Tissue repair material and method for manufacturing the same

Abstract

To provide a tissue repair material that comprises polymeric porous granules with a reduced amount of fine powder and a method for producing the same.SOLUTION: A polymeric porous body to be used for pulverization is controlled to be a fixed size relative to a screen pore size.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a tissue repair material containing polymer porous granules and a method for producing the same. [Background technology]

[0002] Porous materials made of biocompatible polymers such as collagen and gelatin are useful as materials for cell scaffolds, transplantation components, tissue repair materials, etc. Patent Document 1 describes a method for producing recombinant gelatin porous granules. Specifically, a method for producing recombinant gelatin porous granules is disclosed, which involves crushing a porous material obtained by freeze-drying with a screen mill and then classifying it.

[0003] When raw materials such as porous materials are crushed to obtain granules, the granules will contain particles of various sizes. Granules that are significantly different in size from the desired size are undesirable because, in addition to having different physicochemical properties, they can have unexpected effects when used as packing material by being interposed in the packed layer. For example, in the case of perfectly spherical particles, it is known that particles with a radius of 0.29 times or less the main particles can be interposed in any void in the packed layer made up of the main particles.

[0004] From the perspective of ensuring stable quality of tissue repair materials containing polymer porous granules, it is desirable that as many of the granules as possible after grinding fall within the desired particle size range, and that there are few granules smaller than the desired size (fine powder). For example, Non-Patent Literature 1 discloses a method for reducing the amount of fine powder by optimizing the impeller speed and batch input amount. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2014 / 141877 Pamphlet [Non-patent literature]

[0006] [Non-Patent Document 1] Kotamarthy et al., Processes, 2020, p. 683 [Overview of the project] [Problems that the invention aims to solve]

[0007] The object of the present invention is to provide a tissue repair material containing polymer porous granules with a low amount of fine powder, and a method for producing the same. [Means for solving the problem]

[0008] The inventors of this invention conducted intensive research to solve the above problems and found that by setting the size of the polymer porous material added during pulverization to a certain ratio with respect to the screen pore diameter, polymer porous granules with a reduced fine powder ratio can be obtained, thus completing the present invention. Furthermore, they found that the polymer porous granules obtained by reducing fine powder exhibit high performance as a tissue repair material.

[0009] In other words, the present invention includes the following: [1] A tissue repair material containing polymer porous granules, wherein the proportion of fine powder in the polymer porous granules is 10% by mass or less of the total polymer porous granules. [2] A method for producing a tissue repair material according to [1], comprising one or more grinding steps of grinding a polymer porous body with a screen mill, wherein at least one of the grinding steps is a step of introducing a polymer porous body having a size 4 to 6 times the size of the screen pores into the screen mill and grinding it. [Effects of the Invention]

[0010] The present invention provides a bone graft material that exhibits a high bone regeneration rate. Furthermore, a manufacturing method for producing such a bone graft material using a screen mill is also provided. [Modes for carrying out the invention]

[0011] In this specification, the term "step" includes not only an independent step but also a step that cannot be clearly distinguished from other steps as long as the intended effect of this step is achieved.

[0012] In this specification, a numerical range indicated using "~" represents a range that includes the numerical values described before and after "~" as the lower limit value and the upper limit value, respectively.

[0013] In the present invention, the amount of each component in a composition means the total amount of the plurality of substances corresponding to each component in the composition when there are a plurality of substances corresponding to each component in the composition, unless otherwise specified.

[0014] In the present invention, the amino acid sequence of a polypeptide may be expressed using the single-letter notation (e.g., "G" for a glycine residue) or the three-letter notation (e.g., "Gly" for a glycine residue) well-known in the art.

[0015] In the present invention, "% " regarding the amino acid sequence of a polypeptide is based on the number of amino acid (or imino acid) residues, unless otherwise specified. In the present invention, "identity" regarding the amino acid sequences of two polypeptides to be compared refers to the value calculated by the following formula. Note that the alignment of a plurality of polypeptides is performed according to a conventional method so that the number of identical amino acid residues is maximized. Identity (%) = {(Number of identical amino acid residues) / (Alignment length)} × 100

[0016] Hereinafter, the present invention will be described.

[0017] [Polymer] In the present invention, a polymer refers to a molecule with a large molecular weight, having a structure composed of multiple repetitions of units substantially or conceptually obtained from molecules with a small molecular weight. Examples include polyamines, cellulose, amylose, starch, chitin, polypeptides, proteins, DNA, and RNA. The polymer is preferably water-soluble, more preferably polypeptides and proteins. Among polypeptides and proteins, collagen and gelatin are particularly preferred.

[0018] The ratio of hydrophilic repeating units in the polymer is preferably 50% or less, more preferably 30% or less. If the ratio of hydrophilic units is higher than this, the free water around the polymer decreases and freezing is inhibited. Here, the ratio of hydrophilic repeating units refers to the ratio of repeating units having ionic groups and / or hydroxyl groups in the polymer.

[0019] The above gelatin means a polypeptide containing 6 or more consecutive sequences represented by Gly-X-Y, and may have one or more other amino acid residues in addition to the sequence represented by Gly-X-Y in the polypeptide. The sequence represented by Gly-X-Y corresponds to an amino acid sequence derived from a partial amino acid sequence of collagen, and the repetition of this sequence means a sequence characteristic of collagen.

[0020] The plurality of Gly-X-Y may be the same or different from each other. Also, X and Y in the Gly-X-Y sequence are independent for each repeating unit and may be the same or different. In Gly-X-Y, Gly represents a glycine residue, and X and Y represent any amino acid residues other than the glycine residue. As X and Y, it is preferable that many imino acid residues, that is, proline residues or oxyproline residues are contained. The content rate of such imino acid residues preferably occupies 10% to 45% of the whole gelatin. The content rate of Gly-X-Y in the above gelatin is preferably 80% or more of the whole, more preferably 95% or more, and most preferably 99% or more.

