U.S. patent application number 14/280804 was filed with the patent office on 2014-09-11 for method for detoxifying a biological tissue.
This patent application is currently assigned to Sorin Group Italia S.r.I.. The applicant listed for this patent is Sorin Group Italia S.r.I.. Invention is credited to Vincenzo Cassolaro, Marina Strasly.
Application Number | 20140256035 14/280804 |
Document ID | / |
Family ID | 43431091 |
Filed Date | 2014-09-11 |
United States Patent
Application |
20140256035 |
Kind Code |
A1 |
Strasly; Marina ; et
al. |
September 11, 2014 |
METHOD FOR DETOXIFYING A BIOLOGICAL TISSUE
Abstract
Biological tissues may be prepared for use in biological
prostheses. The biological tissue may be fixated with
glutaraldehyde and may be subjected to successive treatment of the
tissue with a solution containing taurine to neutralize excess
aldehyde groups that remain free after fixation.
Inventors: |
Strasly; Marina; (Baldissero
Torinese, IT) ; Cassolaro; Vincenzo; (Saluggia,
IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sorin Group Italia S.r.I. |
Mirandola (MO) |
|
IT |
|
|
Assignee: |
Sorin Group Italia S.r.I.
Mirandola (MO)
IT
|
Family ID: |
43431091 |
Appl. No.: |
14/280804 |
Filed: |
May 19, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13154856 |
Jun 7, 2011 |
8753393 |
|
|
14280804 |
|
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Current U.S.
Class: |
435/325 |
Current CPC
Class: |
A61L 27/3604 20130101;
A61L 27/3687 20130101; C12N 5/0652 20130101; A61L 2430/40
20130101 |
Class at
Publication: |
435/325 |
International
Class: |
C12N 5/077 20060101
C12N005/077 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 9, 2010 |
IT |
TO2010A000486 |
May 26, 2011 |
EP |
11167681 |
Claims
1. A method of treating a biological tissue for use in a biological
prosthesis, the method comprising the steps: treating the
biological tissue with a detoxification solution comprising taurine
at a concentration within the range from 0.10% w/v to saturation at
a temperature between 25.degree. C. and 45.degree. C.; and storing
a treated biological tissue in a phosphate buffer; wherein treating
the biological tissue with the detoxification solution at a
temperature range between 25.degree. C. and 45.degree. C. reduces
calcification of the biological tissue when compared to treating a
biological tissue at 20.degree. C.
2. The method according to claim 1, further comprising immersing
the biological tissue in a fixation solution comprising
glutaraldehyde.
3. The method according to claim 1, wherein the detoxification
solution comprises the taurine at a concentration within the range
of 0.20% w/v to 1.00% w/v.
4. The method according to claim 1, wherein the detoxification
solution comprises the taurine at a concentration of about 0.70%
w/v.
5. The method according to claim 1, wherein the detoxification
solution comprises a buffer selected from a phosphate, citrate,
acetate, HEPES or borate.
6. The method according to claim 1, wherein the detoxification
solution comprises a phosphate buffer.
7. The method according to claim 1, wherein the detoxification
solution has a pH in the range of 4 to 9.
8. The method according to claim 1, wherein the detoxification
solution has a pH in the range of 5 to 8.
9. The method according to claim 1, wherein the detoxification
solution has a pH of about 7.
10. The method according to claim 1, wherein the treatment step is
carried out at a temperature of 40.degree. C.
11. The method according to claim 1, wherein the treatment step is
carried out for a period of 12 to 48 hours.
12. A method of treating a biological tissue for use in a
biological prosthesis, the method comprising the steps: treating
the biological tissue with a detoxification solution at a
temperature between 20.degree. C. and 50.degree. C., the
detoxification solution comprising taurine at a concentration
within the range of 0.10% w/v to saturation; and storing the
biological tissue in a phosphate buffer; wherein treatment of the
biological tissue with the detoxification solution reduces
calcification of the biological tissue when compared to treatment
of a biological tissue using a detoxification solution comprising
homocysteic acid.
13. The method according to claim 12, further comprising immersing
the biological tissue in a fixation solution comprising
glutaraldehyde.
14. The method according to claim 12, wherein the fixing solution
comprises glutaraldehyde at a concentration of about 0.5% v/v.
15. The method according to claim 12, further comprising immersing
the biological tissue in a fixation solution comprising
glutaraldehyde.
16. The method according to claim 12, wherein the detoxification
solution comprises the taurine at a concentration within the range
of 0.20% w/v to 1.00% w/v.
17. The method according to claim 12, wherein the detoxification
solution comprises the taurine at a concentration of about 0.70%
w/v.
18. The method according to claim 12, wherein the detoxification
step is carried out at a temperature of 20.degree. C.
19. The method according to claim 12, wherein the detoxification
step is carried out at a temperature of 40.degree. C.
20. The method according to claim 13, wherein the detoxification
step is carried out at a temperature of 50.degree. C.
Description
RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 13/154,856, filed Jun. 7, 2011, which claims priority to
Italian Patent Application No. IT TO2010A000486 filed Jun. 9, 2010,
and claims priority to European Application No. 11167681, filed May
26, 2011, all of which are incorporated by reference herein in
their entirety.
TECHNICAL FIELD
[0002] The present invention is generally directed to a method for
detoxifying biological tissue for use in biological prostheses such
as prosthetic valves and more particularly to a method for
detoxifying biological tissue for use in prosthetic cardiac
valves.
BACKGROUND
[0003] Biological prostheses are medical devices that utilize
animal tissues. Examples of suitable animal tissues include bovine,
porcine, ovine and equine. Depending on the various medical uses,
the biological tissue include cardiac valves, pericardium, tendons,
ligaments, dura mater, skin, veins, etc.
[0004] The animal tissues used in biological prostheses are formed
primarily of collagen, a protein with a structural unit represented
by three polypeptide chains that associate to form a triple helix.
Collagen molecules assemble to form microfibrils that in turn
assemble to form fibrils that, arranged in corrugated or parallel
bundles, give rise to true collagen fibers. Such tissues have good
resistance to traction and are flexible but substantially
inextensible.
[0005] Animal tissues used in biological prostheses are first
subjected to numerous washings to eliminate traces of blood and a
careful removal of adipose and ligamentous parts. However, cells or
cellular residues from the animal donor can remain trapped in the
structure of the tissue itself. As a result it is possible that the
immune system of the host gives rise to a rejection phenomenon that
can lead to the destruction of the tissue constituting the
biological prosthesis.
[0006] An additional problem is degradation of the collagenous
biological tissue once implanted in the host organism. For this
reason, the biological tissues are subjected to a fixation
treatment with the aim of protecting the tissue from such
degradation phenomena and contributing to preventing the
above-mentioned rejection phenomenon.
[0007] Among the substances used for the fixation of biological
tissues, the most common is glutaraldehyde. This bifunctional
molecule, carrying two aldehyde groups, is capable of stably
binding together free amino groups of the amino acids that
constitute the polypeptide chains both within one collagen molecule
and between adjacent collagen molecules. In this way glutaraldehyde
forms intra-chain and inter-chain bridge structures, causing the
cross linking of biological tissue. Such cross linking protects the
tissue from degradation by the host and confers favorable
mechanical properties such as for example a better resistance to
traction with respect to untreated tissue.
[0008] Glutaraldehyde is a highly bactericidal and virucidal
substance; therefore, in addition to cross linking the tissue, the
fixation step also provides at least a partial sterilization.
[0009] In addition, glutaraldehyde is capable of binding to the
free amino residues of the membrane proteins of the cellular
components still present, masking their antigenic potential and
impeding immune activation phenomena and rejection by the host.
[0010] In spite of widespread use, glutaraldehyde has a
disadvantage of being one of the factors that favors pathological
calcification of implanted tissues. The calcium, present in the
bodily fluids of the host organism, accumulates in proteinaceous
tissue giving rise, for example in the case of biological cardiac
valves, to a process that may represent one of the principle causes
of valve failure. The calcium deposits can reduce the flexibility
of the portion of biological tissue constituting the valve (or the
so-called valve leaflets or cusps) and lead to laceration of the
tissue itself, causing a partial or total loss of valve
function.
[0011] The mechanism responsible for calcification is not yet
completely known and is attributed to numerous factors; however, it
is known that after glutaraldehyde fixation, aldehyde groups
remaining free on the tissue can create binding sites for
calcium.
[0012] In addition, the toxicity of such aldehyde residues can
cause local inflammatory phenomena that lead to the necrosis of
host cells. Destruction of the dead cells gives rise to cellular
debris that, in turn, can constitute binding cites for calcium.
Several types of molecules capable of neutralizing the aldehyde
residues remaining free after the fixation process have been used
to limit the process of tissue calcification. For example, the use
of amino acids has been shown to have an anti calcification effect;
in particular, U.S. Pat. No. 5,873,812 describes the use of amino
carboxylic acids, such as for example homocysteic acid, in the
preparation of aldehyde-fixed biological tissues. However, this
method only partially neutralizes free aldehyde groups and thus
does not resolve the problem.
SUMMARY
[0013] The present invention is directed to improved, more
efficacious solutions that limit the calcification of biological
tissues after implantation in the host.
[0014] In some embodiments, the present invention is directed to a
method for treating a biological tissue for biological prostheses.
The method includes a tissue fixation step and a detoxification
step.
[0015] In some embodiments, the method for treating a biological
tissue includes fixation of the biological tissue via a treatment
with a glutaraldehyde solution and detoxifying the fixed biological
tissues via a treatment with a taurine solution at a temperature
that is in the range of about 30 to about 45 degrees Celsius.
[0016] In some embodiments, the results presented below show that
the method described herein strongly reduces the number of free
aldehyde groups present on fixed tissue, presenting an clear
advantage with respect to methods that envision the detoxification
of tissue by means of immersion in a solution containing
homocysteic acid.
BRIEF DESCRIPTION OF THE FIGURES
[0017] The invention will now be described in detail, by way of
non-limiting example only, with reference to the attached figures,
in which:
[0018] FIG. 1A shows fuchsine staining of a control sample fixed
but not detoxified.
[0019] FIG. 1B shows fuchsine staining of a sample that was fixed
and detoxified according to the known art using a homocysteic acid
solution.
[0020] FIG. 1C shows fuchsine staining of a sample that was fixed
and detoxified with a taurine solution in accordance with
embodiments of the present invention.
[0021] FIG. 2A fuchsine staining of a control sample fixed but not
detoxified.
[0022] FIG. 2B shows fuchsine staining of a sample that was fixed
and detoxified with a taurine solution at room temperature.
[0023] FIG. 2C shows fuchsine staining of a sample that was fixed
and detoxified with a taurine solution at 40 degrees Celsius.
[0024] FIG. 2D shows fuchsine staining of a sample that was fixed
and detoxified with a taurine solution at 50 degrees Celsius.
DETAILED DESCRIPTION
[0025] The invention will now be described in detail, by way of
non-limiting example only, with reference to forming biological
prosthetic cardiac valves. It is clear that the procedure described
herein can be used for the detoxification of any other biological
tissue destined for forming other biological prostheses that use,
for example tendons, ligaments, dura mater, skin, veins, etc.
[0026] In the following description, numerous specific details are
given to provide a thorough understanding of the embodiments. The
embodiments can be practiced without one or more of the specific
details, or with other methods, components, materials, etc. In
other instances, well-known structures, materials or operations are
not shown or described in detail to avoid obscuring certain aspects
of the embodiments.
[0027] Reference throughout the present specification to "one
embodiment" or "an embodiment" means that a particular feature,
structure or characteristic described in connection with the
embodiment is included in at least one embodiment. Thus, the phrase
"in one embodiment" or "in an embodiment" in various places
throughout the present specification are not necessarily all
referring to the same embodiment. Furthermore, the details of
features, structures, or characteristics may be combined in any
suitable manner in one or more embodiments.
[0028] The headings provided herein are for convenience only and do
not interpret the scope or meaning of the embodiments.
[0029] The present invention concerns a method for treating a
biological tissue that includes two steps:
[0030] i) Fixation of the biological tissue via treatment with a
solution containing glutaraldehyde, and
[0031] ii) Detoxification of the previously fixed biological tissue
via treatment with a solution containing taurine.
[0032] In some embodiments, the fixation step includes immersion of
the tissue in a solution containing glutaraldehyde for a period
that varies between one minute and 3 days and a maximum of 13 days.
Fixation cross links the tissue, which confers it with resistance
to degradation and favorable mechanical properties.
[0033] In some embodiments, the detoxification step includes
immersion of the previously fixed tissue in a solution containing
taurine for a period that varies in a range from one minute to a
few hours and a maximum of several days.
[0034] Taurine (also known as aminoethanesulfonic acid) has an
amine group (NH.sub.2) available to bond with the aldehyde groups
of glutaraldehyde that remain free after the tissue fixation step.
In particular, the present inventors discovered that the use of
taurine in the detoxification step in place of homocysteic acid
provides greater efficacy for neutralizing the aldehyde groups that
remain free after glutaraldehyde fixation, therefore reducing in a
highly efficacious way the number of sites of binding and
accumulation of calcium on the biological tissue.
[0035] Biological tissues to be used for biological prosthetic
cardiac valves, or bovine pericardium, equine or possibly
pericardium of other animal species or porcine valves are harvested
from authorized abattoirs and--transported to the
Laboratory--immersed in saline solution maintained on ice to avoid
damage to the tissue before use.
[0036] The tissues are washed with saline solution to eliminate
possible traces of blood, separated from possible adherent
ligamentous and adipose parts, and then carefully selected on the
basis of thickness and on the basis of the absence of evident
defects such as dishomogeneity of thickness, presence of cuts,
abrasions, etc.
[0037] The tissues are initially prefixed in a glutaraldehyde
solution at room temperature in a volume/volume concentration that
is in the range of 0.05% to 0.30% glutaraldehyde in phosphate
buffer at a pH of 7.4 for a period of time that ranges from 3 to 13
hours. In some embodiments, the glutaraldehyde solution includes
0.20% glutaraldehyde
[0038] After the prefixation step, in some embodiments, comes a
step of cutting and shaping the tissue--according to known art
techniques--to form, for example, cardiac valves. This step of
shaping the tissue is irrelevant to the aims of the method object
of the present description.
[0039] The method of fixation follows and is carried out by
immersing the tissue in a solution containing glutaraldehyde at a
concentration in the range 0.30% to 1.00%. In some embodiments, the
solution contains glutaraldehyde at a concentration of 0.5%.
[0040] In some embodiments, the solution containing glutaraldehyde
is an aqueous solution including a buffer selected from phosphate,
citrate, acetate, HEPES, and borate. In some embodiments, the
buffer is phosphate.
[0041] The pH of the glutaraldehyde-based solution is in the range
of 5 to 8. In some embodiments, the pH of the glutaraldehyde-based
solution is 7.4.
[0042] The fixing method is conducted at a temperature in the range
4.degree. C. to 30.degree. C. In some embodiments, the fixing
method is conducted at room temperature (20.degree. C.). The period
of exposure of the tissue to the solution containing glutaraldehyde
can vary in the range 1 to 20 days. In some embodiments, the
exposure time is in the range of 3 to 13 days.
[0043] After the fixation step, the tissue is washed to remove
residual glutaraldehyde that is not conjugated to the tissue. In
some embodiments, the wash solution is a saline solution or a
phosphate buffer pH 7-7.4 and is changed three times. Washing is
performed for a period comprised in the range 30 minutes to 6
hours, with gentle agitation, at room temperature.
[0044] The fixed tissue is then detoxified using an aqueous
solution containing taurine at a concentration w/v comprised in the
range of 0.10% up to saturation of the solution. In some
embodiments, the aqueous solution includes taurine at a w/v
concentration of 0.20% to 1.00%. In some embodiments, the aqueous
solution includes taurine at a concentration of0.70% w/v.
[0045] In some embodiments, the aqueous taurine solution contains a
buffer selected from phosphate, citrate, acetate, HEPES
(4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid), and borate.
In some embodiments, the buffer is phosphate. In some embodiments,
the pH of the solution is in the range of 4 to 9. In some
embodiments, the pH of the solution is 5 to 8. In some embodiments,
the pH of the solution is 7.
[0046] In some embodiments, the detoxification step is conducted at
a temperature in the range from room temperature (20.degree. C.) to
50.degree. C. In some embodiments, the detoxification step is
conducted at a temperature that is in the range of 30.degree. C. to
45.degree. C. In some embodiments, the detoxification step takes
place at a temperature of 40.degree. C. In some embodiments, the
period of immersion of the tissue in the taurine-containing
solution may vary in the range from 2 to 96 hours. In some
embodiments, the immersion period may be in the range of 12 to 48
hours. In some embodiments, the immersion period is 24 hours.
[0047] At the end of the detoxification step, the detoxified tissue
is subjected to washing at room temperature, in phosphate buffer at
pH 7, for about three hours, changing the wash solution three
times.
[0048] The detoxified tissue is finally transferred to a
conservation solution without aldehydes in phosphate buffer pH 7
containing preservatives such as parabens.
[0049] The efficacy of the method described herein, and therefore
the occurrence of the reaction between the amino groups of the
detoxifying molecule and the aldehyde groups present on the fixed
tissue, is evaluated by staining of the aldehyde groups remaining
free. The greater the staining intensity, the more numerous are the
free aldehyde groups and, on the contrary, the weaker (or absent)
the staining, the less numerous (or absent) are the free aldehyde
groups present on the detoxified tissue. An example of a stain
advantageously useful for such determination is fuchsine.
[0050] The results presented below demonstrate that taurine binds
the free aldehyde groups present on fixed tissue more efficaciously
than homocysteic acid. In addition, the present inventors have
observed that better results are obtained by performing the
detoxification step at a temperature above room temperature, that
is, the number of free aldehyde groups present on the fixed tissue
are greatly reduced.
[0051] The various preferred embodiments of the present invention
will be described in detail below.
Materials and Methods
Harvesting and Fixation of Biological Tissue
[0052] The biological tissue, which were pieces of bovine
pericardium harvested from authorized abattoirs, was placed in a
saline solution maintained on ice and transported to the
laboratory.
[0053] The tissues were washed with saline solution to eliminate
possible traces of blood, separated from the any adherent
ligamentous and adipose parts and were selected on the basis of the
correct thickness and the absence of defects, such as
dishomogeneity in thickness, presence of vascularizations, cuts,
abrasions, etc.
[0054] The tissues were prefixed in a glutaraldehyde solution at
0.20% v/v in phosphate buffer pH 7.4, for a time variable in the
range of 3 to 13 hours at room temperature.
[0055] After the prefixation step, the tissues were cut and shaped
according to known art techniques--for making, for example, cardiac
valves. Next the tissues were fixed for a period of time in the
range 3 to 13 days at room temperature, in an aqueous solution of
0.50% v/v glutaraldehyde in phosphate buffer pH 7.4.
Detoxification of Tissue
[0056] The tissue to be detoxified was washed, to remove residual
glutaraldehyde solution, in saline solution or in phosphate buffer
pH 7.4 or pH 7, for about three hours, with gentle agitation, at
room temperature. The wash solution was changed three times, using
about 300 ml of solution each time for each piece of tissue with
dimensions of about 10.times.5 cm.
[0057] The fixed solution was then detoxified using an aqueous
solution containing taurine at a concentration (w/v) of about 0.70%
in phosphate buffer pH 7. The detoxification step was conducted at
room temperature (20.degree. C.), 40.degree. C. and 50.degree. C.,
for a period of time of 24 hours, using about 200 ml of solution
for each tissue.
[0058] At the end of the detoxification step, the detoxified tissue
was washed at room temperature, in phosphate buffer at pH 7, for
about three hours, changing the wash solution three times.
[0059] Finally, the detoxified tissue was transferred to a
conservation solution without aldehydes containing preservatives,
preferably parabens, in phosphate buffer pH 7.
[0060] With the aim of demonstrating the better efficacy of the
detoxifying treatment with taurine compared to the known art, the
present inventors have semi-quantitatively determined the number of
free aldehyde groups in three samples of biological tissue treated
as follows: [0061] a first control sample (FIG. 1A) including
biological tissue fixed with glutaraldehyde but not detoxified;
[0062] a second control sample (FIG. 1B) fixed with glutaraldehyde
and detoxified with a solution containing homocysteic acid at a
concentration (w/v) of 1.00% in phosphate buffer pH 7; [0063] a
third sample (FIG. 1C) fixed with glutaraldehyde and detoxified
with a solution containing taurine at a concentration (w/v) equal
to 0.70% in phosphate buffer pH 7.
[0064] The tissue samples were immersed in the detoxifying
solutions for about 24 hours at room temperature with gentle
agitation.
[0065] To verify the efficacy of the taurine detoxifying treatment
with variation of the detoxifying solution temperature, the present
inventors semi-quantitatively determined the number of free
aldehyde groups in biological tissues treated as follows: [0066] a
first control sample (FIG. 2A) including biological tissue fixed
with glutaraldehyde but not detoxified; [0067] a second sample
(FIG. 2B) fixed with glutaraldehyde and detoxified with a solution
containing taurine at a temperature of 20.degree. C.; [0068] a
third sample (FIG. 2C) fixed with glutaraldehyde and detoxified
with a solution containing taurine at a temperature of 40.degree.
C.; and [0069] a fourth sample (FIG. 2D) fixed with glutaraldehyde
and detoxified with a solution containing taurine at a temperature
of 50.degree. C.
[0070] The samples were immersed in the detoxifying solution for
about 24 hours with gentle agitation both at room temperature and
at 40.degree. C.; the fourth sample detoxified at 50.degree. C. was
maintained immersed for 7-8 hours with gentle agitation.
[0071] About 200 ml of detoxifying solution were used for each
sample.
[0072] At the end of the treatment all samples were washed at room
temperature in phosphate buffer at pH 7, for about three hours,
changing the wash solution three times and using about 300 ml of
solution for each sample at each change.
[0073] All samples were then transferred to a phosphate buffer
solution pH 7 containing preservatives such as parabens.
[0074] Staining of the tissue with fuchsine
[0075] Tissue staining to detect free aldehyde groups uses an
acidic solution of rosaniline hydrochloride (fuchsine). The
staining takes advantage of the formation of bonds between the
NH.sub.2 groups of the dye and the free aldehyde groups on the
tissue.
[0076] At first, the solution is colorless but in the presence of
free aldehyde groups a violet color develops. This is a qualitative
evaluation of the availability of free aldehyde groups, after the
various treatments.
[0077] The samples to stain were cut to obtain cards with
dimensions of about 1.5.times.1.5 cm and successively immersed in
the staining solution, about 10 ml, each card in a separate test
tube.
[0078] The staining solution was 1.00% rosaniline hydrochloride,
4.00% sodium metabisulfite in 0.25 M hydrochloric acid. The samples
remained immersed in the stain for 5 minutes at room temperature,
with gentle agitation.
[0079] Each sample was then transferred to a solution obtained
mixing 8 gr of Na.sub.2SO.sub.3 and 30 ml of 37% hydrochloric acid,
brought to one liter with demineralized water. The samples remained
immersed for 10 minutes in this wash solution, with mild
agitation.
[0080] The samples were then subjected to 2 successive 10 minute
washings, with mild agitation in a wash solution including 700 ml
of ethanol and 30 ml of 37% hydrochloric acid, brought to one liter
with demineralized water.
[0081] The washings removed stain that was non-specifically bound
to the tissue. About 20 ml of wash solution was used at each
change.
[0082] When finished the samples were transferred to phosphate
buffer pH 7 and photographed to document the different
staining.
Reflectance Spectroscopy
[0083] The stained samples were subjected to reflectance
spectroscopy to quantitatively evaluate the different chromatic
characteristics of the fuchsine staining.
[0084] Reflectance spectroscopy is a technique for optical
investigation based on measurement of the spectral reflectance
factor of the surface of a sample as a function of the wavelength
of incident radiation. The reflectance parameter is expressed as
the ratio of the intensity of the reflected radiation and the
incident radiation, as a function of wavelength.
[0085] Reflectance measurements were carried out at a wavelength of
570 nm using a Perkin Elmer Lambda 35 spectrophotometer with a
spherical integrator.
[0086] On a scale of values, a lower reflectance value indicates a
more intense sample staining and on the contrary a higher
reflectance value indicates a weak staining intensity.
[0087] Determination of the contraction temperature
[0088] The contraction temperature is an index of the level of
cross linking of the fixed tissue and was determined on disks of
pericardium of about 5 mm diameter, using a scanning differential
calorimeter (DSC)Q100 TA Instruments with the following parameters:
[0089] nitrogen flow of 50 ml/min, [0090] heating ramp of 5.degree.
C./min, [0091] temperature interval between 65.degree. C. and
95.degree. C.
Results
Detoxification
[0092] The efficacy of the method described herein, and therefore
of the reaction that takes place between the amino groups of the
detoxifying molecule and the aldehyde groups on the fixed tissue,
is demonstrated by staining the aldehyde groups remaining free with
fuchsine; more intense staining indicates numerous free aldehyde
groups and, on the contrary, weaker or absent staining indicates
few or no free aldehyde groups on the detoxified tissue.
[0093] FIGS. 1A-1C show a different intensity of staining of a
sample detoxified with a solution containing homocysteic acid,
sample B, and of a sample detoxified with a solution containing
taurine, sample C. A sample not detoxified, sample A, has an
intense purple color. The intensity of staining of sample B is less
than that of the control sample, but the staining of sample C is
very weak if not absent.
[0094] The number of free aldehyde groups present on the sample
exposed to a solution containing taurine is therefore decidedly
lower than that of a sample not detoxified after fixation.
Furthermore, treatment with taurine is significantly more
efficacious than that carried out with homocysteic acid.
[0095] These observations are confirmed by the reflectance
spectroscopic analysis of the samples. The reflectance of each
sample depends on the staining intensity and by analysis carried
out with a spectrophotometer, it is possible to associate a percent
reflectance value to each sample.
[0096] A higher reflectance value is associated with weaker
staining intensity and vice versa a lower reflectance value is
related to a greater staining intensity.
[0097] As can be seen from the results reported in table 1, the
reflectance analyzed at a wavelength of 570 nm and expressed as a
percent shows a value of 6.5 for the non detoxified sample shown in
FIG. 1A, an intermediate value for the sample detoxified with
homocysteic acid shown in FIG. 1B and a decidedly higher value for
the sample detoxified with a solution containing taurine shown in
FIG. 1C.
TABLE-US-00001 TABLE 1 Staining Reflectance % Sample observed at
570 nm A--non detoxified Very intense 6.5 purple B--homocysteic
acid Violet 9.2 room temperature C--taurine Pale violet 13 room
temperature
[0098] These results confirm that treatment with taurine is more
efficacious than treatment with homocysteic acid in neutralizing
free aldehyde groups on the fixed tissue.
[0099] With reference to FIGS. 2A-2D, it can be seen how modifying
the temperature conditions under which the sample detoxification is
carried out with the solution containing taurine increases the
efficacy of the treatment. The staining intensity of the sample
detoxified at 40.degree. C., sample C, is weaker with respect to
that of the sample detoxified at room temperature, sample B. Even
more evident results are obtained by performing the detoxifying
treatment at a temperature of 50.degree. C.; in fact, sample D has
a staining of decidedly lower intensity indicating that fewer free
aldehyde groups remaining, and therefore sites for binding and
accumulation of calcium, with respect to the other treatments.
[0100] The results of reflectance spectroscopic analysis for the
samples shown in FIGS. 2A-2D are presented in table 2. The highest
reflectance value, equal to 22, is from a sample having a staining
intensity almost absent, that is the sample treated with taurine at
50.degree. C. The non detoxified sample, which has very intense
staining, has the lowest reflectance value. The sample treated with
taurine at room temperature has a higher value than the non
detoxified control, but lower than the sample treated with taurine
at 40.degree. C.
TABLE-US-00002 TABLE 2 Staining Reflectance % Sample observed at
570 nm A--non detoxified Very intense 6.5 purple B--taurine Pale
violet 12 room temperature C--taurine 40.degree. C. Pink 17
D--taurine 50.degree. C. Completely 22 colourless
[0101] Also in this case the reflectance spectroscopy results
confirm that treatment with taurine has a greater detoxifying
capacity when conducted at a temperature higher than room
temperature.
Contraction Temperature
[0102] To verify that the detoxifying treatment does not alter the
cross linking of the biological tissue obtained by means of
immersion in a solution containing glutaraldehyde, the contraction
temperature of tissue detoxified by means of treatment with a
solution containing taurine was compared to the contraction
temperature of control tissue fixed and not detoxified.
[0103] The contraction temperature of tissues immersed in the
solution containing taurine at room temperature or at 40.degree. C.
are indistinguishable from the contraction temperature of the non
detoxified tissue, that temperature being 85-86.degree. C.
Therefore, neither treatment has significant effects on the level
of tissue cross linking.
[0104] The contraction temperature of tissue immersed in the
solution containing taurine at 50.degree. C. remains the same as
that of the non detoxified tissue (85-86.degree. C.) if the
treatment period does not exceed 7-8 hours.
[0105] Without prejudice to the underlying principle of the
invention, the details and the embodiments may vary, even
appreciably, with reference to what has been described by way of
example only, without departing from the scope of the invention as
defined by the annexed claims.
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