U.S. patent application number 13/472173 was filed with the patent office on 2012-09-06 for tissue engineered cartilage, method of making same, therapeutic and cosmetic surgical applications using same.
This patent application is currently assigned to The Government of the United States of America, as represented by the Secretary,Department of Health. Invention is credited to Wan-Ju Li, Rocky S. Tuan.
Application Number | 20120225039 13/472173 |
Document ID | / |
Family ID | 37442076 |
Filed Date | 2012-09-06 |
United States Patent
Application |
20120225039 |
Kind Code |
A1 |
Li; Wan-Ju ; et al. |
September 6, 2012 |
TISSUE ENGINEERED CARTILAGE, METHOD OF MAKING SAME, THERAPEUTIC AND
COSMETIC SURGICAL APPLICATIONS USING SAME
Abstract
Cartilage has been constructed using biodegradable electrospun
polymeric scaffolds seeded with chondrocytes or adult mesenchymal
stem cells. More particularly engineered cartilage has been
prepared where the cartilage has a biodegradable and biocompatible
nanofibrous polymer support prepared by electrospinning and a
plurality of chondocytes or mesenchymal stem cells dispersed in the
pores of the support. The tissue engineered cartilages of the
invention possess compressive strength properties similar to
natural cartilage. Methods of preparing engineered tissues,
including tissue engineered cartilages, are provided in which an
electrospun nanofibrous polymer support is provided, the support is
treated with a cell solution and the polymer-cell mixture cultured
in a rotating bioreactor to generate the cartilage. The invention
provides for the use of the tissue engineered cartilages in the
treatment of cartilage degenerative diseases, reconstructive
surgery, and cosmetic surgery.
Inventors: |
Li; Wan-Ju; (Madison,
WI) ; Tuan; Rocky S.; (Pittsburgh, PA) |
Assignee: |
The Government of the United States
of America, as represented by the Secretary,Department of
Health
Rockville
MD
|
Family ID: |
37442076 |
Appl. No.: |
13/472173 |
Filed: |
May 15, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11922251 |
Apr 23, 2009 |
8202551 |
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PCT/US2006/023477 |
Jun 15, 2006 |
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13472173 |
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60690988 |
Jun 15, 2005 |
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Current U.S.
Class: |
424/93.7 ;
435/398; 435/399; 514/772.3; 514/772.7; 977/788; 977/906;
977/923 |
Current CPC
Class: |
A61P 19/04 20180101;
A61L 27/56 20130101; A61L 2430/06 20130101; A61L 2400/12 20130101;
A61Q 90/00 20130101; A61L 27/3852 20130101; A61L 27/3817 20130101;
A61L 27/18 20130101; A61L 27/18 20130101; C08L 67/04 20130101; A61L
27/3895 20130101 |
Class at
Publication: |
424/93.7 ;
435/398; 435/399; 514/772.7; 514/772.3; 977/788; 977/923;
977/906 |
International
Class: |
A61K 35/32 20060101
A61K035/32; A61K 47/34 20060101 A61K047/34; C12N 5/071 20100101
C12N005/071; A61P 19/04 20060101 A61P019/04 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Research supporting this application was carried out by the
United States of America as represented by the Secretary,
Department of Health and Human Services.
Claims
1. A tissue engineered cartilage, comprising a nanofibrous polymer
support comprising a plurality of polymer nanofibers; and a
plurality of chondrocytes dispersed throughout the polymer support,
wherein the tissue engineered cartilage has a peak compressive
stress (Young's modulus) of greater than 250 MPa.
2-13. (canceled)
14. A method of preparing tissue engineered cartilage comprising
preparing a nanofibrous biocompatible polymer support; contacting a
suspension of cells with the surface of the support to form a
polymer matrix having cells dispersed therein; culturing the
cell-polymer matrix in a bioreactor with a culture medium under
conditions conducive to growth of chondocytes into a tissue
engineered cartilage.
15. A method of preparing tissue engineered cartilage comprising
preparing an nanofibrous biocompatible polymer support; expanding
the nanofibrous polymer support thereby increasing interfiber
distance; contacting a suspension of cells with the support to form
a polymer matrix having cells dispersed therein; compressing the
cell-polymer matrix to create cell-cell contact and cell-polymer
contact; culturing the compressed cell-polymer matrix in a
bioreactor with a culture medium under conditions conducive to
growth of chondocytes into a tissue engineered cartilage having a
peak compressive stress (Young's modulus) of greater than 250
MPa.
16. The method of claim 15, wherein the cells are stem cells or
chondocytes.
17. (canceled)
18. The method of claim 15, wherein the culture medium is
substantially free of serum.
19. The method of claim 15, wherein the nanofibrous polymer support
is dimensionally stable throughout the culturing step.
20. The method of claim 15, wherein the nanoporous biodegradable
polymer comprises polymer nanofibers having a diameter of less than
1 micron.
21. The method of claim 15, wherein the polymer nanofibers have a
diameter of between 100 nm and 1 micron.
22. The method of claim 15, wherein the polymer nanofibers have a
substantially uniform diameter.
23. The method of claim 15, wherein the nanofibrous polymer support
comprises a non-woven mat of electrospun nanofibers having a
diameter of less than 1 micron.
24. The method of claim 23, wherein the nanofibers of the non-woven
mat is randomly oriented.
25. The method of claim 23, wherein the nanofibrous polymer support
is composed of at least one biodegradable polyester or blend
thereof.
26. The method of claim 23, wherein the nanofibrous polymer support
is composed of at least one biodegradable polyester comprising at
hydroxyacid monomer.
27. The method of claim 15, wherein the nanofibrous polymer support
is composed of at least one biodegradable polymer selected from
poly((L)-lactic acid), poly(caprolactone) and blends thereof.
28. The method of claim 15, wherein the compression step comprises
applying a centrifugal force of between 25 g and 500 g to the
polymer-cell matrix for 1 to 10 minutes.
29. The method of claim 28, wherein the compression step further
comprises a step of resting the polymer-cell matrix after the
compression step.
30. The method of claim 28, wherein the compression step comprises
applying the centrifugal force to the cell-polymer scaffold mixture
in a vessel, which vessel has a shape corresponding to the desired
shape of the tissue engineered cartilage.
31. The method of claim 15, wherein the expanded nanofibrous
biocompatible polymer support is formed to form a plurality of
larger pores each of which has a diameter of at least 10
microns.
32. The method of claim 15, wherein the bioreactor suspends the
cell-polymer aggregate or tissue engineered cartilage in a moving
culture medium.
33. The method of claim 32, wherein the bioreactor comprises a
culture chamber having a taurus cross-section in to which the
cell-polymer matrix and culture medium are placed, and wherein the
culture chamber is rotated at a speed sufficient to generate a zero
gravity or low gravity mimicking environment in at least a portion
of the volume of the culture chamber.
34. (canceled)
35. A method of forming cartilage in vivo, the method comprising
the steps of providing a nanofibrous polymer support comprising a
plurality of polymer nanofibers; and inserting the nanofibrous
polymer support into a patient at the position suitable for
formation of new cartilage.
36-54. (canceled)
55. A method of preparing a tissue engineered tissue comprising the
steps of preparing a nanofibrous biocompatible polymer support;
contacting a suspension of cells with the surface of the support to
form a polymer matrix having cells dispersed therein; culturing the
cell-polymer matrix in a bioreactor with a culture medium under
conditions conducive cell growth and differentiation to tissue
engineered tissue.
56. A method of preparing a tissue engineered tissue comprising the
steps of preparing an nanofibrous biocompatible polymer support;
expanding the nanofibrous polymer support thereby increasing
interfiber distance; contacting a suspension of cells with the
support to form a polymer matrix having cells dispersed therein;
compressing the cell-polymer matrix to create cell-cell contact and
cell-polymer contact; culturing the compressed cell-polymer matrix
in a bioreactor with a culture medium under conditions conducive
cell growth and differentiation to tissue engineered tissue.
57-73. (canceled)
74. A tissue engineered cartilage prepared by the method comprising
the steps of: preparing a nanofibrous biocompatible polymer
support; contacting a suspension of cells with the surface of the
support to form a polymer matrix having cells dispersed therein;
culturing the cell-polymer matrix in a bioreactor with a culture
medium under conditions conducive to growth of chondocytes into a
tissue engineered cartilage.
75-81. (canceled)
Description
[0001] The present application claims the benefit of U.S.
provisional application No. 60/690,988, filed Jun. 15, 2005, which
is incorporated herein by reference in its entirety.
FIELD OF INVENTION
[0003] The present invention relates to tissue engineered cartilage
comprising a nanofibrous biocompatible polymer support having
chondocytes dispersed therein, which has compressive strength equal
to natural cartilage, methods of fabricating tissue engineered
cartilage by culturing a mixture of stem cells or chondocytes and a
electrospun nanofibrous polymer substrate in a suitable bioreactor
and methods of treatment comprising implantation of tissue
engineered cartilage into a patient.