[0021] The gelatin described above may be of the natural type or a mutant type that differs from the natural type in at least one amino acid residue. Natural type gelatin means gelatin made from naturally occurring collagen, or a polypeptide having the same amino acid sequence as gelatin made from naturally occurring collagen. Unless otherwise specified, mutant or recombinant gelatin is collectively referred to as recombinant gelatin in this specification. Examples of natural type gelatin or its recombinant gelatin include those derived from animals such as fish and mammals, but it is preferable that it be natural type gelatin or its recombinant gelatin from mammals. Examples of mammals include humans, horses, pigs, mice, rats, etc., and it is more preferable that it be human or pig. Natural type gelatin is preferably derived from pigs or humans, and recombinant gelatin is preferably human-derived recombinant gelatin.

[0022] Furthermore, it is preferable that the gelatin is recombinant gelatin obtained by introducing and expressing, by conventional means, a base sequence or amino acid sequence obtained by modifying one or more bases or amino acid residues in the base sequence or amino acid sequence of the collagen encoding the above collagen having six or more consecutive Gly-XY sequences, into a suitable host. By using such recombinant gelatin, it is possible to enhance the (bone) tissue repair ability and express various properties compared to when natural gelatin is used, and it has advantages such as being able to avoid undesirable effects such as rejection reactions by the body.

[0023] As the recombinant gelatin mentioned above, those disclosed in EP1014176A2, US6992172, WO2004 / 85473, WO2008 / 103041, JP2010-519293, JP2010-519252, JP2010-518833, JP2010-519251, WO2010 / 128672 and WO2010 / 147109, etc., can be used with particular preference. Furthermore, the recombinant gelatin is preferably 2 kDa or more and 100 kDa or less in molecular weight, more preferably 5 kDa or more and 90 kDa or less, and even more preferably 10 kDa or more and 90 kDa or less.

[0024] The recombinant gelatin described above is preferably more biocompatible and contains cell adhesion signals, and more preferably has two or more cell adhesion signals in a single molecule. Examples of such cell adhesion signals include the RGD sequence, LDV sequence, REDV sequence (SEQ ID NO: 2), YIGSR sequence (SEQ ID NO: 3), PDSGR sequence (SEQ ID NO: 4), RYVVLPR sequence (SEQ ID NO: 5), LGTIPG sequence (SEQ ID NO: 6), RNIAEIIKDI sequence (SEQ ID NO: 7), IKVAV sequence (SEQ ID NO: 8), LRE sequence, DGEA sequence (SEQ ID NO: 9), and HAV sequence. Preferably, the RGD sequence, YIGSR sequence, PDSGR sequence, LGTIPG sequence, IKVAV sequence, and HAV sequence are included, with the RGD sequence being particularly preferred. Among the RGD sequences, the ERGD sequence is even more preferred.

[0025] In the recombinant gelatin described above, the arrangement of the RGD sequences is preferably such that the number of amino acid residues between RGD sequences is 0 to 100, and more preferably 25 to 60. Furthermore, it is preferable that the RGD sequences are arranged non-uniformly within this range of amino acid residues. Furthermore, the ratio of RGD sequences to the total number of amino acid residues in the recombinant gelatin is preferably at least 0.4%, and if the recombinant gelatin contains 350 or more amino acid residues, it is preferable that each stretch of the 350 amino acid residues contains at least one RGD sequence.

[0026] The recombinant gelatin described above preferably contains at least two RGD sequences per 250 amino acid residues, more preferably at least three RGD sequences, and even more preferably at least four RGD sequences. However, the sequence of the recombinant gelatin is preferably as follows: (1) it does not contain serine and threonine residues, (2) it does not contain serine, threonine, asparagine, tyrosine, and cysteine ​​residues, and (3) it does not contain an amino acid sequence represented by Asp-Arg-Gly-Asp. The recombinant gelatin may have one of these preferred sequence embodiments (1) to (3) individually, or it may have two or more embodiments in combination. Furthermore, the recombinant gelatin may be partially hydrolyzed.

[0027] The above recombinant gelatin is A-[(Gly-XY) n ] m - Preferably has a repeating structure of B. m represents 2 to 10, preferably 3 to 5. A and B represent any amino acid or amino acid sequence. n represents 3 to 100, preferably 15 to 70, and more preferably 50 to 60.

[0028] Preferably, recombinant gelatin is of the formula: Gly-Ala-Pro-[(Gly-XY) 63 ]3-Gly(In the formula, each of the 63 X's independently represents one of the amino acid residues, and each of the 63 Y's independently represents one of the amino acid residues. Note that there are 3 (Gly-XY) 63 These may be the same or different. ) This is represented by [the symbol].

[0029] It is preferable to bind multiple naturally occurring collagen sequence units to the repeating units of the recombinant gelatin described above. Naturally occurring collagen can preferably be type I, type II, type III, type IV, and type V. More preferably, type I, type II, or type III can be used. The collagen can preferably be derived from humans, horses, pigs, mice, or rats, with human collagen being more preferable. The isoelectric point of the above recombinant gelatin is preferably 5 to 10, more preferably 6 to 10, and even more preferably 7 to 9.5.

[0030] Preferred embodiments of the recombinant gelatin described above include: (1) the carbamoyl group is not hydrolyzed; (2) it does not contain procollagen; (3) it does not contain telopeptides; and (4) it is a substantially pure collagen material prepared with nucleic acids encoding natural collagen. The recombinant gelatin described above may consist of one of these preferred embodiments (1) to (4), or it may consist of a combination of two or more embodiments.

[0031] The above recombinant gelatin can preferably be any of the following (A) to (C) due to its high (bone) tissue repair ability. (A) The polypeptide shown in Sequence ID No. 1 below, GAP(GAPGLQGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGPAGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPP)3G(Sequence ID 1) (B) A polypeptide having a partial sequence having 80% or more sequence identity with the partial amino acid sequence consisting of amino acid residues 4 to 192 in the amino acid sequence of (A) above, and having (bone) tissue repair ability. (C) A polypeptide having (bone) tissue repair ability, consisting of an amino acid sequence in which one or more amino acid residues are deleted, substituted, or added to the amino acid sequence of (A) above.

[0032] Regarding the sequence identity in (B) above, from the viewpoint of the (bone) tissue repair ability of recombinant gelatin, it can more preferably be 90% or more, and even more preferably 95% or more.

[0033] The partial amino acid sequence in the sequence of (B) above is a partial amino acid sequence corresponding to the repeating unit of the sequence shown in Sequence ID No. 1. If there are multiple partial amino acid sequences corresponding to the repeating unit in the polypeptide of (B) above, the polypeptide can contain one, preferably two or more, repeating units with a sequence identity of 80% or more.

[0034] Furthermore, it is preferable that the polypeptide defined in (B) above contains, in total, 80% or more of the total number of amino acid residues, partial sequences having 80% or more sequence identity with the partial amino acid sequence corresponding to the repeating unit.

[0035] The length of the polypeptide defined in (B) above can be between 151 and 2260 amino acid residues. From the viewpoint of degradation after crosslinking, it is preferable to have 193 or more amino acid residues, and from the viewpoint of stability, it is preferable to have 944 or fewer amino acid residues, and more preferably between 380 and 756 amino acid residues.

[0036] Furthermore, the polypeptide defined in (C) above may consist of an amino acid sequence in which one or more amino acid residues are deleted, substituted, or added to the amino acid sequence in (A) above, and may be a polypeptide having tissue repair ability.

[0037] The number of amino acid residues deleted, substituted, or added in the polypeptide defined in (C) above may be one or several, and will vary depending on the total number of amino acid residues in the recombinant gelatin, but for example, it can be 2 to 15, preferably 2 to 5.

[0038] The above-mentioned recombinant gelatin can be produced by genetic recombination techniques known to those skilled in the art, for example, by following the methods described in EP1014176A2, US6992172, WO2004 / 85473, WO2008 / 103041, etc. Specifically, a gene encoding the amino acid sequence of a predetermined recombinant gelatin is obtained, incorporated into an expression vector to create a recombinant expression vector, and introduced into a suitable host to produce a transformant. By culturing the obtained transformant in a suitable medium, recombinant gelatin is produced, and the recombinant gelatin used in the present invention can be prepared by recovering the recombinant gelatin produced from the culture.

[0039] [Polymer aqueous solution] In the present invention, a polymer aqueous solution is an aqueous solution containing one or more polymers. The polymer concentration in the polymer aqueous solution is preferably 0.1% by mass or more, more preferably 1% by mass or more, and particularly preferably 5% by mass or more. If the concentration is lower than 0.1% by mass, it is difficult to maintain the structure of the polymer porous body after the water is removed. The polymer aqueous solution preferably gels at a temperature above the freezing point. The upper limit of the polymer concentration in the polymer aqueous solution is not particularly limited as long as the polymer can be dissolved, but is generally 40% by mass or less, and may also be 30% by mass or less, or 20% by mass or less.

[0040] Aqueous polymer solutions are prepared by purifying and concentrating a solution containing polymers, or by dissolving dried polymers in an aqueous medium. (1) They may be prepared immediately before use, or (2) a pre-prepared solution may be used. (3) The aqueous polymer solution obtained by purification and concentration may be freeze-dried, and the resulting freeze-dried material may be redissolved in an aqueous medium to prepare the aqueous polymer solution. Alternatively, (4) the aqueous polymer solution obtained by purification and concentration may be frozen, and the resulting frozen material may be thawed to prepare the aqueous polymer solution. From the viewpoint of reducing the generation of air bubbles and insoluble matter (undissolved frozen material), it is preferable to thaw the frozen material at 35-40°C for 15-20 hours. From the viewpoint of reducing the effort required for preparation on demand, facilitating transportation and storage, and reducing air bubbles and insoluble matter in the aqueous polymer solution, method (4) above is preferred.

[0041] It is preferable to remove air bubbles and insoluble matter dispersed in the polymer aqueous solution before the freezing process by operations such as filtration, centrifugation, reduced pressure, and degassing. This improves the yield of low-anisotropy polymer aqueous solution freezers. The removal of air bubbles and insoluble matter can be evaluated by turbidity measurement. Alternatively, it can be evaluated by visual inspection with an optical microscope. For example, the number of air bubbles and unwanted matter visible in the field of view of the optical microscope can be calculated and evaluated as the number of air bubbles and insoluble matter in 1 μL of polymer aqueous solution. The number of air bubbles and insoluble matter in the polymer aqueous solution is preferably 0.5 particles / μL or less, more preferably 0.3 particles / μL or less, even more preferably 0.1 particles / μL or less, and particularly preferably 0 particles / μL.

[0042] Non-polymer components may be added to the polymer aqueous solution for the purpose of imparting specific properties. Other such components include, for example, components related to bone regeneration or bone formation, such as bone-inducing agents. Examples of bone-inducing agents include, but are not limited to, BMP (bone morphogenetic factor) and bFGF (basic fibroblast growth factor). Other examples include polypeptide or protein crosslinking agents.

[0043] The aqueous medium for the polymer aqueous solution is not particularly limited as long as it can dissolve the polymer and is suitable for use in biological tissues. Examples include water, physiological saline, phosphate buffer, and other substances commonly used in this field.

[0044] When using gelatin as the polymer, the gelatin concentration in the gelatin solution is not particularly limited, as long as it is a concentration at which gelatin can dissolve. The gelatin concentration in the gelatin solution is preferably, for example, 0.5% to 20% by mass, more preferably 2% to 16% by mass, and even more preferably 4% to 12% by mass. Furthermore, the gelatin solution may be degassed before the freezing process. This makes it easier to uniformly form ice crystals. There are no particular restrictions on the degassing method, but for example, degassing can be done by vacuum centrifugal force at a pressure of 2 to 10 kPa.

[0045] The gelatin solution may be filtered to remove undissolved particles. The filtration method is not particularly limited, but for example, pressure filtration can be performed using a filter with a pore size of 0.22 to 0.45 μm. The material of the filter is also not particularly limited, and polytetrafluoroethylene, polyethersulfone, cellulose acetate, polyvinylidene fluoride, etc. can be used, but cellulose acetate is preferred from the viewpoint of low gelatin adsorption and low elution. The temperature when preparing the gelatin solution is not particularly limited, and a commonly used temperature, for example, 0°C to 60°C, preferably around 3°C to 40°C, is acceptable.

[0046] [Liquid container] In this invention, a liquid container refers to a container for holding an aqueous polymer solution for cooling or freezing. Examples of container shapes include dish-shaped and cylindrical cup-shaped containers. A cylindrical cup-shaped container is preferred. It is preferable that the inside of the container does not have a high curvature. Specifically, it is preferable that the radius is 1 mm or more, and more preferably 2 mm or more (R means radius of curvature). The size of the container is not particularly limited, but in the case of a cylindrical cup-shaped container, the inner diameter is preferably 200 mm or less, and more preferably 150 mm or less.

[0047] A liquid container may have its inner surface coated with a material (coating material) that is the same as or different from the materials that make up the liquid container. Alternatively, a cover material made of the same or different materials that make up the liquid container may be laid on the inner surface of the liquid container, or a cylindrical cover material may be installed. To distinguish it from the coating material and cover material, the materials that make up the liquid container are also called the main materials of the liquid container. There is no restriction on the combination of the main materials, coating material, and cover material of the liquid container. That is, a liquid container may consist of any of the following combinations: (1) the main materials of the liquid container only, (2) the main materials and coating material of the liquid container, (3) the main materials and cover material of the liquid container, or (4) the main materials, coating material, and cover material of the liquid container.

[0048] The inner surface of the liquid container, that is, the surface that comes into contact with the polymer aqueous solution when it is placed in the liquid container, is made of tetrafluoroethylene-hexafluoropropylene copolymer (also called perfluoroethylenepropene copolymer) (FEP). For example, in accordance with (1) to (4) above, (1) the main component of the liquid container is FEP, (2) at least the coating component is FEP, (3) at least the cover component is FEP, and (4) at least the cover component is FEP.

[0049] There are no particular restrictions on the material of the main component of the liquid container; for example, aluminum can be used. When the main component of the liquid container is in contact with a polymer aqueous solution, the main component of the liquid container is FEP. The material of the main component of the liquid container has a linear expansion coefficient (also called thermal expansion coefficient) of 10 × 10 -5 Preferably less than / K, 50 × 10 -6 It is even more preferable that it be less than or equal to / K, which is 25 × 10 -6 It is particularly preferable that the value be less than or equal to / K.

[0050] The cover member only needs to be uniformly laid on the inner surface of the container, and there are no restrictions on the shape or thickness of the cover member. When the cover member is in contact with the polymer aqueous solution, the cover member is made of FEP. The coating member only needs to be uniformly coated on the inner surface of the container, and there are no restrictions on the thickness of the coating film. The coating film is preferably 20 μm or thicker, and more preferably 50 μm or thicker. When the coating member is in contact with the polymer aqueous solution, the coating member is made of FEP.

[0051] [Freezing process] In this invention, the freezing step refers to the step of freezing a polymer aqueous solution placed in a liquid container. There are no particular restrictions on the freezing method; for example, it can be frozen using a refrigerator, freeze dryer, or other device. When freezing with a freeze dryer, it is possible to continuously remove water from the frozen polymer aqueous solution (freeze-drying) using the same device.

[0052] The temperature during the freezing process varies depending on the type of polymer and the concentration of the polymer aqueous solution, but it is preferable that the difference between the temperature of the warmest part of the solution and the temperature of the coldest part of the solution immediately before the generation of solidification heat is 2.5°C or less. Here, "temperature difference immediately before the generation of solidification heat" refers to the temperature difference that is greatest between 1 second and 10 seconds before the generation of solidification heat. Furthermore, it is preferable that the temperature of the coldest part of the solution is -8°C or less, more preferably -10°C or less, and most preferably -15°C or higher.

[0053] [Frozen polymer aqueous solution] In this invention, the term "polymer aqueous solution frozen body" refers to a frozen body obtained by freezing a polymer aqueous solution. In this invention, "anisotropy" of the polymer aqueous solution frozen body refers to the physical properties measured as follows: After freeze-drying the polymer aqueous solution frozen body, the freeze-dried body (polymer porous body) is cut horizontally and vertically near its center. Then, each cross-section is stained, and a certain area (2.0 mm × 2.0 mm or 2.5 mm × 2.5 mm) is observed with an optical microscope. Within the observation area, among the rectangles that circumscribe the area surrounded by the stained material, the circumscribed rectangle with the maximum distance between two opposing sides is selected. The length of the long side of this circumscribed rectangle with the maximum distance between two opposing sides is measured 50 times within the observation area of ​​both the horizontal and vertical cross-sections, and the average of these measurements is taken as the average value of the major axis of the mesh of the frozen body. For each individual mesh in this case, the ratio d2 / d1 obtained by taking the smaller of the two major axes (average value) of the horizontal and vertical cross-sections as d1 and d2 as d2 is defined as "anisotropy". An anisotropy of 3 or less is defined as "low anisotropy".

[0054] [Moisture removal process] In this invention, the moisture removal step refers to the step of removing moisture from the polymer aqueous solution frozen body. There are no particular restrictions on the means of removing moisture, and methods include melting the ice in the polymer aqueous solution frozen body, sublimation (freeze-drying), etc., with freeze-drying being preferred. The freeze-drying period can be, for example, 0.5 hours to 300 hours. There are no particular restrictions on the freeze-drying oven that can be used.

[0055] [Porous polymer material] In this invention, a polymer porous body refers to a porous block made of polymer. A polymer porous body is obtained by removing water from a frozen polymer aqueous solution. The polymer porous body can be further processed by crushing, crosslinking, etc., to form granules of various sizes, which can then be used as cell scaffolds, transplantation components, tissue repair materials, etc.

[0056] From the perspective of enhancing the affinity with the living body, the pore diameter of the polymeric porous body is preferably 10 μm or more and 2500 μm or less, and more preferably 40 μm or more and 1000 μm or less. The pore diameter of the polymeric porous body can be measured as the diameter (equivalent circle diameter) of a circle having the same area as the pore portion when observing the cross-section near the center of the polymeric porous body with a microscope, and can be evaluated as the median value of the equivalent circle diameters of all pore portions within the observation region. The density of the polymeric porous body is 0.01 g / cm 3 or more and 0.3 g / cm 3 or less, preferably 0.05 g / cm 3 or more and 0.1 g / cm 3 or less. The density of the polymeric porous body can be calculated by dividing the mass of the polymeric porous body by the apparent volume of the polymeric porous body including the internal voids.

[0057] From the perspective of enhancing the affinity with the living body, the porosity of the polymeric porous body is preferably 80.00% or more and 99.99% or less, and more preferably 92.01% or more and 99.99% or less. "Porosity" refers to the value calculated by the following formula. Porosity (%) = 100 - 100 × mass (g) ÷ apparent volume (cm 3 ) ÷ true specific gravity (g / cm 3 )

[0058] [Grinding process] In the grinding process, the polymeric porous body is ground to obtain a ground product. For grinding, grinders such as hammer mills and screen mills can be applied. From the perspective of having a small variation in particle size distribution for each test because the ground product is recovered at any time from the material ground to a certain size, a screen mill (for example, Quadro Comil manufactured by Quadro Corporation) is preferred. As the grinding conditions, in order to maintain the structure of the surface of the ground product, a cutting method is more preferable than a crushing method. Also, in order to maintain the structure inside the granules, it is preferable to use a method that does not apply strong compression during grinding. The ground product obtained by grinding the polymeric porous body is contained in the tissue repair material as polymeric porous granules.

[0059] When grinding with a screen mill, from the viewpoint of making the polymer porous granules suitable for use as a tissue repair material (details described later), the pore size of the screen is preferably 0.006 inches or more and 0.25 inches or less, and more preferably 0.04 inches or more and 0.08 inches or less. From the viewpoint of efficient grinding, the rotation speed of the rotating impeller is preferably 100 rpm or more and 6000 rpm or less, and more preferably 1000 rpm or more and 3000 rpm or less. The air velocity in the pores of the screen is preferably 5 m / s or more and 20 m / s or less, and more preferably 7 m / s or more and 15 m / s or less.

[0060] The size of the polymer porous material before being fed into the screen mill for grinding is preferably four times or more the diameter of the screen pores. By keeping the size of the polymer porous material before grinding within the above range, the generation of fine powder (details described later) can be suppressed. Furthermore, the size of the polymer porous material is preferably six times or less the diameter of the screen pores. By keeping the size of the polymer porous material before grinding within the above range, the occurrence of charring due to temperature rise during grinding can be suppressed. The size of the polymer porous material can be defined as the square root of the projected area of ​​the polymer porous material, that is, the length of one side of a square with the same area as the projected area.

[0061] [Classification process] A classification step may be included after the grinding step to achieve uniform particle size. This makes it possible to obtain a polymer pulverized material with a uniform particle size. For example, it is preferable to use a sieve with a mesh size of 300 μm to 1400 μm for classifying the gelatin pulverized material.

[0062] [Polymer porous granules] The size of the polymer porous granules (i.e., the size of the polymer porous material after pulverization) is preferably between 0.01 mm and 10 mm. By keeping the polymer porous granules within this range, a size suitable for tissue repair material can be achieved. It is more preferably between 0.1 mm and 5 mm, and even more preferably between 0.3 mm and 1.4 mm. Note that the size of the polymer porous granules refers to the size of each individual polymer porous granule, and does not refer to a representative value (e.g., mean and median) of the size of multiple polymer porous granules.

[0063] The size of polymer porous granules can be defined by the mesh size of the sieve when the polymer porous granules are sieved. For example, when polymer porous granules are sieved through a 1.4 mm sieve and then the granules that pass through are sieved through a 0.3 mm sieve, the polymer porous granules remaining on the sieve can be defined as polymer porous granules with a size of 0.3 mm or more and 1.4 mm or less.

[0064] When a group of multiple polymer porous granules used as a tissue repair material is fractionated using a test sieve of the R20 / 3 series as defined in ISO 565, the lower sieve of the section with the highest weight ratio ("main section") preferably has a mesh size of 355 μm to 1 mm, and more preferably 710 μm. The fraction contained within the main section is the main fraction. For example, if the lower sieve of the main section has a mesh size of 710 μm, then the "710 μm and above" section is the main fraction.

[0065] The shape of the polymer porous granules is not particularly limited. For example, they may be particulate (granules), amorphous, spherical, powdery, porous, fibrous, spindle-shaped, flattened, or sheet-like. Preferably, they are particulate (granules), amorphous, spherical, powdery, or porous. "Irregular" means that the surface shape is not uniform, for example, those with an uneven surface like a rock.

[0066] [Fine powder] In this invention, polymer porous granules that are classified using sieves with mesh spacing of √2 times the original size and are two or more steps smaller than the main fraction (meaning that the main fraction does not pass through a sieve that passes through a sieve with a mesh spacing of 1 / √2) are defined as "fine powder". The proportion of fine powder in the polymer porous granules is preferably 10% by mass or less of the total polymer porous granules, more preferably 7.5% by mass or less, and even more preferably 5% by mass or less. By setting the proportion of fine powder to 10% by mass or less of the total polymer porous granules, the bone regeneration rate of the tissue repair material containing polymer porous granules is improved.

