U.S. patent application number 11/130018 was filed with the patent office on 2006-02-02 for chemical cleaning of biological material.
This patent application is currently assigned to Organogenesis Inc.. Invention is credited to Ginger A. Abraham, Linda Baker, Robert M. JR. Carr, Paul D. Kemp, Ryan D. Mercer.
Application Number | 20060024380 11/130018 |
Document ID | / |
Family ID | 25315861 |
Filed Date | 2006-02-02 |
United States Patent
Application |
20060024380 |
Kind Code |
A1 |
Abraham; Ginger A. ; et
al. |
February 2, 2006 |
Chemical cleaning of biological material
Abstract
The invention is directed to collagenous tissues which have been
treated to remove non-collagenous components such as cells,
cellular debris, and other extracellular matrix components, such as
proteoglycans and glycosaminoglycans, normally found in native
tissues. Treatment of the tissue with alkali, chelating agents,
acids and salts removes non-collagenous components from the
collagenous tissue matrix while controlling the amount of swelling
and dissolution so that the resultant collagen matrix retains its
structural organization, integrity and bioremodelable properties.
The process circumvents the need to use detergents and enzymes
which detrimentally affect the cell compatibility, strength and
bioremodelability of the collagen matrix. The collagenous tissue
matrix is used for implantation, repair, or use in a mammalian
host.
Inventors: |
Abraham; Ginger A.;
(Braintree, MA) ; Carr; Robert M. JR.; (West
Roxbury, MA) ; Kemp; Paul D.; (Romiley, GB) ;
Mercer; Ryan D.; (Boston, MA) ; Baker; Linda;
(Taunton, MA) |
Correspondence
Address: |
WILMER CUTLER PICKERING HALE AND DORR LLP
60 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Organogenesis Inc.
Canton
MA
|
Family ID: |
25315861 |
Appl. No.: |
11/130018 |
Filed: |
May 16, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10615623 |
Jul 8, 2003 |
6893653 |
|
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11130018 |
May 16, 2005 |
|
|
|
09450577 |
Nov 30, 1999 |
6599690 |
|
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10615623 |
Jul 8, 2003 |
|
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|
08853372 |
May 8, 1997 |
5993844 |
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09450577 |
Nov 30, 1999 |
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Current U.S.
Class: |
424/551 ;
435/6.18 |
Current CPC
Class: |
Y10S 623/92 20130101;
A61L 27/24 20130101; A61L 27/3695 20130101; Y10S 623/917 20130101;
A61K 35/12 20130101; A61L 2430/40 20130101; A61L 27/3687 20130101;
A61K 38/39 20130101 |
Class at
Publication: |
424/551 ;
435/006 |
International
Class: |
A61K 35/38 20060101
A61K035/38; C12Q 1/68 20060101 C12Q001/68 |
Claims
1. A bioremodelable collagenous tissue matrix composition derived
from native tissue, comprising: telopeptide collagen; elastin,
wherein elastin is less than 10% of the total composition based on
dry weight; and non-collagenous and non-elastinous components,
wherein said components are less than 5% of the total composition
based on dry weight; wherein the collagenous tissue matrix is free
of detergent residues, enzymatic modification, endotoxin and cells
and cellular debris; and wherein the native tissue is selected from
the group consisting of dermis, artery, vein, pericardium, heart
valve, dura mater, ligament, bone, cartilage, fascia and intestine;
and wherein the collagenous tissue matrix is sterile.
2. The bioremodelable collagenous tissue matrix composition of
claim 1, wherein the collagenous tissue matrix is sterile.
3. A bioremodelable collagenous tissue matrix composition derived
from small intestine, comprising: telopeptide collagen; elastin,
wherein elastin is less than 10% of the total composition based on
dry weight; and non-collagenous and non-elastinous components,
wherein said components are less than 5% of the total composition
based on dry weight; and wherein the collagenous tissue matrix is
free of detergent residues, enzymatic modification, endotoxin and
cells and cellular debris.
4. The bioremodelable collagenous tissue matrix composition of
claim 3, wherein the collagenous tissue matrix is derived from the
tunica submucosa of small intestine.
5. The bioremodelable collagenous tissue matrix composition of
claim 3, wherein the collagenous tissue matrix is chemically
cleaned.
6. The bioremodelable collagenous tissue matrix composition of
claim 3, wherein the collagenous tissue matrix is sterile.
7. The bioremodelable collagenous tissue matrix composition of
claim 3, wherein the collagenous tissue matrix is layered and
bonded together to form multilayer sheets, tubes, or complex shaped
prostheses.
8. The bioremodelable collagenous tissue matrix composition of
claim 7, wherein the collagenous tissue matrix is crosslinked.
Description
CROSS-REFERENCE SECTION
[0001] This application is a continuation of currently pending U.S.
patent application Ser. No. 10/615,623, filed Jul. 8, 2003, which
is a continuation of U.S. patent application Ser. No. 09/450,577,
filed Nov. 30, 1999, now U.S. Pat. No. 6,599,690, issued Jul. 29,
2003, which is a continuation application of U.S. patent
application Ser. No. 08/853,372, filed May 8, 1997, now U.S. Pat.
No. 5,993,844, issued Nov. 30, 1999.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention is in the field of tissue engineering. The
invention is directed to collagenous tissues which have been
treated to remove non-collagenous components such as cells,
cellular debris, and other extracellular matrix components, such as
proteoglycans and glycosaminoglycans, normally found in native
tissues. Treatment of the tissue with alkali, chelating agents,
acids and salts removes non-collagenous components from the
collagenous tissue matrix while controlling the amount of swelling
and dissolution so that the resultant collagen matrix retains its
structural organization, integrity and bioremodelable properties.
The process circumvents the need to use detergents and enzymes
which detrimentally affect the cell compatibility, strength and
bioremodelability of the collagen matrix. The collagenous tissue
matrix is used for implantation, repair, or use in a mammalian
host.
[0004] 2. Brief Description of the Background of the Invention
[0005] The field of tissue engineering combines the methods of the
engineering with the principles of life sciences to understand the
structural and functional relationships in normal and pathological
mammalian tissues. The goal of tissue engineering is the
development and ultimate application of biological substitutes to
restore, maintain or improve tissue functions. [Skalak, R. and Fox,
C. F., "Tissue Engineering", Alan R. Liss Inc. N.Y. (1988)]
[0006] Collagen is the principal structural protein in the body and
constitutes approximately one-third of the total body protein. It
comprises most of the organic matter of the skin, tendons, bones
and teeth and occurs as fibrous inclusions in most other body
structures. Some of the properties of collagen are its high tensile
strength; its ion exchanging ability, due in part to the binding of
electrolytes, metabolites and drugs; its low antigenicity, due to
masking of potential antigenic determinants by the helical
structure, and its low extensibility, semipermeability, and
solubility. Furthermore collagen is a natural substance for cell
adhesion. These properties and others make collagen a suitable
material for tissue engineering and manufacture of implantable
biological substitutes and bioremodelable prostheses.
[0007] As collagen is one major component of these biological
substitutes, a method for obtaining sufficient quantities of
collagen that is consistent in quality is needed. A need currently
exists for an improved method for the removal of non-collagenous
components such as cells, cellular debris, and other extracellular
matrix components, such as proteoglycans and glycosaminoglycans,
normally found in native tissues to yield a substantially pure
native collagen matrix. Some of these non-collagenous structures
that are present in native tissues are believed to be antigenic and
will elicit a chronic inflammatory response when implanted in a
host. However, in the art there are a variety of methods for the
cleaning of such collagenous tissue which have resulted in
collagenous compositions with different characteristics. The method
used should be one that maintains the biological and physical
properties of collagen and collagenous tissues suitable for use in
tissue engineering.
[0008] In the art of treating a collagenous tissue to yield
essentially a collagenous matrix, detergents and surfactants have
customarily been used in the extraction of cells and lipids from
the tissue. Detergents such as sodium dodecyl sulfate (SDS) are
amphipathic molecules wherein the hydrophobic region binds to
protein and are believed to increase the negative charge of the
protein. When implanted, the increase in charge results in both the
swelling of the tissue due to increased water binding by the
hydrophilic region of the molecule, and decreased thermal stability
in collagen by disrupting hydrogen bonding. Swelling both opens the
structure of the collagen molecule making it susceptible to
cellular enzymes such as collagenase and destabilizes the collagen
matrix to result in a weakened construct. (Courtman, et al. Journal
of Biomedical Materials Research 1994; 28:655-666.) It is further
believed that SDS residues remain bound to the collagen and prevent
cells from migrating into the implant. (Wilson, G J et al. Ann
Thorac Surg 1995; 60:S353-8. Bodnar E, et al. "Damage of aortic
valve tissue caused by the surfactant sodium dodecyl sulfate."
Thorac Cardiovasc Surg 1986; 34:82-85.) Because detergents used in
a chemical cleaning method can undesirably bind to and alter the
bioremodeling capabilities of collagen in the treated tissue, the
inventors have developed a method that eliminates the need for
detergents.
[0009] Chemical cleaning of tissue with enzymes such as trypsin,
pepsin and collagenase is known in the art but their use will
result in chemical modification of the native collagen molecules
and will adversely affect the structural integrity of the
construct. Enzyme treatment of collagenous tissue is known in the
art for removal and/or modification of extracellular matrix
associated proteins. Proteases such as pepsin, trypsin, dispase, or
thermolysin are used in the removal of collagen telopeptides to
yield atelopeptide collagen. Collagen telopeptides are the
non-triple helical portion of the collagen molecule and have been
thought by some researchers to be weakly antigenic while by others
they are thought to be responsible for the strong mechanical
properties of collagen. Limited digestion of collagenous tissue
will remove telopeptides without dissociation of the collagen
matrix of the tissue, while prolonged digestion will dissociate the
collagen fibrils into atelopeptide collagen monomers. It is also
known in the art to modify and remove nucleic acids from the matrix
using enzymes that digest endogenous RNA and DNA through use of
RNAse and DNAse, respectively. As treatment with enzymes can affect
the structural integrity of the collagen, the present method of the
invention circumvents their use.
[0010] Methods for obtaining collagenous tissue and tissue
structures from explanted mammalian tissue, and processes for
constructing prostheses from the tissue, have been widely
investigated for surgical repair or for tissue and organ
replacement. The tissue is typically treated to remove potentially
cytotoxic cellular and noncollagenous components to leave a natural
tissue matrix. Further processing, such as crosslinking,
disinfecting or forming into shapes have also been investigated.
Previous methods for treating collagenous tissue to remove tissue
components from the organized tissue matrix have employed
detergents, enzymes or promote uncontrolled swelling of the matrix.
WO 95/28183 to Jaffe, et al. discloses methods to decrease or
prevent bioprosthetic heart valve mineralization postimplantation.
The disclosed methods provide biological material made acellular by
controlled autolysis. Autolysis is controllably performed using at
least one buffer solution at a preselected pH to allow autolytic
enzymes present in the tissue to degrade cellular structural
components. U.S. Pat. No. 5,007,934 to Stone and, similarly, U.S.
Pat. No. 5,263,984 to Li, et al. both disclose a multiple step
method for chemical cleaning of ligamentous tissue. The method
utilizes a detergent to remove lipids associated with cell
membranes or collagenous tissue. U.S. Pat. No. 5,523,291 to Janzen,
et al. discloses an comminuted injectable implant composition for
soft tissue augmentation derived from ligamentum nuchae. The
ligament is treated with a series soaks in a strongly alkaline
solution of sodium hydroxide followed by hydrochloric acid solution
and then sodium bicarbonate. U.S. Pat. No. 5,028,695 to Eckmayer,
et al. discloses a process for the manufacture of collagen
membranes in which collagenous tissue is repeatedly treated with a
strong alkali and subsequently with a strong acid for a number of
times then further treated with inorganic saline treatment to
shrink the membranes and then with solvent to dry them.
SUMMARY OF THE INVENTION
[0011] Bioremodelable collagenous tissue matrices and methods for
chemical cleaning of native tissue to produce such tissue matrices
are disclosed.
[0012] The present invention overcomes the difficulties in
obtaining bioremodelable tissue matrices that are substantially
collagen. The invention provides tissue matrices that can be used
as a prosthetic device or material for use in the repair,
augmentation, or replacement of damaged and diseased tissues and
organs.
[0013] The chemical cleaning method of this invention renders
biological material, such as native tissues and tissue structures,
substantially acellular and substantially free of non-collagenous
components while maintaining the structural integrity of the
collagenous tissue matrix. As detergents are not used in the
chemical cleaning process, detergent residues that would normally
remain bound to the tissue matrix are not present. As enzymes are
not used, the collagen telopeptides are retained on the collagen
molecules. The method comprises contacting a normally cellular
native tissue with a chelating agent at a basic pH, contacting the
tissue with salt solution at an acidic pH, contacting the tissue
with a salt solution at a physiologic pH, and, then finally rinsing
the resultant chemically cleaned tissue matrix.
[0014] This invention is directed to a chemically cleaned tissue
matrix derived from native, normally cellular tissues. The cleaned
tissue matrix is essentially collagen rendered substantially free
of glycoproteins, glycosaminoglycans, proteoglycans, lipids,
non-collagenous proteins and nucleic acids such as DNA and RNA.
Importantly, the bioremodelability of the tissue matrix is
preserved as it is free of bound detergent residues that would
adversely affect the bioremodelability of the collagen. Further the
collagen is telopeptide collagen as the telopeptide regions of the
collagen molecules remain intact as it has not undergone treatment
or modification with enzymes during the cleaning process.
[0015] The collagenous material generally maintains the overall
shape of the tissue it is derived from but it may be layered and
bonded together to form multilayer sheets, tubes, or complex shaped
prostheses. The bonded collagen layers of the invention are
structurally stable, pliable, semi-permeable, and suturable. When
the matrix material is implanted into a mammalian host, it
undergoes biodegradation accompanied by adequate living cell
replacement, or neo-tissue formation, such that the original
implanted material is ultimately remodeled and replaced by host
derived tissue and cells.
[0016] It is, therefore, an object of this invention to provide a
method for cleaning native tissue resulting in a tissue matrix that
does not exhibit many of the shortcomings associated with many of
the methods developed previously. The method effectively removes
non-collagenous components of native tissue without the use of
detergents or enzymes to yield a tissue matrix comprised
substantially of collagen.
[0017] Another object is the provision of a bioremodelable tissue
matrix material that will allow for and facilitate tissue ingrowth
and/or organ regeneration at the site of implantation. Prostheses
prepared from this material, when engrafted to a recipient host or
patient, concomitantly undergoes controlled bioremodeling and
adequate living cell replacement such that the original implanted
prosthesis is remodeled by the patient's living cells to form a
regenerated organ or tissue.
[0018] Still another object of this invention is to provide a
method for use of a novel multi-purpose bioremodelable matrix
material in autografting, allografting, and heterografting
indications.
[0019] Still a further object is to provide a novel tissue matrix
material that can be implanted using conventional surgical
techniques.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The present invention provides a method for processing
native collagenous tissues for transplantation. The processing
method is designed to generate an implantable, graftable
collagenous biological tissue material, an extracellular matrix
comprising collagen, that serves as a scaffold that can be
bioremodeled by a host in vivo or by living cells in culture in
vitro.
[0021] This invention is further directed to a tissue engineered
prostheses formed from processed native collagenous tissue, which,
when implanted into a mammalian host, can serve as a functioning
repair, augmentation, or replacement body part, or tissue
structure, and will undergo controlled biodegradation occurring
concomitantly with remodeling by the host's cells. The tissue
matrix can be used as a prosthetic material for autografting,
allografting, and heterografting indications. The prosthesis of
this invention, in its various embodiments, thus has dual
properties: First, it functions as a substitute body part, and
second, while still functioning as a substitute body part, it
functions as a remodeling template for the ingrowth of host cells.
Although the prostheses will be illustrated through construction of
various devices and constructs, the invention is not so limited. It
will be appreciated that the device design in its material, shape
and thickness is to be selected depending on the ultimate
indication for the construct.
[0022] The chemical cleaning method of this invention renders
biological material, such as native tissues and tissue structures,
substantially acellular and substantially free of non-collagenous
components while maintaining the structural integrity of the
collagenous tissue matrix. Elastin is sometimes present in native
tissue in small amounts and is not removed by the chemical cleaning
method. The presence of elastin may be desirable for certain
applications. As used herein, the term, "substantially acellular"
means having at least 95% fewer native cells and cell structures
than the natural state of the biological material. "Cells and
cellular structures" refer to cells, living or not living, cell
remnants, cell membranes and membrane structures. By use of the
term, "substantially free of non-collagenous components",
Applicants mean that glycoproteins, glycosaminoglycans,
proteoglycans, lipids, non-collagenous proteins and nucleic acids
such as DNA and RNA comprise less than 5% of the resultant tissue
matrix. As detergents are not used in the chemical cleaning
process, detergent residues that would normally remain bound to the
tissue matrix are not present. As enzymes are not used, the
collagen telopeptides are retained on the collagen molecules.
Further, the chemical cleaning method renders the biological
material both sterile and endotoxin free when processed using
sterile equipment, solutions and aseptic technique.
[0023] The term, "structural integrity", refers to the capacity of
the chemically cleaned collagenous tissue matrix to withstand
forces such as tension, compression, and support. The structural
integrity of the biological material is preserved as swelling is
minimized in the chemical treatment steps even though some swelling
will occur during treatment. Uncontrolled or excessive swelling
both opens the structure of the collagen molecule making it
susceptible to cellular enzymes such as collagenase and
destabilizes the collagen to result in a weakened construct. As
swelling affects the intramolecular structure of the collagen
molecule, it affects the overall structure of the material on an
intermolecular level by disrupting the native crosslinks between
collagen molecules. Together, the structure of the collagen
molecule and the crosslinks between collagen molecules lend
structural integrity to the material.
[0024] Tissue matrix material that maintains much of its native
structural integrity is useful, for instance, when used as a
prosthetic device or as material to construct mulitilayered or
complex devices. The integrity of the material is important if it
is to perform a load bearing function such as a body wall support,
a vascular device, or an orthopedic device. Related to structural
integrity is the term "suturable" which means that the mechanical
properties of the material includes suture retention which permits
needles and suture materials to pass through the prosthesis
material at the time of suturing of the prosthesis to sections of
native tissue, a process known as anastomosis. During suturing,
such prostheses must not tear as a result of the tensile forces
applied to them by the suture, nor should they tear when the suture
is knotted. Suturability of the prosthetic material, i.e., the
ability of prostheses to resist tearing while being sutured, is
related to the intrinsic mechanical strength of the prosthesis
material, the thickness of the graft, the tension applied to the
suture, and the rate at which the knot is pulled closed.
[0025] Biological material as defined in the invention includes but
is not limited to harvested mammalian tissues, and structures
thereof, derived from human, bovine, porcine, canine, ovine,
caprine, and equine organisms. Tissue structures such as dermis,
artery, vein, pericardium, heart valve, dura mater, ligament,
intestine and fascia are all preferred tissue structures that are
able to be cleaned by the methods of this invention to yield a
tissue matrix that is substantially acellular and substantially
free of non-collagenous components.
[0026] A preferred source of mammalian tissue is the tunica
submucosa from small intestine, most preferably from porcine small
intestine. In native small intestine, the tunica submucosa is the
connective tissue layer of the organ and comprises both lymphatic
and blood vessel cells. Methods for obtaining tunica submucosa are
disclosed in WO 96/31157 and is incorporated herein. To obtain
porcine tunica submucosa, also termed "submucosa", the small
intestine of a pig is harvested and mechanically stripped,
preferably by use of a gut cleaning machine (Bitterling,
Nottingham, UK). The gut cleaning machine forcibly removes the fat,
muscle and mucosal layers from the tunica submucosa using a
combination of mechanical action and washing with water. The
mechanical action can be described as a series of rollers that
compress and strip away the successive layers from the tunica
submucosa when the intact intestine is run between them. As the
tunica submucosa of the small intestine is comparatively harder and
stiffer than the surrounding tissue, the softer components from the
submucosa are removed from the tunica submucosa. The result of the
machine cleaning is such that the mesenteric tissues, the tunica
serosa and the tunica muscularis from the ablumen of the tunica
submucosa and as well as the layers of the tunica mucosa from the
lumen of the tunica submucosa are removed from the tunica submucosa
so that the tunica submucosa layer of the intestine solely remains.
The chemically cleaned tissue matrix of the tunica submucosa is
also termed "intestinal collagen layer" or "ICL". It is noted that
in some animal sources, such as carnivores and omnivores, the small
intestine includes a stratum compactum which is also removed by
this mechanical cleaning step.
[0027] Other methods of mechanically stripping layers of the small
intestine are known in the art as described in U.S. Pat. No.
4,902,508 to Badylak, incorporated herein by reference. The method
disclosed by this patent includes mild abrasion of the intestinal
tissue to remove the abluminal layers, including the tunica serosa
and the tunica muscularis, and the inner layers consisting of at
least the luminal portion of the tunica mucosa. The layers that
remain are the tunica submucosa with the attached basilar layer
consisting of lamina muscularis mucosa and, if initially present in
the harvested mammalian tissue, stratum compactum. Intestinal
material obtained by either method can be implanted or first formed
into body wall or vascular device by a number of methods including
suturing, stapling, adhesive compositions, chemical bonding and
thermal bonding.
