JP2023124423A - Grinding method of porous body, and production method of ground product - Google Patents

Abstract

To provide a grinding method of a porous body containing hydrophilic macromolecule, capable of suppressing occurrence of charring; and to provide a production method of a ground product in which occurrence of charring is suppressed.SOLUTION: In a grinding method of a porous body, including grinding of the porous body containing hydrophilic macromolecule, a moisture content of the porous body before grinding is 13.5 mass% or less, and the method of grinding is dry grinding. A production method of a ground product includes steps of: adjusting the moisture content of the porous body containing hydrophilic macromolecule at 13.5 mass% or less: and grinding the porous body by dry grinding.SELECTED DRAWING: Figure 1

Description

The present disclosure relates to a method for pulverizing a porous body and a method for producing a pulverized product.

A pulverized porous material containing macromolecules such as collagen and gelatin is useful as a material for cell scaffolds, graft members, and the like. For example, Patent Document 1 describes a method for producing pulverized recombinant gelatin porous material.

Generally, in the pulverization of a porous polymer material, heat generated during pulverization may burn the material to be pulverized, leading to contamination with colored foreign matter. Techniques for suppressing scorching of pulverized materials include, for example, a method of pulverizing at an ultra-low temperature using liquid nitrogen (Patent Document 2), a method of wet pulverizing by mixing with a solution (Patent Document 3), and a large amount of powder in the pulverizing section. (Patent Document 4), and a method of suppressing heat generation by heat of vaporization by adding water to increase the water content (Patent Document 5).

WO2014/133081 JP-A-55-92667 JP-A-9-176 Japanese Patent Application Laid-Open No. 2004-223500 JP-A-2005-269981

However, the conventional techniques described in Patent Documents 2 to 5 are practically difficult to apply to the pulverization of hydrophilic porous polymeric materials, and effectively prevent the occurrence of scorching during pulverization of porous polymeric materials. There is a fact that it can not be suppressed. In view of such circumstances, the present disclosure relates to providing a method for pulverizing a porous body containing a hydrophilic polymer, which can suppress the occurrence of scorching, and a method for producing a pulverized material in which the occurrence of scorching is suppressed.

Means for solving the above problems include the following aspects.
<1> including pulverizing a porous body containing a hydrophilic polymer,
The water content of the porous body before pulverization is 13.5% by mass or less,
A method for pulverizing a porous body, wherein the method of pulverization is dry pulverization.
<2> The pulverization method according to <1>, wherein the hydrophilic polymer has a 1/IOB value of 0 to 1.0.
<3> The pulverization method according to <1> or <2>, wherein the hydrophilic polymer contains a polypeptide.
<4> The grinding method according to any one of <1> to <3>, wherein the GRAVY score of the polypeptide is negative.
<5> The grinding method according to any one of <1> to <4>, wherein the polypeptide contains recombinant gelatin.
<6> The pulverization method according to <5>, wherein the recombinant gelatin is represented by the following formula.
Formula: A-[(Gly-X-Y) _n ] _m -B
In the formula, A represents an amino acid residue or an amino acid sequence, B represents an amino acid residue or an amino acid sequence, X independently represents an amino acid residue, Y independently represents an amino acid residue, and n is 3 represents an integer of ~100 and m represents an integer of 2-10.
<7> The pulverization method according to <5> or <6>, wherein the recombinant gelatin contains the following polypeptide (A), (B) or (C).
(A) A polypeptide consisting of the amino acid sequence of SEQ ID NO:1.
(B) A polypeptide consisting of an amino acid sequence in which one or several amino acid residues are modified in the amino acid sequence of SEQ ID NO: 1 and having biocompatibility.
(C) consisting of an amino acid sequence having a partial sequence having 80% or more sequence identity with the partial amino acid sequence consisting of the 4th to 192nd amino acid residues in the amino acid sequence of SEQ ID NO: 1, and biocompatibility A polypeptide having
<8> The pulverization method according to any one of <1> to <7>, wherein the pulverization device used for dry pulverization is a screen mill.
<9> The pulverization method according to any one of <1> to <8>, wherein the density of the porous material before pulverization is 0.3 g/cm ³ or less.
<10> The size of the porous body before pulverization is 0.1 mm to 100 mm, and the size of the porous body before pulverization is the square root of the projected area of the porous body, <1> to <9> The pulverization method according to any one of the above.
<11> The size of the porous body after pulverization is 0.01 mm to 10 mm, and the size of the porous body after pulverization is defined by the opening of the sieve obtained by sieving the porous body. <1> The pulverization method according to any one of <10>.
<12> The pulverizing method according to any one of <1> to <11>, wherein pulverizing the porous body is performed in an atmosphere with an absolute humidity of 15 g/m ³ or less.
<13> The pulverization method according to any one of <1> to <12>, wherein the porous body is a medical material.
<14> A method for producing a pulverized material, comprising the steps of: making the water content of a porous body containing a hydrophilic polymer 13.5% by mass or less; and, after the step, pulverizing the porous body by dry pulverization. .
<15> A method for producing a pulverized material, comprising pulverizing a porous body by the pulverizing method according to any one of <1> to <13>.

According to the present disclosure, there are provided a method for pulverizing a hydrophilic polymer-containing porous material that can suppress the occurrence of scorching, and a method for producing a pulverized material that suppresses the occurrence of scorching.

FIG. 1 shows the relationship between the occurrence of scorching and the moisture content of the porous body in Example 1. FIG. FIG. 2 shows a porous body that softens and expands when heated and is charred at the center when pressed against a heat source in Example 2. FIG. FIG. 3 shows a porous body in Example 2 that does not soften and expand when heated and is warped to escape from the heat source. FIG. 4 shows the relationship between the proportion of the porous body softened and expanded upon heating and the moisture content of the porous body in Example 2. FIG.

Hereinafter, modes for implementing embodiments of the present disclosure will be described in detail. However, the embodiments of the present disclosure are not limited to the following embodiments. In the following embodiments, the constituent elements (including element steps and the like) are not essential unless otherwise specified. The same applies to numerical values and their ranges, which do not limit the embodiments of the present disclosure.

In the present disclosure, the term "process" includes a process that is independent of other processes, and even if the purpose of the process is achieved even if it cannot be clearly distinguished from other processes. .
In the present disclosure, the numerical range indicated using "-" includes the numerical values before and after "-" as the minimum and maximum values, respectively.
In the numerical ranges described step by step in the present disclosure, the upper limit or lower limit of one numerical range may be replaced with the upper or lower limit of another numerical range described step by step. . Moreover, in the numerical ranges described in the present disclosure, the upper or lower limits of the numerical ranges may be replaced with the values shown in the examples.
In the present disclosure, each component may contain multiple types of applicable substances. When there are multiple types of substances corresponding to each component in the composition, the content rate or content of each component is the total content rate or content of the multiple types of substances present in the composition unless otherwise specified. means quantity.
In the present disclosure, a combination of two or more preferred aspects is a more preferred aspect.
In the present disclosure, when embodiments are described with reference to drawings, the configurations of the embodiments are not limited to the configurations shown in the drawings. In addition, the sizes of the members in each drawing are conceptual, and the relative relationship between the sizes of the members is not limited to this.

<Method of pulverizing porous material>
The method for pulverizing a porous body of the present disclosure (hereinafter also simply referred to as the "pulverization method of the present disclosure") includes pulverizing a porous body containing a hydrophilic polymer (hereinafter also referred to as a "pulverization step"). The water content of the porous body before pulverization is 13.5% by mass or less, and the pulverization method is dry pulverization. The inventors found that by pulverizing a porous body containing a hydrophilic polymer with a water content of 13.5% by mass or less, scorching can be suppressed. Although the reason is not clear in detail, it is presumed as follows.

As a result of investigations by the inventors, it was found that a porous body containing a hydrophilic polymer tends to soften and expand when heated when the water content is increased. Similar to gelatinization of starch, for example, heating weakens the hydrogen bonds and ionic bonds within and between the molecules of the hydrophilic polymer, and water molecules enter and act as a plasticizer. This is thought to be because the motility of the steel is improved and softened. Furthermore, it is presumed that the porous body expands due to the water vapor pressure generated by heating. Therefore, when a hydrophilic polymer porous material with a high water content is pulverized, the porous material heated by frictional heat softens and expands, and the details of the pulverizer are easily clogged or stuck to the pulverizer. It is thought that the large area makes it easier to come into contact with the crusher. It is presumed that this makes the porous body susceptible to excessive frictional heat and increases the occurrence of scorching. Conversely, a hydrophilic polymer porous material with a low water content does not soften and expand when heated, but rather shrinks as the moisture is removed, and is likely to deform as if escaping from the heat source, making it less likely to receive frictional heat. be done.

The methods described in Patent Documents 2 to 5 are not sufficient for suppressing the occurrence of scorching when pulverizing a hydrophilic porous polymer material. In addition, the method described in Patent Document 2 requires a procedure for cooling the material to be ground to an ultra-low temperature, and the wet grinding described in Patent Document 3 requires a procedure for separating and removing the solution, but the grinding method of the present disclosure. According to the method, it is possible to efficiently pulverize a porous body containing a hydrophilic polymer and suppress scorching. In the method described in Patent Document 4, since a large amount of gas is sent to the pulverizing unit, the pulverization behavior is likely to change due to the air flow. Burning can be suppressed. In the method described in Patent Document 5, buckwheat flour, which is a relatively dense material, is rather hydrated to increase the moisture content, thereby suppressing heat generation due to the heat of vaporization and suppressing scorching. On the other hand, since the porous body has a low density, the water content per volume is small, and the cooling effect due to the heat of vaporization is considered to be insufficient in the water content range within the dry pulverization range. Therefore, it is considered that burning of a porous body containing a hydrophilic polymer can be suppressed by pulverizing the porous body with a low moisture content.

(Pulverization process)
The pulverization method of the present disclosure includes a pulverization step of pulverizing a porous body containing a hydrophilic polymer. "Pulverization" is the fragmentation of solids and aggregates of solids by the application of mechanical energy.

First, a porous body containing a hydrophilic polymer will be described in detail. In the present disclosure, the term "macromolecules" refers to molecules of high molecular weight that have a structure composed of multiple repetitions of units derived substantially or conceptually from molecules of lower molecular weight. "Hydrophilicity" of a polymer means that the polymer has a hydrophilic group. Hydrophilic groups include hydroxyl group, amino group, carboxyl group, sulfonic acid group, thiol group, aldehyde group, amide group, sulfonamide group, phosphoric acid group, phosphine group, phosphine oxide group, pyridinium group, amine oxide group, alkylene Examples include an oxy group and a urethane group.

From the viewpoint that the pulverization method of the present disclosure is particularly useful, the 1/IOB value, which is an index of the hydrophilicity of the hydrophilic polymer, is preferably 0 to 1.0, more preferably 0 to 0.6. More preferably, it is still more preferably 0.01 to 0.4. IOB (Inorganic Organic Balance) is an indicator of hydrophilicity and hydrophobicity based on the organic conceptual diagram showing the polar/non-polarity of organic compounds proposed by Mu Fujita. For details, see "Pharmaceutical Bulletin", vol.2 , 2, pp.163-173 (1954), "Chemical Area" vol.11, 10, pp.719-725 (1957), "Fragrance Journal", vol.50, pp.79-82 (1981), etc. is explained in Briefly, the origin of all organic compounds is methane (CH ₄ ), and all other compounds are regarded as derivatives of methane. , the scores are added to determine the organic value (OV) and the inorganic value (IV), and each value is plotted on a diagram with the organic value on the X axis and the inorganic value on the Y axis. The IOB in the organic conceptual diagram refers to the ratio of the inorganic value (IV) to the organic value (OV) in the organic conceptual diagram, ie, "inorganic value (IV)/organic value (OV)". For details of the organic conceptual diagram, refer to "New Edition Organic Conceptual Diagram - Fundamentals and Applications -" (Yoshio Koda et al., Sankyo Publishing, 2008). In the present disclosure, the hydrophilicity/hydrophobicity is represented by the "1/IOB" value, which is the reciprocal of IOB. The smaller the "1/IOB" value (that is, closer to 0), the more hydrophilic it is.

