Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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A SHAPE-BASED APPROACH FOR SCAFFOLDLESS TISSUE ENGINEERING
STATEMENT OF GOVERNMENT INTEREST
This disclosure was developed at least in part using funding from the National
Institutes of Health, Grant Number ROl AR47839-2. The U.S. government may have
certain
rights in the invention.
CROSS-REFERENCE TO RELATED APPLICATIONS
This application a continuation-in-part of International Application No.
PCT/US2005/24269 filed July 8, 2005, which claims the benefit of U.S.
Provisional
Application Serial No. 60/586,862 filed on July 9, 2004; this application also
claims the
benefit of U.S. Provisional Application No. 60/789,851, filed April 5, 2006,
and also claims
the benefit of U.S. Provisional Application No. 60/789,853, filed April 5,
2006, and also
claims the benefit of U.S. Provisional Application No. 60/789,855, filed April
5, 2006 all of
which are incorporated herein by reference.
BACKGROUND
Tissue engineering is an area of intense effort today in the field of
biomedical
sciences. The development of methods of tissue engineering and replacement is
of particular
importance in tissues that are unable to heal or repair themselves, such as
tissues of the knee
meniscus. The meniscus is a load bearing, fibrocartilaginous tissue within the
knee joint that
is responsible for lubrication, stability, and shock absorption. Regions of
the meniscus,
namely those in the avascular zone, are virtually incapable of healing or
repairing themselves
adequately in response to trauma or pathology. Loss of mechanical function of
the meniscus
is associated with development of degeneration and eventual osteoarthritis.
Because the naturally occurring repair mechanisms are insufficient,
researchers have
proposed various in vitro approaches to the production of fibrocartilaginous
tissue. Generally,
most fibrocartilaginous tissue regeneration strategies have been scaffold-
based. However,
there are disadvantages that come with using either natural or synthetic
scaffold materials.
Many synthetic polymers can induce inflammatory responses or create a local
environment
unfavorable to the biologic activity of cells. On the other hand, the major
problem associated
with natural polymer scaffolds is reproducibility. Moreover, these methods
typically involve
seeding cultured fibrochondrocytes into a biological or synthetic scaffold.
The seeded cells
may migrate from the scaffold to the bottom of the culture vessel or well,
even if the plates
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are not treated to promote cell adhesion. Cells plated on non-tissue-treated
plates may still
eventually attach. Within a week of culture, proteins made by the cells or
supplied in the
medium have usually adsorbed onto the bottom of the wells to promote
attachment. This
results in a reduction in the size of the construct. Another drawback is that
the attached cells
tend to flatten and change to a different phenotype. Those cells compete with
the remaining
fibrochondrocytes for nutrients and do not produce the desired extracellular
matrix proteins
for tissue regeneration.
DRAWINGS
A more complete understanding of this disclosure may be acquired by referring
to the
following description taken in combination with the accompanying figures.
Figure 1 shows the gross appearance (rows 1 and 2) and histological sections
(rows 3
and 4) of 6-mm punched disks from constructs cultured at t=4 wks, 8 wks, and
12 wks over
the agarose substratum. Each mark on the ruler is 1 mm. These constructs were
flat and
smooth. Increases in thickness and opacity over the culture period were
observed. Safranin-
0/fast green staining for GAGs (row 3) and collagen type II
immunohistochemistry (row 4)
were observed throughout the constructs at each time point. Chondrocytes
rested in lacunae
throughout the construct.
Figure 2 shows the gross appearance (rows 1 and 2) and histological sections
(rows 3
and 4) of constructs cultured at t=4 wks and 8 wks on TCP. Each mark on the
ruler is 1 mm.
In contrast to the constructs cultured over agarose, these constructs are
contorted with many
folds. Increases in thickness and opacity over the culture period were
observed. Safranin-
0/fast green staining (row 3) and collagen type II immunohistochemistry (row
4) staining
were observed. The constructs contained both dense and diffuse regions.
Figure 3 shows the total ECM per construct in micrograms. Data are shown as
mean f
standard deviation, and significance is defined as p < 0.05. Significant
groups are separated
by different letters. Constructs cultured over agarose contained significantly
more ECM per
construct than constructs cultured on TCP at the same time points. A) Total
GAG per
construct. Significant increases in GAG per construct were observed for both
treatments. B)
Total collagen per construct. Significant increases in collagen per construct
were observed for
both treatments. Due to the absence of immunohistochemistry staining for
collagen type I,
and also due to gel electrophoresis, most of the collagen produced is
considered type II.
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Figure 4 shows the correlation of aggregate modulus (HA) values of native
articular
cartilage and constructs formed over agarose to GAG/dw and to collagen/dw.
Every point
represents HA plotted against ECM/dw for a specific time point as indicated by
arrows. HA
shows a strong positive correlation with collagen/dw (R2 = 1.00) and a strong
negative
correlation with GAG/dw (R2 = 0.99). Since the ECM composed mainly of collagen
and
GAG, the observed increasing collagen to GAG ratio resulted in decreasing
GAG/dw over
time and a negative correlation of GAG to HA.
Figure 5 shows the pressure chamber assembly consisting of a 1.2 L stainless-
steel
vessel (A) connected to a water-driven piston (B) seated on an Instron 8871
(C). Cells were
placed in heat-sealed bags and placed in the stainless-steel vessel (A). The
vessel was then
placed in an adjacent water bath (not shown). The Instron (C) drove the piston
(B) to
pressurize the fluid within.
Figure 6 shows the gross morphology of the self-assembled constructs at t 4
wks
and t = 8 wks. The cells were seeded without a scaffold and without any ECM at
t 0 wks.
By accumulating ECM produced by the cells, the constructs rapidly reached more
than 1 mm
thickness after 4 wks of culture.
Figure 7 shows the Safranin 0 staining for GAG (top) and immunohistochemistry
staining (bottom) for collagen type II of pressurized constructs and of
controls. Both stains
were observed throughout the constructs from both treatments. The constructs
appeared
denser at t = 8 wks than t = 4 wks for both treatments. By t = 8 wks, most of
the cells were
found to reside in lacunae (arrows).
Figure 8 shows the total GAG per construct over the 8-week culture period for
pressurized and static control samples. Data are represented as mean
standard deviation.
Bars that share the same letter are not statistically different from each
other. Bars that are
under different letters represent statistically significant values (p < 0.05,
n = 4). For example,
a statistically significant decrease was observed from 4 wks to 8 wks in
static samples (bars
do not share the same letter), whereas the decreases found for pressurized
samples over time
was not significant (bars share the letter B).
Figure 9 shows the total collagen per construct over the 8-week culture
period.
Significant increases were observed over time for both treatments. Data are
represented as
mean standard deviation. Bars that share the same letter are not
statistically different from
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each other. Bars that are under different letters represent statistically
significant values (p <
0.05, n = 4).