[0028] Terms pertaining to certain operating parameters are defined
for the entire specification and the examples for amounts, times
and temperatures that can be varied without departing from the
spirit and scope of the invention. As used herein, an "effective
amount" refers to the volume and concentration of composition
required to obtain the effect desired. A preferred effective amount
for the chemical cleaning of tissue is a ratio of 100:1 v/v of
solution to tissue but volumes more or less can be determined by
the skilled artisan when considering the shape, bulk, thickness,
density, and cellularity of the tissue to be cleaned. The time
required for the chemical steps to be effective can be appreciated
by those of skill in the art when considering the cellularity,
matrix density, and thickness of the material to be cleaned.
Larger, thicker, or denser materials will take longer for the
solutions to penetrate and equilibrate in tissue. The temperatures
for the environment and the solutions used in the present invention
is preferably at ambient room temperature, about 25.degree. C., but
can be anywhere in the range of above the freezing temperatures of
the solutions used to less than the denaturation temperature of the
tissue material being treated. Temperatures between about 4.degree.
C. to about 45.degree. C. are sufficient for the cleaning treatment
to be effective. Agitation is meant to be mechanical shaking or
mixing and is used to improve the penetration of the chemical
compositions into the tissue and to reduce the time needed for
chemical treatment to be effective. The term "buffered solution"
refers to an aqueous solution containing at least one agent which
preserves the hydrogen ion concentration or pH of the solution.
[0029] In the preferred method, harvested tissue may need to be
cleaned manually, as by gross dissection, and/or mechanically
cleaned of excess tissues such as fat and vasculature. Manual
cleaning may be necessary for some tissues for handling
manageability during processing or for most effective chemical
treatment.
[0030] The tissue is first treated by contacting the tissue with an
effective amount of chelating agent, preferably physiologically
alkaline to controllably limit swelling of the tissue matrix.
Chelating agents enhance removal of cells, cell debris and basement
membrane structures from the matrix by reducing divalent cation
concentration. Alkaline treatment dissociates glycoproteins and
glycosaminoglycans from the collagenous tissue and saponifies
lipids. Chelating agents known in the art which may be used
include, but are not limited to, ethylenediaminetetraacetic acid
(EDTA) and ethylenebis(oxyethylenitrilo)tetraacetic acid (EGTA).
EDTA is a preferred chelating agent and may be made more alkaline
by the addition of sodium hydroxide (NaOH), calcium hydroxide
Ca(OH).sub.2, sodium carbonate or sodium peroxide. EDTA or EGTA
concentration is preferably between about 1 to about 200 mM; more
preferably between about 50 to about 150 mM; most preferably around
about 100 mM. NaOH concentration is preferably between about 0.001
to about 1 M; more preferably between about 0.001 to about 0.10 M;
most preferably about 0.01 M. Other alkaline or basic agents can be
determined by one of skill in the art to bring the pH of the
chelating solution within the effective basic pH range. The final
pH of the basic chelating solution should be preferably between
about 8 and about 12, but more preferably between about 11.1 to
about 11.8. In the most preferred embodiment, the tissue is
contacted with a solution of 100 mM EDTA/10 mM NaOH in water. The
tissue is contacted preferably by immersion in the alkaline
chelating agent while more effective treatment is obtained by
agitation of the tissue and the solution together for a time for
the treatment step to be effective.
[0031] The tissue is then contacted with an effective amount of
acidic solution, preferably containing a salt. Acid treatment also
plays a role in the removal of glycoproteins and glycosaminoglycans
as well as in the removal of non-collagenous proteins and nucleic
acids such as DNA and RNA. Salt treatment controls swelling of the
collagenous tissue matrix during acid treatment and is involved
with removal of some glycoproteins and proteoglycans from the
collagenous matrix. Acid solutions known in the art may be used and
may include but are not limited to hydrochloric acid (HCl), acetic
acid (CH.sub.3COOH) and sulfuric acid (H.sub.2SO.sub.4). A
preferred acid is hydrochloric acid (HCl) at a concentration
preferably between about 0.5 to about 2 M, more preferably between
about 0.75 to about 1.25 M; most preferably around 1 M. The final
pH of the acid/salt solution is preferably between about 0 to about
1, more preferably between about 0 and 0.75, and most preferably
between about 0.1 to about 0.5. Hydrochloric acid and other strong
acids are most effective for breaking up nucleic acid molecules
while weaker acids are less effective. Salts that may be used are
preferably inorganic salts and include but are not limited to
chloride salts such as sodium chloride (NaCl), calcium chloride
(CaCl.sub.2), and potassium chloride (KCl) while other effective
salts may be determined by one of skill in the art. Preferably
chloride salts are used at a concentration preferably between about
0.1 to about 2 M; more preferably between about 0.75 to about 1.25
M; most preferably around 1 M. A preferred chloride salt for use in
the method is sodium chloride (NaCl). In the most preferred
embodiment, the tissue is contacted with 1 M HCl/1 M NaCl in water.
The tissue is contacted preferably by immersion in the acid/salt
solution while effective treatment is obtained by agitation of the
tissue and the solution together for a time for the treatment step
to be effective.
[0032] The tissue is then contacted with an effective amount of
salt solution which is preferably buffered to about a physiological
pH. The buffered salt solution neutralizes the material while
reducing swelling. Salts that may be used are preferably inorganic
salts and include but are not limited to chloride salts such as
sodium chloride (NaCl), calcium chloride (CaCl.sub.2), and
potassium chloride (KCl); and nitrogenous salts such as ammonium
sulfate (NH.sub.3SO.sub.4) while other effective salts may be
determined by one of skill in the art. Preferably chloride salts
are used at a concentration preferably between about 0.1 to about 2
M; more preferably between about 0.75 to about 1.25 M; most
preferably about 1 M. A preferred chloride salt for use in the
method is sodium chloride (NaCl). Buffering agents are known in the
art and include but are not limited to phosphate and borate
solutions while others can be determined by the skilled artisan for
use in the method. One preferred method to buffer the salt solution
is to add phosphate buffered saline (PBS) preferably wherein the
phosphate is at a concentration from about 0.001 to about 0.02 M
and a salt concentration from about 0.07 to about 0.3 M to the salt
solution. A preferred pH for the solution is between about 5 to
about 9, more preferably between about 7 to about 8, most
preferably between about 7.4 to about 7.6. In the most preferred
embodiment, the tissue is contacted with 1 M sodium chloride
(NaCl)/10 mM phosphate buffered saline (PBS) at a pH of between
about 7.0 to about 7.6. The tissue is contacted preferably by
immersion in the buffered salt solution while effective treatment
is obtained by agitation of the tissue and the solution together
for a time for the treatment step to be effective.
[0033] After chemical cleaning treatment, the tissue is then
preferably rinsed free of chemical cleaning agents by contacting it
with an effective amount of rinse agent. Agents such as water,
isotonic saline solutions and physiological pH buffered solutions
can be used and are contacted with the tissue for a time sufficient
to remove the cleaning agents. A preferred rinse solution is
physiological pH buffered saline such as phosphate buffered saline
(PBS). Other means for rinsing the tissue of chemical cleaning
agents can be determined by one of skill in the art. The cleaning
steps of contacting the tissue with an alkaline chelating agent and
contacting the tissue with an acid solution containing salt may be
performed in either order to achieve substantially the same
cleaning effect. The solutions may not be combined and performed as
a single step, however.
[0034] A preferred composition of the invention is a chemically
cleaned tissue matrix derived from native, normally cellular
tissues. The cleaned tissue matrix is essentially acellular
telopeptide collagen, about 93% by weight, with less than about 5%
glycoproteins, glycosaminoglycans, proteoglycans, lipids,
non-collagenous proteins and nucleic acids such as DNA and RNA.
Importantly, the bioremodelability of the tissue matrix is
preserved as it is free of bound detergent residues that would
adversely affect the bioremodelability of the collagen.
Additionally, the collagen molecules have retained their
telopeptide regions as the tissue has not undergone treatment with
enzymes during the cleaning process.
[0035] Tissue matrices are derived from dermis, artery, vein,
pericardium, heart valves, dura mater, ligaments, intestine and
fascia. A most preferred composition is a chemically cleaned
intestinal collagen layer derived from the small intestine.
Suitable sources for small intestine are mammalian organisms such
as human, cow, pig, sheep, dog, goat or horse while small intestine
of pig is the preferred source. In one preferred embodiment, the
collagen layer comprises the tunica submucosa derived from porcine
small intestine. In another embodiment, the collagen layer
comprises the tunica submucosa and the basilar layers of the small
intestine. The basilar layers consist of lamina muscularis mucosa
and, if present in the native tissue, the stratum compactum.
[0036] The most preferred composition of the invention is the
intestinal collagen layer, cleaned by the chemical cleaning method
of the invention, which is essentially collagen, primarily Type I
collagen, with less than about 5% glycoproteins,
glycosaminoglycans, proteoglycans, lipids, non-collagenous proteins
and nucleic acids such as DNA and RNA. The collagen layer is free
of bound detergent residues that would adversely affect the
bioremodelability of the collagen. The collagen layer is
substantially free of cells and cellular debris, including
endogenous nucleic acids such as DNA and RNA and lipids. Further,
the intestinal collagen layer is both sterile and endotoxin free
when processed using sterile equipment, solutions and aseptic
technique.
[0037] Once the collagenous tissue matrix has been rendered
substantially acellular and free of substantially noncollagenous
extracellular matrix components, prostheses for implantation or
engraftment may be manufactured therefrom. Collagen layers may be
sutured or bonded together by use of any variety of techniques
known in the art. Methods for bonding the layers may employ
adhesives such as thrombin, fibrin or synthetic materials such as
cyanomethacrylates or chemical crosslinking agents. Other methods
may employ heat generated by laser, light, or microwaves.
Convection ovens and heated liquid baths may also be employed.
[0038] Thermal welding of the collagen layers is the preferred
method for bonding together the collagen layers of the invention.
Methods for thermal welding of collagen are described in WO
95/22301, WO 96/31157 and U.S. Pat. No. 5,571,216, the teachings of
which are incorporated herein by reference. The ICL is first cut
longitudinally and flattened onto a solid, flat plate. One or more
successive layers are then superimposed onto one another,
preferably in alternating perpendicular orientation. A second solid
flat plate is placed on top of the layers and the two plates are
clamped tightly together. The complete apparatus, clamped plates
and collagen layers, are then heated for a time and under
conditions sufficient to effect the bonding of the collagen layers
together. The amount of heat applied should be sufficiently high to
allow the collagen to bond, but not so high as to cause the
collagen to irreversibly denature. The time of the heating and
bonding will depend upon the type of collagen material layer used,
the moisture content and thickness of the material, and the applied
heat. A typical range of heat is from about 50.degree. C. to about
75.degree. C., more typically 60.degree. C. to 65.degree. C. and
most typically 62.degree. C. A typical range of times will be from
about 7 minutes to about 24 hours, typically about one hour. The
degree of heat and the amount of time that the heat is applied can
be readily ascertained through routine experimentation by varying
the heat and time parameters. The bonding step may be accomplished
in a conventional oven, although other apparatus or heat
applications may be used including, but not limited to, a water
bath, laser energy, or electrical heat conduction. Immediately
following the heating and bonding, the collagen layers are cooled,
in air or a water bath, at a range between room temperature at
20.degree. C. and 1.degree. C. Rapid cooling, termed quenching, is
required to stop the heating action and to create an effective bond
between the collagen layers. To accomplish this step, the collagen
layers may be cooled, typically in a water bath, with a temperature
preferably between about 1.degree. C. to about 10.degree. C., most
preferably about 4.degree. C. Although cooling temperatures below
1.degree. C. may be used, care will need to be taken not to freeze
the collagen layers, which may cause structural damage. In
addition, temperatures above 10.degree. C. may be used in
quenching, but if the temperature of the quench is too high, then
the rate of cooling may not be sufficient to fix the collagen
layers to one another.
[0039] In the preferred embodiment, the collagenous material is
crosslinked. Crosslinking imparts increased strength and structural
integrity to the formed prosthetic construct while regulating the
bioremodeling of the collagen by cells when the construct is
implanted into a patient. Collagen crosslinking agents include
glutaraldehyde, formaldehyde, carbodiimides, hexamethylene
diisocyanate, bisimidates, glyoxal, adipyl chloride, dialdehyde
starch, and certain polyepoxy compounds such as glycol diglycidyl
ether, polyol polyglycidyl ether and dicarboxylic acid
diglycidylester. Dehydrothermal, UV irradiation and/or
sugar-mediated methods may also be used. Collagen will also
naturally crosslink with age standing at room temperature. However,
crosslinking agents need not be limited to these examples as other
crosslinking agents and methods known to those skilled in the art
may be used. Crosslinking agents should be selected so as to
produce a biocompatible material capable of being remodeled by host
cells. A preferred crosslinking agent is
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC).
The crosslinking solution containing EDC and water may also contain
acetone. Crosslinking with EDC had been described in International
PCT Publication Nos. WO 95/22301 and WO 96/31157.
[0040] In some embodiments, additional collagenous layers may be
added to either the outer or inner surfaces of the bonded collagen
layers, either before or after crosslinking. In tubular constructs,
as in a vascular construct, dense fibrillar collagen may be added
to the luminal surface to create a smooth flow surface for its
ultimate application as described in International PCT Publication
No. WO 95/22301, incorporated herein by reference. This smooth
collagenous layer also promotes host cell attachment, as in the
formation of neointima, which facilitates ingrowth and
bioremodeling of the construct. As described in International PCT
Publication No. WO 95/22301, this smooth collagenous layer may be
made from acid-extracted fibrillar or non-fibrillar collagen, which
is predominantly type I collagen, but may also include other types
of collagen. The collagen used may be derived from any number of
mammalian sources, typically bovine, porcine, or ovine skin or
tendons. The collagen preferably has been processed by acid
extraction to result in a fibril dispersion or gel of high purity.
Collagen may be acid-extracted from the collagen source using a
weak acid, such as acetic, citric, or formic acid. Once extracted
into solution, the collagen can be salt-precipitated using NaCl and
recovered, using standard techniques such as centrifugation or
filtration. Details of acid extracted collagen from bovine tendon
are described, for example, in U.S. Pat. No. 5,106,949,
incorporated herein by reference.
[0041] Heparin can be applied to the prosthesis, by a variety of
well-known techniques. For illustration, heparin can be applied to
the prosthesis in the following three ways. First, benzalkonium
heparin (BA-Hep) solution can be applied to the prosthesis by
dipping the prosthesis in the solution and then air-drying it. This
procedure treats the collagen with an ionically bound BA-Hep
complex. Second, EDC can be used to activate the heparin, then to
covalently bond the heparin to the collagen fiber. Third, EDC can
be used to activate the collagen, then covalently bond protamine to
the collagen and then ionically bond heparin to the protamine. Many
other coating, bonding, and attachment procedures are well known in
the art which could also be used.
[0042] Treatment of the tissue matrix material with agents such as
growth factors or pharmaceuticals in addition to or in substitution
for heparin may be accomplished. The agents may include for
example, growth factors to promote vascularization and
epithelialization, such as macrophage derived growth factor (MDGF),
platelet derived growth factor (PDGF), vascular endothelial cell
derived growth factor (VEGF); antibiotics to fight any potential
infection from the surgery implant; or nerve growth factors
incorporated into the inner collagenous layer when the prosthesis
is used as a conduit for nerve regeneration. In addition to or in
substitution for drugs, matrix components such as proteoglycans or
glycoproteins or glycosaminoglycans may be included within the
construct.
[0043] The collagenous prosthesis thus formed can also be
sterilized in a dilute peracetic acid solution with a neutral pH.
Methods for sterilizing collagen are described U.S. Pat. No.
5,460,962 and are incorporated by reference herein. In the
preferred method, the collagen is disinfected with a dilute
peracetic acid solution at a neutral pH. The peracetic acid
concentration is preferably between about 0.01 and 0.3% v/v in
water at a neutralized pH between about pH 6 and pH 8.
Alternatively, sterilization with gamma irradiation, at typically
2.5 Mrad, or with gas plasma can also be used to sterilize the
collagen. Other methods known in the art for sterilizing collagen
may also be used.
[0044] The following examples are provided to better explain the
practice of the present invention and should not be interpreted in
any way to limit the scope of the present invention. Those skilled
in the art will recognize that various modifications can be made to
the methods described herein while not departing from the spirit
and scope of the present invention.
EXAMPLES
Example 1
Chemical Cleaning of Mechanically Stripped Porcine Small
Intestine
[0045] The small intestine of a pig was harvested and mechanically
stripped, using a Bitterling gut cleaning machine (Nottingham, UK)
which forcibly removes the fat, muscle and mucosal layers from the
tunica submucosa using a combination of mechanical action and
washing using water. The mechanical action can be described as a
series of rollers that compress and strip away the successive
layers from the tunica submucosa when the intact intestine is run
between them. The tunica submucosa of the small intestine is
comparatively harder and stiffer than the surrounding tissue, and
the rollers squeeze the softer components from the submucosa. The
result of the machine cleaning was such that the submucosal layer
of the intestine solely remained. The remainder of the procedure
was performed under aseptic conditions and at room temperature. The
chemical solutions were all used at room temperature. The intestine
was then cut lengthwise down the lumen and then cut into 15 cm
sections. Material was weighed and placed into containers at a
ratio of about 100:1 v/v of solution to intestinal material.
[0046] A. To each container containing intestine was added
approximately 1 L solution of 0.22 mm (micron) filter sterilized
100 mM ethylenediaminetetraacetic tetrasodium salt (EDTA)/10 mM
sodium hydroxide (NaOH) solution. Containers were then placed on a
shaker table for about 18 hours at about 200 rpm. After shaking,
the EDTA/NaOH solution was removed from each bottle.
[0047] B. To each container was then added approximately 1 L
solution of 0.22 mm filter sterilized 1 M hydrochloric acid (HCl)/1
M sodium chloride (NaCl) solution. Containers were then placed on a
shaker table for between about 6 to 8 hours at about 200 rpm. After
shaking, the HCl/NaCl solution was removed from each container.
[0048] C. To each container was then added approximately 1 L
solution of 0.22 mm filter sterilized 1 M sodium chloride (NaCl)/10
mM phosphate buffered saline (PBS). Containers were then placed on
a shaker table for approximately 18 hours at 200 rpm. After
shaking, the NaCl/PBS solution was removed from each container.
[0049] D. To each container was then added approximately 1 L
solution of 0.22 mm filter sterilized 10 mM PBS. Containers were
then placed on a shaker table for about two hours at 200 rpm. After
shaking, the phosphate buffered saline was then removed from each
container.
[0050] E. Finally, to each container was then added approximately 1
L of 0.22 mm filter sterilized water. Containers were then placed
on a shaker table for about one hour at 200 rpm. After shaking, the
water was then removed from each container.
[0051] Treated samples were cut and fixed for histological
analyses. Hemotoxylin and eosin (H&E) and Masson trichrome
staining was performed on both cross-section and long-section
samples of both control and treated tissues. Treated tissue samples
appeared free of cells and cellular debris while control samples
appeared normally and expectedly very cellular.
Example 2
Chemical Cleaning of Porcine Heart Valve
[0052] A porcine heart was procured from a 1 pound piglet and
shipped in physiological pH saline on ice. Within 4 hours, the
heart valves were removed from the heart mass using scalpel and
forceps. Some further gross dissection was performed to remove
excess tissue from around the valves. One valve was retained as a
control with sample pieces cut and fixed for various histological
analyses while the other valve underwent the chemical cleaning
process. The remainder of the procedure was performed under aseptic
conditions and at room temperature. The chemical solutions were all
used at room temperature.
[0053] The valve was placed into 1 L solution of 100 mM EDTA/10 mM
NaOH for about 18 hours while agitating on a shaker platform. The
valve was then placed into 1 L of 1 M HCl/1 M NaCl and agitated for
8 hours. The valve was then placed into 1 L solution of 1 M HCl/10
mM phosphate buffered saline (PBS) and agitated for about 18 hours.
The valve was then rinsed in PBS for between about 24 hours and
then finally rinsed in sterile water for about 1 hour while
agitating. Treated sample pieces were then cut and fixed for
various histological analyses.
[0054] Hemotoxylin and eosin (H&E) and Masson trichrome
staining was performed on both cross-section and long-section
samples of both control and treated valves. Treated valve samples
appeared free of cells and cellular debris while control samples
appeared normally and expectedly very cellular.
Example 3
Chemical Cleaning of Porcine Artery, Pericardium and Fascia
[0055] A segment of femoral artery, the entire pericardium, and
fascia were procured from a 450 lb. sow. The tissues were shipped
in physiological pH saline on ice. The tissues were dissected
further to remove excess tissue. Samples of each tissue were taken
without cleaning for control samples and fixed for various
histological analyses while the remainder of the tissues underwent
the chemical cleaning process. The remainder of the procedure was
performed under aseptic conditions and at room temperature. The
chemical solutions were all used at room temperature.
