 |
Description  |
|
|
This invention relates to a specific form of finely-divided fibrous
collagen and fibrous products derived from collagen particularly adapted
for use in medical and surgical procedures.
Collagen in various treated or prepared forms is useful in surgery and the
treatment of wounds as shown for example, in U.S. Pat. No. 3,157,524 to C.
Artandi and J. F. Prudden, Arch. Surg. 89, 1046-1059, December 1964. E. E.
Peacock, Jr. et al, Ann. Surg. 161, 238-247, February 1965, among others
teach that collagen has hemostatic properties when used as a wound
dressing, and has a low level of antigenicity. It has now been found that
fibrous collagen and fibrous products derived from collagen, when properly
prepared and when wet with blood, will not only demonstrate hemostasis but
also demonstrates an unexpected adhesiveness to severed biological
surfaces in warm blooded animals. Thus, this new form of collagen, unlike
other forms of collagen suitable for use in the treatment of wounds,
demonstrates an unexpected and unique adhesiveness between two severed
biological surfaces and in many instances can actually be used to adhere
severed tissue without the use of sutures as well as to effect hemostasis.
It is a primary object of this invention to provide a finely-divided
fibrous collagen and fibrous products derived from collagen which are
useful in effecting hemostasis of and adhesiveness between two severed
biological surfaces in warm blooded animals.
It is another object of this invention to provide a method of preparing a
finely-divided fibrous collagen and fibrous products derived from collagen
which are useful hemostatic agents and have unique adhesive properties in
contact with a severed biological surface in a warm blooded animal when
wet with blood.
These and other objects are attained in accordance with this invention with
a composition of matter comprising finely-divided fibrous collagen
products having a surface area of at least 1 square meter per gram,
preferably from about 15 to about 30 square meters per gram, and a bulk
density of not more than about 8 pounds per cubic foot, preferably between
about 1.5 and 6.0 pounds per cubic foot. The preferred fibrous collagen
products are partial, ionizable acid salts of collagen containing from
about 50% to 90% of the theoretical stoichiometric bound acid content.
The surface area is determined by the conventional nitrogen adsorption
method using the Perkin-Elmer Sorptometer. The bulk density is determined
by adding the fibrous product to a container of known volume without
compression and weighing the amount of added material.
The fibrous collagen products having the foregoing characteristics possess
unique adhesive properties when wet with blood and these adhesive
characteristics are demonstratable in surgical experiments with animals.
Fibrous collagen products outside the scope of the products of the present
invention do not exhibit the unique adhesive properties. As will be
described herinafter, an in vitro procedure has been devised for measuring
the adhesive properties of the fibrous collagen products when wet with
blood.
The fibrous collagen products may be prepared by mechanically dicing or
chopping wet, undenatured collagen or delimed edible collagen, fiberizing
the diced or chopped collagen, mechanically dispersing the collagen fibers
in an aqueous liquid which controls the swelling of the collagen fibers,
replacing the bulk of the water associated with the fibers by a
water-miscible organic liquid, drying the fibrous collagen product and
finally deaggregating the collagen fibrils or fluffing the dried product.
Alternatively, the wet undenatured collagen or delimed collagen is
mechanically diced or chopped, treated with a water-miscible organic
liquid to remove the bulk of the water, dried and then subjected to the
final deaggregating or fluffing operation.
The wet collagen such as hide is diced or chopped into small fragments of
from 1/4 to 1/2 inch sizes in a cutting or grinding mill, such as, for
example, an Urschel Mill. These fragments may be mixed with crushed ice
and then passed through the Urschel Mill with cutting heads of smaller
dimension to fiberize the collagen into a coarse fibrous product.
If swelling or hydration of the collagen fibers is not controlled during
the subsequent treatment wherein the collagen is subjected to mechanical
shredding or opening in a liquid medium excessive hydrogen bonding or
densification will occur when the material is dried down thereby
effectively preventing the satisfactory deaggregation of the collagen
fibrils during the final mechanical treatment. The initial swelling of the
collagen fibers in the wet state affords many more sites for hydrogen
bonding than is desirable thus leading to a dried material which may be
hornified and difficult to deaggregate into the constituent fibrils or
"fluff". When hornification and densification occurs, the product will not
have the required physical characteristics so as to provide the desired
adhesion to severed biological surfaces in warm blooded animals when wet
with blood nor will it provide the required mechanical properties of the
collagen-blood matrix between the severed surfaces.
The terminology "mechanical properties of the collagen-blood matrix" is
used to designate the property of a mixture of the fibrous collagen
product and blood which holds together the mixture sufficiently and has
sufficient tensile strength to resist separation of the fibers and thereby
seal the wound. The in vitro test procedure to be described is a direct
measure of this mechanical property.
The wet or moist fibrous collagen is mechanically opened and the fibers
dispersed in an aqueous liquid which controls the swelling of the fibers.
The aqueous liquid comprises water and a water-miscible organic liquid
such as low molecular weight alcohols, acetone, and the like. The liquid
may comprise the organic liquid such as methanol, ethanol, isopropanol,
methylethyl ketone, acetone and the like and water in a weight range of
from about 90% of the organic liquid to 10% water to about 50% organic
liquid to 50% water, preferably 75% organic liquid and 25% water. Where
the proportion of water is too high, the collagen fibers swell to such a
great extent that a larger number of sites are provided for hydrogen
bonding and attendant densification during the subsequent drying step.
When this occurs, excessive hornification and/or densification occurs and
it becomes commercially unfeasible to subsequently deaggregate or fluff
the fibrous product and attain the bulk density and the surface area
requirements necessary for the present invention. Although such product
will possess some hemostatic properties, it does not possess the desired
adhesion to severed biological surfaces and will not provide the required
mechanical properties of the collagen-blood matrix between the severed
surfaces.
The bulk of the liquid is drained from the mass and the fibrous collagen
slurried and washed with a water-miscible organic liquid such as the
alcohol or acetone and again the bulk of the liquid is separated from the
partially swollen wet fibers. Preferably, the fibrous material is slurried
in the organic liquid to reduce the water content to a minimum. In
general, the use of three slurrying steps with the organic liquid will
reduce the amount of water present to above 1%. The organic liquid is
removed as by centrifugation and final drying. Drying may be effected
either by oven drying or vacuum drying as at, for example, 40.degree. C.
under a 29 inch vacuum for about 16 hours. In general, this vacuum drying
will reduce the volatile content to under 1%.
Before the final deaggregation into constituent fibers or fluffing
operation to produce the product having the required surface area and bulk
density, the fibrous material is preferably conditioned to contain about
8% to 15% volatiles such as water and/or organic liquid. This conditioning
may be readily effected by allowing the product to remain at normal
atmospheric temperatures and humidities for from about 8 to 24 hours. The
final fiber deaggregation or fluffing operation is necessary to provide
the requisite bulk density and surface area. This operation is, in effect,
an "opening" operation which is somewhat comparable to the deaggregation
of chrysotile fibrils. In this art, this opening separates some fiber
bundles into their ultimate individual chrysotile fibrils. In forming the
product of the present invention, the final fiber deaggregation or
fluffing operation does not separate all of the dried bundles into
ultimate individual fibrils but the product does contain finer fiber
bundles (smaller in diameter) as compared to the coarser fiber bundles
obtained at the end of the drying and conditioning operations. This
deaggregation or fluffing may be effected by apparatus such as a Waring
Blendor or, preferably, a hammer mill type comminution mill such as a Fitz
Mill.
Where it is desired to produce a partial salt of collagen, the required
amount of an ionizable acid may be incorporated in the aqueous liquid
wherein the fibrous collagen is dispersed. The amount of acid incorporated
in the aqueous liquid is such as to provide the product with a bound acid
content of from about 50% to 90% preferably about 60% of the theoretical
stoichiometric bound acid content. After the acid has reacted with the
dispersed collagen, the reaction mass is subjected to slurrying and
washing with the water-miscible organic liquid and the collagen salt
processed as above described.
Alternatively, the partial ionizable salt of collagen may be prepared as
described in the copending application of Orlando A. Battista, Ser. No.
14,709, filed Feb. 9, 1970. In accordance with the method described in
that application, diced or chopped undenatured collagen is introduced into
and mixed in a water solution of an ionizable acid, the pH of the solution
being between about 1.6 and about 2.6 based upon a 1% by weight solids
content of the mixture. The mixture is agitated to permit the desired
reaction between the collagen and acid and produce a partial salt of
collagen containing between about 50% and 90% of the theoretical
stoichiometric bound acid content.
After separating the reacted collagen from the bulk of the solution and
washing it, the fibrous product may be recovered by drying. Because of the
use of the aqueous liquids, hydrogen bonding occurs and the product is
hornified to some extent. A product satisfactory for the present invention
may be obtained by a severe deaggregation procedure and fluffing
operation. For example, a fibrous product prepared in this manner will
require a minimum of two passes through a hammer mill such as a Fitz Mill
in order to provide the requisite bulk density and surface area.
Although the partial salt of collagen may be prepared by the use of the
water-acid solution in a manner as described, the partial salt after its
formation and recovery in a wet state may be slurried and washed several
times with a water-miscible organic liquid as described above and then
dried. Such product will be less hornified than that dried directly from
the water system. In general, such product requires only two passes
through a Fitz Mill to produce a final product having the required bulk
density and surface area.
Alternatively, the wet collagen source material is diced or chopped into
small fragments and then introduced into and mixed in a water-miscible
organic liquid such as ethanol or isopropanol. Mixing is continued for
about one hour so as to permit thorough penetration of the organic liquid
into the small fragments. The bulk of the liquid is then separated as by
draining or centrifuging and the recovered fragments again introduced into
and mixed in the organic liquid for about one hour. Again, the bulk of the
liquid is separated and the procedure repeated. At the end of this period,
the liquid is centrifuged from the mixture and the wet fragments dried as
by oven drying or vacuum drying. The resulting product, after conditioning
as described above, is then subjected to a fiberizing and deaggregation or
fluffing operation. If it is desired to produce a partial ionizable salt
of collagen, the desired amount of acid may be mixed with the organic
liquid in any one of the above described steps. In such instances, the
time of treatment with the organic liquid containing the acid should be
prolonged to permit the required reaction between the acid and the
collagen. Obviously, the time periods may be reduced by operating under
pressure.
The foregoing discussion describes, in general, the preparation of products
under conditions wherein the pH of the treating solution is neutral or
below pH 7 . Products having the required bulk density, surface area and
adhesive characteristics may also be prepared wherein the treating
solution is on the alkaline side; that is, at a pH above 7. Thus, products
with the required properties have been prepared by substituting an alkali
such as sodium hydroxide, potassium hydroxide and ammonium hydroxide for
the acid of the treating solutions. These treating solutions had a pH of
approximately 9. The processing procedures are identical to those
described hereinabove.
The suitability of the collagen product for its intended uses may be
determined by a relatively simple laboratory in vitro procedure which has
been termed a "HAT Test" or "Hemostat-Adhesive Test". In this procedure, a
cone penetration test is performed utilizing a Penetrometer such as used
the testing of lubricating greases and bituminous materials in accordance
with ASTM D 217-67 T. In this test, 1.2 grams of the fibrous collagen
product is mixed with 3.5 mls. of outdated human blood, that is, between
21 and 31 days after it was drawn and stabilized with standard citrate
solutions in accordance with normal procedures.
The blood is transferred to a cavity (0.753" diam., 0.875" deep) in a
plastic block, and the fibrous product slowly added and uniformly mixed
with the blood. It is important that all of the fibers are wetted by the
blood and that the mass is of homogeneous structure. The surface of the
mass is made smooth. Mixing and smoothening are conveniently accomplished
by use of a narrow stainless steel spatula.
The block is inserted loosely in a polyethylene bag, placed in an oven
maintained at approximately 98.degree. F. and held in the oven for 60
minutes 35 one minute. At the end of this period, the block is removed
and placed on the Penetrometer stage.
The head of the mechanism as used in this procedure consists only of the
stainless steel nipple at the end of the normal penetrometer cone. It is
adjusted so as to bring the point of the penetrating cone exactly into
contact with the surface of the sample. The rod supporting the penetrating
cone is then released to allow the cone to penetrate the sample for a
period of 5 seconds at the end of which the supporting rod and cone are
locked into position. A gauge is actuated to measure the change in
position of the rod which is equivalent to the penetration of the cone
into the sample. The standard Penetrometer gauge measures this distance in
tenths of a millimeter. In the testing of the samples, three penetration
tests are applied to each sample at different positions on the surface of
the sample. The results, the average of the three penetration tests, are
expressed as distance in tenths of a millimeter .times. 10 and reported as
"HAT Rating". It is obvious that the greater the adhesion between the
blood wetted fibers the greater the tensile strength of the mass and,
accordingly, the amount of the penetration of the cone into the mass is
inversely proportional to the strength of the mass.
If nubs or gritty particles and/or blobs of fibers are evident during the
initial mixing of the sample and the blood, but no unabsorbed blood is
evident in the cavity, the test is run in the normal manner. However, such
samples should be removed from the cavity at the completion of the
Penetrometer reading, and the plug examined with a spatula to determine if
any of the more dense particles have not been completely soaked through by
the blood. If there appear nubs or particles with cores that are white
indicating nonuniform wetting the blood, the test data is invalidated and
the sample rerun using more thorough blending during the initial mixing
step.
In addition, the sample is considered to have failed this test if after the
initial mixing unabsorbed blood appears freely in the cavity.
While a wide variety of materials exhibit various degrees of hemostatic
properties, it is only a relatively narrow range of the collagen products
of this invention which, in addition to having the hemostatic properties,
also have an adhesive property rendering them satisfactory in adhering
severed tissues. It has been found that there is a direct correlation
between the HAT Test and the in vivo tests in animals involving veins, the
spleen and the liver. In general, the products found to be satisfactory as
both a hemostat and an adhesive exhibit HAT Tests, or HAT Ratings, not
exceedng about 1500, preferably below 900. It has also been observed that,
in general, samples of products which possess the combined hemostatic and
adhesive characteristics when mixed with human blood an incubated for one
hour at 98.degree. F. exhibit a dark red to black color whereas those
products which lack the adhesive properties exhibit a red to bright red
color.
It has also been discovered that in order for the product to exhibit the
desired properties it is preferable that it contain at least about 5% by
weight of fibers having an effective length of not more than 1 mm. and not
over 10% have a length above mm. Where the proportion of short fibers
(not more than 1 mm.) is below the lower limit, there appears to be a
clumping or aggregation of longer fibers with a loss in both hemostatic
and adhesive properties. Where the proportion of short fibers is too high,
there is a loss in adhesive properties. One measure of the fiber length
distribution and the fluffiness of the products is bulk density and the
bulk density of the products satisfactory for the present purposes is not
more than 8 pounds per cubic foot. The bulk density is measured by adding
the fibrous collagen products as initially fluffed to a 100 ml. graduate
cylinder without any compression step and determining the weight of the
added 100 mls of the product.
The finely-divided fibrous collagen product of this invention can be
prepared from an undenatured collagen in the natural state or delimed
edible forms of collagen including, for example, hide, gut, tendon,
cartilage or other high fibrous collagen source material preferably
chopped up for easier handling. The collagen is preferably in a wet and
never-dried state or, if dried, drying has been effected under conditions
which minimize denaturization. Satisfactory raw materials for the collagen
include, for example, fresh cowhides and calfhides, salted down cowhides,
wet moosehide, pigskins, sheepskins and goatskin as conventionally used
for making leather. Special technical hide collagen prepared from hide
splits and possessing a minimal reduced bacteria count is also
satisfactory. The preferred raw material, because of availability is
never-dried cowhide or technical grades of collagen prepared from cowhide
and other animal hides.
In forming the partial salt of collagen, hydrochloric acid is the referred
acid and is used in the examples which flow merely because it is
relatively inexpensive and allows ready flexibility and ease of control.
Other ionizable acids, both inorganic and ionizable organic acids, such
as, for example, sulfuric acid, hydrobromic acid, phosphoric acid,
cyanoacetic acid, acetic acid, citric acid and lactic acid are
satisfactory. Sulfuric acid, for example, is satisfactory, but control of
the action is difficult. Citric acid may be substituted for hydrochloric
acid with about equal results. "Ease of control" has reference to the
ability to arrest the swelling and hydrolysis of the collagen fibers so as
to prevent the rapid degradation of the material to a water-soluble
product.
In the examples which flow, Examples I and II describe the preparation of a
preferred partial salt of collagen and reference will be made to the
product of Example II as a standard for purposes of comparison. In
addition to measuring the bulk density and surface area, a suspension
(1/2% by weight) of each sample was subjected to the action of a Waring
Blendor at high speed for 30 minutes and the pH of the suspension was then
measured. The properties of the fluffy finely-divided fibrous products of
the examples were as reported in Table I.
EXAMPLE I
Raw or green bovine corium was cut into 3 inch strips, the lengths varied
depending upon the position in the original hide, and were then frozen for
future processing. A desired number of strips were subsequently allowed to
thaw for about 1 day and washed several times with water at a temperature
of about 10.degree. C.- 11.degree. C. After the excess water had been
allowed to drain, the wet strips were diced into chips or pieces of 1/4"
to 1/2" size. Specifically, the dicing was accomplished by feeding
manually the strips into an Urschel MIll (Comitrol Model 1300) having a
circular cutting head fitted with bars having a width of 0.030" spaced
apart 0.510". The revolving impeller had a clearance of 0.007" from the
cutting head and was operated at 4140 rpm. The diced material was mixed
with crushed ice and passed through the Urschel Mill fitted with bars
having a width of 0.010" spaced apart 0.040" to fiberize the collagen. The
temperature during dicing and fiberizing was between 10.degree. C. and
about 15.degree.C. and the solids content of the fiberized mass was about
25%. The mass was then centrifuged to a solids content of approximately
30%.
A mixture was prepared containing about 108 pounds of isopropanol and 373
mls. of 12N HCl. To this mixture was added 51.5 pounds of the fiberized
mass. Since the fiberized mass contained about 36 pounds of water, the
aqueous liquid contained approximately 75% isopropanol and 25% water. The
amount of hydrogen chloride was approximately 90% of the theoretical
stoichiometric amount of acid required to react with all amino groups of
the collagen. The solids content (collagen) was about 9.7%. The slurry was
mixed in a Lightnin mixer operating at 115 rpm. for 3 hours and then
centrifuged to a solids content of about 40%, 39 pounds of cake resulting.
The cake was then introduced into about 115 pounds of isopropanol
(containing about 1.5 pounds of water) in a Lightnin mixer and the fibers
slurried for 2 hours. The mass was centrifuged to reduce the cake to a
solids content of about 40%. The cake was reslurried in 117 pounds of
isopropanol (containing about 1.1 pounds of water) and again centrifuged.
All of the liquid treatments were carried out at temperatures between
about 25.degree. C. and 28.degree. C.
The cake was vacuum dried under 28"- 29.5" of mercury at 60.degree. C. for
about 22 hours and resulted in the recovery of 15.25 pounds of fibrous
collagen product. The fibrous product was then conditioned by exposure to
the room atmosphere over a weekend and contained about 11% moisture.
The resulting fibrous mass was then subjected to a deaggregating or
fluffing operation by passing through a hammer mill and screening.
Specifically, the mass was passed through a Fitz Mill (Model DA50-6-5634)
operated at 6250 rpm. and equipped with a #4 screen having openings of
0.243".
The final fluffed product contained 0.658 meq. HCl per gram of collagen
(approximately 84% of the theoretical stoichiometric amount). The bulk
density was 2.5 pounds per cubic foot and the surface area of various
samples varied from 18.1 to 23.0 square meters per gram. The HAT Rating
was 799 with AB+ blood and 850 using A+ blood. Upon disintegrating a
sample of the product in water by subjecting a suspension containing 0.52%
by weight of the fluffy, finely-divided fibers in water to the action of a
Waring Blendor for 30 minutes, a stable dispersion was formed having a pH
of 3.30.
EXAMPLE II
The product used as a standard for the in vivo tests was obtained by
subjecting the product of Example I to screening in a Rotap for 1 hour and
using the material retained on a #14 Sieve. The bulk density was 2.5
pounds per cubic foot, the surface area was 24 square meters per gram and
the HAT Rating was 700 with A- blood.
EXAMPLE III
Example I was repeated but ethyl alcohol was substituted for the
isopropanol of Example I.
EXAMPLE IV
Example I was repeated but methyl ethyl ketone was substituted for the
isopropanol of Example I. The dried conditioned product was deaggregated
or fluffed dry in a Waring Blendor for 1 minute at high speed.
EXAMPLE V
Example I was repeated substituting delimed split corium for the raw corium
of Example I.
EXAMPLE VI
Example III was repeated but no acid was added to the ethanol-water
treating solution. The deaggregated, fluffy product was subsequently
screened through a No. 4 screen, followed by a pass through an 18 mesh
screen (0.039" openings).
EXAMPLE VII
Example III was repeated but no acid was added to the ethanol-water
treating solution and the final deaggregation and fluffing treatment was
altered. The conditioned fibrous mass was first passed through a Fitz Mill
provided with a No 2A screen closed by a polyester film on the back side
of the screen followed by a second pass using a No. 2AA screen having
0.079" openings.
EXAMPLE VIII
Example I was repeated using freeze-dried, green corium in place of the raw
corium, omitting the acid and adjusting the pH of the liquor to pH 9 by
addition of 0.01N NaOH solution. The dried conditioned product was
deaggregated or fluffed dry in a Waring Blendor for 1 minute at high
speed.
EXAMPLE IX
Example I was repeated using freeze-dried, green corium in place of the raw
corium, omitting the acid and adjusting the pH to 8.0 using 0.05N ammonium
hydroxide.
EXAMPLE X
Strips of wet green corium (about 30% collagen) were diced into chips or
pieces 1/4" to 1/2" in size. A mixture was prepared containing about 108
pounds of ethyl alcohol (200 proof) and 51.5 pounds of the wet diced
corium. The solids content (collagen) was about 9.7%. The mixture was
agitated in a Lightnin mixer operating at 115 rpm for 1 hour and then
centrifuged to a solids content of about 40%.
The cake was then added to a mixture of about 115 pounds of ethyl alcohol
and 373 mls. of 12N HCl and the mixture agitated in a Lightnin mixer for
about 20 hours. The mass was then centrifuged to reduce the cake to a
solids content of about 40%. The cake was reslurried in 117 pounds of
ethyl alcohol and again centrifuged. All of the liquid treatments were
carried out at temperatures between about 25.degree. C. and 28.degree. C.
The cake was vacuum dried under 28" -29.5" of mercury at 60.degree. C. for
about 22 hours and resulted in the recovery of 15.25 pounds of the
collagen product. The callagen product was then conditioned by exposure to
the room atmosphere for about 24 hours and contained about 11% moisture.
The resulting conditioned mass was then subjected to a deaggregating or
fluffing operation by passing through a hammer mill and screening.
Specifically, the mass was passed through a Fitz Mill (Model DA50-6-5634)
operated at 6250 rpm. and equipped with a #4 screen having openings of
0.243". It was then subjected to a second pass using No. 2A screen
(0.064").
The final fluffed product contained 0.65 meq. HCl pergram of collagen
(approximately 84% of the theoretical stoichiometric amount). The bulk
density was 1.5 pounds per cubic foot and the surface area was
approximately 1.9 square meters per gram. The HAT Rating was 483 with AB+
blood and 477 using A-blood Upon disintegrating a sample of the product in
water by subjecting a suspension containing about 0.5% by weight of the
fluffy, finely-divided fibers in water to the act of a Waring Blendor at
high speed for 30 minutes, a stable dispersion was formed having a pH of
3.10.
The fibrous collagen products prepared in the above examples were employed
in surgical test procedures designed to prove the efficacy of the material
both as a hemostat and adhesive for severed biological surfaces in a warm
blooded animal when wet with blood. "Severed" biological surfaces for the
purposes of this invention includes cut, sliced, ripped, abraded, torn,
punctured, burned, and tissue severed by any means or method whereby a
fresh biological surface is present. Biological surfaces will include
tissue, cartilage, vesels, bone or other normal organic parts of the warm
blooded animal which may require mending or joining.
The in vivo surgical test procedure in one series of experiments was
carried out on mongrel dogs and involved the makng of a two inch
capsule-to-capsule laceration in the leading edge of the liver and spleen.
The actively bleeding lacerations were packed with as much of the fibrous
collagen product as necessary to effect hemostasis and then compressed
together for 10 to 45 seconds. The capsules were not sutured. If the
adhesive properties were sufficient to maintain the severed surfaces of
the spleen together and arrest bleeding after compression, the collagen
passed the test. If the tissue surfaces would not adhere after compression
for 45 seconds, the callagen failed the test. The results of these tests
each on there different dogs, are reported in the column "In Vivo Tests A"
of Table I. In these procedures, it was generally observed that although
the fibrous mass was set with blood, the mass could be pressed with cotton
or rayon without any apparent adhesion of the cotton or rayon fibers to
the wet mass.
TABLE I
__________________________________________________________________________
pH of
Slurry
Bulk Density
Surface Area In Vivo
Example in H.sub.2 O
#/cu. ft.
sq.m. /gm.
HAT Rating
A B
__________________________________________________________________________
I 3.3 2.50 18.1-23.0
799 - AB+
Passed
I-II
850 - A+
II 3.3 1.62 24.2 700 - A-
Passed
I
III 3.0 3.06 3.9 700 - O+
Passed
II
IV 3.2 3.70 5.6 (1) (1) I
V 3.2 2.50 16.7 1025 - O+
Passed
II
VI 7.0 3.10 4.0 1100 - O+
(1) II
VII 7.0 1.56 2.9 1220 - A+
(1) II
VIII 9.0 2.80 1.5 Failed*
(1) II
IX 9.0 2.75 1.4 Failed*
(1) III
X 3.1 1.50 1.9 477 - A-
Passed
I
483 - AB+
SURGICEL** Failed*
Poor
IV
OXYCEL** Failed*
Poor
IV
GELFOAM Failed*
Poor
IV
Sponge
GELFOAM Failed*
Failed
IV
Powder
__________________________________________________________________________
(1) Not tested
*All blood not absorbed
**Individual fibers pulled from fabrics
In another series of experiments on dogs, cuts were made in cutaneous,
sub-cutaneous and muscle arteries an veins, the largest in the males being
the external pudendals and in the females being the cranial superficial
epigastrics; longitudinal incisions 1/4" to 1/2" in depth and 1" in length
were made in the spleen; and, wedge slices were made in the spleen by
removal of a surface section 1/8" to 1/4" in depth and 1/2" length on each
side of the triangle. Each sample was used a minimum of four times by
applying the sample to the bleedng site, applying pressure over the sample
with a gauze sponge and observing the site each 30 seconds. The time was
noted when the bleeding was controlled; that is, bleeding was arrested.
The testing was "blind"; that is, the investigator was provided with the
fluffy, fibrous product without knowledge of its history except in the
case of the product of Example II.
Generally, an incision was made in the vessels or organs mentioned above
and a gauze sponge applied to the bleeding site for 5 minutes to make
certain that the blood itself did not coagulate and seal the wound within
this period of time. The incision site was then mildly incised to reopen
any vessel which had closed and to insure vigorous bleedng prior to the
application of the fluffy fibrous product A sample of the product of
Example II was then placed in the bleeding site as desribed above and the
time noted when the bleeding had been arrested. After the bleeding has
been arrested, the material was removed from the wound noting the
tenaciousness with which it had adhered to the severed surfaces. The wound
was then reopened to reinitiate free bleeding and one of the other samples
of fluffy fibrous product used in the same manner. Again, the time for the
sample to arrest bleeding and seal the wound wasted and the material
removed. from the wound again noting the tenaciousness with which it
adhered to the severed surfaces. Samles of the fluffy fibrous product of
Example II were used at intervals between the other samples and at the end
of the test samples.
The samples were given a rating by the investigator based upon four
characteristics of the samples: (1) transfer properties which involved the
transfer of the product to the wound and the adhesiveness of the product
to forceps, rubber gloves and gauze sponges, (2) localization of the
sample at the bleeding site, (3) the time for controlling or arresting the
bleeding and (4) the adhesiveness to the severed surfaces. In preliminary
testing of various samples, the investigator decided to use the product of
Exmaple II as a standard or control because of its handling, hemostatic
and adhesive properties and assigned a numerical Rating I to this sample
for future testing. A Rating of II was given to samples which were
satisfactory but were not quite as desirable as the standard or control
samle. In some of these instances, it wasted that although bleeding had
been controlled, there was a tendency toward a breakthrough bleeding which
was arrested by applying additional pressure for 0.5 to 1.0 minute.
Materials which performed about as well as or much like those given a
Rating of II with respect to characteristics (2 ), (3) and (4) but had
poor handling and transfer properties were given a Rating of III. A Rating
of IV indicates that a product was defective in one or more of the
characteristics, that is, it did not have proper transfer properties or
did not arrest bleeding within 5 minutes or did not localize well around
the wound or did not have sufficient adherence to the walls of the wound
to seal the wound. In general, most products given this rating and
considered unsatisfactory contained hornified fibers and gritty-like,
hornified particles as reflected in high bulk densities.
The results of these experiments were as shown in the following table:
TABLE II
__________________________________________________________________________
Bleeding Controlled - Time in Minutes
Cutaneous
Example
Muscle
Pudendals
Epigastrics
Spleen Cut
Spleen Wedge
Rating
__________________________________________________________________________
I 2-2.5 5 3 1.5-2.0
1-1.5 I - II
II 2.4 .+-. 0.5
2.0 .+-. 0.3
Not Tested
1.8 .+-. 0.2
1.7 .+-. 0.2
I
(6 Trials)
(20 Trials) (19 Trials)
(13 Trials)
III 1-3.5 1.5-4 3.5 1.0-1.5
2.5 II
IV 0.5 0.5-1.0
Not Tested
1.5 1.0 I
V 1.0 1.0 Not Tested
2.5 3.0 II
VI 1.0 Not Tested
3.0 1.0 1.0-1.5 II
VII 1.5 Not Tested
3.0 Not Tested
1.0 II
VIII 1.0 1.0 Not Tested
2.0 3.0 II
IX 3.0 3.0 Not Tested
3.0 3.5 II
X Not Tested
2.0 Not Tested
2.5 3.0 I
__________________________________________________________________________
In the production of the fluffy fibrous product to be utilized in human
internal surgical procedures, it is necessary that processing be effected
under conditions that eliminate pyrogenetic substances and microorganisms
such as bacteria, yeasts, molds and the like. The raw material, such as
green corium, is generally received in various sized sheets in frozen
state. The frozen sheets are thawed by placing in hydrogen free, distilled
water containing between 50 and 100 ppm, preferably 50-70 ppm, sodium
hypochlorite, the solution having a pH between 7 and 9, preferably pH 7-8.
The surfaces of the thawed sheets are scraped so as to loosen and/or
remove substances such as dirt, loose protein and any other foreign
material on or embedded in the surfaces while in the hypochlorite
solution. A convenient method involves drawing the corium sheet between
two rotating cylindrical, stiff bristle brushes (stiff nylon bristles)
under a copious stream of hydrogen free, distilled water whch may contain
the hypochlorite.
Following scraping or brush washing, the corium sheets are rinsed with
copious amounts of pydrogen free, distilled water and placed in hydrogen
free, distilled water containing the sodium hypochlorite. The sheets are
then cut into 3"- 8" .times. 8" strips for convenience in subsequent
processing and maintained in the hypochlorite solution until required for
use. The strips are removed from the solution, placed in a centrifuge and
pyrogen free, distilled water added. The strips are agitated in the water
at slow speed for about 1/2 hour and the exceess liquid separated by
operating the centrifuge at high speed. The washing with the pyrogen free,
distilled water is repeated until the effluent shows no detectable
chlorine by the conventional silver choride test. The strips are then
processed into the fluffy, fibrous product as described in Example X. All
processing conducted in a conventional clean room.
In the processing of the corium into the partial salt of collagen, it is
essential that in each step wherein the collagen or partial salt of
collagen is treated in a liquid comprising ethanol, the mixture is
agitated for a period sufficient to equalize the ethanol content of the
liquid in the corium to that of the bulk liquid. Thus, each such treatment
effects an increase in the ethanol content in the corium and prevents
hornification upon final drying. Where the ethanol contains the ionizable
acid, the treating step must be prolonged so as to effect the desired salt
formation.
While the foregoing discussion has been directed to the use of fluffy,
fibrous products, the fibrous products may be converted into non-woven
webs or mats of desired thicknesses in accordance with conventional well
known methods. In using a wet method such as commonly employed in forming
appears and similar products, however, the fibrous collagen products are
slurried in a water-miscible organic liquid to form the furnish. The
water-miscible organic liquid may be of the type used in producing the
collagen products. Alternatively, the web may be formed by an air-laying
method wherein the fibers are blown into a chamber and allowed to deposit
in random arrangement on a formaminous collector such as a screen. In use,
a thin web of the fibrous collagen product is cut, for example, to cover
the spleen wedge as referred to hereinbefore, and pressed over the area in
place of packing the fluffy, fibrous material over the area.
The products may be sterilized by subjecting them to the conventional
ethylene oxide treatment. Alternatively, after packaging, the products may
be sterilized by heating at a temperature of about 120.degree. to
126.degree. C. for 20 to 24 hours. If desired, antiseptics, bactericides,
fungicides, germicides, medicinals and/or antibiotics may be mixed with
the fibrous material. Where such additive is soluble in a water-miscible
organic liquid as used in the production of the fluffy, fibrous product,
the additive may be dissolved in the final wash bath whereby the additive
becomes impregnated into the fibers.
The term "adhesion" as used this specification is used along with
"adhesive" in the mechanical and chemical sense to designate what light be
termed a cementing or gluing action and does not refer to the normal
medical designation.
* * * * *
|
|
 |
|
 |
Description |
|