BACKGROUND OF THE INVENTION
[0004] Diseased or damaged cartilage has often been replaced by an
artificial material, cadaver tissue, or donated, allogenic tissue.
Tissue engineering offers an attractive alternative whereby a live,
natural tissue is generated from a construct made up of a patient's
own cells in combination with a biodegradable scaffold for
replacement of defective tissue.
[0005] Cartilage defects resulting from aging, joint injury, and
developmental disorders cause joint pain and loss of mobility. It
would be desirable to provide a tissue engineering approach
provides a cell-based therapy to repair articular cartilage defects
and to restore joint functions. In prior attempts to tissue
engineer cartilage, chondrocytes and mesenchymal stem cells (MSCs)
have been used for cartilage regeneration, and the choice of cell
type determines the strategy of cartilage tissue engineering in
vitro.
[0006] Existing engineered cartilage and the methods of making same
generate materials which do not possess the mechanical properties
of natural cartilage. Thus, cartilage generated by seeding a
hydrogel or preformed three dimensional polymeric scaffold are less
resistant to compressive force than natural cartilage. Conventional
methods of making cartilage, e.g., using a hydrogel or preformed
three dimensional polymeric scaffold, result in despecification of
the seeded chondocytes, poor intercellular contact between
chondocytes, and/or insufficient mechanical strength.
[0007] The electrospinning process is a simple, economical means to
produce supports or scaffolds of ultra-fine fibers derived from a
variety of biodegradable polymers (Li W J, et al. J Biomed Mater
Res 2002; 60:613-21). Nanofibrous scaffolds (NFSs) formed by
electrospinning, by virtue of structural similarity to natural
extracellular matrix (ECM), may represent promising structures for
tissue engineering applications. We have previously shown that
electrospun three-dimensional NFSs are characterized by high
porosity with a wide distribution of pore diameter, high-surface
area to volume ratio, and morphological similarities to natural
collagen fibrils (Li W J, et al. J Biomed Mater Res 2002;
60:613-21). These physical characteristics promote favorable
biological responses of seeded cells in vitro, including enhanced
cell attachment, proliferation, and maintenance of the chondrocytic
phenotype (Li W J, et al. J Biomed Mater Res 2002; 60:613-21; and
Li W J, et al. J Biomed Mater Res 2003; 67A:1105-14).
[0008] It would be desirable to provide tissue engineered cartilage
materials and methods of making same which are suitable for
preparing cartilage suitable for use in repairing cartilage defects
associated with degenerative joint diseases or in plastic/cosmetic
surgery requiring repair or augmentation of cartilaginous tissue.
More particularly, it would be desirable to provide methods of
making high strength cartilage, tissue engineered cartilage
prepared thereby and methods of treatment using such high strength
tissue engineered cartilage.
SUMMARY OF THE INVENTION
[0009] Described herein are engineered tissues, particularly tissue
engineered cartilage, methods of generating the engineered tissue
cartilage or other tissues, and methods of using the tissue
engineered cartilage or other tissues in various applications
utilizing engineered tissues, including for example, therapeutic or
prophylactic replacement or supplementation of cartilage.
[0010] One aspect is a tissue engineered cartilage having a peak
compressive stress (Young's modulus) of greater than 250 MPa, which
tissue engineered cartilage is composed of a plurality of
chondocytes dispersed in a nanofibrous polymer support comprising a
plurality of polymer nanofibers. In other aspects, the each of the
plurality of chondocytes is in contact with at least one, at least
two, or at a plurality of other chondocytes dispersed in the
polymer support. In certain tissue engineered cartilages provided
herein possess a peak compressive strength of at least about 300
MPa, about 400 MPa or about 500 MPa or a compressive strength of
between about 250-1000 MPa, between about 300-1000 MPa, between
about 300-900 MPa, between about 400-900 Mpa, or between about
600-900 MPa.
[0011] In other aspects, the nanofibrous polymer support of the
tissue engineered cartilage is composed of at least one
biodegradable and biocompatible polymer which can be processed by
electrospinning to form sub-micron fibers. Yet other aspects, the
nanofibrous polymer support comprises polymer nanofibers having a
diameter of less than 1 micron or having a diameter of between 10
nm to 1 micron, 50 nm to 1 micron, 100 nm to 1 micron, or 200 nm to
700 nm.
[0012] In other aspects, the nanofibrous polymer supports comprise
electrospun polyester polymers which have been approved for use in
surgical applications by the FDA or equivalent regulatory agency.
In general, electrospinning is a process of producing nanofibers or
microfibers of a polymer in which a high voltage electric field is
applied to a solution of the polymer. The drawn nanofibers are
collected in on a target covering one of the electrodes. By careful
regulation of inter-electrode distance, voltage, solvent, and
polymer solution viscosity the diameter of the resultant
electrospun fibers can be controlled. Optimization of the
elecrospinning process results in formation of polymer nanofibers
have a substantially uniform diameter. In certain aspects, a two
dimensional static target is used in the electrospinning process to
generate a randomly oriented non-woven mat of fibers deposited onto
the target.
[0013] Another aspect is a method of preparing tissue engineered
cartilage comprising
[0014] (a) preparing a nanofibrous biocompatible polymer
support;
[0015] (b) contacting a suspension of cells with the surface of the
support provided in (a) to form a polymer matrix having cells
dispersed therein;
[0016] (c) culturing the cell-polymer matrix in a bioreactor with a
culture medium under conditions conducive to growth of chondocytes
into a tissue engineered cartilage.
[0017] Yet another aspect is a method of preparing tissue
engineered cartilage comprising
[0018] (a) preparing an expanded nanofibrous biocompatible polymer
support;
[0019] (b) contacting a suspension of cells with the support
provided in (a) to form a polymer matrix having cells dispersed
therein;
[0020] (c) compressing the cell-polymer matrix prepared in (b) to
create cell-cell contact and cell-polymer contact;
[0021] (d) culturing the compressed cell-polymer matrix prepared in
(c) in a bioreactor with a culture medium under conditions
conducive to growth of chondocytes into a tissue engineered
cartilage having a peak compressive stress (Young's modulus) of
greater than 250 MPa.
[0022] Another aspect is a method of forming cartilage in vivo, the
method comprising the steps of
[0023] (a) providing a nanofibrous polymer support comprising a
plurality of polymer nanofibers; and
[0024] (b) inserting the nanofibrous polymer support into a patient
at the position suitable for formation of new cartilage.
[0025] In other aspects, the invention provides methods of
repairing, replacing and/or augmenting cartilage in a patient for
treatment or prevention of diseases or disorders or for cosmetic
purposes. In certain aspects, a method of treating cartilage damage
is provided in which the method comprising the steps of:
[0026] (a) providing a tissue engineered cartilage having a peak
compressive stress (Young's modulus) of greater than 250 MPa, which
tissue engineered cartilage is composed of a plurality of
chondocytes dispersed in a nanofibrous polymer support comprising a
plurality of polymer nanofibers or a tissue engineered cartilage
prepared by the methods provided herein;
[0027] (b) inserting the tissue engineered cartilage into a patient
at the location of damaged cartilage.
[0028] Another aspect is a method for treating cartilage damage,
the method comprising the steps of
[0029] (a) harvesting chondocytes or MSC cells from the
patient;
[0030] (b) preparing tissue engineered cartilage by one of the
methods provided herein, wherein the cells are the chondocytes or
MSC cells harvested from the patient;
[0031] (c) implanting the tissue engineered cartilage in the
patient in the locus having damaged cartilage.
[0032] Yet another aspect is a method for cosmetic or
reconstructive surgery, the method comprising the steps of
[0033] (a) providing a tissue engineered cartilage having a peak
compressive stress (Young's modulus) of greater than 250 MPa, which
tissue engineered cartilage is composed of a plurality of
chondocytes dispersed in a nanofibrous polymer support comprising a
plurality of polymer nanofibers or a tissue engineered cartilage
prepared by the methods provided herein;
[0034] (b) inserting the tissue engineered cartilage into a
patient.
[0035] In another aspect, the invention provides a method for
cosmetic or reconstructive surgery, the method comprising the steps
of
[0036] (a) harvesting chondocytes or MSC cells from the
patient;
[0037] (b) preparing tissue engineered cartilage by a method
provided herein, wherein the cells are the chondocytes or MSC cells
harvested from the patient;
[0038] (c) implanting the tissue engineered cartilage in the
patient.
[0039] Yet another aspect is a methods of preparing a tissue
engineered tissue comprising the steps of
[0040] (a) preparing a nanofibrous biocompatible polymer
support;
[0041] (b) contacting a suspension of cells with the surface of the
support to form a polymer matrix having cells dispersed
therein;
[0042] (c) culturing the cell-polymer matrix in a bioreactor with a
culture medium under conditions conducive cell growth and
differentiation to tissue engineered tissue.
[0043] Still another aspect is a method of preparing a tissue
engineered tissue comprising the steps of
[0044] (a) preparing an expanded nanofibrous biocompatible polymer
support;
[0045] (b) contacting a suspension of cells with the support to
form a polymer matrix having cells dispersed therein;
[0046] (c) compressing the cell-polymer matrix to create cell-cell
contact and cell-polymer contact;
[0047] (d) culturing the compressed cell-polymer matrix in a
bioreactor with a culture medium under conditions conducive cell
growth and differentiation to tissue engineered tissue.