[0056] The tissues were separately placed into 1 L solution of 100
mM EDTA/10 mM and agitated on a shaker platform for about 18 hours.
The tissues were then each separately placed into 1 L solution of 1
M HCl/1 M NaCl and agitated for 8 hours. Next, the tissues were
separately placed into a 1 L solution of 1 M HCl/10 mM phosphate
buffered saline (PBS) and then agitated for about 18 hours. The
tissues were then separately rinsed in PBS for between about 2 to 4
hours and then finally rinsed in sterile water for about 1 hour
while agitating. Treated sample pieces were then cut and fixed for
various histological analyses.
[0057] Hemotoxylin and eosin (H&E) and Masson trichrome
staining was performed on both cross-section and long-section
samples of both control and treated tissues. Treated tissue samples
appeared free of cells and cellular debris while control samples
appeared normally and expectedly very cellular.
Example 4
Differently Ordered Chemical Cleaning
[0058] This procedure was performed under aseptic conditions and at
room temperature and all chemical solutions were used at room
temperature.
[0059] Mechanically stripped porcine intestine was cut into five 15
cm sections as described in example 1.
[0060] To each container was then added approximately 1 L of 0.22
mm filter sterilized solution of 1 M hydrochloric acid (HCl)/1 M
sodium chloride (NaCl). Containers were then placed on a shaker
table for between about 6 to 8 hours at about 200 rpm. After
shaking, the HCl/NaCl solution was removed from each container.
[0061] To each container containing intestine was added
approximately 1 L of 0.22 mm (micron) filter sterilized solution of
100 mM ethylenediaminetetraacetic (EDTA)/10 mM sodium hydroxide
(NaOH) solution. Containers were then placed on a shaker table for
about 18 hours at about 200 rpm. After shaking, the EDTA/NaOH
solution was removed from each bottle.
[0062] To each container was then added approximately 1 L of 0.22
mm filter sterilized solution of 1 M sodium chloride (NaCl)/10 mM
phosphate buffered saline (PBS). Containers were then placed on a
shaker table for approximately 18 hours at 200 rpm. After shaking,
NaCl/PBS solution was removed from each container.
[0063] To each container was then added approximately 1 L of 0.22
mm filter sterilized solution of 10 mM PBS. Containers were then
placed on a shaker table for about one hour at 200 rpm. After
shaking, the phosphate buffered saline was then removed from each
container.
[0064] Finally, to each container was then added approximately 1 L
of 0.22 mm filter sterilized water. Containers were then placed on
a shaker table for about one hour at 200 rpm. After shaking, the
water was then removed from each container.
[0065] Treated sample pieces were then cut and fixed for various
histological analyses. Hemotoxylin and eosin (H&E) and Masson
trichrome staining was performed on both cross-section and
long-section samples of both control and treated tissues. Treated
tissue samples appeared free of cells and cellular debris while
control samples appeared normally and expectedly very cellular.
Example 5
Various Alkaline and Chelating Agents
[0066] The cleaning of mechanically stripped porcine intestinal
submucosa was followed as according to example 1. This procedure
was performed under aseptic conditions and at room temperature and
all chemical solutions were used at room temperature. The chemical
cleaning process of example 1 was followed but with the
substitution of the alkaline chelating agent of step A was
substituted with other alkaline chelating agents of similar
nature:
[0067] A. To each container containing intestine was added
approximately 1 L of 0.22 mm (micron) filter sterilized solution of
either 100 mM ethylenebis(oxyethylenitrilo)tetraacetic acid
(EGTA)/10 mM NaOH; 100 mM EDTA/10 mM Ca(OH).sub.2 (calcium
hydroxide); or, 100 mM EDTA/10 mM K2CO3 (potassium carbonate)
solution. Containers were then placed on a shaker table for about
18 hours at about 200 rpm. After shaking, the alkaline chelating
agents solution was removed from each bottle.
[0068] B. To each container was then added approximately 1 L of
0.22 mm filter sterilized solution of 1 M hydrochloric acid (HCl)/1
M sodium chloride (NaCl) solution. Containers were then placed on a
shaker table for between about 6 to 8 hours at about 200 rpm. After
shaking, the HCl/NaCl solution was removed from each container.
[0069] C. To each container was then added approximately 1 L of
0.22 mm filter sterilized solution of 1 M sodium chloride (NaCl)/10
mM phosphate buffered saline (PBS). Containers were then placed on
a shaker table for approximately 18 hours at 200 rpm. After
shaking, NaCl/PBS solution was removed from each container.
[0070] D. To each container was then added approximately 1 L of
0.22 mm filter sterilized solution of 10 mM PBS. Containers were
then placed on a shaker table for about one hour at 200 rpm. After
shaking, the phosphate buffered saline was then removed from each
container.
[0071] E. Finally, to each container was then added approximately 1
L of 0.22 mm filter sterilized water. Containers were then placed
on a shaker table for about one hour at 200 rpm. After shaking, the
water was then removed from each container. Samples were fixed for
histological analyses.
[0072] Hemotoxylin and eosin (H&E) and Masson trichrome
staining was performed on both cross-section and long-section
samples of both control and treated tissues. Treated tissue samples
appeared free of cells and cellular debris while control samples
appeared normally and expectedly very cellular.
Example 6
Various Acid and Salt Agents
[0073] The mechanically stripped porcine intestinal submucosa of
example 1 was chemically cleaned using a substituted acid agent or
substituted salt agent in step B. This procedure was performed
under aseptic conditions and at room temperature and all chemical
solutions were used at room temperature.
[0074] A. To each container containing intestine was added
approximately 1 L solution of 0.22 mm (micron) filter sterilized
100 mM ethylenediaminetetraacetic tetrasodium salt (EDTA)/10 mM
sodium hydroxide (NaOH) solution. Containers were then placed on a
shaker table for about 18 hours at about 200 rpm. After shaking,
the EDTA/NaOH solution was removed from each bottle.
[0075] B. To each container was then added approximately 1 L of
0.22 mm filter sterilized solution of either 1 M CH3COOH (acetic
acid)/1 M NaCl or 1 M H2SO4 (sulfuric acid)/1 M NaCl solution.
Containers were then placed on a shaker table for between about 6
to 8 hours at about 200 rpm. After shaking, the solution was
removed from each container.
[0076] C. To each container was then added approximately 1 L of
0.22 mm filter sterilized 1 M sodium chloride (NaCl)/10 mM
phosphate buffered saline (PBS). Containers were then placed on a
shaker table for approximately 18 hours at 200 rpm. After shaking,
NaCl/PBS solution was removed from each container.
[0077] D. To each container was then added approximately 1 L of
0.22 mm filter sterilized 10 mM PBS. Containers were then placed on
a shaker table for about one hour at 200 rpm. After shaking, the
phosphate buffered saline was then removed from each container.
[0078] E. Finally, to each container was added approximately 1 L of
0.22 mm filter sterilized water. Containers were then placed on a
shaker table for about one hour at 200 rpm. After shaking, the
water was then removed from each container.
[0079] Treated sample pieces were then cut and fixed for various
histological analyses. Hemotoxylin and eosin (H&E) and Masson
trichrome staining was performed on both cross-section and
long-section samples of both control and treated tissues. Treated
tissue samples appeared free of cells and cellular debris while
control samples appeared normally and expectedly very cellular.
Example 7
Glycosaminoglycan (GAG) Content of ICL Determined by Cellulose
Acetate Gel Electrophoresis and Alcian Blue Assay
[0080] To determine GAG content of ICL, cellulose acetate gel
electrophoresis with subsequent alcian blue stain was performed on
extracts of chemically cleaned ICL.
[0081] Samples of ICL underwent the chemical cleaning regimen
outlined in Example 1, cut into 0.125 cm.sup.2 pieces and placed
into eppendorf tubes. To digest the samples, 100 .mu.l of papain
(0.1 mg/ml papain in 0.1 M sodium phosphate, 0.1 M sodium chloride,
0.005 M EDTA, 0.9 mg/ml cysteine, pH 5.8) was added to each tube
and allowed to incubate for about 18 hours at 60.degree. C.
Standard containing known amounts of GAG (heparin) were prepared in
parallel. Dowex (0.4 g HCl form) and 3 ml water were then added.
After spinning to remove the Dowex resin, 1 ml was removed and
lyophilized. The samples were then rehydrated in 100 .mu.l purified
water and centrifuged for about 5 minutes.
[0082] Samples were separated on cellulose-acetate sheets using the
method of Newton, et al. (1974). Cellulose-acetate sheets were
soaked in 0.1 M lithium chloride/EDTA buffer (pH 5.8) and blotted
gently. Samples (5 .mu.l each) were applied to the sheets at the
cathode end and electrophoresed for 30 minutes at 5 mA.
[0083] Following electrophoresis, the sheets were immersed
immediately in an alcian blue stain solution (0.2% alcian blue 8GX,
0.05 M magnesium chloride, 0.025 M sodium acetate buffer (pH 5.8)
in 50% ethylene alcohol) and placed on a shaker platform for about
30 minutes at room temperature. The sheets were then destained in
at least three washes of destaining solution (0.05 M magnesium
chloride, 0.025 M sodium acetate buffer (pH 5.8) in 50% ethylene
alcohol) for a total of about 30 minutes on a shaker platform. No
detectable GAG staining was observed for papain digested ICL while
as little as 0.005 microgram heparin standard was detectable.
[0084] These results showed that the total amount of GAG remaining
in chemically cleaned ICL is less than 1% (dry weight).
Example 8
Lipid Content of ICL Determined by Methylene Chloride
Extraction
[0085] ICL was laid out flat on plastic plates and air dried for
two hours. Once dried, ICL was cut into smaller pieces of about 1
cm.sup.2 of which 1.100 g were transferred to a soxhlet
thimble.
[0086] To a Kontes brand round bottom flask 24/40 was added 90 ml
methylene chloride. The soxhlet was assembled in the fume hood with
the bottom of the flask in a heated water bath and ice cooled water
running through the distiller.
[0087] Extraction was allowed to proceed for four hours after which
the soxhlet was disassembled. The round bottom flask containing the
solvent and extracted material was left in the heated water bath
until methylene chloride was evaporated until there remained 5 ml.
The methylene chloride was then transferred to a 11.times.13 glass
culture tube and the remaining solvent was boiled off. To the tube
was added 2 ml of methylene chloride and the tube was capped
immediately and the tube placed in a -20.degree. C. freezer.
[0088] The weight of the extracted material was then determined.
The glass tube was placed in an ice bath. The weight of a Ludiag
1.12 ml aluminum weigh boat was tared on a microbalance (Spectrum
Supermicro). 10 .mu.l of resuspended extraction was added to the
weigh boat and the solvent was boiled off by placing the weigh boat
on a hot plate for 45 seconds. The weigh boat was allowed to cool
for about 190 seconds and was placed on the microbalance. The
procedure was then repeated for extract volumes of 20 .mu.l and 30
.mu.l.
[0089] Results indicate that the percentage of lipid is less than
about 0.7% lipid by weight in dry chemically cleaned ICL. In
contrast, non-chemically cleaned ICL contains a higher fraction of
lipid; at least about 1.5% by weight in dry ICL that has not been
chemically cleaned by the method of the invention.
Example 9
Amino Acid Analysis of ICL
[0090] Collagens are proteins characterized by their triple-helical
regions which have a repeating triplet of amino acids glycine-X-Y,
where X is frequently proline and Y is often hydroxyproline.
Hydroxyproline is frequently used as an amino acid to identify and
quantify collagens. Udenfriend, Science, 152:1335-1340 (1966).
[0091] To determine complete amino acid analysis of ICL, PICO-TAG
HPLC was performed on mechanically cleaned (not chemically cleaned)
porcine ICL and chemically cleaned ICL. Hydroxyproline content was
measured for both materials and compared.
[0092] Sample pieces of ICL from each condition weighing about from
0.31 to about 0.36 g were dried further using a CEM AVC80 oven (CEM
Corp.; Matthews, N.C.). Smaller samples were cut from these dried
ICL pieces weighing about 9.5 to about 13.1 mg. Samples were placed
into screw cap culture tubes and the samples were then hydrolyzed
(n=3 for each condition) in 1% phenol in 6 M HCl at 110.degree. C.
for about 16 hours. ICL hydrolysates were then diluted in 0.1 M HCl
to normalize the material concentrations to 1 mg/ml. To labeled
glass tubes (6.times.55), 20 ml of hydrolysates and 8 ml of 1.25
mmol/ml L-norleucine as an internal standard. Samples were then
frozen and lyophilized. Samples were then re-dried by adding 20 ml
of 2:2:1 ethanol:water:triethylamine to the tubes, freezing and
lyophilizing. Samples were then derivatized for 20 minutes at room
temperature by adding 20 ml of reagent (7:1:1:1
ethanol:water:triethylamine:PITC) followed by freezing and
lyophilizing. Samples were finally suspended in 200 ml PICO-TAG
Sample Diluent and aliquoted to HPLC vials.
[0093] Amino acid standards were prepared in the following manner:
0.1 ml of amino acid standard (Product #: A-9531, Sigma) was added
to 1.9 ml 0.1 M HCl. Five serial dilutions at 1:1 were made using
0.1 M HCl. Volumes of 100 ml for each serial dilution and 8 ml of
1.25 mmol/ml L-norleucine were together added to glass tubes
(6.times.55) and then prepared in the same manner as ICL
samples.
[0094] Samples and standards were run on a 3.9.times.150 mm
PICO-TAG Amino Acid column (Part #88131; Waters Corp.; Milford,
Mass.). Injections of 10 ml for samples and 20 ml for standards
were analyzed in triplicate for each.
[0095] Results indicate for chemically cleaned ICL material, the
content of major collagenous amino acids in the material approach
that of purified collagen preparations. Using the hydroxyproline as
a measure of collagen content, the percentage of collagen by weight
in ICL is calculated to be at least about 93% collagen by weight.
In contrast, non-chemically cleaned ICL contains a high fraction of
non-collagenous amino acids; between about 11 to 25% by weight of
ICL is non-collagenous material.
[0096] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
and understanding, it will be obvious to one of skill in the art
that certain changes and modifications may be practiced within the
scope of the appended claims.
* * * * *