of the final weight of the
microparticle.
[0066] Incorporation of the biologically active agent into the
microparticles of the cell/microparticle composition provides a
sustained delivery of the biologically active agent at the
treatment site. It is preferred that the biologically active agent
enhances the primary therapeutic effect resulting from
administration of the cell/microparticle composition. For example,
the biologically active agent can promote tissue growth, inhibit
infection at the treatment site or a combination thereof.
[0067] As used herein, the term "a" or "an" refers to one or
more.
[0068] As used herein, the term "microparticles" refers to
particles comprising biocompatible, biodegradable polymer having a
volume median particle size of between about 1 and 1000
microns.
[0069] A "biocompatible polymer" as that term is used herein refers
to polymer wherein any degradation products of the polymer are
non-toxic to the recipient and also possess no significant
deleterious or untoward effects on the recipient's body, such as a
significant immunological reaction at the injection site.
[0070] "Biodegradable polymer", as defined herein, means the
composition will degrade or erode in vivo to form smaller chemical
species. Degradation can result, for example, by enzymatic,
chemical and physical processes.
[0071] A biocompatible, biodegradable polymer therefor possesses
the characteristics of both a biocompatible and biodegradable
polymer. Suitable biocompatible, biodegradable polymers include,
for example, poly(lactides), poly(glycolides),
poly(lactide-co-glycolides), poly(lactic acid)s, poly(glycolic
acid)s, polycarbonates, polyesteramides, polyanydrides, poly(amino
acids), polyorthoesters, poly(dioxanone)s, poly(alkylene
alkylate)s, copolymers of polyethylene glycol and polyorthoester,
polyurethanes, blends thereof, and copolymers thereof.
[0072] Acceptable molecular weights for polymers used in this
invention can be determined by a person of ordinary skill in the
art taking into consideration factors such as the desired polymer
degradation rate, physical properties such as mechanical strength,
and rate of dissolution of polymer in solvent. Typically, an
acceptable range of molecular weight is of about 2,000 Daltons to
about 2,000,000 Daltons. In a particular embodiment, the polymer is
a poly(lactide-co-glycolide)(hereinafter "PLG") with a
lactide:glycolide ratio of about 50:50 and a molecular weight of
about 5,000 Daltons to about 70,000 Daltons.
[0073] A number of methods are known by which biocompatible,
biodegradable polymer microparticles can be formed. In many cases
the methods are described for embodiments wherein an active agent
is incorporated into the polymer. However, it is to be understood
that the methods described herein can be employed to prepare
microparticles of biocompatible, biodegradable polymer which do not
have an active agent incorporated therein. Suitable methods
include, for example, spray-freeze drying, spray drying, single and
double emulsion solvent evaporation, solvent extraction, phase
separation, and simple and complex coacervation.
[0074] For example, methods for forming a composition for the
sustained release of biologically active agent are described in
U.S. Pat. No. 5,019,400, issued to Gombotz et al., and issued U.S.
Pat. No. 5,922,253 issued to Herbert et al. the teachings of which
are incorporated herein by reference in their entirety.
[0075] In this method, a mixture comprising a biocompatible
polymer, a polymer solvent and in some instances a biologically
active agent is processed to create droplets, wherein at least a
significant portion of the droplets contains polymer, polymer
solvent and if applicable the active agent. These droplets are then
frozen by a suitable means. Examples of means for processing the
mixture to form droplets include directing the dispersion through
an ultrasonic nozzle, pressure nozzle, Rayleigh jet, or by other
known means for creating droplets from a solution.
[0076] Means suitable for freezing droplets include directing the
droplets into or near a liquified gas, such as liquid argon or
liquid nitrogen to form frozen microdroplets which are then
separated from the liquid gas. The frozen microdroplets are then
exposed to a liquid or solid non-solvent, such as ethanol, hexane,
ethanol mixed with hexane, heptane, ethanol mixed with heptane,
pentane or oil.
[0077] The solvent in the frozen microdroplets is extracted as a
solid and/or liquid into the non-solvent to form a polymer/active
agent matrix comprising a biocompatible polymer and a biologically
active agent. Mixing ethanol with other non-solvents, such as
hexane, heptane or pentane, can increase the rate of solvent
extraction, above that achieved by ethanol alone, from certain
polymers, such as poly(lactide-co-glycolide) polymers.
[0078] A wide range of sizes of sustained release compositions can
be made by varying the droplet size, for example, by changing the
ultrasonic nozzle diameter. If the sustained release composition is
in the form of microparticles, and very large microparticles are
desired, the microparticles can be extruded, for example, through a
syringe directly into the cold liquid. Increasing the viscosity of
the polymer solution can also increase microparticle size. The size
of the microparticles which can be produced by this process ranges,
for example, from greater than about 1000 to about 1 micrometers in
diameter.
