U.S. patent application number 11/112156 was filed with the patent office on 2006-06-22 for fibrin material and method for producing and using the same.
This patent application is currently assigned to Baxter International Inc.. Invention is credited to YVES DELMOTTE, James Diorio.
Application Number | 20060134094 11/112156 |
Document ID | / |
Family ID | 35266347 |
Filed Date | 2006-06-22 |
United States Patent
Application |
20060134094 |
Kind Code |
A2 |
DELMOTTE; YVES ; et
al. |
June 22, 2006 |
FIBRIN MATERIAL AND METHOD FOR PRODUCING AND USING THE SAME
Abstract
Abstract of the Disclosure This invention describes a
bioerodible fibrin material which is obtained by mixing fibrinogen
and thrombin reconstituted or diluted with a particular high tonic
strength medium, free of calcium. Such a fibrin-based biomaterial
develops a tight structure with thin fibers and small pore size
suitable for use as an anti-adhesion barrier. In this invention,
thrombin is no longer the variable which governs the tightness and
the porosity of the fibrin material obtained, but still controls
the clotting time. The mechanical behavior, high-water capacity,
and releasable retention properties for therapeutic agents of this
fibrin structure causes the fibrin material to be ideally suited
for use as a drug delivery device, capable of delivering proteins,
hormones, enzymes, antibiotics, antineoplastic agents and even
cells for local and systemic treatment of human and non-human
patients.
Inventors: |
DELMOTTE; YVES; (Tertre,
US) ; Diorio; James; (Antioch, IL) |
Correspondence
Address: |
BAXTER HEALTHCARE CORPORATION
ONE BAXTER PARKWAY
DF2-2E
DEERFIELD
IL
60015
US
|
Assignee: |
Baxter International Inc.
One Baxter Parkway
Deerfield
US
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20050271646 A1 |
December 8, 2005 |
|
|
Family ID: |
35266347 |
Appl. No.: |
11/112156 |
Filed: |
April 22, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09/566,019 |
Nov 15, 2005 |
6965014 |
|
|
11112156 |
Apr 22, 2005 |
|
|
|
09/386,198 |
Oct 8, 2002 |
6461325 |
|
|
09/566,019 |
May 8, 2000 |
|
|
|
08/679,658 |
Nov 23, 1999 |
5989215 |
|
|
EP9600160 |
Jan 16, 1996 |
|
|
|
09/386,198 |
Aug 31, 1999 |
|
|
|
Current U.S.
Class: |
424/94.64 |
Current CPC
Class: |
A61L 31/145 20130101;
A61L 31/146 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 38/4833 20130101; C07K 14/75 20130101; A61K 38/4833 20130101;
A61K 38/363 20130101; A61L 31/046 20130101; A61K 38/363
20130101 |
Class at
Publication: |
424/094.64 |
International
Class: |
A61K 38/48 20060101
A61K038/48 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 16, 1995 |
DE |
19501067.1 |
Claims
1. Cancelled.
2. Cancelled.
3. Cancelled.
4. Cancelled.
5. Cancelled.
6. Cancelled.
7. Cancelled.
8. Cancelled.
9. Cancelled.
10. Cancelled.
11. Cancelled.
12. Cancelled.
13. Cancelled.
14. Cancelled.
15. Cancelled.
16. Cancelled.
17. Cancelled.
18. Cancelled.
19. Cancelled.
20. Cancelled.
21. Cancelled.
22. A method for delivering fibrin hydrogel to a surface comprising
the steps of: providing a liquid solution of fibrinogen; providing
a liquid solution of thrombin; providing a liquid solution of a
calcium chelating agent; providing a spray unit in fluid
communication with the fibrinogen solution, the thrombin solution,
and the chelating agent, the spray unit being capable of atomizing
the fibrinogen solution, the thrombin solution, and the chelating
agent into an aerosol with at least one energy source of a liquid
energy, a mechanical energy, a mechanical energy, and an electric
energy; spraying the fibrinogen solution onto the surface with the
spray unit; spraying the chelating agent onto the surface with the
spray unit; spraying the thrombin solution onto the surface with
the spray unit; and mixing the fibrinogen solution the chelating
agent and the thrombin solution.
23. The method of claim 22 wherein the chelating agent is selected
from the group of EGTA, EDTA, PBS, and citrate.
24. The method of claim 22 wherein the chelating agent is PBS.
25. The method of claim 22 wherein the step of spraying the
fibrinogen solution onto the surface occurs simultaneously with the
step of spraying the thrombin solution onto the surface.
26. The method of claim 22 wherein the step of mixing the
fibrinogen solution and the thrombin solution defines a
substantially homogeneous mixture prior to making contact with the
surface.
27. The method of claim 22 wherein the step of spraying the
fibrinogen solution onto the surface and the step of spraying the
thrombin solution onto the surface occurs sequentially.
28. A method for using a fibrin material to treat a patient, the
method comprising the steps of: providing a first fibrin material
having a pore size from about 2 m to about 10 m; providing a second
fibrin material having a pore size from about 0.1 m to about 5 m;
and being capable of retaining 80-90% of water upon compression by
a force of 1-14 psi; applying the first fibrin material to a
surface; and applying the second fibrin material to the first
fibrin material.
29. The method of claim 28 wherein the step of providing the first
fibrin material comprises the steps of: providing thrombin solution
having a concentration from about 50 IU/ml to about 300 IU/ml;
providing fibrinogen solution having a concentration from about 1.5
mg/ml to about 100 mg/ml; and mixing the thrombin solution with the
fibrinogen solution.
30. The method of claim 28 wherein the step of providing the second
fibrin material comprises the steps of: providing thrombin solution
having a concentration from about 1 IU/ml to about 300 IU/ml;
providing fibrinogen solution having a concentration from about 1.5
mg/ml to about 100 mg/ml; providing a chelating agent as an
antagonist to fibrinopeptide transamidation reactions; and mixing
the thrombin solution, the fibrinogen solution, and the chelating
agent.
31. The method of claim 30 wherein the diluent is phosphate
buffered saline.
32. Cancelled.
33. Cancelled.
34. Cancelled.
35. Cancelled.
36. Cancelled.
37. Cancelled.
38. A multiple layer fibrin material comprising: a fibrin glue
layer; and a fibrin hydrogel layer, the fibrin hydrogel being
capable of retaining from at least about 80% to about 90% of water
by weight of the hydrogel when compressed by a force of about 156
G, and having relatively no cross-linking, wherein the fibrin
hydrogel layer releasably retains a therapeutic agent.
39. The multiple layer fibrin material of claim 38 wherein the
therapeutic agent is selected from the group consisting of
antibiotics, fibrinolytic agents and biological response
modifiers.
40. The multiple layer fibrin material of claim 39 wherein the
therapeutic agent comprises a pharmaceutical compound.
41. The multiple layer fibrin material of claim 39 wherein the
therapeutic agent comprises living cells.
42. A multiple layer fibrin material for treating a patient
comprising: a fibrin glue layer; and a therapeutic fibrin hydrogel
layer.
43. The multiple layer fibrin material of claim 42 wherein the
fibrin glue layer has a pore size from about 2 m to about 10 m.
44. The multiple layer fibrin material of claim 42 wherein the
therapeutic fibrin hydrogel layer has a pore size from about 0.1 m
to about 5 m.
45. The multiple layer fibrin material of claim 42, wherein the
therapeutic fibrin hydrogel layer has a water content of at least
about 90% by weight of the therapeutic fibrin hydrogel layer and
whereby the therapeutic fibrin hydrogel layer retains from about
80% to about 90% of the water upon compression by a force of about
156 G.
46. The multiple layer fibrin material of claim 45 wherein the
retained water comprises a releasably retained diluent.
47. The multiple layer fibrin material of claim 46 wherein the
releasably retained diluent comprises a therapeutic agent.
48. The multiple layer fibrin material of claim 47 wherein the
therapeutic agent is selected from the group consisting of
pharmaceutical compounds, antibiotics, fibrinolytic agents and
biological response modifiers.
49. The multiple layer fibrin material of claim 48 wherein the
therapeutic agent comprises a solution containing living cells.
50. Cancelled.
51. Cancelled.
52. Cancelled.
53. Cancelled.
54. Cancelled.
55. Cancelled.
56. Cancelled.
57. Cancelled.
58. Cancelled.
59. Cancelled.
60. Cancelled.
61. Cancelled.
62. Cancelled.
63. Cancelled.
64. Cancelled.
65. Cancelled.
66. Cancelled.
67. Cancelled.
68. Cancelled.
69. Cancelled.
70. A kit for forming a fibrin hydrogel comprising a container of
proteins comprising fibrinogen; a container of thrombin; a
container of a diluent, wherein the diluent has the ability to
chelate calcium; and a mixing and application apparatus.
71. The kit of claim 70, wherein the container of proteins has a
protein concentration of more than about 30 mg/ml.
72. The kit of claim 70, wherein the container of proteins contains
Factor XIII at a concentration from about 0 IU/ml to about 80
IU/ml.
73. The kit of claim 70, wherein the container of thrombin has
thrombin concentration between about 0.1 IU/ml and about 1000
IU/ml.
74. The kit of claim 70, wherein the mixing and application
apparatus is a syringe.
75. The kit of claim 70, wherein the mixing and application
apparatus is a catheter.
76. The kit of claim 70, wherein the diluent is selected from the
group consisting of: a phosphate buffer, sodium citrate solution,
potassium citrate solution, EDTA, EGTA, and chloride solution.
77. The kit of claim 76, wherein the diluent is the phosphate
buffer.
78. A kit for forming a fibrin hydrogel comprising: a container of
proteins comprising fibrinogen, wherein the protein concentration
is more than about 30 mg/ml; a container of thrombin, wherein the
thrombin concentration is between about 0.1 IU/ml and about 1000
IU/ml: a container of a phosphate buffer solution; and a mixing and
application apparatus, wherein the apparatus is selected from the
group consisting of a syringe and a catheter.
79. The kit of claim 78, wherein the container of proteins contains
Factor XIII at a concentration from about 0 IU/ml to about 80
IU/ml.
80. A fibrin hydrogel material comprising: a fibrin material,
wherein the hydrogel material having has a water content of at
least 90% by weight of the hydrogel material and whereby the
hydrogel material retains from about 80% to about 90% of the water
upon compression by a force of about 156 G.
81. A fibrin hydrogel material comprising: a fibrin material,
wherein the hydrogel material has a water content of at least 90%
by weight of the hydrogel material and whereby the hydrogel
material retains from about 80% to about 90% of the water upon
compression by a force of about 156 G wherein the fibrin hydrogel
material is produced using an essentially calcium free thrombin
solution.
82. A multiple layer fibrin material comprising: a fibrin glue
layer; and a fibrin hydrogel layer, the fibrin hydrogel being
capable of retaining from at least about 80% to about 90% of water
by weight of the hydrogel when compressed by a force of about 156
G, and having relatively no cross-linking.
83. A multiple layer fibrin material for treating a patient
comprising: a fibrin film layer; and a fibrin hydrogel layer, the
fibrin hydrogel having a water content of at least 90% by weight of
the fibrin hydrogel and whereby the fibrin hydrogel retains from
about 80% to about 90% of the water upon compression by a force of
about 156 G.
84. A multiple layer fibrin material for treating a patient
comprising: a fibrin film layer; a therapeutic fibrin hydrogel
layer, the fibrin hydrogel having a water content of at least 90%
by weight of the fibrin hydrogel and whereby the fibrin hydrogel
retains from about 80% to about 90% of the water upon compression
by a force of about 156 G; and a fibrin glue layer attaching the
fibrin film layer to the fibrin hydrogel layer.
Description
Detailed Description of the Invention
Technical Field
[0001] This invention provides a fibrin hydrogel material and
particularly a fibrin hydrogel useful as a drug delivery vehicle
and for the prevention of post surgical adhesion.
Related Applications
[0002] This is a divisional application of U.S. Serial Number
09/566,019, filed on May 8, 2000, which is a continuation-in-part
of U.S. Serial Number 09/386,198, now U.S. Patent No. 6,461,325,
filed on August 31, 1999, which is a continuation-in-part
application of U.S. Serial Number 08/679,658, now U.S. Patent No.
5,989,215, filed on July 12, 1996, which is a continuation-in-part
of PCT application number PCT/EP96/00160 filed Jan. 16, 1996 which
claims priority from German patent application number 195 01 067.1
filed on Jan. 16, 1995. Application Ser. No. 08/679,658 is
incorporated herein by reference and made a part hereof.
Background Art
[0003] One of the major problems in intra-abdominal surgery is the
avoidance of post-operative adhesions. It is well-known that
adhesions contribute to pain, immobility, retarded wound healing,
and in particular to intestinal obstruction which may even be
life-threatening. In the field of gynecological surgery,
post-surgical adhesions involving female reproductive organs may
result in infertility.
[0004] Each surgical procedure necessarily produces various forms
of trauma where the abdominal cavity or other human cavity is
opened for an inspection. Physiologically, the process of wound
closure then starts when bleeding ceases upon formation of a
hemostatic clot at the places where blood vessels are injured. The
clot, at first comprising mainly platelets, is solidified by a
fibrin network resulting from the activation of an enzyme cascade
involving thrombin, factor XIII and calcium. Further steps on the
way to the sealing of the wound are retraction of the hemostatic
clot, invasion of various cell types including fibroblasts into the
wound area and eventually the lysis of the fibrin network.
Adhesions are thought to begin to form when the fibrin clot
covering an injury comes into contact with a bleeding adjacent
surface and the new connective tissue produced by the fibroblasts
attach the two surfaces together.
[0005] The problems associated with adhesions often require a
further operative procedure for removing/lysing the adhesions,
called adhesiolysis, which, like the first operation, principally
bears the risk of forming additional adhesions.
[0006] Accordingly, the prevention of adhesion formation is
medically important. Among the different approaches for prevention
of adhesion formation, one involves the use of materials as a
physical or bio-mechanical barrier for the separation or isolation
of traumatized tissues during the healing process. Both synthetic
materials and natural materials have been used as a barrier to
adhesion formation. Permanent, inert implants like Gore Tex.RTM.
surgical membranes consisting of expanded polytetrafluoroethylene
(PTFE) generally require a second operative procedure to remove
them, while others such as surgical membranes of oxidized
regenerated cellulose are biodegradable, but are thought to elicit
an inflammatory response ultimately leading to adhesion formation
(A.F. Haney and E. Doty, Fertility and Sterility, 60, 550-558,
1993).
[0007] Fibrin sealants and glues are well-known in the art for use
in haemostasis, tissue sealing, and wound healing and have been
commercially available for more than a decade. Use for
anti-adhesion and drug delivery vehicle in glaucoma surgical
procedures is one example. Fibrin glues mimic the last step of the
coagulation cascade and are usually commercialized as kits
comprising two main components. The first component is a solution
comprising fibrinogen with or without factor XIII, while the second
component is a thrombin calcium solution. After mixing of
components, the fibrinogen is proteolytically cleaved by thrombin
and thus converted into fibrin monomers. Factor XIII is also
cleaved by thrombin into its activated form (FXIIIa). FXIIIa cross
links the fibrin monomers to form a three-dimensional network
commonly called "Fibrin Gel."
[0008] As disclosed in the commonly assigned published PCT patent
application, WO 96/22115, a self-supporting sheet-like material of
cross-linked fibrin material can be used.sup.as a bio-mechanical
barrier in the treatment of internal traumatic lesions,
particularly for prevention of adhesion formation as a
post-operative complication. The `115 Application discloses the
mixing of a thrombin and calcium containing solution with a
fibrinogen and Factor XIII containing solution. By using high
thrombin concentrations to catalyze the conversion of fibrinogen
into fibrin, the resulting fibrin material was found to be
sufficiently rigid to be self-supporting and to have sufficiently
small pore size to prevent the ingress of fibroblasts which causes
the formation of adhesions. The resulting fibrin material, however,
did not readily retain water. In fact water could be easily
expelled from the fibrin material by compressing the material by
hand. Thus, this classic type fibrin material could not be used to
deliver drugs to a wound site while being reabsorbed into the body
during the fibrinolytic process.
[0009] This invention overcomes these and other shortcomings in the
prior art devices. Hydrogel fibrin has a tight structure
constituted of thin fibers defined by a low pore size. Water is
trapped in the "void volume" of the structure. The "void volume" is
small, regular, and homogenously distributed through the entire
film material. Water cannot leave the film structure, due to its
internal energy, and is released from the fibrin structure
depending on the fibrinoytic rate of the biopolymer. The release of
a drug incorporated into the water or buffer is regulated by
passive diffusion and, depending upon the molecular weight,
solubility and the fibrinolytic process.
[0010] The removal of calcium from the process of forming a fibrin
structure yields no lateral associations of protofibrils. The lack
of associations of protofibrils corresponds to a high number of
thin fibers per unit of volume, thus conferring a tight pore size
in the fibrin structure. This tight pore size allows for water to
remain trapped in the "void volume."
Summary of the Invention
[0011] Other advantages and aspects of the present invention will
become apparent upon reading the following description of the
drawings and detailed description of the invention.
[0012] This invention provides a medical device for the prevention
of post-surgical adhesion formation and the controlled release of
drugs in human and non-human species. The device comprises a fibrin
hydrogel material having a water content of at least about 90% by
weight of the hydrogel. The fibrin hydrogel has a pore size within
the range of less than 1 micron and preferably less than 0.1 m has
a transparency of less than about 1.0 AUFS, more preferably less
than about 0.8 AUFS when measured with a spectrophotometer at 800
nm. The fibrin hydrogel is substantially free of cross-linking.
[0013] The present invention further provides a multilayer fibrin
material for application to animal tissue. The characteristics of
each layer are determined by the concentrations of the constituents
and the presence of calcium and Factor XIII. In a preferred form,
in addition to a fibrin hydrogel, the material or film includes one
layer of a fibrin glue. The fibrin glue layer has a pore size
within the range of less than 2 to 10 microns. In another
embodiment, the fibrin hydrogel includes a layer of classic fibrin
film. The classic fibrin film layer has a pore size within the
range of 0.1 to 10 microns and is cross-linked.
[0014] In another embodiment, one layer of the multiple layer
fibrin material is a therapeutic fibrin hydrogel material having a
water content of at least 92.5 % by weight of the hydrogel and
whereby the hydrogel retains 90 % of the water upon compression by
a force from 1 to 14 psi. The therapeutic fibrin hydrogel layer of
the multilayer fibrin material releasably retains a diluent whereby
the diluent comprises a therapeutic agent. The therapeutic fibrin
hydrogel layer of the multilayer fibrin material has a pore size
within the range of 0.1 to 1 microns and has an optical clarity of
less than about 1.0 AUFS, more preferably less than about 0.50 AUFS
when measured with a spectrophotometer at 800 nm. The fibrin
hydrogel layer is substantially free of cross-linking. In one
embodiment of the therapeutic fibrin hydrogel layer of the
multilayer fibrin material, the releasably retained therapeutic
agent comprises a pharmaceutical compound. In another embodiment of
the therapeutic fibrin hydrogel layer of the multilayer fibrin
material, the releasably retained therapeutic agent comprises
living cells, such as chondrocytes. Other cell types are
contemplated as well.
Brief Description of the Figures
[0015] Figure 1 depicts the fibrin hydrogel material prepared using
the thrombin concentration of 5 IU.
[0016] Figure 2 depicts the fibrin hydrogel material prepared using
the thrombin concentration of 10 IU.
[0017] Figure 3 depicts the fibrin hydrogel material prepared using
the thrombin concentration of 20 IU.
[0018] Figure 4 depicts the fibrin hydrogel material prepared using
the thrombin concentration of 100 IU.
[0019] Figure 5 depicts the fibrin hydrogel material prepared using
the thrombin concentration of 300 IU.
[0020] Figure 6 depicts a medical device that may be used to form
the fibrin hydrogel material inside and outside the animal
body.
[0021] Figure 7 depicts a pressurized canister housing fibrinogen
and thrombin as powders in separate bags.
[0022] Figure 8 depicts a double syringe system housing fibrinogen
and thrombin in separate chambers.
[0023] Figure 9 depicts a classic fibrin material in gel
electrophoresis.
[0024] Figure 10 depicts a fibrin hydrogel material in gel
electrophoresis.
[0025] Figure 11 is a chart depicting the percent water loss for 4
samples of fibrinogen solutions that were mixed with equal volumes
of thrombin.
[0026] Figure 12A is a chart depicting the percent water loss for 4
samples of fibrinogen and thrombin solutions diluted in PBS.
[0027] Figure 12B is a chart depicting the percent water loss for 4
samples of fibrinogen and thrombin solutions wherein the fibrinogen
is diluted in PBS and the thrombin is diluted in CaCl.sub.2.
[0028] Figure 13A is a chart depicting the percent water loss for 4
samples of fibrinogen and thrombin solution diluted in EDTA.
[0029] Figure 13B is a chart depicting the percent water loss for 4
samples of fibrinogen and thrombin solutions diluted in potassium
citrate.
[0030] Figure 14 is a chart depicting the percent water loss for 4
samples of fibrinogen, which is free of FXIII and thrombin diluted
in PBS.
Detailed Description of the Invention
[0031] While this invention is susceptible of embodiment in many
different forms, there is shown in the drawings and will herein be
described in detail a preferred embodiment of the invention with
the understanding that the present disclosure is to be considered
as an exemplification of the principles of the invention and is not
intended to limit the broad aspect of the invention to the
embodiments illustrated.
[0032] One preferred form of the present invention provides a
self-supporting, biodegradable, fibrin hydrogel material which is
obtained by mixing fibrinogen and thrombin solutions diluted with a
solute inhibiting the action of calcium on fibrinogen in a high
ionic strength medium, both free of calcium. The prior art
discloses that calcium is a critical component to forming a fibrin
material. The resulting hydrogel material has thin fibers and small
pore size and is suitable for use as an anti-adhesion barrier.
I. Fibrin Hydrogel Material
[0033] Preferably the fibrin hydrogel material for anti-adhesion
applications will have a pore size from 0.1- 5 microns, and more
preferably from 0.1- 3 microns. The fibrin hydrogel material
preferably also readily retains water upon compression. Preferably
the hydrogel shall retain 80 - 90 % of its water content upon
compressing the material with a force from 1- 14 psi.
[0034] The hydrogel material has a sufficiently high modulus of
elasticity to be self-supporting. By self-supporting we mean that a
fibrin hydrogel material of 5 cm long by 5 cm wide by 5 mm thick
can be held at one end without the second end deflecting downward
with respect to the held end more than 10 degrees.
[0035] It is also desirable that the fibrin hydrogel be relatively
optically transparent and, in a preferred form, should have an
optical density measured with a spectrophotometer at 800 nm of from
0.1-0.5, more preferably from 0.1-0.4 and most preferably from
0.1-0.2. As can be seen in Figure 1, the fibrin hydrogel has a
network of fibers that should have an average diameter, in a
preferred form, of less than about 5.0 microns, more preferably
from less than about 2.0 microns and most preferably from less than
about 1.0 microns or any range or combination of ranges
therein.
[0036] In one preferred form of the invention, a fibrin hydrogel is
a single layered material. The present invention further provides a
multilayer fibrin material comprising two or more layers.
[0037] In preferred embodiments, the thickness of the fibrin
barrier material is at least 200 m when the barrier is in the wet
state. Preferably the thickness is about 500 m, and most preferably
up to 10,000 m, although it is believed that even material with a
thickness of less than 100 m may be suitable for the purposes of
the invention.
[0038] The hydrogel material must also be capable of being
reabsorbed into the body or be bioreabsorbable. Preferably,
depending upon the concentration of fibrinogen and the quantity
applied, a fibrin hydrogel of 3 cm x 3 cm x 1 cm the hydrogel will
be reabsorbed into the body in its entirety by 14 days, more
preferably within 10 days and most preferably within 5 days.
[0039] The fibrin hydrogel can be distinguished from classic fibrin
material in several ways. First, the fibrin hydrogel can obtain a
lower pore size than that of classic fibrin material with the same
concentration of thrombin. The fibrin hydrogel has a tighter pore
size regardless of the concentration of thrombin used. This has an
advantage over classic fibrin materials in that the fibrinolytic
process is maintained or increased over physiological levels.
[0040] Classic fibrin materials utilize large quantities of
calcium, thus hampering the fibrinolytic process. The fibrinolytic
process is slowed by the presence of gamma-gamma cross-links in the
fibrin material, which are caused by the presence of excess
calcium. The lower level of calcium in the fibrin hydrogel material
inhibits cross-linking, thus allowing for a faster breakdown of the
fibrin hydrogel material.
[0041] The increased time required by classic fibrin materials to
be broken down may also result in a greater degree of adhesion. The
anti-adhesion qualities of the fibrin hydrogel are believed to
result from the thrombin content. The thrombin content of the
fibrin hydrogel allows for a higher fibrinolytic rate than classic
fibrin materials.
[0042] Another distinction is the fibrin hydrogel's ability to
retain water under compression forces. The degree of water
retention in the fibrin hydrogel greatly exceeds the water
retention of the classic fibrin materials. This permeability factor
is a primary distinction between the fibrin hydrogel of the present
invention and classic fibrin materials. The slow rate of water
release by the fibrin hydrogel material allows the hydrogel to act
as a lubricant by release of water, further enhancing its
anti-adhesion properties.
II. Method of Forming a Fibrin Hydrogel
[0043] It has been found by the present inventors that a fibrin
hydrogel material may be formed in the absence of a calcium
containing solution and in the absence of a Factor XIII containing
solution which were previously considered in the art to be
essential components. In a preferred form of the invention a fibrin
hydrogel is obtained by admitting a fibrinogen- containing solution
with a thrombin-containing solution. The fibrinogen solution should
have from 1.5 - 100 mg/ml, more preferably 3 - 70 mg/ml and most
preferably a 45 mg/ml fibrinogen dissolved in a solution containing
components capable of chelating calcium. The chelating component
should also be non-toxic and in a preferred form of the invention
is a phosphate buffer saline solution (PBS) of physiologically
acceptable levels. The chelating agent should be an antagonist to
fibrinopeptide transmidation reaction 5 IU-300 IU.
[0044] Also, contrary to the teachings in the prior art, the
thrombin concentration of the admixed components does not determine
the pore size of the hydrogel fibrin material. As will be discussed
below and as shown in Figures 1-5, fibrin hydrogel materials were
formed having relatively the same pore size notwithstanding the use
of thrombin concentrations from 1 IU to 300 IU. The concentration
of thrombin was still found to control the rate of forming a fibrin
hydrogel.
[0045] It is well known that mixing a first solution containing
fibrinogen with Factor XIII and a second solution of thrombin with
calcium will result in the formation of a fibrin material with
pronounced lateral association and considerable cross-linking among
its thick fibers. It is also well known that thrombin acts as a
protease which will cleave fibrinopeptide A and B from the
fibrinogen molecule and convert it into fibrin. The fibrinopeptides
of vertebrate species reportedly have a large net negative charge.
The presence of these and other negatively charged groups in the
fibrinopeptides are likely actors in keeping fibrinogen apart.
Their release by thrombin gives fibrin monomers a different
surface-charge pattern, leading to their specific aggregation. In
particular, removal of the fibrinopeptides changes the net charge
of the central globular unit from -8 to +5. Each of the terminal
globular units has a net charge of -4. Thus, electrostatic
interactions between the terminal and central globular units
probably stabilize the structure of fibrin.
[0046] It is also known that calcium ions play an important role in
the dissociation of Factor XIII subunit A from Factor XIII subunit
B as Factor XIII is converted to its activated form, Factor XIIIa.
Furthermore, it is know that Factor XIIIa is critical to the
cross-linking of fibrin monomers. It is also known that the widths
of the fibers comprising the fibrin material can be decreased by
increasing the pH and ionic strength of the diluents.
[0047] By removing (chelating) calcium ions bound to fibrinogen,
the inventors have been able to modify the fibrin structure to
obtain further embodiments of the fibrin hydrogel. Accordingly, the
present invention uses a solution capable of scavenging calcium
ions associated with the fibrinogen molecules. In a preferred form
of the invention, the calcium scavenging solution is a phosphate
buffer solution having a concentration similar to physiologically
acceptable levels. The inventors suggest that the resulting
structural modifications of the fibrin hydrogel occur as a result
of a "charge effect" which alters the aforementioned electrostatic
interactions between the terminal and central globular units,
thereby inhibiting the lateral association of fibrin. In further
embodiments of the fibrin hydrogel, modification of Factor XIII
concentration used in the synthesis of fibrin alters the
crosslinking characteristics of the final fibrin material. Thus, in
further embodiments of the invention, the inventors have developed
a fibrin hydrogel that can be synthesized with low concentrations
of thrombin and according to the end user's specifications as to
the lateral association and fiber thickness of the resulting fibrin
hydrogel structure.
[0048] During the formation of a fibrin hydrogel, it is desirable
that all of the fibrinogen be converted into fibrin, as residual
amounts of fibrinogen may lead to adhesion formation upon reacting
with thrombin present in the body. Accordingly, in still further
embodiments of the present invention, the fibrin hydrogel further
comprises less than 5% by weight of fibrinogen, preferably less
than 4% by weight of fibrinogen, preferably less than 3% by weight
of fibrinogen, preferably less than 2% by weight of fibrinogen, and
most preferably less than 1% by weight of fibrinogen, in terms of
the total dry weight of the fibrinogen plus fibrin each time. For
the purpose of determining the fibrin and the fibrinogen content of
the fibrin film, the methods of SDS-Page (SDS-Gel Electro-phoresis)
may be used.
[0049] The medical devices shown in Figure 6 and as further
described in commonly assigned U.S. Patent No. 5,989,215 may be
used topically, in open-type surgeries (for example, laparotomic
surgeries) or minimally invasive surgeries (for example,
laparoscopic surgeries). Of course, there are other types of
open-type surgeries and minimally invasive surgeries as will be
appreciated by one of ordinary skill in the art. The medical device
may be used to form fibrin hydrogel material inside and outside the
animal body.
[0050] The present invention provides a process for preparing a
self-supporting fibrin hydrogel matrix or film outside the body
comprising the steps of;
[0051] (a) mixing a stream of a first, fibrinogen-containing
solution dissolved in PBS with a stream of a second,
thrombin-containing PBS solution;
[0052] (b) applying the obtained mixture to a solid support or
mixing the components of the solid support; and
[0053] (c) incubating the mixture to form the hydrogel matrix.
[0054] In order to obtain a mixture as homogenous as possible (and
thus a homogenous final product) in step (a) a stream of a first,
fibrinogen-containing solution is mixed with a stream of a second,
thrombin-containing solution by simultaneous delivery of the
components. It is also possible to deliver one component to a
surface followed by the other component. Preferably, equal volumes
of the first and the second solution are mixed. In case the
different volumes of the first and the second solution should be
mixed, it will be known in the art which measures have to be taken
in order to ensure that a homogenous mixture is obtained.
[0055] Using the delivery device mentioned above, the resulting
mixture is spread over the surface of a solid support, for example
a petri dish or the like, which is tilted to cover the entire
surface as far as possible before the formation of the fibrin
hydrogel material begins.
[0056] For the purpose of preparing a fibrin hydrogel on mammal
tissue, the inventors propose a process comprising the steps
of:
[0057] providing a first phosphate buffer solution containing
fibrinogen;
[0058] providing a second phosphate buffer solution containing
thrombin;
[0059] mixing the first solution and the second solution before or
after placing the mixture on an animal tissue;
[0060] and obtaining a fibrin hydrogel material with a tight
structure and small pore size suitable for post-surgical adhesion
prevention.
[0061] The fibrinogen and thrombin solutions can be initially mixed
in a delivery device, or be atomized into a spray and mixed while
in the form of spray droplets while in mid air or upon first making
contact with the tissue surface or delivered through a multi-lumen
catheter.
[0062] The fibrin hydrogel may be formed by utilizing constituents
of a kit. A preferred embodiment of the fibrin hydrogel kit
includes:
[0063] a vial of proteins including fibrinogen;
[0064] a vial of thrombin;
[0065] a vial of phosphate buffer solution to serve as a diluent;
and
[0066] appropriate ancillary mixing and application apparatus,
including, but not limited to syringes and catheters.
[0067] One further embodiment of the fibrin hydrogel kit includes a
vial containing the protein cocktail where the protein content is
no less than 30 mg/ml. Another further embodiment of the fibrin
hydrogel kit includes a vial containing the protein cocktail where
the Factor XIII content ranges from 0 IU/ml to 80 IU/ml. Yet a
further embodiment of the fibrin hydrogel kit includes a vial
containing thrombin, where the concentration of thrombin ranges
between 0.1 IU/ml to 1000 IU/ml. In yet another preferred
embodiment of the fibrin hydrogel kit, the constituents supplied
are pre-formulated to ensure that when mixed, the hydrogel achieved
will have a homogeneous structure with tight pore sizes suitable to
act as a prophylaxis to adhesion formation.
[0068] Another preferred embodiment of the fibrin hydrogel kit
includes pre-formulated constituents, supplied to ensure that when
mixed, the hydrogel achieved will have a homogeneous structure with
tight pore sizes suitable to act as a prophylaxis to adhesion
formation and as a drug delivery system. A lack of adhesion can be
seen with in fibrinogen free of FXIII or similar acting component.
The fibrin hydrogel kit includes a vial of fibrinogen mixed with
inactivated thrombin, a vial of suitable buffer, an ancillary, a
device equipped with optical fiber to photoactivate the thrombin,
and a light supply. In another preferred embodiment of the fibrin
hydrogel kit, the fibrinogen, inactivated thrombin, suitable
buffer, and an ancillary are all in one delivery device. Figure 7
illustrates another preferred embodiment where a pressurized
canister houses fibrinogen and thrombin as powder in separate bags.
The canister may be unscrewed to allow for rehydration of the
fibrinogen and thrombin. Increasing the pressure allows both
components to be sprayed through a double tube system or through
concentric channels. Another preferred embodiment, Figure 8,
utilizes a double syringe system with volumes of less than 100, 50,
20 ml each, more preferably less than 20 ml each, and most
preferably less than 10 ml each, but greater than 3.0 ml. This
double syringe system is equipped with a Y-shaped connection to
incorporate a tube. Such devices can be used for veterinary
applications. In another preferred embodiment, the fibrin hydrogel
material can be fabricated into articles selected from the group
consisting of films, tubes, and pellets. These fibrin hydrogel
materials can be fabricated into articles using techniques selected
from the group of extrusion, molding, and thermal forming. These
fibrin hydrogel materials can be sterilized at a temperature below
0.degree.C by gamma radiation, stored at a temperature below
0.degree.C, and used upon demand. The sterilization by gamma
radiation is below - 25.degree. C, at a dosage of at least 25 kGy.
In yet another preferred embodiment of the fibrin hydrogel kit, the
vial of diluent contains a phosphate buffer solution. In another
preferred embodiment of the fibrin hydrogel kit, the vial of
diluent contains a high-ionic strength buffer capable of scavenging
the fibrinogen-linked calcium. Other suitable solutions include;
sodium citrate, potassium citrate, EDTA, EGTA, chloride solutions,
phosphate solutions, or other ions solutions having a strong
affinity for calcium. Fibrinogen, thrombin, and other proteins such
as fibronectin and FXIII may be from a single donor, multiple
donors, pooled donors, Cohn I fraction, or recombinant. In another
preferred embodiment of the fibrin hydrogel kit, the vial of
diluent contains a buffer capable of chelating exogenous
calcium.
III. Therapeutic Fibrin Hydrogel
[0069] The present invention further provides a fibrin hydrogel
that releasably retains a diluent or a therapeutic agent. The
therapeutic agent is retained within the pores of the hydrogel
material and when placed into the body of a mammal is released over
time as the fibrin hydrogel is reabsorbed into the body.
[0070] The therapeutic agent(s) that are contemplated to be
releaseably retained by the therapeutic hydrogel layer comprises,
but is not limited to, pharmaceutical compounds, antibiotics,
fibrinolytic agents, and biological response modifiers, in
particular cytokines and wound repair promoters, preferably in an
amount up to 1 % by weight in terms of the total dry weight of
fibrin plus fibrinogen. Due to the chemotactive properties of
thrombin, low thrombin concentration is preferred for the purpose
of anti-adhesion. However, higher concentrations of thrombin may be
required to hasten clotting time. Clotting time was performed with
a semi-automated BFT II device from Dade Behring on fibrinogen at
25 mg/ml with varying thrombin concentrations of 0.5, 1.0, 2.5,
5.0, and 10.0 IU/ml. PBS was used as a diluent for fibrinogen and
thrombin. The clotting times are listed in the table below.
Table
[0071] This table lists the clotting times. TABLE-US-00001
Fibrinogen Thrombin Clotting Time Concentration Concentration
(Seconds) 25 mg/ml 0.5 IU/ml 430 25 mg/ml 1.0 IU/ml 237 25 mg/ml
2.5 IU/ml 83 25 mg/ml 5.0 IU/ml 37 25 mg/ml 10.0 IU/ml 20
[0072] Examples of fibrinolytic agents include t-PA, -PA,
streptokinase, staphylokinase, plasminogen and the like. These
compounds promote fibrinolysis and thus can be used for controlling
the rate of the degradation of the fibrin film in vivo. The term
"biological response modifiers" is meant to refer to substances
which are involved in modifying a biological response, such as
wound repair, in a manner which enhances the desired therapeutic
effect. Examples include cytokines, growth factors, and the like.
Due to its intrinsic mechanical properties, the fibrin film of the
invention does not require any additional cross-linking agent which
may exert any toxical effects to the human or animal body. Due to
its high level of dilution, it is possible for the fibrin hydrogel
to trap and release water. This is useful for the hydration of
tissues or as a lubricant to assist in the anti-adhesive properties
of the fibrin hydrogel.
[0073] The therapeutic agent can be incorporated into the fibrin
hydrogel material during the formation of the hydrogel. The
therapeutic agent may either water soluble or water insoluble,
antibody, antimicrobial agent, agent for improving
biocompatability, proteins, anti-inflammatory compounds, compounds
reducing graft rejection, living cells, cell growth inhibitors,
agent stimulating endothelial cells, antibiotics, antiseptics,
analgesics, antineoplastics, polypeptides, protease inhibitors,
vitamins, cytokines, cytotoxins, minerals, interferons, hormones,
polysacharides, genetic material, growth factors, cell growth
factors, substances against cholesterol, pain killers, collagens,
stromal cells, osteo-progenitor cells, polylactate, alginate,
C.sub.2-C.sub.24 fatty acids, and mixtures thereof. The delivery of
the therapeutic agent regulated by either or both the passive
diffusion and the fibrinolytic rate. The therapeutic agent can be
dissolved in one or both of the thrombin or fibrinogen solutions.
The therapeutic agent is retained by the hydrogel material as it
forms out of the admixed solution.
IV. Multilayer Fibrin Hydrogel Film
[0074] The present invention further provides a multiple layer
fibrin hydrogel film. The fibrin hydrogel can include a single
additional layer or multiple additional layers of fibrin glue,
classic fibrin film, fibrin hydrogel, therapeutic fibrin hydrogel
film and layers of other synthetic or naturally occurring
materials, such as alginate, polylactic, glycolic, silicon, and
hyluronic compounds. The present invention contemplates this
material being bound to the surface of synthetic polymers by
modifying the surface biomechanically or otherwise altering the
physical retention of the surface. Additionally, partially
premixing the components at the interface between the layers may
allow for bonding to occur. Furthermore, binding to collagen or
other organic material is also contemplated. Collagen and other
organic materials have a chemical affinity for proteins such as
fibrinogen and fibronectin. The present invention contemplates
selecting any combination of the above components and connecting
them together in differing orders based upon the desired function
of the film material. The present invention further contemplates
selecting the individual thicknesses of the individual layers and
the overall thickness of the film based upon its intended function.
The following is a set of non-limiting examples of multiple layered
films. The present invention should not be limited to these
exemplary embodiments.
[0075] The present invention provides a multiple layered fibrin
film having a first layer of a fibrin hydrogel or therapeutic
hydrogel and a second layer of a classic fibrin film. The "classic"
fibrin film is obtained by mixing a thrombin and calcium containing
solution with a fibrinogen and Factor XIII containing solution as
disclosed in detail in PCT Application WO 96/22115 which is
incorporated herein by reference and made a part hereof. The first
and second layers readily adhere to one another. During the
conversion process, the adhesive property of fibrinogen is present
and can allow the layers to stick together. If the fibrinogen is
added too late, the obtained fibrin material is no longer adhesive,
thus resulting in the possibility of delamination. It is
contemplated by the present invention that mechanical retention can
be inhanced by making holes in the first layer where the fibrin
glue can penetrate and adhere the layers.
[0076] The present invention also provides a three-layered film
having a first layer of a fibrin hydrogel, an inner layer of fibrin
glue and an outer layer of a therapeutic hydrogel material. Another
three-layered film includes inner and outer layers of fibrin glue
on opposed surfaces of a layer of fibrin hydrogel material.
Preferably the fibrin glue is obtained by mixing of
fibrinogen-containing solution with an equal volume of a
thrombin-containing solution. The fibrinogen-containing solution
contains fibrinogen and factor XIII (0.1 - 40 IU/ml). The
concentration of fibrinogen is expressed as the total protein
concentration (preferably from about 3 -140 mg/l and more
preferably 30-110 mg/ml) and the percentage of clottable protein
therein.
[0077] It is also preferred that the fibrinogen solution have a
viscosity that allows the solution to be sprayed and preferably
sprayed using pressures generated using a hand-operated syringe.
The fibrinogen solution should have a viscosity of less than 20
centipoise, more preferably less than 10 centipoise, and most
preferably from 1-5 centipoise or any combination or subcombination
of ranges therein. The thrombin-containing solution should have a
thrombin concentration less than 10000 IU thrombin. The fibrin glue
has been preferably made by mixing said fibrinogen-containing
solution with an equal volume of a thrombin-containing solution of
at least 50 IU thrombin, preferably of at least 150 IU thrombin,
and most preferably of at least 300 IU thrombin.
[0078] Yet another example of a multilayered film includes layers
stacked in the order of classic fibrin film/hydrogel/therapeutic
hydrogel/hydrogel/classic fibrin film. In this case the delivery of
the therapeutic agent in the therapeutic hydrogel can be delayed by
the time it takes for the outer layers to be reabsorbed into the
body. Thus, the present invention provides for building into the
structure of the multilayered film time delivery sequences or
schemes as desired.
[0079] Other contemplated embodiments include:
[0080] A multilayered structure composed of a surface layer of
hydrogel material and a bottom layer of membrane. The membrane may
be tissue or fibrin.
[0081] A multilayered structure composed of a surface layer classic
fibrin and a bottom layer of hydrogel material.
[0082] A multilayered structure composed of outer layers of classic
fibrin and an inner layer of hydrogel material.
[0083] A multilayered structure composed of a surface layer of
hydrogel material and an inner layer of fibrin sponge material.
[0084] A multilayered structure composed of outerlayers of hydrogel
material and an inner layer of membrane.
[0085] Beads of hydrogel material between 0.1 mm and 3 mm.
[0086] A hydrogel material anatomically molded.
[0087] The present invention also provides a process for preparing
a multilayer fibrin material. One such process for preparing a
multilayer fibrin material includes the steps of:
[0088] (1) providing a base fibrin hydrogel layer, comprising the
steps of:
[0089] providing a first buffer solution containing fibrinogen;
[0090] providing a second buffer solution containing thrombin;
[0091] providing additional constituents in either the first or
second buffer solutions as required for a specific preparation;
[0092] mixing the first solution, the second solution, and any
additional solutions on a surface such as a petri dish or
tissue;
[0093] and obtaining a fibrin layer with a desired structure and
desired pore size suitable for its designated purpose.
[0094] (2) providing an additional layer by repeating steps 1(a)
through 1(d) wherein the mixing occurs on the earlier formed layer
or layers;
[0095] (3) providing additional layers, if desired, by repeating
step 2;
[0096] (4) providing a final layer by repeating steps 1(a) through
1(d).
[0097] The present invention further provides a fibrin hydrogel
that retains a higher proportion of water than fibrin materials
currently available. The greater degree of water retention is
particularly beneficial to the therapeutic use of the hydrogel. The
retention of water is necessary for the control of the
concentration of therapeutic agents contained within the fibrin
hydrogel, as well as for the effective release of these therapeutic
agents and additives.
[0098] The ability of fibrin hydrogels to retain water while being
subjected to compression forces was tested and compared to the
water retaining capacity of a classic fibrin material. In
particular, compression was applied by centrifugation of the
materials at various rotational speeds and the amount of water
retained was measured. A refrigerated centrifuge (Sorvall RT 6000B)
spun fibrin hydrogels at different speeds:
[0099] -1000 rpms for 30 min. corresponding to 156 G
[0100] -2000 rpms for 30 min. corresponding to 625 G
[0101] -3000 rpms for 30 min. corresponding to 1428 G
[0102] Amicron filter type "centricon 30" was used, corresponding
to a membrane cutoff of 30000 and characterized by a maximum
rotation time of 30 min and sustaining a G-force max of 5000 G. The
Amicron filter is composed of two units. The upper unit contains
the filter component itself and can be attached to the second unit
of the Amicron filter. The second, or lower, unit is the bottom
cup. The bottom cup allows for the collection of water that is
expelled from the fibrin material deposited on the filter of the
upper unit. The water collected in the bottom cup is used to
measure the amount of water released by the fibrin materials at the
various rotational speeds. Once fibrinogen and thrombin solutions
were prepared, a volume of approximately 1 ml of fibrin was applied
to the filter. An appropriate mixing device is required for the
fibrinogen and thrombin mixture to be complete and homogeneous.
[0103] Upper and lower parts are separately weighed before the
fibrin material deposition and after each centrifugation step. A
correction factor is calculated in order to consider that 1 g of
fibrin has been distributed on the filter.
[0104] Separate experiments were conducted to test the effects of
diluents. The procedural steps for each experiment went as
follows:
[0105] The filter and the bottom cup of the Amicron filter are
separately weighed, then the fibrin material obtained by mixing
each fibrinogen solution with a 20 IU/mL thrombin solution is put
on the filter.
[0106] A correction factor is calculated in order to consider that
1 g of fibrin has been distributed on the filter.
[0107] The filter is centrifuged at 1000 rpm for 30 minutes.
[0108] At the end of the centrifugation cycle, the bottom cup is
carefully
[0109] removed, weighed and recorded.
[0110] The bottom cup is connected to the filter and centrifuged at
2000 rpm for 30 minutes, at the end of the cycle, the bottom cup is
weighed and the cumulative value recorded.
[0111] Again the bottom cup is connected to the filter and
centrifuged at 3000 rpm for 30 minutes and the bottom cup weighed
at the end of the cycle.
[0112] In the first experiment, the fibrinogen vial was
reconstituted with 3.5 ml- distilled water to obtain a final
concentration of 100 mg/ml of fibrinogen. Dilutions of the
fibrinogen were performed with water in order to respectively
obtain:
[0113] -dilution 1:2 (50 mg/ml) in water
[0114] -dilution 1:4 (25 mg/ml) in water
[0115] -dilution 1:6 (16.6 mg/ml) in water
[0116] -dilution 1:8 (12.5 mg/ml) in water
[0117] Thrombin (Baxter Hyland) was reconstituted with 3.5 ml of 40
mmol CaCl.sub.2 in order to obtain a concentration of 300 IU/mL. A
dilution is performed with CaCl.sub.2 to obtain a thrombin
concentration of 20 IU/mL.
[0118] Fibrinogen solutions were then mixed with an equal volume of
thrombin (20 IU/mL) to obtain a final concentration of "fibrinogen"
respectively of:
[0119] -Sample 1: diluted 1:4 (25 mg/ml)
[0120] -Sample 2: diluted 1:8 (12.5 mg/ml)
[0121] -Sample 3: diluted 1:12 (8.3 mg/ml)
[0122] -Sample 4: diluted 1:16 (6.25 mg/ml)
[0123] For sample 1, the loss of water was 9.5%, 25%, and 45% at
1000, 2000, and 3000 rpm respectively. Sample 2 showed a loss of
water of 18%, 40%, and 70% at 1000, 2000, and 3000 rpm
respectively. The loss of water in sample 3 was 20%, 40%, and 80%
at 1000, 2000, and 3000 rpm respectively. Sample 4 expressed a
water loss of 20%, 72%, and 90% at 1000, 2000, and 3000 rpm
respectively. See Figure 11.
[0124] The second experiment was a comparison of water retention
between fibrin obtained by mixing fibrinogen and thrombin
respectively reconstituted and diluted in PBS and fibrinogen
reconstituted and diluted in PBS with thrombin reconstituted and
diluted in 40 mmol calcium chloride.
[0125] In these comparisons, a vial of fibrinogen was reconstituted
with 3.5 ml PBS (phosphate buffered saline pH=7.2) to reach a final
concentration of 100 mg/mL of fibrinogen.
[0126] Dilutions were performed from this vial in order to obtain
fibrinogen concentrations of:
[0127] - Sample 1 1:2 (50 mg/ml) in PBS
[0128] - Sample 2 1:4 (25 mg/ml) in PBS
[0129] - Sample 3 1:6 (16.6 mg/ml) in PBS
[0130] - Sample 4 1:8 (12.5 mg/ml) in PBS
[0131] In the experiment depicted in Figure 12A, thrombin (Baxter
Hyland) was reconstituted with 3.5 mL of PBS in order to obtain a
concentration of 300 IU/mL. A dilution was performed with PBS to
obtain a thrombin concentration of 20 IU/mL.
[0132] In the experiment depicted in Figure 12B, thrombin (Baxter
Hyland) was reconstituted with 3.5 ml of 40 mmol CaCl.sub.2
(calcium chloride) in order to obtain a concentration of 300 IU/mL.
A dilution was performed with CaCl.sub.2 to obtain a thrombin
concentration of 20 IU/mL.
[0133] The fibrinogen solutions were then mixed with an equal
volume of thrombin (20 IU/mL), resulting in final concentrations
for fibrinogen of:
[0134] - Sample 1: diluted 1:4 (25 mg/ml) in PBS
[0135] - Sample 2: diluted 1:8 (12.5 mg/ml) in PBS
[0136] - Sample 3: diluted 1:12 (8.3 mg/ml) in PBS
[0137] - Sample 4: diluted 1:16 (6.25 mg/ml) in PBS
[0138] For the experiment depicted in Figure 12A, sample 1
expressed a loss of water that was 6%, 10%, and 14% at 1000, 2000,
and 3000 rpm respectively. Sample 2 showed a loss of water of 10%,
21%, and 35% at 1000, 2000, and 3000 rpm respectively. The loss of
water in samples 3 and 4 was nearly identical at 11%, 20%, and 35%
at 1000, 2000, and 3000 rpm respectively. See Figure 12A.
[0139] The introduction of calcium to the fibrin formulation
through the diluent used for the thrombin dilutions in the
experiment depicted in Figure 12B directly affected the water
retention. The loss of water was not significant at 1000 rpm for
the samples, but water losses increased significantly to
approximately 40% at 2000 rpm for samples 2 through 4. At 3000 rpm,
water loss increased to approximately 65% for samples 2 and 3. A
water loss of 80% for sample 4, similar to the result obtained for
fibrin described in experiment 1, was recorded at 3000 rpm. The
results for these experiments support the hypothesis that fibrin
structures essentially free of calcium ions are also tighter, more
compact, and have a greater resistance to water loss from
compression forces. See Figure 12B.
[0140] As a means of verification for the role of phosphate as a
complexing agent of the remaining calcium ions on fibrinogen
molecules, reproductions of the PBS experiment above were conducted
substituted EDTA for PBS in one trial, and citrate of potassium for
PBS in a second trial. The patterns of water loss for both the EDTA
and citrate of potassium trials were consistent with the results
from the experiment utilizing PBS as a reconstitution agent. See
Figures 13A and 13B. These results sustain the hypothesis that
remaining calcium on fibrinogen reacts with phosphate ions
preventing the collateral association of protofibrils producing a
tight fibrin structure more resistant to water loss than fibrin
structures retaining calcium.
[0141] Another experiment was conducted to determine the impact of
the FXIII present in the formulation on the compaction capability
of the fibrin material obtained with PBS as diluent for both
fibrinogen and thrombin. In this experiment, a vial of fibrinogen
(Tisseel from Baxter Hyland-Immuno lot P5488797D) is reconstituted
with 4.0 ml of PBS (dilution 1:2) to reach a final concentration of
50 mg/mL of fibrinogen.
[0142] Dilutions are performed from this vial in order to obtain a
fibrinogen concentration respectively of:
[0143] - Dilution 1:2 (50 mg/ml) in PBS
[0144] - Dilution 1:4 (25 mg/ml) in PBS
[0145] - Dilution 1:6 (16.6 mg/ml) in PBS
[0146] - Dilution 1:8 (12.5 mg/ml) in PBS
[0147] Thrombin (Baxter Hyland) is reconstituted with 3.5 ml of PBS
in order to obtain a concentration of 300 IU/mL. A dilution is
performed with PBS to obtain a thrombin concentration of 20 IU/mL.
The results of this experiment show that there is no difference
between the Baxter Fibrin sealant containing FXIII (experiment 2)
and the Baxter Hyland-Immuno free of FXIII when submitted to the
compaction test. Results obtained from the compaction tests show
the same behavior for the FXIII free sealant. See Figure 14.
[0148] A table summarizing the water retention data follows
below.
Table 1
[0149] This table summarizes the water retention data.
TABLE-US-00002 Experiment Sample Depicted in % Water Loss % Water
Loss % Water Loss Number Figure at 1000 rpm at 2000 rpm at 3000 rpm
1 11 9.5 25 45 1 12A 6 10 14 1 12B Not significant 1 14 4.2 9.4
14.3 2 11 18 40 70 2 12A 10 21 35 2 12B Not significant 40 65 2 14
15 22 3 11 20 40 80 3 12A 11 20 35 3 12B Not significant 40 65 3 14
23 34 4 11 20 72 90 4 12A 11 20 35 4 12B Not significant 40 80 4 14
14 24 34
[0150] It has been postulated that ionic strength of thrombin
regulates the pore size of fibrin clot structure. By using high
ionic strength thrombin solutions one can achieve a fibrin clot
having a smaller pore size than with lower concentration thrombin
solutions. The present example demonstrates a method for
fabricating a fibrin clot material where the concentration does not
govern the pore size of the fibrin clot. As set forth above, by
using a chelating agent to bind to calcium, calcium concentration
does not affect the pore size. Even when a 4 IU/mL thrombin
concentration and a 250 IU/mL thrombin concentration was used, the
resulting fibrin material had substantially the same pore size.
Ionic strength was measured with an osmometer and correlated with
the measurement of the turbidity at 800 nm with a
spectrophotometer. Observation of the sample network structure was
accomplished by scanning electron microscopy. Table 2 below
summarizes the correlation between final osmolarity of the fibrin
samples and their respective optical densities. The results of this
experiment illustrate that ionic strength, as demonstrated through
osmolarity, does not regulate the structure of the fibrin clot.
[0151] Classic fibrin materials obtained by using water (0 mosm) as
a diluent for fibrinogen and CaCl.sub.2 for thrombin (4 IU/ml), for
example, has a final osmolarity of 539 mosm. This is a result of
fibrinogen reconstituted with water at a concentration of 90 mg/ml
(610 mosm) combined with thrombin reconstituted with CaCl.sub.2 at
a kit concentration of 4 IU/ml (468 mosm). The resulting classic
fibrin material is opaque white with an optical density of 2.8 AUFS
when measured with a spectrophotometer at 800 nm.
[0152] Fibrin materials produced with PBS (286 mosm) have a final
osmolarity of 445 mosm. This is a result of fibrinogen
reconstituted and diluted with PBS at a concentration of 25 mg/ml
(588 mosm) combined with thrombin reconstituted and diluted with
PBS at a concentration of 10 IU/mL (315 mosm). The resulting fibrin
material is optically clear with an optical density of 0.5 AUFS
when measured with a spectrophotometer at 800 nm.
[0153] Fibrin materials produced with a citrate buffer at 0.033 M
(100 mosm) have a final osmolarity of 224 mosm. This is a result of
fibrinogen reconstituted and diluted with citrate at a
concentration of 50 mg/ml (336 mosm) combined with thrombin
reconstituted and diluted with citrate at a concentration of 20
IU/ml (112 mosm). The resulting fibrin material is optically clear
with an optical density of 0.45 AUFS when measured with a
spectrophotometer at 800 nm. The fibrin hydogel material in this
experiment is clear and composed of thin fibers with an ionic
strength of less than 300 mosm, which is considered to be the
physiological level. Fibrin obtained by mixing fibrinogen at 12.5
mg/ml with thrombin at 10 IU remains clear (0.8 AUFS) with an
osmolarity of 167 mosm.
[0154] Fibrin materials produced with a citrate buffer at 0.066 M
(190 mosm) have a final osmolarity of 317 mosm. This is a result of
fibrinogen reconstituted and diluted with citrate at a
concentration of 50 mg/ml (435 mosm) combined with thrombin
reconstituted and diluted with citrate at a concentration of 20
IU/ml (200 mosm). The resulting fibrin material is optically clear
with an optical density of 0.23 AUFS when measured with a
spectrophotometer at 800 nm. The fibrin hydogel material in this
experiment is also clear and composed of thin fibers with an ionic
strength of less than 300 mosm, which is considered to be the
physiological level. Fibrin obtained by mixing fibrinogen at 12.5
mg/ml with thrombin at 10 IU remains clear (0.5 AUFS) with an
osmolarity of 260 mosm.
Table 2
[0155] This table summarizes the correlation between final
osmolarity of the fibrin samples and their respective optical
densities. TABLE-US-00003 Final Osmolarity of Optical Denisty
Buffer Fibrin Material (mosm) (AUFS at 800 nm) Water (CaCl.sub.2)
539 2.8 PBS 451 0.255 Citrate (0.033 M) 224 0.45 Citrate (0.066 M)
317 0.23
[0156] The buffers also play an active role in the permeability,
fiber diameter, and mass length ratio of the fibrin material.
Significant differences can be observed between PBS and NaCl (0.15
M). These buffers have the same osmolarity, yet their effects on
fibrin materials are markedly different. See Table 3 below. For a
thrombin concentration of 2 IU/ml reconstituted with NaCl at 0.15
M, the permeability of the fibrin is 30.6 x 10.sup.-12 at a
fibrinogen concentration of 25 mg/ml and 136 x 10.sup.-12 at a
fibrinogen concentration of 12.5 mg/ml. Using PBS as a buffer
yields a fibrin permeability of 6.9 x 10.sup.-12 at a fibrinogen
concentration of 25 mg/ml and 39.5 x 10.sup.-12 at a fibrinogen
concentration of 12.5 mg/ml. This experiment demonstrates that the
fibrin material utilizing PBS as a buffer is nearly five times less
permeable than classic fibrin materials, thus capable of retaining
more water. The diameters of fibers are also affected by the buffer
selected. For a thrombin concentration of 2 IU/ml diluted with NaCl
at 0.15 M, the fibers have a diameter of 0.107 m at a fibrinogen
concentration of 25 mg/ml and 0.14 m at a fibrinogen concentration
of 12.5 mg/ml. Using PBS as a buffer yields fibers with a diameter
of 0.051 m at a fibrinogen concentration of 25 mg/ml and 0.075 m at
a fibrinogen concentration of 12.5 mg/ml. Additionally, the mass
length ratio is also affected by the buffer selected to
reconstitute the fibrinogen and thrombin. At a thrombin
concentration of 2 IU/ml buffered with NaCl at 0.15 M, the mass
length ratio is 8.1 x 10.sup.12 at a fibrinogen concentration of 25
mg/ml and 13.9 x 10.sup.12 at a fibrinogen concentration of 12.5
mg/ml. The use of PBS as a buffer yields fibers with a mass length
ratio of 1.83 x 10.sup.12 at a fibrinogen concentration of 25 mg/ml
and 4.03 x 10.sup.12 at a fibrinogen concentration of 12.5 mg/ml, a
four-fold reduction under NaCl at 0.15 M.
[0157] For a thrombin concentration of 250 IU/ml (Table 4) diluted
with NaCl at 0.15 M, the permeability of the fibrin is 14.24 x
10.sup.-12 at a fibrinogen concentration of 25 mg/ml and 47.7 x
10.sup.-12 at a fibrinogen concentration of 12.5 mg/ml. Using PBS
as a buffer yields a fibrin permeability of 8.9 x 10.sup.-12 at a
fibrinogen concentration of 25 mg/ml and 41 x 10.sup.-12 at a
fibrinogen concentration of 12.5 mg/ml. This experiment
demonstrates that the fibrin material utilizing PBS as a buffer is
less permeable than classic fibrin materials, thus capable of
retaining more water. The diameters of fibers are also affected by
the buffer selected. For a thrombin concentration of 250 IU/ml
diluted with NaCl at 0.15 M, the fibers have a diameter of 0.073 m
at a fibrinogen concentration of 25 mg/ml and 0.083 m at a
fibrinogen concentration of 12.5 mg/ml. Using PBS as a buffer
yields fibers with a diameter of 0.057 m at a fibrinogen
concentration of 25 mg/ml and 0.077 m at a fibrinogen concentration
of 12.5 mg/ml. Additionally, the mass length ratio is also affected
by the buffer selected to reconstitute the fibrinogen and thrombin.
At a thrombin concentration of 250 IU/ml buffered with NaCl at 0.15
M, the mass length ratio is 3.78 x 10.sup.12 at a fibrinogen
concentration of 25 mg/ml and 4.83 x 10.sup.12 at a fibrinogen
concentration of 12.5 mg/ml. The use of PBS as a buffer yields
fibers with a mass length ratio of 2.36 x 10.sup.12 at a fibrinogen
concentration of 25 mg/ml and 4.24 x 10.sup.12 at a fibrinogen
concentration of 12.5 mg/ml.
[0158] These experiments illustrate that the type of buffer used,
differently effects the permeability factor as shown for NaCl and
PBS (Tables 3 and 4). These experiments demonstrate that the
concentration of thrombin has no effect on the permeability factor,
fiber diameter, and mass length ratio as well when PBS, and not
NaCl, is the buffer. PBS is an admixture composed of 0.13 M NaCl
(800 mg/L), KCl (20 mg/L), anhydrous Na.sub.2HPO.sub.4 (115 mg/L),
and KH.sub.2PO.sub.4. Thus, PBS buffer contains NaCl at a molarity
of nearly the NaCl 0.015 M buffer described in Tables 3 and 4. As
the data shows, phosphate is therefore the complexing agent of the
endogenous calcium. Tables summarizing these experiments are
labeled Table 3 and Table 4 below.
Table 3
[0159] This experiment summarizes the Thrombin 2 IU/ml results.
TABLE-US-00004 Fibrinogen Permeability Fiber mass length Buffer
Concentration (K.sub.s) Diameter .mu.m ratio NaCl 0.15 M 25 mg/mL
30.6 .times. 10.sup.-12 0.107 8.1 .times. 10.sup.12 PBS 25 mg/mL
6.9 .times. 10.sup.-12 0.051 1.83 .times. 10.sup.12 NaCl 0.15 M
12.5 mg/mL 136 .times. 10.sup.-12 0.140 13.9 .times. 10.sup.12 PBS
12.5 mg/mL 39.5 .times. 10.sup.-12 0.075 4.03 .times. 10.sup.12
Table 4
[0160] This experiment summarizes the Thrombin 250 IU/ml results.
TABLE-US-00005 Fiber Fibrinogen Permeability Diameter mass length
Buffer Concentration (K.sub.s) .mu.m ratio NaCl 0.15 M 25 mg/mL
14.24 .times. 10.sup.-12 0.073 3.78 .times. 10.sup.12 PBS 25 mg/mL
8.9 .times. 10.sup.-12 0.057 2.36 .times. 10.sup.12 NaCl 0.15 M
12.5 mg/mL 47.7 .times. 10.sup.-12 0.083 4.83 .times. 10.sup.12 PBS
12.5 mg/mL 41 .times. 10.sup.-12 0.077 4.24 .times. 10.sup.12
[0161] Calculation for permeability (Ks):
[0162] (Flow (ml/sec) x time to clot x viscosity
(10.sup.2))/(Pressure x Surface area of clot)
[0163] Calculation for fiber diameter:
[0164] D.sup.2 = 44.1 x Ks x concentration of fibrinogen
(X.sup.1.3736)
[0165] Calculation for mass length ratio:
[0166] = x D.sup.2 x C/ 4X, where C = 4.36 g/cm.sup.3
[0167] The role of PBS as a complexing agent can also be seen by
gel electrophoresis studies of fibrin materials. When an electric
current is applied to an SDS-polyacrylimide gelelectrophoresis
containing fibrin hydrogel material prepared with PBS, little or no
gamma-gamma banding can be seen while a fibronectin band may be
viewed. This demonstrates that the PBS complexes calcium so that
calcium is not available to collaterally associate fibrin through
cross-linking. Classic fibrin materials show distinctive, strong
gamma-gamma band, and no fibronectin band, when prepared with water
or NaCl as a buffer. NaCl buffer with a molarity of 0.15 cannot
block the action of calcium. The NaCl buffer's inability to block
the action of calcium allows calcium to play its traditional role
in collaterally associating fibrin, thus allowing thrombin to
affect the pore size of the fibrin material. By acting as a
complexing agent for endogenous calcium, PBS substantially removes
thrombin's ability to affect pore size. Figure 9 below illustrates
the classic fibrin material in gel electrophoresis. Figure 6 below
illustrates the fibrin hydrogel material in gel
electrophoresis.
[0168] The fibrin hydrogel materials have also been determined to
contain anti-adhesive properties. Tables 5 and 6 below illustrate
the anti-adhesive properties of the fibrin hydrogel materials.
Table 5 shows the results of a side model study on rats. The caecum
and interfacing parietal wall were abraded sufficiently to cause
bleeding. The bleeding surfaces were cauterized to stop the
bleeding on the injured surfaces in both control and test animals.
Two types of fibrin hydrogel materials were formed between the
injured surface. Fibrin film 1 (FF1) differs from fibrin film 2
(FF2) in that FF2 was compressed to release some water. The results
show that the wounds treated with either normal fibrin hydrogel
material (FF1) or compressed fibrin hydrogel material (FF2) have no
adhesions between the caecum surface and the parietal surface. The
control group for Table 5 had no fibrin hydrogel material applied,
resulting in level 3 (the most severe) adhesions between the caecum
surface and the parietal surface.
[0169] Table 6 shows the anti-adhesion properties of hydrogel
fibrin glue material. This hydrogel material polymerizes within the
body of the animal upon application using a delivery device such as
that shown in Figure 6. The fibrin hydrogel glue was applied
directly to the wound on the caecum surface, as well as to the
parietal surface. All animals that received this fibrin hydrogel
glue treatment were free of adhesion. In a second trial, a pre-cast
fibrin hydrogel material was positioned between injured surfaces.
Using a pre-cast fibrin hydrogel material demonstrated significant
anti-adhesion properties as well.
Table 5
[0170] This table summarizes the results of the side model study on
rats. TABLE-US-00006 Animal Product Applied Result Control Rat 1
None adhesion (level 3) Control Rat 2 None adhesion (level 3)
Control Rat 3 None adhesion (level 3) Control Rat 4 None adhesion
(level 3) Rat 1 FF 1 no adhesion Rat 2 FF 1 no adhesion Rat 3 FF 2
no adhesion Rat 4 FF 2 no adhesion
[0171]
Table 6
[0172] This table summarizes the anti-adhesion properties of
hydrogel fibrin glue material. TABLE-US-00007 Number of Thrombin
Group Type Individuals Concentration Result Control 5 Adhesion
(level 3) Pre-cast 5 100 IU/mL 20% adhesion Hydrogel Fibrin Film
Hydrogel 5 100 IU/mL 0% adhesion Fibrin Glue
[0173] While the specific embodiments have been illustrated and
described, numerous modificaitons come to mind without
significantly departing from the spirit of the invention and the
scope of protection is only limited by the scope of the
accompanying claims.
* * * * *