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BACKGROUND OF INVENTION
1. Field of Invention
The present invention relates to the biological prosthetic materials
including heart valves, vessels, pericardium, dura mater, skin, amniotic
membrane, umbilical cord, muscle tendons, ligaments, fascias and gut of
mammalian origin used as a whole or in a part as grafts for various organs
and tissues. This invention is particularly suitable for replacement or
repair of heart valves, blood vessel, pericardial tissues and other body
membranes.
2. Description of the Prior Art
Bodily tissue replacement surgery has been established over the past 30
years, particularly in the area of valve and organ replacement. The field
has been shared between biological and mechanical replacement in the
repair of tissues and organs. In the area of biological replacement of
heart valves, glutaraldehyde treated porcine heart valves, have been used
as an improvement over mechanical valves. Carpentier A et at., J Torah
Cardiovasc Surg 58:467-82, (1969). Dystrophic calcification is chief cause
of failure of bioprosthetic heart valves derived from
glutaraldehyde-treated porcine aortic valves, Schoen F J et al. Human
Pathol; 16:549-59(1985). Schoen F J et at. Cardiovasc Clin; 18:289-17
(1988). Ibrahim M et al. J Thorac Cardiovasc Surg; 108:221-30(1994).
Calcific deposits in either porcine valves or bovine pericardial valves are
evident in residual connective cells of bioprosthetic tissue within 48
hours of subcutaneous implantation. Initial nucleation sites are located
in cell membranes, cell nuclei, and intracellular organelles, such as
mitochondria. Cell-associated deposits of calcium increase in size and
number over time after implantation. These deposits obliterate cells,
dissect among collagen bundles, and ultimately form gross nodules similar
to those associated with clinical failures. Direct collagen involvement
subsequently occurs. Schoen F J et al. In: Bodnar E, ed. Surgery for heart
valve disease: The proceedings of the 1989 symposium. 679-85 (1990). The
components of a valve can be divided into two parts with respect to their
degree of antigenicity: (1) cells, water soluble proteins,
mucopolysaccharides, and structural glycoproteins which had a high degree
of antigenicity and. (2) collagen and elastin which appear to be less
antigenic. In some instances, collagen can induce an immunological
response. Carpentier A et al. J Thorac Cardiovasc Surg, 58:467-82(1969).
Concerning the prevention of calcification, various chemical techniques
have been used but the results are inconclusive. Inhibition of
mineralization of bioprosthetic valves implanted subcutaneously in small
size animals has been achieved through pretreatment with various chemical
components Carpentier A et al., Circulation; 70 (suppl 1): 165-8(1984).
Lentz D J et al., Trans Am Soc Artif Intern Organs, 28:494-8 (1982) and
with systemic or local controlled release of diphosphonate, Webb C L et
al., Ann Thorac Surg, 46:309-16(1988). Levy R J et al., Circulation
71:349-56(1985). T6 (sodium dodecyl sulfate) processing inhibits the onset
of intrinsic mineralization in glutaraldehyde-fixed xenograft tissue,
Lentz D J et al. Cohn L W, Galluci V, eds. Cardiac biotissue grafts.
Proceedings of the Second International Symposium. New York: Yorke Medical
Books., 306-19(1982). T6 treatment did not significantly affect the onset
and degree of bioprosthetic calcification, Thurbrikar M J et al., Trans Am
Soc Artif Intern Organs, 29:245-9 (1983). Toluidine blue is the calcium
retarding agent employed in the Medtronic-Intact porcine valve. Although
the biochemical mechanism by which the toluidine blue process modifies the
calcification is unknown. It is presumed that toluidine blue treatment
inhibits or retards calcification of collagen, while the calcification of
cellular components remains unaffected Valence M et al., Bodnar E, ed.,
Surgery for heart valve disease: The proceedings of the 1989 symposium,
668-76(1990). Preincubation in aluminum chloride (AICl.sub.3)
significantly inhibits calcification of bovine pericardium in 60 days
after being subcutaneously implanted in rats. Iron also inhibits
calcification of bioprosthetic tissue Schoen F J et at., Bodnar E, ed.