[0079] A further example of a conventional process for producing
microparticles is disclosed in U.S. Pat. No. 3,737,337,
incorporated by reference herein, wherein a solution of a wall or
shell forming polymeric material in a solvent is prepared. The
solvent is only partially miscible in water. If desired a solid or
core material is dissolved or dispersed in the polymer-containing
mixture and, thereafter, the core material-containing mixture is
dispersed in an aqueous liquid that is immiscible in the organic
solvent in order to remove solvent from the microparticles.
[0080] Another example of a process to form microparticles which
can contain a substance is disclosed in U.S. Pat. No. 3,523,906. In
this process a material to be encapsulated is emulsified in a
solution of a polymeric material in a solvent that is immiscible in
water and then the emulsion is emulsified in an aqueous solution
containing a hydrophilic colloid. Solvent removal from the
microparticles is then accomplished by evaporation and the product
is obtained.
[0081] In still another process as shown in U.S. Pat. No. 3,691,090
organic solvent is evaporated from a dispersion of microparticles
in an aqueous medium, preferably under reduced pressure.
[0082] Similarly, the disclosure of U.S. Pat. No. 3,891,570 shows a
method in which solvent from a dispersion of microparticles in a
polyhydric alcohol medium is evaporated from the microparticles by
the application of heat or by subjecting the microparticles to
reduced pressure.
[0083] Another example of a solvent removal process is shown in
U.S. Pat. No. 3,960,757.
[0084] Tice et al., in U.S. Pat. No. 4,389,330, describe the
preparation of microparticles which can contain an active agent by
a method comprising: (a) dissolving or dispersing an active agent
in a solvent and dissolving a wall forming material in that
solvent; (b) dispersing the solvent containing the active agent and
wall forming material in a continuous-phase processing medium; (c)
evaporating a portion of the solvent from the dispersion of step
(b), thereby forming microparticles containing the active agent in
the suspension; and (d) extracting the remainder of the solvent
from the microparticles.
[0085] When a biogically active agent is incorporated into the
biocompatible, biodegradable polymer microparticles, it is believed
that, without being bound by a particular theory, the release of
the biologically active agent can occur by two different
mechanisms. First, the biologically active agent can be released by
diffusion through aqueous filled channels generated in the polymer
matrix, such as by the dissolution of the biologically active
agent, or by voids created by the removal of the polymer solvent
during the preparation of the sustained release composition. A
second mechanism is the release of the biologically active agent,
due to degradation of the polymer. The rate of degradation can be
controlled by changing polymer properties that influence the rate
of hydration of the polymer. These properties include, for
instance, the ratio of different monomers, such as lactide and
glycolide, comprising a polymer; the use of the L-isomer of a
monomer instead of a racemic mixture; and the molecular weight of
the polymer. These properties can affect hydrophilicity and
crystallinity, which control the rate of hydration of the
polymer.
[0086] By altering the properties of the polymer, the contributions
of diffusion and/or polymer degradation to biologically active
agent release can be controlled. For example, increasing the
glycolide content of a poly(lactide-co-glycolide) polymer and
decreasing the molecular weight of the polymer can enhance the
hydrolysis of the polymer and thus, provides an increased
biologically active agent release from polymer erosion.
[0087] The live cell/biocompatible, biodegradable polymer
microparticle composition of the invention can optionally contain a
pharmaceutically acceptable carrier. Pharmaceutically acceptable
carriers are well known to those skilled in the art. The carrier
should not deleteriously affect the cells, microparticles or, when
present, the biologically active agent of the administered
composition. It is preferred that the carrier includes factors
which promote adhesion of the cells to the microparticles of the
composition. For example, the medium in which the cells can be
cultured can be suitable. Acceptable cell culture media are
commercially available and are well known to those skilled in the
art.
Exemplifications
Preparative Methods
General Process for Preparing Microparticles
[0088] Formation of a mixture comprising a biocompatible polymer
dissolved in a suitable polymer solvent and if desired a
biologically active agent.
[0089] Optional homogenization of the mixture.
[0090] Atomization of the mixture to form droplets.
[0091] Freezing of the droplets by contact with liquid
nitrogen.
[0092] Extraction of the polymer solvent from frozen droplets into
an extraction solvent (e.g., -80.degree. C. ethanol) thereby
forming a solid polymer matrix.
[0093] Isolation of the solid polymer matrix particles from the
extraction solvent by filtration.
[0094] Removal of remaining solvent by evaporation.
[0095] Sizing of particles by passage through an appropriately
sized mesh.
EXAMPLE 1
Preparation of Microparticles
[0096] Microparticles were fabricated from PLG (50:50
lactide:glycolide, uncapped (--COOH), M.sub.w.about.10 kDa) using
the General Process outlined above.