A "porous body" means a solid having pores (voids) inside. The porous body used in the pulverization method of the present disclosure contains a hydrophilic polymer. The porous body only needs to contain a hydrophilic polymer, and may or may not contain components other than the hydrophilic polymer. The ratio of the hydrophilic polymer to the total mass of the porous body is preferably 80% by mass or more, more preferably 90% by mass or more, and even more preferably 100% by mass.

The porous body may contain a biocompatible substance. "Biocompatibility" means that it does not cause significant adverse reactions such as long-term and chronic inflammatory reactions when in contact with living organisms. Biocompatible substances include proteins and polysaccharides. The porous body may contain protein.

Hydrophilic polymers are not particularly limited as long as they are hydrophilic polymers, and specific examples include polypeptides, polylactic acid, polyglycolic acid, lactic acid/glycolic acid copolymers, hyaluronic acid, glycosaminoglycans, and proteoglycans. , chondroitin, cellulose, agarose, carboxymethylcellulose, chitin, chitosan, etc., and polypeptides are preferred from the viewpoint that the pulverization method of the present disclosure is particularly useful. Hydrophilic polymers preferably contain biocompatible polypeptides, and specific examples include gelatin, collagen, atelocollagen, elastin, fibronectin, pronectin, laminin, tenascin, fibrin, fibroin, entactin, thrombospondin, retronectin, and the like. is mentioned. The hydrophilic polymer preferably contains collagen or gelatin, more preferably gelatin.

When the hydrophilic polymer is a polypeptide, the grand average of hydrophathicity (GRAVY) score, which is a hydrophilic index of the polypeptide, is preferably negative from the viewpoint that the pulverization method of the present disclosure is particularly useful. It is more preferably 4.5 to -0.5. The GRAVY score is based on Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M.R., Appel R.D., Bairoch A.; Protein Identification and Analysis Tools on the ExPASy Server; (In) John M. Walker (ed. ): The Proteomics Protocols Handbook, Humana Press (2005). pp. 571-607 and Gasteiger E., Gattiker A., Hoogland C., Ivanyi I., Appel R.D., Bairoch A.; in-depth protein knowledge and analysis.; Nucleic Acids Res. 31:3784-3788 (2003).

Gelatin may be either natural gelatin or recombinant gelatin, and recombinant gelatin is preferable from the viewpoint that desired properties can be imparted by adjusting the molecular weight, amino acid sequence, and the like. Examples of natural gelatin and recombinant gelatin include those derived from animals such as fish and mammals, preferably those derived from mammals. Examples of mammals include humans, horses, pigs, mice and rats. When the porous material contains recombinant gelatin, the recombinant gelatin is preferably of human origin.

Hereinafter, amino acid sequences constituting polypeptides are represented by one-letter code (e.g., "G" for glycine residue) or three-letter code (e.g., "Gly" for glycine residue) known in the art. express. In addition, "%" for the amino acid sequence of a polypeptide is based on the number of amino acid (including imino acid) residues unless otherwise specified.

In the present disclosure, recombinant gelatin means a polypeptide or proteinaceous substance having a gelatin-like amino acid sequence produced by genetic recombination technology. Recombinant gelatin can be used, for example, as a tissue repair material that, when implanted in vivo, contributes to tissue formation at the implantation site. The term "tissue repair material" is not limited to materials that contribute to the formation of normal tissue that normally exists at the implantation site, but also includes materials that promote the formation of abnormal tissue including scar tissue. The "tissue" that can be repaired by the tissue repair material may be hard tissue such as teeth and bones, and the following recombinant gelatin is particularly suitable as a base material for bone regeneration.

The recombinant gelatin preferably has repeats of the Gly-XY sequence characteristic of collagen. Here, the plurality of Gly-XY may be the same or different. In Gly-XY, Gly represents a glycine residue and X and Y represent any amino acid residue. X and Y are preferably amino acid residues other than glycine residues, and preferably contain many imino acid residues, ie, proline residues or oxyproline residues. The content of imino acid residues is preferably 10% to 45% of the total amount of gelatin, based on the number of amino acid residues. The content of Gly-XY in gelatin is preferably 80% or more, more preferably 95% or more, and even more preferably 99% or more, based on the number of amino acid residues. .

Examples of recombinant gelatin include EP1014176A2, US6992172, WO2004/85473, WO2008/103041, JP2010-519293, JP2010-519252, JP2010-518833, JP2010-519251, WO2010/1. 28672 and WO2010/147109 et al., but not limited thereto.

The molecular weight of the recombinant gelatin is preferably 2 kDa to 100 kDa, more preferably 5 kDa to 90 kDa, even more preferably 10 kDa to 90 kDa.

From the viewpoint of biocompatibility, the recombinant gelatin preferably further contains a cell adhesion signal, and more preferably has two or more cell adhesion signals per molecule. Cell adhesion signals include the RGD sequence, YIGSR sequence (SEQ ID NO:2), PDSGR sequence (SEQ ID NO:3), LGTIPG sequence (SEQ ID NO:4), IKVAV sequence (SEQ ID NO:5) and HAV sequence. Among them, the RGD sequence is preferred, and among the RGD sequences, the ERGD sequence (SEQ ID NO: 6) is more preferred.

The sequence of the recombinant gelatin preferably satisfies at least one of the following aspects (1-1) to (1-3). The recombinant gelatin may have any one of the following aspects (1-1) to (1-3) alone, or may have two or more aspects in combination.
(1-1) does not contain serine and threonine residues;
(1-2) does not contain serine, threonine, asparagine, tyrosine and cysteine residues;
(1-3) Does not contain the amino acid sequence represented by Asp-Arg-Gly-Asp (SEQ ID NO: 7).

For example, from the viewpoint of being particularly useful as a tissue repair material, the recombinant gelatin is preferably a recombinant gelatin represented by the following formula.
Formula: A-[(Gly-X-Y) _n ] _m -B
In the formula, A represents an amino acid residue or an amino acid sequence, B represents an amino acid residue or an amino acid sequence, X independently represents an amino acid residue, Y independently represents an amino acid residue, and n is 3 represents an integer of ~100 and m represents an integer of 2-10.

m represents 2 to 10, preferably 3 to 5.
n represents 3 to 100, preferably 15 to 70, more preferably 50 to 65.

More preferably, the recombinant gelatin is a recombinant gelatin represented by the following formula.
Formula: Gly-Ala-Pro-[(Gly-XY) ₆₃ ] ₃ -Gly
In the formula, each X independently represents any amino acid residue, and each Y independently represents any amino acid residue. The three [(Gly-XY) ₆₃ ] may be the same or different.

The recombinant gelatin preferably satisfies at least one of the following aspects (2-1) to (2-4). The recombinant gelatin may have any one of the following aspects (2-1) to (2-4) alone, or may have two or more aspects in combination.
(2-1) the carbamoyl group is not hydrolyzed;
(2-2) Does not have procollagen.
(2-3) does not have a telopeptide;
(2-4) A substantially pure collagen-like material prepared with a nucleic acid encoding native collagen.

Recombinant gelatin preferably contains the following polypeptides (A), (B) or (C) because of its high ability to repair tissue.
(A) A polypeptide consisting of the amino acid sequence of SEQ ID NO:1.
GAP(GAPGLQGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGPAGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPP) ₃ G (SEQ ID NO: 1)
(B) A polypeptide comprising an amino acid sequence in which one or several amino acid residues are modified (eg, deleted, substituted or added) in the amino acid sequence of SEQ ID NO: 1 and having biocompatibility.
(C) consisting of an amino acid sequence having a partial sequence having 80% or more sequence identity with the partial amino acid sequence consisting of the 4th to 192nd amino acid residues in the amino acid sequence of SEQ ID NO: 1, and biocompatibility A polypeptide having

In the peptide defined in (B) above, the number of amino acid residues to be modified (e.g., deleted, substituted or added) varies depending on the total number of amino acid residues of the recombinant gelatin, but is, for example, 2 to 15. is preferred, and 2 to 5 is more preferred.

In (C) above, the “sequence identity” with respect to the amino acid sequences of the two peptides to be compared (that is, the peptide (A) and the peptide (C)) is a value calculated by the following formula. Point. The comparison (alignment) of multiple peptides is performed according to a conventional method so that the number of identical amino acid residues is maximized.
Sequence identity (%)
= [(number of identical amino acid residues) / (alignment length)] x 100

In (C) above, the “partial amino acid sequence consisting of the 4th to 192nd amino acid residues” corresponds to the repeating unit in the amino acid sequence shown in SEQ ID NO:1. A "partial sequence" corresponds to a repeating unit in the sequence (C) above. The peptide of (C) above may contain at least one repeating unit (partial sequence) having a sequence identity of 80% or more with the repeating unit in the amino acid sequence shown in SEQ ID NO: 1, and may contain two or more. is preferred.

When the peptide of (C) above contains a plurality of different repeating units, some of the plurality of repeating units have less than 80% sequence identity with the repeating unit in the amino acid sequence shown in SEQ ID NO: 1. may be However, in the peptide of (C) above, the total number of amino acid residues of repeating units (partial sequences) having 80% or more sequence identity with the repeating units in the amino acid sequence shown in SEQ ID NO: 1 is equal to the total number of amino acid residues 80% or more of the cardinal number is preferable.

In addition, the sequence identity between the partial sequence in the peptide (C) and the partial amino acid sequence consisting of the 4th to 192nd amino acid residues in the amino acid sequence of SEQ ID NO: 1 is from the viewpoint of tissue repair ability. Therefore, it is preferably 90% or more, more preferably 95% or more.

The length of the peptide defined in (C) above can be 151 to 2260 amino acid residues, and from the viewpoint of degradability after cross-linking, it is preferable that the number of amino acid residues is 193 or more. Preferably, the number of amino acid residues is 944 or less from the viewpoint of stability. More preferably, the length of the peptide is 380-756 amino acid residues.

The recombinant gelatin described above can be produced by genetic recombination techniques known to those skilled in the art, and may be produced, for example, according to the methods described in EP1014176A2, US6992172, WO2004/85473 and WO2008/103041. Specifically, a gene encoding a predetermined recombinant gelatin amino acid sequence is obtained, the gene is incorporated into an expression vector to prepare a recombinant expression vector, and the recombinant expression vector is introduced into an appropriate host to produce a transformant. make. Recombinant gelatin is produced by culturing the resulting transformant in an appropriate medium, and the recombinant gelatin used in the present disclosure can be prepared by recovering the recombinant gelatin produced from the culture.

A porous body containing a hydrophilic polymer can be obtained, for example, by removing water from a frozen hydrophilic polymer aqueous solution.

The porosity of the porous body is preferably 80.00% to 99.99%, more preferably 92.01% to 99.99%, for example, from the viewpoint of enhancing the affinity with the living body. . "Porosity" refers to a value calculated by the following formula.
Porosity (%)
= 100-100 x mass (g) ÷ apparent volume (cm ³ ) ÷ true density (g/cm ³ )
The apparent volume is the volume of the porous body including internal voids, and can be calculated, for example, by measuring the dimensions of the porous body using vernier calipers. True density can be measured, for example, by a pycnometer method.

The pore size of the porous body is preferably 10 μm to 2500 μm, more preferably 40 μm to 1000 μm, from the viewpoint of enhancing the affinity with the living body. The pore diameter of the porous body is measured as the diameter of a circle having the same area as the pore portion (i.e. equivalent circle diameter) when a cut surface near the center of the porous body is observed with a microscope. It is evaluated as the median equivalent circle diameter of the hole portion.

The size of the porous body before pulverization is preferably 0.1 mm to 100 mm, more preferably 0.1 mm to 50 mm, even more preferably 0.1 mm to 16 mm. By setting the size of the porous body before pulverization within the above range, the time required for pulverization can be shortened. The size of the porous body before pulverization is defined by the square root of the projected area of the porous body, that is, the length of one side of a square having the same area as the projected area.
In addition, in the present disclosure, when referring to the state of the porous body “before pulverization”, “before pulverization” means the time of starting pulverization.

The size of the porous body after pulverization is preferably 0.01 mm to 10 mm, more preferably 0.1 mm to 5 mm, even more preferably 0.3 mm to 1.4 mm. By setting the size of the pulverized porous body within the above range, the size can be made suitable for, for example, a tissue repair material. The size of the porous body after pulverization can be defined by the opening of the sieve used to sieve the porous body. The porous body that remains on the sieve when it is filtered can be a porous body with a size of 0.3 mm to 1.4 mm. The size of the porous body means the size of each porous body, and does not mean the representative value (e.g., average value, median value, etc.) of the sizes of a plurality of porous bodies. . Further, when the pulverization is performed in a plurality of times, the size of the porous body after pulverization means the size of the porous body after the final pulverization.

The density of the porous material before pulverization should be 0.3 g/cm ³ or less from the viewpoint of reducing heat generation due to frictional force by reducing the mass of the pulverized material and, for example, from the viewpoint of enhancing affinity with living organisms. is preferred, and 0.1 g/cm ³ or less is more preferred. From the viewpoint of obtaining sufficient strength, the density of the porous body is preferably 0.01 g/cm ³ or more, more preferably 0.05 g/cm ³ or more. From this point of view, the density of the porous material before pulverization is preferably 0.01 ^g /cm ³ to 0.3 g/cm 3 , more preferably 0.05 g/cm ³ to 0.1 g/cm ³ . is more preferred. The density of the porous body is calculated by dividing the mass of the porous body in a dry state by the apparent volume of the porous body including internal voids.

The water content of the porous body before pulverization is 13.5% by mass or less, and from the viewpoint of better suppression of scorching, it is more preferably 9.5% by mass or less, and 5.0% by mass. % or less. The water content is preferably 0.01% by mass or more. From this point of view, the water content of the porous material before pulverization is preferably 0.01% by mass to 13.5% by mass, more preferably 0.01% by mass to 9.5% by mass, and 0 More preferably, it is 0.01% by mass to 5.0% by mass. The "water content" of the porous body refers to the value calculated by the following formula.
Moisture content (%) = mass of water (g) ÷ sum of mass of water and mass of porous body (g) × 100
The moisture content can be measured, for example, using a Karl Fischer moisture meter.

The method for adjusting the moisture content of the porous body is not particularly limited, and the humidity may be adjusted by allowing the porous body to stand still in a space of constant humidity, or the humidity may be adjusted using a drying device. Examples of the drying apparatus include heat dryers, blower dryers, vacuum dryers, freeze vacuum dryers, constant temperature and humidity chambers, microwave dryers, and the like.

It is preferable that the moisture content of the porous body before pulverization is kept uniform throughout the porous body. For example, it is preferable that the moisture content of the porous body before pulverization is not significantly different between the inside and the outer surface of the porous body. The fact that the water content of the porous body before pulverization does not differ greatly between the inside and the outside surface of the porous body means that, for example, any part inside the porous body and any part including the outside surface of the porous body It can be confirmed by checking that the difference in moisture content measured by cutting out is within 10% by mass, preferably within 5% by mass.

The grinding method of the present disclosure is performed by dry grinding. Dry pulverization is a method of pulverizing in a gas phase (for example, in air or in an inert gas). Dry pulverization is also useful from the viewpoint of saving labor and cost required for drying for separating and removing the liquid. The apparatus used for dry pulverization is preferably an apparatus having a built-in classification mechanism from the viewpoint of obtaining a pulverized product of a desired size at a high yield, and examples thereof include a screen mill and a jet mill. Among them, a screen mill is more preferable from the viewpoint of easily obtaining a pulverized product having a size suitable for a tissue repair material. A screen mill is a pulverizing device in which perforated plates, screens, grit, etc. are arranged.

From the viewpoint of making the porous body suitable for use as a tissue repair material, the hole diameter of the screen in the screen mill is preferably 0.006 inch (0.1524 mm) to 0.25 inch (6.3500 mm), 04 inch (1.0160 mm) to 0.08 inch (2.0320 mm) is more preferable. From the viewpoint of efficient pulverization, the rotational speed of the rotary impeller is preferably 100 rpm to 6000 rpm, more preferably 1000 rpm to 3000 rpm. The air velocity in the holes of the screen is preferably 5 m/s to 20 m/s, more preferably 7 m/s to 15 m/s.
The rotating impeller of the screen mill is preferably round, square, or rake-type, and more preferably rake-type from the viewpoint of efficient pulverization.

From the viewpoint of keeping the water content of the porous body low, the absolute humidity of the atmosphere in the pulverization step is preferably 15 g/m ³ or less, more preferably 11 g/m ³ or less, and 9 g/m ³ or less. It is more preferably 4 g/m 3 or less, most preferably 4 g/m ³ or less. Also, the absolute humidity of the atmosphere in the pulverization step is preferably 0.00001 g/m ³ or more. For example, in the pulverization step, the absolute humidity of the atmosphere inside the pulverizer is preferably within the above range, and more preferably, the absolute humidity of the atmosphere inside the pulverizer and in the charging path is within the above range. It is more preferable that the absolute humidity of the surroundings and the atmosphere of the introduction path is within the above range. The absolute humidity can be calculated by the following formula, which is measured by a hygrometer, or by calculating the saturated water vapor pressure based on the Tetens formula.
Saturated water vapor pressure (hPa) = 6.1078 x 10 ^{(7.5 x temperature (°C) ÷ (237.3 + temperature (°C)))}
Water vapor pressure (hPa) = saturated water vapor pressure (hPa) x relative humidity (RH%)/100
Absolute temperature (K) = 273.15 + temperature (°C)
Absolute humidity (g/ ^m3 )
= Water vapor pressure (hPa) x 100 ÷ (8.31447 x absolute temperature (K)) x 18
The relative humidity of the atmosphere at 23° C. in the pulverization step is preferably 0% RH to 73% RH, more preferably 0% RH to 53% RH, and further preferably 0% RH to 44% RH. Preferably, 0% RH to 20% RH is most preferred. Relative humidity shall be the value measured by a hygrometer.

From the viewpoint of avoiding temperature rise of the porous body and condensation on the porous body, the room temperature in the step of pulverizing the porous body is preferably 15°C to 40°C, more preferably 20°C to 30°C.

From the viewpoint of keeping the water content of the porous body low, the pulverization time is preferably as short as possible, preferably within 1 hour, more preferably within 30 minutes, even more preferably within 10 minutes, and particularly preferably within 5 minutes. Moreover, a drying mechanism may be provided around or inside the pulverizer.

From the viewpoint of suppressing the occurrence of scorching, the porous body and the pulverizing device may be cooled, and the pulverizing device may be provided with a cooling mechanism. The cooling means is not particularly limited, and liquid nitrogen, dry ice, Peltier element, heat pump, or the like may be used.

From the viewpoint of suppressing adhesion to the device and increasing the yield of the pulverized product, the porous body and the pulverizing device may be destaticized, and the pulverizing device may be provided with a static elimination mechanism. The static elimination means is not particularly limited, and radiation irradiation, corona discharge, an antistatic agent, or the like may be used.

In the pulverization step, the pulverization may be performed only once, or may be performed multiple times from the viewpoint of homogeneous pulverization. From the viewpoint of shortening the process and suppressing the occurrence of scorching, the number of pulverization is preferably 1 to 5 times, more preferably 1 to 3 times. When pulverization is repeated, the water content of the porous body may be 13.5% by mass or less before the first pulverization, and the water content of the porous body may be adjusted only before the first pulverization. The moisture content of the porous body may be adjusted for each crushing, and is preferably adjusted for each pulverization. When pulverization is performed using an apparatus having a built-in classification mechanism, the set particle size may or may not be changed for each pulverization, and it is preferable to decrease the set particle size for each pulverization.

The use of the pulverized porous body, that is, the pulverized product is not particularly limited. According to the pulverization method of the present disclosure, from the viewpoint that it is possible to produce a porous body that suppresses the occurrence of scorching and is excellent in safety, functionality, and aesthetics, the porous body is suitable, for example, as a medical material. Used.
The porous material can be used as a tissue repair material that promotes bone regeneration in, for example, orthopedic surgery, dentistry, and the like. Since charring is inferior in terms of in vivo degradability and biocompatibility and has a risk of inhibiting bone regeneration, a production method that suppresses the occurrence of charring is preferable.

The pulverization method of the present disclosure may include other steps in addition to the pulverization step. For example, the pulverization method of the present disclosure may include drying the porous body. The step of drying the porous body is preferably carried out before and during the pulverization of the porous body, and may be carried out either way. From the viewpoint of suppressing electrostatic adhesion of the porous body to the device and improving the yield, the pulverization method of the present disclosure may include a step of neutralizing the porous body. The step of removing static electricity is preferably carried out before and during the pulverization of the porous body, and may be carried out either way.

<Manufacturing method of pulverized product>
The method for producing a pulverized product of the present disclosure comprises the steps of: making the moisture content of a porous body containing a hydrophilic polymer equal to or less than 13.5% by mass; including. The method for producing a pulverized product of the present disclosure may include the aforementioned pulverization method of the present disclosure. Details of the method for making the water content of the porous body containing the hydrophilic polymer 13.5% by mass or less, the porous body containing the hydrophilic polymer, the step of pulverizing the porous body by dry pulverization, and the pulverized material The foregoing may apply.

The method for producing a pulverized product of the present disclosure may further include other steps than the pulverization step. For example, the method for producing a pulverized product of the present disclosure may include a step of further classifying the pulverized porous material in the pulverizing step, and may include a static elimination step of removing static electricity. It may include a chemical or physical cross-linking step to adjust degradability, may include a heating, radiation, or gas sterilization step for use as a medical material, and an aseptic or non-aseptic filling step and may include mixing the pulverized material with other materials or agents.

The percentage of burnt pulverized material after the pulverization process is 0% to 15 in the number of glass vials containing burnt when 89.5 ± 3.0 mg of pulverized material is filled in a 10 mL glass vial. %, more preferably 0% to 10%, even more preferably 0% to 5%.

EXAMPLES Next, the embodiments of the present disclosure will be specifically described with reference to examples, but the embodiments of the present disclosure are not limited to these examples.

[Example 1]
(recombinant gelatin)
In this example, the recombinant peptide CBE3 was used as the recombinant gelatin contained in the porous material. As CBE3, CBE3 described below was used (described in WO2008/103041A1).

Molecular weight: 51.6 kD
Structure: GAP[(GXY) ₆₃ ] _3G
Number of amino acids: 571 RGD sequence: 12 Imino acid content: 33% (based on the number of amino acid residues)
Nearly 100% of the amino acids are GXY repeats.
CBE3 has an ERGD sequence.
The amino acid sequence of CBE3 does not contain serine, threonine, asparagine, tyrosine and cysteine residues.
Isoelectric point: 9.34
Hydrophilic repeating unit ratio in polymer: 26.1%
1/IOB value: 0.323
GRAVY score: -0.682
Amino acid sequence (SEQ ID NO: 1)
_3G

(Container used for freezing)
A cylindrical cup-shaped container made of an aluminum alloy (JIS A5056 alloy) was prepared. The container has a closed side surface and a bottom surface and an open top surface when the curved surface is used as a side surface. The bottom surface has a thickness of 5 mm, and the inner circumference of the bottom surface is chamfered with R2 mm. The inside of the side and bottom surfaces is coated with FEP (tetrafluoroethylene-hexafluoropropylene copolymer), resulting in an internal diameter of 104 mm. This container is hereinafter referred to as a "cylindrical container".

(Preparation of porous body)
After the solution containing the recombinant gelatin was purified, it was concentrated by ultrafiltration to 4.0% by mass at 30°C. After freeze-drying the resulting aqueous gelatin solution, water for injection was added to the freeze-dried product, and the mixture was heated to 37° C. over 30 minutes to re-dissolve, thereby obtaining a 7.5% by weight aqueous gelatin solution. About 20 g of the obtained aqueous gelatin solution was poured into a cylindrical container, and then placed on a 350 x 634 x 20 mm aluminum plate precooled to about -35°C, 14 of which were placed via a 1 mm thick glass plate and covered. A frozen gelatin body was obtained by standing still for 1 hour. The resulting frozen gelatin was freeze-dried using a freeze dryer (DFR-5N-B, manufactured by ULVAC, Inc.) to remove moisture, thereby preparing a freeze-dried product. The freeze-dried body obtained is an example of the porous body of the present disclosure. The freeze-dried product had a porosity of 93.9%, a pore size of 50 μm, and a density of 0.0748 g/cm ³ .

(Measurement of size of porous body)
The above-mentioned porous body is divided by hand into pieces of 20 mm square or less, and the porous body after being divided by hand is provided with gray ruled lines on a black background (both the black background and the gray ruled lines are lower in brightness than the porous body). The images were arranged on a sheet of graph paper and photographed from a direction perpendicular to the paper surface. The photographed image is binarized by image processing so that the porous body region and the rest can be separated, the region corresponding to the porous body (that is, the projected area of the porous body) is specified, and the square root of the projected area is the porous body It was calculated as the size before pulverization. As a result, the median value was 10.6 mm, the minimum value was 6.7 mm, and the maximum value was 14.5 mm. ImageJ (manufactured by National Institutes of Health) was used as image processing software.

(Moisture control of porous body)
The hand-cracked porous body was allowed to stand at 23° C. in humidity environments with relative humidity of 13% RH, 33% RH, 40% RH, and 46% RH, respectively, for 32 hours or more to equilibrate. For each humidity environment, a glycerin aqueous solution was placed in a sealed SUS container, the glycerin concentration was changed to adjust the humidity, and the humidity and temperature were measured with T&D TR-72wf (manufactured by T&D Co., Ltd.). It was confirmed. The equilibrated porous material is placed in an aluminum bag equilibrated in the same humidity environment and heat-sealed using a sealer (T-230K manufactured by Fuji Impulse Co., Ltd.) until it is put into the pulverizer. to keep the moisture content. In the following pulverization, the water content of the porous material immediately before being put into the pulverizer was 9.4% by mass, 11.3% by mass, 12.7% by mass, and 13.0% by mass, respectively, with respect to the above humidity environment. It was 3% by mass. The water content was measured by the coulometric method using a Karl Fischer moisture meter (852 Titrand manufactured by Metrohm).

(Pulverization of porous body)
About 5 g of the porous material that has been conditioned as described above is used in a screen mill (Comil U10, manufactured by Quadro Co., Ltd.), which is a dry pulverizer, and a dust collector (CKU-450AT-HC, manufactured by Chiko Airtech Co., Ltd.) is connected. The porous body was pulverized while being blown with air. Pulverization was performed twice using screens with different pore sizes. The absolute humidity of the experimental environment was between 8.2 g/m ³ and 10.7 g/m ³ .

In the first pulverization, a screen with a hole diameter of 0.079 inch (about 2.0 mm) was used. At this time, the dust collector was operated so that the air velocity in the holes of the screen was 8.5±0.1 m/s, and the rotating impeller was rotated at a rotation speed of 1100±10 rpm to carry out pulverization. The wind speed was measured using a digital anemometer (CW-60 manufactured by Custom Co., Ltd.). A rake type (7A1611) rotary impeller was used. The time required for pulverization was 1 minute.

The first pulverized product was subjected to the second pulverization after being conditioned in the same humidity environment as in the above humidity conditioning of the porous body. In the second pulverization, a screen with a hole diameter of 0.040 inch (about 1.0 mm) was used. At this time, the dust collector was operated so that the air velocity in the holes of the screen was 10.0±0.1 m/s, and the rotating impeller was rotated at a rotation speed of 2200±10 rpm to carry out pulverization. The wind speed was measured using a digital anemometer (CW-60 manufactured by Custom Co., Ltd.). A rake type (7A1611) rotary impeller was used.

After the second pulverization, the pulverized product of the fraction on the sieve with an opening of 1.4 mm and the sieve with an opening of 0.3 mm was collected, and 89.5 in a 10 mL glass vial (barrel diameter: 24.3 mm). ±3.0 mg each was filled.

(Evaluation of burning)
The pulverized material of the porous body filled in the glass vial was observed from the bottom of the glass vial with a magnifying glass, and the presence or absence of scorching (that is, coloring) was visually inspected while shaking the glass vial. Among the 30 glass vials, the number ratio of glass vials in which scorching was observed was 9.4% by mass, 11.3% by mass, 12.7% by mass, and 13.3% by mass of the moisture content of the humidified porous body. They were 0%, 10%, 6.7% and 6.7%, respectively (Fig. 1).

[Example 2]
In order to evaluate the difference in behavior during heating depending on the water content of the porous body, a heating test was conducted on porous bodies with different water contents. A porous body was prepared in the same manner as in Example 1, cut into 1 cm squares with scissors, then conditioned in the same manner, and the moisture content was measured in the same manner. A 12.4% porous body was prepared. For each moisture content, 21 pieces were placed on a hot plate (DP-1L manufactured by AS ONE Corporation) with a surface temperature of 220° C. and heated for 10 seconds. As a result of examining the ratio of the porous bodies that softened and expanded by heating (Fig. 2) and the porous bodies that did not soften and expand (Fig. 3), the ratios of the number that softened and expanded by heating were 0% and 19%, respectively. There was (Fig. 4). The number of softened and expanded porous bodies is the number of porous bodies that swelled at the central portion and that the swollen portion was burned and colored by being pressed against a heat source. FIG. 2 shows a porous body that softens and expands when heated and is pressed against a heat source and burnt at the center. FIG. 3 shows a porous body that does not soften and expand when heated and is warped to escape from the heat source.

[Comparative Example 1]
In order to verify the effect of the water content, the porous material was pulverized in the humidity environment of 23° C. and the relative humidity of 60% RH in Example 1 above. The other apparatus configuration, recombinant gelatin, method for producing a porous body, pulverization method for the porous body, method for measuring the moisture content, and method for evaluating charring are the same as in Example 1 above, so description thereof is omitted. . The water content of the porous body immediately before being put into the apparatus was 14.6% by mass. The number ratio of glass vials in which scorching was observed was 46.7% (Fig. 1).

[Comparative Example 2]
In order to evaluate the difference in behavior during heating depending on the water content of the porous body, a heating test of the porous body was carried out in Example 2 above, with the water content of the porous body set to 15.1% by mass. The other apparatus configuration, recombinant gelatin, porous body preparation method, moisture content measurement method, porous body heating method, and softening expansion behavior evaluation method are the same as in Example 2, so description is omitted. do. The percentage of the number that softened and expanded by heating was 57% (Fig. 4).

From the above, it is presumed that the scorching during pulverization of the porous body is caused by expansion during heating due to the moisture content of the porous body. Thus, it was confirmed that the occurrence of charring could be suppressed. It was also confirmed that when the water content of the porous body before pulverization is 9.5% by mass or less, the occurrence of scorching can be suppressed particularly well.

Claims (14)

including pulverizing a porous body containing a hydrophilic polymer,
The water content of the porous body before pulverization is 13.5% by mass or less,
The grinding method is dry grinding,
A method for pulverizing a porous body. The pulverization method according to claim 1, wherein the hydrophilic polymer has a 1/IOB value of 0 to 1.0. 3. The grinding method according to claim 1 or 2, wherein the hydrophilic polymer comprises a polypeptide. The grinding method according to any one of claims 1 to 3, wherein the GRAVY score of said polypeptide is negative. The grinding method according to any one of claims 1 to 4, wherein the polypeptide comprises recombinant gelatin. 6. The pulverization method according to claim 5, wherein the recombinant gelatin is represented by the following formula.
Formula: A-[(Gly-X-Y) _n ] _m -B
In the formula, A represents an amino acid residue or an amino acid sequence, B represents an amino acid residue or an amino acid sequence, X independently represents an amino acid residue, Y independently represents an amino acid residue, and n is 3 represents an integer of ~100 and m represents an integer of 2-10. 7. The grinding method according to claim 5 or 6, wherein the recombinant gelatin contains the following polypeptide (A), (B) or (C).
(A) A polypeptide consisting of the amino acid sequence of SEQ ID NO:1.
(B) A polypeptide consisting of an amino acid sequence in which one or several amino acid residues are modified in the amino acid sequence of SEQ ID NO: 1 and having biocompatibility.
(C) consisting of an amino acid sequence having a partial sequence having 80% or more sequence identity with the partial amino acid sequence consisting of the 4th to 192nd amino acid residues in the amino acid sequence of SEQ ID NO: 1, and biocompatibility A polypeptide having The pulverization method according to any one of claims 1 to 7, wherein the pulverization device used for the dry pulverization is a screen mill. The pulverization method according to any one of claims 1 to 8, wherein the density of the porous body before pulverization is 0.3 g/cm ³ or less. Claims 1 to 9, wherein the size of the porous body before pulverization is 0.1 mm to 100 mm, and the size of the porous body before pulverization is the square root of the projected area of the porous body. The pulverization method according to any one of the above. Claim 1, wherein the size of the porous body after pulverization is 0.01 mm to 10 mm, and the size of the porous body after pulverization is defined by the opening of a sieve obtained by sieving the porous body. The pulverization method according to any one of claims 1 to 10. The pulverization method according to any one of claims 1 to 11, wherein the pulverization of the porous body is performed in an atmosphere with an absolute humidity of 15 g/m ³ or less. The pulverization method according to any one of claims 1 to 12, wherein the porous body is a medical material. a step of reducing the water content of the porous body containing the hydrophilic polymer to 13.5% by mass or less;
a step of pulverizing the porous body by dry pulverization after the step;
A method for producing a pulverized product, comprising: