Note: Descriptions are shown in the official language in which they were submitted.
CA 022047~3 1997-0~-07
WO 96/14095 PCI~/US95/14308
SY~ C COLLAGEN ORTHOPAEDIC STRUCTURES
SUCH AS GRAFTS, TENDONS AND OTHER STRUCTURES
This is a continuation in part of pending application
Ser. No. 07/990,967, filed Dec. 15, 1992 which is a divisional
application of application Ser. No. 07/297,115, filed Jan. 13,
1989, now U.S. Patent No. 5,171,273. The patent application and
its parent (now the issued patent and referred to collectively
herein as the "parent applications~) are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
This invention relates to high strength reconstituted
collagen fibers which are particularly well suited as grafts for
orthopaedic, dermal, cardiovascular, and dental implants,
prosthesis and other applications particularly in living
subjects, like animals, especially human subjects.
From the description of the prior art it is evident
that a serious and urgent need exists for high strength fibrous
materials suitable for use as a graft that is long lasting and
has biocompatibility with a host and is biodegradable.
The fibers and grafts of the invention overcome many
of the prior art difficulties and problems and have a combinat
of advantageous properties generally absent in the prior al .
The collagenous fibers and grafts of the invention can be
manufactured without sacrifice of the host's tissue. The graft
SUBSTITUTE SHEET (RULE 26)
CA 02204753 1997-0~-07
W O 96/14095 ~rnus95/14308
of the invention quickly incorporates repair tissue, a necessary
characteristic in the design of biomaterials that enhance the
deposition of repair tissue in skin, tendon and the
cardiovascular system. Although high-strength oriented and
unoriented collagenous materials are reported in the literature
no report is known of collagen fibers of small diameter that can
be processed into woven and non-woven textile prostheses which
have the necessary properties that simulate or exceed those of
the natural body part.
The coilagen fibers of the invention are especially
well suited for the repair of soft tissue injuries.
In addition, attempts at arranging fibers in parallel
which retain the strength of the individual fibers when bundled
together for use as prosthetic tendons and ligaments or other
suitable devices for the repair of tissue have not been
successful. Up to 70 to 80~ of the predicted strength of the
bundle is lost when the fibers are bundled in parallel.
This phenomenon has been documented in fields distinct
from that of collagen implants. In rope making, for example,
fibers are bundled and braided in order to retain as much of the
individual strength of the individual fiber as possible. This
approach is, of course, unfeasible, however, in animal bodies,
where tendons and ligaments should be oriented in parallel if
they are to simulate the natural body.
SUBSTITUTE SHEET (RULE 26
CA 022047~3 1997-0~-07
WO96/14095 PCT~S95/14308
EMBODIMENTS OF THE INVENTION
In this description of the invention, the following
~ terms have the following meaning:
~Autograft~ means transferring a tissue or organ by
grafting into a new position in the body of the same individual.
~Implant~' means a graft which is woven into the and
secured in the surrounding tissue.
~Graft~ means anything inserted into something else,
or contacted upon something else so as to become an integral or
associated part of the latter and it includes materials and
substances which are either added to an already intact structure
or serve as a replacement substitute or repair to a damaged or
incomplete structure. Thus a ''graftll is intended to be given the
broadest possible meAnlng and encompasses a prothesis, implant
or any body part substitute for any mAmm~l (animal or human).
As used in this application, the terms llthe same",
uniform", ~parallel~ simultaneously~ and ~coated~ include
~substantiallythesame~ substantially uniform", "substantially
parallelll, 'Isubstantially simultaneously~ and llsubstantially
coated".
~Wet~ when referring to fibers or em-bodiments of the
invention means more than 15~ water content. ~'Dryll means 15~ or
less water content.
SUBSTITUTE SHEET (RULE 26)
CA 022047~3 1997-0~-07
WO96/14095 PCT~S95/14308
The invention provides a high strength collagen fiber
for grafts, as well as a method for the manufacture of the high
strength collagen fibers.
In a particular important embodiment, the invention
further provides a bundle of monofilament collagen fibers
arranged in parallel in which the fibers are tensioned by
stretching so that the bundled fibers retain a high percentage
of their individual strength and so that the fibers fail
simultaneously.
The invention further provides a method for the
production of monofilament collagen fibers arranged or organized
in parallel in which the fibers are tensioned by stretching so
that the bundled fibers retain a high percentage of their
individual strength and so that the fibers fail simultaneously.
The invention further provides a prosthetic device,
such as a tendon or a ligament device, made from a multiplicity
of the tensioned fibers arranged in parallel or from a
multiplicity of the bundles arranged in parallel.
The invention also provides collagen devices like
fibers and grafts for numerous applications, particularly where
high tensile strength and biocompatibility are essential. The
invention also provides collagen proteoglycan fibrous grafts
which have even greater tensile strength than the non-
proteoglycan grafts of the invention. The invention further
provides a method for making improved collagen proteoglycan
fibers for use in such grafts.
SUBSTITUTE SHEET (RULE 26)
CA 022047~3 1997-0~-07
WO96114095 PCT~S9S/14308
The invention provides further implants in which the
collagen grafts are woven and secured into the surrounding
tissue. The surrounding tissue then invades the graft material.
The graft is revascularized and eventually replaced by the host's
tissues.
The invention further provides for grafts with physical
properties that can be manipulated or processed into a variety
of shapes, thicknesses, stiffnesses in woven or non-woven forms.
Other embodiments provided by the invention will become
apparent from the description which follows.
SUMMARY OF THE INVENTION
Several major new embodiments of the invention are
described herein.
