JPH02129224A - Preparation of fibrin - Google Patents

Abstract

PURPOSE:To obtain a fibrin derived from a patient in a short time and under an almost aseptic condition by treating plasma with an enzyme having a thrombin action and polymerizing and solidifying fibrinogen in the plasma. CONSTITUTION:Fibrin is prepd. by treating plasma obtd. by passing a collected blood through a plasma separating membrane and the obtd. plasma with thrombin or an enzyme having a thrombin-like action such as reptilase, thereby polymerizing and solidifying fibrinogen in the plasma. In this process, it is pref. to add a substance which inhibits fibrin degradation such as Ca<2+>, FXIII, aprotinin tranexamate. Polymn. using this enzyme is pref. performed in a buffer soln. close to neutrality.

Description

[Detailed description of the invention] [Industrial use! ] The present invention relates to a method for producing fibrin, which can be used as a medical material with excellent biocompatibility in the medical field.

[Prior Art] A conventional method for producing fibrin involves starting with cryoprecipitate obtained by refrigerated centrifugation, plasma obtained by centrifugation, etc., and adding mainly a thrombin solution to the starting materials, which undergo polymerization and hardening. , a commonly used method for obtaining fibrin. For purification of fibrinogen from whole surfaces, Cohn's ethanol fraction (E.

J, Cohn, J, Am, Chew, Soc.
, 68.459.1946) are well known, and in addition, JP-A-50-71812.5095413.5
As described in No. 3-69819, several methods have been proposed to obtain fibrinogen with good efficiency and purity.

However, recently it has been pointed out that blood products produced from other people's blood are at risk of contamination with viruses such as hepatitis virus and AIDS virus, and the problem of antigenicity has not been eliminated.
Plasma obtained by centrifugation requires large equipment, and for example, in an emergency room or the like, there are problems such as the need to previously install large equipment that takes time.

[Problems to be solved by the invention - Uninvented] In view of these circumstances, Nagisa et al. conducted intensive research on a method for producing fibrin, which is useful as a biomaterial, and produced fibrin in a short time and almost aseptically. The purpose is to provide a method for creating . Another object of the present invention is to provide a method for producing fibnon using blood plasma from a patient as a starting material in the medical field, without the risk of bacterial contamination due to complicated centrifugation and purification operations. . An object of the present invention is to provide a manufacturing method that allows patient-derived fibrin to be obtained in a short period of time and aseptically.

[Means for Solving the Problems] The gist of the present invention is to pass collected blood through a plasma separation membrane to obtain plasma in a short time and with a simple operation,
This method of producing fibrin is characterized by polymerizing and solidifying fibrinogen by treating this plasma with an enzyme having a thrombin-like action such as thrombin or levutylase. In that case, it is preferable to further add Ca'' and FXI[[ when treating with an enzyme having a thrombin-like action such as thrombin or levutylase.

Fibrin is recognized to have many physiological functions, and once it solves the problem of virus contamination and antigenicity, it can also undergo physical modification such as forming a membrane after fibrin formation. Chemical modification to immobilize various physiologically active substances, antibiotics, etc. becomes possible. In other words, if a patient-derived fibrin membrane is created without performing a surgical procedure, it becomes possible to make various modifications to the membrane.

The uses of fibrin produced by the present invention include:
A system called fibrin glue is well known, and it
There is a literature that applies it to nerves and keys (Toshibe Futami: Abstracts of the 6th Biomaterials Society Conference, 10.1984), and a literature that applies it to the transplantation of free skin flaps (Nario Ihara: M
edical Postgraduates, 20.
5.335.1982>, which has shown good results.The tissue healing process is different from that with cyanoacrylate, and the presence of fibrin membrane helps the proliferation of fibroblasts, promotes tissue regeneration, and improves capillary formation. The new growth is said to be good. Furthermore, it can be used to fill dissection cavities of large blood vessels, strengthen bronchial stumps,
It can be used for countless purposes such as repairing the resected surfaces of parenchymal organs such as the lungs and liver, and in particular, there are documents applied to the resected surfaces of the lungs (Osamu Kuwahara; 11 Journals of the Society of Thoracic Surgery; 33.1817.1985) It has shown good results and is said to help preserve lung function by avoiding lung capacity reduction and deformation.

The use of Neubulin membrane as a wound protection material was disclosed in JP-A-58
-185162, No. 57-153645, etc., and is also used as a hemostasis and wound dressing material in JP-A No. 5
In addition to No. 8-36545, an example in which a fibrin film was formed by containing both -1 lagen and fibrinogen and applied as a wound dressing material is disclosed in German published patent application 3.
037513.

Another application of fibrin is to immobilize lysozyme on a fibrin membrane and apply it to a bleeding site to absorb blood, promote tissue repair, and prevent suppuration.

Furthermore, it can then be used as a material that can be digested in the tissue (an example presented is described in JP-A-48-80779). Furthermore, methods for immobilizing enzymes using fibrin membranes as carriers have been researched; for example, for leukemia enzyme therapy, L.
- An example of immobilized asparaginase preparation is published in JP-A No. 52
-151723, and an example in which urokinase is immobilized on a fibrin membrane is described in JP-A-59-93019.

Since the reaction to produce fibrin membranes proceeds under physiological conditions F, L
It is set as 1 point.

In addition to the above-mentioned applied functions, it is well known that one of the physiological functions of fibrin is that activation of plasminogen (PLG) by tissue acrylate (t-PA) is enhanced in the presence of fibrin. It has been described in the literature (Hoyl
aerts, M. J., Biol, Cheap,
257.2912 to 2929.1982>, the mechanism is based on the formation of trimolecular combinations between t-PA, plg, and fibrin membranes. Furthermore, activation of plg by urokinase was believed to be unaffected by the fibrin membrane, but some literature (Takeda et al., Thromb.
os, Haemostas, 42.901-908.
(1979) showed that plasminogen (Glu-plg) is rapidly activated by urokinase in the presence of a buiplin membrane, and furthermore, unlike t-PA, urokinase does not physiologically bind to fibrin membranes. proposed that this activation is the result of a reaction between Glu-pl giants and fibrin membranes.

As mentioned above, fibrin membranes are sometimes found to have the ability to significantly enhance the activation of urokinase by urokinase of plasminogen (Glu-plg), which naturally exists in the body. This shows that the fibrin membrane has a physiological function of increasing fibrinolysis, and that the fibrin membrane prepared according to the present invention can be effectively used.

As in the north, there are still some unknown aspects of fibrin membrane, but it is expected to develop into a major field in the future as a medical material such as the inner coating material for artificial blood vessels and the base material for artificial skin. .

In the method for producing fibrin according to the present invention, the starting material is whole blood, which has been subjected to anticoagulation treatment using a known anticoagulant mainly containing citrate. At this time, it is preferable to use a sterilized container for the whole blood, such as a syringe or a soft medical plastic bag.

For example, blood is collected from a patient into a syringe containing 1/10 volume of 3.8% citrate, and then connected to a blood supply 1 of a plasma separator as shown in FIG. 1, which is composed of a plasma separation membrane. When blood is introduced into the plasma separator while applying an appropriate pressure to the blood, plasma is obtained through the plasma separation membrane 4 and comes out from the plasma membrane 12. At this time, the plasma is placed in a sterilized container, such as a test tube or petri dish, made of a synthetic polymer material and shaped to suit the purpose.

Next, a solution consisting mainly of thrombin solution is added and a fibrin membrane is obtained in a polymerization reaction.

In this case, instead of thrombin, an enzyme having a thrombin-like action such as levutylase or batroxobin can be used to permit polymerization.

In order to polymerize and solidify plasminogen, it is also possible to add substances that inhibit am decomposition such as Ca''' FX■, aprotinin and tranexamic acid when treating with thrombin or an enzyme that has a thrombin-like action such as levutylase. It is.

Depending on the purpose of use of the produced fibrin membrane, crosslinking treatment with a known multifunctional reagent such as geltaraldehyde, freeze drying, natural drying treatment, etc. can be performed.
In addition, depending on the use of the fibrin membrane, before, during, or after the formation of the fibrin membrane, antibiotics, bactericidal substances, enzymes, physiologically active substances, anticoagulants, albumin, Glycoproteins such as fibronectin can also be immobilized.

The fibrin membrane of the present invention is obtained by polymerizing and immobilizing fibrinogen or fibrinogen-containing plasma by treating it with an enzyme having a thrombin-like action such as thrombin or rebutylase.

This enzyme-based polymerization reaction is preferably carried out in a buffer solution near neutrality. This buffer solution and Shiteha, HEP
An ES buffer or the like is used.

Further, an appropriate amount of the Ca' thrombin solution is used, and the thrombin solution is used in an amount of 1 ton or more per 1 g of fibrin, preferably 50 to 350 units. It acts on brinogen and has a net content of about 350, ilj, position/mR.
This is because the physical strength reaches saturation and the effect does not improve even if the strength is increased beyond that point, so it is sufficient to use less than 350 tons.

0 or more fibrin membranes in which a membrane with excellent physiological properties can be obtained by treating the buiplin membrane with a physiological saline solution containing 0.01 to 2% by volume of glutaraldehyde after forming the fibrin membrane. Depending on the purpose of use, it is preferable to treat with physiological saline to also wash away excess unreacted liquid.

As the plasma separation membrane used in the present invention, either a flat membrane or a hollow fiber membrane can be used, and as the material constituting the membrane, known medical polymer materials such as polypropylene and cellulose acetate can be used.

[Function] The "fibrin membrane" created by the present invention is important in the process of wound healing, and unlike collagen etc., it is a protein that does not exist physiologically and appears under non-physiological conditions. is a material with various excellent physiological properties,
It can be found to be applicable in a wide range of medical fields.

Next, the preparation of fibrin according to the present invention will be explained using a specific example, but the present invention is not limited to the following explanation.

[Example 1] 50ml of blood was collected from a normal adult male into a sterilized syringe containing 1/10 volume of 3.8% citrate, mixed well, and the needle of the syringe was removed. The membrane was connected to the blood inlet 1 of a plasma separator (manufactured by Terumo Corporation) consisting of a polypropylene separation membrane 4, and blood was introduced while applying pressure, and then plasma was separated.

At this time, when 50 ml of whole blood was passed through the tube, the amount of collected plasma was about 26 mjj. Also, the time it took was about 6
It was 0 seconds. A sterilized centrifugal sedimentation tube (manufactured by Terumo Co., Ltd., Betray [registered trademark]) is connected to the plasma outlet 2 shown in Fig. 1, and plasma is added thereto, followed by 500 units of thrombin solution (manufactured by Kakashi Seiyaku Co., Ltd., Japanese Pharmacopoeia). When 1000 units of aprotinin (Trasylol [registered trademark]: manufactured by Bayer) were added, a fibrin gel was formed about 2 minutes later.

After leaving the formed fibrin gel at room temperature for 3 hours,
It was fixed in 10% neutral buffered formalin, and paraffin sections were prepared according to the general method for preparing pathological tissue specimens (How to prepare pathological tissue specimens, supervised by Yonosuke Watanabe, Igaku Shoin, 6th edition, 1986). , subjected to HE staining and PTAH staining,
Tissue observation was performed.

As a result, a fibrin fiber network that was stained bright red with HE staining and deep blue with PTAH staining was observed. At this time, it took about 3 minutes to prepare v-pudding from whole blood.

[Example 2] 50 mff1 blood was collected from the carotid artery of a normal rabbit at 1F into a sterilized syringe containing 1/10 volume of 3.8% citrate.
Mixed well. Next, remove the syringe needle from the artery, connect it to the blood inlet 1 of a plasma separator (manufactured by Terumo Corporation) consisting of a polypropylene separation membrane 4 shown in Fig. 1, and insert the collected blood while applying pressure. The plasma was separated and obtained from plasma outlet 2. Plasma was placed in a sterile Petri dish (Terumo Corporation, Betray [registered trademark]) at the plasma outlet 2, and 4 Klobusitzky units of replase (Reptilase [registered trademark] -5° injection, manufactured by Zelya Pharmaceutical Co., Ltd.) was added thereto. aprotinin 10
．． A relatively soluble fibrin gel was obtained by adding a solution containing 000 KIE units (Trasylol (registered trademark), manufactured by Bayer). It took about 5 minutes to prepare fibrin from whole blood.

[Comparative Example] As a control, fibrinogen was purified from rabbit whole blood to produce fibrin. That is, blood was collected from the carotid artery of a rabbit with the addition of 1/10 volume of 3.8% sodium citrate, and 3000
Plasma was obtained by centrifugation at rpm for 15 minutes. Thereafter, the method of Doolittle et al. (D
oolittle, R,F, et al, :Bi
ochem, Acta, 118.456-467,
(1967), fibrinogen was purified. The time required at this time was approximately 4 hours.Next, add 2mM of fibrinogen to the fibrinogen.
of CaCl28, 2mM MgCNx, 150mM NaCj! 5mM HEPES buffer (pH
7,4) was added to produce a fibrin gel.

That is, as described above, in the method for producing buiprin according to the present invention, the time from blood collection to fibrin production can be significantly shortened.

[Effects of the Invention] As detailed above, the present invention has advantages in the medical field.
Effective fibrin membranes are created from plasma that has passed through a plasma separation membrane, and fibrin membranes that can be used effectively in medicine can be produced safely in a short time and without the need for large equipment.
In addition, fibrin membranes can be obtained from self-derived plasma without any concerns about antigenicity. In other words, patient-derived fibrin membranes can be safely and immediately procured on the spot without surgical procedures. In addition, significant technical effects have been obtained, such as providing a method that allows various modifications to be made to the fibrin membrane.

[Brief explanation of the drawing]

FIG. 1 is a schematic cross-sectional view showing one example of a plasma separator of a plasma separation membrane used in the present invention. [Explanation of symbols of main parts]

Claims (2)

[Claims] (1) Fibrinogen contained in the plasma is polymerized and solidified by passing the collected blood through a plasma separation membrane and treating the obtained plasma with an enzyme having a thrombin-like action such as thrombin or levutylase. A method for producing fibrin characterized by: (2) When treated with enzymes with thrombin-like action such as thrombin or levutylase, Ca^2^+, F
The method for producing fibrin according to claim 1, wherein XIII is added.