JPS6010716B2 - Manufacturing method of reinforced fibrin membrane - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for producing reinforced fibrin membranes.

Fibrin is produced by the action of thrombin on fibrinogen during blood coagulation. At this time, fibrin is further polymerized, and the polymerization of fibrin molecules is promoted by a crosslinking reaction by coagulation factor Xm to produce a fibrin polymer (hereinafter simply referred to as fibrin). On the other hand, a method for immobilizing and insolubilizing enzymes using fibrin as a carrier has recently been developed by applying the reaction that occurs when fibrin is produced to the binding of fibrin and enzyme proteins.

The solid iQ enzyme produced in this way can be handled in the same way as a solid catalyst, and since the reaction to generate fipurin proceeds under physiological conditions, there is little denaturation of the enzyme to be immobilized. Since it uses several components, it has the advantage that it does not act as a foreign substance even when applied to medicine. For example, asparaginase, which is known as a therapeutic drug for leukemia, is immobilized on fipurin.
By placing it in the dialysis tube during hemodialysis of leukemia patients, it is possible to prevent the side effects caused by conventional continuous administration of asparaginase. By the way, in order for fibrin to be used for such purposes, it is desired to produce a further strengthened fibrin membrane under conditions that do not cause denaturation of the enzyme to be immobilized.

Conventionally, a known method for producing fibrin membranes is to make fibrin membranes by pressurizing dehydration or heating and molding agar-like so-called clots obtained by treating fibrinogen with trompine. The resulting fibrin membrane is used as a medicine for burns.

However, with this method, the enzyme function is completely lost by heat treatment. On the other hand, once a clot has been collapsed by pressure, it is not easy to re-bond it by pressure, and the pressure-formed membrane has a mechanically discontinuous layer inside and is weak in strength. The method of the present invention is a method in which a fibrinogen solution is treated with thrombin, formed into a thin clot at a temperature of 30 to 40 q0, and then dried until the moisture content is 20% or less at a temperature of about 40 q0 or less. be.

In this method, when applying thrombin,
Preferably, the reaction is carried out in the presence of 5 mmol equivalents or less of calcium ions per 100 fibrinogen solutions. Furthermore, there is a method in which the fibrin membrane thus obtained is stretched in a swollen state, and then dried at a temperature of about 4,000 or less while in the stretched state. The fibrin membrane formed in this manner has no internal discontinuous layer, improves the orientation of fibrin molecules, and significantly increases the strength of the fibrin membrane.

To further explain the method of the present invention, a fibrinogen solution is placed in a flat plate container and a thrombin solution is uniformly sprinkled thereon.

An appropriate amount of thrombin is used, and one or more units, preferably 100 to 30 units, is used per fibrinogen. Gelation begins after spraying, but coagulation factor
In order to sufficiently promote the action of m, the temperature at the time of forming this clot has an influence;
It is preferable to leave the field in a constant temperature room for at least one hour. In addition, it is desirable to carry out the reaction in the presence of calcium ions, and the maximum membrane strength can be obtained by using calcium ions in an amount of 5 mmol equivalent or less, optimally 2 to 3 mmol equivalent, per 100 tablets of fibrinogen solution, and a large excess of calcium ions. Calcium ions have the opposite effect. Calcium chloride is usually used as the calcium ion. A buffer is also used so that the reaction is preferably carried out at a neutral to slightly acidic pH, particularly at pH 6.2. Next, the clot thus obtained is dried by an appropriate means such as air drying or vacuum drying at a temperature below the temperature at which the enzyme causes destruction, that is, below 40 qo, preferably between room temperature and 0°C.

The strength of the fibrin membrane largely depends on the moisture content, and the membrane strength increases rapidly when the moisture content is lower than 20%. By the way, the strength of a film with a moisture content of 20% is approximately 100.
Although it is 0 scale/me, a strength of 4000 gr/me is obtained at a moisture content of 10%. In order to further strengthen the fibrin membrane obtained as described above, the fibrin membrane is briefly immersed in water or a suitable liquid to swell and then stretched.

The dilatation increases the flexibility of the orientation process of fibrin molecules. For stretching, a method is adopted in which both ends of the membrane are fixed and stretched, or a method in which the membrane is passed through a rolling roller while being stretched.

The stretching ratio is about 1.5 times or more. (in extended state)
Dry in the same manner as above to obtain a fibrin membrane. The moisture content of the membrane is preferably 20% or less. Fibrin membranes produced by stretching under this swollen state have a stretching ratio of 1.5 times or more (A, B, C) as a result of improved orientation of fibrin molecules, as seen in the test examples below. An increase in membrane strength was observed at 2 times the stretching ratio.
It has been more than doubled.

In addition, compared to the membrane legs that were stretched twice without being swollen, the orientation of A was improved about 4 times. *Indices corresponding to orientation In the method for producing fibrin membranes of the present invention, if the enzyme is added in the first step, a fibrin membrane with immobilized enzymes can be obtained, and in the method of the present invention, the enzyme can be produced without losing its function. Reinforced fibrin membranes can be produced.

Example 1 Fibrinogen powder of 100 to 2.5 days in a pH 6.2 buffer was dissolved in a stirrer for 3 to 4 hours at about 35 qo, and stirred with oven paper.

CaC12 was added to this solution in an amount of 5 mmol equivalent. Pour this into a flat-bottom container made of methacrylic resin containing 1 x 1 base, take a solution of 50 units of thrombin crystals dissolved in 5 volumes of distilled water with a pipette, and pour it evenly into the above-mentioned fibrinogen solution. is. Although the fibrinogen solution immediately began to gel, it was left to stand in an incubation room at 0.degree. C. for an additional 8 to 1q hours to promote the action of coagulation factor Xm. Dry the molded cloth with the wind of a fan until the moisture content is 10%, and the moisture content is approximately 0.
．． Fibrin membranes with a thickness of 1 to 0.15 ribs were obtained. The fibrin membrane thus produced had a strength of 4000 mm. Example 2 In the method of Example 1, Ca was added to the fibrinogen solution.
A fibrin membrane was produced in exactly the same manner except that C12 was not added.

The fibrin membrane produced in this way has a capacity of 300 female r'
It had a secret strength. Example 3 The fibrin membrane obtained by the method of Example 1 is immersed in water for about 5 minutes, and both ends of the swollen membrane are fixed and stretched to double the size, so that the moisture content becomes 10% in the stretched state. Air-dried until

The strength of the fibrin membrane produced in this way is 000.
Reinforced with nails/pine. Example 4 In the method of Example 1, asparaginase 12m9 was added to the fibrinogen solution.
was added to obtain an asparaginase-encapsulated bonito fibrin membrane.

The activity value of immobilized asparaginase in the obtained membrane was 1
It had the ability to hydrolyze 100 M asparagine per minute per M9. Next, this fibrin membrane was stretched by the method of Example 3 and air-dried to produce a reinforced fibrin membrane. The strength and asparaginase activity of the enzyme-containing reinforced fibrin membrane thus obtained were measured.

Claims (1)

[Claims] 1. Fibrinogen solution is treated with thrombin for 30 minutes.
1. A method for producing a reinforced fibrin membrane, which comprises forming it into a thin clot while maintaining the temperature at ~40°C, and then drying it at a temperature of about 40°C or less until the moisture content becomes 20% or less. 2. The production method according to claim 1, wherein the action of thrombin on the fibrinogen solution is carried out in the presence of 5 mmol equivalent or less of calcium ions per 100 ml of the fibrinogen solution. 3 Apply thrombin to the fibrinogen aqueous solution 3
The fibrin membrane is kept at 0 to 40°C and formed into a thin clot, then dried at 40°C or below until the moisture content is 20% or below, and the obtained fibrin membrane is stretched in a swollen state. A method for producing a reinforced fibrin membrane characterized by drying at about 40°C or lower.