[0067] [Filling process] The process may include filling vials with the ground material after the grinding step. For example, the method of filling the gelatin grind is not particularly limited, but a mass feedback type table feeder can be used. The vials used to fill the gelatin grind are also not particularly limited, but for example, glass vials with a silicone-coated inner surface can be used.

[0068] [Crosslinking process] The process preferably includes a crosslinking step after the grinding step to crosslink the polymers in the resulting pulverized material. Known methods such as thermal crosslinking, enzymatic crosslinking, crosslinking using various chemical crosslinking agents, and UV crosslinking can be used for crosslinking. Crosslinking using chemical crosslinking agents or thermal crosslinking are preferred. In addition to crosslinking (covalent bonding), further higher-order structuring can be achieved through at least one of hydrophobic interactions, hydrogen bonding, and ionic interactions. Those that are already in place are also preferable.

[0069] When performing crosslinking with enzymes, the enzyme is not particularly limited as long as it has a crosslinking effect between biodegradable materials, but preferably transglutaminase and laccase, and most preferably transglutaminase can be used for crosslinking.

[0070] In the present invention, when the pulverized material is gelatin pulverized material, the mixing temperature with the gelatin when treating it with aldehydes or crosslinking agents such as condensing agents is not particularly limited as long as the solution can be uniformly stirred, but is preferably 0°C to 40°C, more preferably 0°C to 30°C, more preferably 3°C to 25°C, more preferably 3°C to 15°C, even more preferably 3°C to 10°C, and particularly preferably 3°C to 7°C.

[0071] After mixing and stirring the crosslinking agent, the temperature can be increased. For example, if the pulverized material is gelatin pulverized material, the reaction temperature is not particularly limited as long as crosslinking proceeds, but considering the denaturation and decomposition of gelatin, it is substantially 0°C to 60°C, more preferably 0°C to 40°C, more preferably 3°C to 25°C, more preferably 3°C to 15°C, even more preferably 3°C to 10°C, and particularly preferably 3°C to 7°C.

[0072] In the case of a crosslinking method using a chemical crosslinking agent, it is more preferable to use glutaraldehyde as the chemical crosslinking agent. When employing a crosslinking method using a chemical crosslinking agent, the chemical crosslinking agent may be added to an aqueous polymer solution and crosslinking may be performed before the drying process.

[0073] The crosslinking temperature applied to the thermal crosslinking method is preferably 100°C to 200°C, more preferably 120°C to 170°C, and even more preferably 130°C to 160°C. By employing the thermal crosslinking method, the use of crosslinking agents can be avoided. The processing time for thermal crosslinking varies depending on the crosslinking temperature, the type of polymer, and the degree of degradability to be maintained.

[0074] For example, when using CBE3, which is used in the examples described later, as a human-derived recombinant gelatin, the thermal crosslinking conditions are as follows: At an actual temperature of approximately 135°C, the conditions are preferably 2 to 20 hours, more preferably 3 to 18 hours, and even more preferably 4 to 8 hours. The thermal crosslinking treatment is preferably carried out under reduced pressure, vacuum, or an inert gas atmosphere in order to prevent oxidation. For example, thermal crosslinking is preferably carried out at 130°C to 150°C under a nitrogen atmosphere for 3 to 7 hours. The degree of reduced pressure is preferably 4 hPa or less. Nitrogen or argon is preferred as the inert gas, and crosslinking under an inert gas atmosphere is preferred over under vacuum in terms of uniform heating. There are no particular restrictions on the heating means, and examples include a vacuum oven such as the DP-43 manufactured by Yamato Scientific.

[0075] The pulverized material after thermal crosslinking may be stored in a container. For example, in the case of a pulverized material made of gelatin, there are no particular restrictions on the container, but for example, a glass vial sealed with a rubber stopper and an aluminum cap can be used. There are no particular restrictions on the size of the glass vial. (The glass vial may be coated with a silicone resin coating using dimethylpolysiloxane, a fluororesin coating, a silica coating, or subjected to dealkalization treatment.) The above container may also be further packaged. There are no particular restrictions on the packaging, but for example, an aluminum pouch can be used.

[0076] [Tissue repair material] In this invention, the tissue repair material is a composition containing polymer porous granules. For example, by using a tissue repair material containing gelatin porous granules, a tissue repair material exhibiting good tissue repair ability is obtained. The tissue to which the tissue repair material is applied is not particularly limited, but bone tissue is an example. To explain this further, it can be considered as follows: That is, the (bone) tissue repair material has a certain degree of biocompatibility and biodegradability, so that it can serve as a replacement site for regenerated (bone) tissue while ensuring strength for the desired period and maintaining the volume of the defect. As a result, it is presumed that the regeneration of bone and other tissues will proceed well by placing the (bone) tissue repair material in the defect. However, this invention is not bound by this theory.

[0077] Since the present invention can provide a tissue repair material with good tissue regeneration ability, methods for tissue repair and methods for treating diseases involving tissue damage are also included in the present invention. Specifically, the tissue repair method in the present invention includes applying the tissue repair material to the site where the target tissue is missing or damaged, and includes other steps as necessary.

[0078] The tissues that can be repaired by the tissue repair material of the present invention are preferably hard tissues such as teeth and bones. In particular, the tissue repair material is suitable as a base material for bone regeneration. The tissue repair material of the present invention can be used alone as a therapeutic agent for bone regeneration. The diseases to which this therapeutic agent can be applied are not particularly limited, as long as they are diseases that require treatment for bone regeneration or bone regeneration.

[0079] The present invention provides a method for treating or repairing damaged tissue, comprising applying a tissue repair material to the site of the defect or damage to the target tissue, and optionally including other steps. Other steps include, for example, applying transplanted cells and / or bone induction agents to the site where the tissue repair material is applied, before, during, or simultaneously with the application of the tissue repair material. When applying the tissue repair material, a spatula, syringe, dappen dish, etc., can be used. The treatment or repair method can be preferably applied to periodontal tissue defects and implant defects in the maxillofacial region; and to GBR (Guided Bone Regeneration), gingival augmentation, sinus lift, or socket reservation as preliminary procedures when implanting an implant.

[0080] [Water absorption rate of tissue repair materials] It is preferable that the tissue repair material has a water absorption rate above a certain level. For example, in the case of a tissue repair material containing gelatin granules, it is preferable that it exhibits a water absorption rate of 300% or more by mass. If the water absorption rate is less than 300%, good (bone) tissue regeneration ability cannot be obtained. The water absorption rate of the (bone) tissue repair material is preferably 400% or more, and more preferably 500% or more, from the viewpoint of blood clot retention during (bone) tissue repair. There is no particular upper limit to the water absorption rate of the (bone) tissue repair material, but it is preferably 4000% or less, more preferably 3000% or less, and even more preferably 2000% or less. If the (bone) tissue repair material contains only gelatin granules, the water absorption rate of the (bone) tissue repair material shall be the water absorption rate of the gelatin granules.

[0081] In this invention, the "water absorption rate" of the (bone) tissue repair material refers to the physical property measured as follows: 10.0 ± 0.2 mg of the test substance is taken into each of three filter cups (each with a volume of 500 μL and equipped with a filter with a pore size of 0.22 μm at the bottom; hereinafter referred to as "containers") whose tare mass has been measured in advance (n=3). A sufficient amount of water is added and mixed until the water absorption by the test substance is saturated (rotate, 2 hours, ambient temperature). Next, the excess water is removed by centrifugation (6000 × g, 1 minute, 25°C), and the mass of the container containing the test substance after water absorption is measured (total mass after water absorption). Separately, three blank tests are performed, and the residual water amount is taken by subtracting the tare mass from the total mass after water absorption without the test substance. The average of the three residual water amounts is taken as the residual water amount for the blank test, and the water absorption rate is corrected. The water absorption rate (%) is calculated by dividing the mass of the test substance after water absorption by the mass of the test substance before water absorption.

[0082] The water absorption rate of bone tissue repair materials varies depending on the components contained in the material, particularly the type of polymer porous granules and the morphology of individual polymer porous granules. However, it can be adjusted, for example, by the temperature and processing time of the freezing or crosslinking process. Generally, increasing the temperature of the freezing process, decreasing the temperature of the crosslinking process, or shortening the crosslinking time tends to increase the water absorption rate.

[0083] [Acid retention rate of tissue repair materials] It is preferable that the tissue repair material exhibits an acid retention rate below a certain level. For example, in the case of a tissue repair material containing gelatin granules, it is preferable that the retention rate is 66% or less by mass after a 3-hour decomposition treatment using 1 mol / L hydrochloric acid. If the retention rate by mass after a 3-hour decomposition treatment using 1 mol / L hydrochloric acid is higher than 66%, the (bone) tissue regeneration ability cannot be said to be sufficient. From the viewpoint of maintaining the volume of the preparation layer at the defect site and replacing it with the regenerating tissue, the acid retention rate of the (bone) tissue repair material is preferably 5% by mass or more, more preferably 20% by mass or more, and even more preferably 34% by mass or more. If the (bone) tissue repair material contains only gelatin granules, the acid retention rate of the (bone) tissue repair material shall be the acid retention rate of the gelatin granules.

[0084] In this invention, the "acid retention rate" of the (bone) tissue repair material refers to a physical characteristic value measured as follows: The mass of a microtube for measurement (product name Mini Super Tube, manufactured by Ibis, capacity 2 ml, hereinafter referred to as the tube) is measured (A). For granular repair material, 15.0 (±0.2) mg is weighed (n=3) without processing in the form to be placed in the tissue defect. For block-shaped repair material, a cylindrical sample with a diameter of 6 mm and a thickness of approximately 1 mm is prepared, its mass is measured (B), and it is filled into the measurement tube. 1.7 ml of 1 mol / L HCl is added to the tube containing the tissue repair material, and it is left to stand at a constant temperature of 37 ± 0.5 °C for 3 hours. After the specified time, the tube is placed on ice to stop the reaction, and it is centrifuged at 10,000 × g for 1 minute in a centrifuge pre-set to 4 °C. After confirming that the tissue repair material has settled, aspirate the supernatant, add ultrapure water that has been pre-chilled on ice, and centrifuge again under the same conditions as above. Repeat the process of aspirating the supernatant, adding ultrapure water again, and centrifugating again under the same conditions as above two more times. After aspirating the supernatant, freeze-dry the tubes. After removing them from the freeze-dryer, quickly cap the tubes to prevent the tissue repair material from absorbing moisture from the air. Measure the mass of each tube (C), and calculate the acid retention rate using the following formula (3). Acid residual rate = (CA) / B×100(%) (3)

[0085] The residual acid content of tissue repair materials varies depending on the components contained in the material, particularly the type and form of the polymeric porous granules, but can be adjusted, for example, by controlling the temperature and processing time during the crosslinking process. Generally, shortening the processing time in the crosslinking process tends to result in a lower acid retention rate. [Examples]

[0086] The present invention will be described in detail in the following examples, but the present invention is not limited to them in any way.

[0087] [Example 1] A bone regeneration substrate according to Example 1 was prepared using recombinant peptide CBE3 as the recombinant gelatin. The CBE3 used was the one described below (as described in WO2008 / 103041A1). CBE3 molecular weight: 51.6kD structure: GAP[(GXY)63]3G Number of amino acids: 571 RGD array: 12 Imino acid content: 33% Almost 100% of the amino acids are in a repeating GXY structure. The amino acid sequence of CBE3 does not contain serine, threonine, asparagine, tyrosine, or cysteine ​​residues. CBE3 has an ERGD sequence. Isoelectric point: 9.34. The ratio of hydrophilic repeating units in the polymer is 26.1%. Amino acid sequence (SEQ ID NO: 1)

[0088] After purifying the solution containing the recombinant gelatin described above, it was concentrated to 4.0% by mass by ultrafiltration at 30°C. The resulting gelatin aqueous solution was freeze-dried, and then sterile water for injection was added to the freeze-dried material and the temperature was raised to 37°C over 30 minutes to redissolve it, thereby obtaining a 7.5% by mass gelatin aqueous solution. This gelatin aqueous solution was filtered through a 0.22 μm cellulose acetate membrane filter and degassed by vacuum centrifugation at 4.0 kPa for 180 seconds using a vacuum degasser (Kurabo Industries Ltd., KK-V300SS-I). The gelatin aqueous solution was sampled into a transparent polystyrene container to a thickness of 2.5 mm and observed using an optical microscope in a 2.5 mm × 2.5 mm field of view, at 100 μm intervals from the bottom to the top of the liquid. Ten fields of view were observed, and the average number of bubbles and insoluble matter was calculated. The average number of bubbles was 0.42 / μL, and the average number of insoluble matter was 0 / μL. Approximately 20g of this gelatin aqueous solution was poured into a cylindrical cup-shaped container made of aluminum alloy (A5056) with an inner diameter of 104mm, a bottom thickness of 5mm, and a bottom circumference chamfered with a radius of R2mm, and the inner surface coated with FEP (Nippon Fluorine, NF-004A). Fourteen of these containers were then placed on a 350×634×20mm aluminum plate pre-cooled to approximately -35°C, with a 1mm thick glass plate in between. The containers were then covered and left to stand for 1 hour to obtain frozen gelatin bodies. The coefficient of linear expansion of the main material of the cylindrical cup-shaped container used (aluminum alloy (A5056)) was 24.3×10⁻⁶. -6 The result was / K. This gelatin frozen material was freeze-dried using a freeze-dryer (ULVAC, DFR-5N-B) to remove moisture, and freeze-dried materials (polymer porous materials) were prepared. When the anisotropy was evaluated, 13 of the freeze-dried materials showed low anisotropy (percentage = 93%).

[0089] [Example 2] The freeze-dried material (polymer porous material) described in Example 1, with low anisotropy, was cut into 11 mm squares and then conditioned overnight at 23°C and 63% RH. It was then ground using a screen grinder (Quadro, Cormill U10) with a 0.079 inch screen, followed by a 0.040 inch screen, to obtain Sample 1. The charring rate and particle size distribution in sample 1 were evaluated as follows.

[0090] (1) Rate of burning Approximately 0.09g of the sample was weighed into 30 glass vials. These were then visually inspected in detail, and vials containing colored particles were classified as "burnt." The number of "burnt" vials out of the 30 was defined as the burnt rate.

[0091] (2) Particle size distribution The sample was classified using analytical sieves with mesh sizes of 1.4 mm, 1.0 mm, 710 μm, 500 μm, and 355 μm, as well as by stacking bread. The weight on each sieve was measured, and the weight ratio of each fraction was determined. The "main fraction" was the one above 710 μm. The fraction below the 500 μm fraction was defined as "fine powder," and the ratio of fine powder to the total granules was defined as the fine powder ratio.

[0092] [Example 3] Sample 2 was prepared in the same manner as in Example 2, except that the polymer porous material was cut into 8 mm squares. The rate of charring and the ratio of fine particles in sample 2 were evaluated in the same manner as in sample 1.

[0093] [Comparative Example 1] Sample 3 was prepared in the same manner as in Example 2, except that the polymer porous material was cut into 17 mm squares. The rate of charring and the ratio of fine particles in sample 3 were evaluated in the same manner as in sample 1.

[0094] [Comparative Example 2] Sample 4 was prepared in the same manner as in Example 2, except that the polymer porous material was cut into 6 mm squares. The rate of charring and the ratio of fine particles in sample 4 were evaluated in the same manner as in sample 1.

[0095] Table 1 summarizes the results for Example 2, Example 3, Comparative Example 1, and Comparative Example 2.

[0096] [Table 1]

[0097] Sample 1 and Sample 4 were treated in a clean oven (Nitto Rika Kogyo, NCO-500A600L-WS) under a nitrogen atmosphere at 135°C for 5 hours to obtain Sample 5 and Sample 6, respectively, as tissue repair materials. The bone regeneration rates for Samples 5 and 6 were determined as follows, and were 80% and 37%, respectively.

[0098] Bone regeneration evaluation In each group of 10 SD rats (male, 10-12 weeks old), a circular bone defect with a diameter of 5 mm was created in the parietal bone, filled with approximately 3.6 mg of sample, and then sutured. Four weeks post-surgery, bone mass was measured in the parietal bone of the rats using micro-CT, and the bone volume inside the defect was measured as the ratio of the defect volume to the defect volume. The average ratio of bone volume to defect volume for the 10 rats was defined as the bone regeneration rate.

Claims

1. A tissue repair material comprising recombinant gelatin porous granules, wherein the proportion of fine powder in the recombinant gelatin porous granules is 10% by mass or less of the total recombinant gelatin porous granules, and the fine powder is recombinant gelatin granules that are two or more steps smaller than the main fraction when classified using a sieve group with a mesh opening spacing of √2 times.

2. A method for producing a tissue repair material according to Claim 1, comprising one or more grinding steps of grinding a recombinant gelatin porous body with a screen mill, wherein at least one of the grinding steps is a step of introducing a recombinant gelatin porous body having a size 4 to 6 times that of the screen pore diameter into a screen mill and grinding it.

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