U.S. patent number 3,927,422 [Application Number 05/424,062] was granted by the patent office on 1975-12-23 for prosthesis and method for making same.
Invention is credited to Philip Nicholas Sawyer.
United States Patent |
3,927,422 |
Sawyer |
December 23, 1975 |
Prosthesis and method for making same
Abstract
A tubular collagen vascular graft prosthesis is subjected to
treatment so that its intimal surface has an increased net negative
surface charge which prevents thrombosis. Treatment with succinic
anhydride is shown as one way of accomplishing this. Valve grafts
are likewise treated.
Inventors: |
Sawyer; Philip Nicholas
(Brooklyn, NY) |
Family
ID: |
23681303 |
Appl.
No.: |
05/424,062 |
Filed: |
December 12, 1973 |
Current U.S.
Class: |
600/36 |
Current CPC
Class: |
A61L
33/0082 (20130101) |
Current International
Class: |
A61L
33/00 (20060101); A61F 001/24 () |
Field of
Search: |
;3/1,DIG.1
;128/334R,335,335.5 ;161/178,226 ;117/95 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
means et al "Chem. Modification of Proteins" Holden-Day, Inc. S.F.,
Califor. pp. 144-148, 74-83. .
Sawyer et al. Trans. ASAIO, Vol. XII, 1966 pp. 183-14 187..
|
Primary Examiner: Truluck; Dalton L.
Attorney, Agent or Firm: Roberts & Cohen
Claims
What is claimed is:
1. A method of improving the performance of a graft prosthesis
comprising making the prosthesis of collagen and increasing the
negative surface charge of the intimal surface thereof by a
succinylation reaction.
2. A method as claimed in claim 1 wherein the collagen is prepared
from ficin digested bovine carotid artery material.
3. A method as claimed in claim 1 wherein the prosthesis is formed
as a vascular prosthesis.
4. A method as claimed in claim 1 wherein the prosthesis is formed
as a valve.
5. A prosthesis made as claimed in claim 1.
6. A method of improving the performance of a graft prosthesis
comprising making the prosthesis of collagen, the prosthesis
including protein with free amino groups, and increasing the net
negative surface charge of the intimal surface thereof by covering
the free amino group.
7. A method of improving the performance of a graft prosthesis
comprising making the prosthesis of collagen and increasing the
negative surface charge of the intimal surface thereof by the
following chemical reaction ##SPC4##
8. A method of improving the performance of a graft prosthesis
comprising making the prosthesis of collagen and increasing the
negative surface charge of the intimal surface thereof, said
collagen being dialdehyde starch tanned collagen which is treated
with succinic anhydride.
9. A method as claimed in claim 8 comprising further modifying said
surface with phenylglyoxal.
10. A method as claimed in claim 8 comprising further modifying
said surface with diethyl pyrocarbonate.
11. A method of improving the performance of a graft prosthesis
comprising making the prosthesis of collagen, said graft being in
the form of a collagen tube, comprising closing off an end of said
tube, inserting said end into a fluid and inserting a liquid
chemical succinylation reactant into the lumen of the tube to
increase the negative surface charge of the intimal surface
thereof.
12. A method as claimed in claim 11 wherein the chemical reactant
is succinic anhydride in a basic solution.
13. A method as claimed in claim 12 wherein a NaHCO.sub.3 buffer
solution is inserted into the lumen in sequential additions of
approximately 10 ml. aliquots to which about 0.1 gm. of crystals of
succinic anhydride were respectively added.
Description
BACKGROUND
It is known to prepare vascular graft prostheses from collagen
materials. Such prostheses are in fact available commercially from
Johnson & Johnson of New Brunswick, New Jersey.
Aside from the publications and articles to be mentioned hereafter,
pertinent material will be found in the following:
Wesolowski, S. A.: The healing of vascular prostheses. Surgery
57:319, 1965.
Wesolowski, S. A., Fries, C. C., Domingo, R. T., Fox L. M., and
Sawyer, P. N.: Fate of simple and compound arterial prostheses:
Experimental and human observations. In: Fundamentals of Vascular
Grafting. McGraw-Hill, New York, 1963, pp. 252-268.
Wesolowski, S. A., Hennigar, G. R., Fox, L. M., Fries, C. C., and
Sauvage, L. R.: Factors contributing to long term failure in human
vascular prosthetic grafts. Presented at Symposium on Late Results
of Arterial Reconstruction. International Cardiovascular Society
Meeting, Rome, September 1963. J. Cardiov. Surg. 5:44 1964.
Sawyer, P. N., and Pate, J. W.: A study of electrical potential
differences across the normal aorta and aortic grafts of dogs.
Research Report NM 007081, 10.06. Naval Medical Research Institute,
Bethesda, Md., 1953.
Sawyer, P. N., and Pate, J. W.: Bioelectric phenomena as etiologic
factors in intravascular thrombosis. Amer. J. Physiol. 175:103,
1953.
Williams, R. D., and Carey, L. C.: Studies in the production of
standard venous thrombosis. Ann. Surg. 149:381, 1959.
Schwartz, S. I., and Robinson, J. W.: Prevention of thrombosis with
the use of a negative electric current. Surg. Forum 12:46,
1961.
Sadd, J. R., Koepke, D. E., Daggett, R. L., Zarnsdorff, W. C.,
Young, W. P., and Gott, V. L.: Relative ability of different
conductive surfaces to repel clot formation on intravascular
prostheses. Surg. Forum 12:252, 1961.
Means, G. E., and Feeney, R. E.: Chemical Modification of Proteins.
Holden-Day, Inc., San Francisco, Calif. 1971, pp. 144-148.
SUMMARY OF INVENTION
It is an object of the invention to provide an improved method for
preparing a collagen vascular graft prosthesis.
Still another object of the invention is to provide an improved
vascular graft prosthetic device which is less susceptible to
thrombosis and the like.
Still another object of the invention is to provide an improved
tubular vascular graft, the intimal surface of which cannot be
recognized by blood platelets for purposes of accumulation.
The above and other objects of the invention are achieved by the
provision of a method in accordance with the invention which
comprises modifying the intimal surface of a vascular graft
prosthesis by increasing the net negative surface charge of the
intimal surface thereof.
Preferably the prosthesis will be of collagen and the intimal
surface thereof will be treated by a succinylation reaction in
order that the free amino groups of the collagen surface be
covered.
Preferably the starting material of the graft will be a collagen
prepared from a ficin digested bovine carotid artery material also
known as a dialdehyde starch tanned collagen.
As will be shown, the graft is generally provided in the form of a
collagen tube one end of which is closed and inserted into a fluid
such as ethanol with a liquid chemical reactant being inserted into
the lumen of the tube to increase the negative surface charge of
the intimal surface thereof. The reactant may preferably be, as
will be shown hereinafter, succinic anhydride in a basic
solution.
More particularly a NaHCO.sub.3 buffer solution is inserted into
the lumen in sequential additions of approximately 10 ml. aliquots
to which about 0.1 gm. of crystals of succinic anhydride are
respectively added.
The above and other objects and features of the invention will
become apparent from the detailed description which follows
hereinbelow.
Before, however, the detailed description of the invention is
presented, there is next given a further brief summary as to how
the study leading to the instant invention was undertaken.
Collagen vascular prostheses prepared by enzymatically digesting
bovine carotid arteries with ficin was obtained. Segments of these
vascular prostheses were modified chemically to alter their intimal
surface electrochemical configuration and, more particularly, for
producing a net positive surface charge, negative surface charge,
or neutral surface charge. These modified vascular grafts, together
with segments of the unmodified collagen vascular prostheses were
implanted into the carotid arteries, external jugular veins,
femoral arteries and femoral veins of mongrel dogs for periods of 1
minute, 15 minutes, 30 minutes and 2 hours. The grafts were then
removed and examined macroscopically for the presence and degree of
thrombotic occlusion. These same prostheses were further analyzed
using a scanning electron microscope in an attempt to determine the
precise nature of the occluding thrombi. It was found that by
increasing the net negative surface charge density on the intimal
surface of these vascular prostheses, thrombotic occlusion could be
substantially averted, while creating a more positive
prostheses-blood interface potential greatly accelerated the
thrombotic events.
Segments of these vascular protheses were again implanted into the
vascular system of mongrel dogs and in vivo arterial and venous
streaming potential measurements were carried out. The polarity of
the streaming potential reflects the interfacial potential between
the vascular prosthesis and the blood flowing through the graft
with a positive streaming potential at the downstream electrode
indicating a net negative surface charge density on the intimal
surface of the graft with respect to the blood elements. The
arterial and venous in vivo streaming potentials obtained from the
"negatively" altered collagen vascular prostheses were uniformly
positive in polarity, while those streaming potentials obtained
from the "positively" modified grafts were uniformly negative. The
streaming potentials obtained from those grafts altered chemically
to neutralize the intimal surface charges at physiological pH were
also found to be positive in polarity but of a lesser magnitude
than the negatively altered grafts. The unmodified ficin digested
bovine carotid arteries had streaming potentials of a slightly
negative polarity. These streaming potential measurements served to
check the chemical modification procedures and assure that the
electrical alteration of surface charge density occurred as
predicted.
BRIEF DESCRIPTION OF DRAWING
FIG. 1 diagrammatically illustrates apparatus for practicing the
invention;
FIG. 2 diagrammatically illustrates a technique for measuring
streaming potential in accordance with the invention; and
FIG. 3 diagrammatically illustrates a portion of FIG. 2 on enlarged
scale.
DETAILED DESCRIPTION
Evidence has been presented implying the formation of a complex
between incomplete carbohydrate chains in collagen and
glucosyltransferase present on the outer surface of platelets as
the primary step resulting in platelet recognition of and
subsequent adhesion to collagen. (Jamieson, G. A., Urban, L. C.,
and Barber, A. J.: Enzymatic basis for platelet: collagen adhesion
as the primary step in haemostasis. Nature New Biology 234:5
(1971): Barber, A. J. and Jamieson, G. A.: Platelet collagen
adhesion characterization of collagen glucosyltransferase of plasma
membanes of human blood platelets. Biochim. Biophys. Acta 252:533
(1971)). The reaction involves the enzymatic coupling of glucose to
galactosyl residues attached to hydroxylysine side chains
incorporated into the collagen peptides. The glucose is supplied by
platelets as uridinediphosphoglucose (UDPG) according to the
following: ##SPC1##
Platelet aggregation activity of collagen in vitro has been shown
to require the structural integrity of the 6-hydroxymethyl group of
galactose and the .epsilon.-amino groups of lysine and
hydroxylysine on the collagen molecule. It has been postulated that
the substrate specificity of the platelet glucosyltransferase is
responsible for these observed platelet aggregation dependent
phenomena (Chesney, C., Harper, E., and Coleman, R. W.: Critical
role of the carbohydrate side chains of collagen in platelet
aggregation. HIEG 72-43 (1972); Wilner, G. D., Nossel, H. L., and
LeRoy, E. C.: Aggregation of platelets by collagen. J. Clin.
Invest. 47:2616 (1968) ).
The use of modified bovine arterial heterografts as vascular
prostheses has previously been reported and their physical and
behavioral characteristics as arterial grafts have been discussed
in detail. In the present studies it was found that increasing the
electronegativity of the inner surface of the prosthesis
significantly contributed to a favorable graft performance.
Some of the above is discussed in the following:
Bothwell, J. W., Lord, G. H., Rosenberg, N., Burrowes, C. B.,
Wesolowski, S. A., and Sawyer, P. N.: Modified arterial
heterografts: relationship of processing techniques to interface
characteristics. In: Biophysical Mechanisms in Vascular Homeostasis
and Intravascular Thrombosis, P. N. Sawyer, Ed.
Appleton-Century-Crofts, New York, 1965, pp. 306-313; Rosenberg,
N., Henderson, J., Douglas, J. F., Lord, G. H., and Gaughran, E. R.
L.: Use of arterial implants prepared by enzymatic modification of
arterial heterografts. II. Physical properties of the elastica and
collagen components of the arterial wall. Arch. Surg. 74:89 (1957);
Rosenberg, N., Henderson, J., Lord, G. H., and Bothwell, J. W.: Use
of enzyme treated heterografts as segmental arterial substitutes.
V. Influence of processing factors on strength and invasion by
host. Arch. Surg. 85:192 (1962); Rosenberg, N., Henderson, J.,
Lord, G. H., and Bothwell, J. W.: An arterial prosthesis of
heterologous vascular origin. JAMA 187:741 (1964); Rosenberg, N.,
Henderson, J., Lord, G. H., and Bothwell, J. W.: Collagen arterial
prosthesis of heterologous vascular origin: physical properties and
behavior as an arterial graft. In: Biophysical Mechanisms in
Vascular Homeostasis and Intravascular Thrombosis, P. N. Sawyer,
Ed. Appleton-Century-Crofts, New York, 1965, pp. 314-321.
Succinylation of solubilized collagen has been demonstrated to
result in an approximately 95% conversion of .epsilon.-amino groups
to free carboxyl groups (Gustavson, K. H.: Akiv. For Kemi 17: 541
(1961)). Furthermore, it has been shown that blockage of the free
amino groups of collagen by deamination, N-acetylation or treatment
with dinitroflurobenzene results in a greater than 90% reduction in
platelet aggregating activity in vitro.
Investigation has now been conducted in order to elucidate the
antiplatelet and antithrombogenic activity of chemically modified
collagen by the in vivo evaluation of ficin digested bovine
arterial heterograft vascular prostheses implanted into the
vascular system of mongrel dogs.
For the chemical modification of ficin digested bovine carotid
arteries, the apparatus devised for the chemical modification of
ficin digested bovine carotid arteries is as appears in FIG. 1. It
includes a receptacle 10 having an open mouth 12 through which
extends the shaft 14 of an electric stirrer 16. A glass stopcock 18
is arranged in the lower end of a ficin digested bovine carotid
artery 20 which is immersed, for example, in 40% ethanol.
In order to increase the net positive surface charge density on the
intimal surface of the collagen vascular prostheses a
carbodiimide-promoted amide formation reaction was employed to
convert the major source of net negative charges (free carboxyl
groups of aspartic and glutamic acid) to amide moieties. The
procedure involves treatment of the protein with excess EDC
(1-ethyl-3,3' -dimethylaminopro pylcarbodiimide HCl) and an amine
(NH.sub.4 Cl) in the presence of a high concentration of a
denaturant (urea). This reaction (Means, G.E., and Feeney, R.E.:
Chemical Modification of Proteins. Holden-Day, Inc., San Francisco,
Calif., 1971, pp. 144-148) has been used for the near quantitative
conversion of protein carboxyl groups to amides for determining
numbers of carboxyl groups in proteins (Hoare, D. G. and Koshland,
D. E.: J. Biol. Chem. 242:2447 (1967)). The chemical reaction
proceeds at room temperature as follows: ##EQU1## wherein:
100 ml of reaction solution at room temperature containing 0.5 M
EDC (7.75 gm/100 ml), 7.5 M urea (45 gm/100 ml), and 5.0 M NH.sub.4
Cl (26.75 gm/100 ml) in triple distilled water were prepared
(adjusted to pH5) and added into the lumen of the vascular
prosthesis in 10 ml aliquots with the implant suspended in 40%
ethanol as shown in FIG. 1. The reaction solution was changed every
hour with the last aliquot remaining within the lumen overnight. At
the conclusion of the 24 hour reaction period the vascular
prosthesis was removed from the modification apparatus and placed
in a pan of sterile saline where the long tubes were cut into
segments of 3.5 cm for implantation studies or 10.0cm lengths for
streaming potential measurements.
In order to increase the net negative surface charge density on the
intimal surface of the collagen vascular prostheses acylation of
the free amino groups (especially those of lysine and
hydroxylysine) of the protein with succinic anhydride in a solution
more basic than a PH of 7.0 was carried out. This reaction not only
covers the major source of free positive charges, but also converts
them into anionic residues (Means, G. E., and Feeney, R. E.:
Chemical Modification of Proteins. Holden-Day, Inc., San Francisco,
Calif., 1971, pp. 74,75). The chemical reaction proceeds as
follows: ##SPC2##
A basic solution of 75 ml of 100% ethanol and 25 ml of 1 M
NaHCO.sub.3 buffer solution was prepared and added into the lumen
of the vascular prosthesis in 10 ml aliquots over a 5 hour period
with the implant suspended in 40% ethanol as shown in FIG. 1. To
each of these 10 ml aliquots of basic solution were added crystals
of succinic anhydride. 1.0 gm of succinic anhydride was equally
divided among the 10 aliquots. At the conclusion of the reaction
period, the vascular prosthesis was removed from the modification
apparatus and placed in a pan of sterile saline where the long
tubes were cut into segments of 3.5 cm for implantation studies or
10.0 cm lengths for streaming potential measurements.
In order to neutralize the electrical surface charges on the
intimal surface of the collagen vascular prostheses, a carbodiimide
promoted internal amide formation reaction was employed to link
together the free carboxyl and amino groups of adjacent peptide
chains. Such a reaction removed the major sources of
electronegativity (carboxyl groups) and electropositivity (free
amino groups). The chemical reaction proceeds as follows:
##EQU3##
A 100 ml solution of 0.5 M EDC (7.75 gm/100 ml) in triple distilled
water was prepared and added into the lumen of the vascular
prosthesis in 10 ml aliquots with the implant suspended in 40%
ethanol as shown in FIG. 1. The reaction solution was changed every
hour with the last aliquot remaining within the lumen overnight. At
the conclusion of the 24 hour reaction period, the vascular
prosthesis was removed from the modification apparatus and placed
in a pan of sterile saline where the long tubes were cut into
segments of 3.5 cm for implantation studies or 10.0 cm lengths for
streaming potential measurements.
As a starting material in the above, there was employed, for
example, dialdehyde starch tanned collagen vascular prostheses
including dialdehyde starch tanned bovine collagen vascular
heterografts which were commercially available and obtained from
Johnson & Johnson, New Brunswick, New Jersey.
Surgical implantation of modified collagen vascular prostheses
involved carotid artery and jugular vein implantation. Mongrel dogs
weighing between 10 and 28 Kg (ave. 18.6 Kg) were anesthetized with
Nembutal (Sodium Pentabarbital), 0.5 cc/Kg, by intravenous
injection. The animals were placed on the operating table in the
supine position and an oblique cervical incision, paralleling the
anterior border of the sternocleidomastoid muscle was made. The
platysma muscle layer was incised along with the anterior layer of
the deep cervical fascia and after achieving hemostasis these
structures were retracted posteriorly to reveal the external
jugular vein. At the angle of the mandible, the posterior auricular
vein, the common facial vein and the retromandibular vein were
identified and a mobilized segment of each was encircled with a
loose ligature of umbilical tape. The posterior external jugular
vein was ligated with 3-0 silk and divided near its entrance. The
external jugular vein was then dissected free of its fascial
attachments down to where it disappears behind the posterior border
of the sternocleidomastoid muscle where another loose ligature of
umbilical tape was secured. All small perforating branches of the
external jugular vein were ligated with 3-0 silk and divided close
to their entrance. The anterior border of the sternocleidomastoid
muscle was retracted posteriorly to expose the dissection of the
fascia and fibroareolar tissue layers that overlie the carotid
sheath. The carotid sheath was incised and, after identification of
the vagus nerve, a loose ligature of umbilical tape was made to
encircle the carotid artery at the level of the third tracheal
ring. The incision in the carotid sheath was continued cephalad to
the bifurcation of the common carotid artery (or the superior
thyroid artery) where another loose ligature of umbilical tape was
secured.
Implantation of the respective modified collagen vascular
prosthesis was carried out as follows: "Booties" were constructed
of 2 cm segments of heavy rubber tubing and placed around the free
ends of the loose ligatures of umbilical tape. These booties were
then drawn down the ligatures to prevent flow in the respective
vessel and held in place with Kelly clamps. Two transverse
incisions approximately 2.5 cm apart were made in the respective
blood vessel with a pair of fine curved Metzenbaum scissors. The
vascular prostheses (3.5 cm in length for macroscopic studies and
SEM evaluation and 10.0 cm lengths for streaming potential
measurements), mounted over hydrochloric acid-cleaned stainless
steel cannulas (1.5 cm in length) doubly ligatured with 3-0 silk
were implanted via their free cannula ends into the rents in the
vessel and secured in place with two ligatures of 3-0 silk. The two
anastomoses thus prepared, the connecting piece of blood vessel was
severed thus permitting the graft to lie flat in its "fascial bed."
The Kelly clamps were then removed relieving tension from the
"booties" over the umbilical tapes (distal to blood flow removed
first) and allowing blood to flow through the vascular prosthesis.
The vascular grafts were left in place for the specified periods of
time with the wound covered with saline-soaked gauze pads.
The internal diameters of the blood vessels and the prostheses were
calculated by measuring the external diameters of the vessels and
implants with blood flowing through the system with a micrometer
and substracting two wall thicknesses from these diameters. The
discrepancies between the internal diameters of recipient and
prosthetic blood vessels are found in Table 1.
TABLE 1
__________________________________________________________________________
DISCREPANCY BETWEEN INTERNAL DIAMETER OF RECIPIENT AND PROSTHETIC
BLOOD VESSELS *I.D. of Recipient *I.D. of Prosthetic Change in
Vessel Vessel (mm) Vessel (mm) Internal Diameter
__________________________________________________________________________
Carotid 4 10 6 Artery 5 9 4 5 10 5 5 8 3 4 10 6 x = 4.6 x = 9.4 x =
4.8 Jugular 8 10 2 Vein 7 9 2 8 10 2 8 10 2 8 10 2 x = 7.8 x = 9.8
x = 2.0 Femoral 4 10 6 Artery 5 9 4 4 10 6 5 8 3 4 10 6 x = 4.4 x =
9.4 x = 5.0 Femoral 7 10 3 Vein 7 8 1 7 9 2 8 10 2 7 10 3 x = 7.2 x
= 9.4 x = 2.2
__________________________________________________________________________
*I.D. = Internal Diameter
For femoral artery and femoral vein implantation, an incision
overlying the femoral triangle was extended caudad over the medial
aspect of the thigh to the level of the knee joint. The superficial
femoral fascia was dissected free from its loose attachment to the
underlying femoral sheath revealing the femoral artery and vein
lying within the sheath at the fossa ovalis. The femoral vessels
were dissected free of their sheath and the branches of the femoral
artery (superficial circumflex iliac artery, external pudendal
artery, medial femoral circumflex artery and lateral femoral
circumflex artery) were identified, ligated with 3-0 silk and
divided close to their origin. A loose ligature of umbilical tape
was encircled about the origin of the femoral artery. The rostral
branches of the femoral vein were likewise identified, ligated with
3-0 silk and divided near their entrance and the femoral vein was
also encircled with a loose ligature of umbilical tape. The
sartorius muscle was retracted laterally and the adductor longus
muscle retracted medially with a pair of self-retaining retractors
thus exposing the descending genicular artery and vein. Blunt
dissection was carefully continued deep to the descending genicular
vessels to expose the caudad continuation of the femoral artery and
vein. Small perforating vessels and muscular branches were ligated
with 3-0 silk and divided close to their origin or entrance. Loose
ligatures of umbilical tapes were secured around the respective
femoral vessel and its descending genicular branch. Implantation of
the respective modified collagen vascular prosthesis was carried
out as previously described..
For streaming potential measurements, a pair of silversilver
chloride electrodes 22 and 24 (FIGS. 2 and 3) were placed into the
lumen 26 of the vascular prosthesis 27 through the center of two
purse-strings 28 and 30 of 5-0 Dacron sewn into its superficial
surface and separated by a distance equivalent to at least 10
radii. Electrodes were attached via conventional shielded leads to
a Kiethley electrometer 32 (Sawyer, P. N., Himmelfarb, E., Lustrin,
I., and Ziskind, H.: Measurement of streaming potentials of
mammalian blood vessels, aorta, and vena cava, in vivo. Biophys. J.
6: 641 (1966)).
For removal, fixation and macroscopic observation, at the
conclusion of the respective time periods, the blood vessels
upstream and downstream from both the arterial and venous implants
were doubly cross-clamped with straight Kelly clamps and divided
between the two clamps. Both prostheses (arterial and venous)
filled with blood and occluded at both ends with the Kelly clamps
were immediately removed and placed into a 10% formalin solution.
While remaining under the fluid level of the formalin, the Kelly
clamps were removed along with the stainless steel cannulas and the
3-0 silk ligatures and the vascular implants were placed in
appropriately labeled tubes containing 10% formalin. The degree of
thrombotic occlusion was graded on a scale of 0 to 4+ by three
independent observers in a single-blind manner. The averages of the
three graded values for each vessel and time period appear in Table
2.
TABLE 2
__________________________________________________________________________
MACROSCOPIC OBSERVATION OF MODIFIED COLLAGEN VASCULAR PROSTHESES
IMPLANTATION* Blood Dialdehyde Time Vessel Unmodified Positive
Negative Neutral Starch Tanned
__________________________________________________________________________
0 min LFA 0 2+ 0 0 0 0 min LFV 0 4+ 0 0 0 15 min LCA 1+ 4+ 0 1+ 0
15 min LJV 2+ 4+ 0 1+ 1+ 30 min RFA 1+ 4+ 1+ 2+ 1+ 30 min RFV 4+ 4+
1+ 2+ 2+ 2 hrs RCA 3+ 4+ 1+ 3+ 2+ 2 hrs RJV 3+ 4+ 1+ 3+ 3+
__________________________________________________________________________
*The degree of thrombotic occlusion graded on a scale of 0 to 4+ by
three independent observers. 0 = macroscopically clean prosthetic
intimal surface; 1+ = junctional thrombi at the stainless steel
cannula-prosthesi interface; 2+ = junctional thrombi plus
macroscopically evident mural thrombi adherent to the prosthetic
wall; 3+ = junctional thrombi plus macroscopically evident mural
thrombi significant enough to cause marked stenosis of the graft
lumen; 4+ = total thrombotic occlusion of the prosthetic vascular
lumen. All scaled observations were carried out "single-blind. (LFA
= Left Femoral Artery; LFV = Left Femoral Vein; LCA = Left Carotid
Artery; LJV = Left Jugular Vein; RFA = Right Femoral Artery; RFV =
Right Femoral Vein; RCA = Right Carotid Artery; RJV = Right Jugular
Vein)
The degree of thrombotic occlusion of the variously modified ficin
digested bovine vascular heterografts is seen in Table 2 above. The
unmodified arterial and venous heterografts did not initially
thrombose when exposed to the blood elements. As the implantation
time increased, however, it can be noted that both the arterial and
to a greater extent the venous vascular prostheses became
thrombotically occluded. A similar sequence of events was found to
occur with the neutrally modified collagen prostheses and the
dialdehyde starch tanned vessels. In the latter case, the
thrombotic events proceeded at a slower pace.
The chemical procedure carried out in order to cause an increase in
the net positive surface charge density on the intimal surface of
the vascular heterografts resulted in an acceleration of the
thrombotic events. It can be noted from Table 2 that the mere
contact of the blood elements with the prosthetic vascular surface
resulted in significant occlusion of the vessel lumen (more marked
in the slower flowing venous implant). Total thrombotic occlusion
occurred in the venous implant immediately and was noted within 15
minutes in the arterial grafts.
The negative modification reaction carried out on the collagen
tubes resulted in a most favorable performance of these implants.
It can be seen from Table 2 that the thrombotic events occurring on
the intimal surface of the vascular prostheses were significantly
prolonged. A macroscopically clean intimal surface was achieved on
both the arterial and venous grafts for up to 15 minutes of blood
flow. After 2 hours of contact with the flowing blood elements only
junctional thrombi at the stainless steel cannula-prosthesis
interface was noted.
The results of the in vivo arterial and venous streaming potential
measurements of modified collagen vascular prostheses implantations
appear in Table 3.
TABLE 3 ______________________________________ IN VIVO STREAMING
POTENTIAL MEASUREMENTS OF MODIFIED COLLAGEN VASCULAR PROSTHESES
VASCULAR ARTERIAL STREAMING VENOUS STREAMING PROSTHESIS POTENTIAL
(mv) POTENTIAL (mv) ______________________________________
Dialdehyde +0.1 -0.1 Starch Tanned +0.2 -0.1 +0.2 +0.1 x = +0.13 x
= -0.03 Positive Ficin -0.8 -0.4 Digested -0.5 -0.4 -0.8 -0.4 x =
-0.7 x = -0.4 Negative Ficin +0.7 +0.3 Digested +0.4 +0.3 +0.7 +0.6
x = +0.6 x = +0.4 Neutral Ficin +0.3 +0.1 Digested +0.2 +0.2 +0.2
+0.1 x = +0.23 x = +0.13 Unmodified -0.2 -0.1 Ficin Digested
______________________________________
The polarity of the streaming potential reflects the interfacial
potential between the vascular prosthesis and the blood flowing
through the graft with a positive streaming potential recorded at
the downstream electrode indicating a net negative surface charge
density on the intimal surface of the graft with respect to the
blood elements.
The polarity of the in vivo arterial and venous streaming potential
measurements obtained from the implantation of the unmodified ficin
digested bovine vascular heterografts was on the order of -0.2 and
-0.1 mv, respectively. These values indicate a slightly positive
interfacial potential exists between the intimal surface of the
vascular graft and the "streaming" blood elements. An even greater
magnitude of negative streaming potential (positive interfacial
potential) was noted with the arterial and venous heterografts
chemically modified to increase the positive surface charge density
(-0.7 mv-arterial; -0.4 mv-venous).
Collagen vascular heterografts chemically modified to neutralize
the electrical surface charges on the prosthetic intimal wall
produced arterial and venous streaming potentials on the order of
+0.23 and +0.13 mv, respectively. These collagen vascular implants
chemically altered so as to increase the negative surface charge
density gave positive streaming potential measurements of greater
magnitude (+0.6 mv-arterial; +0.4 mv-venous). These latter values
indicate that a significant negative interfacial potential was in
fact produced on the intimal surface of the vascular grafts by the
chemical modification procedure.
An ideal vascular prosthesis would possess the recoil
characteristics of an intact elastica plus the strength of a
collagen backbone. The strength of this collagen backbone to
withstand many months of repetitive pulsatile arterial pressures
and function as a prosthetic vascular heterograft has been
documented (Rosenberg, N., Henderson, J., Lord, G. H., and
Bothwell, J. W.: Collagen arterial prosthesis of heterologous
vascular origin: physical properties and behavior as an arterial
graft. In: Biophysical Mechanisms in Vascular Homeostasis and
Intravascular Thrombosis, P. N. Sawyer, Ed.
Appleton-Century-Crofts, New York 1965, pp. 314-321). Furthermore,
many of the desirable features of a vascular prosthesis
(Wesolowski, S. A., Fries, C. C., and Sawyer, P. N.: Some desirable
physical characteristics of prosthetic vascular grafts. In:
Biophysical Mechanisms in Vascular Homeostasis and Intravascular
Thrombosis, P. N. Sawyer, Ed. Appleton-Century-Crofts, New York
1965, pp. 322-336) are present in ficin digested bovine carotid
artery preparations. Enzyme treatment removes the parenchymal
elements subject to necrosis and thus prevents the associated
inflammatory reaction which impairs the integrity of the graft wall
and predisposes to intimal thrombosis. It further produces a degree
of porosity of the prosthetic vascular wall which favors the
orderly invasion by host connective tissue and due to the relative
absence of inflammatory reaction, favors early endothelialization
as well.
Early thrombosis after insertion of these grafts can be inhibited
by increasing the negative surface charge density on their intimal
surfaces. The above results substantiate the importance of the
negative intimal surface charge density in preventing early
thrombotic occlusion of collagen heterograft vascular prostheses.
Chemical modification designed to increase the negative surface
charge density on the inner wall of ficin treated bovine arterial
heterografts was found to prevent luminal occlusion by mural
thrombosis significantly. Chemical modification by a
carbodiimide-promoted amide formation reaction designed to increase
the positive surface charge density on the intimal surface was
found to greatly accelerate the thrombotic events (Table 2).
The polarity of the arterial and venous streaming potentials
measured through the lumens of these vascular prostheses served to
check the chemical modification procedures and assure that the
electrical alteration of surface charge density occurred with the
predicted polarity. It can be seen from Table 3 that the arterial
and venous streaming potentials measured through the lumens of
those grafts modified to increase the intimal surface
electronegativity were uniformly positive in polarity, while those
grafts designed to present a positively charged surface to the
flowing blood elements produced streaming potentials with a
negative polarity. Thus the chemical modification procedures were
effective in modulating the polarity of the interfacial potential
between the luminal surface of the vascular prosthesis and the
blood elements flowing within.
Ficin digested bovine carotid artery vascular heterografts were
also implanted into the vascular system of mongrel dogs in their
unmodified state and in a chemically altered condition designed to
neutralize the electrical surface charges on the graft. It can be
seen from Table 2 that both of these implants were inferior in
their performance when compared with the negatively modified
surface, but superior to the more positively charged graft.
The streaming potential measurements obtained from the unmodified
ficin digested collagen grafts indicate that their interfacial
polarity is slightly positive while that of the neutrally modified
implants is slightly negative, (Table 3). It can be seen from these
results that the chemical procedure designed to neutralize the
slightly positive surface charge on the ficin digested surfaces
(unmodified) actually overshot electrical neutrality and produced a
surface with a slight excess negative charge. Even though the
excess charges on the surfaces of these two vascular prostheses
were opposite in polarity, their funtioning as vascular grafting
material as judged macroscopically was comparable. This apparent
lack of difference in performance between the two can probably be
accounted for on the basis of their relatively small surface charge
density (as compared with the positively and negatively modified
vascular prostheses -- see Table 3).
A further type of ficin digested bovine vascular heterograft was
also evaluated. This dialdehyde starch tanned prosthesis has
previously been reported to perform well in long term (3 years)
studies (Bothwell, J. W., Lord, G. H., Rosenberg, N., Burrowes, C.
B., Wesolowski, S. A. and Sawyer, P. N.: Modified arterial
heterografts: relationship of processing techniques to interface
characteristics. In: Biophysical Mechanisms in Vascular Homeostasis
and Intravascular Thrombosis, P. N. Sawyer, Ed.
Appleton-Century-Crofts, New York, 1965 pp. 306-313). It can be
seen from Table 2 that this grafting material was markedly inferior
to the negatively modified collagen prostheses but comparable to
the unmodified and neutrally altered collagens. The streaming
potential measurements obtained from these implants were quite
ambiguous. The arterial streaming potentials were uniformly
positive in polarity, indicating that the prosthesis presents a
slightly negative surface to the flowing blood. The venous
streaming potentials, however, were not consistent, two of the
three measurements indicating that the graft actually presents a
slightly positive surface to the bloodstream.
The critical role of platelets in intravascular thrombosis has been
well documented in the following:
Mitchell, J. R. A.: Chapter XVI Platelets and Thrombosis, Sci.
Basis Med. Ann. Rev. 266-288 (1968);
Hampton, J. R.: The study of platelet behavior and its relation to
thrombosis. J. Atheroscler. Res. 7:729 (1967);
Mustard, J. F., Packham, M. A. Rowsell, H. C., and Jorgensen, L.:
The role of platelets in thrombosis and aterosclerosis. Thromb.
Diath. Haemorrh. Suppl. 23:261 (1967).
It is believed by some that the initial event leading to the
formation of an intravascular mural thrombus is the unmasking of
subintimal collagen and its subsequent recognition by circulating
platelets with the latter's resulting adhesion and aggregation
(this view is not universally accepted). It is known, however, that
collagen is capable of initiating platelet adhesion to itself and
that this adhesion can result in platelet aggregation. The precise
biochemical mechanism by which platelets recognize collagen and
subsequently adhere to it has been shown to involve the formation
of a complex between incomplete carbohydrate side chains in
collagen and glucosyltransferase present on the outer surface of
platelets (see FIG. 1). The reaction involves the enzymatic
coupling of glucose (supplied by the platelets as
uridinediphosphoglucose) to galactosyl residues attached to
hydroxylysine side chains incorporated into the collagen
peptides.
The reaction employed to increase the net negative surface charge
density on the intimal surface of the collagen vascular prostheses
was a succinylation reaction designed to cover the free amino
groups of the protein. Succinylation of solubilized collagen has
been demonstrated to result in an approximately 95% conversion of
epsilon amino groups to free carboxyl groups (Gustavson, K. H.:
Akiv. for Kemi 17:541 (1961)). This reaction was observed to result
in the production of a vascular prosthesis which when implanted
into the vascular system of mongrel dogs resulted in the least
amount of platelet deposition and subsequent thrombotic occlusion.
It can be seen from the above biochemical data that this
succinylation reaction which covered the epsilon amino groups of
the protein sufficiently altered the substrate of platelet
glucosyltransferase such that the specificity of this enzyme no
longer recognized the heterograft collagen as such. Without
platelet recognition there could be no adhesion to the prosthetic
intimal surface and thus no thrombotic occlusion.
Furthermore, it has been shown that blocking the free carboxyl
groups of collagen results in increased platelet aggregation
activity in vitro. This has been postulated to result from the
potentiation of the effects of the free epsilon amino groups
(Wilner, G. D., Nossel, H. L., and LeRoy, E. C.: Aggregation of
platelets by collagen. J. Clin. Invest. 47:2616 (1968 )). The
results obtained in this study with the reaction designed to
increase the net positive surface charge density on the intimal
surface of the collagen vascular prosthesis by amidation of the
free carboxyl groups is entirely consistent with these in vitro
studies. The positive modification reaction resulted in the rapid
and total thrombotic occlusion of these vascular heterografts when
implanted into the vascular system of mongrel dogs.
A negative intimal surface charge density results in a favorable
vascular heterograft performance by preventing early thrombotic
occlusion of the prosthesis' lumen. A simple chemical modification
procedure is given by which the intimal surface of ficin digested
bovine carotid arteries can be prepared with this added
electronegativity.
In addition to the above, the invention provides a new kind of
prosthesis consisting of a ficin digested artery modified with
anionic dialdehyde starch. This prosthesis will be tanned and will
have had negative charges incorporated into it in one step. Thus
the resulting product will be strengthened as by dialdehyde-starch
tanning and will have an excess of negative charge so as to make a
better non-thrombogenic surface. The dialdehyde-starch derivative
needed is the 6-carboxy-methyl derivative I ##SPC3##
This material is either available or can be made. It is available
as a product called "Water Soluble Anionic Polymeric Dialdehyde"
known as DASOL A available from Miles Laboratories, Inc., which is
of interest as a tanner and suitable charge carrier.
As a dification of an existing prosthesis to introduce negative
charge, a ficin digested dialdehyde starch modified artery can be
further modified with succinic anhydride to introduce negative
charges. The reaction of dialdehyde starch may occur mainly with
the .epsilon.-amino groups of lysine under conditions of tanning
(pH 8.8). Some reaction occurs with the guanidino groups of
arginine as it is known that this group reacts with neighboring
dicarboxyl groups which is one way of looking at dealdehyde-starch.
Be that as it may, there may be sites which are still available for
attack by succinic anhydride. This will result in a tanned,
negatively charged prosthesis.
Further, there is the exhaustive decationization of a succinic
anhydride modified prosthesis. Succinic anhydride reacts mainly
with the .epsilon.-amino groups of lysine introducing a negatively
charged carboxy propeonoyl moeity. Assuming all the lysine groups
are blocked, positive charge sites exist on the arginine residues
and on the histidine residues (some of the imidazole groups of
histidine may have the positive charge removed at pH 7.4).
Modification of the arginine residues can be effected by phenyl
glyoxal which reacts under mild conditions with the positively
charged guanidinum group and results in a neutral derivative.
Histidine can be converted to a neutral derivative by reaction with
diethyl pyrocarbonate to given an ethoxyformyl derivative on the
imidazole ring.
The above techniques involve the negatively charged tanning of
collagen for protection against intravascular thrombosis. It is
appropriate to apply these techniques to other areas in the body
when collagen prostheses and, in fact, heterograft materials are
used which are taken from various species of animals and used in
humans. The most significant of these prosthetic devices are
homograph valves taken from one human and placed in another human
and heterograft valves which conventionally are taken from pigs and
inserted into the aortic valve area. These valves are known to have
certain advantages in that their leaflets permit central flow. The
basic problem with various aspects of their application has always
been that, while they work satisfactorily in the aortic position,
there has always been some question about their utility in the
mitral valve area. The negatively charged tanning of these valves
enhances their utility.
By way of further embodiment of the invention, a heterograft valve
is removed under semi-sterile conditions from the porcine heart. It
is tanned using the negatively charged tanning techniques described
above. The tanning procedure is effected using either succinic
anhydride or dasol to produce a highly negatively charged collagen
surface on the valve which makes the prosthesis more anticoagulent
in character than it would be under conventional circumstances. The
charge characteristic provides not only additional
antithrombogenesis but also increases the life span of the
implanted heterograft valve because of its resistance to fibrin
deposition and destruction. The same also applies to homograph
valves.
There will now be obvious to those skilled in the art many
modifications and variations based on the above. These
modifications and variations will not depart from the scope of the
invention as defined by the following claims.
* * * * *