One such embodiment is an improved biodegradable and
biocompatible reconstituted monofilament collagen fiber which has
increased strength and elasticity of the fiber when compared to
fibers made by other than the method of the invention. The
fibers may be made from soluble or insoluble collagen and may be
crosslinked or not crosslinked. The fibers may be stretched from
2.5~ to 100~ of their length to increase the strength of the
fibers. Generally, the fibers are stretched until the strength
of the fibers reaches a maximum level. The stretching may be
performed in a single stretching application or may be performed
SUBSTITUTE SHEET (RULE 26)
CA 022047~3 1997-0~-07
WO 96/14095 PCT/US95114308
by repeated cycles of stretching, drying, wetting, and stretching
as deemed appropriate under the circumstances. The fibers may
be left uncoated or may be coated with a suitable material,
preferably biocompatible and biodegradable, to protect the fiber
from the environment and from trauma during handling. The
coating is applied as a low weight fraction of the fibers,
generally less than 10~ of the weight of the fibers. The fibers
may or may not be embedded in a matrix compound of a suitable
material, preferably biocompatible and biodegradable, to
lo strengthen the fibers by binding the fibers together in a
fiber/matrix composite. The matrix is applied in a high weight
fraction of the fibers, generally more than 10~ of the weight of
the fibers. Proteoglycans may or may not be incorporated into
the interfibrillar spaces to increase the tensile strength of the
fibers.
A method for the production of the improved
biodegradable and biocompatible collagen fibers is another
embodiment of the invention.
Another embodiment is a bundle of physically associated
collagen fibers arranged in parallel, or substantially parallel,
in which the fibers are tensioned by stretching so that the
fibers each have the same, or substantially same, strength and
fail in, or substantially in, unison at the same load. This
results in a stronger bundle of fibers than could otherwise be
achieved if the fibers had different strengths and consequently
broke individually at different times. The bundles of the
SUBSTITUTE SHEET (RULE 26)
CA 022047~3 1997-0~-07
WOg6114095 PCT~S95/14308
invention retain a high percentage, generally more than about
50~, of the combined strength of the individual fibers. In one
preferred embodiment, in a bundle comprising lO fibers, the load
to failure of the bundle was about 60~ of the calculated
cumulative load to failure of the individual fibers. The fibers
of the bundle may or may not be coated or embedded in a matrix
as described for the monofilament fibers. Proteoglycans may or
may not be incorporated in the bundle as described for the
monofilament fibers.
A process for making bundles of fibers in which the
fibers are tensioned and fail in unison or substantially in
unison at the same or substantially the same load comprises the
following. Fibers made in accordance with the invention, as
disclosed in the parent applications, are attached at each end
to a tensioning device which stretches the fibers from about 2.5
to about lO0.0~, preferably from about 5 to about 7.5~ of their
original length. The stretching results in an increase in
strength of the fibers and, very importantly, results in uniform
strength of the fibers.
Fibers other than those made by the process of the
invention can likewise to treated in the same manner. Another
embodiment of the invention is a prosthetic device for a graft,
such as a tendon or ligament device, which comprises the
tensioned fibers of the invention, aligned in parallel, either
bundled or unbundled. The tendon or ligament prosthetic device
has increased strength, compared with tendon or ligament devices
SUBSTITUTE SHEET (RULE 26)
CA 022047~3 1997-0~-07
WO96/14095 PCT~S95/14308
comprising fibers made by means other than that of the invention,
as the individual fibers of the device are stronger and fail in
unison.
Other embodiments will become apparent as described
further herein.
The invention, as disclosed in the parent applications,
has several embodiments. In one of its embodiments the invention
provides a high strength synthetic collagen graft constituted of
high strength reconstituted crosslinked collagen fibers embedded
0 into a loose uncrosslinked collagen matrix.
The grafts of the invention are biologically compatible
with a host. They simulate the morphological and biomechanical
characteristics of the host~s natural tissue.
In one embodiment of the invention high molecular
weight chondroitin sulfate proteoglycan is added during the
latter stages of collagen fiber synthesis to be incorporated into
interfibrillar spaces and as a result enhances the ultimate
tensile strength of the collagen fibers formed.
A further embodiment of the invention is a process for
the manufacture of the reconstituted collagen fibers.
The fibers of the invention are prepared from soluble
or insoluble collagen. In a preferred embodiment, the collagen
is in solution or dispersion in an acid solution. The solution
or dispersion is extruded through polyethylene tubing into a
fiber formation buffer (~FFB~). The fibers are then successively
bathed in alcohol, to dehydrate the fibers, and then again in
SUBSTITUTE SHEET (RULE 26)
CA 022047~3 1997-0~-07
WO96/14095 PCT~S9S/14308
water, following which the fibers are dried and wound on a
tensioning spool. Prior to drying, as the wet fibers are
collected and tensioned on the spool, the fibers may be
stretched, up to about l00~ of their original length.
It was a surprising finding that for production of
optimum strength in the manufactured fibers a water bath
following the dehydration bath was so highly desirable. Methods
of forming collagen fibers without this water bath yield fibers
of inferior strength.
Other embodiments of the invention will become apparent
to one of average skill in the art to which the invention
pertains.
SUBS~ITUTE SHEET~RULE 26)
CA 022047~3 1997-0~-07
WO 96/14095 PCT/US95/14308
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a diagrammatic example of an automated
method for production of continuous collagen fibers.
Figure 2 is a diagram outlining the various embodiments
of the fibers of the invention.
Figure 3 shows ultimate tensile strength of collagen
fibers which were stretched by varying percentages of the
original lengths of the fibers. Fibers stretched 5.0~ had an
increase in tensile strength of about 50~. Fibers stretched 7.5
had an increase in tensile strength of about 57~.
Figure 4 shows load at failure of collagen fibers which
were stretched by varying percentages of the original lengths of
the fibers. The most dramatic increase in strength occurred at
5.0~ stretch, which resulted in an increase in strength of about
160~. Fibers stretched 7.5~ of their length almost doubled their
strength. Fibers stretched by more than 7.5~ or less than 5.0
had smaller strength gain, although these fibers were still
stronger than control fibers.
Figure 5 shows strain at failure of collagen fibers
which were stretched by varying percentages of the original
lengths of the fibers. All stretched fibers had a higher amount
of strain at maximum (point of breakage). Fibers stretched 5.0
had the highest strain at maximum.
SUBSTITUTE SHEET (RULE 26)
CA 022047~3 1997-0~-07
WO 96/14095 PCT/US95/14308
11 '
Figure 6 shows modulus (stress/strain) at low levels
of applied stress of collagen fibers which were stretched by
~arying percentages of the original lengths of the fibers.
Figure 7 shows modulus (stress/strain) at high levels
of applied stress of collagen fibers which were stretched by
~arying percentages of the original lengths of the fibers.
DETAILED DESCRIPTION OF THE INVENTION
The method for production of the high strength collagen
fibers of the invention is as follows:
Collagen fibers are placed in or passed through a fiber
formation buffer (~FFB~). The collagen precursor fibers which
are placed in the FFB may be soluble fibers in solution or
insoluble collagen in a dispersion in a dilute acid solution.
Before being placed in the FFB, the precursor collagen in the
acid solution may be extruded through tubing with the desired
diameter to yield fibers of the desired thickness.
The FFB may be an aqueous buffer which may contain
neutral salts or it may be composed solely of distilled water.
In a preferred embodiment, the FFB comprises NaCl, TES (N-
Tris(hydroxymethyl) methyl-2-aminoethane sulphonic acid), and
sodium phosphate dibasic. Chemically similar or equi~alent
compounds may also be used as well as other collagen fiber
formation buffers well known in the art.
The temperature of the fiber formation buffer should
be sufficiently high to allow fiber formation to occur but should
SUBST~TUTE SHEET (RULE 26~
CA 022047~3 1997-0~-07
WO 96/14095 PCT/us95/14308
not be so high as to disturb the fiber formation process.
Generally temperatures between about 4~ to about 41~C have been
found to be suitable. The pH of the FFB should be higher than
the isoelectric point. A pH about 7.5 has been found to be
acceptable.
The fibers remain in the FFB for a period of time
sufficient to allow the fibers to become strong enough to support
their own weight when lifted from the FFB. The necessary time
will vary depending on the temperature of the FFB, with lower
temperatures requiring a longer immersion in the buffer. At
higher temperatures, an immersion of 15 minutes may be sufficient
whereas at lower temperatures, an immersion of 8 hours may be
necessary at low FFB temperatures.
After the fiber formation step, the water is removed
from the collagen. water removal can be achieved by immersing
the fibers in a dehydrating solvent, such as an alcohol for a
period of time sufficient to remove of the water from the fibers,
or may be achieved by air drying. Depending on the dehydration
method used, the length of time of for this step will vary.
Using an alcohol, times from 30 minutes to several hours may be
employed. Any alcohol may be used which effects water removal
from the collagen. That is, the solubility parameter of water
in the alcohol used must be high. Examples of alcohols are
suitable for the dehydration step include lower alkanols (up to
6 carbon atoms), like methyl, ethyl, and isopropyl alcohols.
SUBSTITUTE SHEET (RULE 26~
CA 022047~3 1997-0~-07
WO96/14095 PCT~S95/14308
13
Thereafter, the fibers are placed in a water bath for
a time sufficient to remove the excess solvent and other
chemicals and to allow the fibers to align in or near parallel
during the subsequent drying process. The length of time
required varies inversely with the temperature of the water bath.
Generally, water temperatures between about 4~ and about 41~C
have been found to be suitable, with longer immersion times being
required at lower water temperatures. The water is preferably
ion free water which may be distilled water.
Following the water bath, the fibers are dried. The
drying may be by air drying at room temperature or an external
heat source, such as a heat lamp may be used. In a preferred
embodiment, the drying is performed with the fibers under
tension. Drying should preferably continue until the fibers
retain from 0~ to about 30~ moisture by weight. Preferably, the
fibers are dried to retain about 15~ moisture by weight.
Following drying, the fibers are collected. In a
preferred embodiment, the fibers are collected onto a spool under
tension.
The collagen fibers may be formed using a manual method
such as the method described above or by means of a continuous
automated process, such as described in Kato and Silver,
Formation of Continuous Collagen Fibres: Evaluation of
siocompatibility andMechanical Properties, siomaterials, ll:169-
175 (~990) which is incorporated herein by reference.
SUBSTITUTE SHEET (RULE 26)
CA 022047~3 1997-0~-07
WO96/14095 PCT~S95/14308
An automated method for production of continuous
collagen fibers may be performed as follows: A collagen solution
or dispersion is extruded through a continually flowing solution
of FFB with the aid of a syringe pump (Sage Instruments,
Cambridge MA) containing the collagen dispersion. To prevent
pulsatile flow during the process of aggregation and to,
therefore, prevent tensile stress on the freshly extruded
collagen fibers, a micro gear pump (Cole Palmer, Chicago, IL) is
used. A conveyor belt mechanism is used to carry the fibers
through the various processes. The fiber formation buffer is
recycled and reheated to the desired temperature by a pump
system. The extruded collagen flows down a tubing conveyed under
the flow of the fiber formation buffer and is collected on a
conveyor belt. The belt carries the collagen fiber via a pulley
mechanism designed to immerse the belt in solution through an
alcohol bath, followed by a water bath. The fiber is then picked
up by a spool a distance from the conveyor belt, drying under
tension with a heat lamp during the time of travel between the
conveyor belt and the spool. A diagrammatic representation of
an automatic process to produce continuous collagen fibers is
shown in Figure l.
Following the formation of the fibers, the fibers may
be treated in various ways to produce the various embodiments of
the invention. An outline of the treatment of the fibers is
illustrated in Figure 2.
SUBSTITUTE SHEET ~RULE 26
CA 022047~3 l997-0~-07
WO96tl4~5 PCT~S95114308
The collagen fibers, made by a manual or automatic
process, may then be crosslinked using known crosslinking
techniques. U.S. Patent No . 4, 703 ,108 to Silver, et al. which
discloses numerous crosslinking techniques is incorporated he~ein
by reference.
Further details as to suitable methods of crosslinking
are also given herein in Examples I and II.
The bundles of the invention are comprised of a
multiplicity of suitable monofilament collagen fibers bundled
together in parallel alignment. A method of making suitable
collagen fibers is detailed herein, but other suitable methods
can be used to make collagen fibers to be bundled in accordance
with the invention.
Monofilament collagen fibers are placed side to side
in parallel alignment to form a bundle. The bundle may be place~
in a fiber formation buffer solution or may be left dry. The wet
bundle is stretched. The bundle is then air dried at which time
the monofilaments will have become associated to form a single
bundle. The term "associatedl1 in the context of the bundles
means that the fibers are in contact with each other
substantially throughout the entire lengths of the fibers and
that the contact between the fibers is self-sustaining. If
desired, the fibers of the stretched bundle may be crosslinked.
A bundle of collagen fibers of the invention compris~
2s a multiplicity of fibers generally over s and may include several
hundred fibers. For most applications, bundles comprising 7 to
SUBSTITUTE SHEET (RULE 26~
CA 022047~3 1997-0~-07
WO96/14095 PCT~S95/~4308
fibers are suitable. For certain applications bundles
comprising from about 2 fibers to about 100 fibers seem more
suitable; for other applications, bundles comprising loo fibers
to about lO,ooo fibers may be better suited. The number of
fibers in what is termed a 'Ibundlell is suited to the particular
application or use selected.
Any suitable FFB may be used to wet the fibers for
bundling in accordance with the method of the invention. Any of
the FFBs described above to make the collagen fibers may be used
in the bundling process.
The tensioning of the fibers is carried out by any
suitable means, such as a stretching device which may be a screw
driven frame to which parallel oriented supports, which may be
tongue depressors, are clamped at each end using screws. The
ends of collagen fibers to be stretched are attached to the
supports by any suitable means so that the fibers do not slip
when stretched. The supports are separated, thus stretching the
fibers, by turning a screw. The amount of stretching can be
adjusted by number of turns of the screw. Thus, the stretching
device acts like a rack to stretch the fibers. The stretching
device is manufactured from means (mechanical~or otherwise) which
accomplish the desired stretching function.
The fibers are stretched or tensioned to a degree that,
when compared to unstretched fibers, the stretched fibers have
increased strength. The stretching, or tensioning, of collagen
fibers, whether in bundles or as monofilaments, results in an
SUBSTITUTE SHEET (RULE 26)
CA 022047~3 1997-0~-07
WO96/14095 PCT~S95/14308
increase in tensile strength of the fibers, especially when
fibers are stretched from about 2.5 to about lO.0~ of their
original lengths. It has been found, in accordance with the
invention, that the increase in strength of stretched fibers is
optimal when fibers are stretched from 5.0 to 7.5~ of their
original length. See Figure 3. Stretched fibers have higher
load at failure and strain at failure than unstretched fibers,
the increase being most pronounced with stretching from 5.0 to
7.5~ of the fibers~ original lengths. See Figures 4 and 5.
Additionally, stretched fibers are less stiff than unstretched
fibers, especially with higher degrees of applied stress. See
Figures 6 and 7.
When imp~anted in animal bodies, collagen implants
often lose up to 75~ of the initial tensile strength within four
weeks of implantation. It is therefore essential to maximize the
initial collagen fiber tensile strength to compensate for this
rapid loss of tensile strength of implants, especially in
orthopedic applications such as tendon or ligament replacement.
The monofilament and bundled fibers of the invention, having
higher tensile strength than fibers previously known in the art,
are especially well suited for implants requiring long lasting
strength of fibers. As an example, bundles of fibers made in
accordance with the invention were found to retain a high
percentage, approximately 60~, of the predicted strength computed
by multiplying the load at failure of individual fibers times the
number of fibers in the bundle. This compares favorably with
SUBSTITUTE SHEET (RULE 26~
CA 022047~3 1997-0~-07
WO96/14095 PCT~S95/14308
18
other known methods of bundling fibers which result in a loss of
up to 70 to 80~ of predicted strength.
If desired, the bundle may be coated with a suitable
protective material. Individual unbundled fibers may also be
coated. The bundle may be embedded in a matrix. Individual
unbundled fibers may also be embedded in a matrix. Preferably
the coating or matrix material is a biodegradable and
biocompatible polymer, which may be a synthetic or natural
polymer, and may be water or non-water soluble. Suitable
polymers include water soluble polymers derived from natural
sources, such as alginates, pectins, gelatins, and
polysaccharides, and synthetic polymers such as polylactic acid,
polyglycolic acid, polyurethane, and copolymers thereof.
The coating is performed by covering the bundles or the
unbundled fibers with a liquid coating material. Preferably the
coating is performed by dipping the stretched bundles into, or
running the fibers through, the liquid coating material, or
spraying the coating material onto the bundles or fibers until
the bundles or fibers are coated. Generally, less than a lO~
weight fraction of coating material is applied to the fibers.
After coating, the bundles may be dried and crosslinked.
Embedding of the bundled or unbundled fibers in a
matrix is performed in substantially the same way as the coating.
However, a higher weight fraction of matrix compound is used than
is used in coating so that the matrix compound becomes embedded
SUBSTITUTE SHEET (RULE 26~
CA 022047~3 1997-0~-07
Wog6/140s5 PCT~S9Sl14308
19
between the fibers of the bundle. When applying a matrix to
unbundled fibers, the matrix is applied as a coating.
Alternatively, or in addition to coating, the collagen
fibers may be embedded in a biodegradable and biocompatible
polymer matrix to bind the fibers together. The procedure for
producing the matrix/fibers composite may be identical to the
process for coating except that a higher weight portion of the
matrix is used, greater than 10~ of the fibers.
The tendon or ligament prosthetic devices for grafts
of the invention are constructed as follows. The device may be
made in various ways, as illustrated in Figure 2. In one
suitable method, individual fibers made in accordance with the
invention are placed in parallel alignment. The fibers are made
into bundles and the ends of each bundle is secured. A
multiplicity of bundles, each consistiny of a multiplicity of
fibers, for example about 10 fibers per bundle although as few
as two to five fibers and as many as several hundred fibers may
be used per bundle, are joined in parallel to form a tendon or
ligament device. Generally, between 7 and 15 fibers per bundle
will are used to form the prosthetic device of the invention.
In a second method, the parallel fibers are wrapped with a
braided bundle of fibers. The parallel fibers are secured at
each end to form the final device. In a third method, fibers are
made into bundles which are then wetted, stretched, and dried.
The stretched bundle may be crosslinked. The bundles are
arranged in parallel and secured at the ends to form the final
SUBSTITUTE SHEET (RULE 26)
CA 022047~3 1997-0~-07
WO96114095 ~- PCT~S95114308
prosthetic device. In a fourth method, the parallel bundles are
wrapped with a bundle of fibers which may be braided. The ends
of the parallel bundles are secured to form the prosthetic
device.
The graft of the invention has numerous applications
which can assume different physical embodiments or different
geometrical shapes.
The synthetic collagen graft material of the invention
is useful as a mesh, sheet, film, tube, circular casing,
filament, fiber or as a woven or non-woven fabric.
The graft material comprise collagen fibers with a dry
diameter in the range of about 20-60 microns. The diameter of
the wet collagen fibers may be much greater, often l.5 to more
than 2 times as thick.
The collagen fibers used in the grafts of the invention
have tensile strengths in the range of about 30 to about 9l MPa.
It is a noteworthy aspect of the invention that the fibers of the
invention can have ultimate tensile strengths exceeding that of
autograft materials or naturally occurring tendon fibers in
laboratory animals. The fibers of the invention also have
improved strength compared to fibers made by means other than the
method of the invention. The collagen materials of the invention
can have an index of refraction in the range of about l.4 to
about l.7, generally about l.6.
The graft material is biodegradable with the host's
naturally produced repair tissue supplanting the graft material.
SU8STlTtlTE SHEET (RULE 26)
CA 022047~3 1997-0~-07
WO96/14095 PCT~S95/14308
Furthermore, the graft is biologically, morphologically and
biomechanically compatible with surrounding tissue of the subject
treated.
In another embodiment of the invention, proteoglycans
are associated with the collagen fibers of the invention. For
that purpose the extruded fibers are immersed in a fiber
formation buffer containing the proteoglycan. The fibers are
soaked for a sufficient time at an appropriate temperature to
cause the proteoglycan to be incorporated into the fibrous
st--~cture. For instance, the fibers can be soaked for 60 minutes
at 37~C. The fibers are then rinsed with appropriate liquids to
remove excess proteoglycan and dried. Soaking temperature can
be in the range from about 15~C to 50 or 60~C with either longer
or shorter soaking periods, as may be desirable.
Specifically this embodiment of the invention may be
prepared as follows. Proteoglycans in a concentration between
0.01 and 0.02 g/100 ml were added to the fiber formation buffer
and stirred. A 1~ w/v collagen dispersion was placed in a
syringe to which polyethylene tubing of internal diameter 0.58
mm was attached. Fibers were extruded into a fiber formation
buffer. The fiber formation buffer is composed of l3smM NaCl,
30mM TES (N-Tris(hydroxymethyl) methyl -2- aminomethane sulfonic
acid) and 30mM sodium phosphate dibasic. The final pH is
adjusted to about the neutral range such in the range of about
6.5 to 7.5. Chemically similar or equivalent compounds may also
be used as well as other collagen fiber formation buffers well
SUBSTITUTE SH~ IJLE ~6~
CA 022047~3 1997-0~-07
WO 96/140g5 PCT/US95114308
known in the art. The extruded fibers were left in the tray
containing fiber formation buffer for 60 minutes. The buffer was
maintained at 37~C. The buffer was removed and replaced by
isopropanol. The fibers were soaked in isopropanol overnight and
were then soaked in distilled water for 15 minutes. The fibers
were then removed from the distilled water and air dried under
tension. The extruded collagen fibers were then crosslinked by
exposure to glutaraldehyde. Fibers which were formed in the
presence of high molecular weight proteoglycan were found to have
significantly increased ultimate tensile strengths compared to
low molecular weight, chondroitin sulfate, glycosaminoglycans or
controls: Furthermore collagen fibers formed in the presence of
high molecular weight proteoglycans exhibit higher tensile
strength than collagen fibers that are crosslinked. Further
details are given in Example III below.
The high molecular weight proteoglycans which are
generally preferred in the invention are large proteoglycans with
a core protein with a molecular weight greater than about 100,000
and glycosaminoglycans chain with a molecular weight greater than
about 5,000.
These proteoglycan generally have a molecular weight
of the range of about 1,000,000 to about 3,000,000 typically
about 1,200,000. Other proteoglycans desirable for use in the
invention include large proteoglycans from tendon with
chondroitin sulfate chains of average molecular weight of 17,000
and a core protein molecular weight of 200,000. It is not
SUBSTITUTE SHEET (RULE 26)
CA 022047~3 1997-0~-07
WO96/14095 PCT~S95tl4308
23
unlikely that other proteoglycans will also be useful in the
invention providing they impart the desirable properties to the
collagen fibers, in particular the desired tensile strength.
Once the high strength collagen fibers which constitute
the graft material of the invention are formed in accordance with
the various embodiments of the invention, the fibers are
collected. The collected fibers are shaped, pressed or formed
into sheets, tubes and numerous other shapes of varying
dimensions and thickness as desired for the particular
application. The fibers can be processed into woven materials.
They can be packed with various pharmacologically active agents.
These structures then can be directly used as the graft,
prosthesis or implant of the invention depending on the need and
how the particular structure has been prepared. The fibrous
graft can be woven or secured to surrounding tissue as an implant
or graft or topically applied and topically secured.
One skilled in the art will shape the structure to the
desired application.
The following examples are exemplary of the various
embodiments of the present invention discussed hereinabove. They
are not to be construed as limiting but as illustrative of the
process and products of the present invention.
Whenever "means~ or "steps" are disclosed in the
specification, equlvalent "means or "steps" which perform the
same function may be used by one skilled in the art.
SUBSTITUTE SHEET ~RULE 26)
CA 022047~3 1997-0~-07
WO96/14095 PCT~S95/14308
24
EXAMPLE I
Collagen fibers were prepared from a l~ (w/v)
dispersion of insoluble type I collagen derived from bovine
corium in dilute HCl, pH 2Ø This collagen dispersion was
extruded through polyethylene tubing with an inner diameter of
o.28mm into a 37~C bath of aqueous sodium phosphate fiber
formation buffer as described elsewhere. After immersion of 45
minutes, the fibers were placed in isopropanol for at least four
hours. They were then rinsed in distilled water for lS minutes
and allowed to air dry under tension overnight. Fibers were
placed in a sealed desiccator containing l0 ml of a 25~ ~w/v)
glutaraldehyde solution at room temperature and allowed to vapor
crosslink for 24 hours. Alternatively, collagen fibers were
placed in an oven at 110~C in a vacuum of between 50 and l00 m
torr for 72 hours. These fibers were then placed in a sealed
desiccator containing 20 g of cyanamid in 5 ml of distilled water
for 24 hours.
Prostheses containing 200 to 250 individual crosslinked
collagen fibers were coated with a l~ (w/v) collagen dispersion
in HCl, pH 2.0, air dried overnight and then extensively washed
in distilled water. One ml Alcide~ activator and one ml Alcide~
base were added to l0 ml of distilled water and after l0 minutes
diluted with 24 ml of phosphate buffer solution. Each implant
was immersed in this cold sterilant for at least four hours, and
then soaked in one liter of sterile physiological saline prior
to implantation.
SUBSTITUTE SHEET (RULE 26
CA 022047~3 1997-0~-07
WO 96/14095 PCI/US95/14308
EXAMPLE I I
Reconstituted Collagen Fibers
Insoluble collagen type I from fresh, uncured corium
was obtained from Devro, Inc. (Somerville, NJ, USA). The corium
was limed, fragmented, swollen in acid, precipitated, washed with
distilled water and isopropanol, lyophilized and stored at -30~C.
A 1~ (w/v) dispersion of type I collagen in dilute HCl,
ph 2.0 was prepared by adding 1.2g of lyophilized collagen to
120ml of HCl solution in a blender (Osterizer) and mixing at a
speed of 10,000 rev min~l for 4 min. The mixture was allowed to
settle for 10 min and then remixed at 10,000 rev min~1 for 4 min.
The resulting dispersion was placed under a vacuum of 0.01 m torr
at room temperature to remove any trapped air bubbles.
Dispersion was then stored in disposable 30 cc syringes at 4~C.
Collagen fibers were produced by extruding the collagen
dispersion through polyethylene tubing with an inner diameter of
0.28 mm into a 37~C bath of aqueous fiber formation buffer
composed of 135mm NaCl, 30mM TES (N-Tris(hydroxymethyl) methyl -
2- aminomethane sulfonic acid) and 30mM sodium phosphate dibasic.
The final bath pH was adjusted to 7.5 by adding 5.On NaOH drop-
wise. Fibers were allowed to remain in the buffer for 45 min,
and then placed in 500 ml of isopropyl alcohol for at least 4
hours. The fibers were immersed in distilled water for 15-20
min and air dried under tension.
Collagen fibers were crosslinked using glutaraldehyde
or by a combination of severe dehydration and treatment with
SUBSTITUTE SHEET (RULE 26~
CA 022047~3 1997-0~-07
WO96/14095 PCT~S95/14308
26
cyanamid. Glutaraldehyde crosslinking was accomplished by
placing air-dried collagen fibers in a sealed desiccator
containing 10 ml of a 25~ (w/v) aqueous glutaraldehyde solution
in a petri dish. The fibers were placed on a shelf in the
5desiccator and were crosslinked in a glutaraldehyde vapor for 1-4
d at room temperature. Collagen fibers were also cross-linked
by placing in an oven at 110~C and at vacuum of 50-100 m torr for
3 d. Subsequent to dehydrothermal crosslinking (DHT), collagen
fibers were placed on a shelf in a sealed desiccator containing
10a petri dish with 20 g of cyanamide in 5 ml of distilled water.
~ollagen fibers were crosslinked for one day in contact with
cyanamide vapor.
EXAMPLE III
15Extraction of Soluble Type I Collagen
Acid soluble type I collagen was extracted from tail
tendons of young rats. The t~n~ons were stripped from the tails
and dissolved in 0.01 M HCl at 4~C followed by centrifugation for
30 min. at 30,000 X g. The supernatant was sequentially filtered
20through 0.8., 0.65, and 0.45 ~m Millipore filters. The collagen
preparation was analyzed by SDS polyacrylamide gel
electrophoresis and amino acid analysis.
Purification of Insoluble Type I Collagen
25The raw material (bovine corium) was prepared from
fresh uncured bovine hide which was obtained from Devro, Inc.
SUBSTITUTE SHEET (RULE 26~
CA 022047~3 1997-0~-07
W096/14~5 PCT~S95114308
(Somerville, N.J.). The hides were split into two components,
the grain layer (papillary denmis) and the corium (reticular
dermis). Fresh corium was frozen and stored at -20OC until it
was used.
One liter of the frozen raw material was defrosted at
room temperature and placed in an 18 liter Nalgene processing
tank (Consolidated Plastics, Twinsburg, Ohio), equipped with air
and water lines. Distilled water was added until the total
volume of the processing mixture reached 14 liters. Air at a
pressure of 6 psi was introduced into the tank for 5 minutes, to
create a homogeneous mixture. This mixture was then left to
sediment for 2Q minutes. After complete sedimentation occurred,
the liquid phase was drained and fresh distilled water was added
until the total volume reached 14 liters. This procedure was
repeated three times.
Eight liters of 99.8~ of isopropyl alcohol
(Mallinkroft, Inc., Paris, Kentucky) was added to the solid
phase; the sediment was mixed using air in a tank placed on a
gyrotory shaker (New Brunswick Scientific Co., New Brunswick,
N.J.) for 12 hours at a speed of 34 rev/min.
The liquid phase was then removed using a Becton siphon
pump (Consolidated Plastic, Twinsburg, Ohio) and 8 liters of
99.8% isopropyl alcohol was mixed with the solid phase. The
mixture was then placed on the shaker for another 12 hours.
SUBSTITUTE SHEET (RULE 26)
CA 022047~3 1997-0~-07
WO 96/14095 PCT/US95/14308
After removal of the liquid phase, the material was
washed with 2 liters of distilled water, poured into plastic
trays and placed in a freezer until frozen solid.
The frozen material was then placed in the cold trap
of a freeze dryer (Freeze Mobile 12, Virtis, Inc., Gardner, N.Y.)
at -65~C. A vacuum of 10 microns was then applied for 48 to 96
hours. The vacuum was then released and material removed. The
freeze dried collagen was removed from the trays and stored in
air tight bags.
Preparation of Insoluble Type I Collagen
for Fiber Formation
A lN solution of HCl was slowly added to 120.0 ml of
distilled water until the pH was 2Ø A 1.2 g sample of
insoluble type I collagen, extracted by the procedure described
above, was then put into a blender (Osterizer Model, oster
Corporation, Milwaukee, Wisconsin) with the HCl (pH 2.0). This
1~ w/v collagen HCl dispersion was blended at high speed (10,000
rpm) for 3 minutes.
The dispersion was then emptied from the blender into
a 600 ml sidearm flask. A vacuum (Vacuum Pump, Model 150,
Precision Scientific Company, Chicago, Ill.) of 100 microns was
applied at room temperature until the air bubbles were removed
from the dispersion. This procedure required approximately 15
minutes. The vacuum was removed and the dispersion was ready for
making fibers.
SUBSTITUTE SHEET (RULE 26
CA 022047~3 1997-0~-07
WO96tl4095 PCT~S95/14308
29
Glycosaminoglycans and Proteoglycans
Dermatan sulfate (chon~roitin sulfate B from porcine
skin), chondroitin sulfate (type A from whale cartilage),
glycosaminoglycans (GAG) and dextran sulfate (Dexs) were obtained
s from Sigma Chemical Company (St. Louis, Mo.). Chondroitin
sulfate proteoglycan (CS-PG) and dermatan sulfate proteoglycan
(DS-PG) from hypertrophic scar tissue and high molecular weight
proteoglycan from articular cartilage (PGl) were prepared and
characterized as previously described.
~0
FIBRIL ASSEMBLY STUDIES
Turbidity-Time Studies
Lyophilized soluble type I collagen was dissolved at
l mg/ml in HCl, pH 2.0, stirred at 4~C for 24 hours, dialyzed
against HCl, pH 2.0, centrifuged at 1600 g for 60 minutes and the
supernatant was then filtered through a 0.65 ~m Millipore filter.
This collagen stock solution was stored at 4~C for periods of up
to one week.
Fibril formation was initiated by mixing 0.9 ml of a
collagen solution with O.l ml of buffer on ice to give a final
composition of 30 mM n-tris[hydroxymethyl]methyl-2-
aminoethanesulfonic acid (TES), 30 mM phosphate and NaCl to a
final ionic strength of 0.225 at pH 7.3. Cuvettes were filled
with sample, sealed and transferred to a water-jacketed sample
compartment of a Gilford Model 250 spectrophotometer. The
compartment was maintained at the desired experimental
SUBSTITUTE SHEET (RULE 26)
CA 022047~3 1997-0~-07
WO 96/14095 ~ PCT/US95/14308
temperature and the absorbent was recorded as function of time.
Absorbent was defined as the natural logarithm of the ratio of
the incident light and the scattered light intensities.
Absorbent at 131 nm was converted to turbidity by multiplying by
2.303.
Collagen concentrations between 0.20 and 0.45 mg/ml and
proteoglycan concentration between 0.001 and 0.2 g/lOOm were
evaluated at temperatures from 27 to 37~C.
Extrusion of Collagen Fibers
An aqueous fiber formation buffer composed of 135 mM
NaCl, 30-mM TES and 30 mM sodium phosphate dibasic at a final pH
of 7.5 was heated to 37~C in a temperature controlled water bath.
Glycosaminoglycan (concentrations between 0.001 and 0.2 g/100 ml)
or proteoglycan (concentrations between O.01 and 0.02 g/lOOml)
was added to the fiber formation buffer and stirred. A 1~ w/v
collagen dispersion (lg/lOOml) was placed in a syringe to which
a polyethylene tubing (Clay Adams, pE-so) of internal diameter
0.58 mm was attached. A syringe pump (Sage Instruments, Model
341A) at a speed of 7 ml/minute was used to extrude the fibers
into fiber formation buffer. Extruded fibers were left in the
tray containing fiber formation buffer maintained at 37~C for 60
minutes. Fiber formation buffer was then emptied out from the
tray using a vacuum hose and was replaced by isopropanol and left
overnight. Isopropanol was removed and was replaced by distilled
SUBSTITUTE SHEJ (RULE 26~
CA 022047~3 1997-0~-07
WOg6/14095 PCT~S95114308
31
water for 15 minutes. Fibers were then removed from the
distilled water and air dried under tension.
Collagen Fiber Crosslinking
Extruded collagen fibers were crosslinked by exposure
to glutaraldehyde vapor for 24 hours (Glut l) at room temperature
in a sealed desiccator as described previously.
Proteoglycan concentrations present on collagen fibers
were also less than l~ (data not shown). This is another
distinctive characteristic of the fibers which are particularly
useful in the invention.
EXAMPLE IV
Preparation of Collagen Monofilament from Soluble Collagen
Collagen monofilament fibers were prepared from soluble
type I collagen from fetal calf skin. Collagen fibers were
prepared from l~ (w/v) solution of type I collagen in dilute HCl.
The solution was extruded through polyethylene tubing with an
inner diameter of 0.28 mm into a 37~C bath containing aqueous
sodium phosphate fiber formation buffer. The fibers were
extruded into a tray containing fiber formation buffer and then
the fiber was pulled over a transfer device into a bath of
isopropanol and then through a bath of distilled or demineralized
water. The fiber left the last bath and was air dried using a
heat lamp. The fiber was then wound on a tensioning spool.
SUBSTITUTE SHEET (RULE 26~
CA 022047~3 1997-0~-07
WO 96/14095 PCT/US95/14308
32
EXAMPLE V
Formation of Bundles Containing Monofilaments
Ten collagen monofilaments were placed side-by-side in
parallel alignment to form a bundle. The bundle was glued using
an epoxy adhesive onto a support beam (tongue depressor) at each
end of the bundle. The assembly was then placed in phosphate
buffer solution for 25 minutes. The wet bundle with support
beams at each end was then attached to a stretching frame using
screws to secure the beams to the stretching frame. The bundle
was stretched 7 to 7.5~ of its original length and allowed to air
dry overnight. On drying the 10 monofilaments were associated
to form a single bundle which was then removed from the two
support beams. The bundle was then crosslinked for 5 days at
110 ~C .
Bundles are made as described above using 2
monofilament fibers. Results as described in Example VII with
bundles of 2 fibers are similar to results achieved using 10
fibers.
Bundles are made using about 400 monofilament fibers.
Bundles are also made using about 10,000 monofilament fibers.
Results as described in Example VII with bundles of about 400 and
with bundles of about 10,000 fibers are similar those achieved
using 10 fibers.
SUBSTITUTE SHEET (RULE 26
CA 022047~3 l997-0~-07
WO96/14095 PCT~S95114308
EXAMPLE VI
Formation of Coated Bundle
Collagen fibers were produced as in Example IV. Ten
collagen monofilaments were placed side-by-side to form a bundle.
A wet bundle of collagen fibers that was attached to support
beams and stretched to 7 to 7.5~ as described in Example V above
was dipped in a 4~ aqueous solution of sodium alginate at room
temperature. The alginate coated bundle was then air dried and
then crosslinked for 5 days at 110~C.
EXAMPLE VII
Mechanical Testing of Monofilament and Bundled Fibers
Collagen monofilaments and bundles were mechanically
tested while wet using an Instron Model 1122 at a strain rate of
50~ per minute using a 2 cm gauge length. The materials were
mounted on a 2 cm gauge length frame using 2 ton epoxy ~Devron
Corp., Denver, C0). Monofilaments and bundles mounted on paper
fr ~es were immersed in phosphate buffer solution (pH 7.5) for
25 minutes. The paper was cut and the samples were pulled to
failure in tension. Load and strain at failure were found to be
38.0 g and 14.5~ respectively for monofilament and 219 g and
10.5~ for uncoated intermediate bundles.
This bundle retains 219 g of the predicted 380 g or 58~
of the load to failure. The bu.lle breaks uniformly, has a
strain at failure of about lO~ and can be fashioned into a
tendon/ligament device by forming either thin tapes of
SUBSTITUTE SHEET (RULE 26)
CA 022047~3 1997-0~-07
WO96/14095 PCT~S95/14308
34
intermediate bundles, braiding monofilament around a group of
parallel bundles, or by wrapping monofilament around groups of
parallel bundles.
S EXAMPLE VI I I
Formation of Prosthetic Device from Unbundled Fibers
One hundred collagen monofilament fibers are placed
onto spools. The fibers are then tensioned in parallel by
stretching them over a pulley. An outer layer of collagen
fibers is wrapped or braided around the parallel fibers. The
ends of the fibers are secured to stabilize the outer layer
and to preserve the parallel alignment of the fibers.
The above process is performed using 400 and using
10,000 collagen monofilament fibers. The fibers are tensioned
in parallel and wrapped by an external layer of fibers which
may be braided. The ends of the parallel fibers are secured
to stabilize the prosthetic device.
EXAMPLE IX
Formation of Prosthetic Device from Bundled Fibers
Ten stretched crosslinked bundles, each having ten
collagen monofilament fibers are placed in parallel. An outer
layer of collagen fibers is wrapped or braided around the
parallel bundles. The ends of the bundles are secured to
stabilize the outer layer and to preserve the parallel
alignment of the bundles.
SUBSTITUTE SHEET (RULE 26)
CA 022047~3 1997-0~-07
W096/14~5 35 PCT~S95/14~8
The same process is performed using ~00 collagen
monofilament fibers in 40 bundles, each containing 10 fibers.
The process is performed u~ing 10,000 collagen monofilament
fibers in 1000 bundles, each containing 10 fibers. The
bundles are placed in parallel and wrapped by an external
layer of fibers which may be braided. The ends of the
parallel bundles are secured to stabilize the prosthetic
device.
Variations in technique (also ~means" or "steps") of
the type known in the art and understood by those of ordinary
skill to be functional e~uivalents of those disclosed herein
may be substituted as desired, for convenience or for
optimization of yield, or to simplify or improve the cost-
effectiveness of the overall procedure. Therefore, numerous
modifications and variations of the present invention are
possible which are within the scope of the appended claims.
SULSTITUTE SHEET (RULE 26)
CA 02204753 1997-05-07
WO96/14095 36 PCT~S95/14308
K r ~:KL.,. ~, ~S
1. Wang, M.C., Pins, G.D., and Silver, F.H.,, ~Collagen
Fibers with Improved Strength for the Repair of Soft
Tissue Injuries," Biomaterials, 15, 7:508-512, 1994.
2. Kato, Y.P. and Silver, F.H., ~Formation of Continuous
Collagen Fibres: Evaluation of Biocompatibility and
Mechanical Properties," Biomaterials, 11, 169-175.
.
SU~STITUTE SHEET (RULE 26)
