Canadian Patents Database / Patent 1087610 Summary
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|(12) Patent:||(11) CA 1087610|
|(21) Application Number:||256978|
|(54) English Title:||CROSSLINKED COLLAGEN MUCOPOLYSACCHARIDE COMPOSITE MATERIALS|
|(54) French Title:||SUBSTANCES COMPOSEES DE MUCOPOLYSACCHARIDES ET DE COLLAGENE RETICULES|
- Bibliographic Data
- Representative Drawing
- Admin Status
- Owners on Record
|(52) Canadian Patent Classification (CPC):||
|(51) International Patent Classification (IPC):||
|(72) Inventors :||
|(73) Owners :||
|(71) Applicants :|
|(74) Agent:||BORDEN LADNER GERVAIS LLP|
|(22) Filed Date:||1976-07-14|
|(30) Availability of licence:||N/A|
|(30) Language of filing:||English|
|(30) Application Priority Data:|
ABSTRACT OF THE DISCLOSURE
Composite materials are disclosed which are formed by
contacting collagen with a mucopolysaccharide and subsequently
crosslinking the resultant polymer. These composite materials
have a balance of mechanical, chemical and physiological proper-
ties which make them useful in surgical sutures and prostheses
of controlled biodegradability (resorption) and controlled
ability to provoke a foreign body reaction, as well as making
them useful in any applications in which blood compatibility is
THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR
PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A composite material comprising a reaction product formed by
contacting collagen with a mucopolysaccharide under acidic conditions whereby
a collagen-mucopolysaccharide product containing at least about 0.5% muco-
polysaccharide is produced and subsequently covalently crosslinking the col-
lagen-mucopolysaccharide product to a degree which provides significantly in-
creased resorption resistance over native collagen.
2. A composite material of claim 1 having an Mc value of between
about 800 and about 60,000.
3. A composite material of claim 2 wherein collagen is contacted
with a mucopolysaccharide by coating collagen fibrils with a mucopolysaccharide.
4. A composite material of claim 2 wherein collagen is contacted
with a mucopolysaccharide by co-precipitating collagen and a mucopolysaccharide.
5. A composite material of claim 4 wherein covalent crosslinking
is achieved by contacting the collagen mucopolysaccharide product with a
chemical crosslinking agent.
6. A composite material of claim 5 wherein said chemical cross-
linking agent comprises an aldehyde.
7. A composite material of claim 6 wherein said aldehyde comprises
8. A composite material of claim 4 wherein covalent crosslinking
is achieved dehydrothermally.
9. A composite material of claim 8 wherein dehydrothermal cross-
linking is accomplished by subjecting said collagen-mucopolysaccharide product
to a vacuum of at least about 10-5 mm Hg. until said product has the desired
10. A composite material of claim 8 wherein dehydrothermal cross-
linking is accomplished by subjecting said collagen-mucopolysaccharide product
to an elevated temperature of at least about 80°C until said product has the
desired Mc value.
11. A composite material of claim 8 wherein dehydrothermal cross-
linking is accomplished by subjecting said collagen-mucopolysaccharide product
to a vacuum of at least about 0.01 mm of Hg and an elevated temperature of at
least about 95°C until said product has the desired Mc value.
12. A composite material of claims 1, 5 or 8 wherein said mucopoly-
saccharide contains a sulfate group.
13. A composite material of claims 1, 5 or 8 wherein said mucopoly-
saccharide is selected from chondroitin 6-sulfate, chondroitin 4-sulfate,
heparin, heparan sulfate, keratan sulfate or dermatan sulfate.
14. A composite material of claims 1, 5 or 8 wherein said muco-
polysaccharide is selected from chondroitin 6-sulfate, chondroitin 4-sulfate,
heparin, heparan sulfate, keratan sulfate or dermatan sulfate having an Mc
value of between about 5,000 and about 10,000.
15. A composite material of claims 1, 5 or 8 wherein said mucopoly-
saccharide is selected from chondroitin 6-sulfate, chondroitin 4-sulfate,
heparin, heparan sulfate, keratan sulfate or dermatan sulfate having an Mc
value of between about 5,000 and about 10,000, said collagen-mucopolysaccharide
product contains between about 6% and about 15% mucopolysaccharide.
16. A blood-compatible prosthesis formed from a reaction product
produced by contacting collagen and a mucopolysaccharide under acidic condi-
tions and subsequently covalently crosslinking the collagen-mucopolysaccharide
product to an Mc value of between about 800 and about 60,000, said reaction
product containing at least about 0.5% by weight of said mucopolysaccharide.
17. A blood-compatible prosthesis of claim 16 which contains between
about 6% and about 15% by weight of a mucopolysaccharide.
18. A blood-compatible prosthesis of claim 17 wherein said muco-
polysaccharide contains a sulfate group.
19. A blood-compatible prosthesis of claim 18 wherein said sulfate-
containing mucopolysaccharide is selected from chondroitin 6-sulfate, chon-
droitin 4-sulfate, heparin, heparan sulfate, keratan sulfate or dermatan
20. A resorption resistant suture or prosthesis formed from a reac-
tion product produced by contacting collagen and a mucopolysaccharide under
acidic conditions, said reaction product containing between about 6% and about
15%, by weight, mucopolysaccharide and being covalently crosslinked to an Mc
value of below 60,000 which provides significantly increased resorption resis-
tance over collagen.
21. A non-immunogenic suture or prosthesis formed from a crosslinked
polymer of collagen and a mucopolysaccharide reacted under acidic conditions,
said polymer being covalently crosslinked to an Mc value of between about
5,000 and about 10,000 and containing at least about 6%, by weight, mucopoly-
22. A reaction product formed by contacting collagen and a mucopoly-
saccharide in an aqueous solution having a pH of less than about 5 and subse-
quently covalently crosslinking the collagen-mucopolysaccharide product.
23. A reaction product of claim 22 wherein the aqueous solution
comprises an aqueous acetic acid solution.
24. A reaction product of claim 23 wherein said collagen-mucopoly-
saccharide product is formed by coprecipitating collagen and a mucopolysac-
25. A reaction product formed by reacting collagen and a mucopoly-
saccharide under acidic conditions, said reaction product containing at least
about 6%, by weight, mucopolysaccharide and said reaction product being
26. A reaction product formed by reacting noncrystalline collagen
and a mucopolysaccharide and subsequently covalently crosslinking the product
27. A process for preparing a crosslinked collagen-mucopolysaccharide
composite material comprising:
a. contacting collagen and a mucopolysaccharide
under acidic conditions whereby a collagen-mucopoly-
saccharide product is formed; and,
b. covalently crosslinking said collagen-muco-
polysaccharide product to an Mc value of between about
800 and about 60,000.
28. A process of claim 27 wherein said collagen and mucopolysaccharide
are contacted in the presence of acetic acid.
29. A process of claim 27 wherein said covalent crosslinking is
achieved by removing sufficient water to crosslink said collagen-mucopoly-
saccharide product to the desired Mc value.
30. A process of claim 27 wherein said covalent crosslinking is
achieved by employing a chemical crosslinking agent.
31. A process of claim 30 wherein said chemical crosslinking agent
comprises an aldehyde.
32. A process of claim 31 wherein said collagen and mucopoly-
saccharide are reacted in the presence of acetic acid.
3ACKGROUND OF THE INVENTION
1. F;eld of the Invention
. The invention is in the field of materials and more
particularly in the field of composite polymeric materials having
S suitable properties for medical and surg;cal applicati.ons.
2. Description of the Prior Art
Collagen, a major protein constituent.of connective
tissue ~n vertebrate and invertebrate animals, is widely used
in'medical and surgical applications in the fabrication of sur-
gical sutures, blood vessel grafts, and in all forms of surgical
prostheses. Whiie collagen is better'than most materials for
such applications, it does have some significant deleterious
. One such property is co'llagen's low resistance to
resorption since it is a resorbable animal protein which is
degraded by tissue enzymes (collagenases) present at implanta-
tion sites. Attempts.have been made to solve this problem by
crosslinking collagen, but these attempts have turned out to be
only partially successful because rather high degrees of cross- '
linking are required to make collagen non-resorbable. Altho'u'gh
crosslinking solves one problem, it creates another - that is,
~ the tensile strength and other mechanical properties of collagen
`' can suffer significantly when an excessively large degree of
crosslinking is.required to control the resorption to a very
Another deleterious property exhibited by collagen,
insofar as its uses in surgical prostheses and other such appli-
cations are concerned, is that collagen, like most other polymeric -'
- 2 - ~ .:
materials, is non-compatible with blood. To be qualified as blood~ --
compatible, a material must not cause either platelet aggregation or
clotting of red cells. Collagen causes both. Blood platelets are
known to adhere to exposed collagen, such as occurs when blood vessels
are mechanically injured, and this collagen-platelet interaction causes
platelet aggregation. The detailed mechanistic aspects of this inter-
action have been extensively studied and reported in the literature.
See, for example: Muggli, R. and Baumgartner, H. R., Thromb. Res., 3, 715
(1973); and Jamieson, G. A., Urban, C. L. and Barber, A. J., Nature New
Biol., 234, 5 (1971). In addition, collagen has been implicated in
acceleration of blood clotting by activation of Hageman factor (clotting
factor XII). See Wilner, G. D., Nossel, H. L. and LeRoy, E. C., J. Clin.
Invest., 47, 2608 (1968).
Previous efforts to synthesize blood compatible materials
have centered largely around attempts to attach a blood-compatible material
to the surface of a non-compatible material. The most successful materials
were formed by attaching heparin, a known anticoagulant, to the surface
of various synthetic polymers. Attachment of heparin to such surfaces
has been achieved by a variety of techniques, which are generally
classifiable as either ionic interaction or chemical reaction. Both of
these general techniques suffer from disadvantages, however. If the sub-
strate surface is not completely covered, the uncovered portions which
contact blood can cause formation of a thrombus or clot. Additionally,
the possibility-exists that, during the handling or use of such covered
materials, the surface coating of heparin can become detached due to a
mechanical incident or become hydrolyzed or otherwise be attacked chemically
or biochemically by substances found in the blood or in vascular tissue;
the resulting disruption of the surface coating is followed by exposure of
underlying non-compatible substrate. Even more serious, perhaps, is the
difficulty that heparin occasionally desorbs from the substrate and migrates
into the blood where, by virtue of its being a potent anticoagulant, heparin
interferes strongly with the competence of healthy blood to clot, which is
SUMMARY OF THE INVENTION
This invention relates to the synthesis of new composite
materials suitable for a wide variety of medical and surgical uses.
Such materials are formed by intimately contacting collagen with a
mucopolysaccharide and subsequently crosslinking the polymeric product.
Suitable collagen can be derived from a number of animal sources, either
in the form of a solution or in the form of a dispersion, and suitable
mucopolysaccharides include, but are not limited to chondroitin 4-sulfate,
chondroitin 6-sulfate, heparan sulfate, dermatan sulfate, keratan sulfate,
heparin and hyaluronic acid.
Crosslinking can be achieved by chemical, radiation, de-
hydrothermal or any other suitable technique. ~ suitable chemical
technique is aldehyde crosslinking, but other chemical crosslinking
reactants are equally suitable. Dehydrothermal crosslinking, which is
preferred, is achieved by reducing the moisture level of the composites
to a very low level, such as by subjecting the composite material to
elevated temperatures and high vacuum. Dehydrothermal crosslinking
eliminates the necessity to add, and in the case of toxic materials such
as aldehydes, to remove unreacted crosslinking agents; dehydrothermal
crosslinking also produces composite materials containing a wider range
of mucopolysaccharide content.
The products of such syntheses are belived to be comprised
of collagen molecules or collagen fibrils with long mucopolysaccharide
chains attached to them. Crosslinking appears to anchor the mucopoly-
saccharide chains to the collagen so that they will not elute or otherwise
become disengaged. Mechanically, these materials can be thought of as
analogous to fiber reinforced composite materials wherein collagen is the
fiber and mucopolysaccharide is the matrix; therefore, these materials are
sometimes referred to herein as composite polymeric materials.
Crosslinked collagen-mucopolysaccharide composites have been
found to retain the advantageous properties of native collagen. Unexpec-
tedly, however, it has been found that these materials, although relatively :
highly crosslinked, have outstanding mechanical properties. Such materials
can be synthesized, for example, which have ultimate tensile strength,
elongation at break, and other mechanical properties equal to or higher
than collagen crosslinked to the same level of the crosslink density.
In many cases, the mechanical properties of crosslinked collagen-mucopoly-
saccharide composite materials exceed those of native collagen which is
not artificially crosslinked. ~ecause of this, the collagen-mucopoly-
saccharide composites can be crosslinked to provide any desired degree of
resistance to resorption between the low degree exhibited by native collagen
which is not artificially crosslinked up to essentially complete resistance.
The ability to tailor the degree of resistance to resorption without
sacrificing mechanical properties provides a degree of design flexibility
for surgical prostheses, etc. heretofore unavailable with any class of
Surprisingly, most of the crosslinked collagen-mucopoly-
saccharide composites described herein have been found to be compatible
with blood. One such material can be formed from collagen,
a known thrombogenic material, and chondroitln 6-sulfate. Chondroitin
6-sulfate is as soluble in blood as heparin is but, unlike heparin, it
has such a low level of anticoagulant activity that it can be considered
to be inert in this regard. (Tests show, for example, that chondroitin
6-sulfate has between 1/3000 and 1/5000 the anticoagulant activity of
- heparin at equivalent concentrations). Reaction with mucopolysaccharides
appears to suppress essentially the entire procoagulant activity and
thrombogenic nature of native collagen. Thus, most of these composites
do not cause blood platelet aggregation, do not cause clotting, and, with
the exception of the collagen-heparin composite, do not interfere with the
competence of blood to clot. Since the materials are homogeneous, all
problems associated with surface coverage of thrombogenic materials with`
blood-compatible materials are obviated.
DESCRIPTION OF PREFERRED EMBODIMENTS
Collagen is a major protein constituent of connective tissue
in vertebrate as well as invetebrate animals. It is often present in the
form of macroscopic fibers which can be chemically and mechanically
separated from non-collagenous tissue components. Collagen derived from
any source is suitable for use with this invention, including insoluble
collagen, collagen soluble in acid, neutral or basic aqueous solutions,
as well as those collagens which are commercially available. Typical
animal sources include calfskin, bovine Achilles tendon and cattle bones.
Several levels of structuraI organization exist in collagen,
with the primary structure consisting of the sequence of amino acids.
Collagen is made up of 18 amino acids in relative amounts which are well
known for several animal species but in sequences which are still not
completely determined. The total
.. . .
- :: : ~ .
content of acidic, basic and hydroxylated amino acid residues far
exceeds the content of lipophilic residues making collagen a
hydrophilic protein. Because of this, polar solvents with high
'solubility parameters are good solvents for collagen.
" 5 At least two sets of characteristics which differentiate
collagen from other proteins ar'e: (l) the amino acid composition
- which is not only unique but is also distinctive because of its
high content of glycyl, prolyl and hydroxyprolyl residues; and
(2) the wide-angle x-ray diffraction pattern which shows a strong
meridional arc corresponding to a spacing of about 2.9A and a
strong equatorial spot corresponding in moist collagen to a
spacing of about 15A. A more detailed physicochemical definition ' '~
of collagen in the solid state is given in Yannas, I. V.,
- "Collayen and Gelatin in the Solid State", J. Macromol. Sci. -
Revs. Macromol. Chem., C7(1) 49-104 (1972).
The term mucopolysaccharide describes hexnsamine-con-
taining polysaccharid'es of animal origin. Another name often
used for this class of compounds is glycosaminoglycans. Chemically,
mucopolysaccharides are alter~ating copolymers made up of residues `'
of hexosamine glycosidically bound and alternating in a more-or-
less regular manner with ei-ther-hex-uronic acid or hexose moieties.
See Dodgson, K. S., and Lloyd, A. G., in Carbohydrate Metabolism
and its Disorders, e~d. by F. Dickens, et al., vol. 1, Academlc
Press (1968). ''
Some of the better known mucopolysaccharides derived
from animals can be represented by the following structural'
' ' . ' ' ' :
HY~LURONIC ~ ----C~ o
ACID ~¦~o ~ NHAc
COOH HO2SC ~ \ -
4-SULFATE ~ ~OH ~ ~ ~
. ~ NHAc
C,OOH H~ o _
CHONDROITIN ~ \ O ~
6-SULFATE ~ ~ NHAc
HO3SO~ ~ _ O -
DERM~TAN ~ ~ ._ o ~ NHAc
- OH H2COSO3}1
KERATAN HO ~ O o ~ ~ ~ ¦
SULFATE - I \r
2 OOH CH12OH ,COOh
_ ~ _o ~ 0' ~ ~- ~ ~
HO3So N SO3H OE NHSO3H OSO3H
COOH H ~ 3H
HEPARAN I / \1 ~ \~
SULTATE . r ~ ~ ~ ~ _ o _
.. . :
. . : , :, - ~ : .
- . . . , -
- . . . .
. . . , . , :. .
Other mucopolysaccharides are suitable for forming the composite
materials described herein, and those skilled in the art will
either know or be able to ascertain, using no more than routine
experimentation, other suitable mucopolysaccharides. For a more
detailed description of mucopolysaccharides, see the following
reference: Aspinall, G.O., Polysaccharides, Pergamon Press?
Typical sources of heparin include hog intestine, beef
lung, bovine liver capsule and mouse skin. Hyaluronic acid can
be derived from rooster comb and human umbilical cords, whereas
both of chondroitin 4-sulfate and chondroitin 6-sulfate can be
derived from bovine cartilage and shark cartilage. Dermatan
sulfate and heparan sulfate can be derived from hog mucosal tissue
while keratan sulfate can be derived from the bovine cornea.
Collagen can be reacted with a mucopolysaccharide in
aqueous solutions which can be either acidic, basic or neutral.
These reactions can be carried out at room temperature. Typically
small amounts of collagen, such as 0.3% by weight, are dispersed
in a dilute acetic ac;d solution and thoroughly agitated. The
polysaccharide is then slowly added, for example dropwise, into
the aqueous collagen dispersion, which causes the coprecipitation
of collagen and mucopolysaccharide. The coprecipitate is a tangle
mass of collagen fibrils coated with mucopolysaccharide which
somewhat resembles a tangled ball of yarn. This tangled mass of
fibers can be homogenized to form a homogeneous dispersion of five
fibers and then filtered and dried. Collagen-mucopolysaccharide
coprecipitation products have been studied by Podrazky, V.,
Steven, F. S., Jackson, D. S., Weiss, J. B. and Leibovich, S. J.,
Biochim. BiophYs. Acta., 229, 690 (1971).
.. ' ' ' ' , ' .
Although the collagen-mucopolysaccharide reaction pro-
duct coprecipitates from the aqueous medium from which it is formed,
it has been found that the mucopolysaccharide component can dis-
solve in other aqueous solutions. This is particularly true for
more concentrated aqueous salt solutions, such as body fluids.
It is known, for Pxample, that collagen-mucopolysaccharide copre-
cipitates are insoluble in O.OlM NaCl, some~hat soluble in O.lM
NaCl, and quite soluble in 0.4M NaCl - the physiological level is
~ about 0.14M NaCl. Thus, these reac'tion prod'ucts have only limited
0 insolubility and are not suitable, per se, as candidate materials
for implantable surgical prostheses, etc.
While the coprecipitation ~ethod described supra'is
preferred, collagen and mucopolysaccharides can be reacted in
other ways. The essential requirement is that the two materials
be intimately contacted under conditions which allow the muco-
polysaccharides to attach to the collagen chains. Another suitable
technique is to coat collagen with mucopolysaccharide, such as by
dip~ping articles formed from collagen, including sheets, films,
and tubes, into a solution of mucopolysaccharide. A suitable
variation of the latter technique involves prior coating with
collagen of an article,-sheetj film or tube fabricated from a
non-collagenous material, such as a synthetic, natural or modified -
natural polymer, fol'lowed by dipping of the collagen-coated arti-
clej sheet, film or tube i'nto the mucopolysaccharide solution.
Still another' suitable method is tG intimately mix collagen with
mucopolysaccharides, w1th each component in the form of a dry
To those skilled in the art of forming sheets, films,
tubes and other shapes or articles by techniques that are known i-' '"
0'- ' ~n'the!plastics~, elast~merics and~ fiber-forming industries, i-t
- 10 -
. ~ , . . . ..
would be obvious that the co~lagen-mucopolysaccharide product -~
prepared as described above/could also be formed into sheets,
films, tubes and other shapes or articles by such techniques.
To gain any significant increase in resistance to
collagen resorption, it is necessary to have at least about 0.'5
by weight of mucopolysaccharide bound to the collagen chains.
The upper limit may be set by the available sites on collagen for
mucopolysaccharide to attach. For composites wherein the muco-
polysaccharide is chondroitin 6-sulfate, levels of about 28% by
weight have been achieved; with hyaluronic acid, on the other
hand, the upper lim1t achieved is about 25%.
:; Reaction with the~mucopolysaccharides also provi~s
collagen with another valuable property, i,e., inabilitv t.~
provoke an immune reaction (foreign body reaction) from an animal
5~- host. To convert collagen into a material which, when implanted,
would not be recognized as a foreign body requires reacting it
with at l'east about l% by weight of mucopolysaccharide.
- The degree of 1nsol-ubil~ty of the collagen-mucopoly-
saccharidé products can be raised to the desired degree by cross-
D linking these materials. In general, any crosslinking method
- -suitable-for crosslinking col1agen is also suitable for cross-
linking these composite materials. Such crosslinking serves to
prevent dissolution of mucopolysaccharide in aqueous solutions
thereby making the materials~useful for surgical prostheses, etc."~
Crosslinking'also serves another important function by
contributing to raising the resistance to resorption of these ` '
~- ' -mate7riia-l-s. Th'e exact functionro-f- c-rosslinking is n-ot-understood
in this regar~, but it may be that crosslinking anchors the
mucopolysaccharide units to sites on the collagen cha;n which
O - -wo~l'd'normally be attacked by collagenase.
r 11 0
It has been found that the crosslinked composites should
have an M (number average molecular weight between crosslinks) of between
about 800 and about 60000. Materials with M values below about 800 or
above about 60000 suffer significant losses in their mechanical properties.
Composites with an M of between about 5000 and about 10,000 appear to
have the best balance of mechanical properties, and so are preferred
Crosslinking can be achieved by many specific techniques
with the general categories being chemical, radiation and dehydrothermal
methods. An advantage to most crosslinking techniques contemplated,
including glutaraldehyde crosslinking and dehydrothermal crosslinking, is
that they also serve in removing bacterial growths from the materials.
Thus, the composites are being sterilized at the same time that they are
One suitable chemical method for crosslinking the collagen-
mucopolysaccharide composites is known as aldehyde crosslinking. In this
process, the materials are contacted with aqueous solutions of aldehydes,
which serve to crosslink the materials. Suitable aldehydes include formal-
dehyde, glutaraldehyde and glyoxal. The preferred aldehyde is glutar-
aldehyde because it yields the desired level of crosslink density morerapidly than other aldehydes and is also capable of increasing the cross-
link density to a relatively high level. It has been noted that immersing
the composites in aldehyde solutions causes partlal removal of the poly-
saccharide component by dissolution thereby lessening the amount of poly-
saccharide in the final product. Unreacted aldehydes should be removed
from the collagen-mucopolysaccharide materials since residual aldehydes
are quite toxic.
Other chemical techniques which are suitable include
carbodilmide coupling, azide coupling, and diisocyanate cross-
A preferred crosslinking method is referred to
herein as a dehydrothermal process. In dehydrothermal crosslink-
ing, it is not necessary to add external crosslinking agents.
The key is to remove a large percentage of the water in the pro-
duct to be crosslinked. The amount of water which must be removed
- will vary with many factors, but, in general, sufficient water to
achieve the desired density of crosslinking must be removed
Thus, the collagen-mucopolysaccharide product can be subjected
to elevated temperatures and/or vacuum conditions until the mois-
ture content is reduced to extremely low levels. In the absence
of vacuum, temperatures above about 80C, and preferably above
90C, can be used. At 23C, vacuum of at least about 10 5mm.
of mercury, and preferably below 10 6mm. of mercury, are suitabie.
Elevated temperature and vacuum can be also used in combination;
this, in fact, is the most expeditious route and is therefore
preferred. With a vacuum of at least about 10 5mm. of mercury,
it is preferred to use a temperature of at least about 35C.
In general! the materials are subjected to t~he elevated tempera-
tures and vacuum conditions until the degree of insolukility
desired is obtained. The higher the temperature, the lower
is the vacuum required to arrive at a given crosslink density;
and vice versa. A typical crosslinking process to attain an
Mc between about 5000 and 10000 would involve subjecting the
collagen-mucopolysaccharide material to a temperature of 95C
and a vacuum of 0.01mm. of mercury for 24 hours. This dehydro-
thermal crossllnking process overcomes certain disadvantages of
the aldehyde crosslinking method and produces composites having
relatively large amounts of mucopolysaccharide strongly bound
to the collagen chain.
The exact mechanism operating in the dehydrothermal
crosslinking process is not known. However, it may be either
- an amide condensation involving ~-amino groups from collagen
and carboxyl groups from the mucopolysaccharide component, or
esterification involving carboxyl groups from collagen and
hydroxyl groups from the mucopolysaccharide or esterification
involving carboxyl groups from the mucopolysaccharide component
and hydroxyl groups from collagen. Possibly all three mechanisms
- are involved to some extent. For a more detailed description of
dehydrothermal crosslinking, see Yannas, I. V. and Tobolsky, A. V.,
"Crosslinking of Gelatin by Dehydration", Nature, vol. 215, #5100,
pp. 509-510, July 29, 1967, .
To be suitable for vascular prostheses., materials must
~15 have-certain-minimum mechanical properties~ These are mechani-
cal properties which would allow the suturing of candi-
date materials to sections of natural vessel. During suturing, -
such grafts must not tear as a result of the tensile forces
applied to the suture in making the knot. Suturability is related
to the diameter of the suture, the tension applied to the suture,
and the rate at which the knot is pulled closed. Experimentation
performed indicates that the minimum mechanical requirements for
suturing a graft of at least 0.01 inches in thickness are: (1)
an ultimate tensile strength of at least 50 psi; and, (2) an
elongation at break of at least 10~.
The best materials for vascular prostheses should dupli-
cate as closely as possib'le the mechanical behavior of natural
vessels. The most stringent physiological loading conditions
occur in the elastic arteries, such as the aorta, where fatigue
can occur as a result of blood pressure fluctuations associated
with the.systole-diastole cycle. The static mechanical properties
- 14 -
L ., `"~
" ' . ' ' , . . ' ~., .'
- . , ~
. `~. 10~76:10
of the thoracic aorta can be used as a mechanical model. The
stress-strain curve of the thoracic aorta in the longitudinal
direction of persons 20-29 years of age has been determined by
Yamada. See Yamada, H., Strength of Biolo~ical Materials, ed.
F. G. Evans, Chapter 4, Williams & Wilkins (1970). .From this
plot, the mechan.ical properties were calculated and found to be:
(1) an ultimate tensile strength of 360 psi; (2) elongation at
break of 85%; (3) tangent modulus at 1% elongation of 50 psi;
. and.(4) fracture work, i.e., the work to rupture (a measure of
O toughness), of 21,000 psi-%. These four'mechanical pro~erties
serve as a quantitative standard for mechanical properties of
Values for these mechanical properties were determined
for collagen-mucopolysaccharide composites as described herein
using an I.nstron tester. As mig.ht be expected, the mechanical
properties are strongly dependent on the presence of incorporated
mucopolysaccharide, the degree of fibrilla'r'aggregation of the
collagen fibrils and the number of crosslinks per unit volume. - .
For collagen composites with fibril size controlled at a fixed
O level, ~he mechanical behavior becomes a function of the mucopoly-
saccharide content and the degree of crosslinking.
Optimum mechanical properties were obtained for pure
collagen materials with Mc equal to about 5000-10000. The degree
of cross~linking is, of course, the reciprocal of Mc, the average
molecular weight between crosslinks. 'Collagen-mucopolysaccharide
composites prepared by the dehydrothermal crosslinking process
had-superior elongat-ion-at break, strength, and toughness com-
pared to collagen with similar values of Mc. Dehydrothermally
crosslinked compo~ites eas.ily passed the minimum suturability
o r~4~reme~ts and possessed mechanical properties approach-
ing those of the thoracic aorta.
