JPH0260341B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing surgical collagen fibers using Achilles tendon or skin as starting material.

It is known to isolate surgical collagen fibers as sutures or in the form of hemostatic fleeces or as wound dressing materials and other uses. For the surgical applications mentioned above, it is important that collagenous materials, especially collagen fibers, retain their natural structure. For this reason, the method for isolating and purifying collagen fibers from the starting material should extract all non-collagenous components of the original tissue without denaturing the original crystalline structure of the collagen. No non-collagenous contaminants should be present in the final pure collagen fibers unless specified for medical reasons.

Several patent specifications describe methods for isolating microfibril collagen as a hemostatic agent or for reconstituting surgical structures from solubilized collagen gels.

According to the Batista patent (US Pat. No. 3,742,955), fluffy, fine microfibrils of collagen are produced. Since the length of the fibrils ranges on average to 100 μm, further fiber processing is not possible.

Several other patents relate to methods of completely disrupting the fibrillar structure of collagen. These methods involve reconstituting a gel in which collagen molecules are dispersed into fibrillar form by extruding a gel in a complex manner into a precipitation medium [Veis et al., U.S. Pat. No. 2,838,363; Smith et al. ) US Pat. No. 3,527,225; Grisef
U.S. Pat. No. 3,114,372, U.S. Pat. No. 3,114,593 or U.S. Pat. No. 3,511,904 to Nichols et al.; U.S. Pat. No. 3,520,402 to Nichols et al.]. Birkin (Ba
The method according to U.S. Pat. No. 3,374,103 to J.D.-Rkin uses mammalian tendon cut into sections between 10 and 25 mils thick. This step itself prevents the isolation of long fibers, which is an essential feature of the invention. Birkin's method also requires the use of extrusion of acid-swollen collagen solutions to form continuous filaments.

Highteger et al. U.S. Patent No.
Neither the methods of 2234446 and 2234467 produce isolated, parallel aligned collagen fibers that can be processed by fiber technology. The collagen fibers obtained according to the above-mentioned patent are randomly arranged to form sheets of intertwined collagen fiber masses.

In short, none of the prior documents, in whole or in part, alone or in combination, teach or suggest the essential features of the invention described herein. In these methods, the original fibrillar structure of collagen is completely lost, and collagen exists as monomers or dimers in a viscous solution that is extruded and precipitated into continuous artificial collagen fibers. The present invention does not relate to reconstituted collagen, but rather to collagen fibers isolated and purified from starting materials.

Federal Republic of Germany Patent No. 2730623
Another method for producing collagen fibers is described.
The method begins with skin or skin trimmings or tendons treated with alkali or acid. The chopped starting material is first exposed to a strong alkali and then swollen in an acidic environment. The swollen material is then washed and dewatered mechanically by pressing and chemically by various salts or organic solvents to 20-40% by weight of dry matter. This material is then used to isolate the fibers. Contrary to the above-mentioned patent's statement that isolated collagen fibers are free, loose, and do not form knots, in reality, the solubilization of the collagenous material into discrete fibers is incomplete and the insoluble structures are in knots. It produces a high proportion of non-collagen material, which is mainly present in The presence of knots, amorphous material and short fibers makes this material even more difficult to fabricate into threads, fabrics, fleeces etc. using standard fiber techniques which require loose, loose fibers of suitable lengths greater than 3 cm. Interfere with processing into products.

It is an object of the present invention to provide a method for producing surgical collagen fibers in which the isolated fibers are loose and do not form knots. Another object of the invention is to process the collagen material in such a way that the resulting fibers are easily processed by the various machines used in the textile industry. Another object of the invention is to provide the necessary
The purpose is to describe the method of modification of the resulting fibers.

In the present invention, the starting material is Achilles tendon, in particular Achilles tendon or skin from a cow, which contains long collagen fibers arranged in longitudinal arrays in wet or dry conditions. The diameter of these fibers can be further divided mechanically into fibrils that are still arranged in parallel.

The alkaline treatment of the Achilles tendon or skin is continued until the material contains 0.15-0.30 μmol of amide nitrogen per gram of dry weight. This numerical range is essential for the following treatment of collagen. This numerical range of the present invention ranges from 0.30 to
This differs from the range described in DE 27 30 623, which yields collagen with amide nitrogen of 0.40 μmol/g. Since this value relates to the structural stability of this fibrillar protein, the present invention emphasizes the importance of an appropriate content of amide nitrogen in collagen. This number also relates to the purity of collagen free of all non-collagen contaminants. Treatment with alkali can be carried out in any known manner, for example with calcium hydroxide, with standing standing lasting up to 6 weeks. Another known method is treatment with sodium hydroxide in the presence of sodium sulfate, which reduces the treatment period to 2 to 5 days.

During the alkaline treatment of the Achilles tendon or skin, it is advantageous to subject the material to a fluted roll treatment to dehydrate and compact the tendon or skin. After producing collagen with amide nitrogen 0.15-0.30 μmol/g by alkali treatment, the material is washed with hydrochloric acid. Although other organic or inorganic acids can be used to clean alkali-treated tendons, the use of hydrochloric acid is preferred since the sodium chloride that forms during neutralization is more tolerable from a physiological point of view than other salts. It is advantageous because it can be done. During treatment with hydrochloric acid some other non-collagen components are extracted, such as glycosaminoglycans. At the same time, the original rather tight fibers of the tendon or skin are relaxed. During treatment with hydrochloric acid, the PH of the tendon or skin is acidic throughout the entire thickness of the fascicle, and 2
It is desirable to adjust to reach a lower PH.

After acid treatment, the material has a tendon pH of 2-4, preferably
Wash with tap water until pH is 2.5 to 3.5. So far, alkali treatment, acid standing, and washing to the above-mentioned pH have been described. The next step is mechanical treatment. The washed and swollen collagen paste is subjected to mechanical stress on a grooved roll. During this treatment, the collagen fibers are isolated and separated from the adhesive effects of soluble denatured collagen, and at the same time the fibers are partially dehydrated. The mechanical treatment transforms the three-dimensional structure of the tendon into a two-dimensional layered network structure with still constant thickness. This compacted, partially dehydrated layer is hacked to separate the individual fibers without changing their parallel alignment. Advantageously, the hackling treatment is repeated, for example, 2 to 5 times, with successively increasing comb strength between each successive treatment.

As a result of hackling, the parallel-aligned fibers are separated from the bundle of tendon tissue. These individual fibers can be dehydrated in the next step by using various salts or by changing the pH to that of the isoelectric point of collagen, i.e.
Change from 4.5 to 7.0 preferably from 5.5 to 6.5. Dehydration by neutralization, ie, PH change, results in fibers that are less elastic and flexible than when dehydration is achieved using salts such as sodium chloride or sodium sulfate. For the reasons mentioned above, it is preferred to use sodium chloride. Another advantage of using sodium chloride is its easy extractability during the following steps described below. The optimal concentration of sodium chloride used for dehydration is 5~
It is 15% by weight. The released liquid is then removed, for example by centrifugation or other methods of separating the solid phase from the liquid.

The dehydrated fibers are then tanned to reduce or eliminate swelling in water and antigenicity. This treatment allows for fiber processing as described below.

Several tanning agents have been used to crosslink collagen used in various medical applications or in the leather industry. However, experiments have shown that tanning with aldehydes, such as formaldehyde or glutaraldehyde, as in DE 2730623, is unsuitable and leaves cytotoxic residues of the aldehydes within the collagen matrix. I found out. Tanning with hexamethylene diisocyanate has been found to be optimal and yields fibers that are acceptable from a medical-toxicological point of view and from a technologically-textile processing point of view.

Tanning is carried out in an aqueous solution of the salts used to dehydrate the collagen fibers. In this case, the liquid separated from the fibers by the salt is not removed. If this is removed, add the appropriate amount of water to the fibers.

Hexamethylene diisocyanate (HMDIC)
Tanning is carried out using a stoichiometric excess of this tanning agent in the presence of a nonionic or cationic detergent. In practice, the concentration of HMDIC is 10-20% by weight collagen dry matter. Tanning is a rather slow process, with reaction times ranging from 10 to 40 hours, optimally from 15 to 30 hours. By-products formed during tanning, such as hexamethylene diamine, as well as excess detergent and salts still present in large amounts, dehydrate the fibers by separating the liquid and drying the resulting tanned collagen fibers. removed in between.

Dehydration and drying of collagen fibers is accomplished by known methods. Collagen fibers are dried in the presence of various water-miscible high-boiling organic solvents. Note that dehydration with acetone was found to be the most advantageous in practice.

Dried, tanned, longitudinally aligned collagen fibers are processed in a special textile machine. It is clear to those skilled in the art of fiber processing that the special properties of collagen fibers must be taken into account when selecting an appropriate machine. For example, it is known to the expert that cotton is treated with different equipment than wool.

The present invention produces collagen fibers that are significantly longer than those produced by other methods. The length of the fibers is 40 mm to 100 mm, and the diameter is 30 to 80 μm.
This is in contrast to the collagen produced by Batista, US Pat. No. 3,742,955, which states that the average length of the fibers is 100 Å.
It is. This length is too short to permit further fiber processing as described by the present invention.

Said operation is such that the method according to the invention for producing surgical collagen fibers consists of several successive steps,
Some of the individual steps are part of common knowledge, but these individual steps are arranged in a special order,
It is shown that adhering to special conditions results in products with unique properties that are significantly improved over currently known processes.

In order to enable those skilled in the art to better understand how to carry out the present invention, the present invention will now be described in detail based on Examples, but the present invention is not limited thereto.

Example 1 All references to percentage or volume content refer to the wet weight of the tendon-collagen material.

The Achilles tendon excised from the calf is cleaned, any adhering fat is mechanically removed, and then cleaned again. Approximately 250 g of tendon with a dry weight of 30% can be obtained from one calf.

The washed tendon is left standing at 20° C. for 8 days in a solution consisting of 20% sodium sulfate, 2% sodium hydroxide and 150% water. After standing, the tendons are mechanically processed between grooved rolls on days 2, 4 and 6.
On the 8th day, the tendons are thoroughly washed with tap water. The remaining tendon contains 0.23 μmol amide nitrogen/g tissue.

In the next step, the tendon is transferred into a tanning drum and treated with hydrochloric acid. The sample contains 100% water and 10% hydrochloric acid (diluted to 3%). The acid treatment continues for about 4 hours. The tendon is then washed in tap water to a pH of 2.7-3.3 over the entire thickness of the tendon, which now has a dry weight of 14%.

The slightly swollen tendon is exposed to high pressure in a hammer press, during which excess water as well as soluble denatured non-structural proteins are removed. A swollen, compressed tendon has the appearance of a two-dimensional fibrillar reticular layer. This layer is then hacked three times in succession with increasing pressure on the fibrous layer.

The resulting separated fibers are then treated with 10% sodium chloride and 100% water, based on the wet weight of the fibers, for 16 hours. Next, water produced during the dehydration step is removed by centrifugation.

In the next step, the de-swollen material is treated in the presence of 5% sodium chloride (100% relative to the wet weight of the fibers).
Leave it still. Mix a non-ionic detergent with the fibers at a final concentration of 1% and adjust the PH using sodium hydroxide.
Adjust to 6.0. After 30 minutes of mixing, hexamethylene diisocyanate is added to 5% of the wet weight of the fibers. Treatment with this tanning agent lasts for 30 hours with continuous mixing in the drum. Removes toxic vapors. After this treatment, the liquid is removed by centrifugation.

The next step is to dehydrate the now cross-linked material with 100% by weight acetone. Repeat this process 4 times. The dehydrated fibers were dried in a bacterial strainer to minimize contamination with airborne bacteria.

The fibers are separated using a carding machine. The carding machine consists of a pre-carder with two or three working places covered with serrated clothing. A main carder is connected next to a pre-carder with five active locations provided with flexible clothing. Good disentanglement of fibers is obtained by using mutually special cards. A carded web is formed at the end of the machine. By overlapping (folding) this card web, it is possible to obtain an arbitrary thickness. Improvements in the uniformity of the carded web and fleece are obtained by additional carding operations using finer clothing and narrower adjustments. The carded web layered on the fleece can be processed with an embossing roll to form a tight sheet. The fleece obtained in this way (not yet tanned and not cross-linked) exhibits excellent hemostatic properties.

To process threads from separated collagen fibers, the cotton fiber mass treated as described above (unwound into a carded web in a carding machine) is divided into individual strips or threads using a carded web divider. . The resulting cotton fiber strip is twisted by friction to form a preliminary thread that does not yet exhibit strength. The preliminary yarn is drawn and twisted using a condensing ring spinning machine. The number of twists in the yarn depends primarily on the desired yarn strength and fiber length.

Yarns usually present extreme difficulty in weaving or knitting due to the large number of fiber ends protruding from the fiber core. The yarn is thus moistened and the chaff connects the fiber ends to the main fibers. thus,
The fibers were prepared to make cloth or knitted products by conventional methods.

Example 2 Cleft cow skin (without scar tissue and subcutaneous connective tissue) is treated by leather industry methods (softening, liming, depilation, fletching,
skiving). Subsequently, the collagen is further treated with alkali in order to remove physiologically undesirable impurities from the collagen. Furthermore, the skin pieces were exposed to saturated Ca for 25 days.
(CH) ₂ solution and 0.1% sodium hydroxide solution (batch:material solution ratio is 1:3).
Stir the ingredients thoroughly several times daily. This saponifies the fat residue and processes the proteins that are mixed with it.
Wash out. The material is then washed with fresh running water.
The amide nitrogen value is 0.27 mmol/g. After washing,
Treat with 2% hydrochloric acid. Process the material for 4 hours while moving it. After this time, use PH test paper to check whether the skin has become completely acidic. Then wash under cold running water. After about 2 hours, the PH value of the material throughout the thickness is 3.0, and the dry content is 13.8
Weight%. The skin chemically treated with this method
Cut into 10x10cm pieces. It is then compacted between grooved rolls and this compaction is repeated up to eight times. Place the fibrous pieces in a 20% saline solution for deflation. This process can be clearly observed by the color change (glass-like to white). After shrinkage, the saline solution is centrifuged to remove the fibrous debris and the material is dehydrated with acetone; after dehydration, 150% acetone by weight relative to the measured weight of the fibrous material is used three times. The fiber pieces are then dried in the open and processed into a fleece as already detailed in Example 1.

Claims (1)

[Scope of Claims] 1. A surgical collagen fiber preparation comprising decomposing bovine Achilles tendon or skin in an alkaline medium, treating with acid, mechanically treating, de-swelling, cross-linking and drying. In the manufacturing method, the Achilles tendon or skin is mixed in an alkaline medium with a concentration of 0.15 to
After decomposition to an amide nitrogen content of 0.30 μmol/g, treatment with hydrochloric acid, and washing to a pH value of 2 to 4, the thickness of the swollen collagen material was reduced under pressure to form a two-dimensional network structure. the individual long fibers are then exposed by hacking, then after de-swelling the fibers are cross-linked with hexamethylene diisocyanate in the presence of a non-ionic or cationic emulsifier, dehydrated and further processed in a textile machine. 1. A method for producing surgical collagen fibers, comprising a processing step. 2. A method according to claim 1, wherein the reduction in the thickness of the swollen collagen material is carried out using a hammer press or a calender or by means of a grooved roll. 3. Reduce fiber contraction to 5-50% by weight, preferably 7-50% by weight.
3. A method according to claim 1 or 2, which is carried out using a 15% strength by weight sodium chloride solution. 4. The method according to claim 1, wherein the fibers are dehydrated and simultaneously impregnated with a drug.