EXAMPLE 2
Cell Isolation-Chondrocytes
[0097] Bovine articular cartilage was harvested from the
gelnohumeral and humeroulnar joints of neonatal calves and digested
in 0.3% Type II collagenase at 37.degree. C. for 12-16 hours
overnight with shaking. Chondrocytes were passed through a 180 m
filter to remove large particulate material. Cells were washed 3
times with PBS and resuspended in complete medium (Ham's F12, 10%
FBS, 0.3% carboxymethylcellulose, pen/strep/amphotericin B, and
ascorbic acid).
EXAMPLE 3
Chondrocyte/Microparticle Adhesion Assay
[0098] Chondrocytes were mixed with 3.57 mg/mL of PLG (50:50 L:G)
microparticles, prepared as described above, to a final
concentration of 1.times.10.sup.6 cells/mL. Control groups included
cells alone and microparticles alone at the same concentrations as
described above. The mixture of cells and microparticles contained
phosphate buffered saline and cell culture medium.
[0099] The combined cell/microparticle suspension and controls were
incubated on a shaker plate at 37.degree. C. At 0, 2, 4, 16 hours,
1 mL samples were removed and assayed to determine the amount of
cells which had adhered to the microparticles. In order to
determine the amount of cells which had adhered to the
microparticles, each sample was loaded onto a 3 mL histopaque
density gradient and centrifuged for 5 min at 5000 rpm. The
unattached cells were removed by decanting the less dense media
fraction on top of the histopaque (approximately 1 mL). The
remaining portion (approximately 3 mL) contained the microparticles
and attached chondrocytes. The results of the DNA assay provided
data on the extent of attachment of the cells to the microparticles
as well as the kinetics of the attachment of the cells to the
microparticles. The DNA assay employed is described in Kim, Y. J.
et al., "Analytical Biochemistry" Vol. 174, pp. 168-176 (1988).
[0100] The results from assaying for DNA content are shown
graphically in FIGS. 1 and 2. The graphs in FIGS. 1 and 2 show that
the cells exhibited first order binding and at about 16 hours 30%
of the cells in the suspension were attached to the microparticles.
In addition a value of about 5 hours for the exponential time
constant, tau ( ), was determined from FIG. 2. Changes in cell
morphology and matrix production were observed >16 hr.
EXAMPLE 4
Scanning Electron Microscopy
[0101] Scanning electron microscopy was conducted. Samples were
removed from the cell/microparticle suspensions at 0, 2, 4, and 16
hours and analysed by SEM as described below. Glass coverslips were
coated with a thin layer of TissueTack adhesive. Liquid samples
(500 L) of the cell/microparticle suspension were layered onto the
coverslips in a 24 well plate and allowed to settle for 30 minutes.
Samples were then fixed with 2.5% gluteraldehyde, critical point
dried, sputter coated with Au/Pd and visualized via SEM. SEMs of
the cell/microparticle suspension following 2 hours and 8 hours of
incubation are shown in FIGS. 3 and 4 respectively, at the
indicated magnifications. The SEMs showed attachment of cells to
single microparticles at 2 hours, with large clusters of multiple
cells and microparticles present at 16 hours.
In vivo Implantation and Analysis:
[0102] Nude mice (5 per treatment group) were anesthetized with
metofane and each received subcutaneous injections of 150 L of a
suspension of chondrocytes only, microparticles only, and
chondrocytes and microparticles prepared as described above. The
injections were at three individual sites on the dorsal aspect. The
size of the implants were monitored superficially for 4 weeks, at
which time, implants were harvested. Samples were weighed,
photographed for gross morphology, and fixed in 10% buffered
formalin for histology.
[0103] Fixed samples were embedded in paraffin and sectioned to 6 m
thick. Sections were then deparaffinized, rehydrated and stained in
Mayer's hematoxylin and eosin (H&E).
[0104] Gross analysis and H&E revealed formation of small
nodules of tissue resembling cartilage for the group receiving a
mixture of cells and microparticles. Animals receiving an injection
of cells alone or microspheres alone did not yield any new
cartilage growth. The results of H&E staining for animals
receiving a mixture of cells and microparticles is shown in FIG.
5.
[0105] Even though the invention has been described with a certain
degree of particularity, it is evident that many alternatives,
modifications, and variations will be apparent to those skilled in
the art in light of the foregoing disclosure. Accordingly, it is
intended that all such alternatives, modifications, and variations
which fall within the spirit and scope of the invention be embraced
by the defined claims.
[0106] The teachings of all patents, published applications and
references cited herein are incorporated by reference in their
entirety.
[0107] While this invention has been particularly shown and
described with references to example embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *