U.S. patent application number 10/664561 was filed with the patent office on 2004-06-10 for methods and compositions for enhancing fibroblast migration.
Invention is credited to Clark, Richard A., Galanakis, Dennis K., Khan, Azim.
Application Number | 20040110686 10/664561 |
Document ID | / |
Family ID | 32473958 |
Filed Date | 2004-06-10 |
United States Patent
Application |
20040110686 |
Kind Code |
A1 |
Clark, Richard A. ; et
al. |
June 10, 2004 |
Methods and compositions for enhancing fibroblast migration
Abstract
Methods and compositions for enhancing fibroblast migration at a
wound site are disclosed. The method includes contacting the wound
site with fibrinogen that is prepared by a process which includes
precipitating plasma with glycine. The compositions includes a
lipid rich component and fibrinogen.
Inventors: |
Clark, Richard A.; (Poquott,
NY) ; Galanakis, Dennis K.; (Stony Brook, NY)
; Khan, Azim; (Coram, NY) |
Correspondence
Address: |
Rogalskyj & Weyand, LLP
PO Box 44
Livonia
NY
14487-0044
US
|
Family ID: |
32473958 |
Appl. No.: |
10/664561 |
Filed: |
September 19, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10664561 |
Sep 19, 2003 |
|
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09500512 |
Feb 9, 2000 |
|
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60119344 |
Feb 9, 1999 |
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Current U.S.
Class: |
514/13.6 ;
514/16.5; 514/561; 514/9.4 |
Current CPC
Class: |
A61K 38/363 20130101;
A61L 24/106 20130101 |
Class at
Publication: |
514/012 ;
514/561 |
International
Class: |
A61K 038/36; A61K
031/198 |
Goverment Interests
[0002] The subject matter of this application was made with support
from the United States Government under National Institutes of
Health Grant No. AG 1101143-12. The United States Government may
have certain rights in this subject matter.
Claims
What is claimed:
1. A method for enhancing fibroblast migration at a wound site
comprising: contacting the wound site with a fibrinogen
preparation, wherein the fibrinogen preparation includes a lipid
rich component.
2. A method according to claim 1 wherein the fibrinogen preparation
further comprises fibrinogen prepared by a process which comprises
precipitating plasma with glycine.
3. A method according to claim 2 wherein the fibrinogen preparation
further comprises a growth factor, an extracellular matrix
material, or mixtures thereof.
4. A method according to claim 2 wherein the precipitating is
carried out by a process which comprises: adding glycine to plasma
to produce a precipitate and a supernatant; dissolving the
precipitate in a buffer to produce a solution; and precipitating
the solution by adding glycine to the solution.
5. A method according to claim 2 wherein the fibrinogen in prepared
by a process comprising: precipitating plasma with glycine to
produce a first precipitate and a first supernatant; dissolving the
first precipitate in a buffer to produce a first solution;
precipitating the first solution by adding glycine to the first
solution to produce a second precipitate and a second supernatant;
dissolving the second precipitate in a buffer to produce a second
solution; and precipitating the second solution by adding ammonium
sulfate to the second solution to produce a third precipitate and a
third supernatant.
6. A method according to claim 5 wherein the third supernatant
comprises a lipid rich layer.
7. A method according to claim 6 wherein the third supernatant is
further treated to produce the lipid rich component.
8. A method according to claim 7 wherein the third supernatant is
precipitated to produce the lipid rich component.
9. A composition comprising: a lipid rich component and
fibrinogen.
10. A composition according to claim 9 wherein the fibrinogen has a
purity of above 95%.
11. A composition according to claim 9 wherein the fibrinogen has a
purity of about 99%.
12. A composition according to claim 9 wherein the fibrinogen is
prepared by a process which comprises precipitating plasma with
glycine.
13. A composition according to claim 12 wherein the fibrinogen is
prepared by a process which comprises: precipitating plasma with
glycine to produce a first precipitate and a first supernatant;
dissolving the first precipitate in a buffer to produce a first
solution; precipitating the first solution by adding glycine to the
first solution to produce a second precipitate and a second
supernatant; dissolving the second precipitate in a buffer to
produce a second solution; and precipitating the second solution by
adding ammonium sulfate to the second solution to produce a third
precipitate and a third supernatant.
14. A composition according to claim 9 wherein the lipid rich
component is prepared by a process which comprises precipitating
plasma with glycine.
15. A composition according to claim 14 wherein the lipid rich
component is prepared by a process which comprises: precipitating
plasma with glycine to produce a first precipitate and a first
supernatant; dissolving the first precipitate in a buffer to
produce a first solution; precipitating the first solution by
adding glycine to the first solution to produce a second
precipitate and a second supernatant; dissolving the second
precipitate in a buffer to produce a second solution; precipitating
the second solution by adding ammonium sulfate to the second
solution to produce a third precipitate and a third supernatant;
and precipitating the third supernatant to produce the lipid rich
component.
Description
[0001] The present invention is a divisional of U.S. patent
application Ser. No. 09/500,512, filed Feb. 9, 2000 which claims
priority to U.S. Provisional Patent Application No. 60/119,344,
filed Feb. 9, 1999, which is hereby incorporated by reference.
FIELD OF THE INVENTION
[0003] The subject invention is directed to methods and
compositions for enhancing fibroblast migration and promoting wound
healing.
BACKGROUND OF THE INVENTION
[0004] Throughout this application various patents and publications
are referenced, many in parenthesis. Full citations for each of the
referenced publications are provided at the end of the Detailed
Description. The disclosures of each of these patents and
publications in their entireties are hereby incorporated by
reference in this application.
[0005] It is estimated that in 1992, 35.2 million wounds required
major therapeutic intervention in the US (Medical Data
International, Inc. 1993). Surgical incisional wounds are performed
with aseptic technique, and are closed by primary intention. Most
repair and heal uneventfully. Many traumatic wounds and cancer
extirpations, however, must be left open to heal by extirpations,
however, must be left open to heal by secondary intention.
Furthermore, chronic wounds have significant tissue necrosis and
fail to heal by secondary intention. It is estimated that 5.5
million people in the US have chronic, nonhealing wounds and that
their prevalence is increasing secondary to the increase in
age-related diseases, the increase in Acquired-immune Deficiency
Syndrome ("AIDS"), and the increase of radiation wounds secondary
to cancer intervention. In the US, approximately 1.5-2.5 million
people have venous leg ulcers; 300,000-500,000 people have diabetic
ulcers; and 2.5-3.5 million people have pressure ulcers (Callam et
al. (1987); Phillips and Dover (1991); Lees and Lambert (1992);
Lindholm et al. (1992)). These acute and chronic open wounds
require long-term care and procedures that include skin grafting
and tissue flaps, debridement, frequent dressing changes, and
administration of pain medications. This care is costly and labor
intensive. Furthermore, these wounds have a severe impact on the
patients' quality of life. The chronic dermal ulcerations can cost
as much as $40,000 each to heal, and more disappointing is that 50%
reappear within 18 months of healing. Chronic dermal ulcers are
also associated with mortality. As many as 21% of patients in
intermediate-care facilities with pressure ulcers die (Bergstrom et
al. (1994)).
[0006] Although multiple millions of dollars have been spent on the
development of numerous recombinant growth factors (Abraham and
Klagsbrun (1996); Heldin and Westermark (1996); Nanney and King
(1996); Roberts and Sporn (1996)) and organotypic skin replacements
(Boyce et al. (1995)) for use in open wounds over the past decade,
the evidence of cost-effective benefit is meager thus far (Brown et
al. (1989); Robson et al. (1992a); Robson et al. (1992b); Phillips
et al. (1993)).
[0007] Many attempts have been made to produce a composition which
can be used to facilitate wound repair.
[0008] Many of these compositions involve collagen as a component.
U.S. Pat. Nos. 4,950,483 and 5,024,841 each discuss the usefulness
of collagen implants as wound healing matrices. U.S. Pat. No.
4,453,939 discusses a wound healing composition of collagen with a
fibrinogen component and a thrombin component, and optionally
fibronectin. U.S. Pat. No. 4,970,298 discusses the usefulness of a
biodegradable collagen matrix (of collagen, hyaluronic acid, and
fibronectin) for wound healing. Yamada et al. (1995) disclose an
allogeneic cultured dermal substitute that is prepared by plating
fibroblasts onto a spongy collagen matrix and then culturing for 7
to 10 days. Devries et al. (1995) disclose a collagen/alpha-elastin
hydrolysate matrix that can be seeded with a
stromal-vascular-fraction of adipose tissue. Lamme et al. (1996)
disclose a dermal matrix substitute of collagen coated with elastin
hydrolysate. U.S. Pat. No. 5,489,304 and Ellis and Yannas (1996)
each disclose a collagen-glycosaminoglycan matrix.
[0009] There are also numerous compositions which involve
hyaluronic acid ("HA") as a component. Ortonne (1996), Borgognoni
et al. (1996), and Nakamura et al. (1997) each discuss the
usefulness of HA for wound healing. In Nakamura et al. (1997), HA
was combined with chondroitin sulfate in one series of experiments.
In U.S. Pat. No. 5,604,200, medical grade HA and tissue culture
grade plasma fibronectin were used in combination with calcium,
phosphate, uric acid, urea, sodium, potassium, chloride, and
magnesium to create a moist healing environment that simulates the
fetal in utero wound healing matrix. U.S. Pat. No. 5,631,011
discloses a composition of HA and fibrin or fibrinogen.
[0010] Various other compositions have also been explored for their
wound healing capabilities. Kratz et al. (1997) used a gel of
heparin ionically linked to chitosan. Bartold and Raben (1996)
studied platelet-derived growth factor ("PDGF"). Henke et al.
(1996) disclosed that chondroitin sulfate proteoglycan mediated
cell migration on fibrinogen and invasion into a fibrin matrix,
while Nakamura et al. (1997) concluded that chondroitin sulfate did
not affect wound closure in a corneal epithelial wound. Henke et
al. (1996) also disclosed that an anti-CD44 antibody blocked
endothelial cell migration on fibrinogen. U.S. Pat. No. 5,641,483
discloses topical gel and cream formulations containing human
plasma fibronectin for healing cutaneous wounds. Schultz et al.
(1992) discloses a composition of epidermal growth factor ("EGF"),
fibronectin, a synthetic collagenase inhibitor, and Aprotinin.
[0011] Fibrin matrices and components of fibrin matrices have been
investigated for promoting wound healing. Besides being the
ultimate plug of the hemostasis system, fibrin is part of a
provisional matrix that provides tissue cells a scaffold for
repopulation of a wound (Clark et al. (1982a)). Recently, however,
it was discovered that fibroblasts, tissue mesenchymal cells, will
not penetrate a pure fibrin clot (Greiling and Clark (1997)).
Another plasma protein, fibronectin, normally found in blood clots
must be present in the clot for fibroblast migration (Greiling and
Clark (1997)).
[0012] Some fibrinogen preparations have been found to improve
healing. Fibrin sealants, or glues, are topical, biologically
compatible, resorbable tissue adhesive that initiate the last
phases of coagulation during wound healing. The components of
fibrin sealants typically consists of concentrated human fibrinogen
in solution with various amounts of fibronectin and factor XIII, as
well as other components. The fibrin sealants are activated by
addition of thrombin and calcium chloride and subsequently form a
coagulum (clot). Methods of obtaining the concentrated fibrinogen
include precipitation of plasma by cryroprecipitation, polyethylene
glycol or ammonium sulphate (Brennan (1991)). Contradictory results
are obtained, however, for these preparations. In some instances,
the fibrinogen preparations improve healing (Gelich et al. (1995);
Saclarides et al. (1992), in others no improvement in healing was
found (Lasa et al. (1993); Byrne et al. (1992). Further, animal
studies have not been predicative of clinical use. In addition, the
fibrin sealants utilized to date vary in purity levels.
[0013] In view of the severity of the problem of chronic,
nonhealing wounds, new and more effective matrices and methods for
facilitating wound healing, and in particular, fibroblast migration
are needed. The present invention is directed to meeting this
need.
SUMMARY OF THE INVENTION
[0014] The present invention relates to a method for enhancing
fibroblast migration at a wound site. The method includes
contacting the wound site with fibrinogen that is prepared by a
process which includes precipitating plasma with glycine.
[0015] Another aspect of the present invention relates to a method
for enhancing fibroblast migration at a wound site which includes
contacting the wound site with a fibrinogen preparation which
includes a lipid rich component.
[0016] Another aspect of the present invention relates to a
composition which includes fibrinogen and a lipid rich
component.
[0017] Thus, the present invention relates to a method and a
composition for enhancing wound healing by enhancing fibroblast
migration to the wound site. By enhancing fibroblast migration,
wounds that require the rapid formation of new tissue, such as
chronic cutaneous ulcers and fresh surgical and traumatic wounds
that cannot be closed, can be treated. Fibrinogen preparations that
merely act as a blood clotting mechanism cannot achieve this
objective.
DETAILED DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a schematic illustration of the three
sub-fractions of one embodiment of the fibrinogen preparation of
the present invention.
[0019] FIG. 2 is a chromatogram illustrating the fibrinogen peak
and lipoprotein peak of a preparation of the present invention.
[0020] FIG. 3 is a comparison of the effect on fibroblast migration
of fibrinogen isolates C1 (positive control), C2 (negative
control), 2H, 2F, 2HL and 2FL.
[0021] FIG. 4 illustrates the fibroblast migration activity of the
lipoprotein peak of FIG. 2.
DETAILED DESCRIPTION OF THE INVENTION
[0022] The present invention relates to a method for enhancing
fibroblast migration at a wound site. The method includes
contacting the wound site with fibrinogen that is prepared by a
process which includes using glycine to precipitate fibrinogen from
plasma.
[0023] Another aspect of the present invention relates to a method
for enhancing fibroblast migration at a wound site which includes
contacting the wound site with a fibrinogen preparation which
includes a lipid rich component.
[0024] Another aspect of the present invention relates to a
composition which includes fibrinogen and a lipid rich
component.
[0025] As used herein, "enhancing fibroblast migration" is meant to
include any improvement or increase in the movement or mobility of
fibroblast cells in a wound. Fibroblast migration can be measured
by a variety of methods. One particularly advantageous method is
described in U.S. Pat. No. 5,935,850 (the contents of which are
hereby incorporated by reference herein); Greiling and Clark
(1996); and Greiling and Clark (1997). Briefly, organotypic dermal
constructs consisting of human adult dermal fibroblasts embedded in
floating type 1 collagen gels are pasted on 24 well tissue culture
dishes coated with fibrin fibrils. Fibrin gels are then cast around
the "dermal equivalent" forming an "inside-out" wound construct.
The number of fibroblast cells that migrate in the presence and
absence of various soluble biologic response modifiers (such as the
fibrinogen preparations of the present invention) can then be
quantified, for example using a Nikon inverted phase microscope, by
visually counting identifiable fibroblast cell nuclei located
outside of the construct. Preferably, the increase in fibroblast
migration produced by contacting the wound with fibrinogen in
accordance with the present invention (e.g., (A-A.sub.o)/A.sub.o,
where A and A.sub.o are the number of identifiable fibroblast cell
nuclei located outside of the above-described construct in the
presence and absence, respectively, of fibrinogen) is at least
about 0.5, more preferably at least about 1.0, and most preferably
at least about 1.5.
[0026] Further, as used herein, a "wound" and "wound site" are
intended to include both acute and chronic dermal wounds including,
for example, surgical incisional wounds, traumatic wounds, cancer
extirpations, radiation wounds, venous leg ulcers, diabetic ulcers,
and pressure ulcers.
[0027] The plasma employed in the present invention is collected by
conventional methods and, in practice, can be from blood of a
single individual, or, alternatively, it can be pooled from
multiple individuals. Preferably the plasma is from the same
species of animal (e.g., human) as the wound being treated.
[0028] As indicated above, fibrinogen is isolated from the plasma
by precipitation. In particular, the method of precipitation is
achieved with glycine and is carried out in a number of steps.
[0029] As a first step, the plasma is precipitated with glycine to
produce a precipitate and a supernatant in a manner known to those
of ordinary skill in the art. A preferred method is described in
Galanakis (1995). Preferably, precipitation is carried out by
adding glycine to the plasma in an amount such that the final
concentration of glycine in the plasma/glycine mixture is from
about 1.0 to about 2.1 M. Preferably, the glycine is added as dry
glycine to the mixture. Once the addition is complete,
precipitation is allowed to proceed during incubation. Incubation
proceeds at temperatures below room temperature (e.g.,
refrigeration temperatures), preferably from between about
2.degree. C. and about 7.degree. C., more preferably about
5.degree. C. Incubation occurs for from about 30 minutes to about
12 hours, preferably about 1 hour, until the precipitate is formed.
In practice, it is most convenient to conduct the precipitation by
placing the plasma/glycine mixture in a standard refrigerator
(i.e., at about 5.degree. C.). After incubation, a precipitate and
a supernatant are produced, which can be separated by conventional
methods, such as decanting or, preferably, centrifuging at
temperatures from between about 2.degree. C. and about 7.degree.
C., preferably about 5.degree. C. The precipitate will contain
about 90% of the fibrinogen from the plasma. The purity of the
fibrinogen is above 50%.
[0030] If a preparation having high purity content is desired,
fibrinogen is further isolated from the precipitate of the first
step. A high purity content fibrinogen is defined as fibrinogen
having a purity content of about or above 99%.
[0031] A second step is used to isolate fibrinogen from the
precipitate of the first step. As used herein, second step
generally refers to the process of adding buffer to a precipitate
to produce a mixture, adding glycine to the mixture to produce a
precipitate and a supernatant, and separating the precipitate and
supernatant. This second step can be repeated as many times as
desired.
[0032] Typically, the precipitate produced in the first step is
dissolved in a suitable buffer to produce a solution. Preferably,
the buffer has a pH of from about 6 to about 8, preferably from
about 6.2 to about 7.6, most preferably about 6.4. One suitable
buffer for carrying out this process contains about 150 mM of
sodium chloride, about 10 mM sodium phosphate, and 100 mM
epsilon-aminocaproic acid in water, preferably in sterile water
suitable for injection. Other buffers suitable in the present
invention include 10 mM Tris-HCl (pH 7.4) and 150 mM NaCl. The
amount of buffer employed to effect the dissolution is preferably
from about 30% to about 40% of the volume of the original plasma
used in the first step. That is, if the precipitate is precipitated
in the first step from plasma having a volume of V, the buffer used
in this second step preferably has a volume of from about 0.3 V to
about 0.4 V (i.e., between about {fraction (3/10)}'s of V to about
{fraction (4/10)}'s of V). More preferably, the volume of buffer
employed to effect the dissolution of the precipitate is about 35%
of the volume of the plasma used in the first step.
[0033] Glycine, typically dry (as described above), and at a
suitable concentration is then added to the resulting solution in
an amount such that the final concentration of glycine in the
resulting mixture is from about 1.7 to about 2.2 M and, more
preferably, about 2.1 M. The resulting mixture is incubated,
preferably at a temperature of from between about 2.degree. C. to
about 7.degree. C., most preferably at about 5.degree. C., for from
about 30 minutes to about 2 hours, preferably for about one
hour.
[0034] As a result, a precipitate and supernatant form, which are
separated, preferably by centrifugation at from between about
2.degree. C. to about 7.degree. C., most preferably at about
5.degree. C.
[0035] This second step is advantageously repeated several times.
Preferably, the second step is repeated at least twice. The
precipitate at the end of the second step will contain about 60% of
the fibrinogen from the plasma. The fibrinogen is at a purity of
about 90%.
[0036] To produce fibrinogen having a low purity content,
fibrinogen can be additionally or alternatively isolated from the
supernatant (instead of the precipitate) of the original
plasma/glycine mixture of the first step in a manner similar to
that described above. As used herein, low purity fibrinogen means
fibrinogen having a purity content of about or above 95%, but below
99%. The low purity fibrinogen is produced by adding glycine,
typically dry and at a suitable concentration, to the supernatant
of the first step to produce a mixture. The resulting mixture is
incubated, preferably at refrigerator temperatures, for about 1
hour. As a result, a precipitate and supernatant form, which are
separated, preferably by centrifugation at refrigerator
temperatures. The precipitate can then be dissolved in an
appropriate buffer (e.g., the ones described above), glycine added
(preferably to a final glycine concentration of about 2.1 M), the
mixture incubated, and the resulting precipitate separated. This
process can be repeated several times.
[0037] Irrespective of whether the fibrinogen is isolated as a
precipitate from the precipitate or supernatant or both of the
original plasma/glycine mixture described above, the precipitate
can be advantageously further treated to purify the fibrinogen.
Typically, the further treatment includes dissolving the
precipitate resulting from the glycine precipitation(s) described
above in an appropriate buffer (e.g., as described above) to
produce a solution where the precipitate is present in a volume of
1/2 to 1/3 of the original plasma, with 1/3 being especially
preferred, and precipitating this solution. Preferably, the
precipitation is achieved by adding a compound such as ammonium
sulfate to the solution. Typically, the ammonium sulfate is added
as a saturated solution, and the amount of ammonium sulfate in the
solution is about 25 percent of its saturation level. The resulting
solution is redissolved in a suitable buffer (as described above)
and reprecipited, preferably with dialysis in 0.3 M NaCl.
[0038] Using the method of the present invention, a precipitate
fraction of fibrinogen with a purity of greater than 95% (as
ascertained by SDS-polyacrylamide gel electrophoresis ("SDS-PAGE"))
is obtained. When high purity fibrinogen is desired, using the
method of the present invention fibrinogen having a purity of about
or above 99% is obtained.
[0039] In addition, a lipid rich fraction is obtained. The lipid
rich solids are found floating in the final supernatant produced by
either method described above. The lipid rich solids are separated
from the final supernatant (typically by centrifugation), added to
a suitable buffer (as described above) and reprecipited, preferably
with dialysis in 0.3 M NaCl, and treated with 25% saturated
ammonium sulfate (as described above). The precipitate from this
step is discarded and the resultant supernatant is rich in plasma
lipids. This supernatant which includes lipids is called the lipid
rich component. Although not meaning to be bound by theory, the
lipids contained in the lipid rich component result in enhanced
fibroblast migration when applied to a wound. Alternatively,
proteins bound to the lipids of the lipid rich layer result in
enhanced fibroblast migration when applied to a wound.
[0040] Once prepared in the above-described manner, the fibrinogen
and the lipid rich component are stored, preferably at from about
-50.degree. C. to about -80.degree. C. Prior to use, they can be
thawed, for example at 37.degree. C.
[0041] The fibrinogen prepared in accordance with the
above-described methods enhances fibroblast migration during wound
healing and, thus, enhances wound healing. Further, the lipid rich
component enhances fibroblast migration during wound healing.
[0042] Enhancement of wound healing refers to the traditional sense
of wound healing where clean closure of the wound occurs. Since
naturally occurring wound healing involves the movement of
fibroblasts into the wound site, enhancement of wound healing can
be assayed in vitro using the model for cell transmigration
provided in U.S. Pat. No. 5,935,850 (the contents of which are
incorporated by reference herein), Greiling and Clark (1996), and
Greiling and Clark (1997). Briefly, the model provides a contracted
collagen gel containing fibroblasts surrounded by a fibrin gel.
When fibrinogen prepared as described above is contacted with the
fibrin gel, fibroblast movement from the collagen gel into the
fibrin gel is enhanced.
[0043] Accordingly, the above described method of enhancing
migration of fibroblasts at a wound site can advantageously further
include contacting the wound site with other materials which
promote wound healing. Such contacting with other materials that
promote wound healing can occur prior to, during, and/or after the
wound site is contacted with fibrinogen. For example, the wound
site can be contacted with a growth factor (such as
platelet-derived growth factor ("PDGF") (described in, for example,
Seppa et al. (1982) and in Senior et al. (1985)) or an
extracellular matrix material (such as, fibronectin and
hyaluronan). Preferably, the wound site is contacted with
fibronectin in addition to fibrinogen prepared by the process
described above. In a preferred embodiment, fibronectin is added to
the fibrinogen by mixing the fibronectin with the fibrinogen.
Alternatively, the wound site can be contacted with more than one
growth factor, more than one extracellular matrix material, or
combinations of growth factor(s) and extracellular matrix
material(s). Advantageously, the wound site is contacted with the
lipid rich component. The wound site can be contacted with the
lipid rich component either alone or in combination with
fibrinogen, one or more growth factors, one or more extracellular
matrix materials or a combination thereof.
[0044] The fibrinogen prepared as described above (with or without
the lipid rich component) can be contacted with the wound site by
incorporating it in a fibrinogen preparation and then contacting
the fibrinogen preparation with the wound site. The fibrinogen
preparation generally contains fibrinogen which enhances fibroblast
migration. In addition, inert additives may be incorporated into
the fibrinogen preparation. These include preservatives,
dispersants, diluents, and other physiologically compatible
materials. Where the fibrinogen is to be used concurrently with
growth factor(s), extracellular material(s), or combinations
thereof, the growth factor(s) and/or extracellular matrix
material(s) can advantageously be incorporated in the fibrinogen
preparation. Alternatively, the fibrinogen, lipid rich component,
growth factors, extracellular materials, or combinations thereof
can be contacted with the wound site separately.
[0045] In cases where the wound to be treated is bleeding, the
fibrinogen preparation can further include thrombin so that the
fibrinogen preparation, in addition to enhancing fibroblast
migration, also promotes blood clotting. However, unlike
conventional fibrinogen preparations, the fibrinogen preparation of
the present invention (i.e., the fibrinogen preparation containing
fibrinogen prepared as described above) has utility even if it is
substantially free of thrombin, because it is useful for promoting
fibroblast migration in all wounds, including in wounds that are
not bleeding. As used herein, wounds that are not bleeding are
meant to include wounds that may be oozing blood but that would not
be considered by the skilled clinician as requiring intervention to
induce or promote clotting. Fibrinogen preparations that are
substantially free of thrombin are meant to include preparations in
which the thrombin level is below the level which is generally
viewed as being necessary to promote clotting when used in
conjunction with fibrinogen. Examples of fibrinogen preparations
that are substantially free of thrombin include fibrinogen
preparations in which the weight-to-weight ratio of thrombin to
fibrinogen is less than 1, less that 0.8, less than 0.5, less than
0.3, or less than 0.1.
[0046] As indicated above the fibrinogen or fibrinogen preparation
of the present invention is contacted with the wound or wound site.
Contacting can be carried out by any suitable method. For example
the fibrinogen or fibrinogen preparation can be delivered to the
wound site via a syringe or pipet or by spraying or misting
fibrinogen or fibrinogen preparation onto the wound. Lyophilized
fibrinogen or fibrinogen preparation can be used directly in
powdered form, for example by sprinkling the powder on the wound.
Alternatively, the fibrinogen or fibrinogen preparation can be
applied to the wound by incorporating it in a gauze pad, sponge,
collagen or gel-type matrix and then applying the gauze pad,
sponge, collagen or gel-type matrix to the wound. As explained
above, the fibrinogen or fibrinogen preparation of the present
application can be contacted with wounds that are bleeding or with
wounds that are not bleeding. For example, the fibrinogen or
fibrinogen preparation of the present application can be first
contacted with the wound while the wound is bleeding and said
contacting can be continued after bleeding ceases. For purposes of
the present application, the contact which is maintained subsequent
to cessation of bleeding is to be considered to be a contact with a
non-bleeding wound. Alternatively, the fibrinogen or fibrinogen
preparation of the present application can be contacted with a
wound only after bleeding from the wound stops.
[0047] The amount of fibrinogen or fibrinogen preparation to be
applied to the wound depends on a variety of factors, such as the
size of the treatment site, the nature and condition of the wound
in need of treatment, and factors that might be unique to the
patient being treated. The optimal therapeutic amount for a
specific wound can be determined by contacting the wound site with
various concentrations of fibrinogen or fibrinogen preparation and
observing the effect on wound healing.
[0048] The present invention is further illustrated by the
following examples.
EXAMPLES
[0049] Materials and Methods
[0050] Fibrinogen Preparations from Precipitate
[0051] The fibrinogen preparations of the present invention were
prepared from a 1M glycine precipitation formed in the cold
(4.degree. C.) that had been discarded in a previously described
procedure (Galanakis (1995)). In particular, glycine was dissolved
in pooled human plasma to attain 1 M final concentration and the
mixture was allowed to stand on ice for at least one hour. By
subsequent centrifugation at 5.degree. C., two fractions were
obtained: the precipitate and the supernatant. The precipitate
obtained from plasma containing 1 M glycine, 5.degree. C., was
redissolved (pH 6.4) and re-precipitated twice with 2.1 M glycine,
5.degree. C. The precipitate was then dissolved, precipitated with
(NH.sub.4).sub.2SO.sub.4 to 25% saturation, redissolved and
exhaustively dialyzed vs. 0.3M NaCl. The final product was stored
at -80.degree. C. Greater than 95% purity was ascertained by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The gels also
disclosed the expected doublet of fibrinogen bands I and II
indicating that these bands did not differ from those of fibrinogen
in unfractionated plasma.
[0052] Fibrinogen Preparations from Supernatant
[0053] The following preparations were purified from the
supernatant produced during precipitation of the fibrinogen
precipitate described above.
[0054] Sample 1 was isolated as previously described (Galanakis
(1995)). It was the purest fibrinogen isolate. It had virtually all
its alpha chains intact but lacked molecules with gamma chains that
have an extended carboxy terminal which constitute approximately
15% of the fibrinogen in plasma. The extended gamma chains are from
a splicing mRNA variant. It extends the gamma chain by deleting the
last two amino acids of the regular gamma chain and extending it
with a 20 amino acid segment.
[0055] Sample 2 was isolated by a modification (Galanakis (1995))
of the procedure of Mosesson and Sherry (1966). Sample 2 was a
highly pure fibrinogen isolate, but less pure than Sample 1, in
that a minute contaminant of Factor XIII was detectable by biologic
activity. It also had virtually all of its alpha chains intact and
contained molecules with both extended and non-extended gamma
chains.
[0056] Sample 3 was the same as Sample 2, but was enriched with
soluble fibrin. Fibrin monomer was prepared as described in
Galanakis et al. (1987). An amount of fibrin monomer (from a stock
solution of 20 to 30 mg/ml, pH 4.5) was added to exceed 10% (mg/mg)
of fibrinogen in solution, allowed to equilibrate at 37.degree. C.
and any clot that formed was removed. The resulting fibrinogen
solution was termed fibrin-saturated and used. Care was taken to
ascertain that the pH of the fibrinogen solution remained above 6
in storage and during clotting.
[0057] Sample 4 was isolated by a modification (Galanakis (1995))
of the procedure of Mosesson and Sherry (1966). Sample 4 had a
purity similar to that of Sample 3, but contained a major
population of molecules (approx. 20 to 30%) which were clottable
but had degraded alpha chains.
[0058] Sample 5 was isolated by a modification (Galanakis (1995))
of the procedure of Mosesson and Sherry (1966). Sample 5 was
similar to Sample 4 in structure but less pure in that trace, but
detectable, amounts of fibronectin and factor XIII were
present.
[0059] Sample 6 was similar in all respects to Sample 5, with the
exception of being enriched with soluble fibrin. Fibrin monomer was
prepared and added to Sample 5 as described above for Sample 3.
[0060] Sample 7 was isolated from the supernatant produced in
Sample 1 above. It contained only minor amounts of fibrinogen, but
was rich in plasma lipids.
[0061] Normal Human Dermal Fibroblasts
[0062] Primary cultures of human adult dermal fibroblasts, acquired
from Marcia Simon (Living Skin Bank, SUNY at Stony Brook), the ATCC
(Bethesda, Md.), or the NIA (Bethesda, Md.), were cultured in
Dulbecco's Modified Eagle's Medium (DMEM, Life Technologies)
containing 42 mM sodium bicarbonate and supplemented with 100 U/ml
penicillin, 100 .mu.g/ml streptomycin, and 10% fetal bovine serum
(FBS, HyClone, Logan, Utah) at 370 C and 5% CO.sub.2/95% air in a
humidified atmosphere.
[0063] Preparation of Floating Contracted Collagen Gels
[0064] Fibroblast cultures at 80% confluence were harvested by
treatment with a 0.05% trypsin/0.01% EDTA. Trypsin is inactivated
by addition of soy bean trypsin inhibitor in PBS containing 0.2%
BSA. The cells are washed twice with DMEM+2% BSA and resuspended at
a concentration of 1.times.10.sup.6 cells/ml. The fibroblasts are
mixed with neutralized collagen (Vitrogen 100, Celtrix Labs., Santa
Clara, Calif.), 2% BSA, 30 ng/ml PDGF-BB, 30 .mu.g/ml fibronectin,
and concentrated DMEM so that the final concentration of DMEM and
sodium bicarbonate is lx. 600 .mu.l of the cell mixture is added to
the wells of a 24-well tissue culture plate, which has been
precoated with 2% BSA. The collagen is allowed to polymerize at
37.degree. C. The final concentration of collagen is 1.8 mg/ml and
each gel contains 6.times.10.sup.4 cells. After two hours
incubation, the gels are gently detached from the plastic surface
to allow contraction with the addition of 0.5 ml DMEM+2% BSA and 30
ng/ml PDGF-BB per well. The gels are incubated overnight at
37.degree. C. in 100% humidity, 5% CO.sub.2 and 95% air.
[0065] Preparation of the Three-Dimensional Transmigration
Model
[0066] As described in Greiling and Clark (1997), dried fibrin
fibril-coated dishes are washed once with PBS and
fibroblast-contracted collagen gels are placed on the surface.
Fibrinogen, as produced by the method of the present invention (or
as a comparison sample) at a final concentration of 300 .mu.g/ml is
mixed with DMEM and 1.0 U/ml thrombin, added to the wells so that
the solution is level with the top of the collagen gel, and allowed
to clot at room temperature for 30 minutes. When needed, other
supplements, such as 30 ng/ml PDGF-BB, are added to the mixture.
The migration assays are quantified after a 24 hour incubation
period at 37.degree. C. in 100% humidity, 5% CO.sub.2 and 95%
air.
[0067] Evaluation of Cell Migration
[0068] The number of migrated cells was quantified under a Nikon
inverted phase microscope by visually counting identifiable cell
nuclei located outside of the contracted collagen gel in the fibrin
gel. Within a given experiment, each condition was run in
triplicate and means.+-.SD calculated. All experiments were
repeated at least three times. Statistical differences among
conditions can be determined by ANOVA.
Example 1
[0069] The fibrinogen preparations described above were tested to
determine if fibroblast migration was enhanced by the fibrinogen
preparation of the present invention.
[0070] Results
[0071] The preparations were tested using the three-dimensional
transmigration assay described above and in Greiling and Clark
(1997). While preparations of the present invention (i.e. produced
from the precipitate), when clotted, were found to permit
fibroblast migration, most preparations from the supernatant had
little or no activity. A crude preparation of fibrinogen obtained
from Calbiochem was used as a control in these assays. This
commercial laboratory grade fibrinogen was also used in the study
on fibroblast migration through fibrin gels (Greiling and Clark
(1997)). The activity in Sample 4 was intrinsically unstable as it
was lost after 2 freeze/thaw cycles. Although little activity was
found in Sample 5, this fraction had relatively good activity when
saturated with fibrin monomers. Although these data suggest that
the activity is attributable to fibrin monomer, when another
aliquot of the same fibrin monomer preparation was added to Sample
2, no activity was observed. The presence or absence of fibronectin
in these fibrinogen preparations was not responsible for their
differential activity, because fibronectin was always added to the
fibrinogen at the time of assay. In addition, whether Factor XIII
was responsible for the activity was examined. When the plasma
transglutaminase (Calbiochem) was added to Sample 1 fibrinogen (100
nM Factor XIII/M fibrinogen), no discernable activity was observed.
A real possibility is that two or more factors, in addition to
fibronectin, are necessary for fibroblast migration. For example,
perhaps both Factor XIII and fibrin monomers may be necessary for
migration.
[0072] Discussion
[0073] During fibrinogen isolation from plasma, a relatively low
solubility fraction can be harvested with 1M glycine that is
fibrin-rich and contains other proteins with low solubility. In
typical fibrinogen purification schemes, however, this fraction is
usually discarded because it is not readily soluble in physiologic
buffer systems. It has been used, however, to isolate other
proteins including fibronectin, Factor VIII and Factor XIII.
Although there are alternative ways to obtain low solubility
proteins from plasma, (e.g. plasma cryoprecipitation, differential
or graduated ethanol precipitation at very low temperatures,
salting out by various precipitants such as glycine, beta alanine,
and ether) the present invention focuses on the fibrinogen isolate
from the precipitate, since it is the only low solubility protein
fraction from plasma which supports fibroblast migration. In
addition, the present invention is directed to the lipid rich
component, which also supports fibroblast migration.
[0074] Although an explosion of information on the molecular and
cellular biology of wound repair has accumulated over the past
decade (Clark (1996b)), surprisingly little was known about the
induction of new tissue, called granulation tissue, a critical
event in the healing process. Recently several seminal observations
about new tissue formation in wounds were made (Greiling and Clark
(1997); Gailit et al. (1996); Gailit and Clark (1996); McClain et
al. (1996); Xu et al. (1996); Gailit et al (1997)). This
information has been used to develop a fibrin matrix composite that
promotes fibroblast recruitment from an adjacent collagenous matrix
(Greiling and Clark (1997)). This matrix has potential for use in
freshly debrided chronic cutaneous ulcers and fresh surgical and
traumatic wounds that cannot be closed.
[0075] During the first few days after injury, a fibrin clot is
deposited in the wound space (Welch et al. (1990)). The clot
contains fibrin, fibronectin and vitronectin which together provide
a provisional matrix scaffold for the movement of recruited cells
into the wound space (Greiling and Clark (1997); Clark (1993a);
Clark (1993b)). Concomitantly, platelets release a plethora of
growth factors including the potent mesenchymal cell mitogen,
platelet-derived growth factor (PDGF) (Heldin et al. (1996)).
Subsequently, blood leukocytes, especially neutrophils and
monocytes, migrate into the fibrin-rich provisional matrix. As
monocytes mature into macrophages they begin to produce growth
factors, including PDGF-BB, which are added to the wound space
milieu (Rappolee et al. (1988); Shaw et al. (1990)). In response to
these growth factors, fibroblasts in the underlying subcutaneous
tissue, and in the adjacent dermis, to a lesser extent, proliferate
(Clark (1993a)). Endothelial cells within blood vessels adjacent to
the wound also proliferate, causing marked vessel hypertrophy
(Clark et al (1982a); Clark et al. (1982b)). Despite the remarkable
cell proliferation in the tissue surrounding the wound, no
mesenchymal invasion of the fibrin clot was observed for the first
three days after injury (Welch et al. (1990); Clark et al. (1995))
On the fourth day fibroblasts and endothelial cells invade the
fibrin clot-filled wound as an organized tissue construct called
granulation tissue. Fibroblasts of the granulation tissue appear
bound together with a fibronectin meshwork; and endothelial cells
organize into capillaries that intercalate the interwoven
fibroblast aggregate in a vertical array (Clark (1993a)). Despite
the clear clinical importance of this early stage of wound healing,
little is known about the inductive processes leading to
granulation tissue formation.
[0076] Chronic ulcers, in contrast to acute wounds, fail to heal.
Here the problem appears to be a corrupted provisional matrix. The
fibrin provisional matrix in the ulcer bed interstitium fails to
support healing possibly because it becomes partially degraded
(Bini et al. (1989)), excessively crosslinked (Brommer et al.
(1992)) or stripped of other molecules important for wound healing
such as fibronectin (Herrick et al. (1992)). In fact, based on in
vitro data, fibronectin is critical for cell invasion of the fibrin
clot (Greiling and Clark (1997)). To simulate fibroblast movement
from periwound collagenous stroma into provisional matrix-filled
wound space, a contracted collagen gel containing skin fibroblasts
was pasted onto a surface of fibrin fibrils and surrounded by a
fibrin clot (Greiling and Clark (1997)). This forms an "inside-out"
wound environment. To further simulate the in vivo situation, 30
ng/ml PDGF was added to the fibrin clot. Fibroblast appearance in
the translucent fibrin gel was quantified by cell counts. Cell
accumulation in the fibrin gel was attributable to migration rather
than mitogenesis as judged by the movement of nonproliferating,
irradiated cells. Transmigration from the collagen gel into fibrin
required fibronectin in both matrices. In addition, migration was
dependent on both .alpha.5.beta.1 and .alpha.v.beta.3 provisional
matrix integrins (Greiling and Clark (1997)). Thus, the absence of
fibronectin in the wound provisional matrix of chronic ulcers
(Herrick et al. (1992)) may directly hinder tissue cell
repopulation of the wound. This possibility has been supported in
fresh porcine wounds to which exogenous fibrin without fibronectin
has been added. Relatively few cells moved into these wounds
compared to wounds receiving fibrin replete with fibronectin. Thus,
one of the fundamental reasons that a fresh surgical or traumatic
gaping wound heals faster than a chronic ulcer may be that the
former has a fibrin matrix with abundant fibronectin (Clark et al.
(1982)) while the latter has little or no fibronectin in the
provisional matrix (Herrick et al. (1992)).
[0077] Using the 3-dimensional transmigration assay described in
U.S. Pat. No. 5,935,850, (which is hereby incorporated by reference
herein), it was discovered that human dermal fibroblast movement
from a collagen gel to a fibronectin replete-fibrin clot requires a
special preparation of fibrinogen containing fibronectin (Greiling
and Clark (1997)). A number of fibrinogen isolates acquired by
various isolation procedures, were clotted and tested for their
ability to allow fibroblast migration through a 3-dimensional
fibrin clot (Greiling and Clark (1997)). Fibronectin at a 1:10
molar ratio to fibrinogen and 30 to 100 ng/ml PDGF were added to
the fibrinogen just prior to clotting with human thrombin as
previously described to result in maximal cell migration (Greiling
and Clark (1997)).
Example 2
[0078] As a first step toward characterization, quality control
experiments were done on the activity of sequential fibrinogen
isolates from the 1M glycine precipitates produced and examined in
Example 1. Two sets of fibrinogen isolates were prepared from the
1M glycine precipitate. Each isolate was made from 2-5 normal donor
plasma pools. Fibroblast migration occurred in fibrin gels made
from three of six isolates in the first set and four of four
isolates in the second set. In the first set, those with low or no
activity had a low fibrinogen concentration (i.e. <8 mg/ml).
Consequently, they could not be re-tested to confirm their low
activity. In the preparation of the second set of isolates, care
was taken to obtain fibrinogen concentrations >10 mg/ml and
enough material to retest many times. All four preparations from
this set gave fibrin gels that permitted fibroblasts migration. On
first testing, one of the four isolates showed low migration
activity, but another aliquot of this same isolate had activity
equivalent to the other three. The aliquot that did not have
activity had been stored at the top of a -80.degree. C. freezer and
may have undergone repeated partial thawing prior to testing.
Example 3
[0079] Empirical observations have shown that a fibroblast
migration enhancement (FME) property is present in fibrinogen
preparations of relatively low purity and this activity is stable
on freeze thawing of such preparations. Conversely, fibrinogen
preparations that are of the highest possible purity, such as
fibrinogen fraction I-4, DEAEc peak 1 fraction, and others
(produced by methods known to those skilled in the art) either
possess low or no activity. Of particular use is fraction I-4,
which can be prepared in bulk and whose moderate FME activity
progressively decreases on repeat freeze thawing, thus enabling its
use as a negative control. In the discussion below, positive and
negative controls are termed C1 and C2, respectively. Fibrinogen
isolates with high activity are isolated from plasma by procedures
that yield two kinds of active isolates, one enriched with soluble
fibrin and the other lacking fibrin enrichment. The procedures also
yield a lipid or lipoprotein rich (L) component which is enriched
in FME activity.
[0080] Fibrin-Rich Fibrinogen Preparation.
[0081] Glycine is dissolved in plasma to 1 M (or 1 molar)
concentration and allowed to stand at 4.degree. C. overnight. The
precipitate formed is dissolved in phosphate buffered saline, pH
6.4, subjected to reprecipitation with 2.1 M Glycine at 4.degree.
C., and this step is repeated. An additional precipitation step is
performed with either 2.1 M Gly or 25% saturated Ammonium Sulfate.
The fibrinogen isolate thus obtained is dialyzed vs 0.3 M NaCl at
4.degree. C. and termed 2F. As shown below, fibrinogen 2F may be
further separated into sub-fractions. A large amount of insoluble
fibrin gel forms during dialysis, and this is removed by
centrifugation as described below.
[0082] Fibrinogen Low in Fibrin Content Preparation.
[0083] An isolate is obtained from the plasma supernatant of the
initial 1 M Gly step detailed in the paragraph above. For this
purpose, additional Glycine is added and dissolved to achieve 2.1 M
Gly concentration and the precipitate obtained at 4.degree. C. is
subjected to the same precipitation steps described above. The
final isolate is dialyzed as above and termed 2H. As shown below,
fibrinogen 2H may be further separated into sub-fractions. During
dialysis a small amount of insoluble fibrin forms and is removed by
centrifugation as described in the paragraph below.
[0084] Further Sub-Fractionation to Obtain and Characterize
Sub-Fractions.
[0085] Each of the fractions, 2F and 2H, is subjected to
centrifugation using at least 4000 XG for 30 or more minutes at
4.degree. C. The resulting three sub-fractions are illustrated in
FIG. 1. One sub-fraction is a lipid or lipoprotein component which
contains insoluble material floating at the top of the solution.
This subfraction is termed 2FL (from the 2F fibrin rich fraction)
or 2HL (from the 2H low fibrin content fraction). Use of a spatula
or other implement permits harvesting these sub-fractions. The
second sub-fraction is the bulk of isolated fibrinogen and is
referred to herein as 2F sub-fraction (fibrin rich fibrinogen) or
2H sub-fraction (low fibrin content fibrinogen). The third
sub-fraction is a pellet at the bottom of the centrifuged solution,
which consists of insoluble fibrin gel and is discarded. During the
harvest and testing of the lipid and fibrinogen sub-fractions
certain characteristics emerge. One is that fibrinogen 2H
invariably contains major amounts of sub-fraction 2HL, and
fibrinogen 2F, by contrast, tends to contain lower amounts of
sub-fraction 2FL. Such differences are demonstrable by dissolving
the material and measuring its turbidity. Further, some 2FL
sub-fractions are of substantial amounts while others are of very
small amounts compared to those in the 2HL counterparts from the
same starting plasma. A constellation of characteristics of the L
isolates is their self-evident low density and coalescence into
insoluble floating sheets or particles following centrifugation,
their yellowish-white and opaque appearance on visual inspection
(particularly marked in 2HL), their capacity to be readily
dispersed and re-dissolved in fibrinogen containing buffers, and
their high turbidity when re-dissolved and assessed
spectrophotometrically.
[0086] To compare the FME activity of 2HL or 2FL with their 2H or
2F counterparts, fibrin is formed in the assay from the same lot.
Comparison can also be made with the same parent (or uncentrifuged
fibrinogen preparation). In order to test the L moiety of 2FL or
2HL alone, fibrinogen can be removed so that the sub-fraction can
be tested without its parent fibrinogen. To remove its fibrinogen,
ammonium sulfate is added to 25% saturation and the precipitate is
discarded. The floating insoluble material on the top of the
solution is then harvested and dialyzed.
[0087] An alternate method to sub-fractionate 2F or 2H isolates.
The fractions can be sub-fractionated by subjecting them to size
exclusion chromatography, as shown in FIG. 2. This enables removal
of most of the lipid and of the soluble fibrin components, so that
each such component can be tested. Absorbance values of the eluting
fractions are obtained at 280 nm and at 350 nm. Absorbance at 280
nm reflects the presence of protein and that at 350 nm reflects
light scattering and, thus, the opacity caused by the lipid content
of the fibrinogen solution. This chromatographic procedure results
in an early elution peak, labeled peak I. Fractions containing this
peak appear white-opaque, show high absorbance values at 350 nm as
expected, and show the presence of protein by their absorbance
values at 280 nm. When dialyzed against water and freeze dried,
this peak is insoluble in buffer but can be resolubilized at least
in part in fibrinogen or other protein solutions. The second peak,
labeled peak II, consists of fibrinogen and soluble fibrin. This
peak shows absorbance at 280 nm but little or negligible absorbance
at 350 nm, as shown, and constitutes the bulk of protein applied to
the column.
[0088] Fibroblast Enhancing (FME) Activity: 2F, 2H and
non-chromatographic Subfractions.
[0089] Fractions 2F and 2H have ample activity, with 2H showing
moderately higher activity than 2F. Similarly, this activity
remains high, as shown in FIG. 3, in both the L rich subfractions
(2FL, 2HL) and fibrin rich sub-fractions (2H, 2F). Moreover, these
sub-fractions, 2FL and 2HL, show similarly high activity (See FIG.
3 insert) and this activity remains when fibrinogen is removed from
2HL and 2FL (not shown). Because insoluble lipid in 2HL may induce
formation of an abnormal fibrin matrix, care is taken to avoid
insoluble lipid aggregates when using 2HL. This is not the case
with 2FL, which contains a much lower lipid content and lacks such
large insoluble aggregates.
[0090] Fibroblast Enhancing (FME) Activity: Chromatographic
Subfractions.
[0091] Tested as outlined above, both peaks (shown in FIG. 2)
possess substantial fibroblast migration enhancing activity. The
fibroblast migration activity of the lipoprotein (L) peak of FIG. 2
is shown in the FIG. 4. This is consistent with the results from
the post-centrifugation sub-fractions described above, indicating
that the activity is present both in the L rich and the fibrinogen
(L poor) peaks. Considering the small amount of L sub-fraction
required to demonstrate the activity, this implies the lipid in
these isolates contains relatively higher activity than does
fibrinogen per se. That is to say, the activity is more lipophilic
than fibrinogenophilic. Moreover, when fibrin-rich fibrinogen was
isolated from the ascending limb of the chromatogram it too
displayed substantial activity, not shown, consistent with the fact
that fraction 2F is fibrin rich and shows activity comparable to
2H.
SUMMARY AND CONCLUSIONS
[0092] The fibroblast migration enhancement activity of fibrinogen
isolated by the above procedures is associated with three
components or sub-fractions: fibrin rich fibrinogen, lipid rich
fibrinogen, and fibrinogen not enriched with either fibrin or
lipid. Although lipid rich fibrinogen (2FL or 2HL) displays
somewhat higher activity, this activity remained when the lipid
component was rendered free of fibrinogen by chromatography or
other means. Moreover, fibrin rich fibrinogen which is also
rendered lipid poor clearly retains its high activity. In another
set of observations (data not shown), this activity is
progressively lost by freeze thawing of 1-4, and absent in DEAEc
pure fibrinogen. Taken together, these results imply a
non-fibrinogen and possibly non-lipid hydrophobic agent whose FME
activity remains stable in storage of fibrinogen/lipid mixtures.
This explains its stability in fibrinogen isolates of relatively
low purity (i.e. >95% by protein measurements), and enables
potential use of such preparations in situations where enhancement
of wound healing is clinically important. Also, in order for
fibrinogen isolates to possess the highest FME activity they need
be enriched with substantial amounts of a lipid component that
co-isolates with them from normal plasma and results also in
stability of this activity when fibrinogen is stored frozen and
re-frozen. What is more, the lipid component can be isolated and
re-introduced into fibrinogen of high purity or any other
fibrinogen isolate for the purpose of further enriching its FME
activity. This discovery makes it possible to monitor the amounts
of this lipid component in any fibrinogen and/or soluble fibrin
preparations.
[0093] Although the invention has been described in detail for the
purpose of illustration, it is understood that such detail is
solely for that purpose, and variations can be made therein by
those skilled in the art without departing from the spirit and
scope of the invention which is defined by the following
claims.
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