Figure 10 shows the meniscal shaped hydrogel with media and the construct
being
cultured in the bottom of the culture vessel.
Figure 11 shows the meniscal shaped press used to shape the molten hydrogel in
the
culture vessel.
Figure 12 shows the gross morphology of the tissue engineered constructs.
Percentages given refer to the articular chondrocyte content of the culture.
Figure 13 shows a cross-sectional view of the tissue engineered construct
developed
using a culture of 50% articular chondrocytes and 50% meniscal
fibrochondrocytes. Red dye
has been added to the image for ease of visualizing the cross section.
Figure 14 is a graph of the wet weight of the constructs relative to the
percentage of
articular chondrocytes in the culture.
Figure 15 is a graph of the percentage of water in the constructs as compared
to the
percentage of articular chondrocytes in the culture
Figure 16 is a graph of the tensile modulus of the constructs as compared to
the
percentage of articular chondrocytes in the culture.
Figure 17 is a graph of the ultimate tensile strength of the constructs as
compared to
the percentage of articular chondrocytes in the culture.
Figure 18 is a graph of the aggregate modulus of the constructs as compared to
the
percentage of articular chondrocytes in the culture.
Figure 19 is a graph of the cell number per milligram of tissue dry weight of
the
constructs as compared to the percentage of articular chondrocytes in the
culture.
Figure 20 is a graph of percentage of glycosaminoglycans by dry weight of the
constructs as compared to the percentage of articular chondrocytes in the
culture.
Figure 21 is a graph of percentage of collagen by dry weight of the constructs
as
compared to the percentage of articular chondrocytes in the culture.
Figure 22 (A) shows a fabricated cartilage well and a tissue engineered
construct
press-fit into the well. This approach will be used to create an in vitro
model of integration.
Figure 22 (B) shows a 50:50 co-culture made in the shape of the knee meniscus.
Each hash
mark is 0.5 cm.
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Figure 23 shows a negative mold comprised of agarose.
Figure 24 shows a positive mold comprised of agarose. The agarose is saturated
with
culture medium, resulting in the reddish shade.
Figure 25 shows various views of scaffoldless femur constructs made by the
methods
5 of the present disclosure.
Figure 26 shows a comparison of the tissue engineered femur construct to a
femur
shaped piece of plastic. In this case, the construct was formed to resurface
only part of, as
opposed to the entire, femur.
The patent or application file contains at least one drawing executed in
color. Copies
of this patent or patent application publication with color drawing(s) will be
provided by the
Office upon request and payment of the necessary fee.
While the present disclosure is susceptible to various modifications and
alternative
forms, specific example embodiments have been shown in the figures and are
herein
described in more detail. It should be understood, however, that the
description of specific
example embodiments is not intended to limit the invention to the particular
forms disclosed,
but on the contrary, this disclosure is to cover all modifications and
equivalents as illustrated,
in part, by the appended claims.
DESCRIPTION
The present disclosure, according to certain example embodiments, is generally
in the
field of improved methods for tissue engineering. More particularly, the
present disclosure
relates to methods for forming tissue engineered constructs without the use of
scaffolds and
associated methods of use in tissue replacement. As used herein, a "construct"
or "tissue
engineered construct" refers to a three-dimensional mass having length, width,
and thickness,
and which comprises living mammalian tissue produced in vitro.
The methods of this disclosure generally comprise the formation of a tissue
engineered constructs without the use of scaffolds or other synthetic
materials. Generally,
cells are seeded on a shaped hydrogel mold and allowed to self-assemble to
form a construct.
As used herein, "self-assemble" or "self-assembly" refers to a process in
which specific local
interactions and constraints between a set of components cause the components
to
autonomously assemble, without external assistance, into the final desired
structure through
exploration of alternative configurations.
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Among other things, the methods of the present disclosure provide for higher
cell-cell
contact. Chondrocytes are unique in their need to remain in a spherical
morphology to
maintain their phenotype. Since the chondrocytes' only substrate for
attachment is other
chondrocytes in the methods of the present disclosure, this may enhance the
cell to cell
signaling necessary to maintain the chondrocytic phenotype. Another advantage
of the
methods of the present disclosure is that biocompatibility issues of the
scaffold and its
degradation materials are avoided as well as stress-shielding of the seeded
cells by the
scaffold. Cell reaction to the biomaterial, such as dedifferentiation, is also
avoided.
Furthermore, because stress shielding by the scaffold does not occur, the
methods of the
present disclosure may allow for the cells to respond directly to forces which
may aid in
aligning extracellular matrix production. Another advantageous feature of the
present
disclosure is that it allows manipulation of the thickness, geometry, and size
of the resulting
construct.
Formation of Shaped Hydrogel Coated Culture Vessels
The hydrogel used in conjunction with the methods of the present disclosure
may
comprise agarose, alignate, or combinations thereof. A "hydrogel" is a colloid
in which the
particles are in the external or dispersion phase and water is in the internal
or dispersed phase.
Suitable hydrogels are nontoxic to the cells, are non-adhesive, do not induce
chondrocytic
attachment, allow for the diffusion of nutrients, do not degrade significantly
during culture,
and are firm enough to be handled.
In particular embodiments, the hydrogel used in conjunction with the present
disclosure is melted to form a molten hydrogel. The molten hydrogel is
introduced into a
culture vessel and may be shaped using a shaped press. The press may be shaped
to
accommodate the desired shape of the tissue engineered construct. In certain
embodiments,
the press may be in the shape of a ring. In other embodiments, the press may
be a projection
of the medial meniscus rotated through 360 degrees.
The resulting pressed molten hydrogel is allowed to cool around the shape of
the
press. Upon removal of the press, a cooled shaped hydrogel negative mold is
left remaining
in the culture vessel. In certain embodiments, the shape of the resulting
pressed hydrogel is a
projection of the medial meniscus rotated through 360 degrees. In certain
embodiments, a
ring shape of the shaped hydrogel negative mold may aid in the alignment of
the extracellular
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matrix during the formation of the tissue engineered construct by subjecting
the developing
construct to a hoop strain during cell culture.
The Cell Culture
The cells used in conjunction with the methods of the present disclosure may
be
chondrocytes, fibrochondrocytes, or combinations thereof. The chondrocytes may
comprise
articular chondrocytes. Generally, the articular chondrocytes may be from a
bovine or porcine
source. Alternatively if the construct is to be used for in vivo tissue
replacement, the source
of articular chondrocytes may be autologous cartilage from a small biopsy of
the patient's
own tissue, provided that the patient has healthy articular cartilage that may
be used as the
start of in vitro expansion. Another suitable source of chondrocytes is
heterologous
chondrocytes from histocompatible cartilage tissue obtained from a donor or
cell line.