Surgery for heart valve disease: The proceedings of the 1989 symposium.
London: ICR Publishers, 679-85(1990). Other methods to reduce
calcification of biotissue grafts, such as acyl-azide treatment, Petite H
et at., J Biomed Mater Res., 24:179-87(1990) and dye mediated photo
oxidization, Moore M A et al., J Biomed Mater Res, 28:611-8(1994) have
been reported. It also has been reported that L-Glutamic acid
posttreatment significantly reduces the calcification of glutaraldehyde
treated bioprosthetic material subcutaneously implanted in rats
Grabenvorger M et al. J Biomed Mater Res, 26:1231-40(1992). According to
unpublished work by the inventors, it is not apparent that temperature has
a positive effect on calcium mitigation of biotissue grafts, however it
has been reported that pretreatment of porcine aortic valve in
glutaraldehyde at high temperature (50.degree. C.) alone mitigates
calcification in both subcutaneous and circulatory models Carpentier S M
et at., Ann Thorac Surg, 69:S332-8(1995). It has been reported that
.alpha.-amino oleic acid (AOA) posttreatment prevents calcification of
glutaraldehyde treated bioprosthetic heart valves, Girardot M N et al.,
Trans Soc Biomater., 14:114(1991). In all valves implanted in mitral
position in young sheep for 5 months, AOA-treated valves had morphologic
features suggesting generalized tissue degradation, including structural
loosening, surface roughening, and deep cuspal collections of
erythrocytes, Gott J P et al., Ann Thorac Surg, 53:207-16(1992). Moreover,
the treatment with .alpha.-amino oleic acid did not prevent the
calcification of aortic wall in sheep study Chen W et al., Circulation
90:323-9(1994). Researchers have found that the tissue extraction process
significantly reduces the propensity of xenograft calcification in vivo,
Vesely I. et al., Ann Thorac Surg, 60:S359-64(1995). The extent of
glutaraldehyde cross-linking is clearly important, although the specific
mechanisms by which glutaraldehyde fixation facilitates mineralization are
not understood Webb C L et at., Ann Thorac Surg ;46:309-16(1988). The slow
release of residual (unbound) glutaraldehyde from the prosthesis over a
period of time after implantation reinforces the host plasma calcium-acid
bound complex. This acid-plasma calcium complex promotes further
mineralization when glutaraldehyde plays no more role as the primary
factor for nucleation of calcification. Chanda J., Ann Thorac Surg,
60:S339-42(1995)
In cells modified by aldehyde cross-linking or mechanical injury, cell
membranes are disrupted (leading to increased permeability), and
mechanisms for calcium extrusion are no longer fully functional. Moreover,
high energy phosphates (particularly ATP) required to fuel these
mechanisms are unavailable. Thus, calcium accumulation occurs unimpeded
with a dramatic increase in intracellular calcium Schoen F J et al.,
Cardiovasc Clin,; 18/2:28917(1988); Webb C L, et al., Ann Thorac Surg,
46:309-16(1988). When glutaraldehyde treatment is used alone, elimination
of highly antigenic substances such as cellular elements and water soluble
proteins, does not prevent the calcification of glutaraldehyde-treated
biotissue grafts implanted in adult rats. Moreover, further treatment with
glutarahyde does not prevent calcification. Chanda J., Ann Thorac Surg,
60:S339-42(1995). Inactivation of residual glutaraldehyde and unbound
aldehyde moieties on the surface of the tissue gratis, either with amino
compound like chitosan (a biopolymer) or glycine+gentamicin completely
prevents the calcification of glutaraldehyde-treated biotissue grafts
implanted subdermally in adult rats (120-150 g), Chanda J., Ann Thorac
Surg 60:S339-42; Chanda J., Artif Organs, 18:408-10(1994). However
inactivation of free impaired aldehydes either with chitosan, or
glycine+gentamicin, or chitosan in combination with glycine and gentamicin
does not prevent the calcification of glutaraldehydetreated biotissue
grafts when implanted subdermally in weanling (3-week-old, 30-50 g) rats.
Heparin has potent anti-growth effect in smooth muscle cells Hoover R L et
al., Circ Res, 47:578-83(1980). It also has been shown that heparin binds
to the surface of cells Hiebert L M et at., Thromb Res, 8:195-04(1976)
Different reactions used to break down heparin are known Shively J et al.,
Biochemistry, 15:3932-42. (1976). Method for covalent binding of heparin
to artificial surfaces has been developed, Larm O et at., Biomat Med Dev
Art Org, 11:161-73(1983) At present heparin coated tube is widely used as
cardiopulmonary bypass circuit in open heart surgery. Heparin as one of
various forms of glycosaminoglycan may be used in preparation of
artificial skin substitute (wound dressing) U.S. Pat. No. 5,489,304 to
Orgill et al. issued Feb. 6, 1996.
SUMMARY OF THE INVENTION
The present invention, describes a biotissue grafts formed by covalently
binding heparin to glutaraldehyde-treated xenografts or heterografts which
are used in biological tissue grafts. These biological tissue grafts do
not calcify nor are subject to thrombosis after implantation.
The xenografts or heterografts are prepared by treating the tissues removed
from the animal with glutaraldehyde which covalently binds to the tissues.
The glutaraldehyde treated tissues are reacted with amino compound
(chitosan-glycine-gentamicin). The tissues grafts and then reached with
partially degraded heparin which effectively prevents calcification after
implantation of tissue grafts.
Coupling of partially degraded heparin completely prevents the
calcification of glutaraldehydechitosan-treated porcine pulmonary valve,
aortic valve or pericardium implanted in weanling rats for more than 5
months. Heparin bonding after neutralization of residual glutaraldehyde
with amino compound like chitosan, glycine, gentamicin, is essential in
prevention of calcification of glutaraldehyde treated tissue grafts. It is
necessary that the concentration of glutaraldehyde for cross linking the
tissue graft does not exceed 0.25%.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Calcification (accumulation of calcium salt) is the main cause of failure
of bioprosthetic heart valves. This pathological process occurs much
faster in children and young patients than that in older patients (more
than 60 years of age ). The exact mechanism of this calcification process
is unknown. At present all commercially available heterograft heart valve
substitutes are made of glutaraldehyde-treated porcine aortic valves or
bovine pericardium. Highly antigenic substances like easily-extractable
water soluble proteins and cellular elements of grafts can be removed by
treatment with hypertonic solution and proteolytic enzyme like trypsin,
and this can be started immediately after harvesting the grafting
materials. To make the graft non-antigenic (inert), and to stabilize
collagen material glutaraldehyde cross-linking is necessary. At 37.degree.
C. glutaraldehyde cross-links with collagen fibers of grafts much faster.
Stabilization process of collagen fibers with glutaraldehyde is completed
within one month if the concentrations of glutaraldehyde is gradually
increased from 0.1% to 0.25%. No special buffer is necessary for the
preparation of glutaraldehyde solution. Normal saline is sufficient for
required dilution. Posttreatment with amino compounds prevents the
slow-release of residual glutaraldehyde, and inactivates the free aldehyde
moieties on the surface of the glutaraldehyde-treated tissue grafts.
Chitosan, gentamicin, glycine serve these purposes. Due to the presence of
large number of amino termini, one chitosan
[.alpha.(I-4)amino-2deoxy-.beta.-D-glucan] (mol. wt. 2200-86000) molecule
covalently cross-links with free aldehyde moieties on the surface of the
glutaraldehyde-treated biotissue grafts and simultaneously inter-links
other chitosan molecules with the help of residual glutaraldehyde which is
slowly released from the treated tissue. As chitosan alone can not react
with all aldehyde groups of glutaraldehyde, addition of glycine-gentamicin
mixture neutralizes all remaining free aldehyde moieties. Despite complete
inactivation of aldehyde moieties, chitosan-glycine-gentamicin post
treated glutaraldehyde-treated biotissue grafts, do calcify after
implantation subdermally in 3-week-old rats and in systemic circulation in
juvenile sheep. To block the potential binding sites, modify charges and
fill intertropocollagen spaces, heparin is necessary. Free aldehyde
moieties of partially degraded heparin bind covalently with the free amino
groups of chitosan and gentamicin. Further free aldehyde groups of already
coupled heparin are blocked by cross-linking with
chitosan-glycine-gentamicin. Compounds containing aldehyde (CHO--)
functions react with primary amines to yield relatively labile Schiff
bases that can be converted to stable secondary amines by reduction with
sodium borohydride. These biotissue grafts can easily be stored in normal
saline containing glycine and gentamicin. In vivo studies showed that
bioprosthetic material prepared according to the above described method
did not calcify when compared with only glutaraldehyde-treated materials
and can be used in replacement therapy and grafting in mammals including
humans.
EXAMPLE
Porcine aortic and pulmonary valves, and pericardium were washed in 5%
sodium chloride for 24 hours at 4.degree. C. These materials were rinsed
in copious mounts of deionized water, and incubated in trypsin (Trypsin
1:250, Difco Labs, Detroit, Mich., USA) in normal saline (pH 7.4) for 40
minutes at 37.degree. C. Then, all grafts were cross-linked in
glutaraldehyde in normal saline (pH 7.4) with gradually increasing
concentrations of glutaraldehyde (Glutaraldehyde EM 25%, TAAB Laboratories
Equipment Ltd., Reading, UK) from 0.1 to 0.25% at 37.degree. C. for a
period of one month. Glutaraldehyde-treated grafts were placed first in
0.1% chitosan (Sigma Chemical Co., St. Louis, Mo., USA) solution for two
weeks, then in a solution of 0.05% chitosan, 1% glycine (Glycine, Sigma)
and 0.015% gentamicin sulfate (Schering-Plough, Osaka, Japan) for two
weeks, and finally in 1% glycine-0.015% gentamicin sulfate solution for a
week. Heparin (Heparin sodium salt 164.5 I.U./mg, Nacalai Tesque Inc.,
Kyoto, Japan) was partially degraded by nitrous acid generated in situ by
addition of sodium nitrite (Wako Pure Chemical Industries Ltd. Tokyo,
Japan) and hydrochloric acid (Nacalai) to the heparin solution (pH 2.0) at
4.degree. C. for a period of 3 hours. On completion of the partial
degradation, the pH of the solution was adjusted to 7.4 with 1N sodium
hydroxide (Nacalai). All previously treated materials were cross-linked in
a solution containing 0.1% partially degraded heparin for a week. Heparin
bonded grafts were thoroughly washed in normal saline and kept in a
solution of 0.05% chitosan, 1% glycine and 0.015% gentamicin sulfate for
an additional week. Sodium borohydride (Wako) was dissolved in deionized
water containing 1N sodium hydroxide (1 ml/1 liter H.sub.2 O) to titrate
the pH up to 8.4. Treated grafts were placed in 0.06% sodium borohydride
solution for 24 hours at room temperature, followed by washing in normal
saline and 0.1% chitosan solution for 10 minutes. Then all specimens were
kept in a solution containing 0.05% chitosan, 1% glycine and 0.015%
gentamicin sulfate for 3 days and were stored in normal saline containing
0.5% glycine and 0.03% gentamicin sulfate until they were implanted.
Although this invention has been described and illustrated by reference to
certain specific examples, these are exemplary only and the invention is
limited only in scope by the following claims and functional equivalents
thereof.
What is claimed as new and desired to be protected by Letters Patent is set
forth in the appended claims.
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