[0048] Other aspects and embodiments of the invention are discussed
below.
BRIEF DESCRIPTION OF THE DRAWING
[0049] For a fuller understanding of the nature and desired objects
of the present invention, reference is made to the following
detailed description taken in conjunction with the accompanying
drawing figures wherein like reference character denote
corresponding parts throughout the several views and wherein:
[0050] FIG. 1 is a schematic of electrospinning apparatus (10) for
the preparation of nanofibrous polymer supports suitable for use in
the invention;
[0051] FIG. 2. is a scanning electron microscope image of an
electrospun PCL-based nanofibrous scaffold at (A) low and (B) high
magnification. The scaffold contains randomly oriented uniformly
sized fibers of an average diameter of 700 nm (black legend is 30
.mu.m in FIG. 2A and 10 .mu.m in FIG. 2B);
[0052] FIG. 3A-F provides a series of microscopic images of various
nanofibrous scaffolds which are contemplated for use in the methods
and cartilage provided herein (PGA is poly(glycolic acid), PDLLA is
poly(D,L-lactic acid), PLLA is poly(L-lactic acid), PLGA5050 is
poly(D,L-lactide-co-glycolide 50:50), PLGA8515 is
poly(D,L-lactide-co-glycolide 85:15), and PCL is
poly(epsilon-caprolactone));
[0053] FIG. 4A-F provides a macroscopic observation of nanofibrous
scaffolds exposed to a phosphate buffered solution at days 3, 7,
14, 21, and 42 (PGA is poly(glycolic acid), PDLLA is
poly(D,L-lactic acid), PLLA is poly(L-lactic acid), PLGA5050 is
poly(D,L-lactide-co-glycolide 50:50), PLGA8515 is
poly(D,L-lactide-co-glycolide 85:15), and PCL is
poly(epsilon-caprolactone));
[0054] FIG. 5A-F is a series of scanning electron microscope images
of nanofibrous non-woven mats composed of PGA, PDLLA, PLLA,
PLGA5050, PLGA8515, and PCL. Images are provided prior to exposure
to a degradation medium (D0), after one day in the degradation
medium (D1) and after three days in the degradation medium (D3). In
the figure, PGA is poly(glycolic acid), PDLLA is poly(D,L-lactic
acid), PLLA is poly(L-lactic acid), PLGA5050 is
poly(D,L-lactide-co-glycolide 50:50), PLGA8515 is
poly(D,L-lactide-co-glycolide 85:15), and PCL is
poly(epsilon-caprolactone);
[0055] FIG. 6 is a tissue engineered flow chart of one of the
methods of preparing tissue engineered cartilage provided herein
and exemplified in Example 4;
[0056] FIG. 7 is a picture of the tissue engineered cartilage
prepared in Example 4 cultured in the chondrogenic medium
supplemented with different growth factors after 42 days (Control
had no growth factors);
[0057] FIG. 8 is a series of images corresponding to RT-PCR
analysis of cartilage prepared by the method of Example 4 with
different growth factors (control, TGF-.beta.1, IGF-1, and
TGF-.beta.1+IGF-1);
[0058] FIG. 9 is a series of photographs of the H & E staining
histological analysis of cartilage prepared by the method of
Example 4 with Different Growth Factors (control, TGF-.beta.1,
IGF-1, and TGF-.beta.1+IGF-1);
[0059] FIG. 10 is a series of photographs of the Alcian Blue
histological analysis of cartilage prepared by the method of
Example 4 with different growth factors (control, TGF-.beta.1,
IGF-1, and TGF-.beta.1+IGF-1);
[0060] FIG. 11 is a series of photographs showing the
immunohistochemical localization of cartilage-specific ECM in
cartilage prepared by the method of Example 4 with Different Growth
Factors (control, TGF-.beta.1, IGF-1, and TGF-.beta.1+IGF-1);
[0061] FIG. 12 is a bar graph of radioactive sulfate incorporation
in cartilage prepared by the method of Example 4 with different
growth factors (control, TGF-.beta.1, IGF-1, and
TGF-.beta.1+IGF-1);
[0062] FIG. 13 is a bar graph of radioactive proline incorporation
in cartilage prepared by the method of Example 4 with different
growth factors (control, TGF-.beta.1, IGF-1, and
TGF-.beta.1+IGF-1);
[0063] FIG. 14 is a photographic image comparing tissue engineered
cartilage prepared by the dynamic method recited in Example 4 and
tissue engineered cartilage prepared by a static method equivalent
to the method of Example 4;
[0064] FIG. 15 is a bar graph comparing weight increase for tissue
engineered cartilage prepared by the dynamic method recited in
Example 4 (1200% increase) and tissue engineered cartilage prepared
by a static method equivalent to the method of Example 4 (1000%
increase);
[0065] FIG. 16 is a series of images corresponding to RT-PCR
analysis of cartilage prepared by the method of Example 4 under
dynamic conditions and static conditions;
[0066] FIG. 17 is a series of photographs of the H & E staining
histological analysis of cartilage prepared by the method of
Example 4 under dynamic conditions and static conditions;
[0067] FIG. 18 is a series of photographs of the Alcian Blue
histological analysis of cartilage prepared by the method of
Example 4 under dynamic conditions and static conditions;
[0068] FIG. 19 is a graph of a compressive test for cartilage
prepared by the method of Example 4 under dynamic conditions and
static conditions; and
[0069] FIG. 20 is a bar graph of calculated stress for cartilage
prepared by the method of
[0070] Example under dynamic conditions and static conditions under
dynamic conditions and static conditions under dynamic conditions
and static conditions.
DESCRIPTION OF THE PREFERRED EMBODIMENT(S)
[0071] Although a preferred embodiment of the invention has been
described using specific terms, such description is for
illustrative purposes only, and it is to be understood that changes
and variations may be made without departing from the spirit or
scope of the following claims.
[0072] Methods and materials to form tissues, especially cartilage,
are described wherein cells, e.g., chondrocytes or mesenchymal stem
cells, are seeded onto or into a biocompatible, biodegradable,
nanofibrous polymer scaffold which cell-polymer matrix is then
cultured in a rotating bioreactor to form the tissue or cartilage.
The product tissue or cartilage generated in the methods of the
invention is implantation into a patient in therapeutic,
prophylactic or cosmetic procedures.
[0073] In certain aspects, a method of preparing tissue engineered
cartilage is provided in which the method comprises the steps
of:
[0074] (a) preparing a nanofibrous biocompatible polymer
support;
[0075] (b) contacting a suspension of cells with the surface of the
support provided in (a) to form a polymer matrix having cells
dispersed therein;
[0076] (c) culturing the cell-polymer matrix in a bioreactor with a
culture medium under conditions conducive to growth of chondocytes
into a tissue engineered cartilage.
[0077] Yet another aspect is a method of preparing tissue
engineered cartilage comprising the steps of:
[0078] (a) preparing an expanded nanofibrous biocompatible polymer
support;
[0079] (b) contacting a suspension of cells with the support
provided in (a) to form a polymer matrix having cells dispersed
therein;
[0080] (c) compressing the cell-polymer matrix prepared in (b) to
create cell-cell contact and cell-polymer contact;
[0081] (d) culturing the compressed cell-polymer matrix prepared in
(c) in a bioreactor with a culture medium under conditions
conducive to growth of chondocytes into a tissue engineered
cartilage having a peak compressive stress (Young's modulus) of
greater than 250 MPa.
Nanofibrous Polymer Scaffolds
[0082] The nanofibrous polymer scaffold can be manufactured by any
method capable of generating a random web of nanofibers. Preferred
nanofibrous polymer scaffold are composed of one or more
biodegradable and/or biocompatible polymer. In certain aspects, the
nanofibrous polymer scaffold is manufactured from a biodegradable
polymer which is dimensionally stable for the duration of the
tissue engineering process. In certain aspects, nanofibrous polymer
scaffolds comprise nanofibers having a thickness of less than about
1 .mu.m, less than about 750 nm, or a thickness of between about 50
nm and about 800 nm. In certain other aspects, the nanofibrous
polymer scaffold comprises nanofibers having a thickness of between
about 100 nm and about 700 nm or between about 200 nm and about 600
nm. Typically preferred nanofibrous polymer scaffolds comprise a
random web of nanofibers which have an interfiber distance and
nanofiber thickness which approximates the parameters present in
the collagen matrix of natural cartilage.
[0083] In other aspects, the nanofibrous polymer supports comprise
electrospun nanofibers. Nanofibers prepared by electrospinning
provide a nanofibrous polymer support possessing a high surface
area to volume ratio and improved mechanical properties relative to
hydrogels and other polymeric supports. Although not wishing to be
bound by theory, certain nanofibrous polymer supports prepared by
electrospinning mimic the fiber diameter and interfiber
characteristics of collagen and the extracellular matrix of healthy
cartilage.
[0084] In general, electrospinning is a process of producing
nanofibers or microfibers of a polymer in which a high voltage
electric field is applied to a solution of the polymer. The drawn
nanofibers are collected in on a target covering one of the
electrodes. By careful regulation of inter-electrode distance,
voltage, solvent, and polymer solution viscosity the diameter of
the resultant electrospun fibers can be controlled. Optimization of
the elecrospinning process results in formation of polymer
nanofibers have a substantially uniform diameter.
[0085] The term "nanofibrous polymer support" is intended to refer
to materials composed of a plurality of polymeric nanofibers. FIG.
1 is a schematic diagram of an electrospinning apparatus (10),
which consists of a glass syringe containing polymer solution (12),
nanofiber jet (13), copper collecting plate (14), which optionally
has a removable collection layer disposed thereon (not shown), and
power supply (15). In certain embodiments, the nanofibrous polymer
support comprises nanofibers composed of at least one polymer. That
is, the nanofibrous polymer support is composed of nanofibers
composed of a polymer, copolymer, or a blend of polymers or the
nanofibrous polymer support comprises two or more compositionally
distinct polymeric nanofibers. In certain embodiments, the
nanofibrous polymer support is composed of a plurality of uniform
thickness nanofibers prepared by an electrospinning process using a
solution of one or more biocompatible, bioabsorbable or
biodegradable polymers.
[0086] In other aspects, the nanofibrous polymer support of the
tissue engineered cartilage is composed of at least one
biodegradable and biocompatible polymer which can be processed by
electrospinning to form sub-micron fibers. In certain aspects, the
nanofibrous polymer support is composed of one or more
biodegradable biocompatible polyesters. In certain embodiments the
biodegradable polyester is a polymer comprising one or more
monomers selected from glycolic acid, lactic acid, epsilon-lactone,
glycolide, or lactide. By "comprises a monomer" is intended a
polymer which is produced by polymerization of the specified
monomer, optionally in the presence of additional monomers, which
can be incorporated into the polymer main chain. The FDA has
approved poly((L)-lactic acid), poly((L)-lactide),
poly(epsilon-caprolactone) and blends thereof for use in surgical
applications, including medical sutures. An advantage of these
tissue engineered absorbable materials is their degradability by
simple hydrolysis of the ester linkage in the polymer main chain in
aqueous environments, such as body fluids. The degradation products
are ultimately metabolized to carbon dioxide and water or can be
excreted from the body via the kidney.
[0087] In yet other aspects, the nanofibrous polymer scaffold is
composed of bioabsorbable materials selected from biopolymers
including collagen, gelatin, alginic acid, chitin, chitosan,
fibrin, hyaluronic acid, dextran, polyamino acids, polylysine, and
copolymers of these materials. Any combination, copolymer, polymer
or blend thereof of the above examples is contemplated for use
according to the present invention. Such bioabsorbable materials
may be prepared by known methods.
[0088] In certain aspects, the nanofibrous polymer support
comprises nanofibers composed of a biocompatible, biodegradable
and/or bioabsorbable material and at least one bioactive molecule.
Proteins, non-peptidic therapeutic agents and DNA are generally
preferred bioactive molecules for inclusion in the nanofibers of
the nanofibrous polymer support. In certain applications proteins,
such as growth factors, cytokines, and other therapeutic
protein-based drugs, non-protein-based drugs, and DNA, can be
incorporated into the biodegradable nanofibers for the programming
release to enhance cartilage growth. In certain applications, the
bioactive molecules incorporated into the nanofibrous polymer
support continuously supplements growth factors such as TGF-betal
by release of the bioactive molecule from the support by
degradation or absorption of the polymer or leaching of the
bioactive molecule from the support. In certain applications where
the nanofibrous polymer support is composed of a biodegradable
polymer having a growth factor dispersed therein, the growth factor
is released from the polymer during the degradation process to the
surrounding cells endogenously both in vitro and in vivo. The slow
release of the bioactive molecule may beneficially deliver
additional growth factors to engineered tissue incorporated in vivo
in a patient. In certain other applications, incorporating DNA into
the nanofibers of the nanofibrous polymer support can genetically
instruct (transfect or transduce) cells for favorable cell
activities.
[0089] In certain other aspects, the nanofibrous polymer support
comprises nanofibers coated with one or more bioactive molecules.
In certain aspects, electrospun nanofibers can be coated with
bioactive proteins, other peptide sequences, bioactive molecules
and/or DNA to enhance cell adhesion, migration, proliferation and
differentiation. Certain suitable proteins include but are not
limited to fibronectin, vitronectin, collagens, laminin and the
like. Certain other peptide sequences include but are not limited
to RGD (arginine-glycine-aspartic acid). Surface modified
nanofibers can be used alone or in combination with untreated
nanofibers to form the nanofibrous polymer scaffold used in the
engineered tissues or methods of making cartilage provided
herein.
[0090] In other aspects, suitable nanofibrous polymer scaffolds
include those manufactured from biodegradable polymers which
degrade in vivo or in vitro, at a sufficiently slow rate to retain
the desired nanoscale morphology during the tissue culturing
process. In certain applications, the nanofibrous polymer scaffold
is dimensionally stable for at least about 28 days, at least about
35 days, or at least about 42 days.
[0091] In certain embodiments, electrospinning of PCL-based
nanofibers resulted in a scaffold composed of uniform, randomly
oriented fibers of an average diameter of about 700 nm, as seen by
scanning electron microscopy (FIG. 2A-B). Following an 8 week
incubation in culture medium at 37.degree. C., scaffolds maintained
their integrity and three-dimensional structure, while exhibiting
no noticeable change in dry weight over the entire culture
period.
[0092] In certain embodiments, nanofibrous polymer scaffolds are
composed of a biodegradable polymer which is dimensionally stable
for at least the time period required to culture the cartilage or
other tissue formed using the scaffold. FIG. 3A-F is a series of
microscopic images of various nanofibrous scaffolds which are
contemplated for use in the methods and cartilage provided herein.
FIG. 4A-F provides a macroscopic observation of the degradation
profile of various nanofibrous polymer scaffolds exposed to a
phosphate buffered solution after 3, 7, 14, 21, and 42 days. After
42 days, substantial degradation occurred for nanofibrous scaffolds
composed of PGA, PDLLA, PLGA 5050 and PLGA 8515. In contrast,
nanofibrous scaffolds composed of PLLA and PCL retained their
original structure after 42 days in the degradation medium.
[0093] FIG. 5A-F is a series of scanning electron microscope images
of nanofibrous non-woven mats composed of PGA, PDLLA, PLLA,
PLGA5050, PLGA8515, and PCL. Images are provided prior to exposure
to a degradation medium (D0), after one day in the degradation
medium (D1) and after three days in the degradation medium (D3).
Nanofibrous scaffolds composed of PGA, PLLA and PCL retain the
straight and uniform thickness nanofiber morphology after three
days exposure to the degradation medium. PDLLA, PLGA5050, and
PLGA8515 undergo substantial degradation including swelling,
melting and other changes in nanofiber morphology.
[0094] The nanofibrous polymer support of the tissue engineered
cartilage is composed of at least one biodegradable polyester.
Certain preferred polymers include poly((L)-lactic acid),
poly(epsilon-caprolactone) and blends thereof. In certain
applications where additional tensile strength is required,
non-biodegradable biocompatible nanofibers may be incorporated into
the nanofibrous scaffold. In certain methods, including methods of
preparing cartilage which typically take at least 28, 35, or 42
days, the nanofibrous polymer scaffolds are selected from those
composed of PLLA, PCL, and blends thereof, optionally blended with
one or more additional biodegradable or bioabsorbable polymers.
[0095] In yet other aspects, the nanofibrous polymer support
comprises polymer nanofibers having a diameter of less than 1
micron or having a diameter of between 10 nm to 1 micron, 50 nm to
1 micron, 100 nm to 1 micron, or 200 nm to 500 nm.
Cells for Seeding onto the Nanofibrous Polymer Scaffold
[0096] A variety of cells can be used to form engineered tissues.
Chondrocytes, mesenchymal stem cells, and embryonic stem cells are
generally preferred cells for the preparation of cartilage.
Mesenchymal stem cells can be isolated from various tissues,
including but not limited to muscle, blood, bone marrow, fat, cord
blood, placenta, and other tissues known to contain mesenchymal
stem cells.
[0097] Upon administration of chondrocytes to the nanofibrous
polymer scaffold, the cells remain differentiated chondrocyte cells
and begin to form extracellular matrix rich in collagen. Stem
cells, including adult mesenchymal stem cells and embryonic stem
cells, particularly MSC originating from a patient in need of
replacement cartilage are suitable for use in the methods of the
invention and differentiate to chondrocyte cells when the MSC cells
are in contact with the nanofibrous polymer scaffolds used in the
methods of the invention. Other collagen generating cells are also
contemplated for use in the methods of the invention, including but
not limited to tenocytes, ligamentum cells, fibroblasts, and dermal
fibroblasts.
[0098] In certain aspects, the cells seeded on the nanofibrous
polymer scaffold are a mixture of chondrocytes, mesenchymal stem
cells, and/or embryonic stem cells and at least one other cell line
which are beneficial for cartilage growth. In certain other
aspects, the cells seeded on the nanofibrous polymer scaffold are a
mixture of cells selected from chondrocytes, mesenchymal stem
cells, and/or embryonic stem cells which are admixed with a
biocompatible material. Biocompatible materials which are
contemplated for admixing with cells in the preparation of
engineered cartilage include biodegradable and non-biodegradable
polymers and inorganic materials (such as ceramics and metals)
which can be present as a fiber, nanoparticles, microparticle or
mixture thereof. In certain aspects, these materials including
nanofibers, cells, and other materials and agents are dispersed in
the mixture of cells-nanofibers or structured in an organized way
such as the layer-by-layer deposition.
[0099] In certain aspects where the engineered tissue is intended
for implantation into a patient as part of a therapeutic,
preventative, or cosmetic surgical procedure, autologous cells
obtained by a biopsy are used as seed cells in the methods of
engineering tissues or methods of engineering cartilage provided
herein. Cells can be obtained directly from a donor, washed and
suspended in a culture media before contacting the cells with the
nanofibrous polymer support. To enhance cell viability, the cells
are generally added or mixed with the culture media just prior to
incorporation into the nanofibrous polymer scaffold.
[0100] Cell viability can be assessed using standard techniques
including visual observation with a light or scanning electron
microscope, histology, or quantitative assessment with
radioisotopes. The biological function of the cells incorporated
into the nanofibrous polymer scaffold can be determined using a
combination of the above techniques.
[0101] Cells obtained by biopsy are harvested, cultured, and then
passaged as necessary to remove non-cellular contaminants and
contaminating, unwanted cells. Chondrocytes are isolated from
autologous cartilage by excision of tissue, then either enzymatic
digestion of cells to yield dissociated cells or mincing of tissue
to form explants which are grown in cell culture to yield cells for
seeding onto the nanofibrous polymer supports. Mesenchymal stem
cells are isolated from autologous bone marrow. Typically bone
marrow is harvested from the interior of the femoral neck and head
by using a bone curet and then isolated from particulates and other
cells (e.g., non-adherent hematopoietic and red blood cells) by
centrifugation and exchange of culture medium.
Contacting the Cells and the Nanofibrous Polymer Substrate
[0102] In certain methods, a nanofibrous polymer non-woven mat is
electrospun to a desired thickness and then cut to a desired shape
to form the nanofibrous polymer scaffold. In certain embodiments, a
solution of cells is then applied to at least a portion of the
nanofibrous polymer substrate to form a cell-polymer matrix. During
culturing the cells diffuse through the thickness of the
nanofibrous polymer scaffold to form a cell polymer matrix. In
certain embodiments, the cells are selected from chondrocytes,
mesenchymal stem cells, or embryonic stem cells or the cells are
selected from chondrocytes and mesenchymal stem cells.
[0103] In certain instances, a cell culture tube is charged with
the nanofibrous polymer substrate and then a solution of cells is
added to the cell culture tube. The cell-substrate aggregate is
then cultured statically in the tube to generate cartilage. As used
herein, "statically cultured," "cultured in a static environment,"
or like terms are intended to refer to culturing conditions in
which the culture medium is not moving relative to the cell-polymer
matrix. In certain embodiments, the culture medium is a
chondrogenic medium preferably comprising one or more growth
factors. The static culturing is conducted at 37.degree. C. in a
humidified 5% carbon dioxide atmosphere. In certain methods
comprising static culturing, the culture vessel is a conical cell
culture tube, a culture medium and the cell-substrate aggregate are
charged in the cell culture tube, and the mixture maintained at
37.degree. C. under a humidified 5% carbon dioxide atmosphere.
Culturing using a culture tube is referred to herein as "static"
culturing.
[0104] In other methods, a nanofibrous polymer non-woven mat is
expanded to introduce more porosity in the nanofibrous polymer
scaffold. That is, in certain embodiments, an electrospun polymer
mat is plucked, combed, teased or otherwise mechanically treated to
increase the inter-fiber distances in the mat such that the
expanded nanofibrous polymer scaffold has a "cotton ball" or fluffy
appearance. The expanded mat is then contacted with a solution of
cells. Although not wishing to be bound by theory, the increased
inter-fiber distances present in the expanded nanofibrous polymer
scaffold permits creates more apertures through which the cells can
disperse into the expanded nanofibrous polymer scaffold thereby
providing a more uniform distribution of cells throughout the
scaffold after compression.
[0105] In certain methods, after combining the expanded nanofibrous
polymer scaffold and the cell solution for between about 1 minute
and about 8 hours, between about 5 minutes and about 6 hours, or
between about 10 minutes and about 4 hours, the cell-scaffold
aggregate is compressed. In general, any force capable of uniformly
compressing the mixture of the nanofibrous polymer scaffold and the
dispersed cells without causing undue damage to the viability of
the cells is contemplated for use in the methods of the invention.
In certain embodiments, the expanded nanofibrous polymer support is
compressed by mechanical mans, e.g., by compressing between two or
more impermeable objects such as a vessel wall and a non-porous rod
or other implement. In certain other embodiments, application of
centripetal force is used as a compression means. For example, a
solution of the expanded nanofibrous polymer scaffold and dispersed
cells is centrifuged for between 1 and about 10 minutes at between
about 250 g and about 2500 g, or for between 2 and about 8 minutes
at between about 500 g and about 2000 g to form a compressed
cell-polymer matrix. Application of a compressive force compacts
the expanded nanofibrous polymer scaffold entrapping the cells in
the pores thereof. The substrate-solution mixture is then incubated
in a chondrogenic medium for 1 hour to about 1 week under static
conditions to permit the cell-polymer matrix to integrate.
[0106] In certain aspects, a compacted polymer-cell matrix is
obtained by compressing the cell and expanded nanofibrous polymer
scaffold mixture. For example, the cell and expanded nanofibrous
polymer scaffold solution can be centrifuged at between about 250 g
and about 2500 g to the polymer-cell matrix for 1 to 10 minutes.
After centrifugation, the compacted polymer-cell matrix takes on
the three dimensional shape of the bottom of the vessel in which
the solution was centrifuged. That is, the bottom of the
centrifugation vessel functions as a mold for the shape of the
polymer-cell matrix formed during the centrifugation. Thus, for
example, use of a centrifugation vessel having a bottom in the
shape of an ear will result in an ear shaped polymer-cell
matrix.
[0107] In certain embodiments, after centrifugation, the
polymer-cell matrix is cultured for between 1 and about 10 days in
a static environment to generate increased integration of the
polymer-cell matrix. In certain other embodiments the polymer-cell
matrix is cultured in a static vessel for between 2 to 10 days or
between 3 and 7 days. Although not wishing to be bound by theory,
the static culturing period is believed to allow the cells to
generate an extracellular matrix which holds the fibers of the
nanofibrous polymer support in position.
[0108] In certain aspects, after compression and static culturing,
the polymer-cell matrix is transferred to a bioreactor for
additional culturing of up to about 42 days during which time
cartilage is formed. The term "bioreactor" is intended to refer to
vessels suitable for culturing cells or cell-polymer matrixes,
wherein the bioreactor improves delivery of nutrients and removal
of waste products associated with cellular maintenance and
development. Preferred bioreactor devices and vessels in which one
or more biological or biochemical processes can be conducted under
closely monitored and controlled conditions, e.g., environmental
and/or operating conditions can be regulated by an operator.
Certain bioreactors are devices in which the temperature, acidity
(pH), pressure, nutrient supply, atmosphere, and/or removal of
waste can be regulated by an operator or a control device.
Bioreactors suitable for use in the methods of making tissue
engineered cartilage provide a dynamic growth environment. The
terms "dynamic," "cultured in a dynamic environment" and the like
are intended to refer to culturing conditions in which the culture
medium experiences at least one translational, rotational, or other
mechanical force capable of causing the culture medium to flow or
otherwise be translated in the bioreactor culture chamber. In
general, bioreactors which generate movement of the culture medium
relative to the cell-polymer matrix or the tissue engineered
cartilage present in the bioreactor chamber are preferred.
[0109] In certain aspects, the bioreactor is selected from devices
which direct a continuous flow of a culture medium or other fluid
at the cell-polymer aggregate or tissue charged into the bioreactor
culture chamber. In certain embodiments, the bioreactor is selected
from spinner-flask bioreactors, rotating-wall vessel bioreactors,
hollow fiber bioreactors, direct perfusion bioreactors, bioreactors
that apply a controlled direct mechanical force to the cell-polymer
aggregate or tissue, and other bioreactor designs that deliver
continuous fluid flow to the cell-polymer aggregate or tissue. In
certain other aspects, the bioreactor is a rotating bioreactor
having a taurus shaped chamber charged with the cell-substrate
aggregate and culture medium. The bioreactor is rotated about the
central axis of the taurus at a rate sufficient to offset the force
of gravity. In certain aspects, the use of a rotating bioreactor
provides mechanical stresses such as compressive or shear stresses
which contribute to the regulation of chondocytes in cartilage.
