JP2001524619A - Production method of protein fiber and its product - Google Patents

Abstract

  (57) [Summary] The present invention relates to a method for producing a fibronectin and / or fibrinogen-containing protein fiber, an improved fibronectin and / or fibrinogen fiber, and an article containing the protein fiber. The present invention also relates to fibrinogen and fibronectin-containing protein fibers for the manufacture of drugs, bandages or devices for healing wounds and for treatment.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for producing a protein fiber containing fibronectin and / or fibrinogen, an improved fibronectin and / or fibrinogen, and an article containing the protein fiber. The present invention is also for healing wounds,
And fibrinogen and fibronectin-containing protein fibers for the manufacture of therapeutic drugs, bandages or devices.

In the treatment of severe burns and wounds, autologous transplantation techniques are often used to replace damaged skin. This situation is generally less than ideal as it increases the patient's wounds. However, skin substitutes have been developed that attempt to mimic the multilayer structure of the skin or form a cellular sheet over the wound. Many of these are collagen-based so that a close analogy between the surrogate tissue and the natural extracellular matrix is obtained (1,2,3). Cartilage and vascular grafts have also been created from synthetic polymers, poly D-lactic acid, and polyglycolic acid (4,5). These materials have been shown to exhibit excellent adhesion to cells and have the mechanical properties necessary for their intended function. Other techniques include large-scale cell cultures that produce large-area epidermal keratinocytes for transplantation at the site of the recipient to replace the skin.

[0003] Purified fibronectin has long been used to form three-dimensional mats for cell growth. The dimensions of the mat are generally 2cm x 1cm (6,7)
. Such mats are made from a fibronectin solution purified by affinity chromatography in a stirred ultrafiltration cell using a combination of protein concentration and fluid shear. Molded mats consist of very thin strands of protein, which are generally aligned in the ultrafiltration cell by the effect of shear, giving the mat an overall orientation. The mat can be used as a template for oriented cell growth and has found functional use in nerve repair, where it has been found to connect a 1 cm gap in the rat sciatic nerve (8 ). Fibronectin mats have also been used as recruitment points for growth factors such as nerve growth factor (9, 10) and as carriers for epidermal keratinocytes (11) that may be useful in the treatment of bullous epidermis.

[0004] In addition, Wojciak-Storthard (12, 13) discloses that a single thin strand of purified fibronectin, 0.2-5 μm in diameter, has fibroblast and macrophage-like cells along its length. Have been found to promote the orientation and migration of. Cell alignment and orientation is believed to be an important process in reducing wound contracture and enhancing wound repair. However, Woj
The extremely thin fibronectin strands described by ciak-Storthard are difficult to handle.

[0005] It is therefore an object of the present invention to provide an improved method for producing protein fibers containing fibronectin and / or fibrinogen, and to provide fibronectin and / or fibrinogen fibers with improved properties.

The present invention comprises the steps of: adding fibronectin and / or fibrinogen to a solvent to form a solution of fibronectin and / or fibronogen; extruding the solution through an orifice into a coagulation liquid to form the protein fibers. The present invention provides a method for preparing a protein fiber containing fibronectin and / or fibrinogen, comprising: Generally, the solution containing fibronectin and / or fibrinogen is
It is obtained by dissolving a protein solid, for example, a protein precipitate containing fibronectin and / or fibrinogen in a suitable solvent. Since there is no requirement to be in purified form, a mixture of impure proteins can be used as starting material.

[0007] The protein solid preferably contains a mixture of fibronectin and fibronogen. Generally, the ratio of fibronectin to fibrinogen is 9: 1.
１ ： 1: 9, preferably 7.5: 2.5 to 2.5: 7.5, more preferably 6.5.
: 3.5 to 3.5: 6.5. Particularly preferred protein solids are
It contains fibronectin and fibrinogen in a ratio of 6.5: 3.5. Other proteins, such as albumin, may be present in smaller proportions in the protein solids. Generally, fibronectin and / or fibrinogen can be obtained from any source. Preferably, the fibronectin / fibrinogen mixture can be obtained from a cryoprecipitate of plasma, and plasma from other mammalian sources such as calf, pig, or sheep sources may be used, but especially human plasma. Can be obtained from

[0008] A mixture of fibronectin and fibrinogen is described, for example, in Foster, P .;
R. Dickson, A .; J. , McQuillan, T .; A. , Dicks
on, I. H. Keddie, S .; And Watt, J .; G. FIG. Co-authored (1982
) "Control of large-scale plasma thaw
ing for recovery of cryoprecipitate
factor VIII (control of large-scale plasma thawing to recover cryoprecipitate factor VIII) ", Vox Sanguinis, 42: 180-189.

Alternatively, the isolation of fibronectin and fibrinogen from human plasma can be derived from so-called “corn fraction I”, for which a general preparation method is described in “Methods of Plasma Protein Fractiona”.
Tion (Plasma Protein Fractionation Method) "(Culling, JM), Pu
b. Academic Press, London, p. K from 3 to 15
istler, P .; And Friedli, H .; "Et" in co-authoring (1980)
hanol precipitation (ethanol precipitation) ".

It may be possible to make and use fractions of plasma proteins that contain not only what is known as Cohn Fraction I but also additional protein fractions. For example, during the fractionation of plasma, a combined fraction of human plasma proteins represented by I + II + III from which a sub-fraction represented by I + III can be separated (14) from the supply of fibronectin and fibrinogen Can be used as a source. In essence, any fraction of human plasma proteins, including the protein of fraction I, can be used as a source of fibronectin and fibrinogen, regardless of the precise recovery method.

[0011] Therefore, the sub-fraction produced and discarded when producing coagulation factor VIII from a cryoprecipitate mainly containing fibronectin and fibrinogen can also be used. Representative methods that can be used to provide useful protein fractions include US Pat. No. 4,361,509 (Zimmerman,
T. And Fulcher, C .; "Ultrapurification o
f factor VIII activity in whole blue
dor blood plasma (ultra-purification of factor VIII activity in whole blood or plasma) "(1983) and European Patent No. EP 0416983 (Burn).
of-Radosevich, M .; And Burnouf, T .; "Proc
ede de preparation de concentre duc
omplex factor VIII-factor Von Will
lebrand de la coagulation sanguine a
Partir de plasma total "(1991).

Further, fibronectin and / or fibrinogen may be directly extracted from plasma by, for example, ion exchange chromatography or polyethylene glycol (PEG) precipitation. The fibronectin and / or fibrinogen obtained by any of the above methods can optionally be concentrated before being optionally dissolved in a solvent.

[0013] Using a precipitation operation, it is possible to selectively remove fibrinogen from the redissolved cryoprecipitate and then to precipitate fibronectin and the remaining fibrinogen from the supernatant. A typical method used is "Methods of
plasma protein fractionation "(Culling, JM, eds.), Pub. Academic P
res, London, pp. 57-74, Hao, Y .; L. , Ing
ham, K .; C. And Wickerhauser, M .; Co-writing (1980)
"Fractional preparation of proteini"
ns with polyethylene glycol (fractionation and sedimentation of protein by polyethylene glycol) ". It is convenient to carry out the first precipitation operation with 4% (w / v) and the second precipitation operation with 10% (w / v) polyethylene glycol 6000. Alternatively, in order to increase the fibronectin and / or fibrinogen content of the protein fraction used for fiber production, ethanol precipitation in alkali (eg, NaOH) is dissolved, or the protein precipitate is lyophilized to a high concentration in the solvent. Any standard biochemical procedure for precipitating proteins, such as lysis, may be used.

In order to selectively precipitate fibrinogen by denaturation, the cryoprecipitate of the re-dissolved fraction I is raised to + 56 ° C. for up to 30 minutes, and then the fibronectin containing the fraction and the remaining fibrinogen are decanted. The use of heat is also possible by sedimentation from the liquor at + 4 ° C. with 5% (w / v) polyethylene glycol 6000.

[0015] Protein precipitates containing fibronectin and / or fibrinogen generally have a protein in the solvent of more than 50 mg / ml, preferably 70 m / ml.
Dissolve to a concentration above g / ml. However, for some applications, such as large-scale production of ionic fibers, higher concentrations of the solution with protein concentrations above 120 mg / ml and high viscosities may be more suitable. If a more concentrated solution is required, the precipitate may be first lyophilized prior to dissolution in the solvent. The solvent preferably contains a denaturant, which does not substantially irreversibly denature the fibronectin and / or fibrinogen and / or does not result in fibronectin and / or fibrinogen protein properties such as cell adhesion, It acts to denature any other proteins that may be present in protein precipitates, such as viral coat proteins. In addition, this provides a step of virus inactivation that is useful for the process.

[0016] Other modifiers may be used, such as guanidine hydrochloride, but the preferred modifier is urea. Thus, particularly preferred solvents contain 2-8M urea, more preferably 2.5-6.5M urea, for example 6M urea. Such a urea solution may be buffered using, for example, Tris having a pH in the range of 7.0 to 9.0. Since the fibronectin / fibrinogen precipitate contains a significant amount of liquid, dissolving the precipitate in a solvent containing urea will result in diluting the final urea concentration. It is believed that high concentrations of urea could potentially serve as a step in inactivating the virus. One way to maintain a high urea concentration is to add solid urea to the solution in which the protein precipitate is dissolved to maintain the high concentration. However, the addition of solid urea must be controlled to ensure that denaturation does not precipitate the protein as a result of locally high urea concentrations.

Additives may be added during dissolution of the protein to enhance the extrusion properties of the solution and / or to change the properties of the fiber. Representative of these are, for example, sodium salts of carboxymethylcellulose, added at a concentration of 0.1-10%, more preferably 1-5% (w / v), high molecular weight polyethylene glycol, and alginic acid. There is sodium. Such additives increase the viscosity of the solution and, in the case of sodium alginate, reduce the self-adhesion of the final fiber. Further, by including a viscosity enhancer such as sodium alginate, it is possible to reduce the protein concentration to less than the 50 mg / ml required for fiber production. For example, it is possible to produce fibers containing 4.6% protein and 4.8% sodium alginate. The apparent viscosity of such a solution at a shear rate of 1007 s ⁻¹ was 1310 mPas (23 ° C.).

The solution may be degassed prior to being extruded through the orifice. Degassing the solution helps to prevent air bubbles from becoming trapped in the fibers during fiber formation. The orifice from which the solution is extruded may be of any desired size and / or shape. The orifice has a circular cross section with a diameter between 10 μm and 1000 μm, preferably between 50 μm and 500 μm, more preferably between 100 μm and 400 μm.
Conveniently between m. However, much larger diameter fibers up to 15 mm in diameter can be produced.

The extrusion is preferably performed at room temperature (for example, at 18 to 23 ° C.). However, lower temperatures (eg, 4-18 ° C) may be employed if the viscosity of the solution does not increase to a level that makes extrusion impractical. If fibrinogen and / or fibronectin are obtained by cryoprecipitation of plasma and are kept at a low temperature (e.g., 4-18 <0> C), the precipitate may be quite fibrous and difficult to extrude. However, fibrinogen and / or fibronectin obtained from gels or other sources that form non-fibrous precipitates can be extruded at such lower temperatures. The fibers can be extruded over a wide range of extrusion rates. For example, the fibers can be extruded at a rate between 300 μl / min solution and 60 ml / min solution.
However, the extrusion speed will generally depend on the number and size of the holes through which the solution is extruded. It is important that the rate must be such that the protein does not begin to precipitate before it is extruded from the orifice.

The coagulating liquid into which the protein solution is extruded generally contains suitably combined acids and salts to coagulate and precipitate the protein of interest from the solution. Coagulation is generally in a temperature range, preferably between 4 ° C and 25 ° C (ie room temperature)
Happens in.

The isoelectric point of fibrinogen and fibronectin has been reported in the literature to be between pH 5 and pH 6 (15,16). However, surprisingly, the pH solubility curve shows that a mixture of the two proteins, eg, fibronectin 65%: fibrinogen 35%, has minimal solubility at pH 3.0 (see Figure 1). Increasing the proportion of fibrinogen or fibronectin in the mixture results in a shift in the minimum solubility for proteins towards pH 4.0 (see FIGS. 2 and 3). The solubility curve of fibronectin alone is not shown, nor is the migration of the minimum solubility towards pH 4.0-5.0. However, in all cases, very low pH <1.0-1.5 causes precipitation of the protein.

Based on this information, nearly pure fibrinogen or fibronectin can be precipitated at pH <1.0-1.5 or between pH 4-6.
Preferably the fibrinogen / fibronectin mixture has a pH <1.0-1.
Precipitate at pH 5 or between pH 3 and 4 (i.e., around the minimum solubility of a suitable fibronectin / fibrinogen mixture).

In addition, salts such as calcium chloride will precipitate proteins from solution. Figures 5 and 6 show how increasing the concentration of calcium chloride affects the solubility of a fibrinogen / fibronectin solution. Calcium chloride increases the ionic strength of the solution, leading to increased protein precipitation. Further, when calcium chloride is added, the pH decreases due to the generation of hydrochloric acid.

Thus, fibrinogen and / or fibronectin are suitable p
Precipitation from solution can be by H control, high levels of salt, or preferably a combination of the two. The importance of the combination of both salt and pH is shown in Figure 9, where the highest levels of protein precipitation are seen, with both a decrease in pH and an increase in ionic strength.

[0025] Preferred coagulating liquids for the formation of fibrinogen and / or fibronectin fibers are low pH (<pH 1.0 to less than 0.5 M), such as hydrochloric acid or sulfuric acid at a concentration of more than 0.5 M, generally more than 0.1 M or 0.2 M 1.5) strong acid with optionally 1 to 10%, preferably 2 to 7% of a salt such as calcium chloride or sodium sulfate. A particularly preferred solution is 0.25M HCl / 2%
(W / v) CaCl ₂ and 0.05 M H ₂ SO ₄ /1% (w / v) N
a ₂ SO ₄ .

Alternatively, precipitation of the fibronectin / fibrinogen mixture around its minimum solubility (ie, pH 1.5-4.0) involves higher levels of 5-20% salts such as sodium citrate and sodium chloride. Weaker acids, such as between 5 and 20% citric acid and acetic acid, are more preferred. A particularly preferred example is 10
1M citric acid / 1M tri-sodium citrate with 5-10% acetic acid with 2% sodium chloride and 2-10% calcium chloride, pH varies with calcium chloride concentration 2.0- 0.8.

Other materials that can be used for the coagulation liquid include non-ionic polymers such as dextran and polyethylene glycol (eg, 25-50% PEG, pH 4.3-4.8), and organic materials such as ethanol or acetone. There are solvents, ionic polyelectrolytes such as carboxymethylcellulose, and suitable combinations of ions with salts such as sodium tri-citrate, sodium sulfate, or ammonium sulfate.

Once formed, the fibers or fiber bundles can be withdrawn from the coagulating liquid, preferably through a cleaning liquid, to remove contaminants such as acids or salts. Isotonic solution,
A buffered salt solution such as a phosphate buffered salt solution or water can be used as a washing solution. Although the strength and pH of the wash is not critical for fiber formation, it has been found that a preferred solution for fiber bundles having a diameter of up to 200 μm is 0.01 M phosphate buffered saline, pH 7.4. I have. For larger fiber bundles, 200 mM Tris-HCl, pH 7.6 was found to be suitable. It took about 15 minutes to neutralize the fiber in the case of a fiber having a length of 1 cm and a diameter of 5 mm, while the fiber bundle having a length of 10 cm and a diameter of 1.5 cm was safely washed after 30 minutes. Was done. Once washed, the fiber bundles may be further dewatered, if necessary, by immersion in a solvent or solvent / water bath. Suitable solvents include acetone or ethanol.

Once fiber formation has begun by appropriate “topping-up” or replacement of the protein solution and / or coagulating liquid, it is possible to perform essentially a continuous fiber manufacturing process. In this way, tens of centimeters, meters or even hundreds of meters of protein fiber can be produced per operation. For example, when spun from a 350 μm diameter needle, 1 cm of wet fiber, 70 cm, can be produced from 1 ml of a solution containing 100 mg of fibrinogen / fibronectin and 1% of alginate. Fibronectin / fibrinogen, alone or optionally containing 0.5% carboxymethylcellulose, yields 200-600 μm fibers, 120-180 cm. Moreover, such fibers can be further stretched to reduce the diameter.

The continuous fibers were prepared by passing a solution containing 140 mg / mL total protein and 1.3% sodium alginate through a capillary 18 mm in length and 1 mm in diameter with 0.25 M hydrochloric acid and 2% CaCl ₂ , pH It can be formed when spinning into a coagulation bath containing <1.0. Alginate-containing solutions exhibit shear thinning, ie, a decrease in apparent viscosity when shear is applied. Shear according to the fluid in the capillary is proportional to 1 / r ^3. Here, r is the radius of the spinneret or the capillary orifice. Thus, as the radius of the orifice decreases, the shear rate in the capillary increases significantly, resulting in a decrease in the apparent viscosity of the shear-lean solution. High viscosities are generally required for wet spinning solutions, so that a capillary with a large diameter, for example 1 mm, can be used for spinning the fiber bundle. The calculated shear rate in a 18 mm long, 1 mm diameter capillary is 1358 s ^-1 , and 140 mg / mL protein / 1
. The 3% sodium alginate solution had an apparent viscosity of 413 mPas at a shear rate of 1007 s ^-1 and 23 ° C. Thus, a solution having an apparent viscosity of this order can be extruded through the aforementioned capillary to form continuous fibers. Long capillaries increase the residence time of the solution in the spinneret in preparation for coagulation of the oriented protein fibers and can therefore be used to orient the protein molecules in the solution.

In addition, spinnerets with or without smaller diameter orifices (with or without capillaries) can also be used by increasing the protein concentration / viscosity of the solution. The fibers are usually dried in air or stabilized by placing the fibers under the influence of a suitable lyophilization zone (17). Severe drying, such as by lyophilization, is preferred because it is believed that severe drying of the fiber can increase cross-linking in the fiber and / or alter porosity.

[0032] The fibers may be further processed. For example, metal ions such as copper and / or zinc can be incorporated, generally in micromolar concentrations. This can be achieved by soaking the fibers in a solution of the metal salt. It has been observed that the mediation of such salts improves the stability of fibronectin in fibronectin-containing materials in in vitro experiments and alters the cell adhesion / translocation properties of the material.
Different types of cells appear to be resistant to different concentrations of metal ions in the material. Thus, incorporating different concentrations of metal ions in different regions of the fiber can lead to cell separation by elimination by cell type.

The fibers can be cut to a suitable length using a sharp blade, preferably when wet, and optionally a gel or sheet of alginate, hyaluronate to provide a composite material. Film, collagen gel / sponge, agar / agarose sheet, or gelatin sponge and various synthetic matrices such as polymer matrices. Typically, the fibers may be in contact with the substrate while wet and naturally adherent. The fibers may be laid over a flat substrate, for example, a collagen sponge,
Alternatively, it may be wound on a roller covered with a base. Gamma irradiation and / or chemical crosslinking agents can be used to increase the bond strength of the fibers to the substrate if necessary. Gamma irradiation can also provide a potential inactivation step for viruses. As an example, samples were exposed to irradiation for 48 hours to sterilize in vitro cell culture material, for a total exposure of up to about 3 MR.

[0034] Composite materials containing fibers supported on the undersurface can be used to promote wound healing and / or regular regeneration of damaged tissue. The substrate should be a physiologically compatible material and may be a biodegradable or resorbable material that remains in the wound and has virtually no detrimental effect on the patient's body. Preferably, the substrate is also non-toxic and / or non-antigenic. A typical substrate is 1 to
5 mm collagen sponge, with a pore size range of 30 to 1000 μM, porosity or void volume of 30 to 85%, to provide stability that is not cross-linked or can be used as a substrate. It may be chemically crosslinked.

In another aspect, the invention provides a fibrinogen / fibronectin-containing fiber for use in therapy. The invention further provides fibrinogen / fibronectin-containing fibers in the manufacture of a medicament, bandage, or device for use in therapy. This fiber generally contains fibrinogen / fibronectin 9
: 1 to 1: 9, preferably 7.5: 2.5 to 2.5: 7.5, more preferably 6
. It is contained at a ratio of 5: 3.5 to 3.5: 6.5. Particularly preferred fibers contain fibrinogen / fibronectin in a ratio of 3.5: 6.5. This fiber contains a number of substantially parallel fibrils of fibrinogen / fibronectin, and each fiber containing microfine parallel fibrils generally has a diameter of 10 μm to 10 μm.
00 μm, preferably 50 μm to 500 μm, more preferably 100 μm to 40
0 μm. While not wishing to be bound by theory, it is believed that fibronectin and fibrinogen form complexes to form bicomponent fibers. The fibers may be several tens of centimeters in length, and several meters or hundreds of meters in length, and the fibers may be provided so that they can be cut to the appropriate size before use.

[0036] The fibers are used to produce a mat containing any number of fibers, which may generally be arranged in parallel or interwoven intersecting. Besides,
This fiber can be used to form a three-dimensional matrix structure.

The fiber may be twisted to improve tensile strength, which may be important where the fiber is not attached to a substrate. Although not wishing to be bound by theory, twisting of the fibers, which leads to a significant increase in maximum tensile strength, results in the contact of fibronectin binding sites, which were previously separated from each other, and cross-linking within the fiber bundle. Will be increased. Research on electron micrographs of such fiber bundles
Untwisted fiber bundles have a less dense center, as opposed to twisted fiber bundles having a tighter center. In general, the fiber bundles are not twisted, the may be the composition and method of forming the fibers with a tensile strength in the range of 5~39N / mm ^2, whereas the fiber bundle twisted in the 40~85N / mm ² A range of tensile strength can be exhibited. Preferably, twisting of the fiber bundle is performed immediately after fiber formation and before the material loses cohesive properties, such as adhesion.

The fiber bundle and / or substrate coated with fibers can be used as or as part of a wound dressing, or can be applied to a wound that has been opened separately from a normal dressing and guided. Used for nerve regeneration, repair of induced tendons / ligaments, etc. To derive improved strength and / or cosmetic acceptability against purulent wounds, the fibers and / or the substrate coated with the fibers provide penetration in a manner that is arranged along the length of the fiber bundle. It is preferable to apply the fiber bundle to the wound in accordance with the characteristics of the surrounding tissue so as to promote it.

It is to be understood that the ratio of fibrinogen to fibronectin in the fiber can be controlled within the ranges defined herein for a particular application. For example, fibers containing a 1: 1 ratio of fibrinogen / fibronectin were tested compared to a higher percentage of fibronectin or fibrinogen.
It has been demonstrated to increase cell migration rates in vitro against cell types (rat Schwann cells, rat tendons, rat skin fibroblasts, and human dermal fibroblasts). Fibronectin has been found to be important for cell adhesion. While not wishing to settle in theory, an effective `` dilution '' of fibronectin's cell adhesion properties by fibrinogen causes the cells to adhere to the fibers, but is not so strong that they can migrate along protein fibers. It is considered impossible. Therefore, a 1: 1 ratio of fibrinogen / fibronectin appears to provide an excellent balance between migration and adhesion.

In addition, a fibronectin / fibrinogen film can be prepared by mixing a protein solution and a coagulating solution, casting or molding, and drying the mixture. The invention will now be further described, by way of example, with reference to the following non-limiting Examples section and the accompanying drawings.

Example 1 Preparation of a Fibrinogen / Fibronectin Precipitate A fibrinogen / fibronectin precipitate was prepared as described by Foster, P. et al. R
. Dickson, A .; J. , McQuillan, T .; A. , Dickso
n, I. H. Keddie, S .; And Watt, J .; G. FIG. Co-authored (1982)
"Control of large-scale plasma thawa
ng for recovery of cryopricipitate f
actor VIII (control of large-scale plasma thawing for recovery of cryoprecipitate factor VIII) ", Vox Sanguinis, 42 : 180-189.

Example 2 Selective Concentration of Fibronectin by PEG Precipitation Frozen cryoprecipitates were thawed at 4 ° C. overnight, chopped into small pieces, and 5 × at 37 ° C. (
w / v) phosphate buffer (0.01 M Na ₂ HPO ₄ , 0.01 M Na ₂ H
PO ₄ .H ₂ O, 0.015 M NaCl, pH 7.0). When dissolved, the solution contains 16-18 mg / mL total protein, 75-80% of which
Was fibrinogen, and 20 to 25% was fibronectin. The solution was cooled to room temperature and 50% (w / v) PEG 4000 was added to add 4% (v / v).
/ V) was obtained. The solution was stirred for a short time and left at room temperature for 1 hour to precipitate any fibrinogen present. The precipitate was collected at 4500 r. p. m. The solution was separated from the solution containing fibronectin by centrifugation at 15 minutes. The fibrinogen precipitate was discarded and the volume of the remaining supernatant was measured. The supernatant contained fibronectin and fibrinogen in a 2: 1 ratio.

The concentration of PEG 4000 in the solution was increased to 10% (v / v), which was stirred for a short time and left at room temperature for either overnight or 1 hour to precipitate fibronectin. The solution was then added to 4500 r. p. m. For 15 minutes to produce a pellet rich in fibronectin. The pellet contained both fibronectin and fibrinogen in a 2: 1 ratio. Pellets -20 until needed
Stored frozen at ℃. Further concentration of the solution requires 4% and 10% to redissolve the fibronectin pellet, remove more fibrinogen and reprecipitate fibronectin.
% Could be achieved by repeating both PEG cuts.

Example 3 Extrusion of Fibrinogen / Fibronectin Fibers Proteins containing a 2: 1 ratio of fibronectin and fibrinogen as well as polyethylene glycol from the aforementioned precipitation step and small proportions of other plasma proteins such as albumin (not quantified) The precipitate was dissolved in 6 M urea to give a final total protein concentration of> 70 mg / ml. Sodium alginate was added to a concentration of 1% to increase the viscosity and reduce the self-adhesion of the final fiber. The resulting viscous solution was then degassed and extruded through a 350 μm diameter orifice into a coagulation bath where white fibers formed. Next, the fibers are coagulated in a coagulation bath (
Room temperature, pH <1.0, 0.25 M HCl, 2% (w / v) calcium chloride)
And washed with 0.01 M phosphate buffered saline, pH 7.4, before drying at room temperature.

Example 4: Determination of optimal coagulation conditions The PEG precipitate described by Example 3 is dissolved in phosphate buffer to a concentration of 5 mg / ml without adding sodium alginate, and the protein solution is dissolved. It was used for the experiment. Figures 1 to 5 show the percentage of protein remaining in solution after changing either the pH or the ionic strength of the solution with calcium chloride. Protein in solution was measured by the Bradford dye binding assay (Bio-Rad, Munich, Germany). The dashed lines in FIGS. 1-3 indicate that protein solubility decreases as pH decreases below 1.5 or to 1.0. FIGS. 1 and 2 show that the minimum solubility of the fibronectin / fibrinogen mixture is around pH 3.0 and pH 4.0, respectively, and FIG. 3 shows that the minimum solubility of fibrinogen is around pH 4.0.

FIGS. 4 and 5 show that increasing calcium chloride concentration decreases fibronectin / fibrinogen solubility in solution. FIGS. 6, 7, and 8 show that increasing the concentration of salt, ammonium sulfate, ethanol, and PEG decreases fibronectin / fibrinogen solubility in solution. FIG. 9 shows the effect of changing both the pH and the ionic strength of the fibronectin / fibrinogen solution. After determining the effect of pH and salt on fibronectin / fibrinogen solubility, various coagulation bath compositions were tested for fiber formation. A summary of these results is provided in Table 1.

[0047]

[Table 1]

From these results, usually fibronectin / fibrinogen is HCl / C
low pH using a combination of NaCl ₂ and _{H 2 SO 4 / Na 2 SO} 4, i.e. <PH1.5～1.0, and weaker acid, i.e. a combination of citric acid / citrate and acetate / NaCl It can be seen that it will precipitate around the pH of 3.0-4.5 used.

Example 5: Testing the Tensile Strength of Fibronectin / Fibrinogen Fibers 5 mg / ml total protein composed of 3.4 mg / ml fibronectin / 1.3 mg / ml fibrinogen and 0.5 mg / ml albumin A fibronectin / fibrinogen solution having the following formula was prepared according to Example 1.
The protein was precipitated at room temperature by adding 0.1 M citric acid, pH 3.0-3.5, at a protein to acid ratio of 2: 1 (v / v) to the protein solution. The pH of the resulting solution was 4-4.5. The solution was gently agitated to aggregate the protein precipitate, and the protein strand was slowly pulled out of the precipitate using a fine-tipped glass rod.

Stretched and optionally twisted, about 2 cm in length and 100-800 μm in diameter
Is attached to a metal frame with polystyrene rungs by a cyanoacrylate adhesive (RS Components Ltd., Corby, U.S.A.).
K). To prevent contamination of the sample by the adhesive, the strands were glued to the frame in a horizontal position and the adhesive was dried prior to testing so that the adhesive could not cut the coupon. Next, the length of the sample between the two attached points was measured and used as the standard length of the sample. The sample is then operated at a strain rate of 2 mm / min.
estometric 220M tensile tester (The Tensile T
esting Co. Ltd. , Rosdale, UK). Samples tested were splinted to test for variations in strength within the strand and then retested to ensure that the break was away from the glued area. Specimen dimensions were determined using a Nikon Profile Projector. The bulk protein material was made by compressing and dewatering the protein precipitate before cutting into 2 mm wide strips. The wet sample was tested in a sealed moist environment by surrounding the test stand with a plastic cylinder containing wet paper. Relative humidity is measured using a portable probe hygrometer (RS Co.)
components and the moisture content of the material was calculated using the dry weight method.

Applying a constant strain rate of 2 mm / min and a constant relative humidity (> 60%) during a tensile test on a wet specimen (water content> 40%) results in extrusion of the material. I found that. The strand length was found to increase by a factor of 7 with the resulting diameter reduction. Thus, once the strand has been formed, it can be stretched to the required diameter once the relative humidity is at least 60%. Table 2 shows the results of the tensile strength test of the dried test pieces. The tensile strength of the twisted strand was calculated to be 2.5-3.5 g / denier in the dry state.

Twisting the fibronectin material leads to a significant increase in the maximum tensile strength of the strand, possibly by contacting the binding sites of the fibronectin which have been far apart from each other and thus increasing the cross-linking inside the fiber bundle. Electron micrograph studies have shown that twisted strands exhibit a tight center, as opposed to just strands having a tight center, which increases the tensile strength by 15 to 15%.
It is related to the increase to 61 N / mm ² . Twisting must take place immediately after the strand is formed, before the material loses its adhesion. Defects inside the strand were observed when twisting was performed after a loss of self-adhesive capability which would result in voids in the material and lower tensile strength. With defects, the twisted material without defects appears vitreous, as opposed to the appearance of bubbles even under an optical microscope at x10 magnification.

The fracture site of the twisted strand was peculiar to a brittle material, that is, a clean surface. Twisted fibronectin / fibronogen strands
It has a lower average tensile strength than collagen fibers, 61 N / mm ² . This should be considered because collagen is found in load-bearing tissues such as tendons and ligaments. The strand orientation provided by the micron diameter fibrils provides greater tensile strength compared to the bulk material of 22.5 N / mm ² .
Bulk materials do not have an overall orientation, and thus may have reduced intermolecular bonding.

[0054]

[Table 2]

Example 6: Fibers and Fibers Wet Spun Using a Test Apparatus A suitable spinning dope is prepared by dissolving 281 g of freshly thawed protein / polyethylene glycol precipitate in 100 mL of 6M urea. did. Also, sodium alginate (4.0 g) was added to give a solution with a final volume of about 310 mL and a final urea concentration of 2M. Final protein concentration is 1 total protein
40 mg / mL and consists of 50% fibronectin and 50% fibronogen combined with 1.3% (w / v) sodium alginate. During dissolution, the temperature was maintained at 37 ° C. using a water bath and agitation was provided by a single-blade mixer stirring at 500-700 times / min. Dissolution occurred over 4-5 hours and the dope was left overnight at room temperature to degas.

Next, fibers were produced using a pilot apparatus schematically shown in FIG. The prepared solution is put into a stainless steel storage tank (1), and a nitrogen pressure [103.4 to 275.8 kPa (15 to 40) is passed through a 1 cm diameter polythene tube (3).
p. s. i. )] To the flow control valve (5). When the polythene tube (3) is filled, the valve (5) opens and the dope flows toward the 1 mL geared flow control pump (7) per revolution (Slack & Parr, Kwgworth, UK). The dope is pumped to the spinneret (9) at a rate of 10-20 mL / min. The spinneret (9) has an orifice diameter of 1 mm and a conduit length of 18 mm.
Through this, the dope was extruded into a coagulation bath (dimensions 10 × 180 cm) containing 0.25 M HCl and 2% CaCl ₂ , pH <1.0.

A drawn roller made of steel which initially draws the formed fibers (13) by hand in the longitudinal direction of the coagulation bath, picks the fibers from the coagulation bath and transfers them to the washing bath (17) for neutralization of the acid (15) (diameter 10 cm, rotation speed 21 times / minute). A second set of windings are taken to take some of the fiber bundles from the washing bath (17) to a dehydration bath (21) containing 100% acetone and to draw down the fiber bundles to a smaller diameter. A roller (19) (diameter 10 cm, rotation speed 31 times / min) was used.

The tensile strength of the fiber bundle wet-spun was measured with a pilot-scale apparatus, and the results were shown in Table 3.
It was shown to. The average diameter of fibers made of 14% protein / 1.3% alginate was 540 ± 63 and 563 ± 33 μm with and without acetone treatment, respectively.
Comparative results for fibers made of 4.6% protein / 4.8% alginate were 415 ± 36 and 635 ± 17 μm. The results showed a confidence limit of ± 95%.

When the surface of a fibronectin / fibronogen cable extracted from a 5 mg / mL solution was examined using a scanning electron microscope, the surface induced contact to align cells with the surface of the stretched cable. It was found to consist of parallel microfine fibrils believed to provide cues. Examination of the surface of the spun fibers revealed folds approximately 10 μm wide, aligned along the long axis of the fiber bundle made of 4.6% protein / 4.8% alginate. 14% protein /
Fiber bundles made of 1.3% alginate exhibited near micron width folds when tested at high magnification.

[0060]

[Table 3]

The tensile strength was measured as the maximum load per unit cross-sectional area required to break a specimen. Set is the permanent increase in sample length after breakage. The elongation at break is
The percentage increase in sample length at break, expressed as a percentage of the original length. The number of samples tested for each group is shown in parentheses, and all results were ± 95% confidence limits. Reference Materials (1) Yannas, I. et al. V. Burke, J .; F. Gordon, C .; ,
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[Brief description of the drawings]

FIG. 1 is a diagram showing a solubility curve for a solution containing fibronectin / fibrinogen in a ratio of 6.5: 3.5.

FIG. 2 is a diagram showing a solubility curve for a solution containing fibronectin / fibrinogen at a ratio of 2.5: 7.5.

FIG. 3 shows a solubility curve for a substantially pure fibrinogen solution.

FIG. 4 shows a solubility curve for a solution containing fibronectin / fibrinogen in a ratio of 3.5: 6.5, such that the pH of the solution decreases from 7.8 to 4.1 as the calcium chloride concentration increases. It is.

FIG. 5 shows a solubility curve for a solution containing fibronectin / fibrinogen in a ratio of 7.5: 2.5, such that the pH of the solution decreases from 7.3 to 4.6 as the calcium chloride concentration increases. It is.

FIG. 6 shows the solubility curve of a protein solution of 5 mg / ml consisting of 67% fibronectin and 33% fibrinogen, as affected by the addition of various salts.

FIG. 7: Fibronectin / fibrinogen (5
(mg / ml: 67% / 33%).

FIG. 8 is a graph showing the solubility of fibronectin / fibrinogen (5 mg / ml: 67% / 33%) with addition of ethanol or polyethylene glycol 4000.

FIG. 9 shows how the solubility of fibronectin / fibrinogen (5 mg / ml: 67% / 33%) changes when both the pH and the ionic strength of the solution change.

FIG. 10 schematically illustrates a pilot-scale device used to produce the fibers of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 17/02 A61K 37/02 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM ), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, G E, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN , MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZWF term (for reference) 4C076 DD22 DD23 DD24 DD41 DD43 DD54 EE23 EE32 EE36 EE41 GG01 4C081 AA02 AA12 AB19 AC02 BB04 BC02 CD171 4C084 AA02 AA03 BA44 CA36 DA40 DC11 MA63 NA20 ZA892 4L035 AA04 BB03 BB03

Claims (33)

[Claims] 1. adding fibronectin and / or fibrinogen to a solvent to form a solution of fibronectin and / or fibrinogen; extruding said solution through an orifice into a coagulation liquid to form protein fibers. A method for producing a protein fiber containing fibronectin and / or fibrinogen, comprising: 2. The method according to claim 1, wherein the protein fiber contains fibronectin and fibrinogen. 3. The method according to claim 2, wherein fibronectin and fibrinogen are added as solids and the ratio of fibronectin to fibrinogen is in the range of 9: 1 to 1: 9. 4. The method according to claim 1, wherein the ratio of fibronectin to fibrinogen is 7.5: 2.
4. The method of claim 3, wherein the method is in the range of 5-2.5: 7.5. 5. The method according to claim 5, wherein the ratio of fibronectin to fibrinogen is 6.5: 3.
4. The method of claim 3, wherein the number is 5. 6. The method according to claim 3, wherein the ratio of fibronectin to fibrinogen is 1: 1. 7. The method according to claim 1, wherein the fibronectin and / or fibrinogen is obtained from a cryoprecipitate of plasma. 8. The method according to claim 1, wherein the fibronectin and / or fibrinogen is dissolved in the solvent to a concentration of more than 50 mg / ml of the protein. 9. The method according to claim 8, wherein the fibronectin and / or fibronogen is dissolved in the solvent to a protein concentration of more than 120 mg / ml. 10. The preceding claim, wherein the solvent contains a denaturing agent that does not substantially irreversibly denature fibronectin and / or fibrinogen, but acts to denature any other proteins that may be present. A method according to any of the preceding clauses. 11. The method according to claim 10, wherein the denaturing agent is 2-8M urea. 12. The method of claim 1, further comprising adding an additive to the fibronectin and / or fibrinogen solution, wherein said additive increases solution viscosity and / or reduces fiber self-adhesion. The method according to any of the above. 13. The method according to claim 12, wherein the additives are selected from sodium salts of carboxymethylcellulose, high molecular weight polyethylene glycols and / or sodium alginate. 14. A cross section of an orifice for extruding a solution has a diameter of 10 μm to 15 mm.
A method according to any of the preceding claims, wherein the method is circular. 15. The method according to any of the preceding claims, wherein the fibers are extruded at a rate between 300 μl / min of solution and 60 ml / min of solution. 16. The method of claim 1 for producing substantially pure fibrinogen or fibronectin fibers, wherein the coagulation liquid has a pH <1.0-1.5, or between pH 4-6. 17. The method according to any of the preceding claims for producing fibronectin / fibrinogen fibers, wherein the coagulation liquid is at pH <1.0-1.5, or between pH 3-4. 18. The method according to any of the preceding claims, wherein the coagulating liquid contains an acid and / or a salt. 19. The method according to claim 19, wherein the acid is selected from hydrochloric acid, sulfuric acid, citric acid, and acetic acid.
The method according to claim 18. 20. The method according to claim 18, wherein the salt is selected from calcium chloride, trisodium citrate, sodium sulfate and / or ammonium sulfate. 21. The coagulating liquid is 0.25 M HCl / 2% (w / v) CaCl ₂ , 0.05 M H ₂ SO ₄ /1% (w / v) Na ₂ SO ₄ , 10% acetic acid / 10% NaCl, and 1M are selected from citric acid / 1M trisodium citrate / 2-10% of CaCl _2, the method according to claim 18. 22. A method according to any of the preceding claims for producing fibers having a length of more than 10 cm. 23. Fibers containing therapeutic fibrinogen / fibronectin. 24. The fiber according to claim 23, comprising fibrinogen / fibronectin in a ratio of 9: 1 to 1: 9. 25. The fiber of claim 24, comprising fibrinogen / fibronectin in a ratio of 7.5: 2.5 to 2.5: 7.5. 26. The fiber of any one of claims 23 to 25, comprising a plurality of substantially parallel fibrinogen / fibronectin fibrils. 27. The fiber according to claim 26, wherein the fiber has a diameter of 10 μm to 15 mm. 28. The fiber according to any one of claims 23 to 26, wherein the fiber has a length of more than 10 cm. 29. The fiber according to any one of claims 23 to 28, which is twisted. 30. The fiber according to any one of claims 23 to 29 for therapeutic use. 31. A composite material or fiber containing a fiber prepared according to any of the preceding claims in addition to a supporting substrate. 32. A method for healing wounds and / or regenerating damaged tissue.
2. The composite material according to 1. 33. A mat containing any number of fibers according to any one of claims 22 to 29, wherein the fibers are arranged in a generally parallel or interwoven manner.