DE3203957A1 - Method for the production of fine-pore collagen sponges - Google Patents

Abstract

The invention relates to a method for the production of fine-pore collagen fibre networks composed of bovine or porcine collagen, in which the collagen raw materials are decomposed with alkali, acids, hydrogen peroxide and/or proteolytic enzymes, freed from concomitant substances and purified, swollen with an aqueous acid or with aqueous alkali, mechanically comminuted, diluted with water to a degree of dilution of about 2.0% by weight dry content, frozen and dehydrated to stabilise the structure, in which the swelling of the decomposed and purified collagen material is carried out until 500 to 1000% by weight of water has been absorbed and the swollen material, after comminuting and diluting to a solids content of about 2%, is frozen for 30 minutes to 4 hours. In the production of pharmacological or cosmetic active substances and also for improving the resilience and the strength of textile fibres or for the production of bone replacement material, hydroxyapatite and/or calcium phosphate can be admixed with the fine-pore collagen fibre networks prepared according to the invention.

Description

Process for the production of fine-pored collagen

sponges The invention relates to the production of fine-pored collagen sponges. In particular, it relates to a method in which certain known ones are used Carries out procedural measures in a specially controlled manner.

The production of collagen fiber braids (collagen sponges) is known from numerous literature sources.

For example, DE-OS 18 11 290 describes the production of collagen fiber braids described by opening the skin or tendons of animals in an alkaline or acidic manner and mechanically shredded and -... if necessary after adding additives, such as Crosslinking agents, plasticizers or pharmaceuticals - to a homogeneous collagen paste slurries, freezes the collagen slurry, and then drains it. the end this Offenlegungsschrift is also known to foam the comminuted collagen mass and freezing the foamed collagen slurry, storing for 1 to 30 days and then to thaw, whereupon you squeeze out most of the water and the squeezed out collagen fiber mesh dries at room temperature.

A purified collagen material is also known from DE-OS 26 25 289 for the production of collagen fiber braids to dewater by freeze drying. Collagen foams are held according to DE-OS 17 17 118 that one through Digestion of the collagen mass obtained from glue jelly with water to a dry matter content from 0.3 to 4.0 g and then allowed to freeze the homogeneous mass and then removed the water.

It is also known that collagen fiber braids can also be produced in this way can be obtained by freezing collagen dispersions and then with organic, Washes the water gently with water-miscible solvents. In the end It is also known to use crosslinking agents in the manufacture of collagen fiber braids to use in order to achieve stabilization.

In the known prior art methods of manufacture of collagen fiber braids the formation of the structure is essentially through given the type of freeze-drying. The reproducibility of collagen fiber braids with the same constant structure and the same porosity is not guaranteed.

The present invention is based on the knowledge that for the formation of reproducible target layer fiber braids of the source u ;; ysgrad and the freezing process of the swollen, crushed and homogenized material play a crucial role.

The object of the invention is therefore to provide a method for production of fine-pored collagen fiber braids available in which one can reproducibly but contains fine-pored collagen sponges with a large intended variability, which are suitable for numerous medical applications.

This object is achieved by a method according to claim 1 solved.

The subclaims contain preferred and / or advantageous embodiments of the claimed process As a raw material for the production of the invention Cattle or pork hides or cattle achilles tendons are used for collagen fiber braids. These starting materials are pretreated in a known manner. Beef or pig hides are, as is also common in the leather industry, by soaking, ashing, Depilation, flesh removal and splitting pretreated and then carried out in a known manner Exposure to acids, alkali, hydrogen peroxide and / or proteolytic enzymes open minded. This Aufscifuß is still used for the pretreatment of the collagen raw material is counted, serves to rid the collagen of all accompanying substances and to be kept so clean that essentially no more disturbing accompanying substances are present are. This means that accompanying proteins (such as albumins or globulins), glycosamine glycans, Proteoglycans and fats that the animal source material to adhere, to be removed. The chemical treatment is more intensive and more thorough than is usual in the leather industry, because the collagen material subsequently further treated for use for surgical and cosmetic purposes will. The acid treatment results in essentially acid-soluble accompanying substances removed and at the same time loosened the structure of the native collagen.

A measure for chemical cleaning is the determination of the amide nitrogen. The cleaning reduces the value by about 0.3 to 0.5 m mol / g, namely to the value from 2.0 to 4.0 m mol / g.

The chemical pretreatment is basically done with such reagents carried out that can either be completely removed and / or non-toxic are.