Culturing using a rotating bioreactor such as a rotating bioreactor
is referred to herein as "dynamic" culturing.
[0110] In certain aspects, tissue engineered cartilage prepared by
dynamic culturing possesses mechanical and biochemical profiles
which more closely mimic natural cartilage than tissue engineered
cartilage prepared using a static culturing process. In FIG. 14 is
a photographic comparison of tissue engineered cartilage prepared
by the dynamic method recited in Example 4 and tissue engineered
cartilage prepared by a static method equivalent to the method of
Example 4. The dynamically cultured cartilage possesses a smoother
glossier surface than the cartilage prepared using static
culturing. Moreover, the weight gain observed for tissue engineered
cartilage prepared using a dynamic culturing process is greater
than cartilage prepared using a static culture for the same
culturing period (see, FIG. 15). A histological and RT-PCR analysis
comparison of cartilage prepared under dynamic and static culturing
conditions are provided in FIG. 16-18. The mechanical properties
(i.e., Young's modulus (FIG. 20) and compressive test (FIG. 19)) of
dynamically cultured and statically cultured tissue engineered
cartilage indicate that the dynamically cultured sample has greater
strength under compression than statically cultured samples.
Moreover, the dynamically cultured tissue engineered cartilage
possesses a compressive strength and Young's modulus which is
comparable to healthy native cartilage, e.g., a Young's modulus of
between about 500 and about 800 MPa.
[0111] In certain aspects the culture medium is formulated to
support the target engineered tissue. Thus, where cartilage is the
target tissue, the culture medium is a chemically defined
chondrogenic medium appropriate for maintenance of chondrocyte
cells or inducing differentiation of mesenchymal stem cells to
chondrocytes. In certain aspects, chemically defined chondrogenic
media for use in the methods provided herein are substantially
serum-free. Certain chemically defined chondrogenic media comprise
one or more growth factors which regulate and/or promote
chondrocyte formation, development or growth.
[0112] In certain methods provided herein, the culture medium
comprises one or more growth factors suitable for promoting growth
and development of chondrocytes and the differentiation of stem
cells in to chondrocytes. In certain aspects, the growth factors
are selected from transforming growth factors (TGF), insulin-like
growth factors (IGF), bone morphogenic proteins (BMP), fibroblast
growth factors (FGF), and combinations thereof. In certain methods,
the growth factors are selected from IGF-1, TGF-.beta.1,
TGF-.beta.3, BMP-7 and combinations thereof. Thus, for example,
culture medium comprising a mixture of TGF-.beta.1 and IGF-1
provided cartilage having particularly desirable physical and
biological properties, including increased Young's modulus.
Product Cartilage
[0113] One aspect is a tissue engineered cartilage having a peak
compressive stress (Young's modulus) of greater than 250 MPa, which
tissue engineered cartilage is composed of a plurality of
chondocytes dispersed in a nanofibrous polymer support comprising a
plurality of polymer nanofibers. In other aspects, the each of the
plurality of chondocytes is in contact with at least one, at least
two, or at a plurality of other chondocytes dispersed in the
polymer support. In certain tissue engineered cartilages provided
herein possess a peak compressive strength of at least about 300
MPa, about 400 MPa or about 500 MPa or a compressive strength of
between about 250-1000 MPa, between about 300-1000 MPa, between
about 300-900 MPa, or between about 400-900 MPa.
[0114] Certain tissue engineered cartilages provided herein are
prepared by the cartilage tissue engineering methods depicted
schematically in FIG. 6 and described in Example 4. The tissue
engineered cartilage provided herein possess mechanical and
biochemical properties analogous to natural cartilage, including
but not limited to high Young's modulus of about 600-1000 Mpa,
incorporation of lacunae in the cartilage morphology, elevated
expression of cartilage specific proteins, and the like.
[0115] In certain embodiments, tissue engineered cartilage prepared
by the methods of the invention possess lacunae (the voids present
near chondrocytes in cartilage) and an extracellular matrix which
resembles that of natural cartilage. Certain methods produce
cartilage which has a peak compressive strength equivalent to
natural cartilage and possess lacunae, the empty space surrounding
chondocytes in healthy cartilage, which is similar to natural
cartilage.
[0116] A series of tissue engineered cartilages were prepared by
the method recited in Example 4, in which the growth factors added
to the culture medium was varied from no growth factors (control),
TGF-.beta.1 alone, IGF-1 alone, or a combination of TGF-.beta.1 and
IGF-1. The resultant cartilage were analyzed using several
biochemical and mechanical tests.
[0117] FIG. 6 is a tissue engineered flow chart of one of the
methods of preparing tissue engineered cartilage provided herein
and exemplified in Example 4;
[0118] FIG. 7 is a picture of the tissue engineered cartilage
prepared in Example 4 cultured in the chondrogenic medium
supplemented with different growth factors after 42 days (Control
had no growth factors) FIG. 8 is a series of images showing
expression of cartilage specific proteins by RT-PCR analysis in the
cells of the tissue engineered cartilage prepared in Example 4
cultured in the chondrogenic medium supplemented with different
growth factors after 42 days(control, TGF-.beta.1, IGF-1, and
TGF-.beta.1+IGF-1). Histological analysis of cartilage prepared in
Example 4 cultured in a chondrogenic medium supplemented with
different growth factors after 42 days is provided by the images of
FIG. 9 and FIG. 10. More particularly, FIG. 9 provides a series of
photographs of tissue engineered cartilage samples with H&E
staining. FIG. 10 provides a series of photographs of tissue
engineered cartilage samples with Alcian blue staining. FIG. 11 is
a series of photographs showing the immunohistochemical
localization of cartilage-specific ECM in the tissue engineered
cartilage prepared by the method of Example 4 The histology
indicates that the tissue samples possess a cartilage like
extracellular matrix and chondrocytes dispersed in the tissue. FIG.
11 is a series of photographs showing the immunohistochemical
localization of cartilage-specific ECM in cartilage prepared by the
method of Example 4 cultured in a chondrogenic medium supplemented
with different growth factors after 42 days.
[0119] Uptake of radiolabeled sulfate and radiolabeled proline by
the tissue engineered cartilage indicates chondrocyte activity and
development of cartilage specific ECM in the tissue engineered
cartilage. FIG. 12 is a bar graph of radioactive sulfate
incorporation in cartilage prepared by the method of Example 4 with
different growth factors (control, TGF-.beta.1, IGF-1, and
TGF-.beta.1+IGF-1). FIG. 13 is a bar graph of radioactive proline
incorporation in cartilage prepared by the method of Example 4 with
different growth factors (control, TGF-.beta.1, IGF-1, and
TGF-.beta.1+IGF-1).
[0120] In certain aspects, the tissue engineered cartilage provided
herein are compatible with natural cartilage. Thus, for example,
natural cartilage grafted to a tissue engineered cartilage prepared
by the methods of the invention integrate when cultured in vitro
such that after culturing for several weeks the interface between
natural and tissue engineered cartilage fades.
In-Vivo Cartilage Formation
[0121] Another aspect is a method of forming cartilage in vivo, the
method comprising the steps of (a) providing a nanofibrous polymer
support comprising a plurality of polymer nanofibers; and (b)
inserting the nanofibrous polymer support into a patient at the
position suitable for formation of new cartilage.
[0122] In certain aspects, the nanofibrous polymer scaffolds
suitable for use in the in vitro methods of preparing cartilage are
also suitable for implantation into a patient at situs in need of
new cartilage. Suitable nanofibrous polymer scaffolds for
incorporation into a patient include PCL and PLLA and further
include other biodegradable polyesters which provide a
dimensionally stable scaffold for chondrocyte development in the
area requiring new cartilage. Thus, for example, PGA which is
morphologically stable under SEM analysis after three days (FIG.
3), but degrades rapidly between day 21 and day 42 (FIG. 4 and FIG.
5) is also contemplated for use as a nanofibrous polymer support in
the in vivo cartilage formation methods of the invention.
[0123] In certain aspects, the methods of making cartilage are
suitable for producing elastic cartilage, fibrocartilage, or
hyaline cartilage depending upon the cell type used, the method
selected, the growth factors added to the culture medium, the
composition of the support scaffold and the presence or absence of
additional materials in the support structure, such as ceramics,
bone mimetics, high tensile strength bio-compatible fibers and the
like. Cartilage is a specialized type of dense connective tissue
consisting of 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 which is found in articular cartilage, in costal
cartilages, in the septum of the nose, and in the larynx and
trachea. Articular cartilage is hyaline cartilage covering the
articular surfaces of bones. Costal cartilage connects the true
ribs and the sternum. Fibrocartilage is a connective tissue
primarily located in intervertebral disc. Elastic cartilage is
primarily in the epiglottis, the external ear, and the auditory
tube. By harvesting the appropriate chondrocyte precursor cells,
any of these types of cartilage tissue can be grown using the
methods of the invention.
