U.S. patent application number 15/606222 was filed with the patent office on 2017-09-14 for flowable collagen-based hemostat and methods of use.
The applicant listed for this patent is Orthovita, Inc.. Invention is credited to Alice Bachert, Abigail Cohen, Marissa M. Conrad, Jenny E. Morgan, Lauren S. Valdes.
Application Number | 20170258954 15/606222 |
Document ID | / |
Family ID | 46795775 |
Filed Date | 2017-09-14 |
United States Patent
Application |
20170258954 |
Kind Code |
A1 |
Conrad; Marissa M. ; et
al. |
September 14, 2017 |
FLOWABLE COLLAGEN-BASED HEMOSTAT AND METHODS OF USE
Abstract
The invention relates to methods for fabricating a flowable
hemostatic composition. The invention also relates to hemostatic
compositions and methods for promoting wound healing. In various
embodiments, the hemostatic compositions comprise crosslinkable
collagen molecules having a porosity controlled by the ratio of
weight percent collagen solids to weight percent crosslinker when
crosslinking the collagen. In other embodiments, the hemostatic
compositions comprise crosslinkable collagen molecules having a
porosity controlled by the temperature and rate of freezing when
drying the composition during fabrication. In some embodiments, the
compositions contain additional agents, including biological
agents.
Inventors: |
Conrad; Marissa M.;
(Philadelphia, PA) ; Valdes; Lauren S.; (Media,
PA) ; Morgan; Jenny E.; (Phoenixville, PA) ;
Bachert; Alice; (Haverton, PA) ; Cohen; Abigail;
(Huntington Woods, MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Orthovita, Inc. |
Malvern |
PA |
US |
|
|
Family ID: |
46795775 |
Appl. No.: |
15/606222 |
Filed: |
May 26, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15229698 |
Aug 5, 2016 |
9694101 |
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15606222 |
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13411324 |
Mar 2, 2012 |
9447169 |
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15229698 |
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61449292 |
Mar 4, 2011 |
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61449292 |
Mar 4, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/4833 20130101;
A61K 38/4833 20130101; A61K 38/39 20130101; C12Y 304/21005
20130101; A61L 15/00 20130101; A61L 2400/06 20130101; A61L 24/043
20130101; A61L 2300/418 20130101; A61L 15/325 20130101; A61L 24/102
20130101; A61K 9/70 20130101; A61L 15/28 20130101; A61L 24/0015
20130101; A61L 2300/254 20130101; A61L 15/42 20130101; A61L
2300/412 20130101; C07K 14/78 20130101; A61L 2400/04 20130101; A61L
26/00 20130101; A61L 24/0036 20130101; A61L 17/00 20130101; A61P
7/04 20180101; A61K 2300/00 20130101 |
International
Class: |
A61L 15/32 20060101
A61L015/32; A61L 17/00 20060101 A61L017/00; A61K 9/70 20060101
A61K009/70; A61L 15/00 20060101 A61L015/00; A61L 15/28 20060101
A61L015/28; A61L 15/42 20060101 A61L015/42 |
Claims
1-30. (canceled)
31. A flowable hemostatic composition comprising: crosslinked
collagen having a porosity greater than 50% and a surface area of
between 0.5 to 30 m.sup.2/g; and a physiologically acceptable
liquid vehicle selected from the group consisting of water, saline,
calcium chloride, and combinations thereof, wherein the porosity of
the composition is controlled by temperature and rate of freezing
during lyophilization.
32. The composition of claim 31, wherein the crosslinked collagen
comprises at least one material from the group consisting of
fibers, ribbons, ropes, and sheets.
33. The composition of claim 31, wherein the porosity is further
controlled by the ratio of percent collagen solids to percent
crosslinker when crosslinking the collagen.
34. The composition of claim 31, wherein the crosslinked collagen
is fibrillar collagen.
35. The composition of claim 31, further comprising adding at least
one biological agent.
36. The composition of claim 31, further comprising controlling the
porosity by controlling the collagen concentration prior to
freezing the collagen.
37. The composition of claim 31, wherein the controlled temperature
rate of freezing comprises a freezing time of 2 to 6 hours, a
primary drying cycle, and a secondary drying cycle.
38. The composition of claim 37, wherein the primary drying cycle
is performed at temperatures between 0 and 15.degree. C. for 1 to
24 hours and the secondary drying cycle is performed at
temperatures between 20 and 40.degree. C. for 2 to 10 hours
39. The composition of claim 31, wherein the composition can be
dispensed from a syringe having at least a 1.6 mm opening.
40. The composition of claim 31, wherein the composition does not
include thrombin.
41. The composition of claim 31, wherein the composition has a
percent swelling of between 0% and 20% within 10 minutes.
42. A method of fabricating a flowable hemostatic composition
comprising: crosslinking collagen with a crosslinking agent to form
crosslinked collagen, wherein the crosslinked collagen has a
porosity greater than 50% and a surface area between 0.5 to 30 30
m2/g; lyophilizing the crosslinked collagen; and reconstituting the
crosslinked collagen with a physiologically acceptable liquid
vehicle selected from the group consisting of water, saline,
calcium chloride, and combinations thereof.
43. The method of claim 42, the crosslinking agent is
glutaraldehyde.
44. The method of claim 42, wherein the collagen is microfibrillar
collagen.
45. The method of claim 42, wherein the collagen is fibrillar
collagen.
46. The method of claim 42, further comprising controlling the
porosity of the crosslinked collagen by controlling the temperature
and rate of freezing during lyophilization.
47. The method of claim 42, further comprising controlling the
porosity by controlling the collagen concentration prior to
freezing the collagen.
48. The method of claim 42, further comprising controlling the
material structure of the crosslinked collagen by controlling the
temperature and rate of freezing during lyophilization.
49. The method of claim 48, wherein the controlled temperature rate
of freezing comprises a freezing time of 2 to 6 hours, a primary
drying cycle, and a secondary drying cycle.
50. The method of claim 49, wherein the primary drying cycle is
performed at temperatures between 0 and 15.degree. C. for 1 to 24
hours and the secondary drying cycle is performed at temperatures
between 20 and 40.degree. C. for 2 to 10 hours
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation application of
U.S. patent application Ser. No. 15/229,698, filed Aug. 5, 2016,
which is a divisional of U.S. patent application Ser. No.
13/411,324, filed Mar. 2, 2012, now U.S. Pat. No. 9,447,169, which
claims the benefit of the filing date of U.S. Provisional
Application No. 61/449,292 filed Mar. 4, 2011, the disclosures of
which are hereby incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Excessive bleeding or hemorrhaging has always been a
significant issue with many medical or surgical procedures. Because
of this, the medical industry has continuously sought new and
improved products to inhibit bleeding in a patient, and methods of
establishing hemostasis. In general, these hemostatic products and
processes assist in the rapid initiation of a hemostatic plug
formed through platelet activation, aggregation, adhesion and gross
clot formation at a tissue target site.
[0003] A wide variety of hemostatic products are made from
different base materials, such as collagen, gelatin, oxidized
regenerated cellulose, fibers, gauze sponges and fibrin. These
products are used in a wide variety of medical and surgical
procedures. For example, microfibrillar collagen is used
extensively for wide-area parenchyma bleeding and for laparoscopic
procedures. Hemostatic sponges are used in surgical as well as
dermatological applications where adherence to the wound site and
ease of removal are important considerations.
[0004] A number of hemostatic collagen-containing devices have
previously been described. For example, U.S. Pat. Nos. 5,428,024;
5,352,715; and 5,204,382 generally relate to fibrillar and
insoluble collagens that have been mechanically disrupted to alter
their natural physical properties. Injectable collagen compositions
are described in U.S. Pat. Nos. 4,803,075 and 5,516,532.
International application WO 96/39159 describes a collagen-based
delivery matrix made of dry particles in the size range from 5
.mu.m to 850 .mu.m, where the particles are suspended in water and
have a particular surface charge density. A bioactive agent is then
incorporated in the matrix prior to administration to a patient.
U.S. Pat. No. 5,196,185 describes a collagen preparation having a
particle size from 1 .mu.m to 50 .mu.m useful as an aerosol spray
to form a wound dressing. U.S. Pat. No. 7,320,962 describes a
hemostatic composition having a population of crosslinked polymer
(e.g. gelatin or collagen) integrated into a non-crosslinked
polymer (e.g. gelatin or collagen) population, such that the
non-crosslinked collagen dissolves at the wound site, releasing the
crosslinked collagen to form a hemostatic hydrogel. U.S. Pat. Nos.
6,063,061; 6,066,325 and 6,706,690 also describe hemostatic
compositions that include soluble and/or non-fibrillar collagen,
with plasticizers and hemostatic agents, such as thrombin,
integrated within the composition.
[0005] Collagen pads have also been used to improve wound healing
or to stop bleeding, via platelet aggregation and activation, the
formation of thrombin on the surface of activated platelets, and
the formation of a hemostatic fibrin clot by the catalytic action
of thrombin on fibrinogen. Hemostatic agents are typically added to
the collagen pads. For example, in U.S. Pat. No. 4,600,574 a
collagen based tissue adhesive combined with fibrinogen and factor
XIII is described. The fibrinogen and factor XIII are combined with
the collagen by impregnating the flat collagen material with a
solution comprising fibrinogen and factor XIII, and lyophilizing
the material. U.S. Pat. No. 5,614,587 describes bioadhesive
compositions comprising crosslinked collagen using a synthetic
hydrophilic polymer.
[0006] However, because no single device or process can meet the
dynamic applications of the medical industry, there continues to be
a need to provide alternative systems and methods for achieving
hemostasis, particularly compositions that do not require the use
of additional hemostatic agents, such as thrombin. Thus, there
continues to be a need for flowable hemostatic compositions that
can easily be delivered to tissue and establish hemostasis without
delaying or inhibiting tissue repair. There is also a need for
hemostatic compositions that promote wound healing; and hemostatic
compositions that readily adhere to the tissue but not to surgical
materials, such as gauze. The present invention satisfies this
need.
SUMMARY OF THE INVENTION
[0007] The present invention relates to a hemostatic composition.
The composition includes crosslinked collagen, wherein the
composition has a porosity controlled by the ratio of percent
collagen solids to percent crosslinker when crosslinking the
collagen. In one embodiment, the hemostatic composition has a
porosity greater than about 50% and a surface area of between about
0.5 to about 30 m.sup.2/g. In another embodiment, the crosslinked
collagen includes at least one material structure from the group
consisting of fibers, ribbons, ropes and sheets. In another
embodiment, the number of structures is controlled by the ratio of
percent collagen solids to percent crosslinker when crosslinking
the collagen. In another embodiment, the number of structures is
controlled by the collagen concentration prior to freezing the
collagen. In another embodiment, the number of structures is
controlled by the temperature and rate of freezing when the
collagen is lyophilized. In another embodiment, the collagen is
microfibrillar collagen. In another embodiment, the collagen is
fibrillar collagen. In another embodiment, the hemostatic
composition includes at least one biological agent. In another
embodiment, the at least one biological agent comprises thrombin.
In another embodiment, the crosslinked collagen is in a
physiologically acceptable liquid vehicle. In another embodiment,
the liquid vehicle is water, saline, calcium chloride or a
combination thereof. In another embodiment, the composition is
flowable, such that it can be easily dispensed from a syringe
having at least a 1.6 mm opening.
[0008] The present invention also includes a method of fabricating
a flowable hemostatic composition. The method includes the steps of
crosslinking about 0.1-10% collagen with a crosslinking agent at a
ratio between about 7.5:1 to 500:1, lyophilizing the crosslinked
collagen until dried, and reconstituting the crosslinked collagen
at a concentration of about 50-200 mg/mL. In one embodiment, the
crosslinking agent is glutaraldehyde. In another embodiment, the
collagen is microfibrillar collagen. In another embodiment, the
collagen is fibrillar collagen. In another embodiment, the
crosslinked collagen is reconstituted in a physiologically
acceptable liquid vehicle. In another embodiment, the liquid
vehicle is water, saline, calcium chloride or a combination
thereof. In another embodiment, the method further includes the
step of adding at least one biological agent. In another
embodiment, the at least one biological agent comprises thrombin.
In another embodiment, the method further includes the step of
controlling the porosity of the crosslinked collagen by controlling
the temperature and rate of freezing during lyophilization. In
another embodiment, the method further includes the step of
controlling the porosity by controlling the collagen concentration
prior to freezing the collagen. In another embodiment, the method
further includes the step of controlling the material structure of
the crosslinked collagen by controlling the temperature and rate of
freezing during lyophilization. In another embodiment, the material
structure includes at least one from the group consisting of
fibers, ribbons, ropes and sheets.
[0009] The present invention also includes a hemostatic composition
formed by the steps of crosslinking about 0.1-10% collagen with
glutaraldehyde at a ratio between about 7.5:1 to 500:1,
lyophilizing the crosslinked collagen until dried, and
reconstituting the crosslinked collagen at a concentration of about
50-200 mg/mL.
[0010] The present invention also includes a wound healing
composition. The composition includes crosslinked collagen at a
concentration of between about 50-200 mg/mL, wherein the
composition has a porosity controlled by the ratio of percent
collagen solids to percent crosslinker when crosslinking the
collagen.
[0011] The present invention also includes a composition comprising
crosslinked collagen, wherein the composition has a porosity
controlled by the temperature and rate of freezing used to
manufacture the composition.
[0012] The present invention also includes a composition comprising
crosslinked collagen, wherein the composition is flowable such that
it can be easily dispensed from a syringe and where the flowability
is controlled by the presence of at least one material structure
from the group consisting of fibers, ribbons, ropes and sheets.
[0013] The present invention also includes a method of promoting
hemostasis at a bleeding site comprising applying a hemostatic
composition to the bleeding site, wherein the hemostatic
composition includes crosslinked collagen having a porosity
controlled by the ratio of percent collagen solids to percent
crosslinker when crosslinking the collagen.
[0014] The present invention also includes a method of promoting
wound healing at an injury site includes applying to the injury
site a composition of about 1-5% crosslinked collagen that has been
lyophilized and reconstituted at a concentration of about 50-200
mg/mL.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] For the purpose of illustrating the invention, there are
depicted in the drawings certain embodiments of the invention.
However, the invention is not limited to the precise arrangements
and instrumentalities of the embodiments depicted in the
drawings.
[0016] FIG. 1 is an illustration of the formation of insoluble
fibrillar collagen structures from tropocollagen. Triple helical
tropocollagen, which is soluble at a low pH, forms a staggered
fibrillar structure which is substantially insoluble in water as pH
is increased.
[0017] FIGS. 2A-2C are a set of flowcharts for exemplary methods of
fabricating a hemostatic composition of the present invention. FIG.
2A is a general method for fabricating a hemostatic composition;
FIG. 2B is an exemplary method of fabricating a hemostatic
composition using glutaraldehyde as a crosslinker; FIG. 2C is an
exemplary method of fabricating a hemostatic composition using
EDC/NHS as a crosslinker.
[0018] FIGS. 3A-3D are SEM images at a magnification of 500.times.
of 1% collagen samples. FIG. 3A is a non-crosslinked control, and
FIGS. 3B-3D are samples crosslinked with glutaraldehyde (GTA) for 4
hours at 500:1 (FIG. 3B), 250:1 (FIG. 3C) and 100:1 (FIG. 3D),
frozen at -28.degree. C. and lyophilized until dry.
[0019] FIGS. 4A-4C are SEM images at a magnification of 250.times.
of 1% collagen crosslinked at a ratio of 100:1. Collagen pellets of
1% collagen crosslinked at 100:1 were frozen at -28.degree. C.
(FIG. 4A), at -80.degree. C. (FIG. 4B) or in liquid nitrogen (FIG.
4C) and lyophilized until dry.
[0020] FIGS. 5A-5C are SEM images at a magnification of 500.times.
of 1% collagen crosslinked at a ratio of 100:1. Collagen pellets of
1% collagen crosslinked at 100:1 were frozen at -28.degree. C.
(FIG. 5A), at -80.degree. C. (FIG. 5B) or in liquid nitrogen (FIG.
5C) and lyophilized until dry.
[0021] FIGS. 6A-6C are SEM images at a magnification of 1000.times.
of 1% collagen crosslinked at a ratio of 100:1. Collagen pellets of
1% collagen crosslinked at 100:1 were frozen at -28.degree. C.
(FIG. 6A), at -80.degree. C. (FIG. 6B) or in liquid nitrogen (FIG.
6C) and lyophilized until dry.
[0022] FIGS. 7A-7C are SEM images at a magnification of 50.times.
of 1% collagen crosslinked at 100:1, which were ground using
3.times.3 sec pulses, and reconstituted at 150 mg/mL. Samples were
frozen at -28.degree. C. (FIG. 7A), at -80.degree. C. (FIG. 7B) or
in liquid nitrogen (FIG. 7C) prior to lyophilizing (until dry),
grinding and reconstitution.
[0023] FIGS. 8A-8C are SEM images at a magnification of 250.times.
of 1% collagen crosslinked at 100:1, which were ground using
3.times.3 sec pulses, and reconstituted at 150 mg/mL. Samples were
frozen at -28.degree. C. (FIG. 8A), -80.degree. C. (FIG. 8B) or in
liquid nitrogen (FIG. 8C) prior to lyophilizing (until dry),
grinding and reconstitution.
[0024] FIGS. 9A-9C are SEM images at a magnification of 1000.times.
of 1% collagen crosslinked at 100:1, which were ground using
3.times.3 sec pulses, and reconstituted at 150 mg/mL. Samples were
frozen at -28.degree. C. (FIG. 9A), at -80.degree. C. (FIG. 9B) or
in liquid nitrogen (FIG. 9C) prior to lyophilizing (until dry),
grinding and reconstitution.
[0025] FIGS. 10A-10D are SEM images of structural differences in
crosslinked collagen materials. The primary material structures are
fibers (A), ribbons (B), ropes (C) and sheets (D).
[0026] FIGS. 11A and 11B are SEM images of glutaraldehyde
crosslinked microfibrillar collagen materials (250:1) lyophilized
at a freezing rate of -1.degree. C./min (A) and a freezing rate of
-0.5.degree. C./min (B). Images are at 500.times.
magnification.
[0027] FIGS. 12A-12D are SEM images of lyophilized glutaraldehyde
crosslinked microfibrillar collagen materials (250:1) undiluted
(A), and diluted by volume in USP water at 1:1 collagen (B), 1:5
collagen (C) and 1:10 collagen (D).
[0028] FIGS. 13A-13B are SEM images at a magnification of
500.times. of 1% collagen (FIG. 13A) and Surgiflo.RTM. (FIG. 13B)
after hemostasis had been achieved through application of the
material to a bleeding site.
[0029] FIGS. 14A-14C are SEM images of 5% collagen (FIG. 14A,
1500.times.), Floseal.RTM. (FIG. 14B, 1500.times.), and
Surgiflo.RTM. (FIG. 14C, 1000.times.) after hemostasis had been
achieved through application of the material to a bleeding
site.
[0030] FIGS. 15A-15B are SEM images at a magnification of
3000.times. of 1% collagen (FIG. 15A) and 5% collagen without the
addition of thrombin (FIG. 15B) after hemostasis had been achieved
through application of the material to a bleeding site.
[0031] FIGS. 16A-16D are histology sections at a magnification of
20.times. of collagen samples in a liver defect. FIG. 16A is 0.1%
fibrillar collagen crosslinked at a ratio of 40:1, FIG. 16B is 1%
fibrillar collagen crosslinked at a ratio of 25:1, FIG. 16C is 1%
microfibrillar collagen crosslinked at a ratio of 250:1, and FIG.
16D is Floseal.RTM..
[0032] FIGS. 17A-17C are histology sections at a magnification of
10.times. of collagen samples shown at the material/tissue
interface 8 weeks after implantation. FIG. 17A is 1% microfibrillar
collagen crosslinked at a ratio of 100:1, FIG. 17B is 1%
microfibrillar collagen crosslinked at a ratio of 250:1, and FIG.
17C is 0.1% fibrillar collagen crosslinked at a ratio of 10:1.
[0033] FIG. 18A-18E are SEM images of EDC/NHS crosslinked collagen
at 500.times. magnification. FIG. 18A is a EDC/NHS Control; FIG.
18B is material crosslinked at half the EDC/NHS concentration
relative to the control; FIG. 18C is material crosslinked at two
times the EDC/NHS concentration relative to the control; FIG. 18D
is material crosslinked using EDC/NHS chemistry for 2 hours; and
FIG. 18E is material crosslinked for 16 hours using EDC/NHS
chemistry.
DETAILED DESCRIPTION
[0034] The present invention relates generally to hemostatic
compositions and methods for promoting hemostasis. Preferably, the
present invention relates to flowable, hemostatic compositions that
are collagen-based, and provides for the precise, localized
placement of the hemostatic composition (or device) in an actively
bleeding site, such that the device remains localized therein to
establish hemostasis. The invention also relates to hemostatic
compositions and methods for promoting wound healing. In various
embodiments, the hemostatic compositions comprise crosslinkable
collagen molecules suitable for promoting hemostasis or wound
healing. In some embodiments, the compositions optionally include a
biological agent, such as thrombin. In certain embodiments, the
time needed to establish hemostasis is less than about 10 minutes
in an actively bleeding site. In preferred embodiments, the time
needed to establish hemostasis is less than about 2 minutes in an
actively bleeding site. Hemostasis can be established within this
timeframe with or without the use of the additional biological
agents.
[0035] The hemostatic composition of the present invention can
further be used in a variety of applications where known surgical
hemostats and sealants have been used. For example, the present
invention can be used as a surgical hemostat or sealant, a wound
repair adhesive, a soft tissue augmenter and a soft tissue
substitute. In some embodiments, greater surface area is desirable
for establishing rapid hemostasis by creating an optimized platform
for platelet adhesion and clot stabilization. In certain
embodiments, the compositions of the present invention are both
hemostatic and promote wound healing.
[0036] Swelling characteristics of the compositions of the present
invention are dependent not only on the material forming the
composition (collagen), but also on the porosity of the composition
and the extent of crosslinking. Thus, swelling characteristics of
the compositions can be controlled by controlling the porosity of
the composition and the extent of crosslinking. In preferred
embodiments, swelling of the composition is optimized to promote
hemostasis, yet prevent the composition from impinging on the
surrounding tissues.
[0037] The porosity of the composition can be manipulated by the
temperature and/or the rate of freezing during the drying process.
Porosity can also be manipulated according to the crosslinking
ratio used in the crosslinking step during fabrication.
Definitions
[0038] The definitions used in this application are for
illustrative purposes and do not limit the scope of the
invention.
[0039] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e. to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0040] "About" as used herein when referring to a measurable value
such as an amount, a temporal duration, and the like, is meant to
encompass variations of .+-.20%, .+-.10%, .+-.5%, .+-.1%, or
.+-.0.1% from the specified value, as such variations are
appropriate to perform the disclosed methods.
[0041] "Equivalent," as used herein, may refer to a mass ratio. For
example, if using one gram of collagen, then one equivalent would
be one gram of another material, such as a crosslinking agent.
[0042] "Collagen", as used herein, refers to a natural polymer
derived from connective tissue. Although collagen can take many
forms: partially denatured and sometimes partially fragmented;
monomeric with a native triple helical conformation as in
procollagen; polymerized into a five-mer aggregate as in
microfibrillar collagen; or polymerized into higher-ordered
cable-like fibrils as in fibrillar collagen, a "collagen molecule"
may be taken to describe any of these entities or molecular forms
of collagen as described herein throughout.
[0043] Fiber-like structures that are not greater than 30 .mu.m in
diameter are known as "fibers". "Ropes" are bundles of fibers.
"Ribbons" are described as structures that are smooth and flat with
a width that is greater than a fiber width. Structures that are not
fibers, ropes or ribbons that are flat are called "sheets".
[0044] "Glutaraldehyde" (GTA), as used herein, is a compound
containing two equally reactive aldehyde groups that can each react
with an amine to chemically crosslink collagen.
[0045] "N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide
hydrochloride" (EDC) and "N-hydroxysuccinimde" (NHS), as used
herein, refers to two compounds that, when used in combination,
react synergistically to chemically crosslink a carboxylic acid
group and an amine group in collagen.
[0046] "Sodium hydroxide" (NaOH), as used herein, is a base that,
when diluted, can be used to help increase the pH of a
solution.
[0047] "Hydrochloric acid" (HCl), as used herein, is an acid that
can be diluted and used to help lower the pH of a solution.
[0048] "Percent (%) solids", as used herein, is expressed as mg
material per mL. For example, 1% solids is approximately 10
mg/mL.
[0049] "Crosslinking", as used herein, refers to the joining of at
least two molecules (such as, for example, collagen), to each other
by at least one physical or chemical means, or combinations
thereof.
[0050] "Isolated" means altered or removed from the natural state.
For example, a peptide naturally present in a living animal is not
"isolated," but the same peptide partially or completely separated
from the coexisting materials of its natural state is "isolated."
An isolated protein can exist in substantially purified form, or
can exist in a non-native environment.
[0051] "Naturally occurring" as used herein describes a composition
that can be found in nature as distinct from being artificially
produced. For example, collagen present in an organism, which can
be isolated from a source in nature and which has not been
intentionally modified by a person in the laboratory, is naturally
occurring.
[0052] The terms "diminish" and "diminution," as used herein, means
to reduce, suppress, inhibit or block an activity or function by at
least about 10% relative to a comparator value. Preferably, the
activity is suppressed, inhibited or blocked by 50% compared to a
comparator value, more preferably by 75%, and even more preferably
by 95%.
[0053] The terms "effective amount" and "pharmaceutically effective
amount" refer to a nontoxic but sufficient amount of an agent to
provide the desired biological result. The desired biological
result can be reduction and/or alleviation of the signs, symptoms,
or causes of a disease or disorder, the reduction of bleeding,
wound healing or any other desired alteration of a biological
system. An appropriate effective amount in any individual case may
be determined by one of ordinary skill in the art using routine
experimentation.
[0054] An "individual", "patient" or "subject" as used herein,
includes a member of any animal species. Such animal species
include, but are not limited to, birds, humans and other primates,
and other mammals including commercially relevant mammals such as
cattle, pigs, horses, sheep, cats, and dogs.
[0055] As used herein, the terms "peptide," "polypeptide," and
"protein" are used interchangeably, and refer to a compound
comprised of amino acid residues covalently linked by peptide
bonds. A protein or peptide must contain at least two amino acids,
and no limitation is placed on the maximum number of amino acids
that can comprise a protein's or peptide's sequence. Polypeptides
include any peptide or protein comprising two or more amino acids
joined to each other by peptide bonds. As used herein, the term
refers to both short chains, which also commonly are referred to in
the art as peptides, oligopeptides and oligomers, for example, and
to longer chains, which generally are referred to in the art as
proteins, of which there are many types. "Polypeptides" include,
for example, biologically active fragments, substantially
homologous polypeptides, oligopeptides, homodimers, heterodimers,
variants of polypeptides, modified polypeptides, derivatives,
analogs, fusion proteins, among others. The polypeptides include
natural peptides, recombinant peptides, synthetic peptides, or a
combination thereof.
[0056] The term "treatment" as used within the context of the
present invention is meant to include therapeutic treatment as well
as prophylactic, or suppressive measures for the disease or
disorder, the reduction of bleeding, increased rate of wound
healing or any other desired alteration of a biological system.
Thus, for example, the term treatment includes the administration
of a composition prior to or following the onset of bleeding,
thereby establishing hemostasis.
[0057] As used herein, an "instructional material" includes a
publication, a recording, a diagram, or any other medium of
expression which can be used to communicate the usefulness of a
compound, composition, formulation or delivery system of the
invention in the kit for affecting the conditions recited herein.
For example, the instructional material can describe one or more
methods of reducing bleeding at a targeted treatment site. The
instructional material of the kit of the invention can, for
example, be affixed to a container which contains the identified
compound, composition, formulation, or delivery system of the
invention or be shipped together with a container which contains
the identified compound, composition, formulation, or delivery
system. Alternatively, the instructional material can be shipped
separately from the container with the intention that the
instructional material and the compound be used cooperatively by
the recipient.
[0058] Ranges: throughout this disclosure, various aspects of the
invention can be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2,
2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of
the range.
Collagen-Based Hemostatic Compositions
[0059] The hemostatic compositions of the present invention are
formed primarily of collagen crosslinked with a crosslinking agent,
such as glutaraldehyde. In some embodiments, EDC and NHS, or other
carbodiimides may be used to crosslink collagen. In yet other
embodiments, transglutaminase, genipin or an avidin-biotin
interaction may be used, alone or in combination with other
crosslinkers, to crosslink collagen molecules in the compositions.
The compositions may optionally include a biological agent, such as
thrombin. The compositions may also optionally include additional
polymers, such as polyethylene glycol. In a preferred embodiment,
the composition of the present invention is comprised predominantly
of collagen due to the superior hemostatic properties of collagen
versus other materials such as gelatin.
Crosslinkable Collagen
[0060] Collagen, preferably hypoallergenic collagen, is present in
the composition in an amount sufficient to provide hemostatic
activity, as well as to thicken the composition and augment its
cohesive properties. In addition to thickening the composition, the
collagen acts as a macromolecular lattice or scaffold. This feature
gives more strength and durability to the resulting clot. The
collagen may be atelopeptide collagen or telopeptide collagen
(e.g., native collagen). For example, the collagen used as a
starting material may be derived from collagen collected from any
number of mammalian sources, such as bovine, porcine and human. In
another example, the collagen may come from any source, including
corium collagen, tendon collagen, and collagen flour. It should be
appreciated that the present invention is not limited to any
particular type and/or source of collagen.
[0061] The collagen molecule preferably comprises at least one
crosslinkable moiety that is able to form a bond, directly or
indirectly, with another crosslinkable moiety on another collagen
molecule. Any crosslinkable moieties known in the art may be used.
By way of non-limiting examples, the collagen molecules can be
crosslinked by covalent interactions, by non-covalent interactions,
by thermally reversible interactions, by ionic interactions, or by
combinations thereof. These moieties can be crosslinked by
physical, chemical, thermal, or photointiation (e.g., visible, UV)
means, or by any combination thereof. The initial amount of
collagen suitable for the crosslinking steps of the present
invention may be equal to or less than about 5% solids. In other
embodiments, the amount of collagen is between about 0.1-10%
solids. In still other embodiments, the amount of collagen is
between about 0.3-5% solids. In one preferred embodiment, the
amount of collagen is about 1% solids. In another preferred
embodiment, the amount of collagen is about 0.1-5% solids.
[0062] One form of collagen which is employed may be described as
at least "near native" in its structural characteristics. In
various embodiments, the collagen may be characterized as resulting
in insoluble fibers at a pH above 5. In some embodiments, the
collagen comprises fibers having diameters in the range of from
about 10 to about 500 nm and there will be substantially little, if
any, change in the helical structure. In certain embodiments, the
majority of the fibers have diameters in the range of from about 20
to about 100 nm. In addition, the collagen must be able to enhance
gelation in a surgical sealant composition. In preferred
embodiments, the starting collagen material is microfibrillar type
I collagen. In other embodiments, the starting collagen material is
fibrillar collagen. Other form of collagen which are employed may
include microfibrillar collagen mixed with at least partially
denatured collagen or gelatin. Although collagen can take many
forms: denatured and sometimes partially fragmented; monomeric with
a native triple helical conformation as in procollagen; polymerized
into a five-mer aggregate as in microfibrillar collagen; or
polymerized into higher-ordered cable-like fibrils as in fibrillar
collagen, a "collagen molecule" may be taken to describe any of
these entities or molecular forms of collagen as described
hereinthrougout.
[0063] As contemplated herein, microfibrillar collagen may be used
and may further provide several advantages in selected
applications. For example, microfibrillar collagen has a strong
platelet activating activity owing to its ability, via the presence
of glycine-proline-hydroxyproline repeats and integrin binding
sites in its triple helical domain, to ligate and activate platelet
GPVI and .alpha.2.beta.1 integrin receptors. Second, microfibrillar
collagen assembles into collagen fibrils which provide a rigid
substrate and mesh-like network to support platelet adhesion and
clot stabilization. Third, during clot dissolution and wound
healing, microfibrillar and fibrillar collagen bind cells and
growth and differentiation factors, thereby serving as an ideal
substrate for tissue regeneration.
[0064] In another embodiment, collagen in solution is used in the
generation of collagen for the hemostatic composition. Collagen in
solution (CS) consists of triple helical tropocollagen which is
soluble at a low pH (such as around 2). Increasing the pH allows
the collagen fibers to interact through hydrogen bonding, thereby
forming a microfibrillar structure. As a result of adjusting the pH
(using NaOH and/or HCl) to approximately 6.5-8 and optionally
adding calcium ions (or similar charged particles or ions) during
precipitation, the microfibrils begin to associate and form a
staggered fibrillar structure which is substantially insoluble in
water, as depicted in FIG. 1 (adapted from Sweeney, et al, J. Biol.
Chem. 2008, 30, 21187-21197). This is due to a combination of
electrostatic interactions and increasing molecular weight. The
increase in pH upon precipitation causes the carboxylic acid groups
to be deprotonated, which gives an overall neutral charge to
fibrils. The association of the fibers into larger fibrillar
structures that are tightly bound, and have a net charge close to
neutral, increases the repulsion of water thereby helping to
decrease the solubility of collagen fibrils.
[0065] The amount of the collagen in the hemostatic composition can
be varied to provide hemostats of differing viscosities and
strengths, depending on the particular application. In some
embodiments, the collagen is a flowable composition dispersed in
saline to provide a final concentration in the composition
(reconstituted) of less than or equal to about 200 mg/mL. In other
embodiments, the reconstituted concentration is between about
50-250 mg/mL. In still other embodiments, the reconstituted
concentration is between about 100-200 mg/mL. It should be
appreciated that any concentration of collagen may be used,
provided the collagen continues to provide hemostatic activity, and
remains sufficiently flowable so as to be administered via a
syringe, preferably having an opening of at least 1.6 mm Any type
of syringe suitable for carrying 1 cc to 20 cc of material can be
used, such as 5 to 6 cc, and 10 to 12 cc of material.
Crosslinking Agents
[0066] After precipitating collagen and diluting to the desired
concentration, a second processing step is carried out to crosslink
the collagen. Crosslinking, such as with glutaraldehyde (GTA) or
other aldehydes, makes the collagen substantially insoluble by
forming covalent bonds, which are not easily broken, between
collagen fibrils. During this crosslinking step, the GTA forms
covalent bonds between crosslinkable moieties, such as between
amine groups, and possibly some carboxylic acid groups, in the
amino acids in collagen. This forms a matrix of fibrillar collagen,
by formation of covalent bonds that are more difficult to break
than weaker hydrogen bonds, thereby resulting in a substantially
insoluble crosslinked material. Other methods for crosslinking
collagen with glutaraldehyde are described in U.S. Pat. Nos.
4,582,640 and 4,642,117, the disclosures of which are incorporated
by reference herein.
[0067] In some embodiments, a transglutaminase is used to crosslink
collagen molecules. Transglutaminases are known to catalyze the
formation of covalent bonds between a free amine group (e.g.,
protein-bound lysine) and the gamma-carboxamide group of
protein-glutamine. Bonds formed by transglutaminase are highly
resistant to proteolytic degradation. Non-limiting examples of
transglutaminases useful in the compositions and methods of the
invention include Factor XIII and Streptomyces mobaraensis
transglutaminase (e.g., Activa TG.TM.). In other embodiments,
genipin or an avidin-biotin interaction may be used, alone or in
combination with other crosslinkers, to crosslink collagen
molecules in the compositions and methods of the invention. In
certain embodiment, lysyl oxidase (LO) may be employed. LO is a
copper-dependent amine enzyme (oxidase) that crosslinks
extracellular collagen by catalyzing the formation of covalent
bonds between the aldehydes from lysyl and hydroxylysyl side
chains. In this manner, cells could be transfected with LO to
crosslink collagen.
[0068] In additional embodiments, carbodiimides such as
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) may be used to catalyze a
crosslinking reaction between collagen molecules. EDC/NHS is known
to form chemical bonds between a free amine group and a carboxylic
acid group. EDC and NHS facilitate the reaction between the two
functional groups but do not remain in the final crosslinked
material; as a result EDC/NHS is referred to as a "zero length"
crosslinker. This reduces the risk of adverse reactions because the
resultant material is composed only of collagen. The highly water
soluble compounds EDC and NHS are removed during washing steps and
do not remain in the crosslinked collagen material. For example,
crosslinking at a concentration of about 0.4-0.5% collagen can be
achieved with EDC and NHS at a ratio between 850 mg EDC (4.5 mmol)
and 210 mg NHS (1.8 mmol) to 3.7 g EDC (18 mmol) and 840 mg NHS
(7.2 mmol).
[0069] Acyl azide, and diimidoesters (such as
dithiobispropionimidate (DTBP), dimethyl suberimidate (DMS) and
3,3' dithiobispropionimidate (DTBP) are other chemical crosslinking
agents for collagen. Alternatively, dry heat (DHT), UV irradiation
and photochemical crosslinking including photo initiators may be
used separately or in combination with other crosslinkers described
herein. For example, photo initiators may include Rose Bengal or
riboflavin.
[0070] In some embodiments, crosslinking of collagen may be carried
out before use of the formulation for hemostasis; while in other
embodiments, crosslinking may be carried out at the time of
application while the formulation is being used for hemostasis. The
above examples are non-limiting. It should be understood that
various other materials used to crosslink collagen may be used.
Material Fabrication
[0071] As contemplated herein, the collagen-based hemostatic
compositions of the present invention may be manufactured according
to the following steps, and as outlined in the flowcharts of FIGS.
2A-2C. FIG. 2A provides a general method for fabricating a
hemostatic composition. FIG. 2B provides an exemplary method of
fabricating a hemostatic composition using glutaraldehyde as a
crosslinker. FIG. 2C provides an exemplary method of fabricating a
hemostatic composition using EDC/NHS as a crosslinker.
[0072] First, collagen in solution is precipitated using a buffer
then centrifuged to obtain a collagen pellet that can be diluted
with USP water to a desired concentration. Next, the collagen is
crosslinked to create a substantially insoluble material that is
capable of achieving hemostasis. The concentration of collagen used
during the crosslinking step is termed "percent solids" and is
based on the mass of collagen per volume of liquid, for example 10
mg/mL collagen would be 1% solids. Preferably, the process utilizes
a concentration of about 1% solids during crosslinking.
[0073] The ratio of crosslinking agent, such as glutaraldehyde, to
collagen may vary. Prior to the addition of GTA, the pH is adjusted
to between about 6-11, and preferably between about 7-10. In one
preferred embodiment, the pH is adjusted between 8.0-8.2, to create
optimal conditions for the crosslinking reaction. The mass of
glutaraldehyde (GTA) used is relative to the mass of collagen, and
may be written as a ratio of percent solids:percent crosslinker.
For example, 1% solids to 0.01% GTA would be a crosslinking ratio
of 100:1. As contemplated herein, ratios from 7.5:1 to 500:1 may be
used. As described herein, a ratio of 7.5:1 is referred to as a
high crosslink ratio (having a high crosslink density with many
crosslinked molecules); and a ratio of 500:1 is referred to as a
low crosslink ratio (having a low crosslink density with fewer
crosslinked molecules). The crosslink ratio in turn affects the
physical and chemical properties of the material, including
solubility. Table 1 below outlines the mass of each material
required for crosslinking microfibrillar collagen at various ratios
of solids to GTA. Table 2 outlines the mass of each material
required for crosslinking fibrillar collagen at various ratios of
solids to GTA.
TABLE-US-00001 TABLE 1 Mass of materials required for Crosslinking
of microfibrillar collagen Mass Concen- Mass of Mass of 25%
Crosslinking Cross- tration collagen col- GTA ratio linking of
collagen Percent solution lagen Solution (solids:GTA) ratio (mg/mL)
solids (g) (g) (g) 250:1 0.004 10 1 3000 30 0.48 100:1 0.010 10 1
3000 30 1.2 75:1 0.013 10 1 3000 30 1.6 50:1 0.020 10 1 3000 30 2.4
100:1 0.010 50 5 3000 150 6.0 75:1 0.013 50 5 3000 150 8.0 50:1
0.020 50 5 3000 150 12.0
TABLE-US-00002 TABLE 2 Mass of materials required for Crosslinking
of fibrillar collagen. Mass Concen- Mass of Mass of 25%
Crosslinking Cross- tration collagen col- GTA ratio linking of
collagen Percent solution lagen Solution (solids:GTA) ratio (mg/mL)
solids (g) (g) (g) 40:1 0.025 1 0.1 3000 3 0.30 25:1 0.04 1 0.1
3000 3 0.48 10:1 0.1 1 0.1 3000 3 1.2 7.5:1 0.13 1 0.1 3000 3 1.6
40:1 0.025 10 1 3000 30 3.0 25:1 0.04 10 1 3000 30 4.8 10:1 0.1 10
1 3000 30 12.0
[0074] When using EDC/NHS as the crosslinking agents a buffer
solution of 2-morpholinoethane sulfonic acid (MES) is used to
provide the optimal reaction conditions. This solution provides a
pH between 5-6. The volume of buffer used is relative to the mass
of collagen used, preferably a solution concentration of 0.4-0.5%
collagen is created, and more preferably the collagen solution in
MES is between 0.45-0.47%. The mass of EDC and NHS used in the
crosslinking reaction are also relative to mass of collagen used.
The mass equivalents of EDC relative to collagen are between
0.8-3.5 and the mass equivalents of NHS relative to collagen are
between 0.2-0.8. Preferably 0.8-1.7 equivalents of EDC are used and
0.2-0.42 equivalents of NHS are used. After the material is
crosslinked, it is lyophilized (freeze-dried) to remove water. As
contemplated herein, the various structures and properties of the
resulting formulated collagen materials can be customized or
otherwise selected for. For example, altering freezing parameters
such as temperature, temperature gradients, and time of freezing at
one or more temperatures or temperature gradients allows for the
selection and/or control of the final product properties. A
freezing time of 2 to 6 hours at various freezing rates (range of
instantaneous (e.g., liquid nitrogen) to -0.1.degree. C./min) with
a primary drying cycle at temperatures between 0 and 15.degree. C.
ranging from 1 to 24 hours and secondary drying cycle at
temperatures between 20 and 40.degree. C. ranging from 2 to 10
hours.
[0075] The dried material resembles a scaffold that is porous and
can be ground or cut into smaller pieces of a desired size (such as
approximately 2.5.times.2.5 mm cubes or smaller) for filling into
an applicator, such as a syringe. The crosslinked collagen material
is white or may have a yellow tint upon reconstitution in
physiologic fluid. As demonstrated herein, there are four primary
material structures that can affect the reconstitution and handling
properties of the resulting collagen materials of the present
invention. As shown in FIGS. 10A-10C, these structures including
fibers (FIG. 10A), ribbons (FIG. 10B), ropes (FIG. 10C) and sheets
(FIG. 10D). Porosity can also provide a similar effect.
[0076] The collagen pellet can be diluted or reconstituted to
create a flowable composition. In various embodiments, the collagen
is reconstituted in a physiologically acceptable liquid vehicle,
such as an aqueous isotonic vehicle at about a physiologic salt
concentration. Without limitation, the solution can be water, USP
water, saline, calcium chloride or other physiologically acceptable
fluid. The collagen and diluting solution can be mixed using any
methods as would be understood by those skilled in the art, until
the solution is substantially homogeneous. For example, the
collagen can be mixed within the applicator. The final
concentration of reconstituted collagen can vary, and may generally
fall within the range of between about 50-250 mg/mL, and preferably
between about 100-200 mg/mL.
Optional Biological Agents
[0077] A biological agent can optionally be incorporated into the
compositions of the present invention. In some embodiments, the
biological agent is mixed into a solution or suspension comprising
the crosslinkable collagen. In some embodiments, the biological
agent is physically incorporated into the crosslinked collagen
composition just prior to application to the patient or subject. In
other embodiments, the biological agent is incorporated while
reconstituting the lyophilized and cut collagen particles, or even
prior to lyophilization. In still further embodiments, the
biological agent is incorporated in the form of a microsphere.
[0078] Biological agents may be any of several classes of compound.
For example, where the biological agents are proteins, peptides, or
polypeptides, they may be derived from natural materials, or be
materials produced by recombinant DNA technology, or mutants of
natural proteins, peptides, or polypeptides, or produced by
chemical modification of proteins, peptides, or polypeptides. It
should be appreciated that the classes of biological agents listed
herein, and the particular exemplars of each class, are to be
considered as exemplary, rather than limiting. Biological agents
may, for example, be members of the natural coagulation pathway
("coagulation factors"). Such proteins include, by way of
non-limiting examples, tissue factors, factors VII, VIII, IX, and
XIII, fibrin, and fibrinogen.
[0079] A biological agent may also be a protein or other compound
that activates or catalyzes the natural pathways of clotting
("coagulation activators"). These include, for example, thrombin,
thromboplastin, calcium (e.g. calcium glucuronate), bismuth
compounds (e.g. bismuth subgallate), desmopressin and analogs,
denatured collagen (gelatin), chitosan and fibronectin. Vitamin K
may contribute to activation of coagulation. In preferred
embodiments, the addition of exogenous coagulation activators is
not necessary.
[0080] A biological agent may act by activating, aggregating or
stimulating platelets ("platelet activators"), including, for
example, cycloheximide, N-monomethyl L-arginine, atrial naturetic
factor (ANF), small nucleotides (including cAMP, cGMP, and ADP),
prostaglandins, thromboxanes and analogs thereof, platelet
activating factor, phorbols and phorbol esters, ethamsylate, and
hemoglobin. Nonabsorbable powders such as talc, and denatured or
surface-absorbed proteins can also activate platelets.
[0081] A biological agent may act by local vasoconstriction
("vasoconstrictors"), such as, by way of non-limiting examples,
epinephrine (adrenaline), adrenochrome, tetrahydrozoline,
antihistamines (including antazoline), oxymetazoline, vasopressin
and analogs thereof, and cocaine.
[0082] A biological agent may act by preventing destruction or
inactivation of clotting reactions ("fibrinolysis inhibitors"),
including, by way of non-limiting examples, eosinophil major basic
protein, aminocaproic acid, tranexamic acid, aprotinin
(Trasylol.TM.), plasminogen activator inhibitor, plasmin inhibitor,
alpha-2-macroglobulin, and adrenoreceptor blockers.
[0083] Thrombin acts as a catalyst for fibrinogen to provide
fibrin, an insoluble polymer. In some embodiments, thrombin is
present in the composition in an amount sufficient to catalyze
polymerization of fibrinogen present in a patient's plasma.
Thrombin also activates FXIII, a plasma protein that catalyzes
covalent crosslinks in fibrin, rendering the resultant clot
insoluble. In certain embodiments, thrombin may be present in the
composition in a concentration of from about 0.01 to about 1000 or
greater International Units (IU)/mL of activity, and more
preferably about 100 to about 1000 IU/mL. In yet other embodiments,
thrombin may be present in the composition in a concentration of
from about 500 to about 1000 IU/mL or greater of activity. In still
other embodiments, thrombin may be present in the composition in a
concentration of from about 50 to about 500 IU/mL.
[0084] The fibrinogen, thrombin, FXIII or other natural protein
used in the composition may be substituted by other naturally
occurring or synthetic compounds or compositions which fulfill the
same functions, such as a reptilase coagulation catalyzed, for
example, ancrod, in place of thrombin.
[0085] In some embodiments, the hemostatic composition of the
present invention will additionally comprise an effective amount of
an antifibrinolytic agent to enhance the integrity of the clot as
the healing process occurs. A number of antifibrinolytic agents are
well known and include aprotinin, C1-esterase inhibitor and
.epsilon.-amino-n-caproic acid (EACA), for example. EACA is
effective at a concentration of from about 5 mg/ml to about 40
mg/ml of the final adhesive composition, more usually from about 20
to about 30 mg/ml. EACA is commercially available as a solution
having a concentration of about 250 mg/ml. Conveniently, the
commercial solution is diluted with distilled water to provide a
solution of the desired concentration.
[0086] Other biological factors of interest include EGF,
TGF-.alpha., TGF-.beta., TGF-I and TGF-II, FGF, PDGF, IFN-.alpha.,
IFN-.beta., IL-2, IL-3, IL-6, hematopoietic factor,
immunoglobulins, insulin, corticosteroids and hormones.
[0087] In some embodiments, the composition contains at least one
antibiotic. The therapeutic dose levels of a wide variety of
antibiotics for use in drug release systems are well known. See for
example, Collagen, 1988, Vol. III, Biotechnology; Nimni, (Ed.), CRC
Press, Inc., pp. 209-221 and Biomaterials, 1980, Winter et al.,
(Eds.), John Wiley & Sons, New York, pp. 669-676.
Anti-microbial agents are particularly useful for compositions
applied to exposed wound repair sites. In some embodiments,
anti-microbial agents such as silver are useful.
[0088] A biological agent may also include non-protein polymers
that act to thicken or gel, by interaction with proteins, by
tamponnade, or by other mechanisms. Examples include oxidized
cellulose, "Vicryl" and other polyhydroxyacids, chitosan, alginate,
polyacrylic acids, pentosan polysulfate, carrageenan, and
polyorthoesters (e.g., Alzamer).
[0089] A biological agent may be a material that forms a barrier to
leakage by mechanical means not directly related to the natural
clotting mechanisms, such as a "barrier former". Examples of such
agents include oxidized cellulose, ionically or hydrogen-bond
crosslinked natural and synthetic polymers including chitin,
chitosan, alginate, pectin, carboxymethylcellulose, and poloxamers,
such as Pluronic surfactants.
Kits
[0090] The invention also includes a kit comprising a hemostatic,
collagen-based composition as described herein, and an
instructional material which describes, for example, applying the
hemostatic composition of the present invention, to the tissue of a
subject. The kit may optionally include as separate components a
collagen reconstituting solution, a biological agent and/or an
applicator for administering the hemostatic composition. The
applicator may include a rigid tip for accurately delivering the
hemostatic composition to the localized target site. The
instruction material may further describe the admixing, handling
and administration techniques of any such optional components. In
preferred embodiments, the kit includes a delivery device having an
orifice or opening with a diameter of at least 1.6 mm through which
the composition is capable of flowing.
EXPERIMENTAL EXAMPLES
[0091] The invention is now described with reference to the
following Examples. These Examples are provided for the purpose of
illustration only and the invention should in no way be construed
as being limited to these Examples, but rather should be construed
to encompass any and all variations which become evident as a
result of the teaching provided herein.
[0092] Without further description, it is believed that one of
ordinary skill in the art can, using the preceding description and
the following illustrative examples, make and utilize the present
invention and practice the claimed methods. The following working
examples therefore, specifically point out the preferred
embodiments of the present invention, and are not to be construed
as limiting in any way the remainder of the disclosure.
Example 1: Initial Fabrication Studies
[0093] Multiple formulations for the hemostatic compositions of the
present invention were fabricated from collagen starting material.
Table 3 below provides a qualitative summary of these formulations
and the hemostasis testing results. The primary differentiator
between materials relates to characteristics pertaining to the
ability to physically handle the material, such as the ability to
provide precise, localized placement in an actively bleeding
site.
TABLE-US-00003 TABLE 3 Does Efficacy in Stays at not hemostasis
application stick to (w/o Formulation site gauze thrombin)
Non-crosslinked microfibrillar collagen No No Poor suspensions
(20-60 mg/mL) Non-crosslinked gelatin suspensions (20-60 mg/mL) No
No Poor Microfibrillar collagen (20 mg/mL) with Yes No Mild
enzymatic crosslinker (Transglutaminase) Microfibrillar collagen
(20 mg/mL) with No No Mild polymerizing agent (alginate)
Glutaraldehyde crosslinked microfibrillar Yes Yes Moderate collagen
(90 mg/mL) Glutaraldehyde crosslinked gelatin (180 mg/mL) Yes Yes
Moderate Crosslinked fibrillar collagen Yes Yes Moderate
[0094] Based on these preliminary technical evaluations, it was
determined that glutaraldehyde crosslinked collagen formulations
are preferable. The percent glutaraldehyde crosslinking affects
absorption capacity, swelling properties, and in-vivo material
resorption.
[0095] A variety of drying methods can be used, such as dry heat,
vacuum, forced air, lyophilization, microparticle formation
(solvent precipitation), and spray drying, for example. The final
material properties may be dependent on the method of drying,
particularly the exposure time and temperature. For example,
swelling characteristics of the hemostatic composition are
dependent not only on the material forming the composition
(collagen), but also on the porosity of the composition. Thus,
swelling characteristics of the resulting hemostatic composition
can be controlled by controlling the porosity of the
composition.
[0096] Particle formation may be a post-drying process step, or may
be incorporated into the drying step, such as for microparticle
precipitation or spray drying. Particle size and distribution may
affect the absorption capacity and homogeneity of the reconstituted
material. As contemplated herein, the hemostatic compositions of
the present invention are unique from existing devices in their
swelling profiles.
Example 2: Determination of Crosslinking Ratios and Drying
Conditions
[0097] Crosslinking ratios can be determined based on results such
as: number of free amine groups; amount of residual crosslinking
agent; properties of the dried material; swelling; solubility and
various handling properties, such as ease of reconstituting the
material, ease of extruding the material after crosslinking,
stickiness to tissue (greater stickiness preferred) and stickiness
to gauze (less stickiness preferred).
[0098] Initial concentrations of collagen (ranging from about 5% to
about 1% solids, or about 50 mg/mL to 10 mg/mL collagen,
respectively) for crosslinking were explored in greater detail.
Several crosslinking ratios were also explored in greater detail,
ranging from 500:1 up to 50:1 and particularly ratios of about
500:1, 250:1 and 100:1. The extent of crosslinking for all
materials was determined using a trinitrobenzenesulfonic acid
(TNBS) assay. This assay determined the number of free amine
groups, in moles, remaining after the crosslinking reaction. This
number can be converted into a percent (crosslinking extent) or
ratio of non-crosslinked to crosslinked amine groups. Overall the
results show that as the crosslinking extent increases the number
of free amine groups decreases. At very low crosslinking ratios
(500:1) the sensitivity of the assay becomes limited. The results
of this test are provided in Table 4, below.
TABLE-US-00004 TABLE 4 Absorbance (nm) Free Amine Groups %
Crosslinked Non- 0.8027 1.92 .times. 10.sup.-4 0 crosslinked 500:1
0.8363 1.99 .times. 10.sup.-4 0 250:1 0.7760 1.85 .times. 10.sup.-4
3.32 100:1 0.6960 1.66 .times. 10.sup.-4 13.1
[0099] Further, an N-methyl benzothiazolone hydrazone (MBTH) assay
allows the amount of residual crosslinking agent, glutaraldehyde
(GTA) to be determined. The crosslinked collagen solution was
centrifuged, and a sample of the liquid was collected. The
crosslinked pellet was resuspended in USP water, and washed with a
volume of water equal to that of the water in the initial
crosslinking step. This step was repeated, and a total of 3 samples
(reaction water, water from rinse 1 and water from rinse 2) were
obtained for each crosslinking ratio. With higher crosslinking
ratios (above 100:1), low levels of residual GTA were detected in
all samples. Materials made at lower crosslinking ratios (250:1 and
lower) showed no residual GTA after the second rinse step. The
results of this test are provided in Table 5, below.
TABLE-US-00005 TABLE 5 Absorbance GTA (ppm) 500:1 0.052 0 250:1
0.054 0 100:1 0.089 5.89 .times. 10.sup.-4
After crosslinking and rinsing, the crosslinked collagen pellet was
frozen and lyophilized More specifically, samples were lyophilized
for about 2-3 days using a condenser temperature of about -50 to
about -60.degree. C. under vacuum. Several freezing temperatures
were studied to determine how they affected the material
properties. Crosslinked pellets were frozen in liquid nitrogen
(about -196.degree. C.) or at about -80 or about -28.degree. C. SEM
imaging confirms that freezing temperature affects pore size. As
shown in FIGS. 2A-2C, FIGS. 3A-3D, and FIGS. 4A-4C, material frozen
in liquid nitrogen has the most open, porous structure and material
frozen at the highest temperature, -28.degree. C. has fewer pores
and a more "sheet-like" appearance. The properties of the dried
materials were also studied. Materials frozen in liquid nitrogen,
regardless of crosslinking ratio, have a larger static charge
associated with them as compared to the materials frozen at -80 and
-28.degree. C.
[0100] The crosslinked materials were also tested for percent
swelling, which is an indication of how much additional fluid the
material absorbs when placed in a fluid at physiological
conditions. To test for percent swelling, crosslinked collagen
material was reconstituted to the desired concentration, and then
extruded into dialysis tubing. The tubing was incubated in USP
water or PBS (phosphate buffered saline) at 37.degree. C. for 24
hours. The increase in mass for each sample was recorded and used
to determine the percent swelling, according to the formula:
% Swelling = 100 .times. ( weight after swelling - weight before
swelling ) weight before swelling ##EQU00001##
[0101] Preferably, the material should swell sufficiently to absorb
blood at the surgical site and facilitate clot formation,
preferably between about 0% to about 20% within 10 minutes when
reconstituted, and 300-800% for dried material. Further swelling
beyond a short time period is undesirable as expansion of material
in a surgical site can lead to unwanted impingement of the material
on surrounding tissues. The material should preferably reach an
equilibrium swell within about 24 hours. The results of this study
are provided in Tables 6 and 7 below.
TABLE-US-00006 TABLE 6 percent swelling of crosslinked
microfibrillar collagen materials .+-. standard error (in USP
water) Swelling 1% microfibrillar 1% microfibrillar 1%
microfibrillar time point collagen, 100:1 collagen, 250:1 collagen,
500:1 10 min 6.71 .+-. 1.75 8.49 .+-. 5.62 0.93 .+-. 1.27 24 hour
15.54 .+-. 1.43 14.08 .+-. 6.66 9.34 .+-. 1.82
TABLE-US-00007 TABLE 7 percent swelling of crosslinked fibrillar
collagen materials .+-. standard error (in PBS) Swelling 1%
fibrillar time point collagen, 10:1 10 min 4.42 .+-. 0.43 24 hour
2.83 .+-. 1.11
[0102] For reference, the Floseal.RTM. (Baxter) Instructions For
Use document states that the particles of the Floseal.RTM. Matrix
swell approximately 20% upon contact with blood or other fluids;
and that maximum swell volume is achieved within about 10 minutes.
And the Surgiflo.RTM. (Ethicon) Instructions For Use document
states that Surgiflo.RTM. may swell approximately 19% upon contact
with additional fluid. Other studies conducted on the Floseal.RTM.
and Surgiflo.RTM. competitive products show an average percent
swell of about 32% and about 26%, respectively.
[0103] To evaluate handling properties, the collagen material was
cut into 2.5 cm cubes or ground and the crosslinked collagen was
reconstituted at a desired concentration, such as between about
120-160 mg/mL. As the crosslinking ratio increased (e.g., from
500:1 to 250:1 to 100:1), the amount of force required for mixing
also increased. While the amount of force required for mixing the
various crosslinking ratios was distinct for each sample, the
difference in required force for mixing the lowest crosslinking
ratios of 500:1 and 250:1 was minimal
[0104] Once reconstituted, the amount of force necessary to extrude
the material from a syringe was considered. Syringes contained 4 cc
of material reconstituted at 160 mg/mL. The plungers were displaced
0.5'' at a rate of 2''/min. The collagen material crosslinked at
100:1 required the greatest extrusion force, while the collagen
materials crosslinked at 250:1 and 500:1 required a comparable
amount of extrusion force, and significantly less force than the
material crosslinked at 100:1. The results of this study are
provided in Table 8, below.
TABLE-US-00008 TABLE 8 1% collagen, 1% collagen, 1% collagen, 100:1
250:1 500:1 Maximum load, n = 1 7.53 5.06 4.91 (lbf) Maximum load,
n = 2 7.23 5.79 5.63 (lbf) Average maximum 7.38 5.43 5.27 load
(lbf)
[0105] The stickiness of the collagen material to tissue was also
evaluated. Preferrably, it is desirable that the material remain on
the wound (or targeted tissue site) to most effectively stop
bleeding. To test this, crosslinked collagen materials were
extruded onto a chicken liver ex-vivo and a slow, steady flow of 5
cc of blood was passed over the material. In a second test to
determine stickiness to tissue, an incision was made in the liver
and blood was slowly dispensed through the "wound site." Material
was added on top of the wound site as the blood was being expelled,
preferably, the collagen material remains in place, absorbs blood
and remains in a generally cohesive mass. All compositions (e.g.,
500:1, 250:1, 100:1 of 1% crosslinked microfibrillar collagen and
25:1 and 40:1 of 0.1% and 1% crosslinked fibrillar collagen)
performed similarly, showing comparable stickiness to tissue.
[0106] The ability of the collagen material to not stick to gauze
was also evaluated. To test this, pressure was manually applied to
the collagen material using gauze to help pack it into the wound
(or targeted tissue site) and stop bleeding. Pressure was released
and the gauze carefully removed so as not to disrupt the clot. All
materials show similar properties and do not stick to gauze.
[0107] Based on these experiments, it was determined that the 1%
microfibrillar collagen material having about a 250:1 crosslinking
ratio and 0.1% and 1% crosslinked fibrillar collagen having
crosslinking ratios of 25:1 and 40:1 were preferable. The factors
that influenced this determination were that 500:1 and 250:1 and
crosslinked fibrillar collagen were comparable in all
handling/extrusion/bleeding tests and assays. Both materials
crosslinked at 500:1 and 250:1 as well as some crosslinked
fibrillar collagen formulations provided an insoluble crosslinked
material, which was also desirable. However, the 250:1 ratio
demonstrated an appreciably higher percent crosslinking and it also
had no detectable residual GTA after the second rinse.
Example 3: Solubility Testing of Crosslinked Formulations of
Microfibrillar Collagen
[0108] The solubility of various crosslinked formulations was
measured by placing dried, crosslinked and non-crosslinked
materials in PBS, incubating under physiologic conditions and
measuring for the amount of protein dissolved into the surrounding
medium. The following materials were tested: Knox gelatin powder;
non-crosslinked microfibrillar collagen (processed as 1% solids);
microfibrillar collagen crosslinked at a ratio of 100:1 (processed
as 1% solids); microfibrillar collagen crosslinked at a ratio of
250:1 (processed as 1% solids); and microfibrillar collagen
crosslinked at a ratio of 500:1 (processed as 1% solids).
[0109] One hundred (100) mg of each material was placed in a
separate container with 10 mL PBS for 15 minutes at 37.degree. C.
No agitation or vortexing was applied. The tubes were then
centrifuged and the supernatant was removed and filtered with a
0.22 .mu.m filter to remove any non-solubilized material. The
concentration of protein in the collected filtrate was measured
using a bicinchoninic acid (BCA) assay kit. The BCA assay is a
colorimetric assay that uses copper ions and bicinchoninic acid to
react with the peptide bonds in proteins, producing a distinct
color change dependent on the amount of protein present. Protein
concentrations in this study were compared to a standard curve of
bovine albumin protein in PBS. The results of this experiment are
presented in Table 9.
TABLE-US-00009 TABLE 9 Measured concentration of soluble protein
for various crosslinking ratios of collagen and gelatin. 1% solids
1% solids 1% solids microfibrillar microfibrillar microfibrillar
Non- collagen, collagen, collagen, crosslinked crosslinked at
crosslinked at crosslinked at 1% solids Gelatin a ratio of a ratio
of a ratio of microfibrillar powder 500:1 250:1 100:1 collagen
Protein 596.5 .+-. 8.7 0 0 0 31.6 .+-. 3.9 concentration .+-. SD
(.mu.g/mL)
[0110] A second experiment was performed to investigate the
solubility of various test materials from a reconstituted form, as
the reconstituted form is the preferred method of administering the
hemostatic composition to the targeted tissue site. The following
materials were tested: microfibrillar collagen crosslinked at a
ratio of 100:1 (processed as 1% solids), reconstituted at 160
mg/mL; microfibrillar collagen crosslinked at a ratio of 250:1
(processed as 1% solids), reconstituted at 160 mg/mL;
microfibrillar collagen crosslinked at a ratio of 500:1 (processed
as 1% solids), reconstituted at 160 mg/mL; Floseal.RTM. (Baxter),
reconstituted according to manufacturer's instructions using 40
.mu.mol CaCl.sub.2 solution only (no thrombin); and Surgiflo.RTM.
(Ethicon), reconstituted according to manufacturer's instructions
using saline only (no thrombin).
[0111] To approximate adding 100 mg of dried material, 0.625 g of
each reconstituted material was added to separate tubes containing
10 mL PBS. All other procedures used were the same as described
herein. The results of this experiment are presented in Table 10
below.
TABLE-US-00010 TABLE 10 Measured concentration of soluble protein
for reconstituted collagen and gelatin materials. 1% solids 1%
solids 1% solids microfibrillar microfibrillar microfibrillar
collagen, collagen, collagen, crosslinked at crosslinked at
crosslinked at a ratio of a ratio of a ratio of 500:1 250:1 100:1
Floseal .RTM. Surgiflo .RTM. Protein 0 0 0 133.0 .+-. 5.3 552.5
.+-. 12.9 concentration .+-. SD (.mu.g/mL)
[0112] The values of measured protein concentration from dry
material, shown in Table 9, demonstrate that the gelatin powder had
a relatively large portion of soluble protein in comparison to the
non-crosslinked microfibrillar collagen. Table 8 demonstrates that
after reconstitution, both Floseal.RTM. and Surgiflo.RTM., which
are gelatin based commercial hemostats, also had a relatively large
portion of protein that was soluble in PBS. The filtrate from all
three crosslinked formulations of the present invention, however,
did not have a measurable change in absorbance, in either the dry
or reconstituted forms. This confirms that the crosslinked
microfibrillar collagen material of the present invention is
substantially insoluble in these physiologic conditions.
Example 4: Solubility Testing of Crosslinked Formulations of
Fibrillar Collagen
[0113] The solubility of various fibrillar collagen crosslinked
formulations was also measured by placing dried, crosslinked and
non-crosslinked materials in PBS, incubating under physiologic
conditions and measuring for the amount of protein dissolved into
the surrounding medium. The following materials were tested: 0.1%
fibrillar collagen crosslinked at a ratio of 7.5:1; 0.1% fibrillar
collagen crosslinked at a ratio of 10:1; 0.1% and 1% fibrillar
collagen crosslinked at a ratio of 25:1; 0.1% and 1% fibrillar
collagen crosslinked at a ratio of 40:1.
[0114] One hundred (100) mg of each material was placed in a
separate container with 10 mL PBS for 15 minutes at 37.degree. C.
No agitation or vortexing was applied. The tubes were then
centrifuged and the supernatant was removed and filtered with a
0.22 .mu.m filter to remove any non-solubilized material. The
concentration of protein in the collected filtrate was measured
using a bicinchoninic acid (BCA) assay kit. The BCA assay is a
colorimetric assay that uses copper ions and bicinchoninic acid to
react with the peptide bonds in proteins, producing a distinct
color change dependent on the amount of protein present. Protein
concentrations in this study were compared to a standard curve of
bovine albumin protein in PBS. The results of this experiment are
presented in Table 11.
TABLE-US-00011 TABLE 11 Measured concentration of soluble protein
for various formulations of crosslinked fibrillar collagen. Protein
concentration .+-. SD Formulation (.mu.g/mL) 0.1% fibrillar
collagen, 7.5:1 0 0.1% fibrillar collagen, 10:1 0 0.1% fibrillar
collagen, 25:1 0 to 29.58 .+-. 4.5 1.0% fibrillar collagen, 25:1
4.74 .+-. 8.2 0.1% fibrillar collagen, 40:1 58.58 .+-. 6.6 1.0%
fibrillar collagen, 40:1 0
Example 5: Freezing Temperature Study 1% Collagen Crosslinking
Ratio (100:1)
[0115] As explained previously, a variety of drying methods can be
used, such as dry heat, vacuum, forced air, lyophilization,
microparticle formation (solvent precipitation), and spray drying,
for example. The final material properties may be dependent on the
method of drying, particularly the exposure time and temperature.
For example, swelling characteristics of the hemostatic composition
are dependent not only on the material forming the composition
(collagen), but also on the porosity of the composition and the
amount of crosslinking. Thus, swelling characteristics of the
resulting hemostatic composition can be controlled by controlling
the porosity of the composition. As contemplated herein, the
porosity of the composition can be manipulated by the temperature
and/or the rate of freezing during the drying process. Porosity can
also be manipulated according to the crosslinking ratio used in the
crosslinking steps during fabrication. The total porosity of the
hemostatic composition is preferably greater than about 50% with
interconnected pores. The interconnectivity of the pores
facilitates quick fluid uptake and retention via capillary action.
In preferred embodiments, the porosity is between 70-90%. Macro,
meso and microporosity are also desirable, preferably with the
majority of the pores being micro or meso pores. As defined herein,
macroporosity is defined by pores having pore diameters greater
than about 100 .mu.m, mesoporosity is defined by pores having pore
diameters between about 10 .mu.m to 100 .mu.m, and microporosity is
defined by pores having pore diameters less than about 10
.mu.m.
[0116] As shown in FIGS. 3A-3D, the porosity of the material and
the pore size decreases as the crosslinking ratio increases.
Non-crosslinked material has a very open, interconnected pore
structure with many collagen fibers visible. In materials
crosslinked at 500:1 and 250:1 some fibers can be distinguished,
and many pores are still observed, however there is an increase in
the sheet-like surfaces present. Whereas the material crosslinked
at 100:1 appears more sheet-like.
[0117] Freezing temperatures were also studied to help evaluate the
structural effects (such as porosity) of the resulting lyophilized
collagen materials (or scaffolds). Collagen materials or scaffolds
of 1% collagen crosslinked at 100:1 were frozen in liquid nitrogen
(10 min), at -28.degree. C. (2-3 hours) and at -80.degree. C. (2-3
hours). Samples were then lyophilized for about 2-3 days using a
condenser temperature of about -50 to about -60.degree. C. under
vacuum. It was observed that higher freezing temperatures produced
larger pore sizes. It was also observed that higher freezing
temperatures (e.g., -28.degree. C. and -80.degree. C. compared to a
liquid nitrogen temperature of about -196.degree. C.), produced
less open structure (more sheet-like with less surface area),
whereas the scaffolds frozen in liquid nitrogen had a more open
structure (more surface area), and smaller pores. Further, it was
observed that the freezing temperature affects the amount of static
charge. Particularly, the higher the freezing temperature, the less
static charge.
[0118] In certain embodiments, greater surface area is desirable,
as this additional surface area may create a better platform for
platelet adhesion and clot stabilization, and establish hemostasis
(see Example 6 and FIGS. 10A-10D, FIGS. 11A-11B, FIGS. 12A-12D,
below). In other embodiments aimed at wound healing, less surface
area and larger pores may be desirable. Generally, a surface area
of between about 0.5 to about 30 m.sup.2/g is preferred.
[0119] As shown in FIGS. 4A-4C, FIGS. 5A-5C, and FIGS. 6A-6C, the
scaffolds frozen at -28.degree. C. and -80.degree. C. appear like
"sheets" of material, where some pores are observed. However,
scaffolds frozen in liquid nitrogen have a more open structure,
many small pores, with lots of individual collagen fibers observed.
It should be noted that at higher magnifications, fibers can be
seen for all freezing conditions.
[0120] Freezing temperatures were also studied to help evaluate the
physical effects of the collagen material after reconstitution.
Collagen materials or scaffolds of 1% collagen crosslinked at 100:1
from samples frozen in liquid nitrogen (10 min), at -28.degree. C.
(2-3 hours) and at -80.degree. C. (2-3 hours) were ground using
3.times.3 sec pulses, and reconstituted in 200 .mu.M CaCl.sub.2 at
150 mg/mL of crosslinked material. It was observed that the
collagen material frozen in liquid nitrogen was thick, the material
frozen at -80.degree. C. was slightly watery, and the material
frozen at -28.degree. C. had fluid come out initially, indicating
that the material held less fluid and began to phase separate.
Regarding the handling properties for each sample, all collagen
materials for each sample extruded well, and none of the collagen
materials were too sticky to gloves or gauze. All material samples
absorbed blood.
[0121] As shown in FIGS. 7A-7C, FIGS. 8A-8C and FIGS. 9A-9C, the
surface appearance of the scaffolds frozen at -28.degree. C. and
-80.degree. C. appeared like crumpled paper, with smooth surfaces
and peaks, and few large pores. The scaffolds frozen in liquid
nitrogen appeared to have a smoother and flatter surface, with no
peaks and valleys. At higher magnifications, fibers were observed.
Notably, the collagen materials, regardless of freezing conditions,
were more similar after reconstitution, in that there was less open
space and the materials are more of a cohesive mass. However, it
was observed that the thickness of the collagen material
reconstituted at the same concentration increases with decreasing
freezing temperature. This suggests that the absorption capacity of
a given material is higher when lower freezing temperatures are
used.
[0122] From this, it appears that the rate of drying can help
control pore size and material thickness, resulting in the ability
to optimize and/or select for the properties of the resulting
hemostatic composition related to these features.
Example 6: Freezing Rate for Lyophilizing Material
[0123] As explained in previous examples, the final material
properties may be dependent on the freezing rate of the material,
particularly morphology due to crystal formation during the
lyophilization process. Approximately forty (40) grams of 1%
microfibrillar collagen was crosslinked in glutaraldehyde at a
ratio of 250:1 for 4 hours. The collagen was centrifuged at 10,000
RPM for 8 minutes. The material was rinsed twice with additional
centrifugation between each rinse. The pellet was then spread
evenly in a reservoir, between a 2-3 mm thickness, for
lyophilization. The samples were frozen at -50.degree. C. for 4-5
hours, then subjected to a programmed lyophilization cycle run for
a duration of approximately 30 hours. Samples lyophilized using a
first freezing rate (-1.degree. C./min) had mostly sheets with
slight fraying. Samples lyophilized using a slower freezing rate
(-0.5.degree. C./min) had more ribbon-like materials with moderate
fraying, as shown in the SEM images below. Collagen materials were
ground and reconstituted in saline at 150 mg/mL of crosslinked
material. As listed in Table 12, blood absorption was 27.88% for
the faster freezing rate and 15.28% for the slower freezing rate as
summarized in the table below. The results from this experiment
demonstrate that the rate of freezing rate can affect both the
morphology and the ability of the material to absorb blood. As seen
in FIGS. 11A and 11B, the faster freezing rate resulted in more
sheet-like materials compared to the slower freezing rate which
resulted in ribbons.
TABLE-US-00012 TABLE 12 Blood absorption of collagen materials
lyophilized using two different freezing rates Average Blood
Formulation Description absorption (%) .+-. SD Glutaraldehyde
crosslinked 27.88 microfibrillar collagen. 250:1 @ a freezing rate
of -1.degree. C./min Glutaraldehyde crosslinked 15.28
microfibrillar collagen. 250:1 @ a freezing rate of -0.5.degree.
C./min
Example 7: Blood Absorption of Hemostatic Materials
[0124] The ability of various crosslinked formulations to absorb
blood was investigated by partially submerging (.about.40%) the
reconstituted materials in porcine blood for 2 minutes. The mass of
each material was measured before and after placement in blood and
used to determine % absorption by mass, according to the
formula:
% Blood Absorption = Hydrated mass - initial mass of material
initial mass of material .times. 100 ##EQU00002##
The results from the experiment are summarized in Table 13,
below.
TABLE-US-00013 TABLE 13 Blood absorption of various formulations of
collagen materials. Average Blood absorption (%) .+-. Description
SD Baxter Floseal .RTM.: 38.24 .+-. 1.87 Reconstituted according to
Instructions For Use 1% microfibrillar collagen, 27.88 (n = 1) GTA
crosslinked at a ratio of 250:1 @ a freezing rate of -1.degree.
C./min, non-sterilized 0.1% fibrillar collagen GTA 0.12 (n = 1)
crosslinked at a ratio of 40:1, gamma sterilization
[0125] From these experimental results, it was determined that the
gelatin material had the highest absorption followed by
microfibrillar collagen and then fibrillar collagen. Overall, the
glutaraldehyde crosslinked microfibrillar collagen had higher blood
absorption properties compared to fibrillar crosslinked collagen
materials. This suggests that blood absorption can be affected by
both the crosslinking agent and raw material.
Example 8: Starting Concentration of Material for
Lyophilization
[0126] The starting concentrations of the material undergoing
lyophilization were also studied to evaluate the effects on the
structure of the resulting lyophilized collagen materials (or
scaffolds). The water content of the starting material has a direct
effect on crystal formation during the freezing portion of the
lyophilization cycle. Forty (40) grams of 1% microfibrillar
collagen crosslinked in glutaraldehyde at a ratio of 250:1 for 4
hours (undiluted collagen material) and diluted collagen materials
(1:1; 1:5; 1:10 by volume in USP water for a total of 10 mL for
each sample) were frozen at -50.degree. C. for 4-5 hours and then
were subjected to a programmed lyophilization cycle run for a
duration of approximately 30 hours. As shown in FIGS. 12A-12D, SEM
images reveal that starting materials with higher volumes of water
had larger pore sizes and more open structures. There was a trend
of increasing pore size with more diluted samples as again depicted
in FIGS. 12A-12D.
Example 9: Quantification of Surface Area
[0127] Surface area analysis was performed using the BET (Brunauer,
Emmet, and Teller) theory to calculate the surface area of a sample
of 1% microfibrillar collagen crosslinked at a ratio of 250:1.
Briefly, approximately 4 g of sample was prepared by degassing
overnight to remove impurities. The sample was then cooled with
liquid nitrogen and analyzed by measuring the volume of krypton gas
adsorbed at specific pressures. The amount of adsorbed gas was used
to calculate the total surface area of the material by a
multi-point method. Results show a surface area of approximately
0.7809.+-.0.0040 m.sup.2/g.
[0128] Based on these results, further surface area testing was
performed using the BET theory. The following materials were
tested: 1% microfibrillar collagen crosslinked at a ratio of 250:1;
1% fibrillar collagen crosslinked at a ratio of 25:1; 0.1%
fibrillar collagen crosslinked at a ratio of 40:1; and 1.0%
fibrillar collagen crosslinked at a ratio of 40:1. Briefly,
approximately 0.1-0.5 g of each ground sample was prepared by
degassing overnight to remove impurities. The samples were then
cooled with liquid nitrogen and analyzed by the volume of nitrogen
gas adsorbed as specific pressures. The amount of adsorbed gas was
used to calculate the total surface area of the material by a
multi-point method. Results are shown in Table 14.
[0129] Table 14. Measured surface area of glutaraldehyde
crosslinked collagen formulations.
TABLE-US-00014 Surface area (m.sup.2/g) .+-. Sample Type standard
deviation 1% microfibrillar collagen, crosslinked at a 1.9674 .+-.
0.0418 ratio of 250:1 1% fibrillar collagen, crosslinked at a ratio
0.4180 .+-. 0.0258 of 25:1 0.1% fibrillar collagen, crosslinked at
a 2.1472 .+-. 0.0421 ratio of 40:1 1.0% fibrillar collagen,
crosslinked at a 2.4881 .+-. 0.0478 ratio of 40:1
[0130] Within each formulation type, samples exhibiting a higher
surface area tended to have better handling properties such as ease
of mixing and material consistency. The investigation of freezing
temperatures demonstrates that materials frozen at the lowest
temperatures have a more open structure with many small pores and
fibers. The surface area results suggest that an increased surface
area, such as that visually observed in the freezing temperature
study, may contribute to a material's absorption capacity.
Example 10: Hemostasis Testing
[0131] A porcine bleeding model was used to assess the hemostatic
abilities of various crosslinked formulations. The following
materials were tested: 1% collagen crosslinked at a ratio of 100:1;
5% collagen crosslinked at a ratio of 500:1; Floseal.RTM.; and
Surgiflo.RTM.. Crosslinked collagen materials were prepared by
grinding lyophilized scaffolds and reconstituting with saline to a
concentration of between 120 to 140 mg/mL. Commercial gelatin
products were prepared according to the manufacturer's
instructions. For collagen test materials of the present invention
prepared with thrombin, material was reconstituted using thrombin
with a concentration of 1000 IU/mL. For the competitive product
materials, the manufacturer's instructions were followed. The
concentration of thrombin for Floseal.RTM. was 500 IU/mL. The
concentration of thrombin for Surgiflo.RTM. was 320-480 IU/mL
(based on using 2 mL of 800-1200 IU/mL thrombin per the
manufacturer's instructions). A 6 mm biopsy punch was used to
create a defect approximately 7 mm deep in the kidney, liver, or
spleen. The resulting tissue flap was removed using either scissors
or a scalpel. Test material was applied to the bleeding site and
pressure was held using gauze for 60 seconds. The wound was then
observed for bleeding. If hemostasis had not been achieved,
additional material was applied and/or pressure was held for
another 30 seconds. This was repeated until no bleeding could be
observed. Time from material application to hemostasis was
recorded. The results of the experiment are presented in Table
15.
TABLE-US-00015 TABLE 15 Time to hemostasis for crosslinked collagen
materials and commercial gelatin hemostats in a non-heparinized
bleeding model. Average Time to Hemostasis (seconds) .+-. Sample
Type standard error 1% microfibrillar collagen, 89 .+-. 36
crosslinked at a ratio of 100:1 with 1000 IU/mL thrombin 1%
microfibrillar collagen, 157 .+-. 87 crosslinked at a ratio of
100:1 5% microfibrillar collagen, 78 .+-. 34 crosslinked at a ratio
of 500:1 with 1000 IU/mL thrombin 5% microfibrillar collagen, 52
.+-. 15 crosslinked at a ratio of 500:1 Floseal .RTM. 170 .+-. 70
Surgiflo .RTM. 121 .+-. 45
[0132] These results can also be seen in FIGS. 13A-13B, FIGS.
14A-14C, and FIGS. 15A-15B. For example, FIGS. 13A-13B are SEM
images at a magnification of 500.times. of 1% collagen (FIG. 13A)
and Surgiflo.RTM. (FIG. 13B) after hemostasis had been achieved
through application of the material to a bleeding site. FIGS.
14A-14C are SEM images of 5% collagen (FIG. 14A, 1500.times.),
Floseal.RTM. (FIG. 14B, 1500.times.), and Surgiflo.RTM. (FIG. 14C,
1000.times.) after hemostasis had been achieved through application
of the material to a bleeding site. FIGS. 15A-15B are SEM images at
a magnification of 3000.times. of 1% collagen (FIG. 15A) and 5%
collagen without the addition of thrombin (FIG. 15B) after
hemostasis had been achieved through application of the material to
a bleeding site. In a second set of tests, a porcine bleeding model
was used to assess the hemostatic abilities of various crosslinked
formulations. The following materials crosslinked with
glutaraldehyde were tested: 1% microfibrillar collagen crosslinked
at a ratio of 100:1; 1% microfibrillar collagen crosslinked at a
ratio of 250:1; 0.1% fibrillar collagen crosslinked at a ratio of
10:1; 0.1% fibrillar crosslinked at a ratio of 25:1; Surgiflo.RTM.;
and Floseal.RTM.. Crosslinked collagen materials were prepared by
grinding lyophilized scaffolds and reconstituting with saline to a
concentration of 120 to 140 mg/mL. Commercial products were
prepared according to the manufacturer's instructions. The
concentration of thrombin for Floseal.RTM. was 500 IU/mL. The
concentration of thrombin for Surgiflo.RTM. was 320 to 480 IU/mL
(based on using 2 mL of 800-1200 IU/mL thrombin per the
manufacturer's instructions). A 6 mm biopsy punch was used to
create a defect approximately 7 mm deep in the kidney, liver, or
spleen. The resulting tissue flap was removed using scissors or a
scalpel. Test material was applied to the bleeding site and
pressure was held using gauze for 60 seconds. The wound was then
observed for bleeding. If hemostasis had not been achieved,
additional material was applied and/or pressure was held for
another 30 seconds. This was repeated until no bleeding could be
observed. Time from material application to hemostasis was
recorded. The results of the experiment are presented in Table
16.
TABLE-US-00016 TABLE 16 Time to hemostasis for crosslinked collagen
materials and commercial gelatin hemostats in a non-heparinized
bleeding model. Average Time to Hemostasis (seconds) .+-. Sample
Type standard error 1% microfibrillar collagen, 143 .+-. 37
crosslinked at a ratio of 100:1 1% microfibrillar collagen, 77 .+-.
17 crosslinked at a ratio of 250:1* 0.1% fibrillar collagen,
crosslinked 102 .+-. 15 at a ratio of 10:1 0.1% fibrillar collagen,
crosslinked 127 .+-. 15 at a ratio of 25:1 Surgiflo .RTM. 78 .+-.
18 Floseal .RTM. 60 .+-. 0 *1/4 sites did not achieve hemostasis
within 10 minutes.
[0133] In a third set of tests,_a porcine bleeding model was used
to assess the hemostatic abilities of various crosslinked
formulations. The following materials were tested: 0.1% fibrillar
collagen crosslinked with glutaraldehyde at a ratio of 40:1; 1%
fibrillar collagen crosslinked with glutaraldehyde at a ratio of
25:1; crosslinked fibrillar collagen; and Floseal.RTM.. Crosslinked
collagen materials were prepared by grinding lyophilized scaffolds
and reconstituting with saline to a concentration of 120 to 130
mg/mL. Commercial products were prepared according to the
manufacturer's instructions. The concentration of thrombin for
Floseal.RTM. was 500 IU/mL. The concentration of thrombin for
Surgiflo.RTM. was 320 to 480 IU/mL (based on using 2 mL of 800-1200
IU/mL thrombin per the manufacturer's instructions). A 6 mm biopsy
punch was used to create a defect approximately 7 mm deep in the
kidney, liver, or spleen. The resulting tissue flap was removed
using scissors or a scalpel. Test material was applied to the
bleeding site and pressure was held using gauze for 60 seconds. The
wound was then observed for bleeding. If hemostasis had not been
achieved, additional material was applied and/or pressure was held
for another 30 seconds. This was repeated until no bleeding could
be observed. Time from material application to hemostasis was
recorded. The results of the experiment are presented in Table
17.
[0134] The same model was also performed in heparinized animals.
The following materials were tested: crosslinked fibrillar
collagen; 1% fibrillar collagen crosslinked with glutaraldehyde at
a ratio of 25:1; and Floseal.RTM.. The concentration of thrombin
for Floseal.RTM. was 500 IU/mL. The results are presented in Table
18.
TABLE-US-00017 TABLE 17 Time to hemostasis for crosslinked collagen
materials and commercial gelatin hemostats in a non-heparinized
bleeding model. Average Time to Hemostasis (seconds) .+-. Sample
Type standard error 0.1% fibrillar collagen, crosslinked 194 .+-.
33 at a ratio of 40:1 1% fibrillar collagen, crosslinked at 211
.+-. 44 a ratio of 25:1* 1% microfibrillar collagen, 122 .+-. 24
crosslinked at a ratio of 250:1 Floseal .RTM. 126 .+-. 27 * 1/10
sites did not achieve hemostasis within 10 minutes.
TABLE-US-00018 TABLE 18 Time to hemostasis for crosslinked collagen
materials and commercial gelatin hemostats in a heparinized
bleeding model. Average Time to Hemostasis (seconds) .+-. Sample
Type standard error 1% fibrillar collagen, crosslinked at 166 .+-.
70 a ratio of 25:1* Floseal .RTM.* 143 .+-. 83 *1/5 sites did not
achieve hemostasis within 10 minutes.
Representative histology images of each material following
application to a bleeding liver defect are shown in FIGS. 16A-16D.
The fibrillar collagen has a disorganized appearance with red blood
cells trapped between pieces of collagen (FIGS. 16A and 16B). The
microfibrillar collagen has a more ordered configuration with red
blood cells trapped between collagen fibers (FIG. 16C).
Floseal.RTM. can be observed as large granules of gelatin particles
with red blood cells in the interstitial spaces between material
particles (FIG. 16D).
Example 11: In Vivo Biocompatibility and Degradation Testing
[0135] A rabbit degradation model was used to assess tissue
biocompatibility and in vivo degradation of various crosslinked
formulations. The following materials crosslinked with
glutaraldehyde were tested: 1% microfibrillar collagen crosslinked
at a ratio of 100:1; 1% microfibrillar collagen crosslinked at a
ratio of 250:1; and 0.1% fibrillar collagen crosslinked with
glutaraldehyde at a ratio of 10:1. Crosslinked collagen materials
were prepared by grinding lyophilized scaffolds and reconstituting
with saline to a concentration of 110 to 160 mg/mL. An incision was
created in the paraspinal muscle and blunt dissection was used to
create an intramuscular pocket. Into each pocket, approximately 1
cc of each crosslinked collagen formulation was applied. At 8
weeks, sites were excised and placed en bloc in fixative solution.
The tissue samples were processed using standard histological
techniques and sections were stained with hematoxylin & eosin
and Masson's trichrome. All sites displayed normal healing at
necropsy. The amount of material present and appearance of the
tissue and implanted collagen material are described in Table 19.
Representative histology images are shown in FIGS. 17A-17C.
TABLE-US-00019 TABLE 19 Microscopic analysis of crosslinked
collagen materials at 8 week implantation time. Amount of Material
Appearance of Sample Type Present tissue/material 1% microfibrillar
Minimal to marked Cellular and tissue collagen, 100:1 infiltration,
organized muscle tissue formation 1% microfibrillar Minimal to
moderate Large degree of cellular collagen, 250:1 infiltration,
redevelopment of muscle tissue 0.1% fibrillar Marked Cellular
infiltration only on collagen, 10:1 the outside of material
Example 12: Determining Crosslinking Conditions Using EDC/NHS
Chemistry
[0136] Crosslinking can be used to tailor properties of the
crosslinked material such as handling properties, blood absorption,
material consistency, ability to hold shape, and flexibility. The
most preferred material will be able to hold its shape and be
extruded through a syringe.
[0137] In order to crosslink collagen using EDC/NHS chemistry, the
general procedure of Wissink (2000) was modified as follows. The
concentration of collagen used was between 4-5 mg/mL (0.4-0.5%) and
more preferably between 0.45-0.47%. The effect of reaction
conditions on material properties were explored in greater detail.
Collagen was crosslinked using 1.740 g EDC per 1 g of collagen and
0.42 g NHS per 1 g of collagen. The solution was allowed to stir at
room temperature for 4 hours. The mass to mass ratio of EDC to NHS
was always kept between 4.0:1 and 4.5:1. The resultant material
properties were analyzed using SEM for fiber size and connectivity
(See FIGS. 18A-18E), Fourier-Transform infrared spectroscopy (FTIR)
to determine if changes in chemical structure could be observed
after crosslinking, blood absorption, and differential scanning
calorimetry (DSC) to determine if the stability of the material
increased as a result of crosslinking.
[0138] The structure of the material is altered as reaction
conditions such as concentration and time are varied (See Table 21,
below). The control material is shown in FIG. 18A. All materials
are compared to this material. Materials crosslinked at lower
concentrations; such as about half the concentration of EDC and NHS
relative to the control (FIG. 18B) or materials crosslinked for a
shorter time, such as about two hours (FIG. 18D), have a more
closed, less porous structure. These materials have mostly a
sheet-like appearance with a few fibers that are visible. Materials
crosslinked at a higher concentration, for example about two times
more EDC and NHS were used compared to the control material (FIG.
18C). The increase in concentration appears to result in a more
interconnected porous structure with small sheets of material. The
reaction time can be increased from about 4 hours for the control
material to about 16 hours to provide material that has a more open
and porous structure with a few sheets of material and an increase
in the number of fibers that form the porous structure (FIG.
18E).
TABLE-US-00020 TABLE 21 Mass Mass Reaction Material EDC (g) NHS (g)
Time (h) Control 1.74 0.42 2, 4, or 16 1/2 Control Concentration
0.87 0.42 4 2 Times Control Concnetration 3.65 0.42 4
[0139] The ability of the crosslinked collagen material to absorb
blood was also monitored. The results are shown in Table 20. By
either decreasing the concentration of EDC and NHS or decreasing
the reaction time, which allows fewer crosslinks to be formed, the
ability of the crosslinked material to absorb blood can be
decreased. This affords the ability to control absorption of blood,
and to a certain extent swelling is also controlled by controlling
the reaction parameters. Thus the material properties can be
tailored to provide the most desirable hemostatic or wound healing
device.
TABLE-US-00021 TABLE 20 Percent of Blood Absorbed by EDC/NHS
Crosslinked Collagen. Material % Blood Absorption Control 96.14 1/2
x 47.25 2 x 84.58 2 h rxn 70.62 16 h rxn 61.96
The EDC/NHS crosslinked material has an increased capacity to
absorb blood compared to GTA crosslinked collagen. This is
potentially because EDC/NHS attaches two or more collagen chains
during crosslinking and does not alter the overall chemical
composition of the collagen. GTA crosslinking, however, inserts a
linker that can disrupt the spacing and interactions between
collagen chains as well as alter the charge of the collagen thereby
decreasing the amount of blood that can be absorbed.
[0140] While the invention has been disclosed with reference to
specific embodiments, it is apparent that other embodiments and
variations of the invention may be devised by others skilled in the
art without departing from the true spirit and scope of the
invention. The appended claims are intended to be construed to
include all such embodiments and similar variations.
[0141] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety.
* * * * *