The fibrochondrocytes used in conjunction with the methods of the present
disclosure
may comprise meniscal fibrochondrocytes. Generally, the meniscal
fibrochondrocytes may
be from a bovine or porcine source for in vitro studies. Alternatively if the
construct is to be
used for in vivo tissue replacement, the source of meniscal fibrochondrocytes
may be
autologous fibrocartilage from a small biopsy of the patient's own tissue,
provided that the
patient has healthy meniscal fibrocartilage that may be used as the start of
in vitro expansion.
Another suitable source of fibrochondrocytes is heterologous fibrochondrocytes
from
histocompatible fibrocartilaginous tissue obtained from a donor or cell line.
In certain embodiments, the chondrocytes and fibrochondrocytes used in
conjunction
with the methods of the present disclosure may be derived from mesenchymal or
skin cells.
The fibrochondrocytes, chondrocytes, or a co-culture of the two are suspended
in
media. An example of suitable media may be DMEM with 4.5 g/L-glucose and L-
glutamine(Biowhittaker), 10% fetal bovine serum (Biowhittaker), 1% fungizone
(Biowhittaker), 1% Penicillin/ Streptomycin (Biowhittaker), 1% non-essential
amino acids
(Life Technologies), 0.4 mM proline (ACS Chemicals), 10 mM HEPES (Fisher
Scientific),
50 g/mL L-ascorbic acid, (Acros Organics) supplemented with 20% FBS and 10%
DMSO.
In certain embodiments, the cells may comprise 50% fibrochondrocytes and 50%
chondrocytes. The cells may be seeded in a shaped hydrogel negative mold or a
hydrogel
coated culture vessel and allowed to self-assemble. In certain embodiments,
the cells may be
seeded at a density in the range of about 10 x 106 cells per cm2 to 90 x 106
cells per cmZ of
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hydrogel coated surface. In certain embodiments, the suspension of
fibrochondrocytes and
chondrocytes is seeded at a density of 24 x 106 cells/ cm2 of hydrogel coated
surface. In other
embodiments, the cells may be seeded at a density of about 29 x 106 cells/ cm2
of hydrogel
coated surface.
Self-Assembly of the Seeded Cells
The cells seeded on hydrogel coated culture vessels or hydrogel negative molds
are
allowed to self-assemble. Self-assembly may result in the formation of non-
attached
constructs on the hydrogel surfaces. It is preferable to use hydrogel coated
surfaces instead of
tissue culture treated surfaces since articular chondrocytes seeded onto
standard tissue culture
treated plastic (TCP) readily attach, spread, and dedifferentiate. In certain
embodiments, the
self-assembly process may occur in culture vessels that are shaken
continuously on an orbital
shaker and then pressurized. In certain embodiments, the pressurization of the
cells may
occur in a pressure chamber. Pressurization of the samples during the self-
assembly process
may aid in increased extracellular matrix synthesis and enhanced mechanical
properties. In
certain embodiments, the cells may be pressurized to 10 MPa at 1Hz using a
sinusoidal
waveform function. In other embodiments, the cells are pressurized during
culture of the self-
assembled cells. In particular embodiments, a loading regimen (e.g.
compressive, tensile,
shear forces) may be applied to the cells during self-assembly based on
physiological
conditions of the native tissue in vivo. Loading of the cells during self-
assembly and/or
construct development may cause enhanced gene expression and protein
expression in the
constructs.
In particular embodiments, the cells may be treated with staurosporine, a
protein
kinase C inhibitor and actin disrupting agent, during the self-assembly
process to reduce
synthesis of aSMA, a contractile protein. Reducing aSMA in the constructs via
staurosporine
treatment may reduce construct contraction and may also upregulate ECM
synthesis.
In other embodiments, the cells may be treated with growth factors to increase
construct growth and matrix synthesis. Suitable examples of growth factors
that may be used
with the methods of the present disclosure include, but are not limited to,
TGF-01 and IGF-I.
The dosing of the growth factors may be intermittent or continuous throughout
the period of
the self-assembly process. One of ordinary skill in the art, with the benefit
of this disclosure,
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will be able to determine the appropriate dosing regimen and amount and type
of growth
factor to provide to the developing constructs.
Hydrogel Molds
In certain embodiments, the cells used in conjunction with the methods of the
present
disclosure may be seeded on a hydrogel coated culture vessel and allowed to
self-assemble
for about 1 to about 7 days before being transferred to a shaped hydrogel
negative mold.
Alternatively, rather than seeding the cells on a hydrogel coated culture
vessel, in certain
embodiments, the cells may be seeded directly onto a shaped hydrogel negative
mold. The
shaped hydrogel negative mold may comprise agarose. Other non-adhesive
hydrogels, e.g.
alignate, may be used in conjunction with the methods of the present
disclosure. In other
embodiments, the hydrogel mold may be a two piece structure comprising, a
shaped hydrogel
negative mold (See for example, Figure 23) and a shaped hydrogel positive mold
(See for
example, Figure 24). The shaped hydrogel negative and positive molds may
comprise the
same non-adhesive hydrogel or may be a comprised of different non-adhesive
hydrogels. In
certain embodiments, the cells may be seeded on a hydrogel coated culture
vessel and
allowed to self-assemble into a first construct. The first construct may be
transferred to a
shaped hydrogel negative mold. A shaped hydrogel positive mold may be applied
to the
negative mold to form a mold-construct assembly. The mold-construct assembly
may then
further be cultured to form a second construct. As used herein, the term "mold-
construct
assembly" refers to a system comprising a construct or cells within a shaped
positive and a
shaped negative hydrogel mold.
In certain embodiments, the molds may be shaped from a 3-D scanning of a total
joint
to result in a mold fashioned in the shape of said joint. In other
embodiments, the molds may
be shaped from a 3-D scanning of the ear, nose, or other non-articular
cartilage to form molds
in the shapes of these cartilages. In certain embodiments, the mold may be
shaped to be the
same size as the final cartilaginous product. In other embodiments, the molds
may be shaped
to be smaller than the final cartilaginous product. In certain embodiments,
the molds may be
fashioned to a portion of a joint or cartilage so that it serves as a
replacement for only a
portion of said joint or cartilage (See Figures 25 and 26).
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Analysis of the Constructs
The properties of the constructs may be tested using any number of criteria
including,
but not limited to, morphological, biochemical, and biomechanical properties,
which also
may be compared to native tissue levels. In this context, morphological
examination includes
5 histology using safranin-O and fast green staining for glycosaminoglycan
(GAG ) content, as
well as picro-sirius red staining for total collagen, immunohistochemistry for
collagens I and
II, and confocal and scanning electron microscopies for assessing cell-matrix
interactions.
Biochemical assessments includes picogreen for quantifying DNA content, DMMB
for
quantifying GAG content, hydroxyproline assay for quantifying total collagen
content, and
10 ELISA for quantifying amounts of specific collagens (I and II).
Constructs also may be evaluated using one or more of incremental tensile
stress
relaxation incremental compressive stress relaxation, and biphasic creep
indentation testing to
obtain moduli, strengths, and viscoelastic properties of the constructs.
Incremental
compressive testing under stress relaxation conditions may be used to measure
a construct's
compressive strength and stiffness. Incremental tensile stress relaxation
testing may be used
to measure a construct's tensile strength and stiffness. Additionally,
indentation testing under
creep conditions may be used to measure a construct's modulus, Poisson's
ratio, and
permeability.
Without wishing to be bound by theory or mechanism, although both collagen II
and
GAGs are excellent predictors of biomechanical indices of cartilage
regeneration, typically
only collagen II exhibits a positive correlation. Though seemingly this
hypothesis is
counterintuitive for compressive properties, as GAG content is usually thought
to correlate
positively with compressive stiffness, our results show that in self-assembled
constructs,
GAG is negatively correlated with the aggregate modulus (R2=0.99), while
collagen II is
positively correlated (RZ=1.00).
The constructs of the present disclosure may be assessed morphologically
and/or
quantitatively. Quantitatively, the constructs of the present disclosure may
be evaluated using
a functionality index (FI) as described in Eq. 1. The functionality index is
an equally
weighted analysis of ECM production and biomechanical properties that includes
quantitative
results corresponding to the constructs' salient compositional characteristics
(i.e., amounts of
I
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collagen II and GAG) and biomechanical properties (compressive and tensile
moduli and
strengths).
FI=1 1 + 1
4 GQt Ca, 2 2 Ec,.,
2 ffl", 2 at
Eq. (1)
In this equation, G represents the GAG content per wet weight, C represents
the
collagen II content per wet weight, ET represents the tensile stiffness
modulus, Ec represents
the compressive stiffness modulus, ST represents the tensile strength, and Sc
represents the
compressive strength. Each term is weighted to give equal contribution to
collagen, GAG,
tension, and compression properties. The subscripts nat and sac are used to
denote native and
self-assembled construct values, respectively. The aggregate modulus is not
used in Eq. 1, as
it is expected to mirror the compressive modulus obtained from incremental
compressive
stress relaxation. Similarly, the amount of collagen I is not be used in Eq.
1, as this type of
collagen may not appear in a measurable fashion; however, if the amount of
collagen I is
non-negligible, FI may be altered accordingly to account for it.
Each term grouped in parentheses in Eq. 1 calculates how close each construct
property is with respect to native values, such that scores approaching 1
denote values close
to native tissue properties. Equal weight is given to GAG, collagen II,
stiffness (equally
weighted between compression and tension), and strength (also equally weighted
between
compression and tension). This index, FI, will be used to assess the quality
of the construct
compared to native tissue values, with a lower limit of 0 and an unbounded
upper limit, with
a value of 1 being a construct possessing properties of native tissue.
However, the FI can
exceed 1 if optimization results in constructs of properties superior to
native tissue.
Methods of Using the Tissue Engineered Constructs
A hydrogel coated culture vessel or shaped hydrogel negative mold is seeded
with
cells to produce new tissue, such as tissue of the knee meniscus, tendons, and
ligaments. The
hydrogel coated culture vessel or shaped hydrogel negative mold is typically
seeded with
cells; the cells are allowed to self-assemble to form a tissue engineered
construct. In certain
embodiments, applications of the tissue engineered construct include the
replacement of
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tissues, such as the knee meniscus, joint linings, the temporomandibular joint
disc, tendons,
or ligaments.
The constructs may be treated with collagenase, chondroitinase ABC, and BAPN
to
II'~ aid in the integration of the constructs with native, healthy cartilage
surrounding the desired
location of implantation. The integration capacity of a construct with native
tissue is crucial
to regeneration. A wound is naturally anti-adhesive, but debridement with
chondroitinase
ABC and/or collagenase removes anti-adhesive GAGs and enhances cell migration
by
removing dense collagen at the wound edge. BAPN, a lysyl oxidase inhibitor,
may cause the
accumulations of matrix crosslinkers and may, thus, strengthen the interface
between the
construct and native tissue at the desired location of implantation.
The tissue engineered constructs may be implanted into a subject and used to
treat a
subject in need of tissue replacement. In certain embodiments, the constructs
may be grown
in graded sizes (e.g. small, medium, and large) so as to provide a resource
for off-the-shelf
tissue replacement. In certain embodiments, the constructs may be formed to be
of custom
shape and thickness. In other embodiments, the constructs may be devitalized
prior to
implantation into a subject.
To facilitate a better understanding of the present disclosure, the following
examples
of specific embodiments are given. In no way should the following examples be
read to limit
or define the entire scope of the disclosure.
EXAMPLES
Example 1: Isolation and Seeding of Chondrocytes and Fibrochondroc es
Chondrocytes were isolated from the distal femur of week-old male calves
(Research
87 Inc.) less than 36 hrs after slaughter, with collagenase type I
(Worthington) in culture
medium. The medium was DMEM with 4.5 g/L-glucose and L-
glutamine(Biowhittaker),
10% fetal bovine serum (Biowhittaker), 1% fungizone (Biowhittaker), 1%
Penicillin/
Streptomycin (Biowhittaker), 1% non-essential amino acids (Life Technologies),
0.4 mM
proline (ACS Chemicals), 10 mM HEPES (Fisher Scientific), and 50 g/mL L-
ascorbic acid
(Acros Organics). Chondrocytes were frozen in culture medium supplemented with
20% FBS
and 10% DMSO at -80 C for 2 wks to a month before cells from two donor legs
were pooled
together. Cells from each leg were counted on a hemocytometer, and viability
was assessed
I
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using a trypan blue exclusion test. Each leg yielded roughly 150 million
cells, and viability
was greater than 99% for both legs. After thawing, viability remained greater
than 92%.
Fibrochondrocytes were harvested from the medial meniscus of approximately 1-
wk
old male calves (Research 87, Boston, MA) less than 36 hrs after slaughter,
with collagenase
in the culture medium. The medium was DMEM with 4.5 g/L-glucose and L-
glutamine, 10%
FBS, 1% fungizone, 1% Penicillin/Streptomycin, 1% non-essential amino acids,
0.4 mM
proline, 10 mM HEPES, and 50 g/mL L-ascorbic acid. Cells were frozen at -80 C
in culture
medium supplemented with 20% FBS and 10% DMSO for 2 to 4 wks before cells from
donor
legs can be pooled together.
Example 2: Formation of the Hydrogel Molds
A silicon positive die consisting of 5 mm diameter x 10 mm long cylindrical
prongs
has been constructed to fit into a 6-well plate. To construct the agarose
mold, sterile, molten
2% agarose will be introduced into a well fitted with the silicon positive
die. The agarose will
be allowed to gel at room temperature for 15 min. The agarose mold will then
be separated
from the silicon positive die and submerged into two exchanges of culture
medium. The
agarose mold will thus be completely saturated with the culture medium by the
time of cell
seeding. To each agarose well, 5.5 x 106 cells will be added in 50 l of
culture medium. The
cells will self-assemble within 24 hrs in the agarose wells and will be
maintained in the same
wells for 3 days. These self-assembled constructs will then be placed into
larger agarose
wells with 3 mL of medium, exchanged once every 3 days. Constructs will be
cultured for the
specified amount of time; t=0 will be defined as 24 hrs after seeding.
Example 3-9: Analysis of Constructs Formed on Hydrogel Coated Surfaces and
Tissue
Culture Treated Surfaces
Example 3: Self Assembly and Culture of the Tissue engineered Constructs
Each well of a 96 well plate was coated with 100 l of 2% molecular biology
grade
agarose (Sigma). The plates were tilted to spread the agarose along the walls,
and then
inverted to shake out the excess agarose. To each well, 5.5 million
chondrocytes in 300 l of
culture medium were introduced. Within 24 hrs, the cells formed non-attached
constructs at
the bottom of each well, and these constructs were maintained in the 96 well
plates for 4 wks
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before being transferred to agarose coated 46 well plates. Each day 500 l
medium was
changed (250 l twice daily). Time zero (t=0) was defined as 24 hrs after
seeding.
Self assembly on tissue culture treated plates without hydrogel coating was
also
assessed. To each well of a 96-well TCP plate, 5.5 million cells in 300 l of
culture medium
were introduced. Within 24 hrs, the cells formed attached constructs at the
bottom of each
well, and these constructs were maintained in the 96 well plates for 4 wks,
and were then
transferred to tissue culture treated 46-well plates. Each day 500 l medium
was changed
(250 l twice daily). Time zero (t=0) was defined as 24 hrs after seeding.
After seeding chondrocytes on either TCP or over an agarose substratum, the
chondrocytes formed cohesive constructs within 24 hrs (defined as t=0 wks). At
t=0 wks, the
constructs could be manipulated in the medium but were not testable
mechanically. Thus,
histological, biochemical, and biomechanical data were collected at t=4 wks
and 8 wks. Since
constructs cultured over agarose consistently outperformed constructs cultured
on TCP in
terms of biochemistry and biomechanics (significance defined as p < 0.05),
they merited an
extended culture period to t=12 wks.
Constructs from both treatments increased in opacity over time. After 24 hrs,
cells on
agarose formed one cohesive nodule that was not attached to the substratum.
Other than the
single nodule, the agarose surface did not have any other attached cells or
nodules. In
contrast, the control cells readily attached to the bottom of the TCP wells
and formed nodules
that adhered to TCP and detached from the constructs as time progressed.
Constructs cultured
over agarose appeared smooth, flat, and hyaline-like in appearance. Disks 6-mm
in diameter
were punched out of the center of the constructs cultured over agarose for
mechanical testing,
and these are shown in Figure 1, rows 1 and 2. Unlike the constructs formed
over agarose, the
constructs cultured on TCP became contorted with folds (Figure 2).
The constructs cultured over agarose assumed a bowl shape and increased in
diameter
from an initial 5 mm to more than 1 cm at t=12 wks. The thickness of
constructs also
increased significantly over time, from 460 78 m at t=4 wks, to 770 75 m at
t=8 wks, and
to 950 80 m at t=12 wks. The constructs were not of uniform thickness
throughout, and the
thickness of the thinnest portion (the 6-mm punched out disks) is reported,
since this was the
mechanically tested region. The constructs cultured on TCP also significantly
increased in
thickness over time, from 344 39 m at t=4 wks to 663 34 m at t=8 wks. The
constructs
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formed over agarose were significantly thicker than those formed over TCP for
these time
points.
The constructs cultured over agarose displayed many similarities to native
tissue.
Even within the loose shell (Figure 1), the chondrocytes were in lacunae, as
compared to the
5 TCP constructs, where the loose shell contained no lacunae and fewer cells.
In addition, cells
appeared to rest in lacunae that were mostly elongated in the z-direction
(thickness) for
constructs formed over agarose at t=4 wks. Where more than one cell rested in
the same
lacuna, these cells were also stacked in the z-direction. The curl of the bowl-
shaped
constructs suggests a pre-tensed state. At t=4 wks, many lacunae were aligned
in the z-
10 direction, which may indicate an organization (Figure 1) yet to be reported
by studies using
scaffolds. The constructs formed over agarose showed increases in staining
intensity and
coverage for safranin-O from t=4 wks to t=8 wks. Over this time the constructs
also matured
as to be devoid of the loosely organized shell. On TCP, however, the cells did
not appear to
organize in any particular direction, and staining intensity did not increase
over time.
15 Example 4: Histology and Immunohistochemistry of the Tissue engineered
Constructs
Samples were frozen and sectioned at 14 gm. Safranin-O and fast green staining
were
used to examine GAG distribution. Slides were also processed with IRC to test
for the
presence of collagen type I (COLI) and collagen type II (COL2) on a Biogenex
i6000
autostainer. After fixing in chilled acetone, the slides were rinsed with IRC
buffer
(Biogenex), quenched of peroxidase activity with hydrogen peroxide/methanol,
and blocked
with horse serum (Vectastain ABC kit). The slides were then incubated with
either mouse
anti-COLI (Accurate Chemicals) or mouse anti-COL2 (Chondrex) antibodies. The
secondary
antibody (mouse IgG, Vectastain ABC kit) was then applied, and color was
developed using
the Vectastain ABC reagent and DAB (Vector Laboratories ).
To assess DNA content, GAG content, and collagen content, samples were
digested
i
with 125 gg/mL papain (Sigrna) n 50 mM phosphate buffer ~H= 6.5) containing 2
mM N-
acetyl cysteine (Sigma) and 2 mM EDT A (Sigma) at 65 C overnight. Total DNA
content
was measured by Picogreen Cell Proliferation Assay Kit (Molecular Probes).
Total sulfated
GAG was then quantified using the Blyscan Glycosaminoglycan Assay kit
(Biocolor), based
on 1,9-dimethylmethylene blue binding. After being hydrolyzed by 2 N NaOH for
20 min at
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110 C, samples were assayed for total collagen content by a chloramine- T
hydroxyproline
assay.
For constructs cultured over agarose, at t=4 wks the constructs displayed two
distinct
regions (Figure 1, rows 3 and 4). A porous, diffuse outer shell with low
safranin-0 and low
collagen type II staining, indicating low amounts of GAG and collagen in this
area. In
contrast to the positive collagen type II staining, collagen type I staining
was not observed at
any time. A protein gel (data not shown) further confirmed the presence of
collagen type II
alpha 1 chains and the absence of collagen type I alpha 1 and alpha 2 chains,
indicating
maintenance of the chondrocytic phenotype. As a control, sections stained with
safranin-
0/fast green for GAG and with Immunohistochemistry for collagen type II on
constructs
cultured over TCP were used. As with t=4 wks constructs cultured over agarose,
at t=8 wks, a
loosely organized shell was seen around the constructs cultured on TCP. Matrix
from this
shell easily peeled off as sheets. This was in stark contrast to the cohesive
constructs formed
over agarose.
Example 5: Quantitative Biochemistry of the Constructs
Samples (self assembled without pressurization) were digested with 125 g/mL
papain (Sigma) in 50 mM phosphate buffer (pH = 6.5) containing 2 mM N-acetyl
cysteine
(Sigma) and 2 mM EDTA (Sigma) at 65 C overnight. Total DNA content was
measured by
Picogreen Cell Proliferation Assay Kit (Molecular Probes). Total sulfated GAG
was then
quantified using the Blyscan Glycosaminoglycan Assay kit (Biocolor), based on
1,9-
dimethylmethylene blue binding. After being hydrolyzed by 2 N NaOH for 20 min
at 110 C,
samples were assayed for total collagen content by a chloramine-T
hydroxyproline assay.
Constructs cultured over agarose gained mass over the culture period, and, at
each
time point, these constructs contained significantly larger mass than
constructs cultured on
TCP. Wet weights (ww) for constructs cultured over agarose were 39.1 4.3 mg at
t=4 wks,
53.1 4.2 mg at t=8 wks, and 99.0 5.7 mg at t=12 wks. The ww of TCP constructs
increased
from 28.1 3.1 mg at t=4 wks to 39.1 4.3 mg at t=8 wks. For the constructs
formed over
agarose, the total cell number did not show significant changes during the
culture period and
ranged from 5.8 1.2 to 7.1 1.2 million per construct. The number of cells in
these constructs
was more, though not significantly, than the constructs cultured over TCP,
which showed an
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increase in cell number from 4.5 1.2 to 6.3 1.4 million cells per construct
over the culture
period.
The constructs formed over agarose contained significantly higher GAG and
collagen
per sample at each time point when compared to control (Figure 3). For
constructs cultured
over agarose, the total GAG per sample increased significantly from t=4 wks at
640 100 gg
to 1700 210 g at t=12 wks (Figure 3A). Total GAG per construct also increased
significantly for constructs cultured on TCP, from 480 40 g at t=4 wks to 650
60 g at t=8
wks (Figure 3A). The total collagen per construct cultured over agarose
significantly
increased from 280 40 g at t=4 wks to 1840 170 g at t=12 wks (Figure 3B).
For constructs
cultured on TCP, total collagen per construct increased significantly from 93
16 g at t=4
wks to 480 50 g at t=8 wks (Figure 3B).
To compare the ECM produced in the constructs to bovine articular cartilage
(BAC),
the biochemical data were normalized to dry weight (dw). Both treatments
produced
significantly more GAG/dw at all time points compared to BAC. GAG/dw of
agarose
constructs displayed a decreasing trend with time, from 0.29 0.05 (g GAG/g
construct) at t=4
wks, to 0.26 0.03 at t=8 wks, to 0.23 0.03 at t=12 wks. Collagen/dw increased
from
0.13 0.04 at t=4wks, to 0.21 0.02 at t=8 wks, to 0.23 0.03 at t=12 wks. At
t=12 wks,
GAG/dw of construct formed over agarose was 2/3 higher than BAC, while
collagen/dw
reached more than 1/3 the level of BAC (Figure 4). Collagen/dw provided a
strong positive
correlation (R2=1.00) to Hn values (Figure 4), and may serve as an excellent
predictor of
construct stiffness.
Example 6: Mechanical Analysis of the Constructs
For mechanical analysis, samples were evaluated with an automated indentation
apparatus. Each specimen was attached to the sample holder by use of
cyanoacrylate glue,
and was submerged in saline solution. The specimen was positioned under the
load shaft of
the apparatus so that the sample surface test point was perpendicular to the
indenter tip. The
specimen was automatically loaded with a tare mass of 0.4 g (0.004 N), using a
1.67 mm-
diameter rigid, flat-ended, porous indenter tip. Samples were allowed to reach
tare creep
equilibrium, which was defined as deformation<10-6 mm/s or a maximum creep
time of 10
min. When tare equilibrium was reached, a step mass of 2.34 g (0.023 N) was
applied.
Displacement of the sample surface was measured until equilibrium was reached
or a
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maximum creep time of 1.5 hrs elapsed. At that time, the step load was
removed, and the
displacement was recorded until equilibrium was again reached. Preliminary
estimations of
the Young's modulus of the samples were obtained using the analytical solution
for the
axisymmetric Boussinesq problem with Papkovich potential functions. The
intrinsic
mechanical properties of the samples were then determined using the linear
biphasic theory.
Calf tissue from the tibial plateau was tested to yield an Ha of 139 141 kPa
(n=5).
Constructs from both groups were not mechanically testable at t=0 wks.
Starting from
t=4 wks, constructs from both treatments were tested biomechanically under
conditions of
creep indentation. Constructs cultured over agarose consistently outperformed
constructs
cultured over TCP.
For constructs cultured over agarose, Boussinesq-Papkovich estimates of the
Young's
modulus ranged from 70-75 kPa at t=4 wks, 65-101 kPa at t=8 wks and 78-121 kPa
at t=12
wks. Boussinesq-Papkovich estimates of the Young's modulus for constructs
cultured over
TCP ranged from 39-61 kPa at t=4 wks, and did not significantly increase by
t=8 wks. Using
the biphasic theory, the aggregate modulus (HA) of the t=4 wks constructs
formed over
agarose was 19 3 kPa, and this significantly increased to 43 13 kPa at t=8
wks. "Aggregate
modulus" is a conventional measurement used in characterizing cartilage.
Ultimately, the
samples reached an HA of 53 9 kPa after 12 wks (See Table 1 below). Control
constructs
were significantly softer at each time point, ranging from 13f4 kPa at t=4 wks
to 19 3 kPa at
t=8 wks (See Table 1 below). The permeability and Poisson's ratio values were
not
significantly different across the two treatments. At t=8 wks, constructs
cultured on TCP
reached 14% of the stiffness of calf articular cartilage, whereas constructs
on agarose reached
31%. By t=12 wks, constructs cultured over agarose increased their stiffness
to almost 40%
of the stiffness of native tissue. By t=8 wks, the permeability values of
constructs cultured on
TCP and over agarose were not significantly different from native tissue. The
Poisson's ratios
of constructs from both groups were initially greater than native tissue,
though these values
decreased over time to approach native tissue. The results of mechanical
analysis can be seen
in Table 1 below.