Methods of Tissue Engineering
[0124] The tissue engineering methods described supra are directed
to the preparation of tissue engineered cartilage, the methods are
equally suited to the preparation of other tissue engineered or
engineered tissues. Thus, for example, substituting cells obtained
from other tissues for chondrocytes, or replacing the chondrogenic
medium with media suited for other tissues in the methods of tissue
engineering and methods of preparing cartilage recited supra, will
generate other tissues.
[0125] Certain methods of engineering tissue are suitable for use
in forming bone, muscle, tendon, ligaments, and other tissues.
Thus, the methods of cartilage formation provided herein are
modified by (a) contacting the nanofibrous polymer support with
cells appropriate for the desired tissue, and (b) adding tissue
appropriate growth factors to the culture medium. In certain
aspects, including for example, methods of making tendon and/or
ligament, the use of a non-biodegradable nanofibrous polymer
support is desirable to provide additional tensile strength to the
engineered tendon or ligament.
III. Methods of Treatment
[0126] In other aspects, the invention provides methods of
repairing, replacing and/or augmenting cartilage in a patient for
treatment or prevention of diseases or disorders or for cosmetic
purposes. In certain aspects, a method of treating cartilage damage
is provided in which the method comprising the steps of:
[0127] (a) providing a tissue engineered cartilage having a peak
compressive stress (Young's modulus) of greater than 250 MPa, which
tissue engineered cartilage is composed of a plurality of
chondocytes dispersed in a nanofibrous polymer support comprising a
plurality of polymer nanofibers or a tissue engineered cartilage
prepared by the methods provided herein;
[0128] (b) inserting the tissue engineered cartilage into a patient
at the location of damaged cartilage.
[0129] Another aspect is a method for treating cartilage damage,
the method comprising the steps of
[0130] (a) harvesting chondocytes or MSC cells from the
patient;
[0131] (b) preparing tissue engineered cartilage by one of the
methods provided herein, wherein the cells are the chondocytes or
MSC cells harvested from the patient;
[0132] (c) implanting the tissue engineered cartilage in the
patient in the locus having damaged cartilage.
[0133] Yet another aspect is a method for cosmetic or
reconstructive surgery, the method comprising the steps of
[0134] (a) providing a tissue engineered cartilage having a peak
compressive stress (Young's modulus) of greater than 250 MPa, which
tissue engineered cartilage is composed of a plurality of
chondocytes dispersed in a nanofibrous polymer support comprising a
plurality of polymer nanofibers or a tissue engineered cartilage
prepared by the methods provided herein;
[0135] (b) inserting the tissue engineered cartilage into a
patient.
[0136] In another aspect, the invention provides a method for
cosmetic or reconstructive surgery, the method comprising the steps
of
[0137] (a) harvesting chondocytes or MSC cells from the
patient;
[0138] (b) preparing tissue engineered cartilage by a method
provided herein, wherein the cells are the chondocytes or MSC cells
harvested from the patient;
[0139] (c) implanting the tissue engineered cartilage in the
patient.
[0140] In one embodiment, the present invention provides methods of
treating disease and/or disorders or symptoms thereof which
comprise administering a nanofibrous polymeric support, an
engineered tissue or engineered cartilage provided herein to a
subject (e.g., a mammal such as a human). More particularly, the
present invention provides methods of treating damaged or destroyed
cartilage by inserting tissue engineered cartilage herein at the
locus of cartilage damage or destruction in the patient. Thus, for
example, a patient suffering from arthritis of the knee may have
damaged or destroyed some or all of the cartilage interposed
between the femur, the fibula, and/or the patella. The methods of
the invention provide for treatment by inserting tissue engineered
cartilage or inserting a nanofibrous polymer support in the knee at
the point of damage to replace or repair the damaged cartilage.
[0141] In certain other aspects, engineered cartilage provided
herein is administered to a subject (e.g., a mammal such as a
human) to provide desirable reconstructive or cosmetic benefit to
the patient. Thus, for example, a patient sustained an injury which
caused damage or destruction of the cartilage of the ear or nose.
The methods of the invention provide for reconstruction or cosmetic
enhancement of the ear or nose by inserting a formed engineered
cartilage into the damaged nose or ear thereby improving the
function or aesthetics of the nose or ear.
[0142] As used herein, the terms "treat," treating," "treatment,"
and the like refer to reducing or ameliorating a disorder and/or
symptoms associated therewith. It will be appreciated that,
although not precluded, treating a disorder or condition does not
require that the disorder, condition or symptoms associated
therewith be completely eliminated.
[0143] As used herein, the terms "prevent," "preventing,"
"prevention," "prophylactic treatment" and the like refer to
reducing the probability of developing a disorder or condition in a
subject, who does not have, but is at risk of or susceptible to
developing a disorder or condition.
[0144] As used "cosmetic surgery" or "reconstructive surgery" is
intended herein to refer to surgical procedures intended to modify
or improve the appearance of a physical feature, irregularity, or
defect.
[0145] The invention will be further described in the following
examples. It should be understood that these examples are for
illustrative purposes only and are not to be construed as limiting
this invention in any manner.
EXAMPLE 1
Isolation and Culture of Bone Marrow-Derived hMSCs
[0146] With approval from the Institutional Review Board of Thomas
Jefferson University, bone marrow-derived hMSCs were obtained from
the femoral heads of patients undergoing total hip arthroplasty,
and processed as previously described (Noth U, et al. J Orthop Res
2002; 20:1060-9; Haynesworth S E, et al. Bone 1992; 13:81-8; and
Wang M L, et al. J Orthop Res 2002; 20:1175-84). Briefly, whole
bone marrow was curetted from the exposed cutting plane of the
femoral neck, washed extensively in Dulbecco's Modified Eagle's
medium (DMEM; BioWhittaker, Walkersville, Md.), separated from
contaminating trabecular bone fragments and other tissues using a
20-gauge needle attached to a 10-cc syringe, and cultured in DMEM,
10% fetal bovine serum (FBS; Premium Select, Atlanta Biologicals,
Atlanta, Ga.) from selected lots (Caterson E J, et al. Mol
Biotechnol 2002; 20:245-56), and antibiotics (50 .mu.g/mL
streptomycin, 50 IU/mL of penicillin; Cellgro, Herndon, Va.) at a
density of 4.times.10.sup.5 cells/cm.sup.2. Six hours post-plating,
tissue culture flasks were washed twice with phosphate-buffered
saline (PBS) to remove non-adherent cells. Medium changes were made
every 3-4 days. Subconfluent cell monolayers were dissociated using
0.25% trypsin (Gibco BRL, Life Technologies, Grand Island, N.Y.)
and either passaged or utilized directly for study.
EXAMPLE 2
Fabrication of Electrospun Nanofibrous PCL Scaffolds
[0147] Nanofibrous scaffolds were fabricated according to an
electrospinning process described previously (Li W J, et al. J
Biomed Mater Res 2003; 67A:1105-14). Briefly, PCL polymer was
dissolved in an organic solvent mixture (1:1) of tetrahydrofuran
(THF; Fisher, Pittsburgh, Pa.) and N,N, dimethylformamide (DMF;
Fisher, Pittsburgh, Pa.) at a final concentration of 0.14 g/mL. The
polymer solution was delivered through the electrospinning
apparatus at a constant flow rate of 0.4 mL/h under an applied 0.6
kV/cm charge density, resulting in a 144 cm.sup.2 mat with an
approximate thickness of 1 mm. To remove residual organic solvent,
the non-woven polymer mat was placed within a vacuum chamber for 48
h, and subsequently stored in a desiccator. Prior to cell seeding,
squares measuring 10 mm.times.10 mm.times.1 mm were fashioned from
the electrospun mat, sterilized by ultraviolet irradiation for 30
min per side in a laminar flow hood, and pre-wetted for 24 h in
Hanks' Balanced Salt Solution (HBSS; BioSource International,
Camarillo, Calif.).
EXAMPLE 3
Seeding and Differentiation of hMSCs on PCL Scaffolds
[0148] Pre-processed nanofibrous PCL scaffolds were placed in
24-well tissue culture plates (Corning Glass Works, Corning, N.Y.)
coated with 0.3% poly(2-hydroxyethyl methacrylate) (poly HEMA;
Polysciences, Warrington, Pa.) to prevent normal cell attachment to
tissue culture polystyrene. Cellular scaffolds were incubated at
37.degree. C. for 4 h to allow MSCs to diffuse into and adhere to
the scaffold before the addition of 2 mL of culture medium to each
well. During the 4 h incubation, 20 .mu.L of serum containing
culture medium was applied every 30 min to each cellular scaffold
to prevent the constructs from drying. For chondrogenic
differentiation studies, 4.times.10.sup.5 hMSCs were seeded per PCL
scaffold and maintained in a chemically defined medium containing
serum-free DMEM, 50 .mu.g/mL ascorbate, 0.1 .mu.M dexamethasone, 40
.mu.g/mL L-proline, 100 .mu.g/mL sodium pyruvate, ITS-plus
(Collaborative Biomedical Products, Cambridge, Mass.), antibiotics,
and 10 ng/mL recombinant human transforming growth factor-.beta.1
(TGF-.beta.1; R&D Systems, Minneapolis, Minn.) (Johnstone B, et
al. Exp Cell Res 1998; 238:265-72; Yoo J U, et al. J Bone J Surg Am
1998; 80:1745-57; and Mackay A M, et al. Tissue Eng 1998;
4:415-28). Control cell scaffolds were maintained without the
addition of TGF-.beta.1. For osteogenesis and adipogenesis, hMSCs
were seeded at a density of 2.times.10.sup.5 cells/scaffold.
