U.S. patent application number 12/842820 was filed with the patent office on 2010-11-11 for tissue graft.
This patent application is currently assigned to CRYOLIFE, INC.. Invention is credited to Kirby S. Black, Steven Goldstein, Jeremy D. Ollerenshaw.
Application Number | 20100285587 12/842820 |
Document ID | / |
Family ID | 26874499 |
Filed Date | 2010-11-11 |
United States Patent
Application |
20100285587 |
Kind Code |
A1 |
Ollerenshaw; Jeremy D. ; et
al. |
November 11, 2010 |
Tissue Graft
Abstract
A method of preparing a tissue graft material is disclosed. The
disclosure also relates to a multipurpose tissue graft material and
to methods of using same as a replacement for vascular and
non-vascular tissue.
Inventors: |
Ollerenshaw; Jeremy D.;
(Marietta, GA) ; Goldstein; Steven; (Atlanta,
GA) ; Black; Kirby S.; (Acworth, GA) |
Correspondence
Address: |
SUTHERLAND ASBILL & BRENNAN LLP
999 PEACHTREE STREET, N.E.
ATLANTA
GA
30309
US
|
Assignee: |
CRYOLIFE, INC.
Kennesaw
GA
|
Family ID: |
26874499 |
Appl. No.: |
12/842820 |
Filed: |
July 23, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11049291 |
Feb 3, 2005 |
7763081 |
|
|
12842820 |
|
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|
09769769 |
Jan 26, 2001 |
6866686 |
|
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11049291 |
|
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|
60178632 |
Jan 28, 2000 |
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Current U.S.
Class: |
435/378 |
Current CPC
Class: |
A61L 31/005 20130101;
A61F 2/06 20130101; A61F 2/04 20130101; A61L 2430/40 20130101; A61L
27/3691 20130101; A61L 27/3687 20130101; A61L 27/3683 20130101;
A61L 27/3604 20130101 |
Class at
Publication: |
435/378 |
International
Class: |
C12N 5/07 20100101
C12N005/07 |
Claims
1. A method of preparing a decellularized tissue for implantation
comprising: contacting a harvested tissue with water or an aqueous
hypotonic solution; contacting the tissue with one or more nuclease
enzymes; and contacting the tissue with a detergent; wherein said
contacting produces the decellularized tissue which comprises a
tissue matrix suitable for acting as a scaffold for spontaneous
repopulation by host cells in vivo.
2. The method of claim 1, further comprising washing the tissue
treated with the nuclease and the detergent in a sterile aqueous
solution.
3. The method of claim 1, wherein the one or more nuclease enzymes
comprise an endonuclease.
4. The method of claim 1, wherein the tissue comprises a human or
animal ureter.
5. The method of claim 1, further comprising contacting the tissue
with an antimicrobial solution.
6. The method of claim 5, wherein the antimicrobial solution
comprises vancomycin.
7. The method of claim 5, wherein the antimicrobial solution
comprises lincomycin.
8. The method of claim 1, further comprising contacting the tissue
with Tris[hydroxymethyl]aminomethane hydrochloride and a magnesium
salt while contacting the tissue with the one or more nuclease
enzymes.
9. The method of claim 8, further comprising contacting the tissue
with a calcium salt while contacting the tissue with the one or
more nuclease enzymes.
10. The method of claim 1, further comprising sterilizing the
tissue with gamma radiation.
11. The method of claim 1, wherein greater than 95% of all cellular
material is removed from the tissue.
12. The method of claim 1, wherein the detergent comprises Triton
X-100.
13. A method of preparing a decellularized tissue for implantation
comprising: treating a tissue material by contacting the tissue
material with water or an aqueous hypotonic solution, one or more
nuclease enzymes, and a detergent; and washing the treated tissue
material in a sterile aqueous solution or solutions to remove the
detergent from the tissue material; wherein the treating and
washing produce a decellularized tissue which comprises a tissue
matrix suitable for acting as a scaffold for spontaneous
repopulation by host cells in vivo.
14. The method of claim 13, wherein the nuclease comprises an
endonuclease.
15. The method of claim 13, wherein the tissue material comprises a
human or animal ureter.
16. The method of claim 13, further comprising contacting the
tissue with an antimicrobial solution.
17. The method of claim 16, wherein the antimicrobial solution
comprises vancomycin.
18. The method of claim 17, wherein the antimicrobial solution
comprises lincomycin.
19. The method of claim 13, further comprising contacting the
tissue with Tris[hydroxymethyl]aminomethane hydrochloride and a
magnesium salt while contacting the tissue with the one or more
nuclease enzymes.
20. The method of claim 19, further comprising contacting the
tissue with a calcium salt while contacting the tissue with the one
or more nuclease enzymes.
21. The method of claim 13, further comprising sterilizing the
tissue with gamma radiation.
22. The method of claim 13, wherein greater than 95% of all
cellular material is removed from the tissue.
23. The method of claim 13, wherein the detergent comprises Triton
X-100.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 11/049,291, filed Sep. 1, 2005, which is a
continuation of U.S. application Ser. No. 09/769,769, filed on Jan.
26, 2001, which claims the benefit of Provisional Application No.
60/178,632, filed Jan. 28, 2000, all of which are incorporated
herein by reference in their entirety.
TECHNICAL FIELD
[0002] The present invention relates to a method of preparing a
tissue graft material. The invention also relates to a multipurpose
tissue graft material and to methods of using same as a replacement
for vascular and non-vascular tissue.
BACKGROUND
[0003] In general, biological tissues have a better functional
performance than equivalent synthetic devices when used as a body
implant. Tissue grafts are presently largely limited to autologus
and allograft tissues that have inherent supply constraints and
logistic concerns of harvest, transportation and serologies.
Accordingly, there is a need for additional sources of biological
tissue grafts. Animal tissues represent such a source. Animal
tissues can be relatively easily obtained from slaughterhouses in
large quantities. Prior to use, however, these tissues must be
treated to remove antigenic proteins that elicit a rejection
response by the host following implantation.
[0004] Removal of antigenic proteins can be achieved by processing
the donor tissue in a manner such that the cellular component of
the donor tissue is removed. Many antigenic proteins are present on
cellular membranes. Therefore, removal of cells also removes these
proteins. After decellularization, the tissue can be packaged and
sterilized for use as a biological graft. Grafts can be implanted
into humans and other animals to repair, augment or replace natural
structures, systems or existing prosthetic devices. These include
but are not limited to, cardiovascular, vascular, urogenital,
neurological, gastrointestinal and orthopedic systems. Grafts can
also be used to provide hemodialysis access.
[0005] The present invention provides a method of processing animal
tissue so as to render it suitable for implantation into a human
(or non-human) host. The invention also provides a method for
processing human tissue for use as an allograft implant.
SUMMARY OF THE INVENTION
[0006] The present invention relates to a method of preparing a
tissue graft material and to the resulting multipurpose graft
material. The invention also relates to a method of using the
tissue graft as a replacement for vascular or non-vascular
tissue.
[0007] Objects and advantages of the present invention will be
clear from the description that follows.
DETAILED DESCRIPTION OF THE INVENTION
[0008] The present invention relates to a method of preparing an
animal or human tissue in a manner so as to render it suitable for
use in vascular and non-vascular graft applications. Tissue
prepared in accordance with the method of the present invention
exhibits physical and biological properties that render it
particularly well adapted for tissue graft applications.
[0009] Tissue suitable for use in the present invention can be
obtained from human cadavers or from bovine, porcine or other
animal, for example, under abattoir conditions. Tissue can be
transported to the point of tissue preparation under conditions
necessary to keep the tissue at a desired temperature. Tissues can
be transported, for example, immersed in a physiological salt
solution and, upon arrival, inspected, washed, for example, in a
physiological salt solution, and cleaned (dissected) free of
unwanted adherent material, such as connective tissue and fat.
[0010] In a preferred embodiment, an isolated ureter is the tissue
graft material. However, other tissues can be used including
arteries, veins, tendons, heart valves, fascia lata, pericardium
and nerves.
[0011] After collection and dissection, the transplant tissue is
advantageously, first washed, for example, with phosphate buffered
saline (PBS), to reduce microbial bioburden. The tissue is then
incubated (e.g., at about 37.degree. C. for about 18 hours) in a
solution containing one or more antimicrobial agents, for example,
an antibiotic or an antifungal agent, or mixture thereof, to
further reduce the bioburden. Preferred antibiotics include
amakacin, lincomycin, cefotaxime, vancomycin, rifampin, diflucan
and amphotericin B. Advantageously, a mixture of these antibiotics
is used. The tissue can then be cryopreserved for further
processing at a later time or immediately subjected to
decellularization.
[0012] Decellularization is preferably accomplished by incubating
the tissue in a solution effective to lyse native cells in the
tissue. Advantageously, the tissue is incubated (e.g., at about
37.degree. C.) in sterile water (for example, for about 4 hours in
the case of ureters), however an aqueous hypotonic buffer or low
ionic strength buffer can also be used. If desired, the
decellularizing solution can include other agents, such as protease
inhibitors ((e.g., chelators such as EDTA)).
[0013] After decellularization, the resulting tissue matrix is
treated with an enzyme (e.g., nuclease) cocktail to degrade nuclear
material. Nucleases that can be used for digestion of native cell
DNA and RNA include both exonucleases and endonucleases. Other
nucleases are suitable for use in this step of the process and are
commercially available. For example, a cocktail can be used
comprising DNAse I (SIGMA Chemical Company, St. Louis, Mo.) and
RNAse A (SIGMA Chemical Company, St. Louis, Mo.).
[0014] Preferably, the nucleases are present in a buffer solution
that contains magnesium and calcium salts (e.g., chloride salts).
The ionic concentration and pH of the buffered solution, the
treatment temperature and the length of treatment are selected to
assure the desired level of effective nuclease activity. In the
case of ureters, the buffer is preferably a Tris buffer at pH 7.6.
Preferably, the nuclease cocktail contains about 0.1 .mu.g/ml to 50
.mu.g/ml, preferably 17 .mu.g/ml, of DNAse I, and about 0.1
.mu.g/ml to 50 .mu.g/ml, preferably 17 .mu.g/ml, of RNAse A. The
nuclease treatment can be effected at, for example, about
20.degree. C. to about 38.degree. C., preferably at about
37.degree. C., for about 1 to 36 hours. In the case of ureters,
nuclease treatment for about 19 hours is typically sufficient.
[0015] Subsequent to decellularization and nuclease treatment, the
resultant tissue matrix can be treated (washed) to assure removal
of cell debris which may include cellular protein, cellular lipids,
and cellular nucleic acid, as well as extracellular debris, such as
extracellular soluble proteins, lipids and proteoglycans. Removal
of cellular and extracellular debris reduces the likelihood of the
transplant tissue matrix eliciting an adverse immune response from
the recipient upon implant. For example, the tissue can be
incubated in a buffer (e.g., PBS) or in a detergent solution such
as a solution of Triton X-100 in water. The composition of the
solution, and the conditions under which it is applied to the
tissue matrix can be selected to diminish or eliminate the activity
of the nuclease utilized during nuclease processing and to remove
cell debris. The process can include incubation at a temperature of
between about 2.degree. C. and 42.degree. C., with 37.degree. C.
being preferred. The tissue matrix can be incubated in the
detergent solution for up to 7 days, about 24 hours being
sufficient in the case of a ureter matrix. When buffer is used
rather than a detergent solution, the tissue matrix can be
incubated for up to 30 days, about 14 days being sufficient in the
case of a ureter matrix.
[0016] If used, the detergent solution can be washed out of the
tissue matrix using multiple washes in a sterile aqueous solution
(e.g., water). Optimum wash number and times can be readily
determined, however, about 4 30 minute washes are preferred in the
case of ureter matrices.
[0017] After washing, the tissue matrix can then be packaged,
sterilized and/or stored prior to implantation. Advantageously,
packaged tissue matrix is maintained in a non-frozen state,
preferably at a temperature between 0.degree. C. and 40.degree. C.,
more preferably, between 0.degree. C. and 20.degree. C., most
preferably between 2.degree. C. and 8.degree. C. up to and during
sterilization using, for example, the approach used in Example 11.
After sterilization, the tissue matrix can be maintained at room
temperature. If desirable, the tissue matrix can be cryopreserved
before or after sterilization for later use. Techniques of
cryopreservation of tissue are well known in the art. Brockbank, K.
G. M. Basic Principles of Viable TissuePreservation. In:
Transplantation Techniques and use of Cryopreserverd Allograft
Cardiac Valves and Vasular Tissue. D. R. Clarke (ed.), Adams
Publishing Group, Ltd., Boston. pp 9-23, discusses cryopreservation
of tissues and organs and is hereby incorporated by reference.
[0018] Tissue matrices of the invention, whether or not previously
cryopreserved, can be sterilized using art recognized sterilization
techniques. Advantageously, sterilization is effected using gamma
irradiation at a dose of between 10 kGy and 100 kGy, preferably
between 20 kGy and 40 kGy, more preferably between 25 kGy and 40
kGy. Alternative modes of sterilization include iodine peracetic
acid treatment or electron beam. After sterilization, the tissue
matrix can be stored frozen or unfrozen prior to implantation. If
stored cryopreserved, for example, in liquid nitrogen, the tissue
matrix is stable for at least 5 years. Prior to using a frozen
tissue matrix, the matrix is thawed using a protocol designed to
elute cryoprotectant solutions. For example, the matrix can be
thawed rapidly to 4.degree. C. in a waterbath at a temperature of
37-42.degree. C. The matrix can then be quickly transferred to a
growth medium such as Dulbeccos' Modified Eagles Medium (DMEM)
containing mannitol (e.g., at about 0.5%). Mannitol and residual
cryoprotectants can be removed by serial dilution (washing) with
0.5, 0.25 and 0.0M solutions of mannitol in DMEM. Following the
washes, the tissue is ready to be used. For tissues not stored
frozen, alternate washing protocols can be used, for example,
washing in PBS or DMEM. Art-recognized implantation procedures can
be used and the procedure selected is dependent on the tissue
matrix used and site of implantation.
[0019] The tissue matrix resulting from the above-described
process, particularly a ureter matrix, can be used as a conduit
(tubular) graft. For example, a ureter matrix can be used as a
vascular graft, nerve guide, or replacement for any tubular
structure, including a ureter. When used as a conduit graft, the
diameter of the graft should generally be about the same as the
diameter of the native structure. The grafts of the invention
demonstrate favorable characteristics as hemodialysis access
grafts.
[0020] For use in other graft applications, a conduit graft (e.g.,
ureter tissue matrix) resulting from the above process can be cut
longitudinally and rolled out to form a patch of tissue. The entire
decellularization/nuclease treatment procedure described above can
be carried out on patches of tissue (e.g., ureter tissue) prepared
by cutting the segment longitudinally and unrolling it to form a
pre-graft patch. The prepared graft patches can be utilized, for
example, as a skin graft material or for repair of other body
tissue defects lending themselves to surgical application of a
tissue graft patch having the physical and functional
characteristics of the present graft composition.
[0021] The tissue matrix of the present invention acts as a
scaffold for spontaneous repopulation by host cells in vivo leading
to tissue reconstruction and stabilization. The result is a fully
functional, non-immunogenic, viable construct containing autologous
cells expressing contractile proteins. The better patency rate and
lack of infection seen in the present grafts may be attributable to
the early incorporation, recellularization and remodeling of the
matrix with host cells.
[0022] Certain aspects of the present invention are described in
greater detail in the non-limiting Examples that follow.
EXAMPLE 1
Implantation of Decellularized Bovine Ureter as a Peripheral
Vascular Graft in the Dog
[0023] Methods
[0024] Fresh bovine ureters were collected from slaughterhouses
within 2 hours of death and shipped to the processing facility at
4.degree. C. in a solution of phosphate buffered saline (PBS) to
prevent tissue degradation in transit. Upon receipt of the tissue,
a gross tissue inspection was made and ureters of 4 mm outside
diameter and 30 to 40 cm in length were selected for processing.
Selected ureters were dissected using sterile instruments to remove
unwanted adherent material such as connective tissue and fat.
Ureters were then placed in 300 ml capcity polypropylene containers
and washed four times with 250 ml sterile PBS to reduce microbial
bioburden. Ureters were then taken through steps to remove the
tissue cell and antigen content.
[0025] Decellularization was initiated by incubation in a cocktail
of antibiotics and antimycotic agents which consisted of a solution
of antibiotics. This mixture contained the following: amakacin (34
.mu.g/ml), lincomycin (160 .mu.g/ml), cefotaxime (18 .mu.g/ml),
vancomycin (136 .mu.g/ml), rifampin (82 .mu.g/ml), diflucan (120
.mu.g/ml) and amphotericin B (0.5 .mu.g/ml). Incubation was for 18
hours in a shaking incubator at 37.degree. C. For cell lysis, the
antibiotic solution was replaced with 250 ml of sterile water and
incubation was allowed to proceed for 4 hours in a shaking
waterbath at 37.degree. C. This was followed by incubation for 19
hours at 37.degree. C. in an enzyme cocktail to degrade the nuclear
material now exposed by lysing the cell organelles. This cocktail
contained DNAse I (47 Kunitz U/ml) and RNAse A (1 Kunitz U/ml) in a
solution containing magnesium chloride (1 .mu.g/ml) and calcium
chloride (3 .mu.g/ml) and buffered using
Tris[hydroxymethyl]aminomethane hydrochloride (50 .mu.g/ml) at pH
7.6. (The DNAse I and RNAse A were obtained from Sigma Chemical
Company (D-5025 and R-5000) and both were used at a concentration
of 17 .mu.g/ml.) Subsequently, the tissue was placed in a 3.5 mM
solution of Triton X-100 detergent in sterile water to remove cell
debris. This incubation was carried out for 24 hours at 37.degree.
C. in a shaking waterbath. The detergent solution was then washed
out of the tissue by four washes in sterile water at 37.degree. C.
for 30 minutes each. After these washes, the resulting matrix was
packaged, cryopreserved, sterilized and placed in storage prior to
use as a vascular graft.
[0026] For preservation, the tissue was packaged in sterile
packages containing Dulbeccos' Modified Eagles Medium solution
(DMEM) and 10% dimethyl sulfoxide (DMSO) with 10% fetal bovine
serum. Cryopreservation was performed using a controlled rate
freezer to reduce the package temperature to -80.degree. C. at
0.5.degree. C. per minute. When the tissue temperature had reached
-80.degree. C. each package was removed and placed in liquid
nitrogen at -196.degree. C. for long term storage. Sterilization of
tissue was performed in the frozen state using a 25-30 kGy dose of
gamma radiation. Following sterilization, the tissue was stored at
-196.degree. C. in liquid nitrogen until use.
[0027] Prior to implantation, the tissue matrix was thawed to
remove the cryoprotectant solution from the tissue. The grafts were
thawed rapidly to 4.degree. C. in a waterbath at a temperature of
37-42.degree. C. Tissue was quickly transferred to DMEM containing
0.5% mannitol. Mannitol and residual cryoprotectants were removed
by dilution with 0.5, 0.25 and 0.0M solutions of mannitol in DMEM.
Following these washes, implantation of acellular bovine conduit
(that is, the resulting tubular tissue matrix) was performed as an
end-to-side interpositional graft in the left and right carotid and
left femoral arteries of an adult mongrel dog. The graft lengths
were between 9 and 12 cm and the internal diameter of the grafts
was between 5 and 10 mm. Implantation was made using standard
surgical techniques for vascular graft implantation in these
positions. An oral anticoagulant regimen of 325 mg Aspirin and 75
mg Persantin was administered daily to the animal beginning two
days prior to the surgical procedure.
[0028] At two weeks and at four weeks after surgery, arteriograms
were performed to determine the patency of the implanted grafts.
The animal was sacrificed and the grafts explanted immediately
following the second arteriogram at four weeks after the surgery.
Explanted grafts were evaluated for patency and gross appearance
and further examined histologically to determine graft microscopic
integrity.
[0029] Results
[0030] During the four weeks duration of the study, the animal
behaved normally and there were no complications following surgery.
At two weeks and at four weeks after surgery, all three bovine
ureter grafts were determined to be fully patent on angiographic
examination. At four weeks after surgery, gross analysis of the
explanted graft tissue indicated there to have been a healing
response that had stabilized the grafts into the surgical site and
the patency of all the grafts was confirmed by observing flow
through the graft prior to placing the grafts in formalin for
fixation. After fixation each graft was cut into seven separate
samples for histological analysis. Samples were taken from the
native artery at both proximal and distal ends away from the graft.
Sections of the proximal and distal anastamosis sites were taken
along with the proximal, middle and distal portion of each graft.
Following processing, paraffin embedding and sectioning, graft
samples were stained using a standard hematoxylin and eosin stain.
Microscopic analysis revealed the grafts to be structurally intact.
The matrix of the bovine ureter had begun to become re-vitalized
through the movement of cellular components from the outer edges of
the surgical area. Through this remodeling the grafts were taking
on the appearance of natural arterial blood carrying conduits.
EXAMPLE 2
Implantation of Decellularized Porcine Ureter as a Peripheral
Vascular Graft in the Dog
[0031] Methods
[0032] Porcine ureter tissue was collected and prepared and
preserved exactly as described in Example 1 with the exception that
the dimensions of tissues selected for processing were 3 mm in
internal diameter and 25 cm in length. Implantation of treated
porcine ureters was made in an adult mongrel dog as an end-to-end
interpositional vascular graft in the left and right femoral
arteries and in the left and right carotid arteries. All grafts
were examined by arteriogram two weeks after surgery. The animal
was sacrificed four weeks after surgery and the grafts were
explanted for gross examination and histological evaluation of
performance. Histology samples were taken and stained as described
in Example 1.
[0033] Results
[0034] Arteriograms performed two weeks after implantation showed
the grafts to be patent. On explantation, gross examination
indicated the grafts to be fully patent and difficult to
distinguish from the native blood vessel. Histological examination
showed the grafts to have become partly recellularized with
spindle-shaped cells. This recellularization likely represents the
first stages of remodeling into a fully-functional blood-carrying
conduit that would be indiscernible from native tissue.
EXAMPLE 3
Implantation of Decellularized Bovine Uterine Artery as a
Peripheral Vascular Graft in the Dog
[0035] Methods
[0036] Bovine uterine artery tissue was collected and prepared and
preserved exactly as described in Example 1. Implantation of
treated bovine uterine artery was made in an adult mongrel dog as
an end to side interpositional graft in the carotid artery. After 4
weeks of implantation, the grafts were explanted and taken for
histological analysis.
[0037] Results
[0038] At four weeks after implantation, the grafts were patent.
Histology indicated these grafts to have begun to take on cells
from the host animal.
EXAMPLE 4
Implantation of Decellularized Bovine Gastric Artery as a
Peripheral Vascular Graft in the Dog
[0039] Methods
[0040] Bovine gastric artery tissue was collected and prepared and
preserved exactly as described in Example 1. Implantation of
treated bovine uterine artery was made in an adult mongrel dog as
an end to side interpositional graft in the carotid artery. After 4
weeks of implantation, the grafts were explanted and taken for
histological analysis.
[0041] Results
[0042] At four weeks after implantation, the grafts were patent.
Histology indicated these grafts to have begun to take on cells
from the host animal.
EXAMPLE 5
Determination of Burst Strength Characteristics of Decellularized
Bovine Ureter
[0043] Methods
[0044] Three segments of bovine ureter tissue were collected and
prepared and preserved exactly as described in Example 1. Using
compressed nitrogen gas, the pressure required to burst the graft
was determined by slowly increasing the head-pressure of nitrogen
applied to the graft. The gas was contained within the graft using
standard high-pressure pipe fittings and cable ties. Each graft
segment was tested in duplicate and average burst strength for each
graft was calculated.
[0045] Results
[0046] The three grafts were found to burst at 3361, 2456 and 2327
mm Hg, the average being 2715 mm Hg. This magnitude represents a
burst strength of around 1.5 times that of the human fresh
saphenous vein which is commonly used in bypass surgical
procedures.
[0047] EXAMPLE 6
Determination of Burst Strength Characteristics of Decellularized
Porcine Ureter
[0048] Methods
[0049] Six segments of porcine ureter tissue were collected and
prepared and preserved exactly as described in Example 2. Using
compressed nitrogen gas, the pressure required to burst the graft
was determined by slowly increasing the head-pressure of nitrogen
applied to the graft. The gas was contained within the graft using
standard high-pressure pipe fittings and cable ties. Each graft
segment was tested in duplicate and the average burst strength for
each graft was determined.
[0050] Results
[0051] The six grafts were found to burst at 2068, 5171, 6722,
6722, 5688 and 3620 mm Hg, the average being 4999 nm Hg. This value
represents a burst strength of almost 3 times that of human fresh
saphenous vein which is commonly used in bypass surgical
procedures.
EXAMPLE 7
In Vitro Recellularization of Decellularized Bovine Ureter with
Vascular Conduit Cells
[0052] Methods
[0053] Three segments of bovine ureter tissue were collected and
prepared and preserved exactly as described in Example 1. Each
piece of tissue was placed into a 75 cc tissue culture flask
containing DMEM and supplemented with 10% fetal bovine serum. Each
graft was seeded with endothelial cells or smooth muscle cells to
enable cell growth into the graft segments. Cultures were fed using
fresh serum-supplemented DMEM two times each week for 4 weeks.
After 4 weeks, the tissues were extracted from the culture system
and examined using histological sectioning of the tissue and
H&E staining
[0054] Results
[0055] After four weeks of cell culture, endothelial cells were
observed growing on the surface of graft tissue but not internally.
In addition, smooth muscle cells were found growing on the surface
of the grafts and in the wall of the graft material once they could
gain access on the surface of the graft.
EXAMPLE 8
Tissue Graft Derived from Ureter as Aortic Graft in the Dog
[0056] Methods
[0057] Bovine ureters were used to provide the conduit matrix for
the vascular tissue graft. These tissues are available in lengths
and diameters suitable for a number of vascular applications and
they do not contain valves in the lumen or possess tributaries that
require ligation. Ureters were obtained from U.S. Department of
Agriculture approved slaughterhouses. The tissues were washed in
physiological salt solution and transported for tissue preparation
on ice within 24 hours of harvest. Ureters were first dissected
free of adherent connective tissue and fat and only segments with a
6 mm internal diameter were taken for further processing.
[0058] Initial processing consisted of bioburden reduction using a
solution of multiple antibiotics as described in Example 1. Removal
of greater than 95% of all cellular material was achieved in
several steps. First, incubation in sterile water produced
hypotonic cell lysis. The resulting tissue matrix was then
equilibrated in buffer (PBS) and treated with a solution containing
ribonuclease and deoxyribonuclease (see Example 1). An isotonic
washout over several days completed the cellular protein removal.
Removal of cellular debris was monitored using hematoxylin and
eosin staining of histological sections. Tissue matrices were then
sterilized by gamma irradiation (25 kGy to 40 kGy) prior to use and
analysis of sterility was carried out on each processing batch.
[0059] Eight mongrel dogs weighing 50 to 60 lb were anesthetized
with sodium thiopental, endotracheally intubated and placed on
inhaled isoflurane. The abdomens were prepared and draped in
sterile fashion. A midline incision was made and the abdominal
aorta distal to the renal arteries was isolated in each dog.
Vascular conduits were prepared by washing the tissue matrix in 100
ml of sterile HEPES-buffered Dulbecco's Modified Eagle Medium and a
segment approximately 6 cm in length and 6 mm in internal diameter
was inserted as an aortic interposition graft using interrupted
prolene sutures to construct proximal and distal, end to end,
anastomoses. All animals received 325 mg aspirin and 75 mg
dipyridamole p.o. daily for 2 days prior to, and for 14 days
following, surgery.
[0060] Patency and structural stability were observed with
angiographic examination following surgery every 6 weeks in the
longer survivors and once immediately prior to euthanasia. Two
animals were sacrificed at 3 weeks, 3 at 6 weeks, and 1 animal at
13 weeks after surgery. The 2 remaining animals were last evaluated
at 43 weeks and are still living. After sacrifice, grafts were
removed in bloc incorporating proximal and distal anastamoses
inspected grossly and processed for histological analysis.
[0061] Following harvest, grafts were fixed in 10% buffered
formaldehyde solution. The whole of the graft along with
anastamotic sites and proximal and distal native aorta was divided
into 7 tissue segments and placed in paraffin blocks for
processing. Hematoxylin and eosin-stained sections of these tissues
were examined and immunohistochemical analysis was carried out
using specific antibiotics to identify the presence of smooth
muscle a-actin (a-SMA), desmin and vimentin contractile
filaments.
[0062] Results
[0063] After processing, vascular tissue grafts prepared from
bovine ureter showed removal of greater than 95% of bovine cellular
material. The remainder consisted of cellular debris and not intact
cells. Conduit graft sterility and pyrogen levels of below 20
endotoxin units were demonstrated. Implantation of these
interposition grafts into the canine infrarenal aorta was
uncomplicated and handling properties of the grafts were similar to
normal vascular tissue.
[0064] Arteriograms performed on each of the dogs indicated grafts
to be fully functional over the 43-week implant period without the
appearance of dilation or stenosis. Gross evaluation of all
explanted grafts after 3, 6 and 13-weeks of implantation confirmed
fully patent grafts. Histologic examination showed a healing
response around the graft adventitia with recellularization of the
media. A layer of cells on the lumenal surface resembled
endothelium. All cells found in the graft were presumed to have
originated from the host because the original graft material was
acellular. The extent of medial recellularization was approximately
20% at 3-weeks, 30% at 6-weeks and 50% at 13-weeks. Revitalization
of the graft media appeared to occur from the adventitial area
towards the lumen and as recellularization progressed, there was
circumferential organization of cells growing perpendicular to the
flow of blood in the conduit.
[0065] Analysis of anastomotic sites showed intimal hyperplasia to
be minimal and cellular overgrowth was evident at the suture-line
creating a smooth transition from native aorta to graft.
Histologically, there was no evidence of hyperplastic reaction
narrowing the lumen in the graft explanted after 13-weeks. Also,
narrowing was not observed angiographically up to 43-weeks after
implantation.
[0066] Immunohistochemistry staining was used to identify the type
of cells present in the recellularized grafts. The proportion of
cells expressing smooth muscle contractile proteins were
demonstrated using stains containing antibodies to a-SMA, desmin
and vimentin. A very large percentage of medial cells at 3, 6 and
13 weeks, were A-SMA positive. Vimentin was also commonly expressed
by A-SMA positive cells. Desmin positive cells were less abundant
but present in a sub-population. Most of the cells present in the
grafts stained positive for at least one of these contractile
proteins. As no intact cells were present in the graft conduits
prior to implant, all cell-specific immunostaining demonstable for
A-SMA, desmin and vimentin was present on cells that had originated
from the host.
EXAMPLE 9
Use of Tissue Graft Derived from Ureter as an Arterio-Venous
Fistula in the Dog
[0067] Methods
[0068] Nine segments of treated bovine ureter tissue graft conduit,
prepared as described in Example 9, (20 cm.times.6 mm ID) were
implanted as arteriovenous grafts in the carotid artery (CA) and
jugular vein (JV) (n=5), or in the femoral artery (FA) and femoral
vein (FV) (n=4) in 6 adult dogs. A control group of 7 dogs received
11 (6 mm ID) polytetrafluoroethylene (PTFE) grafts (7 in the CA and
JV, and 4 in the FA and FV). All grafts were matured for 14 days
and then sham-accessed once weekly with two 17-gauge hemodialysis
needles. Routinely over a 6-month period, patency was assessed and
blood was drawn to monitor CBC and clotting factors. Histological
analysis was performed in a sub-group of explanted grafts at 2, 4,
10 and 24 weeks.
[0069] Results
[0070] 27% (3/11) of the PTFE grafts became infected, while none of
the tissue graft conduits prepared in accordance with the present
invention became infected during the study. The patency rate of the
tissue graft conduit was 86% compared to 72% for the PTFE grafts.
The white blood cell count was not elevated in either group at 2
and 7 weeks and blood clotting factors were also unchanged. The
hemostasis times after sham sticking of the grafts was longer (mean
10 minutes) in the PTFE grafts compared to the tissue graft
conduits (mean 3 minutes). Histology at 10-weeks showed tissue
graft conduits to have undergone recellularization of the tunica
media with host spindle shaped cells as well as excellent
incorporation into surrounding tissues as evidenced by capillary
ingrowth into the tunica adventitia. PTFE grafts showed no
significant cellular ingrowth and an absence of luminal
endothelium.
EXAMPLE 10
Packaging and Sterilization of Tissue Graft
[0071] Packaging
[0072] Tissue product is packaged in heat sealed clear polyester
pouches containing phosphate buffered saline and stored at 40
centegrade for up to 7 days prior to sterilization.
[0073] Shipping
[0074] Three frozen 2 lb cold gel bricks are placed in the bottom
of a pre-chilled 19''.times.14.5''.times.22'' cardboard container
insulated with 2'' polyurethane foam. Two cardboard separators are
placed on top of these.
[0075] Three cold 2 lb gel bricks are added followed by product
load (1,100 cubic inches).
[0076] Two filler bags are used as temperature indicators on which
various temperature indicator strips are present, the filler bags
are placed among the samples. A seven-day mechanical temperature
recorder is also placed among the samples.
[0077] Three cold 2 lb gel bricks are place on top of the product
load followed by a cardboard separator and three frozen 2 lb gel
bricks. A foam plug is placed on top of the last layer of bricks
and the box is closed.
[0078] The box is shipped to sterilization facility for
sterilization by gamma irradiation at 25-40 kGy. The box is then
returned and product is unpacked and stored at room temperature
until use. The total tissue time between packing and unpacking is
advantageously less than 100 hours and the temperature is
maintained throughout this period at 2.degree. C.-8.degree. C.
[0079] Storage
[0080] The tissue graft can be stored at room temperature for 2
years.
[0081] All documents cited above are hereby incorporated in their
entirety by reference.
[0082] One skilled in the art will appreciate from a reading of
this disclosure that various changes in form and detail can be made
without departing from the true scope of the invention.
* * * * *