U.S. patent application number 12/238958 was filed with the patent office on 2009-01-22 for method for inhibiting platelet interaction with biomaterial surfaces.
Invention is credited to Brian Pederson.
Application Number | 20090023004 12/238958 |
Document ID | / |
Family ID | 39795689 |
Filed Date | 2009-01-22 |
United States Patent
Application |
20090023004 |
Kind Code |
A1 |
Pederson; Brian |
January 22, 2009 |
Method for Inhibiting Platelet Interaction with Biomaterial
Surfaces
Abstract
A method for passivating a biomaterial surface includes
modifying proteinaceous material disposed at the biomaterial
surface. The passivation may be effectuated by exposing the
biomaterial surface to therapeutic electrical energy in the
presence of blood or plasma.
Inventors: |
Pederson; Brian; (Plymouth,
MN) |
Correspondence
Address: |
Haugen Law Firm PLLP
1130 TFC Tower, 121 South 8th Street
Minneapolis
MN
55402
US
|
Family ID: |
39795689 |
Appl. No.: |
12/238958 |
Filed: |
September 26, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12057729 |
Mar 28, 2008 |
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12238958 |
|
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60908576 |
Mar 28, 2007 |
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Current U.S.
Class: |
428/478.2 |
Current CPC
Class: |
A61L 2300/42 20130101;
A61L 33/128 20130101; A61N 1/37512 20170801; A61N 1/375 20130101;
Y10T 428/31768 20150401; A61L 33/0094 20130101; A61N 1/05 20130101;
A61L 33/126 20130101; A61L 33/0041 20130101; A61L 33/062
20130101 |
Class at
Publication: |
428/478.2 |
International
Class: |
B32B 9/02 20060101
B32B009/02 |
Claims
1. A method for passivating a biomaterial surface, said method
comprising obtaining a qualitatively modified fibrinogen at said
biomaterial surface.
2. A method as in claim 1 wherein said qualitatively modified
fibrinogen includes an increased level of .beta.-chain
fibrinogen.
3. A method as in claim 1 wherein said qualitatively modified
fibrinogen includes a decreased level of .alpha.-chain and
.gamma.-chain fibrinogen.
4. A method as in claim 1 wherein said biomaterial surface is a
portion of a medical article.
5. A passivated biomaterial surface including a qualitatively
modified fibrinogen.
6. A passivated biomaterial surface as in claim 5 wherein said
biomaterial surface has an electrical resistivity of less than
about 5 ohms.
7. A passivated biomaterial surface as in claim 6 wherein said
biomaterial surface is selected from pyrolytic carbon, titanium,
nitinol, stainless steel, platinum, or iridium.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 12/057,729, filed on Mar. 28, 2008 and
entitled METHOD FOR INHIBITING PLATELET INTERACTION WITH
BIOMATERIAL SURFACES, which itself claims priority from U.S.
provisional patent application Ser. No. 60/908,576, filed on Mar.
28, 2007 and entitled METHOD FOR INHIBITING PLATELET INTERACTION
WITH IMPLANTED MEDICAL DEVICES, the contents of which are
incorporated herein in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to providing biomaterial
surfaces with thromboresistivity generally, and more particularly
to materials and methods for passivating a biomaterial surface so
as to inhibit blood platelet interaction therewith.
BACKGROUND OF THE INVENTION
[0003] Since the year 2000 alone, more than 1,000,000 vascular
prosthetic devices have been implanted worldwide. From stents to
artificial heart valves and ventricular assist devices, a wide
range of devices are being used to treat patients often expected to
live for many years after the procedures. Since biomaterials
promote surface-induced thrombotic phenomena to some extent, an
ever-increasing pool of patients reliant upon indefinite
anticoagulant therapy has been created. This is unfortunate, as the
use of drugs like heparin, warfarin and clopidogrel carries a
serious risk of side effects like bleeding, bruising and serious
internal hemorrhage.
[0004] The study of thrombogenetic sequence originates at platelet
response to endothelium damage. Although platelets can be activated
in suspension, they are by nature adhesive elements which perform
their hemostatic function under flow conditions. Whereas platelets
will not interact with the endothelial layer that covers the
vascular tree, they will rapidly respond to a mechanically damaged
vessel. Within several minutes after injury, the exposed surface
will be covered by a continuous layer of platelets.
[0005] The sequence of events developing after the endothelium
becomes damaged is well established. Studies with blood circulating
through vascular segments mounted in specially designed chambers
have clearly established that initial platelet attachment is
mediated through the interaction of insoluble von Willebrand factor
(VWF) bound to subendothelium with the platelet glycoprotein Ib-IX
complex (GPIb-IX). Additional interactions of platelet GPIIb-IIIa
(known also as integrin .alpha..sub.2b.beta..sub.3) with the amino
acid sequence Arg-Gly-Asp-Ser (RGDS) present on several adhesive
proteins (fibrinogen, VWF and fibronectin) will play a major role
on platelet spreading and aggregate formation.
[0006] All major receptors on the platelet membrane are connected
via GTP regulatory proteins to cytoplasmic
second-messenger-generating enzymes. Coupling of receptors with
their specific agonist will generate a second messenger that raises
the free calcium level in platelet cytoplasm. Increased levels of
Ca++will result in the amplification of activation mechanisms with
cytoskeletal assembly, internal contraction, fusion and release of
the alpha granules and expression of activation dependent antigens
(CD-62P) that would facilitate crosstalk interactions with
leukocytes. During this process of activation anionic phospholipids
will become externalized at the membranes of activated platelets.
These phospholipids will further facilitate mechanism of blood
coagulation.
[0007] Blood contacting biomaterial surfaces in particular, have
been shown to adsorb a layer of proteins from blood and to attract
platelets. Build-up of blood components on the surface of implanted
devices may reduce their effectiveness, and in many cases will lead
to serious adverse complications or operational failure.
Thrombogenesis presents a major problem associated with the
clinical use of all kinds of prosthetics, and the prevention of
unwanted clotting without the side effects incurred through the use
of blood thinning drugs would be a major advancement in the field
of biomaterials.
[0008] One method for securing biomaterials against unwanted
thrombosis is to modify the biomaterial surface itself. For
example, anti-thrombogenic materials have been covalently bonded
onto the blood-contacting biomaterial surfaces. Additionally, the
biomaterial has been treated to give its surface a fixed charge
which can affect the biocompatibility of the material. In other
cases, the surface has been polished to an extremely high degree.
None of these techniques, however, have been completely effective
in deterring platelet adhesion to the biomaterial surface.
[0009] Platelets will avidly interact with any foreign surface
including any kind of artificial material. Mechanisms responsible
for the interaction of platelets with artificial surfaces are
mediated by the same glycoproteins described above, though
functions of these glycoproteins are not identical to those
described in the previous section. It is fully accepted that the
presence of proteins adsorbed on the artificial surface play a
crucial role in mediating the initial interactions of platelets
with the surface, and the composition of the synthetic surface is a
key determinant on the rate and nature of the protein adsorbed.
Vroman and Cols demonstrated the effect named as "Vroman effect",
describing that a first protein was deposited on the surface after
the initial contact of blood with a polymer surfaces, that initial
protein was sequentially replaced by another protein. The nature of
the adsorbed proteins has a critical influence on further platelet
deposition. Albumin is known to inhibit platelet deposition on
artificial surfaces in vitro. Contrarily to albumin, fibrinogen,
fibronectin and von Willebrand factor enhance platelet interactions
with the artificial surface. Two regions of the fibrinogen alpha
chain that contain an RGD motif, as well as the carboxyl-terminus
of the fibrinogen gamma chain, represent potential binding sites
for GPIIb-IIIa in the fibrinogen molecule.
[0010] In essence, while the initial attachment of platelets with
vascular subendothelium is initiated through interactions of
GPIb-IX with vWF bound to collagen, the interaction of platelets
with artificial surfaces may be considered to be mainly driven by
GPIIb-IIIa and fibrinogen adsorbed onto the surfaces.
[0011] It has been theorized that promoting adhesion of albumin to
the detriment of fibrinogen at the blood-contacting surface could
be effective in altering the thrombogenicity of various materials.
In fact, Grunkemeier et al., Biomaterials, November, 2000 pp.
2243-2252, and Tsai et al., Journal of Biomedical Materials
Research Dec. 15, 2003, pp. 1255-68, found that the amount of
adsorbed fibrinogen was the chief determinant of the degree of
platelet adhesion, although platelets were most attracted to a
surface when a combination of proteins was residing on the surface,
including Von Willebrand factor. No preadsorption of particular
blood proteins has yet been shown to prevent clotting entirely. It
is very difficult to prevent fibrinogen from adhering to the
biomaterial surface, and only a small amount of adhered fibrinogen
is necessary to start a chain reaction leading to thrombosis.
[0012] Some materials coated with anticoagulant agents such as
heparin have had limited success in preventing thrombosis. However,
heparin coatings will eventually dissolve over time. Drawbacks to
agent-eluting surfaces have also been realized. A study by
Pfisterer et al., Journal of American College of Cardiologists,
Dec. 19, 2006 pp. 2592-5 regarding the Base1 Stent Kosten
Effektivitats Trial, Late Thrombotic Events, suggested that between
7 and 18 months after implantation, the rates of nonfatal
myocardial infarction, death from cardiac causes, and
angiographically documented stent thrombosis were higher with
drug-eluting stents than with bare metal stents.
[0013] Overall, there have been no recognized clinical advancements
that could warrant replacing traditional anticoagulation therapy.
At this time, only consistent maintenance of a regimen of blood
thinning agents is clinically proven to prevent the dangerous
thrombotic events associated with implants.
[0014] It is a primary object of the present invention to inhibit
and/or prevent thrombogenesis and blood platelet adhesion on a
biomaterial surface.
[0015] It is another object of the present invention to inhibit
and/or prevent blood platelet adhesion and thrombogenesis on an
electrically conductive, blood-contacting surface of an implantable
device.
[0016] It is a further object of the present invention to provide
an anti-thrombogenic characteristic to biomaterial surfaces by
providing certain blood proteins thereat.
[0017] It is a further object of the present invention to provide
an anti-thrombogenic characteristic to biomaterial surfaces by
providing conformationally-modified blood proteins thereat.
[0018] It is a still further object of the present invention to
provide a method to pre-treat biomaterials such as pyrolytic
carbon, titanium, nitinol, and stainless steel using therapeutic
electrical energy so as to prevent blood platelet adhesion to the
pre-treated biomaterials.
SUMMARY OF THE INVENTION
[0019] By means of the present invention, biomaterial surfaces may
be provided with a thromboresistant characteristic, such that
blood-contacting surfaces of a biomaterial inhibits blood platelet
interaction and adhesion therewith. Such passivation of the
biomaterial surface is effectuated through a passivating procedure,
which may involve application of therapeutic electrical energy
and/or deposition of certain proteinaceous materials thereat.
Biomaterial surface passivation may be accomplished in vivo,
ex-vivo, or in vitro, and may be done prior to, or subsequent to
implantation of a biomaterial in a patient.
[0020] In one embodiment, a method for passivating a biomaterial
surface involves exposing the biomaterial surface to therapeutic
electrical energy in the presence of blood or plasma.
[0021] Another method for passivating a biomaterial surface
includes modifying proteinaceous material disposed thereat.
[0022] The biomaterial surface may also be passivated by exposing a
quantity of blood or plasma to therapeutic electrical energy and
subsequently depositing proteinaceous material from such quantity
of blood or plasma on the biomaterial surface.
[0023] A biomaterial surface effective in inhibiting blood platelet
adhesion thereto includes conformationally modified fibrinogen.
[0024] A still further method for passivating a biomaterial surface
includes modulating a preferential adsorption of blood proteins to
the biomaterial surface.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 is a schematic diagram of a testing apparatus used in
the testing of the materials and methods of the present
invention;
[0026] FIG. 2 is a partial cut-away view of a portion of the
testing apparatus illustrated in FIG. 1;
[0027] FIG. 3 is a process flow diagram of the testing procedure
for testing the methods and materials of the present invention;
[0028] FIG. 4 is a SEM image comparison between a first control
test article exposed to whole human blood and a second control test
article not exposed to blood;
[0029] FIG. 5 is a SEM image comparison between a third control
test article exposed to whole human blood in an unstimulated
environment, with a fourth test article exposed to whole human
blood in a stimulated environment;
[0030] FIG. 6 is a series of gel electrophoresis images comparing
surfaces of test articles in a stimulated environment with test
articles in an unstimulated environment;
[0031] FIG. 7 is a radioactive count chart illustrating platelet
concentrations at test articles exposed to different test
environments; and
[0032] FIG. 8 is a schematic representation of the molecular
structure of fibrinogen.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0033] The objects and advantages enumerated above together with
other objects, features, and advances represented by the present
invention will now be presented in terms of detailed embodiments.
Other embodiments and aspects of the invention are recognized as
being within the grasp of those having ordinary skill in the
art.
[0034] The present invention is drawn to techniques and materials
which have been found to be useful in inhibiting platelet
interaction with biomaterial surfaces. Such interaction may
include, for example, adhesion, aggregation, thrombosis, clotting,
and/or coagulation of blood platelets at a biomaterial surface
exposed to such platelets. For the purposes of this application,
the terms "passivate", "passivated", or "passivating" shall refer
to a surface that exhibits anti-thrombogenic properties so as to
inhibit thrombosis thereat. In some instances, such term may
further connote improving the biocompatibility of the surface, such
as through thromboresistant properties. For the purposes of this
application, "thromboresistant" and "anti-thrombogenic" may be used
interchangeably.
[0035] A variety of biomaterials may be passivated through the
present invention. Most commonly, however, biomaterials include
those materials thought to be useful in the fabrication of medical
articles, such as implantable medical articles. However, the
techniques and materials of the present invention may indeed
facilitate the use of "biomaterials" which, in the absence of the
techniques and materials of the present invention, would not
typically be considered in medical applications, such as in
implantable medical articles. Accordingly, as used herein,
"biomaterials" is intended to include any native, natural, and/or
artificial material used in a biological application, such as in
the contacting of blood, plasma, or other biological fluids.
Example biomaterials may include metal such as stainless steel,
nitinol, and titanium, plastics such as polyolefins, polyesters,
polystyrenes, polyurethanes, polyamides, polytetrafluoroethylenes,
polysiloxanes, polyimides, phenolics, amino-epoxy resins,
polyacrylonitriles, polymethacrylates, silicones, and silicone
rubbers, as well as other materials such as pyrolytic carbon and
ceramics. In some embodiments of the invention, electrically
conductive materials, such as those having an electrical
resistivity of less than about 5 ohms may be utilized, though such
resistivity threshold may be overcome by using higher voltage
potentials. The biomaterials may be used in medical articles
including vascular stents, grafts, heart valves, heart diaphragms,
catheters, implantable pacemakers, defibrillators, and related
leads, sutures, needles, tubing, dialysis membranes, filters, and
the like.
[0036] The Applicant has focused studies on the thromboresistant
properties exhibited by proteinaceous materials contained in or
derived from blood. Example such proteinaceous materials that have
been addressed in this effort include albumin and fibrinogen. One
thromboresistant factor that has been developed in the present
studies is a conformationally altered fibrinogen. In its standard
form, fibrinogen includes three distinct "conformations", each
having slightly different molecular weights. These may be referred
to as alpha, beta, or gamma fibrinogen. Such distinct conformations
can be viewed through gel electrophoresis, wherein three distinct
bands are prevalent at about 50 kD. As shown in FIG. 8, fibrinogen
contains three putative platelet interaction sites, namely the
sequence Arg-Gly-Asp-Phe (RGDF) at A.alpha..sup.95-98, the sequence
Arg-Gly-Asp-Ser (RGDS) at A.alpha..sup.572-575, and the
dodecapeptide sequence
His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (HHLGGAKQAGDV) at
.gamma..sup.400-411. Both the RGDS, and the dodecapeptide sequence
play a key role in platelet aggregation.
[0037] Subsequent to exposure to the electrical energy as described
herein, the relative concentrations of the fibrinogen conformations
at the biomaterial surface are modified. In one embodiment, at
least one of the three fibrinogen conformations exposed to the
therapeutic electrical energy is found at significantly higher
concentrations at a surface exposed to the electrical energy than
the concentration of such fibrinogen conformation at a surface not
exposed to the therapeutic electrical energy. In addition, at least
one fibrinogen conformation concentration is significantly
decreased upon electrical stimulation.
[0038] The inmovilization and the conformational change in the
fibrinogen molecule exposes specific sites for the GPIIb-IIIa
receptor in the platelet membranes [31]. It has been postulated
that the presence of two .gamma.-chain carboxyl-terminal domains in
the dimeric fibrinogen molecule may influence the adhesion of
nonstimulated platelets when the ligand is immobilized onto a
surface [32]. If these interpretations are correct, the
.gamma.-chain of the fibrinogen would play a more important role
mediating the interaction of platelets with fibrinogen bound to
surfaces.
[0039] Interestingly, the evidence provided by FIG. 6 indicates
qualitative changes in the fibrinogen molecule deposited on the
stimulated surfaces. The 3 bands around 50 kD corresponding to the
alpha, beta and gamma chains of fibrinogen are well preserved in
surfaces non exposed to the electrical field technology.
Application of specific electrical energy to the same surface
resulted in an increased presence of the .beta. chain of fibrinogen
with an evident reduction in the presence of .alpha. and .gamma.
chains of fibrinogen. These observations may reflect a
conformational alteration of fibrinogen. It is hypothesized that
the binding factor of fibrinogen to blood platelets is modified or
eliminated through the fibrinogen conformational adjustment
described above. As such, the conformationally altered fibrinogen
has little or no adhering interaction with blood platelets, thereby
effectuating a thromboresistant characteristic.
[0040] Another thromboresistant factor of the present invention is
the preferential promotion of albumin adhesion to a biomaterial
surface. Biomaterials having a relatively high surface
concentration of albumin have been shown to inhibit fibrin cascade
and platelet attachment, potentially through disruption of electric
charge-related platelet interactions. Typically, however,
fibrinogen is often the dominant protein adsorbed from protein
mixtures such as blood, blood serum, or plasma. Because fibrinogen,
in its standard form, is known to promote platelet adhesion at a
surface, preferential albumin adsorption acts to inhibit platelet
adhesion both through the thromboresistant properties of albumin
and through the reduction of fibrinogen presence at the
surface.
[0041] One technique for effectuating one or more of the
thromboresistant factors described above at a biomaterial surface
involves the application of electrical energy to proteinaceous
material found in blood and/or the application of electrical energy
to a biomaterial surface while in the presence of such
proteinaceous material. The application of electrical energy, such
as a magnitude of electrical energy deemed therapeutic, to blood or
plasma has surprisingly been found to cause thromboresistance in
biomaterial surfaces contacted with the treated blood or plasma. It
is theorized that the thromboresistance generated at the
biomaterial surface is derived from one or both of the presence of
conformationally altered fibrinogen and the disproportionately high
concentration ratio of albumin to standard fibrinogen at such
biomaterial surface. Moreover, it is theorized that the existence
of such thromboresistant factors at the biomaterial surface is
created through the application of therapeutic electrical energy to
proteinaceous material contained in blood, blood serum, or plasma,
wherein such proteinaceous material includes albumin and/or
fibrinogen. Through experimentation, Applicant has determined that
application of electrical energy, in the therapeutic magnitudes
described herein, establishes an environment for the creation of a
thromboresistant, passivated biomaterial surface characteristic.
Applicant contemplates, however, that alternative methods may be
employed to establish the thromboresistant factors described
herein, and to provide a biomaterial surface with one or more of
such factors.
[0042] In one embodiment, a biomaterial surface may be passivated
by exposing such biomaterial surface to therapeutic electrical
energy in the presence of blood or plasma. In another embodiment, a
biomaterial surface may be passivated by exposing such biomaterial
surface to blood or plasma which has been treated with therapeutic
electrical energy. In a further embodiment, a biomaterial surface
may be passivated by adsorbing at such surface blood proteins
treated with therapeutic electrical energy. In another embodiment,
a thromboresistant biomaterial surface may be achieved through the
provision of a conformationally altered fibrinogen thereat. Other
embodiments in addition to those described above are also
contemplated as being within the scope of the present
invention.
[0043] Investigations have been conducted into the prevention of
biomaterial surface/platelet interaction with the application of
electric current to various materials. Initial studies have focused
on the reaction of pyrolytic carbon, stainless steel, nitinol, and
titanium. Currently marketed cardiac and vascular stents are
primarily made of stainless steel (also carbon coated), and
nitinol. In initial experiments, pyrolytic carbon was chosen due to
previous experience with this material. The type and magnitude of
electrical energy (frequency and current) needed to provide
thromboresistance on the surface of carbon have been investigated
using an in-vitro blood perfusion system, as described below.
Assessment of the reactions has been accomplished through scanning
electron microscopy (SEM), electrophoresis, Indium (radioactive)
platelet labeling, protein assay assessment, and
Fluorochrome-labeled antibody staining.
[0044] Test System
[0045] A blood perfusion system (BPS) was developed for the
evaluation of the biomaterial surfaces and its reaction to blood,
and was designed to hold any one of the biomaterials of interest. A
schematic diagram of blood perfusion system 10 is illustrated in
FIG. 1, and includes a sample reservoir 12, a test chamber 14, and
a fluid pump 16 for pumping sample fluid throughout system 10. An
electrical power supply 18 is electrically coupled to test chamber
14 through electrical leads 20, 22, and may controllably apply
electrical energy to test chamber 14. System 10 further includes
fluid conduit sections 30, 32, 34 for transporting the sample fluid
throughout system 10.
[0046] Sample reservoir 12 of system 10 may be any type of
reservoir for the fluids utilized in the test procedure. By way
example, such fluids may include whole blood, platelet-rich plasma,
or platelet-poor plasma. In some cases, a suspension such as sodium
citrate or sodium heparin may be added to the fluid to inhibit
spontaneous clotting. Sample reservoir 12 in the test apparatus was
a 0.5 liter glass bottle.
[0047] Both a pulsatile pump and a roller pump were utilized as
pump 16 of system 10. The roller pump, which was a Model 323 pump
manufactured by Watson Marlow was utilized in continuous flow
regimes at a flow rate of 600 ml per minute. A MOX106 pulsatile
pump manufactured by Waters Instruments was calibrated to mimic a
beating human heart, wherein a pump surge rate of 70-80 surges per
minute was set with an output volume of 50 ml per surge. Pump 16
pumped the sample fluid throughout conduit sections 30, 32, 34,
which comprise silicon rubber tubing. In particular, pump 16 pumps
the sample fluid from sample reservoir 12 to test chamber 14, and
then back to sample reservoir 12.
[0048] Test chamber 14 is illustrated in greater detail in FIG. 2,
wherein test chamber 14 includes a polycarbonate housing 42, a top
lid 44 and a bottom lid 46. The top and bottom polycarbonate lids
44, 46 are sealingly engageable with housing 42 via O-rings 48. The
sample fluid is supplied to test chamber 14 at fluid inlet 50, and
is removed from test chamber 14 at fluid outlet 52. Valves 54 were
positioned at fluid inlet and fluid outlet 50, 52 for additional
control of fluid flow through test chamber 14. The biomaterial
surface analyzed in test chamber 14 was a prosthetic bi-leaflet
heart valve 56 fabricated from pyrolytic carbon, and further
provided with a fabric suture cuff 58 in conventional fashion. The
prosthetic heart valve was a 25 mm ATS Open Pivot.TM. aortic valve
having a leaflet surface area of about 12.4 cm.sup.2.
[0049] To suspend the valve prosthesis within test chamber 14, a
titanium pin retainer 60 with electrically insulative plastic
covers 62 were retained at apertures 43 of housing 42, and pierced
the fabric suture cuff 58 of valve prosthesis 56. At least one pin
retainer 60 was placed into contact with the pyrolytic carbon body
of valve prosthesis 56 so as to make electrical contact to at least
the valve body of valve prosthesis 56. In addition, such at least
one pin retainer includes an exposed extension portion 64 to which
electrical connection may be made. This pin retainer 60 thus forms
an electrode for establishing direct electrical contact with valve
prosthesis 56. A further electrode 70 is provided through an
aperture 45 in top lid 44, with titanium electrode 70 extending
into the chamber defined by housing 42 and into contact with the
valve leaflets of valve prosthesis 56. In this manner, direct
electrical contact to valve prosthesis 56 could be established by
connecting an electrical lead to connection end 72 of electrode 70.
A set screw 76 was utilized in order to adjust the vertical
position of electrode 70 within test chamber 14, and particularly
into and out from electrical contact with valve prosthesis 56.
[0050] A still further electrode 80 was provided in test chamber
14, wherein titanium electrode 80 is exposed to the sample fluid
13, but is spaced from valve prosthesis 56. Electrical connection
to electrode 80 could be made at connection end 82 thereof. As
illustrated in FIG. 2, the level of sample fluid 13 was above valve
prosthesis 56, such that valve prosthesis 56 was submerged in the
sample fluid 13 during the test procedure.
[0051] Power supply 18 was a combination of a Tektronix.TM. AFG310
arbitrary waveform generator which is capable of producing multiple
electrical waveforms (sin, triangular, square, and pulsatile) and a
custom precision voltage to current converter capable of delivering
various current levels. Electrical leads from power supply 18 were
connected to respective ones of the electrodes 60, 70, 80 during
the test procedure. In some cases, positive polarity was coupled to
both electrodes 60 and 70 while negative polarity was coupled to
electrode 80. In other cases, positive polarity was coupled only to
electrode 70 while negative polarity was coupled to electrode 80.
Electrical connection was established at the terminus of the
electrical leads through conventional electrical clips.
[0052] Test Procedure
[0053] The evaluation procedure was as follows:
[0054] A test article was inspected and cleaned with alcohol, and
then placed in test chamber 14 as described above. A 250 ml
reservoir of saline was placed in a water bath at 37.degree. C.
Once the saline reached equilibrium temperature, the open ends of
conduit sections 30, 34 were placed in the reservoir. Pump 16 was
activated and adjusted to a flow rate of 600 ml per minute to pump
the saline through system 10 for ten minutes to rinse the system
and to test for potential leakage.
[0055] In "direct connection" tests, positive polarity electrical
connections are made to electrodes 60, 70, and a negative polarity
electrical connection is made to electrode 80. Moreover, in "direct
connection" tests, electrode 70 is positioned so as to make direct
contact with at least a portion of the test biomaterial article. In
the case of valve prosthesis 56, electrode 70 may be placed in
direct contact with the pyrolytic carbon leaflets when conducting a
"direct connection" test.
[0056] In "electrical field" tests, positive polarity electrical
connection is made to electrode 70 and negative polarity electrical
connection is made to electrode 80. Electrode 70 is vertically
positioned within test chamber 14 so as to be out of contact with
the test article during the application of electrical energy.
[0057] Power supply 18 was calibrated to provide a signal having
positive going (2.25 V/2.25 mA DC offset, a 4.5 V peak pulse which
correlates to a 4.5 mA current. The current is derived by making a
differential measurement of the signal across a precision 1
k.OMEGA. resistor. A duty cycle of 41.6% was assigned (25 ms ON
(+4.5 V) and 60 ms OFF (0 V)).
[0058] Pump 16 is then turned off and system 10 drained of the
saline. A 250 ml reservoir of sample fluid (human whole blood,
animal whole blood, blood serum, platelet rich plasma, platelet
poor plasma, etc.) replaces the saline reservoir in the water bath
set to 37.degree. C. Pump 16 is again activated to expose system 10
to the sample fluid. Upon completion of the test period, the sample
fluid is drained from system 10 and system 10 is then immediately
flushed with saline through the process described above.
[0059] The test article is then removed from test chamber 14,
rinsed in saline, and placed in a solution of gluteraldehyde to
arrest further cell action and interaction. The test article is
then dehydrated with ethanol to enable assessment of the article
surface within the scanning electron microscope vacuum chamber.
EXAMPLES
[0060] The invention is further and more specifically illustrated
by the following examples and tests.
Example 1
[0061] A control experiment was conducted using whole human blood
donated within three hours of testing. The whole human blood was
pumped through system 10 in the absence of applied electrical
energy, and was contacted with a pyrolytic carbon heart valve
prostheses at test chamber 14. This test was continued for 30
minutes.
[0062] FIG. 4 illustrates two scanning electron microscope (SEM)
slides taken at 1000.times. magnification. A photograph of a clean,
untested pyrolytic carbon valve prosthesis leaflet is shown on the
left, and a pyrolytic carbon valve prosthesis leaflet taken from
the test article following the control test is shown on the right.
It is clear from this control sample that blood platelets are
adhered and spread across the surface of the pyrolytic carbon under
typical blood exposure conditions, such as those found in vivo. The
conditions of the control experiment substantially replicate
conditions experienced in vivo for implantable medical
articles.
Example 2
[0063] A sample of whole human blood was separated into two
aliquots, with a first aliquot being tested through the "direct
connection" procedure described above for 45 minutes. The second
aliquot of whole human blood was cycled through system 10 and
contacted with a pyrolytic carbon test article in the absence of
electrical energy application as a control for 45 minutes. The SEM
slides of FIG. 5 demonstrate an image of the control test article
surface on the right, and an image of the test article surface used
in the "direction connection" test on the left.
[0064] It is clear from visual comparison of the SEM slide that the
test article surface exposed to the electrical energy is
substantially clear of adhered platelets, while the control test
article exhibits significant platelet confluency at its surface. A
graphical pixilation analysis was performed to derive a
quantitation of blood platelet cell presence at the respective test
article surfaces. The graphical pixilation analysis was performed
by colored pixel count of the SEM images, wherein individual pixel
colors other than black were considered adhered platelet cells. The
graphical pixilation count analysis of test article surface exposed
to electrical energy revealed about 2.5% platelet adhesion, while
the control test article surface exhibited about 59.1% platelet
cell confluency.
Example 3
[0065] A sample of human platelet rich plasma (PRP) was separated
into three aliquots with a first aliquot being tested through the
"direct connection" procedure described above in a "stagnant" flow
regime, wherein the test articles are exposed to a stagnant volume
of test fluid for the test period. A first pyrolytic carbon test
article was exposed to the first aliquot of PRP in the presence of
the electrical energy application described above for 15 minutes.
The first pyrolytic carbon test article was then exposed to whole
human blood from the PRP donor in a pulsed flow regime for 45
minutes in the absence of applied electrical energy.
[0066] A second pyrolytic carbon test article was tested similarly
to the first pyrolytic carbon test article, except that the second
test article was exposed to PRP in the presence of applied
electrical energy for 30 minutes prior to exposure to whole human
blood from the PRP donor for 45 minutes in the absence of applied
electrical energy.
[0067] Control pyrolytic carbon test articles were exposed to PRP
in the stagnant chamber for 15 and 30 minutes, respectively,
without applied electrical energy, and then exposed to whole human
blood from the PRP donor in a pulsed flow regime for 45 minutes in
the absence of applied electrical energy.
[0068] The test article surfaces were assessed with SEM, and the
first and second test articles exhibited significantly less adhered
platelets than the amount of adhered platelets observed on the
control test article.
Example 4
[0069] A sample of human platelet poor plasma (PPP) was separated
into three aliquots for testing in connection with three test
articles. A first pyrolytic carbon test article was exposed to the
first aliquot of PPP in the presence of direct connection
electrical energy for 15 minutes. The first pyrolytic carbon test
article was then exposed to whole human blood from the PPP donor in
a pulsed flow regime for 45 minutes in the absence of applied
electrical energy.
[0070] A second pyrolytic carbon test article was exposed to PPP in
the presence of applied electrical energy for 30 minutes prior to
exposure to whole human blood from the PPP donor for 45 minutes in
the absence of applied electrical energy.
[0071] Control pyrolytic carbon test articles were exposed to PPP
in the stagnant chamber for 15 and 30 minutes, respectively,
without applied electrical energy, and then exposed to whole human
blood from the PPP donor in a pulsed flow regime for 45 minutes in
the absence of applied electrical energy.
[0072] The test article surfaces were assessed with SEM, and the
first and second test articles exhibited significantly less adhered
platelets than the amount of adhered platelets observed on the
control test article.
Example 5
[0073] Bovine blood platelets labeled with indium-111 were used as
the sample fluid in a test to determine the ability of passivated
test article surfaces to remain effective in the prevention of
platelet adhesion over time without continued electrical
stimulation. In this "pre-treatment" exercise, four pyrolytic
carbon test articles were exposed to the bovine blood for 60
minutes. One of such test articles was electrically stimulated for
the entire 60 minute test period. Two test articles were stimulated
for 30 minutes during the bovine blood exposure, and then
disconnected from the electrical energy for the remaining 30
minutes of the test period. One test article was unstimulated
throughout the entire 60 minute test period. The electrical
stimulation was conducted at the parameters described above.
[0074] The chart of FIG. 7 illustrates radioactive counts for each
of the three test groups described above, wherein the radioactive
counts are indicative of platelet concentration at the respective
test article surface. As demonstrated therein, it appears that
pre-treatment of the test article with stimulation in the presence
of blood is also effective in inhibiting platelet adhesion even in
the absence of continued electrical stimulation. Specifically, the
"Group 2" test article surfaces, which were electrically stimulated
only for the first 30 minutes of the 60 minute test period,
exhibited post-test platelet concentrations similar to the
post-test platelet concentrations at the test article surfaces of
"Group 1", which received electrical stimulation throughout the 60
minute test period. By contrast, the "Group 3" test article
surfaces, which were exposed to blood in the absence of electrical
stimulation, exhibited post-test platelet concentrations several
fold higher than the platelet concentrations exhibited by either of
the stimulated group test articles.
[0075] Analysis
[0076] A gel electrophoresis analysis was performed on the test
article surfaces regarding the blood proteins present thereat. Gel
electrophoresis was performed through the use of the microplates
procedure of a BCA.TM. protein assay kit available from Pierce, a
division of Thermo Fisher Scientific, Inc., of Rockford, Ill.
Proteins taken from 6 test articles, 3 of which were tested in a
"stimulated" environment, and the remaining 3 were tested in an
"unstimulated" environment by being exposed to blood in the absence
of applied electrical energy.
[0077] FIG. 6 illustrates results for "stimulated" samples (those
tested with exposure to therapeutic electrical energy) versus
unstimulated samples (control). The dark bands between 65 and 75 kD
indicate the presence of albumin. It appears that the presence of
albumin is enhanced by 5-10.times. in the stimulated group, based
on the results of the protein assay. It is well understood that
albumin stabilizes charges on materials thereby preventing electric
charge-related platelet interactions. The presence of albumin at
the surface therefore plays a role in inhibiting platelet
interaction/adhesion with the test article surface. It is a
surprising result of the above-described tests, however, that
application of the utilized levels of electrical energy modulates
the preferential adsorption of at least albumin to the test article
surface, in that albumin adsorption appears to be significantly
preferentially promoted. Such preferential promotion of albumin
adsorption is demonstrated by the enhanced albumin presence in the
gel electrophoresis slides illustrated in FIG. 6, as well as in the
protein assay analyses. It is theorized that the preferential
promotion of at least albumin adsorption on the stimulated article
surface is caused by the electrical charge provided at the surface
through the applied electrical energy.
[0078] FIG. 6 further illustrates a conformational alteration of
fibrinogen in the stimulated group, as compared to the fibrinogen
found on the unstimulated test article surfaces. As described
above, the three bands around 50 kD represent alpha, beta, and
gamma fibrinogen. The unstimulated article surfaces exhibit all
three fibrinogen conformations with approximately similar intensity
response through gel electrophoresis. The stimulated test article
surfaces, however, exhibited a significantly higher concentration
of beta fibrinogen, and a lower concentration of at least alpha
fibrinogen and possibly a lower concentration of gamma fibrinogen
as well. Of the three fibrinogen conformations, alpha fibrinogen is
the lowest molecular weight, and gamma fibrinogen is the highest
molecular weight. The concentration changes illustrated by the gel
electrophoresis intensity changes in FIG. 6 likely reflects a
conformational alteration of the fibrinogen that is related to or
induced by electrical stimulation.
[0079] The protein assay described above further confirms a
substantial increase in fibrinogen concentration at the stimulated
test article surfaces, as compared to the fibrinogen concentrations
found on the unstimulated test article surfaces. The fibrinogen
detected at the surfaces at the stimulated group, however, was
conformationally altered as described above. It was determined that
the fibrinogen concentration of the stimulated group was
5-10.times. greater than the fibrinogen concentration of the
unstimulated group, thus evidencing a preferential promotion of at
least conformationally altered fibrinogen adsorption on the
stimulated article surface.
[0080] It is theorized that the alteration of fibrinogen receptors
caused by the exposure to the therapeutic electrical energy
inhibits the binding of further fibrinogen to the surface. The
alpha chain of fibrinogen contains the RGDS sequence necessary for
platelet interactions. The gamma chain of fibrinogen holds the
dodecapeptide sequence (including the RGDS) that can be used for
platelet aggregation. Reducing or eliminating the presence of alpha
and/or gamma fibrinogen, as seen in the gel electrophoresis images
of the stimulated group, may therefore correspondingly inhibit
platelet adhesion and aggregation. One explanation for this effect
may be that the reduced reactivity of the stimulated surfaces could
be explained through an interference of the electrical current with
the Vroman effect resulting in: 1) alterations of the molecule of
fibrinogen deposited; 2) modifications on the characteristic
conformational changes that occur after the adsorption of
fibrinogen to the surface; and 3) albumin related cross-linking
mechanisms altering the properties of the adsorbed fibrinogen.
[0081] Commentary
[0082] In view of the above examples and analysis, Applicant has
determined that, in one embodiment, therapeutic electrical energy
applied to a biomaterial surface, while such surface is exposed to
a blood protein-containing fluid such as whole blood or plasma, can
passify such biomaterial surface, at least against thrombosis.
These studies have further shown that biomaterial surface
passivation may be accomplished in a "pre-treatment" arrangement,
wherein the biomaterial surface undergoes a passivating procedure
and is subsequently placed into a blood platelet-contacting
environment. The passivated biomaterial surface exhibits ongoing
thromboresistant properties even in the absence of continuing
surface passivation. In effect, therefore, a biomaterial surface
may be passivated in advance of implantation, with the passivated
biomaterial surface remaining effective, at least in the case of
thromboresistance, for a significant length of time subsequent to
implantation. Accordingly, the methods and materials of the present
invention may be utilized, for example, to prevent thrombosis
formation in devices such as vascular stents by pre-treating
blood-contacting surfaces thereof in the presence of a relatively
small amount of the respective patient's blood or plasma prior to
device implantation into the patient.
[0083] The electrical stimulation described above with reference to
the examples, is merely representative of various electrical energy
magnitudes that may be useful in passivating biomaterial surfaces.
For example, the applied electrical current in the above examples
of electropositive 4.5 mA directed to a pyrolytic carbon material
having a surface area of about 12.4 cm.sup.2 provides a current
density of about 0.35 mA/cm.sup.2. As described in our co-pending
patent application Ser. No. 11/402,463 entitled "System for
Conditioning Surfaces in Vivo", the content of which being
incorporated herein by reference, an electropositive current
density of between about 0.001 and about 1.0 mA/cm.sup.2 may also
be useful in the present application. Applicant believes that a
current density of at least about 0.1 mA/cm.sup.2 may be most
beneficial for the purposes described herein, depending upon the
electrical conductivities of the biomaterials at issue. An upper
threshold on the electropositive current density provided at the
biomaterial surface for therapeutic conditioning thereof in the
present application may be limited only by the current density
threshold above which undesired and/or permanent damage to such
biomaterial or interfacing material may occur.
[0084] For the purposes of this application, therefore, the term
"therapeutic electrical energy" shall mean electrical energy that
is effective in generating an electropositive current density at
the subject biomaterial surface at a magnitude sufficient to
passivate the biomaterial surface through exposure of the
biomaterial surface to the electrical energy in the presence of
blood or plasma. In one embodiment, such therapeutic electrical
energy results in an electropositive current density at the
biomaterial surface being treated of at least about 0.1
mA/cm.sup.2.
[0085] As also described above, a further aspect of the present
invention is the surprising finding that the deposition of certain
blood proteins and/or blood protein concentrations at a biomaterial
surface is effective in passivating such biomaterial surface. One
passivating material of the present invention is a conformationally
altered fibrinogen, and specifically a fibrinogen with a relatively
high concentration of beta fibrinogen and/or relatively low
concentrations of alpha fibrinogen and/or gamma fibrinogen.
Applicant has determined that the presence of such modified
fibrinogen at the biomaterial surface at a concentration of at
least about 5-10.times. the concentration of unmodified fibrinogen
at an unstimulated surface exposed to blood or plasma for at least
about 15 minutes may be effective in passivating the biomaterial
surface. As such, Applicant envisions a variety of techniques for
depositing a passivating agent, such as conformationally modified
fibrinogen, at the biomaterial surface for passivating such
biomaterial surface. For example, such a passivating agent may be
isolated and used as needed, such as by depositing the passivating
agent at a biomaterial surface prior to biomaterial implantation
into a patient. Such treatment of the blood-contacting surface of
the biomaterial may be performed immediately prior to implantation
or significantly prior to implantation, with the passivating agent
retaining its passivating properties for a significant period of
time subsequent to implantation.
[0086] An overall impact, therefore, of the present invention is
the prevention of platelet adhesion and thrombogenesis on
biomaterial surfaces, including artificial implants, transplants,
and native tissue, through the provision of certain modified and/or
unmodified blood proteins at such surfaces. In one embodiment, such
blood proteins may be provided at the biomaterial surfaces through
the application of therapeutic electrical energy to the surface
while the surface is in the presence of blood or plasma. Other
techniques, however, for the provision of effective passivating
agents on target surfaces are envisioned in the present
invention.
[0087] The invention has been described herein in considerable
detail in order to comply with the patent statutes, and to provide
those skilled in the art with the information needed to apply the
novel principles and to construct and use embodiments of the
invention as required. However, it is to be understood that various
modifications to the invention can be accomplished without
departing from the scope of the invention itself.
* * * * *