Osteogenic induction was accomplished using DMEM supplemented with
10% FBS, 50 .mu.g/mL ascorbate, 10 mM .beta.-glycerophosphate, 0.1
.mu.M dexamethasone, and antibiotics (Pittenger M F, et al. Science
1999; 284:143-7). Finally, adipogenesis was induced using DMEM
supplemented with 10% FBS, 1 .mu.M dexamethasone, 0.5 mM
3-isobutyl-1-methylxanthine, 1 .mu.g/mL insulin, and antibiotics
(Pittenger M F, et al. Science 1999; 284:143-7). Control cultures
were maintained without osteogenic and adipogenic supplements,
respectively. All cell scaffolds were maintained for 21 days in a
humidified incubator at 37.degree. C. and 5% CO2 with medium
changes every 3-4 days.
EXAMPLE 4
Preparation of Tissue Engineered Cartilage using Dynamic
Culturing
4.1. Electrospin Nanofibers
[0149] 1.6 gm of tissue engineered, biodegradable PLLA is dissolved
in 10 mL of chloroform and 2 mL of DMF for overnight. The PLLA
polymer solution is placed in a 10 mL syringe with a metal needle
and the tip of needle is 10 cm away from the collecting plate. 16
kV of voltage is applied to the polymer solution. After 8 hour of
electrospinning, 12.times.12 cm of nanofibrous mat is produced.
4.2. Expand the Nanofibers to Form an Expanded Nanofibrous Polymer
Support
[0150] Use two metal specula to manually loosen and fluff
nanofibers. Weigh 30 mg of fluffy nanofibers and place it in a 50
mL tissue culture conical tube. 20 mL of 100% of ethanol is added
in the tube and shake the tube to further separate and disperse
individual nanofibers in the ethanol suspension. Replace the
ethanol liquid phase with a balanced salt solution by a gradient
fluid exchange, e.g., exchange the 100% ethanol with 70% ethanol,
then with 30% ethanol, then with 100% pure water, and then finally
with balanced salt solution.
4.3. Combine Cells and Fluffy Nanofibers
[0151] Chondrocytes grown on a monolayer culture is trypisnized and
10 million of cells in 1 mL of 10% FBS containing medium is placed
in a nanofiber containing tube. Cells and nanofibers are well mixed
by gently shaking the tube. The cell-nanofiber mixture is left in
an incubator for 1.5 hour, packed tightly using a long bar with a
flat surface at the end, and centrifuged at the speed of 1500 g for
5 min. The cell-nanofiber aggregation is cultured in the 50 mL
conical tube for additional 7 days before it is removed to a
dynamic culture system.
4.4. Culture Cell-Polymer Aggregate in a Rotating Vessel Wall
Bioreactor
[0152] The cell-nanofiber composite is placed in a rotating vessel
wall bioreactor for next 42 days. The rotation speed of a
rotating-wall vessel bioreactor is controlled to maintain the
cell-nanofiber composite stay in the situation of floating in the
medium. The cell-nanofiber composite is cultured in the
chondrogenic medium supplemented with 10 ng/mL of transforming
growth factor-beta 1 (TGF-1) and 50 ng/mL of insulin-like growth
factor and half the volume of the cell culture medium is replaced
every three days.
EXAMPLE 5
Physical and Biochemical Analysis Methods
[0153] 5.1. Scanning Electron Microscopy (SEM)
[0154] For each condition, two cell-polymer constructs were fixed
in 2.5% glutaraldehyde, dehydrated through a graded series of
ethanol, vacuum dried, mounted onto aluminum stubs, and sputter
coated with gold. Samples were examined using a scanning electron
microscope (S-4500; Hitachi, Japan) at an accelerating voltage of
20 kV.
[0155] 5.2. Reverse Transcription Polymerase Chain Reaction
(RT-PCR) Analysis
[0156] Total cellular RNA was extracted using Trizol Reagent (Gibco
BRL, Life Technologies, Grand Island, N.Y.) according to the
manufacturer's protocol. For efficient yield, six cell-scaffolds
from the same culture condition were first briefly homogenized in
Trizol Reagent using a pestle (Kontes, Vineland, N.J.).
Concentrations of RNA samples were estimated on the basis of
OD.sub.260. RNA samples were reverse transcribed using random
hexamers and the SuperScript First Strand Synthesis System (Gibco
BRL, Life Technologies, Grand Island, N.Y.). PCR amplification of
cDNA was carried out using AmpliTaq DNA Polymerase (Perkin Elmer;
Norwalk, Conn.) and the gene-specific primer sets listed in Table 1
of Li, et al., Multilineage Differentiation of human mesenchymal
stein cells in three-dimensional nanofibrous scaffold,
Biomaterials, 2005. These genes included adipose specific genes-,
lipoprotein lipase (LPL) and peroxisome proliferator-activator
receptor-.gamma.2 (PPAR .gamma.2); cartilage specific genes-,
aggrecan (AGN), collagen type II (Col II), and collagen type X (Col
X); and bone specific genes-, alkaline phosphatase (ALP), bone
sialoprotein (BSP), collagen type I.alpha.2 (Col I.alpha.2), and
osteocalcin (OC). Thirty-two cycles were used for all genes, and
consisted of 1-min denaturation at 95.degree. C., 1-min annealing
at 57.degree. C. (AGN, Col II, and Col X) or 51.degree. C. (all
remaining genes), 1-min polymerization at 72.degree. C., followed
by a final 10-minute extension at 72.degree. C. The housekeeping
gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was used as
a control for RNA loading of samples. PCR products were analyzed
electrophoretically using the Agilent 2100 Bioanalyzer (Agilent
Technologies, Palo Alto, Calif.).
[0157] 5.3. Cryoembedding and Sectioning
[0158] For each condition, two of each adipogenic, chondrogenic,
and osteogenic cellular constructs were harvested following 21 days
of culture, fixed in 4% PBS-buffered paraformaldehyde for 15 min,
washed three times with PBS, infiltrated with 20% sucrose, embedded
with Tissue-Tek O.C.T Compound (Sakura Finetek USA, Inc., Torrance,
Calif.), and cryosectioned at 8 mm thickness using the Leica CM
1850 (Bannockburn, Ill.) cryostat microtome.
[0159] 5.4. Histological Analysis
[0160] Control and adipogenic cell-polymer constructs were
incubated in 60% isopropanol for 5 min, followed by Oil Red 0 stain
for 5 min. The cells were then rinsed with tap H2O and
counterstained with hematoxylin for 1 min. Cell-polymer constructs
maintained in chondrogenic medium with or without TGF-.beta.1 (10
ng/mL) were stained with alcian blue (pH 1.0), as previously
described (Denker A E, et al. Differentiation 1999; 64:67-76).
Control and osteogenic cell-polymer constructs were stained
histochemically for alizarin red, as previously described (Puchtler
H, et al. J Histochem Cytochem 1969; 17:110-24), and alkaline
phosphatase (Sigma Cat. No. 86-C) according to the manufacturer's
protocol. Two samples from each condition were prepared for this
analysis.
[0161] 5.5. Immunohistochemical Analysis
[0162] Immunohistochemistry was used to detect aggrecan, collagen
type II, and link protein, in control and treated chondrogenic
cell-polymer constructs, and bone sialoprotein, and collagen type I
in control and treated osteogenic cell-polymer constructs. Sections
were pre-digested for 15 min at 37.degree. C. in 1.5 U/mL of
chondroitinase A/B/C before they were incubated for 1 h at
37.degree. C. in 10 mg/mL of aggrecan primary antibody (1-C-6;
Developmental Studies Hybridoma Bank, Iowa City, Iowa). Bone
sialoprotein and collagen type I were detected using primary
antibodies at a 1:500 dilution (BSP; Chemicon International,
Temecula, Calif.) and 15 .mu.g/mL (SP1.D8; Developmental Studies
Hybridoma Bank), respectively, for 1 h at 37.degree. C.
Antigen-antibody complexes were detected colorimetrically using the
Broad Spectrum Histostain-SP Kit (Zymed Laboratories, Inc., South
San Francisco, Calif.); sections were counterstained with
hematoxylin.
INCORPORATION BY REFERENCE
[0163] All patents, published patent applications, and other
references disclosed herein are hereby expressly incorporated by
reference in their entireties by reference.
Equivalents
[0164] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *