Note: Descriptions are shown in the official language in which they were submitted.
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Enhanced Submucosal Tissue Graft Constructs
Field of the Invention
The present invention relates to intestinal tissue derived tissue grafts
and their use in repairing damaged or diseased tissues. More particularly,
this
invention is directed to intestinal submucosal tissue grafts that have been
seeded with
a preselected population of cells to enhance the repair capabilities of the
tissue graft
construct.
Background and Summary of the Invention
The present invention is directed to vertebrate submucosa-derived
collagenous matrices in combination with preselected cell population as tissue
graft
construct for the use in the repair of damaged or diseased tissues. The
collagenous
matrices for use in accordance with the present invention comprise highly
conserved
collagens, glycoproteins, proteoglycans, and glycosaminoglycans in their
natural
configuration and natural concentration. The extracellular collagenous matrix
for use
in this invention is derived from submucosal tissue of a warm-blooded
vertebrate.
In accordance with the present invention the submucosa is isolated
from warm-blooded vertebrate tissues including the alimentary, respiratory,
intestinal,
urinary or genital tracts of warm-blooded vertebrates. The preparation of
intestinal
submucosa is described and claimed in U.S. Patent No. 4,902,508, the
disclosure of
which is expressly incorporated herein by reference. Urinary bladder submucosa
and
its preparation is described in U.S. Patent No. 5,554,389, the disclosure of
which is
expressly incorporated herein by reference. Stomach submucosa has also been
obtained and characterized using similar tissue processing techniques. Such is
described in U.S. patent application No. 60/032,683 titled STOMACH SUBMUCOSA
DERIVED TISSUE GRAFT, filed on December 10, 1996. Briefly, stomach
submucosa is prepared from a segment of stomach in a procedure similar to the
preparation of intestinal submucosa. A segment of stomach tissue is first
subjected to
abrasion using a longitudinal wiping motion to remove the outer layers
(particularly
the smooth muscle layers) and the luminal portions of the tunica mucosa
layers. The
resulting submucosa tissue has a thickness of about 100 to about 200
micrometers, and
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consists primarily (greater than 98%) of acellular, eosinophilic staining (H&E
stain)
extracellular matrix material.
Preferred submucosal tissues for use in accordance with this invention
include intestinal submucosa, stomach submucosa, urinary bladder submucosa,
and
uterine submucosa. Intestinal submucosal tissue is one preferred starting
material, and
more particularly intestinal submucosa delaminated from both the tunics
muscularis
and at least the tunics mucosa of warm-blooded vertebrate intestine.
As a tissue graft, submucosal tissue undergoes remodeling and induces
the growth of endogenous tissues upon implantation into a host. It has been
used
successfully in vascular grafts, urinary bladder and hernia repair,
replacement and
repair of tendons and ligaments, and dermal grafts. The preparation and use of
submucosa as a tissue graft composition is described in U.S. Patent Nos.
4,902,508;
5,281,422; 5,275,826; 5,554,389; and other related U.S. patents. When used in
such
applications the graft constructs appear not only to serve as a matrix for the
regrowth
1 S of the tissues replaced by the graft constructs, but also promote or
induce such
regrowth of endogenous tissue. Common events to this remodeling process
include:
widespread and very rapid neovascularization, proliferation of granulation
mesenchymal cells, biodegradation/resorption of implanted intestinal
submucosal tissue
material, and lack of immune rejection. The use of submucosal tissue in sheet
form
and fluidized forms for inducing the formation of endogenous tissues is
described and
claimed in U.S. Patent Nos. 5,281,422 and 5,275,826, the disclosures ofwhich
are
expressly incorporated herein by reference.
Submucosal tissue can be obtained from various sources, including
intestinal tissue harvested from animals raised for meat production,
including, for
example, pigs, cattle and sheep or other warm-blooded vertebrates. This tissue
can be
used in either its natural configuration or in a comminuted or partially
digested
fluidized form. Vertebrate submucosal tissue is a plentiful by-product of
commercial
meat production operations and is thus a low cost cell growth substrate,
especially
when the submucosal tissue is used in its native layer sheet configuration.
The submucosa tissue graft constructs prepared in accordance with the
present invention are a substantially acellular matrix that provides a
superior cell
growth substrate resembling the matrix environment found in vivo. The natural
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composition and configuration of submucosal tissue provides a unique cell
growth
substrate that promotes the attachment and proliferation of cells.
It has been reported that compositions comprising submucosal tissue of
the intestine of warm-blooded vertebrates can be used as tissue graft
materials in sheet
or fluidized form. U.S. Patent No. 4,902,508 describes tissue graft
compositions that
are characterized by excellent mechanical properties, including high
compliance, a high
burst pressure point, and an effective porosity index. These properties allow
such
compositions to be used for vascular and connective tissue graft constructs.
When
used in such applications the preferred graft constructs serve as a matrix for
the in vivo
regrowth of the tissues replaced by the graft constructs. U. S. Patent No.
5,275,826
describes use of fluidized forms of vertebrate submucosal tissues as
injectable or
implantable tissue grafts.
The present invention is directed to submucosa tissue graft constructs
and a method of enhancing or expanding the functional properties of vertebrate
submucosal tissues as an implantable or injectable tissue graft construct. The
improved tissue graft constructs are prepared by seeding the submucosal tissue
in vitro
with preselected or predetermined cell types prior to implanting or injecting
the graft
construct into the host.
Detailed Description of the Preferred Embodiments
The present invention is directed to an improved tissue graft construct
comprising vertebrate submucosa delaminated from both the external smooth
muscle
layers and the luminai portions of the tunica mucosa. The improvement
comprises the
addition of a preselected population of cells to the substantially acellular
submucosa
matrix. The cells to be combined with the submucosa are selected based on the
cell
type of the intended tissue to be repaired. In one embodiment the preselected
cells
comprise primary cells isolated from epithelial, endothethial or cartilage
tissues.
There are certain areas of the body that contain a combination of
complex differentiated structures for which regeneration has never shown to be
possible. These areas typically heal with great difficulty and damage to these
structures creates significant morbidity and often mortality. Examples of such
areas
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include the esophagus, the central nervous system, skin and its appendages,
among
others.
The combination of the preselected population of cells with the submucosa
matrix provides an improved tissue graft construct that shows surprising
improved
wound healing capabilities and better restoration of tissue function when
compared to
the use of either component alone as a therapeutic agent. Furthermore, the
composition comprising submucosa seeded with added cells can be cultured prior
to
the implantation of the construct into the affected region. Intestinal
submucosa is
capable of supporting the proliferation and growth of a wide variety of cells,
including
primary cells that are normally difficult to culture in vitro. The ability of
submucosa to
provide a substrate that supports the growth of such cells provides the
opportunity to
expand a population of cells prior to implantation into a host. In one
embodiment the
submucosa is seeded with autologenous cells isolated from the patient to be
treated.
There is provided in accordance with this invention a method and
1 S composition for supporting the proliferation and inducing tissue
differentiation of
eukaryotic cells cultured in vitro. Generally the method comprises the step of
contacting eukaryotic cells, in vitro, with a vertebrate submucosa-derived
collagenous
matrix under conditions conducive to eukaryotic cell growth. The term
"contacting"
as used herein with reference to cell culture is intended to include both
direct and
indirect contact, for example in fluid communication, of the submucosal tissue
and the
cultured cells. The term "conditions conducive to eukaryotic cell growth" as
used
herein refers to the environmental conditions, such as sterile technique,
temperature
and nutrient supply, that are considered optimal for eukaryotic cell growth
under
currently available cell culture procedures. Although optimum cell culture
conditions
used for culturing eukaryotic cells depend somewhat on the particular cell
type, cell
growth conditions are generally well known in the art. However a number of
differentiated cell types are still considered difficult to culture (i. e.
islets of Langerhans,
hepatocytes, chondrocytes, osteoblasts, etc.).
The collagenous matrix component of the present cell culture substrate
is derived from vertebrate submucosa and comprises naturally associated
extracellular
matrix proteins, glycoproteins and other factors. Preferably the collagenous
matrix
comprises intestinal submucosal tissue of a warm-blooded vertebrate. The small
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intestine of warm-blooded vertebrates is a particularly preferred source of
the cell
culture substrate for use in this invention.
Suitable intestinal submucosal tissue typically comprises the tunica
submucosa delaminated from the tunica muscularis and at least the luminal
portion of
the tunica mucosa. In one preferred embodiment of the present invention the
intestinal
submucosal tissue comprises the tunica submucosa and basilar portions of the
tunica
mucosa including the lamina muscularis mucosa and the stratum compactum which
layers are known to vary in thickness and in definition dependent on the
source
vertebrate species.
The preparation of submucosal tissue for use in accordance with this
invention is described in U.S. Patent No. 4,902,508, the disclosure ofwhich is
expressly incorporated herein by reference. A segment of vertebrate intestine,
preferably harvested from porcine, ovine or bovine species, but not excluding
other
species, is subjected to abrasion using a longitudinal wiping motion to remove
the
outer layers, comprising smooth muscle tissues, and the innermost layer, i.e.,
the
luminal portion of the tunica mucosa. The submucosal tissue is rinsed with
saline and
optionally sterilized; it can be stored in a hydrated or dehydrated state.
Lyophilized or
air dried submucosa tissue can be rehydrated and used in accordance with this
invention without significant loss of its cell proliferative activity.
The submucosa component of the present invention can be sterilized,
prior to the addition of the preselected cells, using conventional
sterilization techniques
including glutaraldehyde tanning, formaldehyde tanning at acidic pH, propylene
oxide
treatment, gas plasma sterilization, gamma radiation, electron beam, peracetic
acid
sterilization. Sterilization techniques which do not adversely affect the
mechanical
strength, structure, and biotropic properties of the submucosal tissue is
preferred. For
instance, strong gamma radiation may cause loss of strength of the sheets of
submucosal tissue. Preferred sterilization techniques include exposing the
graft to
peracetic acid, 1-4 Mrads gamma irradiation (more preferably 1-2.5 Mrads of
gamma
irradiation) or gas plasma sterilization; peracetic acid sterilization is the
most preferred
sterilization method. Typically, the submucosal tissue is subjected to two or
more
sterilization processes. After the submucosal tissue is sterilized, for
example by
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chemical treatment, the tissue may be wrapped in a plastic or foil wrap and
sterilized
again using electron beam or gamma irradiation sterilization techniques.
The submucosal tissue specified for use in accordance with this
invention can also be in a fluidized form. Submucosal tissue can be fluidized
by
comminuting the tissue and optionally subjecting it to protease digestion to
form a
homogenous solution. The preparation of fluidized forms of submucosa tissue is
described in U. S. Patent No. 5,275,826, the disclosure of which is expressly
incorporated herein by reference. Fluidized forms of submucosal tissue are
prepared
by comminuting submucosa tissue by tearing, cutting, grinding, or shearing the
harvested submucosal tissue. Thus pieces of submucosal tissue can be
comminuted by
shearing in a high speed blender, or by grinding the submucosa in a frozen or
freeze-
dried state to produce a powder that can thereafter be hydrated with water or
a
buffered saline to form a submucosal fluid of liquid, gel or paste-like
consistency. The
fluidized submucosa formulation can further be treated with a protease such as
trypsin
or pepsin at an acidic pH for a period of time sufficient to solubilize all or
a major
portion of the submucosal tissue components and optionally filtered to provide
a
homogenous solution of partially solubilized submucosa.
The viscosity of fluidized submucosa for use in accordance with this
invention can be manipulated by controlling the concentration of the submucosa
component and the degree of hydration. The viscosity can be adjusted to a
range of
about 2 to about 300,000 cps at 25 ° C. Higher viscosity formulations,
for example,
gels, can be prepared from the submucosa digest solutions by adjusting the pH
of such
solutions to about 6.0 to about 7Ø
Applicants have discovered that compositions comprising submucosal
tissue can be used for supporting growth or proliferation of eukaryotic cells
in vitro.
Submucosal tissue can be used in accordance with this invention as a cell
growth
substrate in a variety of forms, including its native sheet-like
configuration, as a gel
matrix, as an addition for art-recognized cell/tissue culture media, or as
coating for
culture-ware to provide a more physiologically relevant substrate that
supports and
enhances the proliferation of cells in contact with the submucosal matrix. The
submucosal tissue provides surfaces for cell adhesion and also induces cell
differentiation. The submucosal tissue is preferably sterilized prior to use
in cell
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culture applications, however nonsterile submucosal tissue can be used if
antibiotics
are included in the cell culture system.
In one preferred embodiment cells are seeded directly onto sheets of
vertebrate submucosal tissue under conditions conducive to eukaryotic cell
proliferation. The porous nature of submucosal tissue allows dii~'usion of
cell nutrients
throughout the submucosal matrix. Thus, for example, cells can be cultured on
either
the luminal or abluminal surface of the submucosal tissue. The luminal surface
is the
submucosal surface facing the lumen of the organ source and typically adjacent
to an
inner mucosa layer in vivo whereas the abluminal surface is the submucosal
surface
facing away from the lumen of the organ and typically in contact with smooth
muscle
tissue in vivo.
Cells cultured on solid sheets of vertebrate submucosal tissue display a
different growth pattern, and exhibit different interactions with the
submucosal growth
substrate, depending on which side of the submucosal sheet the cells are
grown.
Histological examination of tissue/cells cultured on intestinal submucosal
tissue sheets
in accordance with this invention reveals that cells that are seeded onto the
abluminal
surface not only grow/proliferate along the surface of the submucosal tissue,
but they
also more readily migrate into and proliferate within the submucosal tissue
itself. The
luminal surface comprises a more dense matrix than the abluminal side and thus
cells
are less likely to penetrate the luminal side. Cells that are seeded onto the
luminal
surface attach to the matrix but generally do not penetrate the surface.
However
certain cell types are capable of penetrating both the abluminal and luminal
surfaces (eg
squamous carcinoma cells and fibroblasts). In addition, certain cell types,
such as fetal
rat cells, when seeded on the luminal side proliferate to form a polylayer of
cells. Cells
ZS of this polylayer can differentiate to perform functions characteristic of
cells in vivo
and indicative of their position in the polylayer.
In one embodiment of the present invention, cell growth substrates in
accordance with the present invention are formed from fluidized forms of
submucosal
tissue. The fluidized submucosal tissue can be gelled to form a solid or semi-
solid
matrix. Eukaryotic cells can then be seeded directly on the surface of the
matrix and
cultured under conditions conducive to eukaryotic cell proliferation.
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The cell growth substrate of the present invention can be combined
with nutrients, including minerals, amino acids, sugars, peptides, proteins,
or
glycoproteins that facilitate cellular proliferation, such as laminin and
fibronectin and
growth factors such as epidermal growth factor, platelet-derived growth
factor,
transforming growth factor beta, or fibroblast growth factor. In one preferred
embodiment fluidized or powder forms of submucosal tissue can be used to
supplement standard eukaryotic culture media to enhance the standard media's
capacity
for sustaining and inducing the proliferation of cells cultured in vitro.
In accordance with the present invention there is provided a cell culture
composition for supporting growth in vitro of an eukaryotic cell population in
combination with submucosaI tissue of a warm-blooded vertebrate. The
composition
comprises nutrients, and optionally growth factors required for optimal growth
of the
cultured cells. The submucosa substrates of the present invention can be used
with
commercially available cell culture liquid media (both serum based and serum
free).
When grown in accordance with this invention, proliferating cells can either
be in
direct contact with the submucosal tissue or they can simply be in fluid
communication
with the submucosal tissue. It is anticipated that the cell growth
compositions of the
present invention can be used to stimulate proliferation of undifferentiated
stems cells
as well as differentiated cells such as islets of Langerhans, hepatocytes and
chondrocytes. Furthermore the described cell growth composition is believed to
support the growth of differentiated cells while maintaining the
differentiated state of
such cells.
It has been well documented that submucosal tissue is capable of
inducing host tissue proliferation, remodeling and regeneration of appropriate
tissue
structures upon implantation in a number of microenvironments in vivo (e.g.,
tendon,
ligament, bone, articular cartilage, artery, and vein). The use of such tissue
in sheet
form and fluidized forms for inducing the formation of endogenous tissues is
described
and claimed in U.S. Patent Nos. 5,281,422 and 5,275,826, the disclosures of
which are
expressly incorporated by reference.
In one embodiment of the present invention the tissue replacement
capabilities of graft compositions comprising submucosal tissue of warm-
blooded
vertebrates are further enhanced or expanded by seeding the tissue with
various cell
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types, prior to implantation. For example, submucosal tissue may be seeded
with
endothelial cells or keratinocytes and used as a vascular graft or skin
replacement,
respectively. In one embodiment the subrnucosal tissue is seeded with islet of
langerhans cells for use as an auxiliary pancreas. Alternatively, the
submucosal tissue
can be seeded with mesenchymal cells (stem cells) initially for expansion of
the cell
population and thereafter for implantation into a host. Submucosal tissue can
also
serve as a delivery vehicle for introducing genetically modified cells to a
specific
location in a .host. The submucosal tissue for use in accordance with this
embodiment
can either be in a fluidized form or in its native solid form. Optionally,
after the
submucosal tissue has been seeded with eukaryotic cells, the graft composition
can be
subjected to conditions conducive to the proliferation of eukaryotic cells to
further
expand the population of the seeded cells prior to implantation of the graft
into the
host.
In one embodiment, compositions comprising submucosal tissue and a
proliferating cell population can be encapsulated in a biocompatible matrix
for
implantation into a host. The encapsulating matrix can be configured to allow
the
diffusion of nutrients to the encapsulated cells while allowing the products
of the
encapsulated cells to diffuse from the encapsulated cells to the host cells.
Suitable
biocompatible polymers for encapsulating living cells are known to those
skilled in the
art. For example a polylysine/alginate encapsulation process has been
previously
described by F. Lim and A. Sun (Science Vol. 210 pp. 908-910). Indeed,
vertebrate
submucosa itself could be used advantageously to encapsulate a proliferating
cell
population on a submucosal matrix in accordance with this invention for
implantation
as an artificial organ.
Submucosal tissue advantageously provides a physiological
environment that supports the differentiation of cells cultured in vitro on
the
submucosal tissue. Thus, cell culture substrates comprising submucosal tissue
can be
used in combination with standard cell culture techniques known to those of
ordinary
skill in the art, to produce tissue grafts, in vitro, for implantation into a
host in need
thereof. The cells of such a tissue perform their proper natural function
based on cell
type and position within the submucosal tissue graft construct.
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The method of forming a tissue graft in vitro comprises the steps of
seeding eukaryotic cells onto a cell growth substrate comprising submucosal
tissue of a
warm-blooded vertebrate and culturing the cells in vitro under conditions
conducive to
proliferation of the eukaryotic cells. Advantageously the synthesis in vitro
of a tissue
graft construct, wherein the cells of the tissue perform their proper natural
function,
allows the generation of tissue grafts from an initially small cell population
that can be
expanded in vitro prior to implantation.
In accordance with one embodiment of the present invention an
improved tissue graft construct is provided. The tissue graft construct
comprises
tunics submucosa delaminated from both the tunics muscularis and at least the
luminal
portion of the tunics mucosa of vertebrate intestinal tissue combined with a
preselected
population of cells. In one embodiment the preselected population of cells
includes
connective tissue precursor cells. Intestinal submucosa can induce the
differentiation
of precursor cells into cells that assist in the repair of damaged tissues.
1 S Advantageously, submucosa seeded with a population of precursor cells can
be
implanted into a variety of different in vivo locations and the precursor
cells will
differentiate into the appropriate cell type for the environment. For example,
implantation of the composition adjacent to cartilage or bone will result in
the graft
construct remodeling into cartilage or bone.
In accordance with one embodiment vertebrate submucosa is combined
with primary cells to form an improved vertebrate submucosa tissue graft
construct. In
one embodiment, the improved tissue graft construct comprises vertebrate
submucosa
delaminated from both the external smooth muscle layers and the luminal
portions of
the tunics mucosa and added primary cells. More particularly, in one
embodiment the
vertebrate submucosa comprises tunics submucosa delaminated from both the
tunics
muscularis and at least the luminal portion of the tunics mucosa of vertebrate
intestinal
tissue. The improved graft construct of the present invention are implanted
into an in
vivo site in need of repair to enhance the repair of the endogenous tissues.
The
primary cells can be selected from the group consisting of endothelial,
keratinocytes,
chondrocytes, epithelial and mesenchymal cells. Typically, the submucosa will
be in a
solid form, however in an alternative embodiment the submucosa utilized is
fluidized
submucosa. The submucosa can be fluidized by comminuting the tissue and/or
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digesting the submucosa with an enzyme for a period of time sufficient to
solubilize the
submucosa.
In one embodiment, the improved tissue graft construct of the present
invention comprises tunics submucosa delaminated from both the tunics
muscularis
and at least the luminal portion of the tunics mucosa of vertebrate intestinal
tissue and
a population of primary cells selected from the group consisting of
endothelial cells,
keratinocytes and mesenchymal cells. Furthermore, the preselected cell type
may
include cells that have been genetically modified. For example, the cell may
be
modified by including genes that express proteins that enhance the repair of
the
damaged or diseased tissues.
The present invention further provides a method for enhancing the
capabilities of a submucosa graft construct to repair articular cartilage
defects. The
method comprises the step of seeding the vertebrate submucosa with
chondrocytes
prior to implanting or injecting the graft construct into a host. Accordingly,
in one
embodiment of the present invention a composition for the repair of articular
cartilage
defects comprises tunics submucosa delaminated from both the tunics muscularis
and
at least the luminal portion of the tunics mucosa of vertebrate intestinal
tissue and
added primary chondrocyte cells.
The present invention also provides a method for enhancing the
capabilities of vertebrate subrnucosa graft construct to repair epithelial
defects (such as
periodontal structures or the esophagus), said method comprising the step of
seeding
the submucosa with primary epithelial cells prior to implanting or injecting
the graft
construct into a host. The method of repairing these tissue can further
comprising the
step of subjecting the seeded graft construct to conditions conducive to the
proliferation of the cells prior to implanting or injecting the graft material
into the host.
Accordingly, in one embodiment of the present invention a composition for the
repair
of periodontal structures or the esophagus comprises tunics submucosa
delaminated
from both the tunics muscularis and at least the luminal portion of the tunics
mucosa
of vertebrate intestinal tissue and added primary epithelial cells, and more
particularly
primary epithelial cells selected from the group consisting of primary gingiva
epithelial
cells and primary esophageal epithelial cells.
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Example 1
Sterilization of Submucosal Tissue
Because cell culture techniques must be performed under strict aseptic
conditions, if antibiotics are not included in the culture system, the
submucosa tissue
must be prepared in a sterile manner for use as a cell culture substrate.
Numerous
sterilization methods have been investigated to assess the effect of
sterilization on the
biotropic properties of submucosal tissue. Sterilization techniques which do
not
significantly weaken the mechanical strength and biotropic properties of the
tissue are
preferred. The following sterilization methods for intestinal submucosa have
been
evaluated: peracetic acid sterilization, 2.5 Mrad gamma-irradiation, 1.0 Mrad
gamma-
irradiation, Exspor (Alcide, Norfolk, CT) sterilization and various
combinations of
these sterilization methods. Gamma-irradiation was performed on hydrated
submucosal tissue using a 6°Cobalt-gamma chamber. Exspor sterilization
was
performed according to manufacturer's specifications using a sterilant volume
(ml) to
intestinal submucosa (g) ratio of 10 to 1.
Various cell types (e.g., IlVm-90, FR, HT-29, RPEC) were seeded upon
the sterilized submucosa and their growth characteristics were analyzed at
1,3,7 and 14
days. Results obtained for all cell types showed that submucosal derived
growth
substrates sterilized by gamma irradiation or peracetic acid treatments
supported some
degree of adherence and growth of cells. However, cells seeded onto peracetic
acid
sterilized submucosal derived substrates showed increased adherence, increased
survival, and enhanced rates of proliferation and differentiation; peracetic
acid appears
to be the preferred sterilization technique for preparation of submucosa as a
cell
culture substrate.
Example 2
Sterilization of Submucosal Tissue with Peracetic Acid
Submucosal tissue is soaked in a peracetic acid/ethanol solution for 2
hours at room temperature using a ratio of 10:1 (mls peracetic solution: grams
submucosal tissue) or greater. The peracetic acid/ethanol solution comprises
4%
ethanol, 0.1 % (volume: volume) peracetic acid and the remainder water. The
0.1
peracetic acid component is a dilution of a 35% peracetic acid stock solution
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commercially available and defined as in table 1. Preferably, the submucosal
tissue is
shaken on a rotator while soaking in the peracetic acid solution. After two
hours, the
peracetic acid solution is poured off and replaced with an equivalent amount
of
lactated Ringer's solution or phosphate bui~ered saline (PBS) and soaked (with
shaking) for 1 S minutes. The submucosal tissue is subjected to four more
cycles of
washing with lactated Ringer's or PBS and then rinsed with sterile water for
an
additional 15 minutes.
Table 1~ Chemical Composition of the 35% Peracetic Acid
Solution
Composition, % by weight
Peracetic acid 35.5
Hydrogen peroxide 6.8
Acetic acid 39.3
Sulfuric acid 1.0
Water 17.4
Acetyl peroxide 0.0
Stabilizer 500 PPM
Typical active oxygen analysis, % by weight
Active Oxygen as peracid 7.47
Active Oxygen as H20z 2.40
Total active oxygen 10.67
In order to promote a further understanding of the present invention
and its features and advantages, the following specific Examples are provided.
It will
be understood that these specific Examples are illustrative, and not limiting,
of the
present invention.
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Example 3
Growth Characteristics Of Various Cell Types On Sterilized Submucosa
Small intestinal submucosa was harvested and prepared from freshly
S euthanatized pigs as described in U.S. Patent No. 4,902,508. Following
sterilization
via various techniques (gamma irradiation, peracetic acid, etc.), the
submucosal tissue
was clamped within a polypropylene frame to create a flat surface area (50
mmz) for
cell growth. The frame was submerged in culture medium to allow access of
medium
nutrients to both surfaces of the submucosal tissue. Various cell types were
seeded {3
x 104 cells/submucosal tissue section) on the submucosal tissue and then
placed in a
5% CO2, 95% air incubator at 37°C. Following various periods of time,
the seeded
submucosal tissue was fixed in I O% neutral buffered formalin, embedded in
paraffin,
and sectioned (6 um). Various histological and immunohistochemical staining
procedures were then applied to determine the cell growth characteristics.
To date, the growth characteristics of the following cell lines have been
studied using submucosal tissue as a growth substrate:
CELL LINE CELL LINE DESCRIPTION
CHO Chinese hamster ovary cells
3T3 Swiss albino
mouse embryo fibroblasts
C3H10T1/2 C3H mouse embryo, multi-potential
FR Fetal rat skin
(Sprague Dawley)
IMR90 Human fetal lung fibroblasts
HT-29 Human colon adenocarcinoma, moderately
well
differentiated, grade II
RPEC Rat pulmonary endothelial cells
HUVEC Human umbilical vein cells
SCC-12 Squamous Cell Carcinoma
Table 2 summarizes various cell types and the corresponding specific
medium conditions used to culture on the submucosa derived cell culture
substrates.
The medium chosen represents optimal or near optimal conditions for
propagation of
each cell type under standard cell culture conditions (i.e., plastic tissue
culture flasks).
All cell preparations were incubated at 37° C in a humidified
atmosphere of 5%
COZ/air.
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Table 2: Cell types and corresponding culture conditions investigated using
Intestinal Submucosal Tissue as a cell
growth matrix
CELL TYPE MEDIUM
3T3 (American Type Culture DMEM (Dulbecco's modified Eagle's
Collection (ATCC), medium) with
CRL 1658) Swiss mouse embryo 1.5 g/L NaHCO,, 10% NNCS (neonatal
fibroblasts calf
serum), 100 U/mI penicillin,
100 ug/ml
stre tomycin, 2 mM L- lutamine
FR (ATCC, CRL 1213) cell lineDMEM, 10% NNCS, 100 U/ml penicillin,
developed from a 100
skin biopsy of a fetal (18 ug/ml streptomycin, 2 mM L-glutamine
day gestation) germ-free
S ra ue Dawle rate
HT-29 (ATCC, HTB 38) cell McCoy's, 10% NNCS, 100 U/ml
line derived from penicillin, 100
human colon adenocarcinoma a ml stre tomycin, 2 mM L-
lutamine
HUV-EC-C (ATCC, CRL 1730) F12 K medium, 10% FBS (fetal
endothelial cell bovine serum), 100
I line isolated from human umbilicalug/ml heparin, 50 ug/ml endothelial
5 vein cell growth
supplement, 100 U/ml penicillin,
100 ug/ml
stre tom ein, 2 mM L- lutamine
IMR-90 (ATCC, CCL 186) human McCoy's SA medium, 20% NNCS,
diploid 100 Ulml
fibroblasts penicillin, 100 ug/ml streptomycin,
2 mM L-
lutamine
RPEC (J.P. Robinson, Purdue RPMI 1640, 5% NCS (newborn
University) calf serum) 5%
endothelial cell line derivedFBS (fetal bovine serum), 100
from rat pulmonary U/ml penicillin, 100
endothelial cells a ml stre tomycin. 2 mM L-
lutamine
C3H10T1/2 (ATCC, CCL 226) BME (basal medium Eagle), 10%
mouse embryo FBS, 100 U/ml
fibroblasts penicillin, 100 ug/ml streptomycin,
2 mM L-
lutamin
SCC-12 (VJ. Crreenlee, PutdueDMEM, 5% FBS (fetal bovine
University) serum), 4 mM L-
s uamous cell carcinoma lutamine, 1 mM sodium ruvate
CHO (Chinese Hamster Ovary F12 Medium 10% FBS with antibiotics
Cells) eom cin)
The cellular growth on both the luminal and abluminal sides of intestinal
submucosal tissue has been investigated. Intestinal submucosal tissue as a
growth
substrate exhibits sidedness; that is, the cell/matrix interactions are
dii~erent when the
cells are cultured on the abluminal versus the luminal side of the intestinal
submucosal
tissue. When selected cell types, such as rat FR cells are seeded on the
luminal side,
the cells attach to the matrix surface and proliferate to form a cellular
polylayer.
Alternatively, when FR cells are seeded on the abluminal side, the cells not
only grow
along the surface but also migrate into the submucosal matrix.
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The stratum compactum of the luminal side of vertebrate intestinal
submucosal tissue provides a dense connective tissue matrix and more readily
supports
monolayer or polylayer formation of select cell types (i.e. endothelial and
epithelial
cells). Alternatively, the abluminal side represents a more loose connective
tissue
structure that more readily supports migration of cells within the matrix
structure (i.e.
fibroblasts).
IIVIft-90 fibroblasts, when seeded upon the abluminal or luminal sides of
the intestinal submucosal tissue, quickly became adherent and proliferated
throughout
the matrix components. These cells illustrated their characteristic spindle
shape and
large vesicular nucleus within the extracellular matrix components. However,
3T3
fibroblasts showed minimal adherence and growth potential when seeded upon the
intestinal submucosal tissue.
Endothelial cells formed a confluent monolayer of spindle-shaped cells
along the stratum compactum surface of the intestinal submucosal tissue within
3 days.
1 S At later times the monolayer became more dense and some cells intercalated
down into
the matrix components. Interestingly, some endothelial cells that penetrated
into the
matrix components formed a lining along the lumen of structures representing
original
blood vessels of the native intestine.
To date, the growth characteristics of the following primary cell strains
have been studied using intestinal submucosal tissue as a growth substrate:
CELL STRAIN
Rat Cardiac Muscle
Porcine Smooth Muscle (aorta)
Porcine Endothelial (aorta)
Rabbit Smooth Muscle (aorta)
Rabbit Endothelial(aorta)
Porcine Smooth Muscle and Endothelial (mixed & co-cultured)
Human Osteoblasts Human Endothelial Cells
Primary cell strains are cells that have been harvested from an organism
and placed in culture. Subsequent passages of these cells (from 2-3 times)
using
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standard in vitro cell culture techniques (to increase the number of cells)
were frozen
for later use. Each of the above listed cell strains was thawed, cultured in
the presence
of intestinal submucosal tissue and examined histologically. Each of the
cultured cell
strain populations proliferated and retained their dii~erentiated appearance
as
determined by histological examination. For example, after 7-14 days of
culture on
intestinal submucosal tissue: the human osteoblast cells continued to
accumulate
appetite crystals and respond to osteogenic stimuli such as hormones; rat
cardiac
muscle cells retained their contractile properties; porcine smooth muscle
cells retained
smooth muscle actin; and porcine endothelial cells made factor eight.
Example 4
Intestinal Submucosal Cell Culture Substrates as a Tumor Cell Growth Model
Svstem
The morphology and invasive properties of an established cell line from
a human squamous cell carcinoma of the face known as SCC-12 (obtained from W.
Greenlee, Purdue University) cultured in vitro were studied. When grown under
standard cell culture conditions for skin cells (e.g., gamma-irradiated or
mitomycin C-
treated feeder layer of Swiss 3T3 mouse fibroblasts) a monolayer of flattened
cells is
formed. However SCC-12 cells when seeded upon the abluminal surface of
intestinal
submucosal tissue, showed, upon histological examination active degradation of
the
submucosal matrix components and invasion of the intestinal submucosal tissue.
SCC-12 cells were seeded (3 x 104 cells/0.8 cm2 of intestinal
submucosal tissue) on either the abluminal or luminal surface of sterilized
intestinal
submucosal tissue and floated in growth medium consisting of DMEM containing
5%
fetal calf serum, 4mM L-glutamine, and 1mM sodium pyruvate. At timepoints
representing 3, 7, 14, and 21 days, the growth characteristics were analyzed
using
standard histologic techniques. On day 3, the cells were strongly adherent and
appeared to form a continuous layer (1-2 cells thick) along surface of the
intestinal
submucosal tissue. Morphologically, the cells were round and actively
producing
extracellular matrix products. After 7 days, a significant difference was
noted in the
cells' ability to invade the abluminal versus the luminal surface of the
intestinal
submucosal tissue. The layer of cells along the luminal surface of the
intestinal
submucosal tissue appeared to only increase in density. Alternatively, those
cells
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seeded upon the abluminal surface, showed active degradation of the submucosal
matrix components and penetration up to 30 um. At longer durations, there was
an
increasing number of cells at greater depths of penetration and a greater
extent of
intestinal submucosal tissue degradation. Although the SCC-12 cells actively
invade
intestinal submucosal tissue from both the abluminal and luminal surfaces, the
observed
invasion rate was greater when SCC-12 cells were placed on the abluminal side.
Example 5
Intestinal Submucosal Tissue Supports Cytodii~erentiation
FR Epithelial cells form a stratified polylayer when cultured on the
luminal (stratum compactum) side of intestinal submucosal tissue. Cells
adjacent to
the intestinal submucosal tissue were columnar in shape and became
progressively
more flattened near the surface of the polylayer. After 14 days, structures
resembling
desmosomes were identified and the cellular layer stained positively for
cytokeratin
with a pan cytokeratin antibody. In addition, it appeared that the epithelial
cells
produced supporting matrix products (potentially basement membrane) as they do
in
vivo under normal healthy conditions. These findings suggest that the
intestinal
submucosal tissue supports natural epithelial cell maturation and
differentiation
processes.
The observed stratification of FR cells grown on the luminal side
(stratum compactum) of a submucosal growth substrate provides evidence that
the
intestinal submucosal tissue supports and induces cellular differentiation in
vitro. To
verify the induction of cytodifferentiation of the FR cells,
immunohistochemical and
immunofluorescence analyses were performed for detecting the production of
cytokeratin .by FR cells cultured in the presence and absence of intestinal
submucosal
tissue. Cytokeratin is a predominant intracellular structural protein produced
by
terminally differentiated epithelial cells known as keratinocytes.
Immunohistochemistry was performed on the protease-digested, formalin-fixed,
parafl"m embedded sections of FR cells grown on intestinal submucosal tissue
using an
anti-pan cytokeratin (C2931, Sigma, St. Louis, MO) as the primary antibody.
Immunodetection was performed using the avidin-biotin complex (ABC) method and
the Biogenex supersensitive StriAviGen kit (Vector Laboratories, Burlingame,
CA).
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Tissue sections representing rat skin biopsies and HT29 cells grown on
intestinal
submucosal tissue were included in the analysis as positive and negative
controls,
respectively.
Results indicated a gradation of cytokeratin staining along the FR
cellular polylayer with those cells at the surface of the polylayer staining
most
intensely. A similar positive staining pattern was observed in the cells
forming the
epidermal layer of the rat skin. However, no cytokeratin was detected in the
specimens representing HT29 cells cultured on intestinal submucosal tissue.
An immunofluorescence analysis for cytokeratin was performed using
flow cytometry to determine if the FR cell line expressed the differentiation
product
cytokeratin under standard culture conditions (in the absence of intestinal
submucosal
tissue). Swiss 3T3 Fibroblast (3T3) and squamous cell carcinoma (SCC-12) cell
lines
were included in the analysis as negative and positive controls respectively.
Cells were
harvested from tissue culture flasks, permeabilized using a cold methanol
pretreatment,
and incubated in the presences of anti-pan cytokeratin antibody at various
dilutions
(including the absence of anti-pan cytokeritin antibody to serve as a
control). A goat
anti-mouse antibody conjugated with fluorescein isothiocyanate (GAM-FITC) was
then applied to facilitate immunodetection. The cell preparations were then
analyzed
on a EPICS Elite flow cytometer (Coulter Corp., Hialeah, FL) using 488 nm
excitation
produced by an air-cooled argon laser. Fluorescence emissions were measured at
525
nm with a bandpass filter. Untreated cells and cells treated only with GAM-
FITC were
also analyzed to establish background fluorescence levels. Table 3 represents
the
relative percentage of FITC fluorescence for each cell type following indirect
immunofluorescence staining. As the data indicate only the positive control
SCC-12
cell line expresses cytokeratin and the FR cell line does not express
cytokeratin under
standard culture conditions in the absence of submucosal substrate.
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Table 3 Indirect Immunotluorescence Analysis for
Cytokeratin SCC-12, 3T3 and FR Cells
Dilution of Anti- Percent GAM-FITC
Cell Tyne Pan Cytokeratin Fluorescence
SCC-12 0(control) 2%
SCC-12 1:100 72%
SCC-12 1:400 74%
SCC-12 1:1000 76%
SCC-12 1:4000 72%
3 T3 0{control) 11
3T3 1:100 10%
3T3 1:400 18%
3T3 1:1000 8%
3T3 1:4000 5%
FR 0(control} 6%
FR 1:100 11%
FR 1:400 6%
FR 1:1000 4%
FR 1:4000 4%
Example 6
Isolation Of Hamster
Pancreatic Islets
Hamster pancreatic
islets were
isolated
as previously
described
by
Gotoh et al. (Transportation Vol. 43, pp. 725-730(1987)). Briefly, 6-8 week
old
Golden hamsters (Harlan, Indianapolis, IN) were anesthetized via inhalation of
Metofane (Methoxyflurane; Pitman-Moore; Mundelein, IL). The common bile duct
was cannulated under a stereomicroscope with a polyethylene catheter (PE-10
tubing;
CMS; Houston, TX), through which approximately 3-4 mls of ice cold M-199
medium
(commercially available from Gibco BRL) containing 0.7 mg/ml of collagenase P
was
injected slowly until whole pancreas was swollen. The pancreas was excised and
digested at 37° C for approximately 50 minutes in M-199 medium
containing 100
~g/ml of penicillin G and 100 pg/ml of streptomycin (no additional
collagenase). The
digest was washed three times in ice cold M-199 medium and passed sequentially
through a sterile S00 pm stainless steel mesh, then a 100 pm mesh. Following
purification by centrifugation through a ficoll density gradient (1.045,
1.075, 1.085 and
1.100} at 800 g for 10 min, islets were recovered from the top two interfaces.
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Culturing of Pancreatic Islet Cells on Intestinal
Submucosal Tissue
Islets of Langerhans (islet cells) were cultured on submucosal cell
growth substrates at 37°C in an incubator supplemented with 5% CO and
95% air.
The islet cells were cultured in the presence of various forms of intestinal
submucosal
tissue using the following procedures:
1. Direct Contact: Intestinal submucosal tissue and the cultured cells
physically contact one another.
2. Indirect Contact: Intestinal submucosal tissue and the cultured cells
are separated by a stainless steel mesh.
3. Solubilized intestinal submucosal tissue is added to the culture media
4. Cells are cultured on solubilized intestinal submucosa coated culture
plate. The coating was applied by placing 1 ml of solubilized intestinal
submucosaI
tissue in a 35mm culture plate, heated at 37°C for 2 hours, removing
the excess
I5 intestinal submucosal tissue fluid by aspiration and washing the coated
plates once with
culture media.
In direct contact culture method, an intestinal submucosa membrane of
approximately 1x1 cm was placed on top of stainless steel mesh with the
stratum
compactum side facing up. Isolated islets were then placed onto the membrane
and
continuously cultured in M-199 medium (commercially available from Gibco BRL)
for
7 days. Cell proliferation was examined every second day under a
stereomicroscope
and was compared with the control group (cultured in the absence of submucosa
tissue).
Sterilization of Submucosal Tissue Before Co-culturing
1. Intestinal submucosal tissue derived cell culture substrates were
sterilized by several different means: peracetic acid treatment or gamma
irradiation.
Gamma irradiated and the native (no further treatment after isolation of the
intestinal
submucosal tissue) membranes can be used directly as cell culture substrates
provided
they have been sufficiently rehydrated with the culture media prior to the co-
culture
(native membranes must be cultured in the presence of antibiotics). Peracetic
acid
sterilized membranes, must first be washed to remove residual peracetic acid
prior to
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culturing since peracetic acid residue may be toxic to the cells. Typically
peracetic
acid sterilized tissues were soaked in a large quality of medium for 24 hours
followed
by extensive washing with the same medium.
2. Solubilized forms of intestinal submucosal tissue were sterilized by
dialyzing against 6.5% chloroform in either O.1M acetic acid (AA-submucosa) or
phosphate buffered saline (PBS-submucosa) for 2 hours at room temperature. The
exterior surface of the dialysis tubing is sterilized by rinsing the outside
of the tubing
with 70% alcohol prior to removal of the intestinal submucosal tissue. The
dialysis
tubing has a molecular weight cut-off of 12,000-14,000; thus, proteins
retained inside
tubing are those with molecular weight greater than 14,000.
Results
In the control group (islets cultured in the absence of submucosa tissue)
examination of seven day cultures revealed that fibroblast cells had overgrown
the islet
1 S cells.
When islet cells were cultured on growth substrates comprising
intestinal submucosal tissue, overgrowth of the islet cells by fibroblast
cells did not
occur. In intestinal submucosal tissue direct culture systems, the islets
became loosely
packed with many cells surrounding the islet capsule. Cells migrated from the
capsule
and cell proliferation occurred on top of the membrane in the absence of
fibroblast
overgrowth. Culturing islet cells on intestinal submucosal tissue coated
culture ware
also appeared to facilitate migration of epithelioid cells out of the islet
capsule.
Further attachment to the coating surface and the formation of a monolayer of
epithelioid cells was observed.
These data indicate that submucosal substrates can be used to stimulate
growth of islet cells in vitro without overgrowth of fibroblast cells. Islet
cells can thus
be isolated from pancreatic tissue and grown in vitro in contact with a cell
growth
substrate comprising intestinal submucosal tissue of a warm-blooded vertebrate
under
conditions conducive to the proliferation of the islet cells and without
concurrent
growth of fibroblasts. These islet cell culture compositions remain
substantially free of
fibroblast overgrowth.
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Example 7
The ability of intestinal submucosa to augment wound healing and
promote tissue remodeling has been previous disclosed. However, certain
tissues
repair slowly after tissue damage, or have been damaged to such a great extent
(for
example in burn patients) that submucosal tissue alone fails to provide the
desired
speed of recovery. The combination of a preselected population of cells with
the
submucosa matrix has been found to enhance the repair capabilities of the
submucosa
tissue graft constructs. In one embodiment the submucosa tissue is combined
with
primary cell cultures specific for damaged or diseased tissues to be repaired
in the
body. The combination of the preselected population of cells with the
submucosa
matrix provides an improved tissue graft construct that shows surprising
improved
wound healing and subsequent better restoration of function when compared to
the use
of either component alone as a therapeutic agent.
A study was conducted in which the gingiva (gums) and deeper
periodontal structures were completely removed from several teeth in a canine
model.
The defect areas, which extended down to the alveolar bone, were treated with
either
intestinal submucosa alone, Alloderm (a commercial product derived from human
dermis), or a composite of intestinal submucosa plus primary autologous
gingiva
epithelial cells. Results showed that the sites treated with the composite
intestinal
submucosa plus primary epithelial cells healed best with an intact epithelial
cell
population consisting of the cultured epithelial cells and a sub-epithelial
connective
tissue layer which replaced the missing connective tissue support structures.
The sites
treated with intestinal submucosa alone or Alloderm formed a more typical scar
tissue
type response with a lesser epithelial cell component than was seen in the
sites treated
with the composite structures. This study showed the superior healing of the
composite.
In addition, primary esophageal epithelial cells have been grown on an
8-layer intestinal submucosa laminate structure which has shown utility as an
esophageal repair device. The preparation of mufti-laminate submucosa
structures is
fully described in US Patent 5,711,969, the disclosure of which is expressly
incorporated herein.
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Example 8
A large study recently completed in pigs shows that intestinal
submucosa when used in combination with a split thickness skin graft (which
essentially represents an autograft) results in improved "take" rates for the
split
thickness skin graft (STSG) when compared to use of the STSG alone. This
combination intestinal submucosa plus thin STSG fills a specific need in the
treatment
of full thickness skin wounds: specifically, thin (less than 0.010 inch) STSGs
can be
harvested and used with the intestinal submucosa carrier to treat these types
of wounds
instead of needing to harvest a thick (greater than 0.010 inch) STSG which
leaves a
deep wound at the graft harvest site with significant morbidity and subsequent
scarring. This type of study shows the utility of the composite intestinal
submucosa
plus primary epithelial cells and its superiority over the use of either
alone.
Example 9
Use of Intestinal Submucosa in the Repair of Articular Cartilage Defects
As articular cartilage has a limited ability for repair, once it has been
damaged by trauma or disease, it may deteriorate, resulting in an
osteoarthritic joint
Although joint replacement with a prosthesis is the treatment of choice, these
have a
limited lifespan, and replacement of a failed implant is a difficult
procedure.
Consequently, biological resurfacing has been developed as a way to treat
localized, or
early cartilage damage in order to delay or preferably prevent the onset of
osteoarthritis.
Recently, there has been increasing interest in the transplantation of
chondrocytes, either alone or within a carrier into cartilage defects. Early
studies
demonstrated that isolated chondrocytes were incorporated into defects;
however,
there was a 28-61 % failure rate due to poor fixation. Oversewing the
chondrocyte
implant with periosteum has increased the success rate, although the technique
is
technically demanding and may damage the adjacent cartilage. Seeding the
chondrocytes within carriers has been used to help improve the retention of
cells in the
defect. Recent studies by Freed et al. using cartilaginous tissue polyglycolic
acid
polymer composites to repair defects in rabbits showed promising results, but
the
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defects were in the non-weight-bearing part of the joint and only short-term
studies
were performed.
Porcine small intestinal submucosa is a resorbable biomaterial that upon
implantation induces tissue remodeling. This material was investigated for use
as a
suitable substratum for the formation of cartilage in vitro, which could then
be used to
resurface damaged joints. The submucosa--cartilaginous tissue composites were
implanted into full thickness articular cartilage defects. After 4 weeks, the
composites
had survived and retained their hyaline-like appearance, although
fibrovascular and
fibrocartilaginous tissue was also present. Defects receiving intestinal
submucosa
alone contained predominantly fibrovascular tissue, whereas ungrafted defects
were
filled with fibrocartilage. submucosa-cartilaginous tissue-resurfaced defects
scored
significantly better than submucosa-filled defects, but were no different from
the
unfilled defects. However, the repair tissue in the composite-filled defects
scored
significantly higher than that in the unfilled defects. This pilot study
suggests that
submucosa-cartilaginous tissue grafts may be useful for joint resurfacing.
In one embodiment chondrocytes are cultured in vitro to forma cell
layer on the surface of intestinal submucosa and then implanted to support
bone
ingrowth while the submucosa is bioabsorbed. Such a construct promotes graft
fixation without jeopardizing the integrity of the overlying cartilage.
MATERIALS AND METHODS
Chondrocyte Culture
Intestinal submucosa was prepared as a two-layer woven sheet and was
stored at -20 ° C. One week prior to use, the intestinal submucosa was
soaked in three
changes of antibiotics (10,000 U/ml penicillin G, 10 mg/ml streptomycin
sulphate,
25 pg/ml fungizone; GIBCO BRL, Burlington, Canada), rinsed three times with
Ham's
F12 (GIBCO BRL) cut into 4-mm discs using a biopsy punch (Premier Medical
Products, Norristown, PA), and either used for direct implantation or for cell
culture.
To obtain chondrocytes, the stifle joints of New Zealand White (NZW)
rabbits (male, 2 kg) were opened, and the superficial portion of the articular
cartilage
was dissected off and discarded. The remaining cartilage (lower third) was
harvested
and the cells isolated by sequential digestion with 0.25% proteinase (Sigma
Chemical
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Co., St Louis, MO) for 1 h, followed by 0.1% collagenase (Collagenase A,
Boehringer
Mannheim, Laval, Canada) for 4 h.2' The chondrocytes were resuspended in Ham's
F12 supplemented with S% normal rabbit serum (NRS) and plated on intestinal
submucosa (2 X 105 cells/disc of woven sheet Intestinal submucosa). The rabbit
serum
was obtained by intracardiac puncture of NZW rabbits (University of Toronto,
Toronto, Canada), heat inactivated (60°C for 30 min), and filter
sterilized prior to
being stored at -20°C.
On day S, the medium was changed to Dulbecco's modified Eagles
medium (DMEM; GIBCO BRL), supplemented with 20% NRS and 100 pg/ml
ascorbic acid (AA). The medium was changed every 2-3 days. On day 14, 10 mM ~i-
glycerophosphate (~i-GP) was also added to the medium. The cultures were
harvested
at various time points up to 8 weeks for analysis.
Histological Evaluation of Cultures
Cultures were fixed in 10% formalin and then parafl'm embedded. Five-
micron sections were stained with H&E to assess cellularity, toluidine blue to
assess
for the presence of sulfated proteoglycans, or von Kossa to assess for
calcification.
The thickness of 8-week-old cultures was determined morphometrically using the
QSOOMC image analysis system (Leica Canada Ltd., Willowdale, Canada). A
minimum of 10 points per section and three sections per culture were measured.
DNA and Proteoglycan Quantitation
The amount of DNA and proteoglycan in 8-week-old intestinal
submucosa-cartilage cultures, 4-mm discs of Intestinal submucosa, both "as
received,"(
having been stored at -20 ° C) and after 8 weeks in culture, and rabbit
deep cartilage
were quantitated. Samples were rinsed with PBS and lyophilized, and their dry
weights were measured. They were then digested with papain (80 pg/ml in 20 mM
ammonium acetate, 1 mM EDTA, 2 mM dithiothreitol; Sigma, St. Louis, MO) for at
least 18 h at 65 ° C.
The DNA content in the digests was measured using Hoescht 33258
dye (Polysciences Inc., Warrington, PA) and fluorometry using techniques known
to
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those skilled in the art. Calf thymus DNA (Pharmacia, Montreal, Canada) in PBS
was
used to generate the standard curve.
The amount of proteoglycans was estimated by measuring the
glycosaminoglycan (GAG) content of the papain digests using the
dimethylmethylene
S blue dye (Polysciences Inc.) binding assay using techniques known to those
skilled in
the art. The assay was performed in 96-well plates, and the absorbance at 525
nm was
measured by an automatic plate reader (Titrek Multiscan, InterSciences Inc.,
Finland).
Chondroitin sulfate (Sigma) was used to generate the standard curve. All
values were
normalized to dry weight.
The GAG and DNA content of the cartilaginous tissue formed on the
intestinal submucosa was estimated by correcting for the intestinal submucosa
contribution (submucosa cultured for 8 weeks in the absence of cells).
Proteoglycan Analysis
To examine proteoglycan size, proteoglycans (PG) were extracted from
8-week-old cultures, deep articular cartilage and intestinal submucosa with 4
M
guanidine HCl (50 mM sodium acetate, pH 5.8) containing protease inhibitors (5
mM
N ethylmaleimide, 50 mM benzamidine HCI, 10 mM EDTA, 0.1 M 6-aminohexanoic
acid), and precipitated with three volumes of ice cold ethanol. Thirty
micrograms of
each sample were separated using a 0.8% submerged horizontal agarose gel. The
gels
were stained with 0.002% toluidine blue in 0.1 N acetic acid for 30 min and
destained
with 0.1 N acetic acid.
Collagen Analysis
Representative cultures were labeled with ['4G]proline (10 uCi/ml, L-
['4C(U)]proline; Dupont NEN, Boston, MA) for 24 h. The collagen was pepsin
extracted ( 100 pg/ml 0.1 N acetic acid; Worthington Biochemical Corp.,
Freehold,
NY) for 24 h at 4°C. The pepsin extracts were separated on a 5% sodium
dodecyl
sulfate-polyacrylamide gel (SDS-PAGE) and either prepared for autoradiography
or
transferred to nitrocellulose for Western blot analysis. The presence of type
I or type
II collagen was determined by Western blot using antibodies reactive with type
I
collagen (polyclonal; Southern Biotechnology Associates Inc., Birmingham, AL)
or
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type II collagen (monoclonal antibody CIICI; Developmental Studies Hybridoma
Bank, Baltimore, MD). Reactivity was detected using affinity purified,
secondary
antibodies conjugated with alkaline phosphatase (BioRad, Mississauga, Canada).
Nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate were added for
S substrate and colour reaction (GIBCO BRL).
In Vivo Resurfacing Studies
Thirteen NZW rabbits (females, 4.5 kg) were divided into two groups:
seven experimental and six control. The rabbits were anesthetized with
Acepromazine
(1 mg/kg s.c.) and Somnotol (25 mg/kg i.v.). The stifle joints were exposed
using a
lateral longitudinal parapatellar incision, and the patella was dislocated.
Bilateral
incisions were made in the experimental group and a unilateral incision in the
control
group. A 4-mm diameter defect was made in the articular cartilage of the
trochlear
groove of the distal femur using a disposable biopsy punch in order to
minimize
1 S damage to the surrounding cartilage. The defect was then extended into the
subchondral bone using an electric drill (Variable speed rotary drill, Sears
model no.
924375).
Animals in the experimental group received a intestinal submucosa-
cartilaginous tissue transplant in one knee and intestinal submucosa only in
the
opposite knee. The intestinal submucosa-cartilaginous tissue composites had
been in
culture for 8 weeks prior to transplantation. The transplants were press-fit
into the
defect so that the surface of the transplant was level with the articular
surface. The
defects created in the control group of animals were left unfilled. The
patella was
reduced, and the capsule and skin were sutured closed. The animals were
allowed free
activity. Food and water were provided ad libitum. The rabbits were sacrificed
at 4
weeks with an overdose of T61 and the knee joints harvested.
Histological Evaluation of the Defects
The distal femurs were fixed in I O% buffered formalin, processed
undecalcified in graded ethanols, and plastic embedded in Spurr resin. Five-
micron-
thick sections were cut and stained either with H&E, or toluidine blue. The
histological features were assessed using a scoring method (maximum score =
13)
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adapted from O'Driscoll et al., J. Bone Joint Surg. [Am.] 70-A, 595 (1988) and
Ben-
Yishay et al., Tissue Eng. 1, 119 (1995) See Table 1. Briefly, the features
assessed
were (a) the tissue filling the defect (type of tissue, staining
characteristics and
appearance of the surface of the repair tissue) (b) fusion with the adjacent
cartilage and
bone, (c) filling of the defect, and (d) whether the adjacent tissue showed
degenerative
changes. Differentiation between hyaline-like and fibrocartilaginous tissue
was based
on histological features and whether collagen fibers could be visualized by
polarized
light microscopy, which is a feature of fibrocartilage. Data were analyzed
statistically
using the Student's t test and significance was assigned at ap value of <0.05.
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TABLE 1. HISTOLOGICAL SCORING SCHEME
FOR REPAIR OF CARTILAGE DEFECTS
Tissue in defect
Type of the predominant tissue
Hyaline
Hyaline/fibrocartilage 2
Fibrocartilage I
No cartilage 0
Staining of matrix
Normal 3
Moderate
Slight 1
None 0
Surface
Smooth
Slight disruption I
Severe disruption/fibrous 0
Filling of defect
Equal with adjacent cartilage 1
Depressed/raised 0
Fusion to surrounding tissue
Complete
Partial
One side 1
None 0
Adjacent cartilage
Normal 1
Degenerative changes 0
Maximal score 13
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RESULTS
Histological Appearance of intestinal submucosa-Cartilaginous Tissue Formed In
Vitro
The intestinal submucosa-cartilaginous tissue composites were
examined histologically after 3, 6, and 8 weeks in culture. The chondrocytes
attached
to the woven intestinal submucosa, accumulated extracellular matrix, and
formed
cartilaginous tissue. The chondrocytes were round and in lacunae. The
extracellular
matrix stained metachromatically with toluidine blue, indicating the presence
of
sulfated proteoglycans. Some cells had infiltrated into the intestinal
submucosa, and
these were surrounded by small amounts of matrix. Von Kossa staining revealed
the
presence of focal deposits of calcification in the cartilaginous tissue matrix
adjacent to
the intestinal submucosa, and in some cultures these formed a continuous layer
of
mineral by 8 weeks. By 8 weeks in culture, the cartilaginous tissue had an
average
thickness of 153.4 ~ 60.7 pm (mean ~ SE).
Ouantitation of DNA and Proteoglycan Content of the Cartilaginous Tissue
Formed
In Vitro
The amount of DNA and proteoglycan was measured in 8-week-old
intestinal submucosa-cartilage cultures and was compared to the in vivo rabbit
deep
cartilage (Table 2). The cartilaginous tissue formed on the intestinal
submucosa was
estimated to contain 0.86 t 0.2 pg DNAImg dry weight (mean ~ SE) and 86.5 ~
5.5
pg GAG/mg dry weight (mean ~ SE), whereas the rabbit deep cartilage contained
0.83
t 0.2 pg DNA/mg dry weight (mean t SE) and 109.64 ~ 7.8 pg GAG/mg dry weight
(mean ~ SE). Although there was no significant difference in the amount of
DNA,
there was significantly more GAG present in the in vivo tissue than the
cartilaginous
tissue formed in vitro (p = 0.03). The "as received" intestinal submucosa
samples had
a DNA content of 2.90 t 0.2 pg/mg dry weight (mean ~ SE) and a GAG content
25.35 ~ 5.0 p.g/mg dry weight (mean t SE), whereas the intestinal submucosa
analyzed
after 8 weeks of culture {"post culture") had DNA and GAG values of 2.04 ~ 0.
l and
8.66 ~ 0.6 pg/mg dry weight (mean ~ SE}, respectively.
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Analysis of the Proteoglycans and Collagen
As large proteoglycans and type II collagen are the major
macromolecules present in the matrix of cartilage, the proteoglycan and
collagen
present in the cartilaginous tissue formed in vitro was analyzed to determine
whether
the chondrocyte phenotype was retained under these culture conditions. Agarose
gel
electrophoresis showed that the 8-week intestinal submucosa-cartilage cultures
synthesized a population of large proteoglycans that were similar in size to
those
extracted from rabbit deep articular cartilage, although some of the
proteoglycans were
larger than those extracted from the in vivn tissue. Proteoglycans extracted
from
intestinal submucosa were much smaller and ran as two broad bands near the
bottom
of the gel.
Pepsin extracts of 8-week intestinal submucosa-cartilaginous tissue
composites were analyzed by SDS-PAGE and autoradiography. A band similar in
size
I 5 to the a, (I) chain of type II collagen was seen. No band, corresponding
to a,~ chain of
type I collagen, was present. A higher molecular weight band, likely
representing
dimers of the al(H) chains, was also present. Western blot analysis of these
extracts
confirmed the presence of type II collagen. Type I collagen was detected in
both the
pepsin extracts of intestinal submucosa-cartilaginous tissue composites and
from
intestinal submucosa alone. No type II collagen was detected in the intestinal
submucosa extracts by Western blot analysis.
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TABLE 2. DNA AND GLYCOSAMINOGLYCAN QUANTITATION
Dry weightDNA GAG
Tissue (mg) (mglmg dry weight)(mglmg dry
weight)
Rabbit deep cartilageNa 0.83 t 0.2 109.64 t 7.8
Intestinal submucosa 0.65 t 2.90 f 0.2 25.35 t 5.0
"as 0.02
received"
Intestinal submucosa 0.43 t 2.04 t 0.1 8.66 t 0.6
"post 0.02
culture"
Intestinal submucosa-2.33 t 1.14 f 0.2 70.65 t 4.0
0.3
cartilaginous tissue
Cartilaginous tissue 1.90 t 0.86 t 0.2 86.46 t 5.5"
in vitro 0.6
1$ eNA = not applicable. The pieces of rabbit deep cartilage analyzed ranged
from 1.37 to 3.65 mg.
bSignificant difference between rabbit deep cartilage and cartilaginous tissue
in vitro (p = 0.03).
The dry weight, and the amount of DNA and glycosaminoglycan (GAG) were
determined as
described in Materials and Methods. The data for the cartilaginous tissue
formed in vitro were
estimated by subtracting the mean dry weight or mean total DNA and GAG for
intestinal submucosa
after 8 weeks in culture ("post culture"), from the dry weight and total DNA
and GAG respectively of
each Intestinal submucosa-cartilaginous tissue sample. The analysis vas
performed on at least two
different samples from each of three dift'erent experiments. The data are
expressed as mean t SE.
In Vivo Studies
The joints of animals that had received intestinal submucosa-
cartilaginous tissue grafts showed no synovial effusion, although in two
rabbits the
joints were enlarged due to the presence of osteophytes. The defects were all
filled
with white tissue, and the adjacent cartilage appeared normal in five joints
and
fibrillated in two.
Of the seven animals for which intestinal submucosa alone had been
used to repair the defect, four developed osteophytes; and one had a synovial
effusion.
One animal developed a unilateral septic arthritis with a purulent effusion,
and the joint
was eliminated from the study. The defects in the remaining six rabbits were
filled with
whitish granular tissue. The adjacent cartilage showed signs of degeneration
in all but
3 S one animal.
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Of the six rabbits that received no transplant, four of the joints were
unremarkable grossly and two had synovial effusions. No osteophytes were seen.