Therefore one will only use reagents which are themselves free from disturbing impurities are. Hydrochloric acid has proven itself for acid treatment, so that you basically use hydrochloric acid for this.

The analytical assessment of the result of the chemical pretreatment consists essentially in the recording of 2 factors: a) Degree of purification (maturity) of collagen and b) Degree of swelling of collagen The degree of purification is determined by the usual analytical procedures established. The degree of swelling is very important of collagen. The pretreatment, i.e. the breakdown of the collagen, must be carried out in this way that the collagen has a swellability which is equivalent to uptake of 650 to Corresponds to 800% by weight of water. At a pH of 3, as found in the digestion with Hol ceases, the collagen must absorb 6 to 8 times the amount of water, based on its weight. One maximum swelling with HCl is achieved at a pH of 2.0, however, the swelling value is usually set at the pH value of 3.0 because the material digested with HCl has this p-value.

The swelling of the collagen depends on a number of factors and is influenced by these: Young connective tissue, i.e. material from young animals swells more than that of old animals. In addition to the chemical pretreatment at the cleaning and digestion of the material also plays a role here mechanical treatment plays a major role.

A heavily chemically pretreated material swells more than it does Less heavily pretreated material and a shredded material swells more as a non-comminuted material.

As already mentioned, the swellability also depends on the pH and the Dependent on the maturity of the collagen.

The swelling of the collagen is expedient according to the so-called Kubelka method determined (KUBELKA, K: The collagen tissue of the skin (Czech.) BRATISLAVA 1961), whereby the decrease in the amount of a source liquid provided is determined.

The one swollen with an aqueous acid or with an aqueous alkali Collagen is then mechanically crushed. This comminution can, for example by chopping it up once or several times with a meat grinder. One to strong comminution, i.e.

Shortening the fibers is not advisable, so the mechanical one is used Crushing preferably up to an average diameter of approx. 6mm. Instead of of a meat grinder, other shredding devices, vie Toothed disk mills are used.

The crushed material is then diluted with water to a degree diluted with a dry content of approx. 2.0% by weight. A dry content between 1 to 3%, in particular 1.3 to 2.6% is possible, but the best values in the further Production of fine-pored collagen fiber braids with a dry content of approx. 2% can be achieved.

The diluted, crushed and swollen material can then, depending be further homogenized according to the intended use, e.g. B. in a colloid mill.

Then, if necessary, it is homogenized in a colloid mill Material frozen. The freezing must take place under precisely controlled conditions be carried out, in particular the freezing time must be strictly observed. This is half an hour to four hours and preferably one and a half to two and a half hours Hours for a material 2.5 cm thick. One can freeze with the help from brine or a suitable freezer.

Slow freezing or freezing at relatively high temperatures results in coarser ice crystals and a coarse internal structure. Brief freezing leads a finely crystalline product with very fine pores. Depending on the intended Using the Ma- Materials for certain surgical or prosthetic or cosmetic purposes, the freezing conditions are selected.

The frozen product is then used to stabilize the structure drains. This drainage can be done in different ways and essentially also depends on whether and to what extent beforehand, if applicable chemical crosslinking and stabilizing agents have been added. To this Crosslinking agents will be pointed out later. Are chemical crosslinking agents has been used, dehydration is not carried out by freeze-drying. Much more the material is thawed after the frozen product has been conditioned and squeezed the water. Not stabilized by chemical crosslinking agents Products are expediently made by freeze-drying in a manner known per se drains and stabilizes.

So far, the basic measures are required for the production of fine-pored Collagen fiber braids have been described, with emphasis on playing a role was on the fact that the degree of swelling is set in a special way and also the product is frozen in a specially monitored manner.

To fine-pored collagen fiber braids the special purposes anztlfit, a number of measures, some of which are known per se, are now possible or required: For the production of bone substitute materials one can use the collagen material add the additives that are important for bone replacement, for example calcium phosphate and hydroxyapatite.

This admixture takes place after homogenization but before freezing. The bone substitute materials must be produced in the alkaline range Two processes are carried out for this purpose: a) At the isoelectric point of the material the additives important for bone replacement are mixed in.

b) The pH is then adjusted to the desired level.

For example, a mixture for making bone substitutes can be 10 contain up to 70% calcium phosphate.

The optimum swelling in such a product takes place at pH values from 9.5 to 11.5, the amide nitrogen content of the collagen raw material being 0.30 to 0.40 wt.% Should be. In the production of bone substitute material takes place the stabilization of the structure expediently by freeze-drying.

It is also possible to use textile fibers before the collagen mass is frozen to mix in. This can be done, for example, to the finished collagen fiber braid to convey a better jump or greater strength. Suitable fibers are, for example, those that are already used for synthetic sutures have been used and also those that have so far mainly been used for textile or industrial applications Purposes. Fibers made of polyester can be mentioned here, for example, Polyamides, polyvinyl alcohol, wool, cotton, cellulose and viscose. The length the fiber is very variable. It can be between about 3 to 50 mm. The amount , based on the dry weight of the collagen fibers1 is generally about 10 to 60% by weight.

The fibers are mixed together at the isoelectric point of the material. The pH is then adjusted to the desired level.

A number of additives can be added to the collagen material before freezing be admitted. Particular mention should be made of crosslinking agents here. Because most of them Crosslinking agents are not physiologically harmless, this is essentially the case only special products in question. These include in particular aliphatic diisocyanates, which react with the amino groups to form urea groups and physiologically are harmless. Due to the type and amount of diisocyanate used, you can significantly influence the physiological properties of the end product.

The use of diisocyanates as crosslinking agents is fundamental known. It is also known that the use of diisocyanates is nonionic Must use emulsifiers.

Other suitable crosslinking agents are those that are already used in Preparations of blood plasma have been used, namely glyoxal and succinyl anhydride. Glyoxal is expediently used in quantities of 6 to 10% based on the dry content of the collagen product, applied and mixed in at acidic pH (about 2.5 to 4.0).

Such a crosslinked product is suitably about 3 to Frozen for 12 days, the crosslinking reaction being slow in the frozen state expires.

If succinyl anhydride is used as a crosslinking agent, then is the amount here preferably about 5 to 30%, based on the dry weight of the Collagen material. In this case, too, the product will last longer after freezing Time, for example, a week or more left, with the networking realization then expires.

The exact amount of crosslinking agent used is related with the type of crosslinking agent and the intended use of the collagen fiber braid. The crosslinking agents influence the physical and chemical properties and the appearance of the finished material. It is important that the crosslinking reaction the swelling state of the shredded collagen is precisely adapted. The crosslinking reaction itself should not run too quickly to avoid premature swelling of the To bring about collagen and thus the formation of hard, film-like structures. Crosslinking that takes place very slowly, especially at low temperatures takes longer. The amount of crosslinking agent must be selected in such a way that when the material is later stored, unreacted crosslinking agent no post-crosslinking and thus no unintentional Change of material is expiring. As mentioned earlier, these are the crosslinked materials not freeze-dried, but the water is mechanically abye-squeezed.

The crosslinking agents are added to the collagen slurry before freezing mixed with cosmetic active ingredients, perfumes or pharmacologically active substances.

Particularly interesting is the effect of various emulsifiers that have a positive influence on the properties of the mesh. This influence can can be compared with the effect of fatty substances in the leather industry.

Collagen acts like an ion exchanger in the body and binds electrolytes, Metabolites as well as drugs.

Because these properties are found in the finished thin-pored collagen fiber braid are preserved, the braids can be used advantageously as a carrier of active ingredients will.

Example 1: 1.1. Raw material 50 kg columns from beef necks after one In the usual methods of leather production softened, ashed, fleshed out, dehaired and split. The dry weight of the material is 20% by weight.

1.2. Alkaline treatment 110 1 water, 0.8 1 NaOH (50 wt%); 0.3 kg H202 (35 wt. er, slowly stirred, treatment time 12 to 24 h.

1.3 Wash for 1 hour in cold running water.

1.4. Analytical control of the amide nitrogen content is 0.43 m mol / g.

1.5. Acid treatment Then 90 * minute pockets in flowing cold water, subsequent treatment in the tanning barrel: collagen material, 5 kg HCl (35% by weight), 30 kg of water; 6 hours of intense exercise; the columns are in full thickness leavened. Then wash in running water for 4 hours. pH after washing 3.0; Amide nitrogen 0.41 m mol / g. Swelling of the material is 714% about 14 Weight% dry weight (determined by the Kubelka method) 1. 6. Comminution Das Material is first cut into pieces 20 x 20 cm and then in a meat grinder ground twice (perforated disc - 20 - 10 mm).

1.7. Dilution The approx. 14% by weight mass is mixed with distilled water Diluted to a concentration of 1.8 to 2.2% by weight. Then follows: the homogenization by passing through a colloid mill three times. The dry weight of the mass is set to exactly 2.0. The pH value is raised again with dilute EICl 3.0 set.

1.8. Adjusting the mass The well-homogenized mass becomes 600 g non-ionic emulsifier added (e.g. ethoxylated Sóktylphenol from 9 to 10 alkoxy groups) and then 2.5 kg of hexamethylene diisocyanate, based on dry matter, mixed into the suspension and due to the risk of gelation within 10 minutes in the forms introduced. Layer height in the forms 2.5 cm.

1.9. Freezing The filled forms are now with the help of a brine frozen (freezing time 2 1/2 hours.) temperature of the brine - 15 "C. Then the material is removed from the mold 1. 10. Conditioning After freezing, a further storage (conditioning) in a cold room. Temperature - 50C, time 7 days.

1.51. Thawing and washing after conditioning is finished the sponges are thawed in heated water (approx. 30 ° C). This is followed by the squeezing and washing out with running water. This process is repeated 4 times.

1.12 Drying and dressing After washing the sponges at room temperature dried. The dried sponges will be Split to the desired thickness, punched and packaged and are used as dressing material can be used for wound treatment.

Density: 0.02 Example 2: 50 kg of beef necks were produced according to Example 1 chemically and mechanically treated, diluted and applied with dilute hydrochloric acid pH adjusted to 3.0. The dry weight of the homogenized mass is exactly 2.0; Amide nitrogen 0.39 m mol / g. The swelling of the material is 729% by weight.

The homogenized collagen mass is deaerated in a vacuum vessel, in Filled molds and frozen in a brine bath at a temperature of - 15 ° C. Freezing time is 2 hours. The 2.5 cm thick collagen plates thus obtained are then freeze-dried. The freeze-dried plates are on Split, punched and packaged to the desired thickness.

The material can be used as a hemostatic agent.

The density of this sponge is comparable to the sponge that in Example 1 has been described.

Example 3: 10 kg slices of beef necks are chemically and mechanically Treated according to Example 1, diluted and adjusted to pH with dilute hydrochloric acid set. The dry weight is exactly 2.0%; Amide nitrogen 0.38; the water absorption of the material is 732 total%.

The well homogenicicrtc mass is now with 5 of the above sodium hydroxide solution adjusted to pH 5.5. After briefly stirring, si.ch separate the de-swollen collagen fibers from the spring water. Then 60 parts of calcium phosphate (on dry weight of Collagen calculated) stirred into the mass. The raw material can be homogenized well. The pH is then slowly adjusted to 11.5. The collagen fibers are now fully swollen again. The pulp is made by running through the Colloid mill homogenized. The homogenized collagen mass is filled into molds and frozen in a brine bath at a temperature of - 150C. Freezing time is 150 minutes and the thickness of the layer 2 1/2 cm. Then the Freeze-dried plates. After freeze-drying, the plates are placed on the desired thickness split, punched, sterilized and packaged.

Claims (5)

Process for the production of fine-pored collagen spong PATENT CLAIMS 1. Process for the production of fine-pored collagen sponges from bovine or Pig collays, in which the collagen raw materials are mixed with alkali, acids, hydrogen peroxide and / or proteolytic enzymes, freed from accompanying substances and on purifies an amide nitrogen content of 3.0 to 4.0 mmol / g with an aqueous acid (with HCl to a pH of about 3) or with aqueous alkali (with NaOH to a pH of about 12.5) swells, mechanically comminuted, with water to a degree of dilution of approx. 2.0 Ge. Dry content dilutes, freezes and stabilizes the structure freeze-dried or treated with tanning agent and conditioned and dehydrated, thereby G e k e n n n -z e i c h n e t that one is the swelling of the digested and purified Collagen material up to the absorption of 650 to 800% by weight of water, measured at pH 3 or p11l215 and the material swollen in this way after crushing and Dilute to one Solids content of approx. 2% 30 minutes to 4 Freezes for hours. 2. The method according to claim 1, characterized in that g e k e n n -z e i c h n e t that you can freeze in 1 1/2 to 2 1/2 hours to complete freezing performs. 3. The method according to any one of the preceding claims, characterized g e it is not indicated that one can additionally use a pharmacologically non-interfering Stabilized crosslinking agent. 4. The method according to claim 1, characterized in that g e k e n n -z e i c h n e t that you can use the purified starting material to improve the length and the The strength of the collagen fiber braids at the isoelectric point is mixed with textile fibers. 5. The method according to claim 1, characterized in that g e k e n n -z e i c h n e t that one can produce bone substitute material at the isoelectric point add hydroxyapatite and / or calcium phosphate before freezing.