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TABLE 1: Results of Mechanical Analysis
H~ (kP~) k (10""Ma~~~) v
Week 4, ovez ap~;ar~se ~ 9 3 17-~ 6 0.23 0.08
Week 8a over agztrose 43 13 40 21 0.11 0.08
Week lZ, Ã~~~~ a(yar~~~ ~~~ 9 22~24 0.03 0.0 5
Week 4, on. TC"P I3 4 24 10 0.22=0.11
Week 8, on TCP 19 3 33 21 0.07 0.09
Native ari;icularcartila~~~ 139 41 42 28 0.01 0,01
The strong correlations of HA to ECM/dw are linear and, as shown in Figure 4,
are in
a linear relationship to native tissue values. This is an exciting finding as
it suggests that the
tissue produced in this study develops in a manner analogous to native
articular cartilage.
Extended culture periods, bioactive agents, or mechanical stimuli may aid this
tissue to
further progress down this pathway towards native tissue-like functionality.
The results of the above examples comparing hydrogel coated and TCP surfaces
show
that, indeed, chondrocytes attached and flattened onto the TCP. In addition,
constructs
formed over agarose were smooth in appearance, thicker, contained more ECM,
and were
stiffer than those formed on TCP. When seeded on TCP, cells formed numerous
distinct
nodules that did not contribute in forming one uniform cohesive construct. In
contrast, cells
on agarose did not spread, but rather self-assembled immediately into one
large nodule that
increased in diameter and thickness over time. The self-assembled cartilage
construct formed
over agarose contained spherical cells with a chondrocytic phenotype. This
tissue engineered
product also contained 2/3 more GAG/dw than native tissue. Collagen/dw reached
1/3 the
level of native tissue, and the stiffness reached more than 1/3 that of native
tissue. Based on
these observations, it is suggested that the scaffold-free self-assembling
process over an
agarose substratum may provide a feasible culture methodology to produce
functionally
relevant tissue analogues. Further experimentation involving pressurization of
the samples
during self assembly were then performed on hydrogel coated surfaces.
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Example 7: Self Assembly with Pressurization
Agarose molds were constructed out of agarose with 3mm diameter wells. To each
well, 5.1 million chondrocytes were seeded and allowed to self-assemble. After
self-
assembling for 24 hr, defined as t= 0 wk, the constructs were transferred to
agarose coated
5 100 mm diameter petri dishes. An equivalent of 3 mL of medium was exchanged
per
construct every 2 days. The petri dishes were shaken continuously on an
orbital shaker at 60
rpm beginning at t = 0. At t = 2 wk of culture, constructs were divided into
pressure and
control groups.
Both control and pressure group constructs were loaded into heat sealable bags
10 (Kapak) previously sterilized by ethylene oxide. To each bag, medium was
added, and the
bags were tapped gently to release any residual bubbles adhering to the bottom
of the bag.
The bags were heat-sealed without any bubbles inside.
Control specimens were placed into an opened pressure chamber, while pressure
specimens were placed into a pressure chamber (Parr Instrument Company),
filled with
15 water, and sealed underwater without any bubbles inside. The pressure
chamber is a 1.2 L
stainless-steel vessel capable of withstanding pressures upwards of 13 MPa
(Figure 5, A). It
is connected to a water-driven piston (PHD Inc.) (Figure 5, B) via a stainless-
steel %" hose
(Dunlop) rated for pressures up to 40 MPa. The piston is connected to an
Instron 8871
(Figure 5, C), controlled using the Instron WaveMaker software. For 5
consecutive days a
20 week, the specimens were pressurized to 10 MPa at 1 Hz using a sinusoidal
waveform for 4
hrs. After the execution of the desired regimen, the pressure chamber was
disassembled, and
the pouches were sterilized with 70% ethanol. In a sterile culture hood, the
pouches were
opened with autoclaved instruments and the samples were then returned to
orbitally shaken
culture dishes.
The pressure set-up assembled in this study applied intermittent hydrostatic
pressure
at 10 MPa, 1 Hz, 4 hrs a day consistently over an 8-week period. Articular
chondrocyte
constructs subjected to this loading regimen were shown to withstand the
repeated
mechanical stimulus.
At t = 4 wks and t = 8 wks, samples from both pressurized and controls were
frozen
and sectioned at 14 m. Safranin-O and fast green staining were used to
examine GAG
distribution. Slides were also processed by immunohistochemistry to test for
the presence of
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collagen type II (COL2) on a Biogenex i6000 autostainer. After fixing in 4 C
acetone, the
slides were rinsed with immunohistochemistry buffer (Biogenex), quenched of
peroxidase
activity with hydrogen peroxide/methanol, and blocked with equine serum
(Vectastain ABC
kit). The slides were then incubated with either mouse anti-collagen type I
antibody
(Accurate Chemicals) at 1:1500 dilution in PBS or mouse anti-collagen type II
antibody
(Chondrex) at 1:1000 dilution on PBS. The secondary antibody (antimouse IgG,
Vectastain
ABC kit) was then applied, and color was developed using the Vectastain ABC
reagent and
DAB (Vector Laboratories). Slides stained with mouse IgG 1/2a12b (Accurate
Chemicals)
served as negative controls.
The gross appearance of the 3-D culture is shown in Figure 6. After 4 wks of
culture,
the pressurized samples reached thicknesses of 2.01 0.04 mm. Likewise, the
controls
reached thicknesses of 1.98 0.51 mm. The thicknesses of the constructs were
maintained
for the remainder of the culture period, and did not differ significantly
between treatments.
By t = 8 wks, both pressurized and control constructs stained positive for
collagen type II
throughout the thickness of the construct. Safranin 0 staining for GAG was
also observed
throughout the constructs (Figure 7). At this time, the cells were round and
rested in lacunae
(Figure 7, arrows).
Example 8: Quantitative Biochemistry of the Constructs after Pressurization
Samples (self assembled with pressurization) were digested with 125 g/mL
papain
(Sigma) in 50 mM phosphate buffer (pH = 6.5) containing 2 mM N-acetyl cysteine
(Sigma)
and 2 mM EDTA (Sigma) at 65 C overnight. Total DNA content was measured by
Picogreen Cell Proliferation Assay Kit (Molecular Probes). Total sulfated GAG
was then
quantified using the Blyscan Glycosaminoglycan Assay kit (Biocolor), based on
1,9-
dimethylmethylene blue binding. After being hydrolyzed by 2 N NaOH for 20 min
at 110 C,
samples were assayed for total collagen content by a chloramine-T
hydroxyproline assay.
By 4 wks, pressurized constructs reached a wet weight (WW) of 87.5 7.5 mg,
and
the wet weight remained steady, reaching 92.7 9.0 mg at 8 wks. The same WW
range was
observed with control samples. Control sample WW was 92.3 5.6 mg at 4 wks
and 83.9
11.7 mg at 8 wks. This decrease was not statistically significant. Total GAG
per construct
significantly decreased in the control samples, while the pressurized samples
showed an
insignificant decrease (Figure 8). For the pressurized samples, GAG content
decreased from
I
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1590 230 g at t = 4 wks to 1200 140 g at t = 8 wks (Figure 8), though
this decrease
was not significant. GAG per construct for the control decreased significantly
from 1600 80
g at t = 4 wks to 840 220 g at t = 8 wks (Figure 8). Total collagen content
increased
significantly for pressurized samples only from 430 130 g at t = 4 wks to
770 100 g at t
= 8 wks (Figure 9). Collagen per construct for the control also increased from
430 130 g
at t 4 wks to 660 150 g at t = 8 wks (Figure 9), though this increase was
not significant.
For pressurized samples, GAG/DW decreased from 31 % 5% at t= 4 to 28% 2%
at t 8 wks, though this decrease was not significant. Collagen/DW increased
significantly
from 8% 1% at t = 4 wks to 17% ~ 4% at t = 8 wks. GAG/DW observed in
controls was
47% 19% at t = 4 wks and 22% ~ 6% at t = 8 wks. This significant decrease
was not
observed in the pressurized samples. Collagen/DW of constructs observed in
controls was
14% 6% at t = 4 wks and 17% 4% at t= 8 wks, and this increase was not
significant.
In the pressurized samples, the total number of cells per construct was found
to
increase from 3.6 0.8 million at t 4 wks to 4.5 1.3 million at t = 8 wks.
Total cell
numbers in controls ranged from 4.1 ~ 2.7 million at t = 4 wks to 4.2 1.2
million at t = 8
wks in controls. The number of cells at t = 4 wks were significantly fewer
than the 5.1 0.1
million seeded, as cell loss was observed in orbital culture.
Example 9: Mechanical Analysis of the Constructs followim Pressurization
For mechanical analysis, samples were evaluated with an automated indentation
apparatus. Each specimen was attached to the sample holder by use of
cyanoacrylate glue,
and was submerged in saline solution. The specimen was positioned under the
load shaft of
the apparatus so that the sample surface test point was perpendicular to the
indenter tip. The
specimen was automatically loaded with a tare mass of 0.4 g (0.004 N), using a
1.67 mm-
diameter rigid, flat-ended, porous indenter tip. Samples were allowed to reach
tare creep
equilibrium, which was defined as deformation<10-6 mm/s or a maximum creep
time of 10
min. When tare equilibrium was reached, a step mass of 2.34 g (0.023 N) was
applied.
Displacement of the sample surface was measured until equilibrium was reached
or a
maximum creep time of 1.5 hrs elapsed. At that time, the step load was
removed, and the
displacement was recorded until equilibrium was again reached. Preliminary
estimations of
the Young's modulus of the samples were obtained using the analytical solution
for the
axisymmetric Boussinesq problem with Papkovich potential functions. The
intrinsic
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mechanical properties of the samples were then determined using the linear
biphasic theory.
Calf tissue from the tibial plateau was tested to yield an Ha. of 139 41 kPa
(n=5).
The aggregate modulus of pressurized samples reached 20 5 kPa at t = 4 wks
and
maintained this level to the end of the culture period. The stiffness of the
controls was not
significantly different, reaching 22 7 kPa at t = 4 wks. As with pressurized
samples, the
stiffness of the controls also remained constant to t = 8 wks. Permeability of
the samples at t
= 4 wks ranged from 10 2(10-15) m4/Ns in pressurized samples to 13 6(10-
15)m4/Ns in
controls. The permeability of the samples also remained constant throughout
the culture
period. The Poisson's ratio values of constructs ranged from 0.006 to 0.015
across treatments
and were not significantly different over culture time.
The previous examples involving pressurization show, for the first time, that
long-
term culture of tissue engineered articular cartilage construct benefits from
intermittent
hydrostatic pressure and positively affects ECM synthesis in the chondrocyte
constructs..
Although the specific loading regimen applied the aforementioned examples did
not result in
improved mechanical properties over the control, such differences may manifest
themselves
over time.
Example 10-11: Analysis of Tissue engineered Constructs formed on Pressed
Hydrogel
Coated Surfaces
Example 10: Formation and Analysis of the Shaped Constructs
Cell suspensions were seeded on the cooled, pressed hydrogel coated surfaces.
See
Figure 10, Figure 11, and Figure 12. 100% articular chondrocytes and 100%
meniscal
fibrochondrocytes were seeded on the hydrogel coated surfaces. Co-cultures of
the two were
also seeded comprising: 75% articular chondrocytes and 25% meniscal
fibrochondrocytes,
50% articular chondrocytes and 50% meniscal fibrochondrocytes, and 25%
articular
chondrocytes and 75% meniscal fibrochondrocytes. Figure 13 is an image of the
developing
shaped construct. See also Figure 22.
Quantitative biochemical analysis indicates that glycosaminoglycans and
collagen
type II were present in all of the constructs. (Figure 20and Figure 21). In
addition, wet weight
and dry weight compositions of the constructs were analyzed. See Figure 14,
Figure 15, and
Figure 19.
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Example 11: Mechanical Analysis of the Constructs
Mechanical testing of the representative constructs formed from different co-
culture
compositions was performed. All constructs formed from co-cultures had a lower
aggregate
modulus than either the construct formed from 100% articular chondrocytes or
the construct
formed from 100% meniscal fibrochondrocytes (See Figure 18).
The tensile modulus of the developing constructs were analyzed using known
techniques. The tensile modulus appears to increase with increasing
fibrochondrocyte
composition (See Figure 17).
The ultimate tensile strength of the developing constructs were analyzed using
know
techniques. The ultimate tensile strength of the constructs also appears to
increase with
increasing fibrochondrocyte composition. (See Figure 16).
Notwithstanding that the numerical ranges and parameters setting forth the
broad
scope of the invention are approximations, the numerical values set forth in
the specific
examples are reported as precisely as possible. Any numerical value, however,
inherently
contain certain errors necessarily resulting from the standard deviation found
in their
respective testing measurements.
Therefore, the present invention is well adapted to attain the ends and
advantages
mentioned as well as those that are inherent therein. While numerous changes
may be made
by those skilled in the art, such changes are encompassed within the spirit of
this invention as
illustrated, in part, by the appended claims.