. . - 15 -
~ any of the collagen-mucopolysaccharide composite materials
described herein have been found to have outstanding compatibility with
blood. This is in contrast to most materials, particularly synthetic
polymers, which have been found to be almost universally non-compatible
with blood. As used herein, "blood-compatible" means that a material compares
- favorably with human blood vessels in three regards: (1) tendency not to
cause platelet aggregation; (2) tendency not to cause clotting of red blood
cells; and preferably, (3) tendency not to interfere with the competence
of healthy blood to clot.
Both in vitro and in vivo testing has established that many
of the collagen-mucopolysaccharide composite materials can be synthesized
to be blood-compatible. For example, composites containing either
chondroitin 6-sulfate or heparin have been prepared which have whole blood
clotting times (WBCT) exceeding sixty minutes, which values are comparable
- to normal endothelium. The WBCT test is a well-known in vitro procedure
for qualitatively evaluating the affect of materials on (1) blood coagulation,
(2) platelet aggregation, and (3) red blood cell aggregation; this test
is described in more detail in the Examples herein. Other in vitro tests,
including the thrombin time (TT), activated partial thrombin time (APTT)
prothrombin time (PT), and platelet aggregation tests, as well as in vivo
testing, corroborate the blood-compatible nature of many of the materials
It does appear from the testing done that a certain minimum
level of mucopolysaccharide has to be present to achieve blood-compatibility.
This minimum amount is believed to be about 6% by weight based upon the
total weight of the composite to produce materials having blood~compatibility
significantly better than native collagen. Additionally, the presence of
one or more sulfate groups on the mucopolysaccharide appears to be
.~ , .
- 16 -
important, as evidenced by the low WBCT values for composites
formed from hyaluronic acid, to provide a material with non-
` ' clotting characteristics. Nevertheless, hyaluronic acid compo-
sites have not been found to cause platelet aggregation. :
Based upon resistance to resorption, freedom from
foreign body reaction, mechanical pro~ertie~ and blood-compati-
bility, crosslinked composites containing at least about 0.5%
bound mucopolysaccharide are preferred. Those composites con-
taining betw~een about 6% and-about 15% of a sulfate-containing
mucopolysaccharide are particularly preferred because of their
outstanding blood-compatibility. The percentage of mucopoly-
- - -sac~cha~ride specified h~ereln is -that obtained when the measurement
is made immediately after crosslinking. Chondroitin 6-sulfate is
an especially good mucopolysaccharide in such composites because,
even if eluted into the~bl~odstream, it does not interfere with
normal coagulation processes, as eluted heparin does.
~ The crosslinked collagen-mucopolysaccharide composite
; ' materials described herein have- outstandin.g properties for many
. . utilities. Primarily, they are useful in medical and surgical
applications, specifically for surgical sutures, blood vessel
' ' grafts, and in gen'eral, the fabrication of surgical prostheses.
Addltionally, they are usefui in the fabrication of blood-compat-
ible components for the fabrication of artificial organs which
pump blood such as artificial'kidneys and in the fabrication of
blood compatible equipment such as blood oxygenators as wel1 as
in the fabrication of miscell.aneous equl:pment for the handl'ing
and storage of bl~od such as pumps, tubes and storage bags.
Materials other than collagen could probably be con-
tacted with chondroitin 6-sulfate and other mucopolysaccharides
to yield blood-compatible materiais. Such materials could
- 17 - .
include synthetic polymers such as the segmented polyurethanes, hydroxyethyl
methacrylate and other "hydrogels", silicones, polyethylene terephthalate
and polytetrafluoroethylene or modified natural polymers such as cellulose
acetate or natural polymers such as elastin ~the fibrous, insoluble, non-
collagenous protein found in connective tissues such as the thoracic aorta
- and ligamentum nuchae) or pyrolytic carbon and other carbons which may
have been treated thermally or by an electric arc. Such composites could
be formed either by intimate mixing of the powdered solids or mixing of
compatible solutions or dispersions of the two components or by coating
with a mucopolysaccharide one of the materials mentioned in this paragraph.
Irrespective of the method used to contact the mucopolysaccharide with the
other material, the two components could be covalently bonded to form a
material from which the mucopolysaccharide cannot be dissolved or extracted
by contact with mucopolysaccharide solvents such as aqueous electrolytic
solutions. Covalent bonding could be effected by a radiation grafting co-
polymerization technique using, for example, Y-radiation from a cobalt-60
source. In all such procedures, chondroitin 6-sulfate or other mucopoly-
. . .
saccharides which do not interere with normal blood clotting if accidentally
- eluted out of the composite material during use are clearly preferred over
heparin which strongly interferes with normal blood clotting.
It is also quite probable that blood-compatible materials
could be prepared by bonding, using an adhesive, the crosslinked collagen-
mucopolysaccharide composite in the form of a sheet, film, granulated
solid or powder or other form onto a variety of substrates. Such substrates
would include synthetic polymers such as the segmented polyurethanes,
hydroxyethyl methacrylate and other "hydrogels", silicones, polyethylene
.: . .
- 18 -
. . .
polytetrafluoroethylene or modified natural polymers such as
cellulose acetate or natural polymers such as elastin or pyroly-
tic carbon and other carbons which may have been treated ther-
mally or by an electric arc or metals such as vitalium, titanium
and various steels. A suitable adhesive would, for example, be
- a silicone rubber adhesive~
The invention is further and more specifically
illustrated by the following Examples.
PREPARATION OF COLLAGEN DISPERSIONS
AND MUCOPOLYSACCHARIDE SOLUTIONS
The collagen used was prepared by precutting limed
calf hides into strips 3/8" wide and then into thin pieces. These
thin pieces of hide were contacted with three parts of water con-
taining 0.3% propionie acid and 0.1% benzoic acid. Equilibrium
- was established after four hours at which time the solution had
a pH approaching 5.3. The collagen slurry was separatea from the
water and qround to products of different particle sizes and struc-
tures with a centrifugally acting cutter-grinder. The calf hide
eollagen slurry (1:1 water-to-hide weight ratio) had a gelatin
eontent of about 2%. Additionally, it contained about 0.41~
ealcium and about;0.041% magnesium. Physieally, the slurry was
composed of highly entangled fibrillar aggregates.
J The ea~ hide eollagen slurry was purified by a
; repeated preeipitation from a turbid dispersion in 0.05M aeetie
aeid with 0.2 M sodium dihydrogen diphosphate, NaH2PO4. After -
purifieation, collagen was dispersed in 0.05M acetic aeid or in
a eitrie aeid-buffer solution at pH 3.2 (0.1 M citrie aeid,
0.2 M sodium dihydrogen diphosphate).
, .. -. . .
. - . -, : :
the dispersion was thoroughly homogenized in a Waring Blender
until the abosrbance at 440 millimicrons of a 0.3% (W/V) collagen
dispersion was about 0.5 as measured on a spectrophotometer
(Coleman Junion II A, Maywood, Illinois). The resulting colla-
gen dispersions were stored at 4C until further processing was
Mucopolysaccharide solutions were prepared from
sodium heparin, hyaluronic acid and chondroitin 6-sulfate. So-
dium heparin from hog intestinal mucosa, 143 U.S.P. units of ac-
tivity per milligram, was purchased from Abbott Laboratories,North Chicago, Illinois. Hyaluronic acid, from rooster comb was
prepared by the method of Swann, D.A., Biochim. Biophys. Acta,
156, 17 (1968) . The resulting hyaluronic acid contained 47.1
hexuronic acid and 42.6% hexosamine.
Chondroitin 4-sulfate from bovine nasal cartilage
was prepared by the method described by Roden, L., Baker, J.R.,
Cifonelli, J.A. and Mathews, M.s.~ in Methods of Enzymology,
V. Ginsburg, ed., vol, 28s, Academic Press, New York, p. 73.
Heparan sulfate and dermatan sulfate were both extracted from hog
mucosal tissues and purified by the methods described by Cifonelll,
J.A. and Roden, L., Biochemical Preparations, 12, 12 (1968).
; Chondroitin 6- sulfate, from shark cartilage -
Grade B, was purchased from Calbiochem, San Diego, California. It
contained 2.66% nitrogen, 37.2% glururonic acid and 5.61
Heparin, hyaluornic acid, chondroitin 4-sulfate,
heparain sulfate, dermatan sulfate and chondroitin 6- sulfate
were dissolved (1% W/V) in a citric acid-phosphate buffer pH
3.2,` The mucopolysaccharide solutions were stored at 4C.
~: . . ` '
PREPARATION OF COLLAGEN-HEPARIN AND
COLLAGEN-HYALURONIC ACID COPRECIPITATES
Collagen 0.3% (W/V) dispersed in O.05 M acetic acid was
5 ' thor'oughly agitated with a Teflon stirrer at 23C. While the di.s.-
persion was mixing, heparin or hyaluronic acid 1% (W/V) in 0.05 M
acetic acid was added drop wise from a buret at the rate of about
'O'.l'ml. per second. The addi~tio-n of mucopolysacchari-de caused
collagen to coprecipitate forming a tangled mass of collagen
fibrils coated with muçopolysaccharide which somewhat resembled
a tangled ball of yarn. When 90% by weight of collagen was co-
precipitated in this manner with 10% by weight mucopolysaccharide,
a systematic mass balance showed that about 95% of the original
' muco'po'lysaccharide was coprecipitated.
'15 After coprecipitation, the tangled mass of fibrils was
homogenized in a Waring Blender until the fibrils were about 1 mm.
. in length. The m'ixture of fibrils in 0.05 M acetic acid sepa-
. - rated into two phases when left unagitated for more than five
', m;nutes, so that m;x;ng was required before filtration. Filtra-
tion was performed by filtering the collagen-mucopolysaccharide
dispers;on under vacuum through a Buchner funnel conta;ning
Schleicher and Schuell (Keene, New Hampshire) filter paper No. 576.''
The copreci.pitate was allow'ed 'to dehydrate under atmospheric con-
ditions until the moisture content was about 20% by weight.
. ~ - 21 - .
PREPARATION OF COLLAGEN-CHONDROITIN 6-SULFATE COPRECIPITATES
Collagen 0.3% (W/V) dispersed in a citric acid-phosphate
buffer solution pH 3.2 at 23C was coprecipitated with a lg (W/V)
chondroitin 6-sulfate buffer solution pH 3.2 at 23C. The copre-
cipitate was homogenized, filtered and allowed to dry in the
atmosphere as described in Example 2.
ALDEHYDE CROSSLINKING OF A COLLAGEN-
~10 CHONDROITIN 6-SULFATE COMPOSITE
Coprecipitated collagen - chondroitin 6 - sulfate as
prepared~in Example 3 was crosslinked by immersing it in a 0.02 M
solution of glutaraldehyde. This treatment effectively immobilizeG
a fraction of .the polysaccharide component on the collagen fibrils
or molecules. Cr~s-si-inkin;g was--evidenced by the inability to
remove the polysaccharide from the aldehyde-treated film by pro-
longed washing with a phosphate buffer solution containing 0.4 M :
sodium chloride, pH 7.4,-which is a well known solvent of chon-
. droitin 6-sulfate. Unreacted.aldehydes were removed by treatment
: 20 with a solution of 5,$-dimethyl-1,3-cyclohexane dione (dimedone).
Evaporation of the water left behind a film containing up to
about 10% by weight polysaccharide. .
~ , ~
-- 22 -
DEHYDROTHERMAL CROSSLINKING OF COLLAGEN-CHONDROITIN 6-SULFATE
The product of Example-3 was placed in a vacuum oven
and exposed to a temperature of ll5C and a vacuum of at least
0.3mm. Hg. for 48 hours. At-the end of this treatment, less than
lO weight percent of the polysaccharide originally incorporated
into the film could be removed by 48-hour immersion in distilled
- - water,-a s-olvent for chondroitin 6-sulfate.
HEXOSAMINE ANALYSIS AND MOLECULAR WEIGHT BETWEEN CROSSLINKS
; ~ Since mucopolysaccha-rides are hexosamine-containing
polymers, the level of'hexosamine is directly related to the
amount of a specific mucopolysaccharide in a composite material.
Onc`e a relationship is establis-hed b~etween the hexosamine content
and weight for each individual mucopolysaccharide, the determina-
tion is straightforward. This analysis is described in detail
; by Huang, C., Sc. D. Th~esis, Mech. Eng. Dept., M.I.T., Cambridge,
Mass., Chaps. 3, 4 (lg74). The method is summarized as follows.
A known weight of a vacuum'dried (48 hours at l05C) composite is
placed in a 5 ml. ampule and l ml. of 8 M HCl is added. The
ampule is evacuated and flushed with nitrogen gas followed by
sealing under vacuum. Hydrolysis is initiated when the ampule
is placed in a circulating air oven at 95C. After 4 hours at
95C, the ampule is cooled with tap water to lOC. The contents
of the tube are then evaporated to dryness at 40C until only the
~ dr~ hydrolyzate remains. The hydrolyzate is dissolved in distilled
- - 23 -
- , ,
water to give a concentration of about 50-150 mg. of mucopoly-
saccharide per ml. of water. One ml. of the hydrolyzate solution
is added to 1 ml. of an 8% (V/V) solution of acetylacetone in
1 M Na2CO3. After heating at 95C for 1 hour? hexosamine contained
i in the hydrolyzate reacts with acetylacetone in alkaline solùtion
to form derivatives of pyrhole. Upon cooling the solution to
10C, 5 ml. of 95% ethanol and 1 ml. of Ehrllch reagent (prepared
by dissolving 1.339. of p-dimethylamino-benzaldehyde, DAB, in
50 ml. of 6 M HCl to which-50 ml. of 95% ethanol is added) are
) added followed by thorough mixing. The-reaction between DAB and
derivatives of pyrhole results in the formation of a chromophore
which colors the product an intense red. After the mixture is
allowed to stand for 2 hours, the absorbance is measured at 527
millimicrons against a reagent blank using a Coleman Junior II A
j spectrophotometer. The results of the analysis are compared to
standard calibration curves for each mucopolysaccharide.
The results of hexosamine analysis on several cross-
linked collagen-mucopolysaccharide materials prepared by the
methods of previous Examples are presented in Table 1. The
0 mucopolysaccharide content before crosslinking was determined
to be about 10~ for each composite listed in Table 1. It appears
that during glutaraldehyde crosslinking and subsequent washing
; steps, large quantities of mucopolysaccharide were lost. This
implies that the solubility of uncrosslinked mucopolysaccharides
S is high in the aqueous glutaraldehyde solution in which the
former are immersed for the purpose of crossl`inking them. For
the dehydrothermally crosslinked composites, only 10~, at most, of
a mucopolysaccharide was eluted, versus up to 61~ for the glutar-
O The mechanical properties of the composite mater~als
` is strongly influenced by the number of crosslinks per polymer
chain. The molecular weight between crosslinks (M ) is inversely
proportional to the number of crosslinks per unit volume. By measuring
the stress-strain behavior of thermally denatured collagen-mucopolysaccharide
composites, values of M can be determined. The technique is described
by Treloar, L.R.G., The Physics of Rubber Elasticity, Second Edition,
Clarendon Press (1958). A summary of experimental results for several
collagen-mucopolysaccharide composites is also presented in Table I.
Material Crosslinking % MPS Mc (+ 10%)
Collagen G (24, 7.4) 0.0 1,500
Collagen-H G (24, 3.2) 5.7+1.29,400
Collagen-H G (48, 7.4) 5.5+1.31,200
Collagen-H G (24, 3.2 4.0_1.01,800
Collagen-H D (48, 90C) 9.7+1.02,800
Collagen-CS-6 G (24, 3.2) 3.9+.36,800
Collagen-Cs-6 D (48, 90 C) 9.6+1.11,200
Collagen-HA G (24, 7.4) 2.3+.42,200
Collagen-HA D (48, 90C) 9.0+.52,500
G = Glutaraldehyde at 23C (hours, pH)
D = Dehydrothermal (hours, temp.)
H = Heparin
CS-6 = Chondroitin 6-sulfate
HA = Hyaluronic Acid
- 25 -
: -' , : : ' . '-
COMPOSITES FORMED BY COATING COLLAGEN WITH MUCOPOLYSACCHARIDES
.. . .
Mucopolysaccharide solutions were prepared by dissolving
40 mg. of the mucopolysaccharide in 20 ml. citric acid-phosphate buffer
(pH 3.2). A length of an insoluble collagen film was then added to the
mucopolysaccharlde solution and maintained at a constant temperature of
37 C and allowed to incubate for about 24 hours. Glutaraldehyde was then
added to the solution to give a resultant concentration of 0.025 M of
aldehyde. The collagen was kept in this solution for another 24 hours
and was subsequently transferred to a 0.025 M solution of glutaraldehyde
maintained at a pH of 7.4. The latter step was done in order to insure
efficient crosslinking of collagen. After 24 hours in the glutaraldehyde
solution, the collagen fibers were rinsed three times with distilled water
and transferred to a 0.2 weight percent solution of dimedone in order to
remove excess, unreacted aldehydes. After another 24 hours in the dimedone
solution, the fibers were rinsed five times with distilled water and kept
in a citric acid-phosphate buffer solution at pH 7.4 at 4C. The weight
percent of mucopolysaccharide attached to the collagen was determined by-
hexosamine analysis. The molecular weight between crosslinks, Mc, was
determined using the procedure described in Treloar, L.R.G., The Physics
of Rubber Elasticity, 2nd ed., Clarendon Press (1958). The results are
preseneed in Table II.
- 26 -
.; . ~ , .
`' . . ' - ' '~ ' ~
`l' , , .
Material . ~ MPS (+0.5) Mc (+500)
Collagen 0 3800
,- Collagen-CS-6 11.3 4100
Collagen-CS-4 8.7 4000
. ~ Collagen-HA 8.2 . 4200
Collagen-DS. 8.2 3900
Collagen-H 8.7 3800
. . Collagen-KS . 10.5 3800
CS-6 = Chondroitin 6-sulfate DS = Dermatan Sulfate
CS-4 = Chondroitin 4-sulfate - H = Heparin
HA = Hyaluronic Acid KS = Keratan Sulfate
. . .
. ' :
- . . - 27 -
- , :
ENZYMATIC DEGRADATION OF COMPOSITES FORMED
FROM COLLAGEN COATED WITH MUCOPOLYSACCHARIDE
A study of the enzymatic degradation of composites
formed by coating a mucopolysaccharide onto collagen fibers as
described in Example 7 was made. The mucopolysaccharide-coated
collagen films, in the form of tape, were extended to a strain
of 4.0+0.5% in the presence of a solution of collagenase
(40 units/ml.) and the force induced on the tape was recorded
as a function of time. The force was found to be representable
by a single negative exponential of the time and hence a plot of
the logarithmic force versus time yields a straight line. The
slope of the straight line yields l/r - a value which is taken
as a measu~re of the rate of enzymatic degradation of the collagen
by the collagenase. The results are presented in Table III.
- l/r X 104 (+0.07)
Material % MPS (+0.5) (min-l)
Collagen O 8.48
Collagen-CS-6 11.3 1.46
) Collagen-CS-4 . 8.7 0.88
Collagen-HA 8.2 S.38
Collagen-DS 8.2 0.90
Collagen-H , -~ 8.7 0.98,
Collagen-KS 10.5 1.10
~' . ' '
- 28 - `
. . ,
ENZYMATIC DEGRADATION OF COMPOSITES FORMED
FROM COPRECIPITATED COLLAGEN AND CHONDROITIN 6-SULFATE
Crosslinked composites of collagen and chondroitin
- 6-sulfate prepared according to the method of Example 4 were
tested for their susceptibility to collagenase degradation.
The technique used is described in~ the previous Example
except that the strain imposed was 20+2X. The results are
presented in Table IV.
1 /T X 1 o2 ( +0 . 009 )
%CS-6 (+0.2)Mc (+1000) (min-l)
.~ 0 15000 0.255
1.8 14000 0.149
3.0 12000 0.153
4~8 13000 0.093
6.5 11000 0.084
8.6 9000 0.049
11.2 1000~ 0.052
13.3 . 12000 0.047
. 14.9 11000 0.064
16.0 14000 0.067
MECHANICAL PROPERTIES OF CROSSLINKED COLLAGEN-
MUCOPOLYSACCHARIDE COMPOSITE MATERIALS
Mechanical testing was done on an Instron tester using
a B-type load cell. Dumbell -shaped specimens 0.25 in. wide and
about 0.01 in. thick were prepared for each candidate material.
The top end of the specimen was attached to the load cell of the
Instron while the lower end was attached to the crosshead through
a clamping device. The strain on the specimen was calculated
based on the crosshead movement. All measurements were conducted
at a constant elongation rate of 50%/minute in tension at 37C
in a citric acid-phosphate buffer solution at pH 7.4.
Values of the force per unit area at rupture or ultimate
tensile stress (U.T.S.), tangent to the stress-strain curve at 1%
elongation (1% tangent modulus), elongation at break (E.B.), and
~work required to fracture (fracture work) were calculated for each
material from the experimental stress-strain curve.
The results are presented in Table V below.
., , . . :
~ , .
,. ' ' ~.
~' ' ' , .
- - 30--
o . o o o o o o o o o o o o o o
a) g O o o o o O O O O N U-) O O
~ ~ ~ ~ O U) ~ ; ,_ ~ ~7 ,_ 1.
rd V~ C~
U'~ r~ Ln C~l ~ C~J C~l ~ (~) N N t~) ~ ~ ~
~ ~ +l +l +l +l +l +l +l +l +l +l +l +l +l -
1~1 O ~) U'~ O ~ ~ D .-- O O . C
~ ~ r-- I C~J ~ ~) ~ N ~ ~ N C~ O
o ~n ~ ~ o o o o o o o ~ o
O U~ O O O O O N ~ o I
(IJ V) O Lrl O O O O O O NO ~ n O r~
c~ ~, + I + I + I + I + I + I '+ I + I '+ I '+ I $ 1 + I + I
1-- ~ ~ U~L~'' g o l~ "J
~0-- ~ ~ N ~ ~)cn ~ ~ O
cl o o o o o o o o o o o o o
~ ~ ~ o o o o o ooooo o oo o o
~J O N t~)
r1 ~ O . . + ¦ + ¦ + I+ I + ~ D O
~ CL -
. ~ ~) _ _ e~ _ _
~ ~ ~ O~) ~
v O`\ O~ ^ I~ ~ ~ cn 1-- t~~) 1~ al a~ O
.~. ~ 0 d- ~ 0~ 0 d ~CO
O _ _ ~ J N ~t t~!d' d' C,l r~
. ~ ~
O V7 V7 U~
~1 I ~ C C CC ~ o
~ d n ~ a.a
In O ~
-- 3 1 -- . -
- . ' ,
EXAMPLE ll -
INCREASED TOUGHNESS DUE TO
INCORPORATION OF MUCOPOLYSACCHARIDES
Comparison of specimens of collagen and crosslinked
collagen-mucopolysaccharide composites at similar crosslinking levels
suggests that the presence of the mucopolysaccharide significantly increases
toughness of collagen. For example, the fracture work at similar levels
of crosslinking from materials taken from the preceding Table are presented
in Table VI below.
Material % MPS Mc (psi-%) ~10%
Collagen 0.0 1200 1900
Collagen-CS-6 9.6~1.1 1200 7100
As can be seen, the incorporation of about 10 weight percent
chondroitin 6-sulfate in collagen increases the fracture work from about
1900 to about 7100 psi-%. Since the fracture work is m simal at an Mc
level of about 6500, it appears likely that a collagen-chondroitin 6-sulfate
composite with about 10 weight percent of the mucopolysaccharide and an
Mc equal to about 6500 might possess a fracture energy greater than about
11,000 psi-%, the maximum fracture energy recorded for pure collagen under
the conditions of these tests.
- 32 _
... , :
: " ''''' '' ' :
EXA~PLE 12 -
IN VIT~O BLOOD~COMPATIBILITY OF COMPOSITES ~REP~RED
USING DIFFERENT CROSSLINKING ~ETHODS
The WBCT test provides an in vitro method for qualitatively
evaluating the effects of materials on (1) blood coagulation, (2) platelet
aggregation, and (3) red blood aggregation. This test is based upon the
fact that blood isolated in a venous segment, lined by normal endothelium,
shows signs of clotting within an hour, and within two to eight hours
completely coagulates into a solid gel. Even normal endothelium cannot
prolong the coagulation time of blood indefinitely when it is deprived
of the protective effects of flow and natural filtration mechanisms. Thus,
candidate non-thrombogenic materials can be considered to duplicate the
effect of normal endothelium if, when in contact with blood, they do not
cause clotting in less than 60 minutes. Blood so tested, however, must
have a finite clotting time since prolongation of the clotting time of
blood longer than 60 minutes raises the suspicion of artifactual delays
such as protein adsorptlon or denaturation and is not conclusive in
determining the surface effects of factor XII activation. If either
denaturation of one or more of the protein factors of the coagulation
process or some other form of anticoagulation (e.g., inhibition of a co-
agulation factor) is involved in whole blood clotting time (WBCT) prolonga-
tion, blood placed in contact with the test surface for 60 minutes normally
will not clot even when transferred to an active surface such as glass.
If, however, transferred blood does clot then WBCT prolongation must be
due primarily to the surface and not to protein adsorption, denaturation,
or permanent anticoagulation. In summary then, the WBCT test is used to
qualitatively evaluate the effect of candidate materials on (1) blood
- 33 ~
platelet aggregation, and (3) red blood cell aggregation. Blood
in contact with the candidate materials with WBCTs greater than
one hour is transferred to glass and analyzed for heparin or
heparin-like anticoagulants to clearly demonstrate that protein
adsorption, denaturation, or permanent anticoagulation is not
- involved in prolongation of the clotting time. For further
details of the WBCT test, See Lee, R. I., and White, P. D., Am.
J. Med. Sci., 145, 495 (1913).
Specifically, tubes of each test material, 4 cm. long
and 0.7 cm. in diameter, were clamped at the bottom with a hemo-
stat. One to two ml. of freshly drawn human blood was poured into
each tube. To obtain a control clotting time, blood was also
poured into glass tubes having similar dimensions as the test
tubes. Each tube was placed on a heating block at 37C and was
tilted every 30 seconds to observe the fluidity of the blood. The
clotting time end point was arbitrarily taken to be the time at
which the blood was totally transformed into gel.
The thrombin time (TT) test was used to detect low levels
of the anticoagulant heparin in blood. Heparin is known to inter-
fere with the catalysis, by thrombin, of the polymerization of
fibrinogen to fibrin. When thrombin is added to citrated plasma,
the conversion of fibrinogen to fibrin is inhibited in the presence
of heparin. Generally, this test is performed by exposing plasma
to the test surface in the presence of bovine thrombin under
standardized conditions until coagulation is detected with a
fibrometer. Plasma unexposed to a test surface is used as a
control. For a more detailed descr;ption, see Biggs, R. and
MacFarlane, R. G., Human Blood Coagulation and Its Disorders,
~ - 34 -
Specifically, thrombin time tests were carried out by placing
blood in tubes of each test material for 60 minutes at 37C and antico-
agulating the blood with 10% (V/~) of 3.8% (W/V) sodium citrate. The
plasma was then separated from the cellular components by centrifugation
at 23C, and then kept on ice until the plasma was tested.
- Control plasma (0.1 ml.), unexposed to a test surface, was
coagulated with 0.1 ml. of bovine thrombin (Parke - Davis, Detroit,
Michigan). The bovine thrombin activity was adjusted by dilution with
normal saline, until the coagulation time was 20 seconds as measured with
a fibrometer (Baltimore Biological Lab., Baltimore, Maryland). Plasma
previously exposed to each test surface, was coagulated in the same manner
as the adjusted control (20 second thrombin time). Adjusted bovine
activities ranging between 0.7 and 3.5 units per ml. were used in different
phases of these tests. When heparin was present in blood exposed to a
test surface, the thrombin time was found to be greater than 20 seconds.
The exact heparin concentration in the exposed plasma was found by protamine
; sulfate neutralization. See Hardisty, R. ~. and Ingrim, G.I.C., in
Bleeding Disorders, Blackwell Scientific Publications, Oxford (1965). One
mg. of protamine sulfate neutralizes the activity of about 85 units of
Protamine sulfate was added in varying amounts to the exposed
plasma until the thrombin time was again 20 seconds. The level of protamine
sulfate required to neutralize the activity of heparin quantitatively
identifies the level of solubilized heparin in the sample. Once the
number of units of heparin in each sample was known, the concentration
of heparin in units per ml. in each whole blood sample was calculated by
dividing the number of units by the sample volume and multiplying by 0.65,
the volume fraction
of plasma in whole blood. The thrombln time test only reveals defects
in the mechanism for converting fibrinogen to fibrin. Selective adsorption
of a plasma protein could in fact prevent whole blood from clotting when
exposed to a surface. By transferring blood exposed to a test surface
to an active surface such as glass, any coagulation defects caused by
protein adsorption or anticoagulation became apparent if the blood failed
The results are presented in Table VII below.
_ 35 _
- , ..
_ u~ m
~ ~ u~
~ ~ ~ c~l o u~ o ~
- ~ ~
~ . ..
o ~ + + + +
E-- o o c,~ ~ N tl~
s E ~ ,_ ~ ~ 3 ,
i_ c a) E
C O ~ C r~ ~ C C
C I I c ~ ,~ ~,
~- E n~ o
o ~ ~ o
. ~ a
~ ? + + + + ~ N I C L ~
3 _ L~) OO O O ~ O d- _ ~ O ~ C _
~ L C ~ tl tl +l +l ~, t o
E~ o _u~o L~) . u7 u7 Ir~ o 1~ o ~ o C~l
r~ ~ ~ ~ ~)
o~ ~ o ~ o ~ In
.. ~ o ,+1 +1 +1 +1 l '+I l l
~ ~ .
C~ ~ N ~ E
C o n
.,^ ^ ^ ^ ^. ^ ^ ^ ^ r~l 5 ~
n ~ ~ ~ ~ ~J N ~t C~J ~ O n
L ~ ~ ~ Cl C~ C:~ ~ C~ c .,_
~, E .~ ~
a) o ~-
~ ~ ~ ~ ~ o c C L
a~ I I ~ ~ ~ I ~ L S,--
:~~ ~ r -- ' ' ' '_ '_ ~~ 11 11 ~
O O O O O O OO ~
-- 37 --
IN ~ITRO BLOOD~COMPATIBILITY OF COMPOSITES PREPA~ED
FROM DIFFERENT MUCOPOLYSACCHARIDES
Collagen was extracted from rat tail tendon with 0.05M
acetic acid following the method of Piez, K. A. et al., J. Biochim.
- Biophys. Acta, 53, 596 (1961). A stock solution was stored under
refrigeration at 4C.
Chondroitin 6-sulfate from shark cartilage was purchased
from Calbiochem, San Diego, CA. Hyaluronic acid from rooster comb was
prepared according to method of Swann, D. A., Biochim. Biophys. Acta,
56, 17 (1968).
Films of crosslinked lonic collagen-mucopolysaccharide com-
plexes were prepared as follows. A solution or dispersion of collagen was
mixed with a solution of one each of the mucopolysaccharides at pH 3.2
with stirring. The resulting precipitate, a collagen-mucopolysaccharide
ionic complex, was collected as a film-like residue which was air dried
and immersed in a 0.02M solution of glutaraldehyde, pH 7.4, over 48 hours
at 23C. Unreacted aldehydes were removed by reacting with a solution of
Standardized in vitro hematological tests were carried out
using freshly drawn human venous blood. Whole blood clotting time (WBCT)
and thrombin time ~TT) were determined as described in the previous Example.
; The activated partial thromboplastin time (APTT) was
determined by incubating citrated plasma with kaolin and cephalin
(Thrombofax, Ortho, Raritan, New Jersey), a partial thromboplastin, in
; tubes made from the test material and observing coagulation time with a fibrometer after recalcifying; a control consisted
.. : - ~ . : ' . : . .: .
of repeating the procedure in the absence of the test surface.
This test is described in more detail in Proctor, R. R. and
Rapaport, S. I., Am. J. Clin. Path., 36, 212 (1961)
The prothrombin time (PT) was determined as the clotting
- time following recalcification of plasma containing a tissue
extract thromboplastin (Hyland, Costa Mesa, CA) and previously
placed in contact with the test material. This test is described
in more detail in Thomson, J. M., A Practical Guide to Blood
Coagulation and Haemostasis, Churchill, London (1970).
Platelet aggregation was studied by stirring the test
material (in powder form) in platelet-rich plasma inside an aggre-
gometer (Chrono-Log, Broomall, PA) and recording the optical
density of the system as a function of time. Platelet aggregation
was accompanied by an increase in transparency (decrease in optical
density) of the originally turbid medium.
The result of all but the platelet aggregation test are
presented in Table VIII below.
WBCT APTT TT PT
Material % MPS (m;n.) (sec.) (sec.) (sec.)
Control - 3.5+536.0+0.519.5+0.5 13.0+0.2
Collagen - 25+5 57+3 20.5+0.5 25+4
Collagen-CS-69.6+1.1 >60 35.8+0.421.0+0.5 13.0+0.2
Collagen-H 9.7+1.0 ~60 >180 >180 13.0+0.3
- ~ ~
- ' , . ' .' .. ~
In the platelet aggregation test, the optical density of
collagen had dropped from an initial value of about 9 to a value of below
4 after 4 minutes whereas that of all of the composites containing muco-
polysaccharides had only slightly dropped to a value of around 8.75 in
the same time.
IN VI~O BLOOD CO~P~TIBILITY TESTING OF CO~POSITES
Crosslinked collage-mucopolysaccharide materials were sutured
with little or no inconvenience. Little or no tearing was observed during
suturing and no leakage was observed when tubular prostheses fabricated
from collagen-chondroitin 6-sulfate were implanted as arterial grafts in
sheep and dogs. Post surgica] arterial flow observation using an ultra-
sonic signal detector showed that the collagen-chondroitin 6-sulfate tubular
prosthesis grafted to the carotid artery of a lamb was capable of sustaining
substantial steady arterial flow. This observation was repeated with the
same results two weeks later just before removing the graft. Upon removal,
the proximal lumen of the graft was found to be partially narrowed by the
presence of thrombus; about 50% of the lumen was clear at that site. The
distal end contained very little thrombus, while about 90% of the lumen
was free and available for flow at that site. Thrombus appeared to
initiate at the proximal suture line and extend to about 50~ of the pros-
thetic length. When the graft was cut open longitudinally along its axis,
the existing thrombus separated readily from the surface of the graft
and did not appear to be attached to it. Initial observations made with
the optlcal microscope indicated that
,., . , : - . .
neither large platelet clumps nor fibrinogen were attached to
the implant lumenal surface. Microscopie observations also
showed that a dense layer of granulation tissue was deposited on
the exterior surface of the implant.
IN VIVO TESTING OF RESORPTIQN RESISTANCE OF AND
ABSENCE OF FOREIGN BODY REACTION TOWARDS COMPOSITES
In this Example, crosslinked collagen-mucopoly-
saccharide membranes, prepared both by coating collagen with eachof the mucopolysaccharides as described in Example 7 as well as
by coprecipitating collagen with each of the mucopolysaccharides
as deseribed in Examples 2 and 3, were implanted subcutaneously
in guinea pigs as described below.
The collagen-mucopolysaccharide membranes had been ~`
sterilized by the process used to crosslink them. Immersion in
; an aldehyde bath (and, in particular, in a glutaraldehyde bath)
over several hours as described in Example 4, as well-known as
an effective means of chemical sterilization of a variety of
materials prior to implantation or other surgical procedures.
It is also known that exposure to temperatures in excess of 100C
over several hours is an alternative method of sterilization of
materials that will be implanted or otherwise used in surgery. In
addition, however, if the materials have been prepared eonsidera-
bly prior to grafting lt is preferable to disinfect them just
before grafting by immersion in 70/30 isopropanol/water for 24
hours at 23C. Immersion in the latter medium does not alter
either the erosslink density or other important structural fea-
tures of collagen-mucopolysaccharide composites.
' ~. ' ' ~ ' ' ,.':
Subcutaneous implantation was carried out under aseptic
conditions. White, Hartley, female guinea pigs, weighing approximately
400 grams, were used as subjects. For 7 days prior to implantation, a
weight change history was recorded for each animal. Shortly before im-
plantation, the back of each animal was sheared with electric clippers
over an area of ca. 6 cm x 5 cm and loose hair clippings were carefully
removed with vacuum suction. The animal was then anesthesized by exposure
to a mixture of oxygen and halothane, and its back was washed with 70/30
~ one-inch incision was made on one side of the back of the
animal. The incision was made such that a pocket between the dermis and
the panniculus carnosus was created. The specimen was inserted into this
pocket such that the whole specimen lay flat within the pocket. The
incision was then sutured with nylon sutures. ~ total of about 5 to 6
stitches were made to close the incision. The procedure was repeated with
the other side of the guinea pig back, using an identical specimen. The
right side was subsequently used for histological studies while specimens
from the left side were, after explantation, used for physicochemical
On the 4th, 10th, and 20th postimplantation days, the animals
were sacrificed by placing them in a desiccator containing ether. From
~ both the left and right implantation sites, l-l/2~' X 1-1/2" squares of the
`~ tissues were cut below the subcutaneous layer such that the implanted
specimens remained in the tissue. The tissue from the right~side was placed
in a 10% formalin solution and was subsequently used for histological
studies as described below. The tissue from the left side was immersed
in sterile Dulbecco's solution (50 ml) containing a few drops of
~ - 42 - `
, , . . . . , . ,:
, - : . . . .
, ., :, : . ' '.: ,` - ' .~ :
chloroform (which acts as a bacte~icide) and stored under ref~igeration --
for not more than 24 hours before the sample within it was removed.
Removal of the sample within the tissue was done by placing
the tissue on the stage of a low powered microscope (equipped with a
camera) and stripping the subcutaneous tissue from the dermis in such a
way that the state of the sample within the tissue could be examined
clearly with the microscope. This could be achieved by first cutting
between the dermis and the subcutaneous tissue and gently separating the
two parts by means of forceps. When viewed on the microscope, the state
of the tissue and the sample embedded within it could be examined to
reveal such features as the attachment of tissues to the implanted material.
Removal of the sample from the tissue was done on the microscope stage by
means of a forceps. After the materials had been removed from the tissue
they were stored in Dulbecco's solution at 4C until required to determine
the following physicochemical properties:
1. The fractional weight change of the sample ~W/Wi. This
was obtained by determining the dry weight of the samples (after dehydration
at 105C at a pressure of lO 3 mm Hg. for 48 hours). The fractional weight
change was then calculated as
A WlWi = We Wi
where We = dry weight of the explanted sample, and Wi = dry weight of
the sample prior to implantation (the latter was determined ~y use of a
2. Tensile modulus, E~ (in dynes/cm ). This was obtained
` by the method described in Example 10 except that the
. . .
- 43 -
. ~ .
modulus was determined as the slope of the straight portion of
. ~r the stress-strain curve.
3. Molecular weight between crossl1nks, Mc. This was
measured as described in Example 4.
The characteristics of collagen-mucopolysaccharide speci- :
mens just before implantation as well as on the 4th, 10th and 20th
' , days followi,ng impla,ntation are presented in Table IX for materials
that were prepared by coati-ng-collagen with various mucopolysac-
charides prior to crossllnking and in Table X for materials pre-
pared by coprecipitating collagen with mucopolysaccharides prior
. . ' , ':
. ~ ' .' .
- .,: .: . ~ .
., . - . . -
',, '~ :;
- 44 - , '
. ~, - -- .' . : : : :
- . . . . . . .. . . . . .
Y ~ ;
d o . ~ .
: ~ 4 ~ n ' ~ .
+~ l ~+l
~u ~ r,u
- 4 5
, . ,,..... . . , . ~ .
?~ ~ i ; ~ ; ~
~ ~ . o o o +, o o U70
Oo~ ; ~ o~.
1 ~' ~
a C v t~ O . ~ W ~ r
W ~ . 1~, ~
m ~ u 3 x a ,u ~ ~ u ¦ ¦ w ,,u
- 46 - -
. o o~ o
~ ~ ,o+
~n ~ o
o ~' ~ ~ ~
. . ' ':
~' ~ X ~ o~ O O
., ~ . Q)
. ~ ~ .
~ 'O o' O
' .P. ~S~ .
0 ~ ,i
~ O a . K ~
:~ . u~ , . ..
-- 47 --
.~' ' - .
W 0~ o o ~ ') t
3: C ~ R ~ ~ o ~
~i ~ D o o ~ ~ . o
H H . ..
X U D ~ D ~ ¦ o ,,
E~ U ~ . O ~1 ~ . O ~i ~i O :
~ ~ l r~ '
O H O o o ~) O +~ J ~ ~ +l
UR I o ,~ o _ ~ , o
~ ,1 lo ~.~1 3 3rl~loo ~ 3 3
O ¦ U ~ <, C ~U ~ ~ X U
- 48 -
~ . . o ~ ~ .
+l o +o~
O O O
~ ~ ~' ~r +
rl U) .
U) ~ .
~ ~ o
,~ ~ ~ ~ +, o o
o o o o ~ ~ ~
, ~ ~ o. . +
~ . ~ ~
, U ~ ~ ~ +ol o o
~ o o ~o + ~r
.~ . X +, +, o
~ ~ .
~ l ~
~ ~ ~ ~ o
E~ O. O .,~ oO , O
.~ +, +, o
~ ~o ~ ~ o
P~ i ,, ~ .
. ~ ~ CO~ ~o
. 1 ~ a) I ~ lo ~ ~ 10
, U ~ ~ ~ U~ X
~ X ~~ X ~ ~t) ,
It is clear from Tables IX and X that, in almost all
- - cases where collagen wa-s crosslinked with a mucopolysaccharide,
either after being coated or coprecipitated with a mucopoly-
saccharide, the fractional weight loss was arrested indicating
that the degradation of collage-n~had been effectively abolished
by reaction with the mucopolysaccharide. The only exceptions
were collagen coated with hyaluronic acid (a nonsulfated muco- ;
polysacchari?de) and collagen copr-ecipitated with only 1.8 weight
percent chondroitin 6-sulfate; in the latter case, the fractional
weight loss of collagen was delayed rather than arrested completely
In a-ll other cases, an occ-asional very small initial weight loss,
possibly due to deswelling of the implanted specimen, was reversed -~
usually by the l0th day until, by the 20th day, the implant was
i - he7a-v-ier than when implante~d.'~The increase in weight of the im-
plant was found to be due to adherence of somè of the surrounding
tissue onto the implant as the latter was removed from the animal.
The tissue adhering on the implant was analyzed and found to be
constituted a1most entirely of collagen, an observation showing
that new collagen had been synthesized on the implant by cells
in the surrounding tissues. Th-us, not only did reaction with the
sulfated mucopolysaccharides prevent degradation of collagen but
also yielded a composite material capable of eliciting synthesis
of new connective tissue on its surface by cells in the surround-
The protection from resorption afforded to collagen by
reaction with sulfated mucopolysaccharides is also evident in the
prevention of the substantial decrease in modulus E and decrease
in crosslink-density (increase in Mc) which is observed with
~ollagen itself or with a collagen-hyaluronic acid composite.
The maintenance of E and Mc to relatively steady levels (within
.. . .
- 50 -
the experimental uncertainty) up to 20 days of implantation for
composites of collagen and one each of the sulfated mucopoly-
saccharides is indicative of a crosslinked macromolecular network
which remains intact for at least 20 days in the tissue of the
Histological studies were performed on the tissue/implant
block removed from the right side of the animal on the 4th, 10th
and 20th days. The standardized p~ocedure used in preparing the
specimens for histological examination was the following:
1. The tissue was fixed in 10% formalin (Fischer
Scientific Co., NJ) for at least 24 hours at room temperature.
2. It was then dehydrated by sequential immersion in
water-ethyl alcohol mixtures containing 50%, 70%, 85%, 95% and
100% alcohol, th'e time of im'me'`rsion being 1 hour per mixture.
3. The tissue was then immersed in dioxane for 2 hours
before it was embedded in a tissue-embedding medium (Paraplast,
Mpt. 56-57C; Curtin Scientifi'c'''~"o~.', Houston, Texas). Embedding
was achieved by first placing the tissue in the molten paraffin
kept at 58C for 4 hours, with hourly exchanges for the paraffin.
Finally, the tissue was placed i'n a mould and embedded with a
fresh supply of paraffin.
4. The paraffin block containing the tissue was then
cooled to 0C in a bath containing chipped ice for 20 minutes and
was then mounted on a microtome (Minot Custom Microtome; Interna-
; 25 tional Equipment Co., Needham Heights, MA). Slices of the paraf-
fin containing the tissue were microtomed to thicknesses of about
5. The microtomed specimen was then mounted on a clear
microscope slide and deparaffinization was achieved by immersing
the mounted specimen in two exchanges of xylene for 3 minutes each.
6. The specimen was then rehydrated by sequential
immersion ;n water-ethyl alcohol mixtures containing 100~, 95%,
85%, 70%, 50X and 0% alcohol, the time of immersion being 1 hour
per mixture. The specimen was finally rinsed thoroughly with
7. The specimen was then stained with hematoxylin for
5 minutes and rinsed briefly with distilled water. Excess stain
was removed by rinsing the specimen with 0.5% acid alcohol (70% :
. ethyl alcohol in concentrated-hydrochloric acid). The acid alcohol
0 was finally removed by rinsing the specimen and immersing it in
water for 1/2 hour.
8. The specimen was then stained with 0.5% aqueous
eosin for 3 minutes and then rinsed wit.h 5 exchanges of water.
9. The specimen was dehydrated as in (2) above and ~
then rinsed a few times with xylene.
10. It was then mounted on a clean. cover slip with a
; .permanent mounting medium (Harleco Synthetic Resin; Hartman-
Leddon Co.,~Philadelphia, PA).
~i 11. The cover slip containing the stained specimen was
0~ examined with a microscope.
~ The histological studies revealed that the extent and .
severity of chronic inflammation in the tissue surrounding the
collagen implant decreased steadily as the content of chondroitin
6-sulfate in a series.of implants based on coprecipitated
5~ collagen-chondroitin 6-sulfate composites increased in the range
1.8 to 11.2 weight percent. These results showed that while the
collagen used in the composite materials provoked, when used by ~ .
itself, a moderate immune response, reaction of the collagen with
chondro~t~n.6-sulfate led to practlcally complete suppression of -
10;1; this immune response. These findings were also made when the
- 52 -
~ ~ .............. .. .. .
- - . .. . .. :: ' .. . ' . ~ : , ' : .'
implant was based on composite materlals prepared by coating collagen with
one of the sulfated mucopolysaccharides. In summary, the histological
observations showed that the ability of implanted collagen to provo~e a
foreign body reaction from the animal host could be controlled and suppressed
by reaction with one each of the sulfated mucopolysaccharides.
Those skilled in the art will know, or be able to ascertain
by no more than routine experimentation, many equivalents to the specific
embodiments expressly described herein. These are within the scope of
this invention and are intended to be covered by the appended claims.
- 53 -
- : . .. .. - : . .
:: , :' ' '
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|Forecasted Issue Date||1980-10-14|
|Fee Type||Anniversary Year||Due Date||Amount Paid||Paid Date|
|Current Owners on Record|
|MASSACHUSETTS INSTITUTE OF TECHNOLOGY|
|Past Owners on Record|