JP6454125B2 - Method for producing collagen gel - Google Patents

Description

  The present invention relates to a method for producing a collagen gel and a collagen gel.

  Conventionally, as a technique for orienting collagen fibers, techniques using shearing, liquid crystallizing, a magnetic field, or an electric field are known (see, for example, Patent Document 1 and Non-Patent Documents 1 to 4). Among these techniques, shearing is the only method that can provide sufficient orientation for the living body to recognize the orientation structure and can be scaled up and made efficient for mass production as a medical device.

  As conventional collagen fiber orientation techniques using shearing, for example, techniques described in Patent Documents 2 to 3 and Non-Patent Documents 5 to 7 are known. Patent Document 2 discloses a technique in which a high pH collagen solution having a collagen concentration of 0.2% is extruded from a nozzle onto a substrate and the substrate is moved in a direction opposite to the moving direction of the nozzle. Patent Document 3 discloses a technique in which a “nano loom” is used to extrude a collagen solution from a hole having an inner diameter of only several tens of μm, and the periphery of the hole is heated, thereby causing fibrosis and orientation.

  Non-Patent Document 5 discloses a technique in which a collagen solution having a collagen concentration of 0.08% or less is introduced into a microchannel, and is fibrillated in the middle to attach oriented collagen fibers on a substrate. Non-Patent Document 6 discloses a wet spinning technique in which a collagen solution having a collagen concentration of 0.6% is extruded from a nozzle into a coagulation liquid to form a fiber. The coagulation liquid promotes collagen fibrosis. In order to do so, it is described that polyethylene glycol is added. In Non-Patent Document 7, a collagen solution having a collagen concentration of 0.3% or less is introduced into a channel having a micro-sized (100 to 750 μm) diameter provided between glass plates, and is made to fibrosis in the middle. A technique for attaching oriented collagen fibers on a substrate is disclosed.

International Publication No. 2009/064437 Special table 2012-519537 gazette International Publication No. 2007/038601

Giraud-Guille et al., Liquid crystalline assemblies of collagen in bone and in vitro systems.J. Biomechanics 36, 1571-1579 (2003) Guo and Kaufman, Flow and magnetic field induced collagen alignment, Biomaterials 28, 1105-1114 (2007) Torbet and Ronziere, Magnetic alignment of collagen during self-assembly, Biochem. J. 219, 1057-1059 (1984) Cheng et al., An electrochemical fabrication process for the assembly of anisotropically oriented collagen bundles, Biomaterials 29, 3278-3288 (2008) Lanfer et al., Aligned fibrillar collagen matrices obtained by shear flow deposition, Biomatrials 29, 3888-3895 (2008) Zeugolis et al., Engineering extruded collagen fibers for biomedical applications, J. Appl. Polym. Sci. 108, 2886-2894 (2008) Saeidi et al., Dynamic shear-influenced collagen self-assembly, Biomaterials 30, 6581-6592 (2009)

However, the conventional collagen fiber orientation techniques described in Patent Documents 2 to 3 and Non-Patent Documents 5 to 7 are similar to artificial tendons for the reasons listed in (1) to (3) below. It is not sufficient as a method for producing such an implant.
(1) In order to produce an implant, it is necessary to make oriented collagen fiber bodies into “fiber bundles” that are three-dimensionally shaped in a size of mm to cm. However, the conventional technique for producing oriented fibers attached to a substrate or the like, as described in Patent Document 3 and Non-Patent Documents 5, 7, and 8, provides an extremely thin planar oriented collagen fiber sheet. Only. Therefore, in order to manufacture a three-dimensionally shaped “fiber bundle” with a size of mm to cm scale, a large number of planar oriented collagen fiber sheets must be laminated and combined with their orientation axes aligned. There is a problem that an implant cannot be formed.
(2) Although the wet spinning technique described in Non-Patent Document 6 has high mass productivity, the degree of orientation of collagen fibers is insufficient.
(3) Although the technology of “nano loom” as described in Patent Document 3 is also high in mass productivity, the resulting collagen gel is extremely thin and mechanically fragile, so they are combined to produce a fiber bundle Is difficult.

  The present invention has been made in view of the above circumstances, and provides a method for producing a collagen gel that can non-destructively produce a “fiber bundle” that is three-dimensionally shaped on a large scale, and a collagen gel thereof. For the purpose.

  As a result of intensive studies to achieve the above object, the present inventors have found that the rate of increase in shear stress accompanying collagen fibrillation is in a specific range in the step of applying shear to a collagen aqueous solution having a specific concentration. At that time, it was found that by setting the shear rate within a specific range, an oriented collagen fiber bundle three-dimensionally shaped on a large scale can be obtained non-destructively, and the present invention has been completed.

That is, the present invention is as follows.
[1] A step of orienting collagen fibers by applying a shear rate in the range of 0.20 s ^{−1 to} 30 s ^{−1 to} an aqueous collagen solution containing collagen at a concentration of 0.50 mass% to 3.0 mass%. A method for producing a collagen gel comprising the steps of 2 to 120 seconds wherein the shear stress is increased at a rate of 1% to 30% per second due to the formation of collagen fibers. A method for producing a collagen gel, wherein collagen fibers are oriented.
[2] The method for producing the collagen gel described above, wherein the collagen fibers are formed by heating the collagen aqueous solution from a temperature of 25 ° C. or lower to 32 to 42 ° C.
[3] The method for producing a collagen gel as described above, wherein the collagen aqueous solution contains an inorganic salt, and the ionic strength of the inorganic salt is 0.40 to 1.0.
[4] The method for producing a collagen gel as described above, wherein the inorganic salt contains sodium hydrogen phosphate and sodium chloride.
[5] The method for producing a collagen gel as described above, wherein the collagen aqueous solution contains a crosslinking agent.
[6] The method for producing a collagen gel as described above, wherein the aqueous collagen solution has a pH of 6.0 to 9.0.
[7] The method for producing a collagen gel as described above, wherein the collagen contains mammal-derived telopeptide-removed collagen.

  According to the present invention, it is possible to provide a method for producing a collagen gel that can non-destructively produce a “fiber bundle” that is three-dimensionally shaped on a large scale, and the collagen gel.

Schematic illustration of the appearance of the disc-shaped gel obtained between the sensor part and the sensor part of the dynamic viscoelasticity measuring device used in the examples and comparative examples, and the positions of various test pieces cut out from the disc-shaped gel and the shapes of the test pieces FIG. It is a figure which shows typically the heating profile and shear condition of collagen aqueous solution in an Example. It is a figure which shows typically the method of calculating a shear stress increase rate using Formula (1) from the time change curve of a shear stress. In an Example, it is a chart which shows the shear stress change of the collagen aqueous solution (collagen gel) with progress of the time which gave the rotational shear to the collagen aqueous solution after reaching 37 degreeC. In an Example and a comparative example, it is the graph which plotted the birefringent phase difference of collagen gel with respect to the period (time after reaching | attaining 37 degreeC) which provided the rotation shear to the collagen aqueous solution. In Examples and Comparative Examples, when the period during which rotational shear was applied to the collagen aqueous solution (time after reaching 37 ° C.) was 20 seconds, the birefringence phase difference of the collagen gel was plotted against the shear rate. It is a graph. It is a scanning electron microscope image inside a gel in an Example and random collagen fiber gel (gel produced without giving rotation shear). It is the figure which showed the typical stress-strain curve obtained from a tension test about an Example and a random collagen fiber gel (gel produced without giving rotational shear). It is the figure which compared the various physical-property values obtained from a tension test in an Example and a random collagen fiber gel (gel produced without providing rotational shear). It is a figure which shows the result of having tracked the gelatinization of the aqueous solution when raising the temperature of collagen / genipin aqueous solution from 23 degreeC to 37 degreeC by the micro vibration test using a dynamic viscoelasticity measuring apparatus.

  Hereinafter, a mode for carrying out the present invention (hereinafter simply referred to as “the present embodiment”) will be described in detail with reference to the drawings as necessary. However, the present invention is limited to the following embodiment. It is not a thing. The present invention can be variously modified without departing from the gist thereof.

In the method for producing a collagen gel according to this embodiment, a shear rate in the range of 0.20 s ^{−1 to} 30 s ^{−1 is} applied to an aqueous collagen solution containing collagen at a concentration of 0.50 mass% to 3.0 mass%. A method for producing a collagen gel comprising a step of orienting collagen fibers, wherein the step increases shear stress at a rate of 1% to 30% per second due to the formation of collagen fibers for 2 seconds to 120 It includes a process of seconds. The method for producing a collagen gel of this embodiment may include a step of preparing an aqueous collagen solution.

  The collagen aqueous solution contains collagen at a concentration of 0.50 mass% to 3.0 mass%. Collagen is water-soluble, but preferably contains telopeptide-removed collagen that hardly progresses to fibrosis around room temperature, and more preferably substantially comprises telopeptide-removed collagen. Telopeptide-removed collagen is a telopeptide that collagen molecules have at both ends enzymatically decomposed and removed by proteolytic enzymes. For example, telopeptide that collagen molecules have at both ends is decomposed and removed by pepsin digestion. It is a thing. Among telopeptide-removable collagens, mammalian-derived telopeptide-removable collagen that is approved as a raw material for medical devices is preferable, and telopeptide-removable collagen derived from pig skin that has already been clinically applied and has excellent thermal stability. More preferably used.

  Collagen is not particularly limited as long as it has collagen-forming ability (fibrogenic collagen). Among fibrogenic collagens, type I which is collagen constituting bone, skin, tendon and ligament, type II which is collagen constituting cartilage, type III contained in living tissue composed of type I collagen, etc. It is preferably used from the viewpoints of availability, abundant research results, and similarity to the biological tissue to which the produced gel is applied. Collagen may be obtained by extraction and purification from a living tissue by a conventional method, or a commercially available product may be obtained. Collagen may be purified from each type or a mixture of multiple types.

  The denaturation temperature of collagen is preferably 32 ° C. or higher, more preferably 35 ° C. or higher, and further preferably 37 ° C. or higher. When the denaturation temperature is 32 ° C. or higher, the fluidity of the collagen aqueous solution at room temperature can be maintained for a longer time, and collagen denaturation is less likely to occur in vivo. The upper limit of the denaturation temperature of collagen is not particularly limited, but is preferably 50 ° C. or lower, more preferably 45 ° C. or lower, and further preferably 41 ° C. When the denaturation temperature is not more than the above upper limit value, collagen fibrillation due to temperature rise is accelerated, collagen fibers are more easily oriented due to shearing, and the bioabsorbability of collagen is better maintained. The denaturation temperature of collagen is measured by a conventional method, that is, a change in circular dichroism, optical rotation, or viscosity with an increase in temperature of the collagen aqueous solution. The denaturation temperature of collagen may be adjusted by selecting collagen having a denaturation temperature within the above numerical range.

  In the collagen aqueous solution of the present embodiment, the collagen concentration is preferably 0.50% by mass to 3.0% by mass, and 0.75% by mass to 2.0% by mass based on the total amount of the collagen aqueous solution. And more preferred. When the collagen concentration is 0.50% by mass or more, gel breakage is suppressed in the collagen gel production process, the mechanical strength of the resulting gel can be further increased, and the degree of orientation of the collagen fibers is also improved. Can be increased. On the other hand, when the collagen concentration is 3.0% by mass or less, the fluidity of the collagen aqueous solution at room temperature can be improved, and shearing can be more easily performed.

  The aqueous collagen solution of the present embodiment preferably contains an inorganic salt from the viewpoint of obtaining ionic strength and pH in the preferred ranges described below. The inorganic salt is not particularly limited. For example, sodium chloride, potassium chloride, sodium phosphate, sodium hydrogen phosphate (generic name for sodium dihydrogen phosphate and disodium hydrogen phosphate), and potassium hydrogen phosphate (phosphoric acid) For example, potassium dihydrogen and dipotassium hydrogen phosphate). An inorganic salt is used individually by 1 type or in combination of 2 or more types. Of these inorganic salts, the pH of the aqueous collagen solution can be easily adjusted to the preferred range described below, and the aqueous collagen solution contains sodium hydrogen phosphate and sodium chloride (salt) from the viewpoint of being harmless to the living body. preferable.

  In addition, the aqueous collagen solution may contain a neutral isotonic solution as a solvent in order to exert two effects of minimizing damage to cells and living tissue and causing collagen fibrosis. The neutral isotonic solution may be phosphate buffered saline (PBS) that is used in cell washing operations and can actively fibrillate collagen.

  The ionic strength of the inorganic salt contained in the collagen aqueous solution is preferably 0.40 to 1.0, and more preferably 0.60 to 0.80. In addition, the ionic strength of the inorganic salt in the present embodiment refers to the ionic strength of the plurality of inorganic salts as a whole when the collagen aqueous solution includes a plurality of inorganic salts. The responsiveness to the temperature of collagen fibrillation increases as the ionic strength increases, but the responsiveness is further improved when the ionic strength of the inorganic salt is 0.40 or more. In addition, when the ionic strength of the inorganic salt is 1.0 or less, fibrosis at a low temperature is suppressed, and the stability of the aqueous collagen solution at room temperature increases (that is, the state of the solution without fibrosis). Can be held for a longer time.) In this specification, the ionic strength of the inorganic salt is obtained by adding the product of the molar concentration of each ion and the square of the charge for all ionic species derived from the inorganic salt contained in the collagen aqueous solution. It is calculated by multiplying by 2.

  The pH of the aqueous collagen solution of the present embodiment (pH at 23 ° C., the same in the present specification) is preferably 6.0 to 9.0, and more preferably 6.5 to 8.0. It is known that collagen fibrosis occurs actively near neutrality. When the pH is 6.0 or more, the fibrosis of collagen can be further promoted. Moreover, when the pH is 9.0 or lower, collagen fibrosis can be further promoted. The pH can be adjusted by a conventional method. For example, the concentration of inorganic salt contained in the aqueous collagen solution, preferably the concentration of sodium chloride and sodium hydrogen phosphate, or a strong acid such as hydrochloric acid or sodium hydroxide, and It is possible to adjust the pH by adding a strong alkali. In the present specification, pH is measured with a pH meter (for example, trade name “NAVIh F-71” manufactured by HORIBA).

  The concentration of sodium chloride when the aqueous collagen solution contains sodium chloride (salt) is not particularly limited as long as the pH and ionic strength of the aqueous collagen solution are in a desired range. For example, the concentration of sodium chloride is preferably 200 mM to 400 mM and more preferably 250 mM to 350 mM with respect to the total amount of the collagen aqueous solution. By setting the concentration of sodium chloride within such a range, the ionic strength of the inorganic salt is set within the range of 0.40 to 1.0 while the pH of the collagen aqueous solution is within the range of 6.0 to 9.0. It becomes easier.

  Further, the concentration of sodium hydrogen phosphate in the case where the collagen aqueous solution contains sodium hydrogen phosphate is not particularly limited as long as the pH and ionic strength of the collagen aqueous solution are within a desired range. For example, the concentration of sodium hydrogen phosphate is preferably 70 mM to 180 mM and more preferably 90 mM to 140 mM with respect to the total amount of the collagen aqueous solution. By setting the concentration of sodium hydrogen phosphate in such a range, the pH of the collagen aqueous solution is in the range of 6.0 to 9.0, and the ionic strength of the inorganic salt is in the range of 0.40 to 1.0. It will be easier.

Formation of collagen fibers from an aqueous collagen solution gradually takes several hours or more and increases the strength of the gel. Therefore, if the recovery of the collagen gel is accelerated, the gel tends to be broken. Therefore, from the viewpoint of increasing the strength of the collagen gel early and recovering the collagen gel in a shorter time, the aqueous collagen solution preferably contains a crosslinking agent. The crosslinking agent is not particularly limited, and one kind can be used alone or two or more kinds can be used in combination. However, the plant-derived genipin, which is considered to have low cytotoxicity of the crosslinking agent itself, or a crosslinking agent between collagen molecules. 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (hereinafter referred to as “EDC”) which is removed by washing with water and N-hydroxysuccinimide (NHS) which is a crosslinking aid thereof Preferably used. Genipin is an aglycone of geniposide, and can be obtained, for example, by oxidation, reduction and hydrolysis of geniposide or by enzymatic hydrolysis of geniposide. Geniposide is an iridoid glycoside contained in Rubiaceae gardenia and is obtained by extraction from gardenia. Genipin is represented by a molecular formula of C ₁₁ H ₁₄ O ₅ , and may be synthesized by a conventional method or a commercially available product may be obtained. Further, genipin may be derivatized to the extent that the crosslinking effect is ensured to such an extent that the achievement of the object of the present invention is not hindered. EDC is a kind of water-soluble carbodiimide, and any water-soluble carbodiimide can be used as a cross-linking agent regardless of its type. Among them, EDC is particularly preferably used because it is inexpensive and highly safe. A water-soluble carbodiimide is used individually by 1 type or in combination of 2 or more types. EDC may be used alone or in combination with NHS. It is known that the crosslinking activity of EDC is improved by mixing NHS.

  When the collagen aqueous solution of the present embodiment contains a crosslinking agent and the crosslinking agent is genipin, the concentration of genipin is preferably 0.5 mM to 5.0 mM, based on the total amount of the collagen aqueous solution, It is more preferable that it is 4.0 mM, and it is still more preferable that it is 2.0 mM-3.0 mM. When the concentration of genipin is 0.5 mM or more, the strength of the collagen gel can be increased earlier, and it is difficult for the collagen gel to be destroyed during recovery, thereby improving the yield. On the other hand, when the concentration of genipin is 5.0 mM or less, the fluidity of the collagen aqueous solution at room temperature can be better maintained, and the cytotoxic effect of genipin can be further suppressed.

  When the collagen aqueous solution of this embodiment contains a crosslinking agent and the crosslinking agent is EDC, the concentration of EDC is preferably 1.0 mM to 20 mM, based on the total amount of the collagen aqueous solution, and is 2.0 mM to 10 mM. More preferably, it is 3.0 mM to 8.0 mM. The effect of having an EDC concentration in the range of 1.0 mM to 20 mM is the same as that of genipin. It is preferable to mix NHS with EDC since the crosslinking activity is increased. The molar ratio of NHS to EDC (EDC: NHS) is preferably in the range of 10: 1 to 1: 1. By being in this range, the crosslinking activity of EDC is enhanced, and the influence of cytotoxicity due to the remaining NHS can be further suppressed.

  In addition, the collagen aqueous solution of the present embodiment may further contain various solvents and additives used in conventional collagen aqueous solutions. Examples of such solvents and additives include acids such as dilute hydrochloric acid, citric acid, and acetic acid, and buffers such as HEPES and Tris.

  The said additive and solvent are used individually by 1 type or in combination of 2 or more types. Moreover, the content rate of the said additive and solvent in collagen aqueous solution will not be specifically limited if it is a range which does not inhibit achievement of the objective of this invention.

The method for producing a collagen gel according to the present embodiment includes a step of increasing the shear stress generated when shearing the aqueous collagen solution by forming collagen fibers (hereinafter also referred to as “fiber formation step”). In at least a part of the fibril formation process, shearing in a range of a shear rate of 0.20 s ^{−1 to} 30 s ⁻¹ is given to the collagen aqueous solution. And while giving the shear of such a shear rate, it has the process for which the speed | rate (ratio) which increases the shear stress of collagen aqueous solution becomes the range of 1-30% per second for 2 second-120 second. Here, the increase rate of shear stress per second (hereinafter referred to as “shear stress increase rate”) is calculated by the following equation (1).
Shear stress increase rate (%) = (τ1-τ0) / τ0 / T × 100 (1)
In Equation (1), τ0 represents the shear stress at the start point of the period for calculating the shear stress increase rate, τ1 represents the shear stress at the end point of the period for calculating the shear stress increase rate, and “T” is Indicates the length (unit: second) of the period for calculating the shear stress increase rate, and is calculated in an arbitrary range of 5.0 ≦ T ≦ 120. Even if the increase in shear stress increases momentarily only at a certain time, it is not sufficient for the orientation of collagen, so the rate of increase in shear stress is calculated over a period of 5.0 seconds or more. On the other hand, since shear is applied in a period of 120 seconds or less, the period for calculating the shear stress increase rate is also 120 seconds or less.

  In the fiber formation step according to this embodiment, the formation of collagen fibers is performed, for example, by heating a collagen aqueous solution. When the pH of an aqueous collagen solution containing an inorganic salt is 6.0 to 9.0 and the ionic strength is 0.40 to 1.0, the collagen molecules are self-organized by hydrophobic and electrostatic interactions by heating. To form fibers. When the collagen aqueous solution is heated, it is preferable to form collagen fibers by heating the aqueous solution from a temperature of 25 ° C. or lower to 32 to 42 ° C. By heating the collagen aqueous solution from a temperature of 25 ° C. or lower, it is possible to further suppress the formation of collagen fibers before starting the heating. On the other hand, by heating the collagen aqueous solution to a temperature of 32 ° C. or higher, the formation rate of collagen fibers can be further increased, and by heating to a temperature of 42 ° C. or lower, the thermal denaturation of collagen is more effective and reliable. Can be prevented. When collagen fibers are formed by heating, the increase in shear stress may start later than the increase in temperature, and after reaching the target temperature for heating (for example, 32 to 42 ° C.), the temperature is maintained at that temperature. Shear stress may increase during the process.

  In the method for producing a collagen gel according to this embodiment, shearing is applied to a collagen aqueous solution having a shear stress increase rate in the range of 1% to 30% to orient collagen fibers. By applying shear in a period in which the rate of increase in shear stress is 1% or more, collagen fibers can be arranged in a uniaxial direction and collagen gel can be immobilized continuously, and collagen collagen can be prevented while breaking down. It becomes possible to further increase the orientation of the fibers. On the other hand, by applying shear in a period in which the rate of increase in shear stress is 30% or less, it is possible to suppress local fibrillation of the collagen aqueous solution to which shear is applied. It is possible to prevent the collagen gel from being fixed before it is sufficiently generated. For example, even when the shear stress is increased by heating a collagen aqueous solution under shear, by applying shear during a period in which the shear stress increase rate is 30% or less, the heat source to be heated A difference in the degree of fibrosis between the near part and the far part becomes difficult to occur.

The shear rate at the time of imparting shear to the collagen aqueous solution is at least partially 0.20 s ^{−1 to} 30 s ⁻¹ , preferably 0.50 s ^{−1 to} 10 s ⁻¹ . When the shear rate is 0.20 s ⁻¹ or more, the orientation of the collagen fibers can be made sufficiently high. On the other hand, when the shear rate is 30 s ^-1 or less, the shear stress can be effectively transmitted to the collagen aqueous solution in the process of gelation, and the flow of the collagen aqueous solution is disturbed and the degree of orientation of the collagen fibers is reduced. It is possible to prevent lowering, and to prevent gel breakage more effectively and reliably.

  The time during which the shear stress is increased at a rate of 1% to 30% per second by applying shear to the collagen aqueous solution is 2 seconds to 120 seconds, preferably 5 seconds to 60 seconds. By setting this time to 2 seconds or more, the orientation of collagen fibers can be made sufficiently high. On the other hand, by setting the time during which the shear stress increases at the above-mentioned speed within 120 seconds, it is possible to prevent destruction of oriented collagen fibers that have progressed to gelation.

  The method of shearing when applying shear to the collagen aqueous solution is not particularly limited, for example, a method of generating shear between rotating parallel plates, a method of generating shear between parallel plates shifted in a uniaxial direction, and in a cylinder There is a method in which a cylinder is rotated coaxially and shear is generated in the gap between the cylinder and the cylinder. Among these, a method of causing shear between rotating parallel plates (rotational shearing) is preferable from the viewpoint that collagen gel can be easily collected and shearing can be continuously applied.

  As a device used for applying various kinds of shear to the aqueous collagen solution, a known device for applying shear to the solution or gel can be used. In order to quickly increase the temperature of the collagen aqueous solution to the temperature at which collagen fibrillates and to obtain a high degree of orientation, it is preferable to have a heat source that heats the portion in contact with the collagen aqueous solution. As such an apparatus, for example, a temperature control type rheometer equipped with a Peltier controller can be used.

  The collagen gel of this embodiment is produced by the method for producing a collagen gel of this embodiment. This collagen gel contains oriented collagen fibers. More specifically, the collagen gel of this embodiment has a birefringence phase difference of 30 ° to 90 ° per 1 mm optical path length and 1 mass% collagen concentration. A collagen gel exhibiting such a birefringence phase difference can be regarded as one in which collagen fibers are sufficiently oriented, for example, oriented as a fiber bundle of mm to cm scale. The birefringence phase difference of fully uniaxially oriented collagen fibers does not exceed 90 ° as a standardized value as described above, and the upper limit is preferably 80 °, more preferably 70 °, and even more preferably 60 °. . The birefringence phase difference of the collagen gel is measured by the method described in the examples below.

  By producing a collagen gel using the production method of the present embodiment described above, the thickness of the obtained collagen gel is 0.2 mm to 5 mm in a state where collagen fibers are oriented over the entire layer in the thickness direction. It is possible to be in the range. When the thickness of the collagen gel is 0.2 mm or more, it becomes possible to produce an implant that can be used for animals or humans, and when it is 5 mm or less, the uniformity of collagen fiber orientation inside the gel is improved. Can do. From the same viewpoint, the thickness of the collagen gel is more preferably 0.5 mm to 3 mm. In addition, the collagen gel fibers are “orientated over all layers in the thickness direction” of the collagen gel, on the surface that appears by cutting the collagen gel perpendicularly to the thickness direction at any position in the thickness direction, This can be confirmed by the orientation of the collagen gel fibers.

  In the collagen gel of this embodiment, the concentration of collagen is preferably 0.50% by mass to 3.0% by mass, and 0.75% by mass to 2.0% by mass, based on the total amount of the collagen gel. And more preferred. When the collagen concentration is 0.50% by mass or more, the mechanical strength of the collagen gel can be further increased, and the degree of orientation of the collagen fibers can be increased. On the other hand, when the collagen concentration is 3.0% by mass or less, the collagen gel of the present embodiment can be obtained more uniformly and more easily.

  Although the use of the collagen gel of the present embodiment is not particularly limited, it can be used as an artificial tendon because of its size, because it can obtain a biological implant, particularly collagen fibers oriented as a fiber bundle of mm to cm scale. It is expected. Moreover, according to the present embodiment, such an oriented collagen fiber can be formed nondestructively and efficiently (for example, within 20 minutes). In addition, when using the collagen gel of this embodiment as implants for living bodies such as artificial tendons, a plurality of collagen gels obtained by a plurality of production methods may be laminated and combined as necessary. A plurality of collagen gels may be combined to form a composite.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples.

(Examples 1-13, Comparative Examples 1-8)
[Preparation of collagen solution]
A collagen solution made of pig skin having a concentration of 1.0% by mass (telopeptide-removable collagen, Nippon Ham Co., Ltd., collagen denaturation temperature: 40 ° C.) was prepared as a collagen stock solution. The collagen solution was concentrated using an evaporator (water temperature: 29 ° C.) to obtain collagen solutions having concentrations of 1.2% by mass and 2.4% by mass. A collagen solution having a concentration of 0.6% by mass was obtained by diluting a collagen stock solution with a dilute hydrochloric acid aqueous solution (pH = 3).

[Preparation of genipin aqueous solution]
Using a buffer solution containing sodium hydrogen phosphate and sodium chloride as a solvent, genipin (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved therein to prepare a genipin aqueous solution. The concentrations of sodium hydrogen phosphate, sodium chloride, and genipin were adjusted so that their concentrations in the finally obtained collagen aqueous solution were as shown in Table 1.

[Preparation of EDC / NHS aqueous solution]
Using a buffer solution containing sodium hydrogen phosphate and sodium chloride as a solvent, EDC (manufactured by Dojindo Laboratories) and NHS (manufactured by Wako Pure Chemical Industries, Ltd.) were dissolved therein to prepare an EDC / NHS aqueous solution. The concentrations of sodium hydrogen phosphate, sodium chloride, and EDC / NHS were adjusted so that their concentrations in the finally obtained collagen aqueous solution were as shown in Table 1.

[Preparation of aqueous collagen solution]
6 g of the collagen solution prepared as described above was placed in a 15 mL centrifuge tube, and allowed to stand in a refrigerator whose internal temperature was adjusted to 4 ° C. A magnetic stirrer (10.8 g, inner diameter 10 mm × 39 mm) for promoting stirring was accommodated in the tube. Then, 2 mL of room temperature genipin aqueous solution or EDC / NHS aqueous solution was sucked up with a micropipette, added to a centrifuge tube containing a collagen solution, and the centrifuge tube was vigorously shaken and stirred. The centrifuge tube after stirring for about 30 seconds is set in a predetermined position of the centrifuge, centrifuged at 3200 rpm for 1.5 minutes, air bubbles are collected on the liquid surface, and an aqueous collagen solution containing a crosslinking agent Got. This collagen aqueous solution preparation work was completed within 4 minutes at room temperature. Table 1 shows the results of measuring the pH of the collagen aqueous solution with a pH meter (trade name “NAVIh F-71” manufactured by HORIBA) and the ionic strength of the inorganic salt in the collagen aqueous solution.

[Preparation of collagen gel by micro vibration]
The aqueous collagen solution prepared as described above is allowed to flow on a bottom plate (23 ° C.) of a dynamic viscoelasticity measuring apparatus (product name “HAAKE MarsIII”, manufactured by Thermo Fisher Scientific, Inc.) equipped with a parallel plate type sensor having an inner diameter of 60 mm. The collagen aqueous solution was sandwiched between the bottom and the upper plate (gap = 1 mm) (see FIG. 1A). Next, by applying a constant shear strain of 0.005 to the collagen aqueous solution at a frequency of 1 Hz, the temperature was adjusted while microvibrating, and the time change of the storage elastic modulus (G ′) was followed. In FIG. 2A, the solid line shows the time change of G ′ up to 180 seconds after the start of measurement, and the broken line shows the heating profile of the collagen aqueous solution up to 180 seconds after the start of measurement. Moreover, (B) of FIG. 2 shows the period which provided the minute vibration and the rotation shear by 180 seconds after the measurement start. The shear rate of the rotational shear was changed in the range of 0.10 s ^{−1 to} 50 s ⁻¹ . The result shown in FIG. 2A is based on the condition I in FIG. First, after 60 seconds at 23 ° C., the heating profile was raised to 37 ° C. in 30 seconds, and then kept constant at 37 ° C. Immediately after the temperature reached 37 ° C., G ′ increased rapidly and exceeded 1000 Pa within 60 seconds. As shown in FIG. 2 (B), after the minute vibration for 180 seconds was continued, the minute vibration at 37 ° C. was further continued for 14 minutes, and the discotic collagen gel produced between the plates was collected. The obtained collagen gel was subjected to analysis as a sample prepared without applying rotational shear based on the preparation method of the present invention. Such a collagen gel is called a “random collagen fiber gel”.

[Production of collagen gel by rotational shearing]
In the same manner as the preparation of the collagen gel by the above minute vibration, the collagen aqueous solution is sandwiched between the bottom and the upper plate (gap = 1 mm), and the temperature profile shown in FIG. ) Rotational shear was applied under the conditions II, III, IV, V, and VI shown in FIG. After completing the rotational shear, it shifted to micro vibration. Even when rotational shearing was performed, a disk-shaped collagen gel was produced non-destructively between the plates as in the case of microvibration (see FIG. 1B). Table 2 shows the conditions for heating and rotational shearing.

  As described above, the rotational shear condition shown in FIG. 2B was changed to II, III, IV, V, and VI, and the period for applying the rotational shear was changed. FIG. 4 shows typical shear stress changes obtained by applying shear under conditions III, IV, V and VI to a 1.8 mass% collagen aqueous solution containing 2.5 mM genipin and an inorganic salt having an ionic strength of 0.65. Shown in When rotational shear was applied until condition II, that is, until the temperature was raised from 23 ° C. to 37 ° C., no significant increase in shear stress was observed, and the viscosity sometimes decreased slightly (see Table 2). .) In condition III, an increase in shear stress was observed in only 10 seconds after reaching 37 ° C. When the period for applying rotational shear was further increased, the rate of increase in shear stress increased rapidly, and the rate of increase in shear stress reached a maximum between conditions IV and V. However, in the case of the condition VI in which the period for giving the rotational shear is further increased, a decrease in the shear stress increase rate was observed as compared with the case of the condition V.

  Here, FIG. 3 shows T, τ0, and τ1 that are used when the shear stress increase rate is calculated using the equation (1) from the time change curve of the stress. In addition, Table 2 shows the period for calculating the shear stress increase rate in each example (calculation period T, 5.0 ≦ T ≦ 120). In the case of Condition II, a period during which the temperature was raised from 23 ° C. to 37 ° C. over 30 seconds was defined as a calculation period T. In the case of conditions III, IV, V, and VI, a calculation period T was defined as an arbitrary period in which the increase in shear stress was particularly significant after reaching 37 ° C. Table 2 also shows the results of the shear stress τ0 and τ1 at the start point, start point, and end point of the period for calculating the shear rate increase rate, and the shear rate increase rate.

  In order to compare the degree of orientation of collagen fibers in the obtained collagen gel, the birefringence phase difference of the gel was measured. A helium-neon laser beam having a wavelength of 633 nm is modulated by a polarizing element to produce light of a known polarization state, which is incident in the thickness direction of the collagen gel sample, and the transmitted light intensity obtained through the polarizing element is analyzed. The birefringence phase difference of the collagen gel was determined by the polarization modulation method (this is referred to as “birefringence phase difference (A)”). Since the birefringence phase difference (A) increases as the collagen concentration increases and the optical path length increases, the standardized value obtained by dividing the birefringence phase difference (A) by the collagen concentration and the thickness of the sample is expressed as “birefringence phase difference”. (B) ". The respective values are shown in Table 3.

In order to evaluate the mechanical properties of the gel with collagen fibers oriented, a tensile test of the collagen gel of Example 2 was performed. As a control, a random collagen fiber gel was used. The sample preparation method and test method are described below. First, from the edge of the disc-shaped collagen gel produced between the plates of the dynamic viscoelasticity measuring device, as shown in FIG. 16 mm). Since the shearing direction is concentric in the disc-shaped gel, the specimen cut out from the positions x and y has collagen fibers oriented substantially parallel and perpendicular to the major axis direction, respectively. The obtained test piece is placed on a frame of 10 mm × 10 mm filter paper (frame width 4 mm), and both ends of the test piece and the filter paper are bonded with an instantaneous adhesive (trade name “Aron Alpha”, manufactured by Toagosei Co., Ltd.). did. It was housed in a plastic container as it was and allowed to stand at 4 ° C. for 1 hour to complete the adhesion under a moist environment where gel drying did not occur. After that, both ends of the test piece are fixed to the chuck of the strength tester (product name “TA.XTplus”, manufactured by Stable Micro Systems) together with the filter paper at a distance of 14 mm between the chucks and parallel to the long axis of the test piece ) The filter paper frame was cut with scissors. The tensile test was performed at a deformation rate of 0.2 mm / sec, and Young's modulus (inclination of a linear region of strain 0.01 to 0.04), breaking stress, and breaking strain were calculated from the obtained stress-strain curve. All physical property values were calculated assuming that the cross-sectional area of the test piece was constant (4 mm ² ).

  In order to evaluate the improvement in the mechanical properties of the gel due to crosslinking, a similar tensile test was performed on the test piece obtained by adding EDC / NHS crosslinking to the collagen gel of Example 2. The disk-shaped collagen gel produced between the plates of the dynamic viscoelasticity measuring apparatus was immersed in an EDC / NHS aqueous solution (50 mM / 10 mM) using PBS as a solvent at room temperature for 3 hours to add cross-linking. The gel was thoroughly washed with PBS, and unreacted cross-linking agent was washed and removed. Then, a test piece was prepared in the same manner as described above, and a tensile test was performed.

  Table 4 shows the collagen gel preparation conditions, cut-out sites, and cross-linking contents of each test piece. Each physical property value was expressed as an average value ± standard deviation (number of tests = 6). A significant difference between groups was determined by Tukey method, which is one of multiple comparison methods, and a case where p value was less than 0.05 was regarded as significant difference.

  In order to observe the nanostructure of the gel in which collagen fibers are oriented, the collagen gel of Example 2 was observed with a scanning electron microscope (SEM). A random collagen fiber gel was used as a control. The sample preparation method and test method are described below. First, a rectangular sample (7 mm × 12 mm) was cut out from the edge of the disc-shaped collagen gel produced between the plates of the dynamic viscoelasticity measuring apparatus as shown in FIG. After immobilization with glutaraldehyde aqueous solution, it was successively immersed in 20%, 50%, 70%, 80%, 90%, and 99.5% ethanol aqueous solution for dehydration. Furthermore, after being immersed in a 99.5% ethanol aqueous solution three times, it was immersed in t-butyl alcohol three times. This was dried with a vacuum dryer (model number “VFD-21S”; manufactured by Vacuum Device Inc.) for freeze-drying t-butyl alcohol. A part of the dried sample was peeled off with tweezers, the inside of the sample was exposed, and gold was deposited to obtain a sample for SEM observation. This sample was subjected to SEM observation at an accelerating voltage of 15 kV using a Miniscope TM3000 (trade name, manufactured by Hitachi High-Technologies Corporation).

* In condition II, the rotational shearing was stopped when the temperature reached 37 ° C., so the calculation period was 30 seconds when the temperature was raised from 23 ° C. to 37 ° C.

The birefringence phase difference (A) of the random collagen fiber gel was 2.0 ° ± 0.8 °.

* Collagen gel produced from a collagen aqueous solution containing genipin using a dynamic viscoelastic device is introduced with genipin cross-linking.

In Examples 1 to 13, the shear stress increased at a rate of 1% to 30% per second due to the formation of collagen fibers with respect to the collagen aqueous solution containing collagen at a concentration of 0.50% by mass to 3.0% by mass. The shear rate in the range of 0.20 s ^{-1 to} 30 s ^{-1 was} applied during 2 seconds to 120 seconds. As a result, a collagen gel having collagen fibers with a high degree of orientation (birefringence phase difference (B)) could be obtained non-destructively. On the other hand, in Comparative Examples 1 to 4, since the shear stress was not increased or the shear was stopped when the increase was small, the degree of orientation of the collagen fibers was not sufficient, and the birefringence phase difference (B) was 30 °. It did not exceed. Furthermore, in Comparative Example 5, since the shear rate was less than 0.20 s ⁻¹ , the degree of orientation of the collagen fibers did not increase, and the birefringence phase difference (B) did not exceed 30 °. In Comparative Example 6, as a result of the shear rate exceeding 30 s ⁻¹ , the shear stress increase rate of the collagen aqueous solution was less than 1%, the degree of orientation of the collagen fibers was not increased, and the birefringence phase difference (B) was 30. It did not exceed °. In Comparative Examples 7 and 8, since the collagen concentration was less than 0.50 mass%, the degree of orientation of the collagen fibers did not increase, and the birefringence phase difference (B) did not exceed 30 °.

  Of the obtained results, Examples 2, 6, 8 and 9 show changes in the shear stress of the aqueous collagen solution (collagen gel) with the passage of time after applying rotational shear after reaching 37 ° C. 4 shows. Further, based on the results of Examples (Actual) 2, 6, 8, and 9 and Comparative Example (Ratio) 1, the birefringence phase difference (A) is determined with respect to the rotational shearing application time after reaching 37 ° C. The plotted figure is shown in FIG. As is apparent from these results, the degree of orientation of the collagen fibers was increased by applying rotational shear when the shear stress was increased. When the temperature rises from 23 ° C. to 37 ° C., the shear stress hardly increases, and when the rotational shear is stopped when the temperature reaches 37 ° C., the degree of orientation of the collagen fibers is not so high because the shear stress increase rate is low. (See also Comparative Examples 1 to 4 in Table 2.) It was also found that the application of rotational shear after the shear stress began to decrease did not affect the degree of collagen fiber orientation.

Further, for Examples (actual) 1 to 5 and Comparative Examples (ratio) 5 and 6, a diagram (a semilogarithmic graph) in which the birefringence phase difference (A) is plotted against the shear rate is shown in FIG. From this figure, it was found that the degree of orientation of the collagen fibers is particularly high when the shear rate is 0.20 s ^{−1 to} 30 s ⁻¹ .

  An SEM image of the collagen gel obtained in Example 2 is shown in FIG. Moreover, the SEM image of the random collagen fiber gel produced without giving rotational shear is shown in FIG. In the random collagen fiber gel, amorphous entanglement of collagen nanofibers was observed, whereas in the collagen gel obtained in Example 2, the direction of rotational shear (the direction indicated by the arrow in FIG. 7A) ) In parallel with the collagen nanofibers was observed.

  FIG. 8 shows representative stress-strain curves obtained from the tensile test of the collagen gel of Example 2, the collagen gel obtained by subjecting the collagen gel of Example 2 to additional crosslinking, and the random collagen fiber gel. In addition, Table 4 shows the collagen gel preparation conditions, cut-out sites, and cross-linking contents of each test piece. The collagen gel of Example 2 and the collagen gel obtained by subjecting the collagen gel of Example 2 to additional cross-linking have a tension parallel to the direction of rotational shear (i.e., the orientation direction of collagen fibers) rather than a perpendicular tension. High stress was shown. That is, it was found that mechanical anisotropy was generated by the orientation of the collagen fibers, and the mechanical properties higher than that of the random collagen fiber gel were obtained by the anisotropy. Moreover, when additional cross-linking was performed with EDC / NHS on a collagen gel cross-linked only with genipin, the stress increased in both test pieces at the cut-out site and the strain until breakage decreased. From this, it was found that the mechanical properties of the collagen gel of the present invention can be adjusted by a known crosslinking agent.

  FIG. 9 shows physical property values of Young's modulus, breaking strain, and breaking stress calculated from the stress-strain curve. The Young's modulus and rupture stress of Rot-Gx were higher than those of Osc-G and Rot-Gy. Similar results were obtained from a comparison of Rot-ENx, Rot-ENy, and Osc-EN. From this, it was found that the above-mentioned mechanical anisotropy based on FIG. 8 is statistically significant. Moreover, about the fracture | rupture distortion, the tendency different from the mechanical anisotropy based on FIG. 8 was seen (Rot-Gy> Rot-Gx = Osc-G). From this, it was found that the orientation of collagen fibers shows high flexibility in tensile deformation in the direction perpendicular to the orientation direction. However, this flexibility advantage was lost due to EDC / NHS crosslinking. That is, it was found that EDC / NHS cross-linking is effective in increasing the elastic modulus and strength of a collagen gel composed of oriented collagen fibers, but reduces the flexibility of the gel.

[Effect of genipin concentration on collagen gel hardening]
A collagen aqueous solution containing genipin was obtained in the same manner as in Example 1 except that the concentration of genipin was changed from 2.5 mM to 0 mM, 1.25 mM, 3.75 mM or 5.0 mM. About the obtained collagen aqueous solution, the change of the storage elastic modulus G 'on the conditions I was tracked using the dynamic viscoelasticity measuring apparatus (Brand name "HAAKE MARS III", the product made from Thermo Fisher Scientific). The obtained gelation curve is shown in FIG. In FIG. 10, the curve “1” is 0 mM, the curve “2” is 1.25 mM, the curve “3” is 2.5 mM, the curve “4” is 3.75 mM, and the curve “5” is 5. It is a gelation curve by the collagen aqueous solution of 0 mM genipin concentration. It has been found that when genipin is added to the collagen aqueous solution according to the present invention, the increase rate of the storage elastic modulus G ′ is increased, and a high-strength collagen gel can be obtained in a short time. Such a high-strength collagen gel is preferable from the viewpoint of improving production efficiency because it can suppress breakage when the gel is recovered.

  According to the present invention, it is possible to non-destructively produce collagen fibers oriented as a three-dimensional fiber bundle of mm to cm scale, so that the present invention can be used industrially as an implant material for living bodies such as artificial tendons. There is.

Claims (7)

Collagen having a step of orienting collagen fibers by applying a shear rate in the range of 0.20 s ^{-1 to} 30 s ^{-1 to} an aqueous collagen solution containing collagen at a concentration of 0.50 mass% to 3.0 mass% A method for producing a gel comprising:
A method for producing a collagen gel, wherein the step includes a step of 2 to 120 seconds in which the shear stress increases at a rate of 1% to 30% per second due to the formation of collagen fibers, thereby orienting the collagen fibers. .   The method for producing a collagen gel according to claim 1, wherein the collagen fibers are formed by heating the collagen aqueous solution from a temperature of 25 ° C or lower to 32 to 42 ° C.   The method for producing a collagen gel according to claim 1 or 2, wherein the aqueous collagen solution contains an inorganic salt, and the ionic strength of the inorganic salt is 0.40 to 1.0.   The method for producing a collagen gel according to claim 3, wherein the inorganic salt contains sodium hydrogen phosphate and sodium chloride.   The said collagen aqueous solution is a preparation method of the collagen gel of any one of Claims 1-4 containing a crosslinking agent.   The method for producing a collagen gel according to any one of claims 1 to 5, wherein a pH of the collagen aqueous solution is 6.0 to 9.0.   The method for producing a collagen gel according to any one of claims 1 to 6, wherein the collagen contains a mammal-derived telopeptide-removed collagen.