All
the defects were filled with a white tissue, and no degeneration of the
adjacent
cartilage was visible.
Histological examination of the defects that had received intestinal
submucosa-cartilaginous tissue transplants showed that graft tissue was
present in six
of seven defects. The grafts maintained their appearance of hyaline cartilage.
All the
transplants were surrounded by variable amounts of fibrovascular tissue
towards the
joint surface and fibrocartilage tissue above or below the implant. When the
graft
approximated the adjacent host cartilage or bone, there was fusion between the
two
tissues. Fusion between the transplant and the under lying fibrocartilage also
occurred.
The one defect from which the graft had fallen out was filled entirely with
fibrocartilage.
1 S Histologically, the defects that received intestinal submucosa grafts
were filled with fibrovascular tissue. There was no or minimal amounts of
fibrocartilage. If fibrocartilage was present, it was present at the edge of
the defect.
Residual intestinal submucosa was only seen in one defect. Although the
articular
cartilage surrounding the defect merged with the fibrovascular tissue filling
the defect,
it was possible to distinguish the margins of the articular cartilage from the
reparative
tissue. In contrast, the joint defects that had not received a transplant were
entirely
filled with fibrocartilage, which showed variable amounts of bonding to the
adjacent
cartilage and bone. The fibrocartilage could be easily differentiated from the
adjacent
articular cartilage as it was more cellular, the matrix was fibrous in
appearance, and
collagen fibers could be visualized under polarized light. The surface of the
fibrocartilage showed degenerative changes with loss of proteoglycans.
Scoring the histological appearance of the defects (Table 3)
demonstrated that the joints resurfaced with the intestinal submucosa-
cartilaginous
tissue grafts or which had received no transplant were significantly better
than those
which had been implanted with intestinal submucosa alone (p = 0.004). There
was no
difference between the histological scores of the intestinal submucosa-
cartilaginous
tissue transplanted group and those defects that were left unfilled. However,
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comparison of the scores obtained for tissue type and staining of the repair
tissue in the
defect showed that the intestinal submucosa-cartilaginous tissue grafted group
was
significantly better than that of the no graft group (p = 0.019).
TABLE 3. HISTOLOGICAL SCORES OF DEFECTS
Graft Type and staining of tissue in defect° Total°
Intestinal submucosa- 3.7 t 0.8d 6.1 t 1.2°
cartilaginous tissue
Intestinal submucosa 1.0 f 0.7 3.8 t 0.8°
None 2.6 t 0.6d 6.210.8
'Values out of 6 points, expressed as mean t SD.
bValues out of a total of 13 points, expressed as mean t SD.
'Significant difference between Intestinal submucosa-cartilaginous tissue and
intestinal submucosa graft (p = 0.004).
°Significant difference between Intestinal submucosa-cartilaginous
tissue graft and
no graft (p = 0.019).
DISCUSSION
Rabbit chondrocytes attached to Intestinal submucosa, accumulated
extracellular matrix, and formed cartilaginous tissue in vitro. The
chondrocytes under
these conditions maintain their phenotype and synthesize large proteoglycans
and type
II collagen, macromolecules characteristic of hyaline cartilage. There was
mineralization of the extracellular matrix near the Tntestinal submucosa. In
contrast to
the cartilaginous tissue formed by chondrocytes obtained from the deep zone of
bovine
articular cartilage that consistently showed a continuous layer of
mineralization when
cultured on filter inserts, the tissue on the intestinal submucosa more
commonly had
only focal areas of mineralization. This discontinuous mineralization was also
observed when rabbit deep chondrocytes were cultured on filter inserts (data
not
shown) suggesting that intestinal submucosa was not affecting tissue
mineralization.
One explanation for the variation in the extent of mineralization may be due
to the
difference in the in vivo cartilage thickness between the rabbit and calf.
Bovine
cartilage is much thicker, and isolation of the deep cells is less likely to
be
contaminated with chondrocytes from the superficial and mid portion of the
cartilage.
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This is supported by the finding that the rabbit chondrocytes did not
synthesize
detectable amounts of type I collagen, unlike chondrocytes isolated from the
deep zone
of bovine cartilage, but similar to chondrocytes isolated from the full
thickness of
cartilage. Type I collagen was detected by Western blot, but was not seen on
autoradiographs suggesting that the type I Collagen was derived from the
Intestinal
submucosa. However, it is possible that small amounts of newly synthesized
collagen
were not detected by autoradiography.
The thickness of the cartilaginous tissue that formed on the intestinal
submucosa in vitro appeared to be influenced by the method of intestinal
submucosa
processing (data not shown). intestinal submucosa woven into a sheet supported
cartilaginous tissue formation most consistently, which is why it was used in
these
experiments. When chondrocytes were placed on intestinal submucosa processed
in
the standard way, there were areas of the mucosal surface of the intestinal
submucosa
that had thin or no cartilage formation. As the seeding density of
chondrocytes is
critical for maintenance of chondrocyte phenotype and matrix synthesis, and as
the
nonwoven intestinal submucosa curled or provided an irregular surface, it is
likely that
the initial distribution of cells over the surface of the intestinal submucosa
was uneven,
which may explain the variability.
Comparison of the cartilaginous tissue formed in vitro with the rabbit
deep cartilage demonstrated that, while there was no difference in the amount
of DNA,
the amount of GAG in the cartilaginous tissue was approximately 79% of the in
vivo
tissue. It is possible that this difference was due to the way in which the
contribution
of the intestinal submucosa to the composite was determined. intestinal
submucosa
that had been stored at -20°C upon receipt ("as received"} was found to
contain
approximately 25 pg GAG per mg dry weight similar to values obtained by Hodde
et
a1.33 However, intestinal submucosa cultured for 8 weeks in the absence of
cells lost
66% of its initial dry weight and the GAG content declined from 2.5% to 0.8%
of the
dry weight. To estimate the GAG content of the cartilaginous tissue formed on
the
intestinal submucosa, we assumed that the composition of intestinal submucosa
in the
intestinal submucosa-cartilage culture was similar to that of the intestinal
submucosa
after 8 weeks of cell free culture. However, as chondrocytes can produce a
variety of
matrix degrading enzymes, it is possible that the amount of intestinal
submucosa
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remaining was overestimated. Alternatively, as the intestinal submucosa-
cartilaginous
tissue composite became thicker, it is possible that nutrient diffusion was
limited,
which may have influenced chondrocyte biosynthesis and matrix accumulation.
The size of the proteoglycan population synthesized by the
chondrocytes cultured on intestinal submucosa overlapped that isolated from
deep
articular cartilage. However, the in vitro population also contained larger
proteoglycans. In a previous study, rabbit chondrocytes cultured on
extracellular
matrices were stimulated to synthesize larger proteoglycans than when they
were
cultured on tissue culture polystyrene. Furthermore, intestinal submucosa
contains
I O fibroblast growth factor (FGF-2) and transforming growth factor ~3 (TGF-
Vii) related
protein, both of which have been shown to stimulate chondrocytes to synthesize
larger
proteoglycans. It is therefore possible that the intestinal submucosa
influenced the
chondrocytes to synthesize larger proteoglycans in vitro.
Although the joint defects resurfaced with intestinal submucosa--
1 S cartilaginous tissue composites contained viable transplants, the repair
was not optimal
at 1 month. This was likely due to technical reasons, including inadequate
graft
fixation, which resulted in pannus formation. Good graft fixation is
considered critical
for graft incorporation. Although pressfit as a method of fixation has been
used by
others, in this study it resulted in the grafts having a concave geometry in
the defect.
20 The pannus that formed may then have further exacerbated the problem. It is
possible
that other methods of fixation might result in better graft incorporation. For
example,
in one study, perichondral grafts were sutured to a bone core and then
pressfit into a
defect. Using this approach to provide the intestinal submucosa with a more
rigid
support may keep the transplant level with the adjacent cartilage surface.
25 Although there was no difference in the overall histological scores
between the intestinal submucosa-cartilaginous tissue transplanted defects and
the
unfilled self repaired defects, the score obtained for the type and staining
of the tissue
in the defect was significantly higher in the intestinal submucosa-
cartilaginous tissue
group. This was due to the hyaline-like appearance of the transplant, while
the unfilled
30 defects contained only fibrocartilaginous repair tissue. Fibrocartilage is
considered
inferior as it undergoes degeneration over time.
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The intestinal submucosa alone, when placed in the defects, was not
replaced by bone, although 1 month may have been too short a period of time
for this
to occur. The absence of fibrocartilage filling of the defect, as was seen in
the
ungrafted defect, suggests that intestinal submucosa may be inhibiting or
preventing
the formation of this tissue. It is well accepted that, when a joint defect
breaches the
subchondral bone, the mesenchymal stem cells present in the marrow infiltrate
into the
defect and differentiate to produce fibrocartilaginous repair tissue.'~3~42 It
is possible
that the intestinal submucosa acted as a barrier preventing this infiltration.
In summary, intestinal submucosa supports the formation of
cartilaginous tissue in vitro. intestinal submucosa-cartilaginous tissue
grafts survive
upon transplantation into a joint and are able to fi~se with the adjacent
cartilage.
