DE1694509B - Process for the production of a collagen dispersion as a starting material for strand-like collagen products. Eliminated from: 1417368 - Google Patents

Description

high percentage of pure collagen, which is homogenized with materials measuring pump made of stainless steel (e.g. a Zenith that does not swell in acidic solution. pump), whereby the pressure is controlled and between about 14.06 at of collagen in an acidic solution to produce a start and 4.22 to 5.62 at at the end of the homogeneous homogeneous dispersion of collagen fibers. It is very well kept. At the end of the homogeneity it is important that the collagen discs are not allowed to grow together during the sizing stage, the pressure between the pump and the swelling stage. If the 0.13 cm wide nozzle is fairly constant and the collagen swells up, it becomes sticky, and if one is at 4.22 to 5.62 at nearly 450 cm ⁸ / minute.
the inside of the mass obtained does not come into play. After the homogenization, the dispersion contains the swelling solution. It is therefore advisable to prevent the individual tendon material from growing together, which would clog the multi-hole nozzle, in order to ensure a homogeneous fiber and must be removed. This becomes easy by getting dispersion in a reasonable time. causes the dispersion to pass under pressure

In a dispersing vessel 15 provided with a stirrer, a plate filter presses through it, which removes the non-collagenous

the collagen discs are slowly retained in the acidic substance.

dividing medium stirred, whereby the collagen slices The collagen fiber dispersion is immediately after

absorb the acidic solution with swelling. the filtration referred to as "green dispersion" because

The temperature is a critical factor after attempts to spin this product without post-curing.

the addition of the acid to the tendon disks, since the ao nen, excessive breaking of the spun end-

Collagen in the presence of acids at about 30 ⁰ C result in loose thread. However, if the

and is degraded above it. Because of this, dispersion too long at room temperature

If all the handling following the addition of acid is necessary, the collagen will break down and the

treatment stages at a temperature below about 25 ⁰ C dispersion forms threads of lower tensile strength if

be performed. ₂₅ she's too old. Under optimal conditions, the

Perfluoric acid of the formula collagen solution at room temperature (approx. 25 ^° C.) serves as the swelling solution

CF (CF) _n COOH aged for a period of approximately 24 hours and

then kept in the refrigerator at 5 ° C until the

in which η is an integer between 0 and 6. Spinning is used. The collagen dispersion will

It is best to use perfluoric acids 30 in the refrigerator for 3 to 4 weeks until spinning.

with at least 2 but not more than 8 carbon preserved.

atoms to make the collagen dispersion. The preparation of the dispersion from pure swollen dispersed Collagen is less resilient than the above against decomposition when a perfluoric acid is carried out with fewer procedures to remove all impurities is used as 4 carbon atoms; on the other hand 35 eliminate because of some lack of connection If the perfluoric acid is more than 6 carbon in mass, breaks in the individual threads during the Atoms the water solubility of the perfluoric acid thus leads to the spinning process. Even tiny air bubbles are reduced, That methanol be added to the solution causes breaks in the monofilaments, and it is therefore In order for the swelling by the perfluoric acid to be necessary, all of the air from the dispersion must be briefly removed increase. Perfluorobutyric acid has proven to be very useful. 40 to be removed before use. This can be in be-die Amount of acid can quemer be effected with the equivalent weight by the fact that the the acid and its ionization constants vary. Dispersion just before spinning into a large one Generally, however, an acidity level of about vacuum desiccator is set and 2 or 3 hours 0.20 to about 1%. based on the total weight the vacuum of about 15 mm of mercury applies. Solution, used. The appropriate pH is 2 45. The presence of a liquid with a low to 3. Vapor pressure in the aqueous dispersion, such as. B.

It is very difficult to produce more than 2% collagen with a collagen dispersion from methanol, which helps remove air bubbles, since more concentrated methanol is an excellent additive solvent. Dispersions have an extremely high viscosity. because of its low specific weight. Approximately when the used collagen 50 50 percent by volume methanol is advantageously added to the collagen dispersion for pressing or spraying endless threads dispersion. If the amount of tendon in the swelling is used, larger amounts which lead to difficulties in loosening, should preferably be 1%. A dispersion swells up the collagen fibers and leads to one made of collagen fibers with a solids content under dispersion that is difficult to homogenize and 0.8% ^is difficult to ponder. The other hand has a 55 squirt is. An aqueous dispersion without an additional concentration of collagen fibers ^{in excess of 1 u} / o in solvent requires a longer time to make a suspension more difficult to completely deaerate under vacuum,
splash is. The difficulty is of equal importance. -I ₁
ability to get a homogeneous dispersion if the example
Total solids content is too high. It is also extremely important that the collagen to be injected is homogeneous in a thickness of fibril dispersion perpendicular to its longitudinal axis, as a slight change - about 0.038 to 0.064 cm and after being broken down in the solids concentration of the To be sprayed on the total solids content with a substance large differences in the cross section of the final enzyme solution treated to dissolve the elastin. The product entails. 65 Enzyme solution is made by stirring 40 parts of MaIz-

After the swelling in the dispersion container to diastase with 400 parts of water in 10 minutes

to a large extent is done, the suspension is put through. The homogeneous dispersion is made with 2000 rev /

repeated pumping by means of a rotating centrifuged min. 20 minutes and the clear

aqueous solution of the centrifugation stage filtered under vacuum through an inert analytical ceramic filter material. The filtrate, which is usually slightly acidic, is adjusted to pH 7 with a few drops of dilute sodium hydroxide solution. Then distilled water is added to the neutral enzyme solution to bring the total volume to 1200 parts, 400 parts of the sliced tendon are immersed in this solution, which is then covered with a layer of toluene to prevent mold growth, The tendon enzyme- mixture is kept in the incubator at 37.5 ⁰ C overnight (15 to 20 hours).

After the incubator treatment, the tendon disks are washed 3 or 4 times by decanting with distilled water and then treated with 1000 parts of water containing 4 g of ethylenediaminetetra-sodium tetraacetate. This mixture is kept almost 2 hours at 37.5 ⁰ C in the incubator, remove soluble proteins and lipids to. Following this treatment with ethylenediaminetetra-sodium tetraacelal, the pH is adjusted back to 7, if necessary, since the tendon discs are easier to treat in a neutral solution (less swelling and hydration). The tendon discs are washed again by decanting with 5 to 5 changes of distilled water.

The swelling solution consists of 50% aqueous methanol of about 0.35% based on the total solvent weight of perfluorobutyric acid. In general the collagen dispersion with a solids concentration of about 1% is easy to treat, and the Amount of acidic swelling solution can easily be determined from the weight and the solids content of the used Tendon to be calculated. So have z. B. the cut tendons used to prepare the present Solution 33% solids (67% moisture), and the total weight of the collagen and solids Contamination is nearly

400 parts-33% = 132 parts.

When calculating the amount of tendon required to prepare the dispersion of known concentration the weight of the tendon solids (on a dry basis) must be a factor of 1.1 are multiplied to correct the non-collagenous substances contained in the tendon. This Material is not swollen by the acidic solution and must be removed from the dispersion. The The total weight of a 1% dispersion of 132 parts of tendon is therefore

132 parts - - ^ - = 12,000 parts.

1.1%
The total weight of the swollen solution is

12,000 parts - 132 parts = 11,868 parts.

As a result of the water content of the tendon, which at this stage of the process has a wet weight of 2145 parts an excess of methanol over water is required to produce a true 50% solution. The acidic solution is made by mixing

6000 parts of methanol

3987 parts of distilled 42 parts of perfluorobutyric acid (12000 0.35%) were added,

The acidic aqueous methanol solution is cooled below 25 °, a dispersing tank of sufficient

Capacity supplied and the treated collagen slices also to the dispersing container added while the stirrer works at about 60 rev / min. It is important that the remaining procedural steps carried out at temperatures below 25 °

ίο be and that the temperature of the collagen dispersion does not rise above this temperature.

Stirring is continued for 3 hours, during which the individual collagen slices are swollen. the Dispersion is then repeated by pumping through the rotating stainless steel measuring pump, homogenized as described above, having in-line nozzles made of stainless steel openings of 0.076 and 0.102 cm. During the homogenization the stirrer is in the dispersion tank moved continuously.

The pressure on the higher pressure side of the homogenization nozzles falls to 4.921 atm and remains constant after 3 hours, which essentially indicates complete homogenization. The dispersion is then forced through 0.127 cm and 0.102 cm nozzles into a plate filter with three stainless steel screens (No. 316). These sieves are separated from one another by 0.32 cm spacers and the mesh size decreases so that the dispersion first passes through a 0.036 cm sieve, then a 0.023 cm sieve and finally a 0.010 cm sieve. During the filtration stage, the pressure on the filter is constantly maintained below 2.812 atm.
The dissolved collagen fiber dispersion contains nearly 11,000 parts (0.9% solids) after filtration. 600 parts of the material are retained on the filter. The dispersion forms an opaque, thixotropic mass that takes on a very viscous, slowly flowing state at room temperature. At 15 °, the viscosity of the dispersion, determined using a "Piastigraph" viscosity meter from the Brabender Corporation, has 440 Brabender units. The dispersion can be compared with glycerine, which has a viscosity of 51 poises at 7.5 ° and shows a value of 420 Brabender units in the "Plastigraph" device. Viscosity determinations of this dispersion at other temperatures are listed in the table below. By plotting the logarithm of the consistency against the reciprocal of the absolute temperature, one can see that there is some irreversible physical change between 30 and 35 °.

Con temperature Logarithmic XIT 10 ' sistance ⁰ C consistency 440 15th 2,644 34.70 360 20th 2.556 34.11 319 25th 2.504 33.54 283 30th 2,452 32.99 123 35 2.090 32.45 33 40 1.518 31.93

)
lated water

(6000 parts - [2145 parts - 132 parts])
manufactured. This aqueous methanol mixture who

65 The activation energy of the dispersion according to this example, calculated from the data in the table, is 5.3 kcal per mole below the transition

7 8

temperature of about 34 °. The temperature of the dispersion is above 34 ° during the homo activation energy 40 kcal per mol. Genization stage kept below 25 °.

The collagen dispersion of this example is The resulting dispersion is overnight at

Freeze dried and left to stand under the electron 23 ° without stirring. The next morning

photographed microscope. A characteristic infrared 5 is the dispersion ¹ I ₂ hours at 40 rev / min.

Spectrum of the dispersion obtained according to this example is stirred and then passed through a plate filter with 0.038 cm,

is pressed by casting a thin film and passing through 0.023 cm and 0.014 cm screens. While

Determination of the propagation of the air-dried this filtration stage exceeds the pressure on the

Films with a Perkin-Elmer infrared spectrophoto filter not 2.813 at. The dispersion from the filter

meter. ίο (pH 2.80) is deaerated under vacuum. The homogeneous

The dispersion obtained can be nized under mild conditions and pressed through sieves in

conditions are dried to very pure collagen spun a methyl ethyl ketone spinning bath, which is 8 cm ³

fibers. of analytically pure ammonium hydroxide reagent (28

B is ρ ie 1 2 to 30 ° / ₀ NH ₃ ) in 10 liters of methyl ethyl ketone.

15 The pump runs at 8.9 rev / min. operated to

A collagen dispersion is prepared from about 2.64 cm ^{3 of} the dispersion per minute.

1500 parts of tendon disks injected with 15000 parts.

a solution of 15 parts of ficin, 3.63 parts of Di-Example 3
sodium salt of ethylenediaminetetraacetic acid and

1.95 parts of ethylenediaminetetra-sodium tetraacetate be ao be treated according to the method of the foregoing. The pH of this solution is 5.1 before game prepared collagen dispersion is added with 15000 Teidem of the tendon discs; after adding the len of an aqueous solution (pH 6.0) from 3 parts of discs, the pH of the enzyme solution is 6.3. After (0.2%) ficine, 3.63 parts of disodium acid of ethylene standing for 17 hours at room temperature 24.4 °, diamine tetraacetic acid and 1.95 parts of ethylene diamine are decanted and the disks are treated with tetra sodium tetraacetate. The tendon discs with 15,000 parts of water, the 50 parts of 30% strength contain 35.3 weight percent dry solids; Hydrogen peroxide are added, stirred. The total weight of tendon solids, based on the hydrogen peroxide solution, is the dry weight of the tendon disks, is 529.5 g.
decanted after about 20 minutes. After standing at room temperature for 17 hours

The swelling solution consists of 148.1 parts of Per- 30 the ficin solution is decanted from the disks fluorobutyric acid and a mixture of 17019 parts and the disks for 20 minutes with 1500 parts of water and 24132 parts of methanol. Perfluorobutter stirred, the 50 cm ^{3 of} 30% hydrogen peroxydic acid is held in an amount of 19.5 percent by weight. The pH of this solution is 6.0 to 6.3 after the dry tendon solids, equal to 0.264% of the disks are added. The total weight of hydrogen peroxide applied. The calculated dry solution is decanted; the tendon disks dried ken solids are 1.1% of the total weight and 29150 parts of methanol, 22079.5 parts of water and (solids and swelling solution). 156.3 parts of perfluorobutyric acid were added. Trains-

The dried tendon disks are made of perfluorobutyric acid is 19.5 percent by weight

fluorobutyric acid swelling solution was added and adjusted to 20% of the dry solids or 0.266% of the

cooled and the solution I ¹ I ₂ hours by air 40 total weight of the mixture. The mix will

blow in stirred by the mixture. The mixture was stirred for 1 hour while the temperature was below

is then 1 hour at 40 rev / min. stirred, 25 ° is kept.

while the aqueous acidic methanol solution is below 25 ° The swollen tendon discs are through

is held. The swollen suspension is pumped into a 1.27 cm tube and the cooled disperser

Tendon discs are then pumped around the 45 sion through a 0.32 cm opening, a

Suspension homogenized through a 1.27 thick tube. 0.152 cm wide opening and one 0.127 cm wide opening

Since the viscosity of the dispersion thus obtained is pumped. Eventually the dispersion is made twice (approximately 1.1% solids) is still too high are passed through a 0.102 cm opening. the the total solids to about 0.9% by adding dispersion is stored at 23 ° overnight and of 5400 parts of water, 5400 parts of methanol, and 50 then through a three-sieve plate filter to 0.038. 9.7 parts perfluorobutyric acid reduced. The 0.023 and 0.014 cm filtered. The filtration pressure over-thinned Dispersion will then pass through the 0.32-cm nozzles, not 13.6 at, and the temperature will be pumped, then through a 0.152 cm nozzle, then held during this time at 15 to 23 °. The air will removed from the dispersion under vacuum through a 0.127 cm nozzle and finally one. The end 0.102 cm nozzle pumped (two full passes). The pH is 2.75 and the solids content is 0.90%.

Claims (2)

