Method For Removing Noncollagenous Matter From Mammalian Gut

Abstract

Gut strands used to manufacture sutures, tennis racquet stringing and such are treated at least once with an aqueous nonionic surfactant solution consisting essentially of from about 0.2 to about 5 percent surfactant to remove a portion of the noncollagenous matter therefrom. Subsequent treatment of the gut with aqueous alkaline solutions removes the remaining portion of the noncollagenous matter to produce a collagen strand substantially free from noncollagenous matter (i.e., fats and noncollagenous protein). Strands so treated are of light color and high strength. Such strands may be uniformly tanned. The strands resist undesirable color intensification at elevated temperatures and do not discolor suture tubing fluids.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

Cleaning and degreasing of serosa and submucosa layers of mammalian intestine for collagen strand preparation.

2. Description of the the Prior Art

The serosa layer of beef intestine and the submucosa layer of sheep intestine are widely used collagen source in the preparation of absorbable surgical sutures. These layers typically contain significant amounts of naturally occurring noncollagenous matter such as lipids, fats, pigments, greasy components, noncollagenous proteins, elastin, and other complex components. It is essential to remove as much noncollagenous matter as possible since its presence in the final suture product is objectionable for several reasons. One problem is the undesirable yellow to orange color imparted to nonchromicized (i.e., plain) sutures. This color intensifies when sutures are subjected to high temperatures such as normally occur in a heat sterilization process. Furthermore, as the amount of noncollagenous matter within the gut varies, sutures produced from the gut will exhibit corresponding variations in the color so that within a given suture lot, there may appear a color variation from white to deep orange. Such nonuniformity of color is highly undesirable in a product directed to the medical profession.

A further problem associated with both nonchromicized and chromicized sutures arises when sutures containing large amounts of noncollagenous matter are stored in tubing fluids such as alcohol-water mixtures. The tubing fluid extracts a portion of the noncollagenous matter and is thereby transformed from a colorless liquid to a pale yellow to deep orange liquid depending upon the amount of noncollagenous matter extracted. Since preferred suture packages are transparent, an obviously unpleasant aesthetic effect results.

Another result of noncollagenous matter in the suture is a noticeable reduction in the in-vivo tensile strength of chromicized sutures. This reduction is believed due to the difficulty of uniformly tanning serosa or submucosa which contains large amounts of noncollagenous matter.

Among additional problems associated with the presence of noncollagenous matter are poor ply adhesion, the resultant tissue irritation which occurs when the sutures are implanted in living tissue and difficulty in uniformly dyeing the sutures.

When gut is to be used for tennis racquet stringing, light color, uniformity of color, and high strength are of prime importance, all of these factors being adversely effected by the presence of noncollagenous matter in the gut.

A variety of methods have been used to remove noncollagenous matter. One common method involves treating the intestinal layers with one or more aqueous alkaline solutions such as sodium carbonate or sodium hydroxide solutions. This treatment, while effective in removing noncollagenous protein, is not particularly effective in removing a sufficient amount of the fatty components to eliminate many of the undesirable properties listed hereinabove. Aqueous solutions of trypsin and other enzymes having mucase and/or elastase activity have been used to remove noncollagenous matter. Block et al. in U.S. Pat. No. 2,750,251 teach the use of aqueous solutions of the tetrasodium salt of ethylenediamine tetra-acetic acid as a method of removing noncollagenous matter from mammalian gut. The patent further discloses that the addition of nonionic detergents to the solution facilitates and accelerates the removal of fat. Klevens in U.S. Pat. No. 3,071,477 discloses a method for removing noncollagenous matter from beef tendon one of whose dimensions does not exceed 15 to 30 mils in a regenerated collagen process by treating same with aqueous solutions of sodium carbonate and salts of polyphosphoric acid such as sodium hexametaphosphate and the like. Klevens further discloses that the incorporation of a nonionic detergent into the solution is an aid in cleaning the collagen without detrimentally affecting it.

SUMMARY OF THE INVENTION

This invention relates to a method for removing naturally occurring noncollagenous matter from the portions of mammalian intestine used to prepare natural collagen strands for use as surgical sutures, tennis racquet stringing, and the like. The pertinent portions of intestine, hereinafter sometimes called "gut" for simplicity, are the serosa and submucosa layers of beef and the submucosa layer of sheep inclusive of the noncollagenous matter ordinarily associated with the gut following its separation from the bulk of the intestine. Noncollagenous matter is composed of two fractions: a fatty fraction hereinafter sometimes called "fats," and a noncollagenous protein fraction. The fatty fraction is composed of lipids, pigments, greasy components, and other complex components having fatty characteristics. The fats are believed to cause the undesirable color in the final collagen strand and are known to reduce strand strength and to interfere with uniform tanning of the strands. The noncollagenous fraction contains components such as muscle, myosin, elastin, hexosamines, and such, which are nonfats, but which must nevertheless be removed since their presence reduces strand strength and is known to cause irritation of living tissue.

This invention more particularly relates to a method for removing noncollagenous matter from mammalian gut which comprises contacting the gut at least once with an aqueous solution of one or more nonionic surfactants, prior to bleaching or tanning the gut, for a period of time sufficient to remove a portion of the noncollagenous matter from the gut. Generally, treating the gut with a solution consisting essentially of from about 0.2 to about 5 percent nonionic surfactant for at least about 16 hours is sufficient to remove the aforementioned amount of noncollagenous matter from the gut. The remaining portion of the noncollagenous matter may be removed either before or after the surfactant treatment by contacting the gut at least once with an alkaline aqueous solution of sodium peroxide, sodium hydroxide, sodium carbonate or such under controlled conditions to produce gut which is substantially free from noncollagenous matter.

The surfactant treatment is of primary utility in removing fats which resist removal in alkaline treatments. Gut subjected solely to alkaline treatment, for example, ordinarily contains about 2 to 4 percent residual fats measured as alcohol extractables. However, gut subjected sequentially to a surfactant and alkaline treatment ordinarily contains about 1.2 percent or less residual fats measured as above. Gut containing 2-4 percent fats exhibits most of the undesirable properties listed hereinabove while gut containing 1.2 percent or less is virtually free from these undesirable properties. This reduction in residual fats of at least about 40 percent occasioned by the surfactant treatment is therefore an essential step in preparing acceptable gut for use in sutures and tennis racquet stringing.

The alkaline treatment is primarily a step for removing noncollagenous protein although appreciable amounts are also removed in the surfactant treatment. For example, the surfactant treatment will typically remove about 30 to about 40 percent and the alkaline treatment about 60 to about 70 percent of the noncollagenous protein (measured by primary amino group analysis as described hereinbelow) ultimately removable by the combined process steps.

It is an important aspect of this invention that the surfactant treatment and the alkaline treatment be separate. When surfactant is added directly to the alkaline baths in an attempt to achieve single-step removal of noncollagenous matter, the amount of fat and noncollagenous protein removed is noticeably diminished as compared to separate bath processing.

It is, therefore, an object of this invention to provide a method for removing from mammalian gut that portion of noncollagenous matter not susceptible to removal by alkaline treatment, by directly treating the gut with an aqueous nonionic surfactant solution.

It is another object of this invention to provide a method for removing the remaining portion of noncollagenous protein from mammalian gut by alkaline-treating gut which has been separately surfactant treated thereby producing gut which is substantially free from noncollagenous matter.

It is another object of this invention to produce nonchromicized sutures of reduced fat content and hence lighter color and improved resistance to color intensification upon exposure to elevated temperature.

It is another object of this invention to produce tennis racquet stringing of improved strength and uniformly lighter color.

It is a further object of this invention to produce more uniformly chromicized sutures of improved package and in-vivo properties.

It is a still further object of this invention to produce both nonchromicized and chromicized sutures which are nonirritating to living tissue and which may be packaged in colorless tubing fluids without discoloring same.

These and other objects of this invention will be apparent from a total reading of this specification.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts schematically a flowsheet for processing sheep or beef gut into a collagen strand for use as a surgical suture or tennis racquet stringing. Stages in the process at which the surfactant bath may be conveniently inserted are depicted by broken lines.

FIG. 2 is a graph depicting the cumulative removal of noncollagenous protein (measured as gram moles of primary amino groups per 10 gallons of process bath) from beef serosa occurring as the serosa progresses through a typical alkaline cleaning process, and as it passes through the same process after having been pretreated with an aqueous surfactant bath.

DESCRIPTION OF THE PREFERRED EMBODIMENT

A variety of methods are used to process gut into sutures, tennis racquet stringing, or the like. Variety of method is particularly prevalent in the early stages of processing which attempt to remove as much noncollagenous matter as possible from the gut. For purposes of further illustrating the invention and its flexibility in the processing of the gut in a clear manner, the invention will be discussed in relation to the process outlined schematically in FIG. 1 although the invention is in no way limited to this particular process.

The collagenous raw material for preparing absorbable surgical sutures, tennis racquet stringing, or the like, is usually the serosa or submucosa intestinal layer of beef or the submucosa layer of sheep, with beef serosa and sheep submucossa the preferred source of collagen. Beef serosa or submucosa is obtainable as an abattoir byproduct; it is separated mechanically from the bulk of the intestine and cut into thin ribbons 20 to 25 feet long which are then either frozen or salted for storage. Sheep submucosa is similarly prepared except it is not cut into ribbons but is stored as a casing cut to lengths of 20 to 25 feet.

The gut is usually not pure serosa or submucosa tissue since in separating the gut from the intestine, muscle tissue or other extraneous tissue is frequently left attached to the gut. The serosa layer, for example, usually contains some longitudinal, and on occasion, radial muscle tissue attached to it; serosa in this form is referred to in the art as "gold-beater" ribbon to distinguish it from pure serosa tissue.

Referring to FIG. 1, the frozen or salted gut from the abattoir is stored 1 as received until needed. Frozen gut is then immersed in warm water until completely thawed 2. Salted intestine is similarly treated 3 to leach out the salt. The beef serosa or submucosa ribbons are immersed in a dilute (about 0.15 to about 0.23 percent) aqueous solution of sodium peroxide 4 having a pH of about 13 to about 14, this bath being known in the art as the "slime". After the slime is completed, the beef serosa or submucosa is successively immersed in two baths of stronger (about 0.4 to about 0.6 percent, pH of about 13 to about 14) aqueous sodium peroxide, 5, 6 known in the art as the "strongs". The prime purpose of the slime and strongs is to remove noncollagenous matter from the gut.

Sheep submucosa must be given a milder slime and strongs since the sodium peroxide baths discussed above would cause undesirable excessive alkaline hydrolysis of the sheep submucosa. Sheep submucosa therefore is subjected to a dilute (about 0.12 to about 0.25 percent, pH of about 9 to about 11) aqueous sodium carbonate slime 7 and more concentrated sodium carbonate (about 0.4 to about 0.6 percent, pH of about 9 to about 11) strongs, 8, 9. Alternatively, a solution containing sodium carbonate and a minor amount of sodium peroxide (relative to the sodium carbonate) may be utilized in both the slime and the strongs provided solution pH is maintained about about 9 to 11. Following slime 7 the mild hydrolysis which has occurred within the submucosa imparts a greasy texture to the submucosa casing. This is therefore a particularly convenient time to split the tube into two or more ribbons for further processing by sliding the tube over a conventional cutting horn 10 and then treating the ribbons in the sodium carbonate strongs 8, 9.

Following the slime and strongs, both sheep and beef ribbons are water rinsed 11 to a pH below about 8.5 and 9.5 respectively. This can require as many as four separate rinses. At this point, tennis gut and ribbon destined for nonchromicized sutures are immersed in a bleaching bath 12 of dilute aqueous hydrogen peroxide. Ribbon destined for chromicized sutures is not bleached but is immersed in a conventional tanning bath 13 until the desired degree of chromicizing is achieved.

The bleached and tanned ribbons are water-rinsed 14 to remove residual bleach and tanning compounds. The ribbons are looped and measured 15 to obtain the desired ply structure, twisted 16 into strands, and dried 17. Suture strands are then cut to the desired length 18, polished 19, packaged 20, and sterilized 21. Ribbon destined for tennis racquet stringing, i.e., tennis gut, is subjected to somewhat different processing following the drying step 17. It is polished 22, manually lacquered with shellac or a polyester lacquer 23, cut to size 24, coiled 25, inspected for quality and graded 26, tied 27, and packaged 28.

The gut may be contacted with the aqueous nonionic surfactant solution in accordance with the process of this invention at a variety of stages in the process prior to the tanning 13 or bleaching 12 step. Aqueous nonionic surfactant solutions containing less than about 0.2 percent surfactant remove noncollagenous matter at extremely slow rates and are therefore of little practical interest. On the other hand, surfactant solutions containing more than about 5 percent surfactant have low surfactant efficiency as compared to more dilute solutions and needlessly magnify the problem of removing the surfactant from the gut.

The surfactant treatment may be employed singly or in combination. For example, similar results are achieved by contacting the gut with a single 1 percent surfactant solution or with a series of two 0.2 percent surfactant solutions. The time required to remove noncollagenous matter depends, among other variables, upon the surfactant concentration, the bath temperature, and the amount of noncollagenous matter present in the gut. Generally, a period of at least about 16 hours is required for satisfactory operation. Elevated surfactant bath temperature is desirable although an upper temperature limitation of about 120.degree. F. is usually imposed since collagen hydrolyzes above this temperature. A temperature range of 90.degree.-100.degree. F. has been found particularly suitable.

A variety of nonionic surfactants are operable although the preferred species are water-soluble at the bath temperatures of interest to facilitate preparing a bath of uniform surfactant concentration. Among the suitable nonionic surfactants are included such diverse members as the ethoxylated fatty amides and ethoxylated fatty acids such as those sold under the trademarks "Ethomid" and "Ethofat" respectively by the Armour Industrial Chemical Co., the ethoxylated alkylphenols such as those sold by the Dow Chemical Co. and Jefferson Chemical Co. under the respective trademarks of "Dowfax" and "Surfonic", and the ethoxylated polyoxypropylenes sold by Wyandotte Chemical Co. under the trademark "Pluronics."

A preferred nonionic surfactant is an ethoxylated nonylphenol of the formula:

An aqueous solution containing about 1 percent of the above surfactant has been found to be particularly suitable.

Inorganic detergent builder compositions such as one or more of the water-soluble salts of silicic acid and polyphosphoric acid and the like may be included in the aqueous surfactant solutions. Among the preferred builder compositions are sodium silicate and sodium tripolyphosphate. As builder concentration increases, surfactant concentration can usually be correspondingly reduced.

Referring to FIG. 1, there are a variety of locations during the initial processing of the gut where the aqueous surfactant bath 29 may be conveniently inserted. These locations are indicated in FIG. 1 by dotted lines. A particularly effective and highly preferred point at which to insert the surfactant treatment is immediately after the thawing bath 2 in cases where frozen gut is employed. The surfactant may also be added directly to the thaw bath 2 with the advantage that removal of the fats occurs concurrently with a necessary process step thereby obviating the need for a separate surfactant treatment.

The surfactant bath may be inserted at any point before, during, or after the alkaline baths (i.e., the slime and strongs) but prior to bleaching 12 or tanning 13. The surfactant baths are preferably inserted at some point prior to the water rinse 11 to assure adequate removal of residual surfactant.

The amount of noncollagenous protein removed by the surfactant treatment is readily shown by the data of Table I wherein the concentration of primary amino groups in various process baths is presented for surfactant treated and nonsurfactant treated beef serosa. Primary amino group concentration is an indication of the noncollagenous protein extracted from the serosa by the various process baths and is not necessarily a quantitative determination of the amount of noncollagenous protein removed. However the primary amino group analysis is a useful figure for comparative purposes. The data were gathered following the procedure of Example 1 presented hereinbelow. ##SPC1##

Although surfactant pretreatment of serosa prior to alkaline treatment does not noticeably enhance removal of noncollagenous protein during the subsequent alkaline treatment, the surfactant bath alone, based on primary amino group analysis, removed 37 percent of the noncollagenous protein ultimately removed. The surfactant-alkaline treatment removed about 70 percent more noncollagenous protein than the alkaline treatment alone.

FIG. 2 is a graphical representation of the data of Table I contrasting the cumulative effect of noncollagenous protein removal from beef serosa given only an alkaline treatment with that of serosa given a combined surfactant and alkaline treatment. Similar results are obtainable with beef and sheep submucosa.

The following illustrative examples present several preferred embodiments of the invention.

EXAMPLE 1

This example illustrates the treatment of beef serosa with surfactant solution prior to alkaline treatment of the serosa.

Ten knots of frozen beef serosa were immersed in 95.degree. F. water (a knot represents 80-100 serosa ribbons each about 221/2 feet long). The serosa contained fats, color pigments, muscle, noncollagenous protein, and other complex components not removed when the serosa layer is separated from the rest of intestine. When the serosa was thawed (15-36 hours), a cord was looped around each knot for easy handling and the serosa knots were immersed in a 1 percent aqueous solution of a surfactant of the formula:

at a temperature of 95.degree. for about 16 hours. The knots were then successively immersed in three 95.degree. F. aqueous baths containing 0.19 percent, 0.5 percent, and 0.5 percent (by weight) sodium peroxide respectively for periods of time of about 0.5 hour, 1 hour, and 1 hour respectively. The knots were water rinsed to a pH of less than 9.5. If chromicized sutures were desired, the serosa was immersed in any of a variety of known tanning baths such as a dilute aqueous solution of basic chromium sulfate and pyrogallol for up to 2 hours. If nonchromicized sutures were desired, the tanning step was omitted and the serosa immersed in a 95.degree. F. aqueous bleaching solution containing about 2 percent by volume hydrogen peroxide and traces of formaldehyde and sodium silicate. The chromicized or nonchromicized serosa was dried to produce a collagen strand suitable for use as a suture or as tennis gut. Suture strands were then cut to length, polished, packaged and sterilized. Tennis gut was polished, lacquered, cut to length, coiled, graded, and then tied.

Experimental control was achieved in this and the following examples by subjecting gut of like quality to identical processing except omitting the surfactant treatment.

Surfactant treated sutures were invariably of lighter and more uniform color than controls, and showed improved resistance to color intensification during heat sterilization. More uniform tanning was noticed with the treated sutures. Fats measured as ethanol extractables showed the treated sutures to contain at least 40 percent less fats than the control. Removal of noncollagenous protein was measured by analyzing the primary amino content of the surfactant, sodium peroxide, and water washes employing the ninhydrin colorimetric technique of McPhee, described in Textile Research Journal, Vol. 28, pp. 303-314 (1958). About 70 percent more noncollagenous protein was removed from the treated suture than from the control. Surfactant treated tennis gut showed better color and ply adhesion, and improved durability and strength.

Typical comparative suture package data are shown below for chromicized material. ---------------------------------------------------------------------------

Average Tensile Strength (lb.) __________________________________________________________________________ Surfactant Treatment Plys Size Diameter Straight Knot (mils) Pull Pull __________________________________________________________________________ Yes 1 2/0 16.2 12 6.3 No 1 2/0 16.2 10.7 6.3 Yes 2 0 19.5 14.6 8.0 No 2 0 19.5 14.3 7.7 __________________________________________________________________________

In-vivo tensile strength was determined by implanting chromic sutures in rabbits, sacrificing the animals at perscribed intervals (7 and 15 days below) following implantation, removing the sutures, and measuring their tensile strength. ---------------------------------------------------------------------------

Straight Pull (lb.) __________________________________________________________________________ Surfactant Treatment SIZE DAY 7 DAY 15 __________________________________________________________________________ Yes 00 4.4 2.6 No 00 3.5 2.2 __________________________________________________________________________

The above data indicate that comparable package properties are obtained with treated and nontreated sutures. However, significantly improved in-vivo strength is noted with surfactanttreated sutures. Although in-vivo strength retention is markedly improved, complete digestion of the treated suture was observed within 60-90 days.

EXAMPLE 2

This example illustrates treatment of beef serosa with surfactant solution containing inorganic detergent builders at an intermediate point in the alkaline treatment of the serosa.

Ten knots of frozen beef serosa were thawed as in Example 1. The thawed serosa was immersed in a 95.degree. F. 0.19 percent aqueous sodium peroxide solution of 0.5 hour followed by immersion in a 95.degree. F. 0.5 percent aqueous sodium peroxide solution for 1 hour. The gut was then immersed in a 95.degree. F. solution containing 2 percent sodium tripolyphosphate, 0.4 percent sodium silicate, and 1 percent of the surfactant shown in Example 1 for about 16 hours. The gut was then immersed in a 95.degree. F. 0.5 percent aqueous sodium peroxide solution for 1 hour, water-rinsed to a pH below 9.5, tanned or bleached according to Example 1, water-rinsed, looped and measured, twisted and dried.

About 56 percent more fats were removed from the surfactant-treated suture than from the control which had received identical processing except that the surfactant treatment was omitted; treated plain sutures were of noticeably lighter and more uniform color. Added comparative data are tabulated below ##SPC2##

The above data clearly show the significantly improved in-vivo strength retention of the surfactant-treated sutures. Despite the improved strength retention, complete digestion of the treated sutures was observed within 60-90 days.

EXAMPLE 3

This example illustrates the treatment of beef serosa with two highly dilute surfactant solutions containing detergent builders at interim stages in the alkaline treatment.

Ten knots of serosa were thawed as in Example 1. The thawed serosa was immersed in a 95.degree. F. aqueous 0.19 percent sodium peroxide solution for 0.5 hour and then in a 95.degree. F. aqueous solution containing 0.4 percent sodium tripolyphosphate, 0.08 percent sodium silicate and 0.2 percent of the surfactant of Example 1 for 16 hours. The serosa was then immersed for 1 hour in a 95.degree. F. 0.5 percent aqueous solution of sodium peroxide after which it was reimmersed in a fresh surfactant solution as described immediately above for 16 hours. Thereupon serosa was immersed in a second 95.degree. F. aqueous 0.5 percent sodium peroxide bath for 1 hour, water-rinsed to a pH of below 9.5, tanned or bleached as in Example 1, looped and measured, twisted, and dried.

About 58 percent more fats were removed from the treated suture than the control. Other suture properties were similar to those shown in Examples 1 and 2.

EXAMPLE 4

This example illustrates the treatment of beef serosa by incorporation of the surfactant directly into the thawing bath.

The identical procedure of Example 1 is employed except that the surfactant bath is omitted. In its place, sufficient surfactant as shown in Example 1 is added to the thaw water prior to immersion of the frozen serosa therein, to produce a 1 percent solution. Suture properties are substantially those shown in Examples 1 and 2.

EXAMPLE 5

This example illustrates the use of a concentrated aqueous surfactant solution.

Following substantially the same procedure as in Example 1, except that the surfactant concentration in the solution was 5 percent instead of 1 percent, sutures having substantially the same properties as shown in Example 1 are obtained.

EXAMPLE 6

This example illustrates the use of other nonionic surfactants.

Following substantially the same procedure as in Examples 1, 2 or 3, the following nonionic surfactants are successfully employed in place of the ethoxylated nonylphenol adduct. ##SPC3##

EXAMPLE 7

This example illustrates the treatment of sheep submucosa with surfactant solution prior to alkaline treatment.

Fifty frozen sheep submucosa casings are immersed for thawing in 95.degree. F. water for 16 to 36 hours. The thawed submucosa is immersed in a 1 percent surfactant solution containing a surfactant of the formula:

for 16 to 36 hours. The gut is then immersed successively in a 95.degree. F. aqueous 0.2 percent sodium carbonate bath after which the gut casing is run over a splitting horn severing it into two ribbons (for a total of 100 ribbons) which are then bound for easy handling. The ribbons are immersed successively in two 95.degree. F. 0.5 percent aqueous sodium carbonate baths whereupon they are rinsed, chromicized or bleached as desired, looped and measured, twisted, and dried.

The sutures so produced exhibit properties similar to the beef serosa sutures shown in Examples 1 and 2.

Claims

I claim:

1. A method for removing noncollagenous matter from long strips of beef serosa prior to bleaching or tanning said serosa consisting essentially of contacting long strips of beef serosa at last once with an aqueous nonionic surfactant solution consisting essentially of from 0.2 percent to about 5 percent of a nonionic surfactant which is an ethoxylated nonylphenol adduct having 9 moles of ethylene oxide per nonylphenol of the formula:

and 0.08 percent to 0.4 percent sodium silicate and 0.4 percent to 2 percent sodium tripolyphosphate at a temperature between about 90.degree. F. and about 100.degree. F. for a period of time sufficient to remove a portion of noncollagenous matter therefrom, including at least about 40 percent of residual fats and then immersed successively in the following aqueous alkaline solutions:

a. sodium peroxide, about 0.15 percent to about 0.23 percent

b. sodium peroxide, about 0.4 percent to about 0.6 percent

c. sodium peroxide, about 0.4 percent to about 0.6 percent

for a period of time sufficient to remove remaining noncollagenous matter, thereby obtaining said serosa substantially free from noncollagenous matter.