1 2 PaientansDPüohe · previously used swelling agents, e.g. B. in dilute μ 'hydrochloric acid is the case. Accordingly, this also shows in 1. A method for producing a thread material dispersion obtained for the subsequent spinning of the collagen-mounted collagen dispersion suitable according to the invention for spinning collagen threads, which thread material dispersion has the substantially more favorable degree of strength from animal tendons.
Recovered collagen eyeglasses in as little as possible. Particularly good results are obtained from a treatment that contains degraded form and in which, for swelling, an aqueous solution of perfluoro-purpose uses the comminuted animal material, for example at butyric acid at a concentration of 0.2 to about 1%, room temperature in an aqueous acid solution based on the total weight of the solution, left to swell, dispersed and homogenized to a mechanical pH of 2 to 3, optionally with the addition of pathways, whereby methanol is used, the content denoting that swelling and animal tendons is preferably 1%.
Dispersion in the aqueous solution of a per- In the following, the invention is described with reference to some fluoric acid with 2 to 8 carbon atoms of the For- exemplary embodiments, the mel 15 preceding the production of the collagen dispersion first CF ₃ (CFa) _n COOH pretreatment of the raw material is outlined. in which η means an integer between 0 and 6, Mammals are primarily used as raw material will. tendons, but also whale parts, as well as pork, 2. The method according to claim 1, characterized in that sheep and ox tendons are suitable. The best draws that an aqueous solution of swelling results have been obtained so far by using perfluorobutyric acid at a concentration of 0.2 cattle lower flexor tendon,
up to about 1 ⁰ I ₀ , based on the total weight of the The ox tendons are supplied from the storage solution, with a pH value of 2 to 3, optionally in a frozen state, in order to use a disintegration with the addition of methanol, 25 To prevent settling or destruction, and thawed, the content of animal tendons, preferably around the tendon of fat, non-collagenous protein and wise is 1%. to clean other foreign matter. The cleaned tendon is frozen in slices of about Cut 0.025 to 0.064 cm. Thicker slices swell The invention relates to a method for 30 len slowly in aqueous acidic solution and are difficult Production of collagen dispersions from dispersing. Thinner slices disperse easily swollen collagen fibrils, from which essentially ter, but the dispersion has low tear strength, the sterile collagen thread material for surgical use when injected. Preferably the tendon Purposes. cut transversely to the main axis, since cutting in When producing homogeneous dispersions, such a slow swelling occurs in the longitudinal direction swollen collagen fibrils leading to the strand. An aliquot sample of the cut tendon pressing and spinning are suitable, it is important that the total solids analysis is now carried out since the in that the natural impurities contained in the mammal- the tendon of various moisture tendons are removed and the severance of the natural suppliers and not constant at different times borrowed collagen fibers under such conditions. It follows that a solution or breakdown of the collagen- The sliced tendon is then using fibrils is avoided. The tensile strength of the treated from an enzyme solution to the elastin (elastic pressed product is of the retention of the structural protein of the tendon and blood vessel walls) depending on the natural collagen fiber structure. to loosen, which surrounds the natural collagen fibers For the gentle processing of animal skin - 45 and holds together. This treatment makes the or tendon material, the crushed elastin has already been loosened and can be de-animalized Material can be removed at around room temperature. It can be proteolytic with advantage aqueous acid solution swell, dispersed and enzymes of vegetable or animal origin dissipate homogenized mechanically, whereupon they are turned. The pancreatin is an enzyme that for Dispersion by extrusion or spinning 50 removal of the elastin is effective. It can be deformed would. As an acid for dispersing the NEN also enzymes from plants, such as. B. Ficin, ver Materials usually hydrochloric acid was used, wherefore. Another enzyme that works with this Although thermal and chemical degradation processes are met, what is certain to buy through extraction Dimensions could be pushed back without licher malt diastasis can be produced with water that it would have been possible to find the fibrils in a 55 (USA.-Pharmacopoeia IX). The tendon-enzyme mixture will be 15 to 20 times undesirable for the strength properties of the final products Measures its natural shape and its kept at room temperature. Through these original ones To maintain mechanical properties, essentially all of the elastin is used th. natural collagen fibers separated. It has now been found that it is possible to reduce the tendon use by 60 after the enzyme treatment washed by perfluoric acid with 2 to 8 carbon disks with water. Soluble proteins and Atoms of the formula lipoids are formed by treating the disks with CF (CF) COOH of a dilute aqueous solution of a chelate. the reagent, e.g. B. Ethylenediamine tetra sodium tetra- in which η is an integer between 0 and 6, 65 acetate, removed. After this treatment, the When swelling and dispersing collagen dispersions, tendon discs are washed again to get adherent manufacture in which significantly less degradation removes traces of the chelating reagent, of the collagen occurs when it is in the acidic. The cleaned tendon disks contain one