Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/14—Macromolecular materials
  • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
  • A61L27/222—Gelatin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/56—Porous materials, e.g. foams or sponges

Definitions

• the present disclosure relates to a tissue repair material and a method of manufacturing the tissue repair material.
• Regenerative medicine is a new medical treatment that uses three factors: cells, scaffolds, and growth factors to recreate the form and function of the original tissue in living tissues that cannot be recovered using only the body's natural healing ability. It's technology.
• bone regeneration in the field of orthopedics or dentistry is an area that is attracting much attention in the field of regenerative medicine. If the bone disease occurs in the legs or lower back, the bone loss caused by the disease will make it impossible to walk, and if the bone disease is in the periodontal tissues, it will be difficult to eat, so the bone disease is significant. It causes a decline in QOL (Quality of Life).
• WO 2013/137268 discloses a tissue repair material containing crosslinked gelatin.
• tissue repair materials are required to have excellent tissue repair ability, and for example, after applying the tissue repair material to a defective or damaged area, the tissue repair rate is high after about 4 weeks have elapsed. (hereinafter also referred to as tissue repair properties) is required.
• a problem to be solved by an embodiment of the present disclosure is to provide a tissue repair material with excellent tissue repair properties and a method for manufacturing the tissue repair material.
• the present disclosure includes the following aspects. ⁇ 1> In the elution curve of the cross-linked gelatin containing cross-linked gelatin and obtained by gel permeation chromatography under the following conditions, an absorption peak with an absorbance of 0.025 or more is present in a molecular weight range of 14 kDa or more and 51 kDa or less. At least one tissue repair material is present.
• tissue repair material according to ⁇ 1> above which has an absorption peak in a molecular weight range of 14 kDa or more and less than 28 kDa, and a molecular weight range of 28 kDa or more and less than 43 kDa.
• ⁇ 4> According to any one of ⁇ 1> to ⁇ 3> above, wherein the residual rate of the crosslinked gelatin is 60% or less after a 3-hour decomposition treatment using 1 mol/L (liter) of hydrochloric acid. tissue repair material.
• the gelatin is a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1; a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 with one or several amino acids deleted, substituted, or added, and A peptide that has biocompatibility; and an amino acid sequence that has 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 set forth in SEQ ID NO: 1;
• the tissue repair material according to any one of ⁇ 1> to ⁇ 4> above, which is one or more selected from the group consisting of peptides having affinity.
• the ratio of the area where the molecular weight is 14 kDa or more and 43 kDa or less to the area where the molecular weight is more than 43 kDa and 51 kDa or less is 2.5 or more, The tissue repair material according to any one of the above.
• a method for producing a tissue repair material comprising: ⁇ 8> The method for producing a tissue repair material according to ⁇ 7> above, wherein the radiation is a gamma ray or an electron beam.
• the molecular weight is The tissue repair according to ⁇ 7> or ⁇ 8> above, which has an absorption peak in a range of 14 kDa or more and 51 kDa or less, a molecular weight range of 14 kDa or more and less than 28 kDa, and a molecular weight range of 28 kDa or more and less than 43 kDa.
• Method of manufacturing wood is The tissue repair according to ⁇ 7> or ⁇ 8> above, which has an absorption peak in a range of 14 kDa or more and 51 kDa or less, a molecular weight range of 14 kDa or more and less than 28 kDa, and a molecular weight range of 28 kDa or more and less than 43 kDa.
• the gelatin is a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1; a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 with one or several amino acids deleted, substituted, or added, and A peptide that has biocompatibility; and an amino acid sequence that has 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 set forth in SEQ ID NO: 1;
• the method for producing a tissue repair material according to any one of ⁇ 7> to ⁇ 12> above, which is one or more selected from the group consisting of: peptides having affinity;
• tissue repair material with excellent tissue repair properties and a method for manufacturing the tissue repair material.
• FIG. 1 shows the elution curve obtained from the tissue repair material of Example 1.
• FIG. 2 shows the elution curve obtained from the tissue repair material of Example 2.
• FIG. 3 shows the elution curve obtained from the tissue repair material of Example 3.
• FIG. 4 shows the elution curve obtained from the tissue repair material of Example 4.
• FIG. 5 shows the elution curve obtained from the tissue repair material of Example 5.
• FIG. 6 shows the elution curve obtained from the tissue repair material of Comparative Example 1.
• a numerical range indicated using “ ⁇ ” indicates a range that includes the numerical value written before “ ⁇ ” as the lower limit value and the numerical value written before “ ⁇ ” as the upper limit value.
• the upper limit or lower limit described in a certain numerical range may be replaced with the upper limit or lower limit of another numerical range described step by step.
• the upper limit or lower limit described in a certain numerical range may be replaced with the value shown in the Examples.
• amino acid sequences constituting gelatin are expressed using the one-letter notation (for example, "G” for a glycine residue) or the three-letter notation (for example, "Gly” for a glycine residue) well known in the art.
• G for a glycine residue
• Gly for a glycine residue
• Gly-XY Gly represents a glycine residue
• X and Y represent any amino acid residue other than the glycine residue.
• crosslinked gelatin means gelatin that is crosslinked with each other.
• the amount of each component in the composition refers to the total amount of the corresponding multiple substances present in the composition. means.
• step includes not only independent steps but also steps that cannot be clearly distinguished from other steps if the intended purpose is achieved.
• the absorption peak of crosslinked gelatin is confirmed as follows. First, a tube with a capacity of 15 mL is filled with 5 mg of tissue repair material, 5 mL of water for injection is added, and then incubated at 37° C. for 24 hours. Next, after mixing by inversion, 1 mL of the supernatant is collected using a low adsorption tube. The fractionated solution is filtered using a 0.22 ⁇ m pore size filter and used as a measurement sample. Gel permeation chromatography is performed on 5 ⁇ L of the measurement sample under the following conditions to obtain an elution curve with absorbance on the vertical axis and elution time on the horizontal axis.
• a substance with a known molecular weight such as a polystyrene standard substance, is measured at the same or different timing than the measurement sample, and the relationship between elution time and molecular weight in the elution curve is determined. The presence or absence of an absorption peak is confirmed from the elution curve obtained as described above. Note that in the present disclosure, the absorption peak includes not only a waveform at which the absorbance is maximum, but also a waveform having a shoulder portion indicated by the symbol X in FIG. 1 . As the column, ACQUITY UPLC Protein BEH SEC Column, 200 ⁇ , 1.7 ⁇ m, 4.6 mm x 150 mm, or a column with equivalent performance can be used.
• the heating temperature of gelatin refers to the set temperature of the device used for heating, and is not the temperature of gelatin to be heated.
• the water absorption rate of the tissue repair material is measured as follows. First, a filter cup whose mass (w0) has been measured is set in a microtube (hereinafter referred to as tube). Next, 500 ⁇ L of water is added to the filter cup and stirred on a rotator for 2 hours. After stirring, the tube is centrifuged at 25° C. and 6000 ⁇ g for 1 minute to confirm that water has moved from the filter cup into the tube. The mass (w1) of the filter cup is measured again, and the amount of remaining water is calculated using the following formula.
• the acid decomposition residual rate of the tissue repair material is measured as follows.
• a microtube (hereinafter referred to as tube) is prepared and its mass (A) is measured.
• 1.7 mL of 1 mol/L hydrochloric acid is added to the tube containing the tissue repair material, and heated for 3 hours using a heat block set at 37°C. After heating, the tube is placed on ice to stop the acid decomposition reaction, and centrifuged at 10,000 xg for 1 minute using a centrifuge preset at 4°C.
• the molecular weight of crosslinked gelatin is a molecular weight calculated using polystyrene as a standard substance, and is measured by the gel permeation chromatography under the above conditions.
• the radiation dose is measured by the following method. Multiple vials filled with cross-linked gelatin are placed in a cardboard box, and a polymethyl methacrylate dosimeter is attached to the side of the cardboard box. After irradiation with radiation in this state, the dose of radiation measured by the dosimeter is taken as the dose of radiation irradiated to the crosslinked gelatin.
• tissue repair material contains crosslinked gelatin, and in the elution curve of the crosslinked gelatin obtained by gel permeation chromatography under the following conditions, the molecular weight is in the range of 14 kDa or more and 51 kDa or less, and the absorbance is 0.025. At least one of the above absorption peaks exists.
• the tissue repair material is sometimes required to have a high tissue repair rate (hereinafter also referred to as tissue repair rate) after approximately two weeks have passed after being applied to a defective or damaged site.
• tissue repair rate tissue repair rate
• the presence of at least one absorption peak with an absorbance of 0.025 or more in a molecular weight range of 14 kDa or more and 51 kDa or less tends to improve the tissue repair speed of the tissue repair material.
• the absorbance of the absorption peak is preferably 0.025 or more, more preferably 0.03 or more, and even more preferably 0.035 or more.
• the upper limit of the absorbance of the absorption peak is not particularly limited, and can be 0.06 or less.
• the absorption peaks preferably exist in a range where the molecular weight is 14 kDa or more and less than 28 kDa, and a range where the molecular weight is 28 kDa or more and less than 43 kDa, and when the molecular weight is 43 kDa or more. , 51 kDa or less, a molecular weight of 28 kDa or more and less than 43 kDa, and a molecular weight of 14 kDa or more and less than 28 kDa.
• the ratio of the area where the molecular weight is 14 kDa or more and 43 kDa or less to the area where the molecular weight is more than 43 kDa and 51 kDa or less is preferably 2.5 or more. , more preferably 3.0 or more, and still more preferably 7.0 or more.
• the upper limit of the above area ratio is not particularly limited and can be 30.00 or less.
• the area where the molecular weight is more than 43 kDa and 51 kDa or less, and the area where the molecular weight is 14 kDa or more and 43 kDa or less are determined from the elution curve obtained as described above. Specifically, this is performed by determining the area of the region surrounded by the elution curve and the baseline in a range where the molecular weight is greater than 43 kDa and less than 51 kDa, or in a range where the molecular weight is greater than or equal to 14 kDa and less than 43 kDa.
• the baseline refers to a straight line where the absorbance becomes 0.
• the water absorption rate of the tissue repair material of the present disclosure is preferably 450% or more, more preferably 500% or more, and even more preferably 600% or more.
• the upper limit of the water absorption rate of the tissue repair material is not particularly limited, but is preferably 9900% or less, more preferably 5000% or less, still more preferably 3000% or less.
• the water absorption rate of the tissue repair material can be adjusted by the components contained in the tissue repair material, the type of crosslinked gelatin, and the form of the crosslinked gelatin.
• the water absorption rate can be adjusted by adjusting the freezing step, the temperature of the crosslinking step, the crosslinking treatment time of the crosslinking step, etc. in the method for producing the tissue repair material.
• water absorption tends to increase when the temperature of the freezing step is raised, the temperature of the crosslinking step is lowered, or the crosslinking treatment time of the crosslinking step is shortened.
• the tissue repair material of the present disclosure has a residual rate (acid decomposition residual rate) after a 3-hour decomposition treatment using 1 mol/L (liter) of hydrochloric acid on a mass basis. It is preferably 60% or less, more preferably 55% or less, even more preferably 50% or less.
• the acid-decomposed residual rate of the tissue repair material is preferably 5% by mass or more, more preferably 20% by mass or more, from the viewpoint of maintaining the volume at the defect site and replacing it with tissue to be regenerated.
• the content is preferably 40% by mass or more, and more preferably 40% by mass or more.
• the acid-decomposed residual rate of the tissue repair material can be adjusted by the components contained in the tissue repair material, the type of crosslinked gelatin, and the form of the crosslinked gelatin. Further, the acid decomposition residual rate can be adjusted by the freezing step, the temperature of the crosslinking step, the crosslinking treatment time of the crosslinking step, etc. in the method for producing a tissue repair material. Generally, when the temperature of the freezing step is raised, the temperature of the crosslinking step is lowered, or the crosslinking treatment time of the crosslinking step is shortened, the acid decomposition residual rate tends to decrease.
• the tissue repair material of the present disclosure contains crosslinked gelatin obtained by subjecting gelatin to heat treatment, crosslinking agent treatment, and the like.
• cross-linked gelatin is preferably obtained by heat-treating gelatin, and in the present disclosure, heat-treated cross-linked gelatin is also referred to as dehydrated cross-linked gelatin.
• Dehydrated crosslinked gelatin is preferable from the viewpoint of biosafety because it does not require the use of a crosslinking agent or only uses a small amount of crosslinking agent, and is less likely to cause cytotoxicity, inflammation, etc.
• the gelatin used to produce crosslinked gelatin may be a natural gelatin, or may be a mutant or recombinant gelatin that differs from the natural gelatin in at least one amino acid residue.
• natural gelatin refers to 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 gelatins are collectively referred to as recombinant gelatin in this disclosure.
• natural gelatin or recombinant gelatin examples include those derived from animals such as fish and mammals, but natural gelatin or recombinant gelatin derived from mammals is preferred. Examples of mammals include humans, horses, pigs, mice, rats, etc., and humans or pigs are more preferable.
• the natural gelatin is preferably a pig-derived or human-derived natural gelatin, and the recombinant gelatin is preferably a human-derived recombinant gelatin.
• gelatin refers to a polypeptide containing six or more consecutive amino acid sequences represented by Gly-XY, and includes other amino acid residues in the polypeptide in addition to the amino acid sequence represented by Gly-XY. It may have one or more groups.
• Gly-XY Gly represents a glycine residue
• X and Y represent any amino acid residue other than the glycine residue.
• the amino acid sequence represented by Gly-XY is a sequence corresponding 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.
• the plurality of Gly-XYs in one molecule of gelatin may be the same or different. Further, in the Gly-XY sequence, X and Y are independent for each repeating unit and may be the same or different.
• X and Y contain a large amount of imino acid residues (specifically, proline residues or oxyproline residues).
• the content of such imino acid residues is preferably 10% by mass to 45% by mass in one molecule of gelatin.
• the content of Gly-XY in one molecule of gelatin is preferably 80% by mass or more, more preferably 95% by mass or more, and most preferably 99% by mass or more.
• Gelatin is made by changing one or more bases or amino acid residues to the base sequence or amino acid sequence of a gene encoding collagen that has six or more consecutive amino acid sequences represented by Gly-XY.
• the gelatin is a recombinant gelatin obtained by introducing and expressing a gene having a base sequence or an amino acid sequence into a suitable host by a conventional method.
• Examples of recombinant gelatin include EP1014176A2, US6992172B1, WO2004/85473A2, WO2008/103041A1, Special Table of Contents No. 2010-519293, Special Publication No. 2010-519252, Special Publication No. 2010-518833, and Special Publication No. 2010- Publication No. 519251, Those disclosed in WO2010/128672A1, WO2010/147109A1, etc. can be particularly preferably used.
• the molecular weight of gelatin is preferably 2 kDa to 100 kDa, more preferably 5 kDa to 90 kDa, and even more preferably 10 kDa to 90 kDa.
• gelatin preferably further contains a cell adhesion signal, and more preferably has two or more cell adhesion signals in one molecule.
• cell adhesion signals include RGD sequence, LDV sequence, REDV sequence, YIGSR sequence, PDSGR sequence, RYVVLPR sequence, LGTIPG sequence, RNIAEIIKDI sequence, IKVAV sequence, LRE sequence, DGEA sequence, and HAV sequence.
• RGD sequence, YIGSR sequence, PDSGR sequence, LGTIPG sequence, IKVAV sequence or HAV sequence and more preferably RGD sequence.
• the ERGD sequence is more preferred.
• the number of amino acid residues between RGD sequences is preferably 0 to 100, more preferably 25 to 60. Further, the RGD sequence is preferably arranged non-uniformly within the above range of the number of amino acid residues.
• the ratio of RGD sequences to the total number of amino acid residues in gelatin is preferably at least 1.2%, and the recombinant gelatin contains 250 or more amino acid residues. of amino acid residues, each stretch of 250 amino acid residues preferably contains at least one RGD sequence. More preferably, the gelatin 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.
• the gelatin sequence preferably satisfies at least one of the following aspects, but is not limited thereto.
• Gelatin may be partially hydrolyzed.
• the gelatin has the amino acid sequence A-[(Gly-XY) n ] m -B.
• A represents one or more arbitrary amino acid residues
• B represents one or more arbitrary amino acid residues
• Gly represents a glycine residue
• n Xs represent a glycine residue.
• the n Y's each independently represent an arbitrary amino acid residue.
• m represents an integer of 2 to 10, preferably an integer of 3 to 5.
• n represents an integer of 3 to 100, preferably an integer of 15 to 70, more preferably an integer of 50 to 65.
• the m Gly-XYs may be all the same, partially the same, or different from each other.
• the recombinant gelatin has the amino acid sequence Gly-Ala-Pro-[(Gly-XY) 63 ] 3 -Gly.
• the 63 X's each independently represent any amino acid residue
• the 63 Y's each independently represent any amino acid residue. All of the 63 Gly-XYs may be the same, some of them may be the same, or they may be different from each other. It is preferable that the repeating unit of gelatin is formed by forming a part of the amino acid sequence of naturally occurring collagen into one unit and bonding a plurality of such units.
• the naturally occurring collagen mentioned here preferably includes type I collagen, type II collagen, type III collagen, type IV collagen, and type V collagen, and type I collagen, type II collagen, or type III collagen is more preferable.
• the collagen is preferably human, horse, pig, mouse, or rat-derived collagen, and more preferably human-derived collagen.
• the isoelectric point of gelatin is preferably from 5 to 10, more preferably from 6 to 10, even more preferably from 7 to 9.5. Note that the isoelectric point of gelatin is calculated based on the amino acid composition of gelatin.
• Gelatin preferably satisfies at least one of the following aspects, but is not limited thereto.
• gelatin is a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 below (hereinafter also referred to as specific peptide A); A peptide consisting of an amino acid sequence (hereinafter also referred to as specific amino acid sequence B) in which five amino acids are deleted, substituted, or added and has biocompatibility (hereinafter also referred to as specific peptide B); and SEQ ID NO: 1 consists of an amino acid sequence (hereinafter also referred to as specific amino acid sequence C) that has 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 described in It is preferable that the peptide is one or more selected from the group consisting of peptides having affinity (hereinafter also referred to as specific peptide C).
• GAP GAP(GAPGLQGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGERGAAGLPPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGPAGAPGAPGLQGMPGERGAA GLPGPKGERGDAGPKGADGAPGKDGVRGLAGPP) 3 G (SEQ ID NO: 1)
• the number of amino acid residues to be deleted, substituted or added may be one or several, and can be from 2 to 15, preferably from 2 to 5. .
• the sequence identity of specific peptide C is preferably 90% or more, more preferably 95% or more.
• the partial amino acid sequence is a partial amino acid sequence corresponding to a repeating unit of the sequence shown in SEQ ID NO:1.
• the specific peptide C may contain two or more specific amino acid sequences C.
• the ratio of the number of amino acid residues included in the specific amino acid sequence C to the total number of amino acid residues included in the specific peptide C is preferably 80% or more.
• Recombinant gelatin can be produced by genetic recombination techniques known to those skilled in the art. For example, it can be manufactured according to the method described in EP1014176A2, US6992172B1, WO2004/85473A2, WO2008/103041A1, etc.
• the number of amino acid residues contained in gelatin can be 151 to 2260, and from the viewpoint of degradability after crosslinking, it is preferably 193 or more, and from the viewpoint of stability, it is 944 or less.
• the number is preferably 380 to 756, and more preferably 380 to 756.
• crosslinked gelatin is not particularly limited, and examples include forms such as sponge, film, nonwoven fabric, particles, and mesh. Among these, from the viewpoints of cell invasion, tissue repair speed, and tissue repair properties, the crosslinked gelatin is preferably in the form of particles (hereinafter, particulate crosslinked gelatin is also referred to as crosslinked gelatin particles).
• the crosslinked gelatin particles are preferably particles that pass through a 4 mm sieve, more preferably particles that pass through a 1400 ⁇ m sieve, and 1000 ⁇ m sieves. It is more preferable that the particles pass through a sieve, and it is particularly preferable that the particles pass through a sieve having an opening of 710 ⁇ m. From the viewpoint of the elasticity of the layer formed by arranging the tissue repair material of the present disclosure in a predetermined space (hereinafter also referred to as a formulation layer), it is important that the crosslinked gelatin particles remain on a sieve with an opening of 75 ⁇ m. is preferable, and more preferably remains on a sieve with an opening of 300 ⁇ m.
• a test sieve according to ISO3310 standard is used to sieve the crosslinked gelatin particles, and the sieving method is in accordance with the sieving method described in Method 2 of Section 3.04 of the 16th edition of the Japanese Pharmacopoeia. That is, shaking for 5 minutes was performed multiple times intermittently, and after shaking, the mass of the particles remaining on the sieve was 5% or less of the mass of the particles on the sieve before shaking. When it ends.
• the term “pass through” means that the particles remaining on the sieve at the end point are no more than 10% by weight of the total mass before the sieve.
• the term “remains” means that the particles remaining on the sieve at the end point are 95% by weight or more of the total mass before sieving.
• crosslinked gelatin is preferably a porous material.
• the porosity of crosslinked gelatin is preferably 80% to 99.99%, more preferably 95.01% to 99.9%. preferable.
• the porosity of crosslinked gelatin is determined using the bulk density ( ⁇ ) of crosslinked gelatin, the true density ( ⁇ c) of crosslinked gelatin, and the following formula.
• the bulk density ( ⁇ ) is calculated from the dry mass and volume of the crosslinked gelatin, and the true density ( ⁇ c) of the crosslinked gelatin is determined by the pycnometer method using a Gay-Lussac type pycnometer.
• Porosity P (%) (1- ⁇ / ⁇ c) ⁇ 100
• the tap density ( ⁇ t) is determined by the method described below.
• the true density ( ⁇ c) is determined by the pycnometer method using a Hubbard pycnometer.
• the crosslinked gelatin particles may have communicating pores. Because the crosslinked gelatin particles have communicating pores, voids are continuous from the outside to the inside of the tissue repair material, allowing cells that have come into contact with the outside of the tissue repair material to disperse or diffuse into the inside of the tissue repair material. .
• the diameter of the communicating hole is preferably 10 ⁇ m to 2500 ⁇ m, more preferably 50 ⁇ m to 2500 ⁇ m, and even more preferably 100 ⁇ m to 1000 ⁇ m, in order to exhibit the above function.
• the tap density of the crosslinked gelatin particles is preferably 10 mg/cm 3 to 500 mg/cm 3 , more preferably 30 mg/cm 3 to 450 mg/cm 3 . It is more preferably 50 mg/cm 3 to 420 mg/cm 3 , particularly preferably 140 mg/cm 3 to 280 mg/cm 3 .
• tap density is a value that represents how many particles can be densely packed into a certain volume, and the smaller the value, the more complex the structure of the formulation layer tends to be, and the smaller the value, the more complex the structure of the formulation layer, and the smaller the value tends to be wide, and the particles tend to be coarsely packed.
• the tap density measurement method is performed as follows. First, a cylindrical (volume: 0.616 cm 2 ) container (hereinafter referred to as a cap) with a diameter of 6 mm and a length of 21.8 mm is prepared, and the mass of only the cap is measured (wt). Next, the cap and the funnel are connected, and the crosslinked gelatin particles are poured through the funnel so that they accumulate in the cap. After pouring in a sufficient amount of crosslinked gelatin particles, tap the cap 200 times on a hard surface such as a desk, remove the funnel, and scrape off the crosslinked gelatin particles that have risen beyond the edge of the cap with a spatula. The mass of the crosslinked gelatin particles completely filled in the cap is measured (wg).
• the content of crosslinked gelatin with respect to the total mass of the tissue repair material of the present disclosure is preferably 70% by mass or more, more preferably 80% by mass or more, and 90% by mass or more. It is more preferably at least 95% by mass, particularly preferably at least 95% by mass.
• the upper limit of the content of crosslinked gelatin is not particularly limited, and may be 100% by mass.
• the tissue repair material of the present disclosure may contain components other than crosslinked gelatin, including components related to bone regeneration or new bone formation, such as osteoinductive agents.
• osteoinductive drugs include bone morphogenetic protein (BMP), basic fibroblast growth factor (bFGF), and the like.
• the method for producing a tissue repair material of the present disclosure includes a step of heating gelatin at 100° C. to 170° C. for 2 hours to 24 hours to obtain crosslinked gelatin (hereinafter referred to as a crosslinking step); A step of irradiating radiation at a dose of 5 kGy to 50 kGy (hereinafter referred to as a radiation irradiation step).
• the method for producing a tissue repair material of the present disclosure includes, before the step of obtaining crosslinked gelatin, a step of preparing a gelatin solution containing gelatin dissolved in an aqueous medium (hereinafter referred to as a gelatin solution preparation step), and a step of freeze-drying the gelatin solution.
• a gelatin solution preparation step a step of preparing a gelatin solution containing gelatin dissolved in an aqueous medium
• a step of freeze-drying the gelatin solution may include a step of obtaining a freeze-dried product (hereinafter referred to as a freeze-drying step) and a step of pulverizing the freeze-dried product to obtain a pulverized product (hereinafter referred to as a crushing step).
• the gelatin solution in order to obtain crosslinked gelatin, which is a porous body, the gelatin solution is cooled to below the ice crystal formation temperature (hereinafter referred to as ice crystal formation temperature) before the freeze drying step. ) is preferable.
• the method for producing a tissue repair material of the present disclosure includes the step of heating gelatin at 100° C. to 170° C. for 2 hours to 24 hours to obtain crosslinked gelatin. Since the type of gelatin to be crosslinked has been described above, the description thereof will be omitted here. Further, the gelatin to be crosslinked may be a pulverized freeze-dried product of a gelatin solution, which will be described later.
• the heating temperature is preferably 120°C to 170°C, more preferably 120°C to 150°C, and even more preferably 130°C to 140°C.
• the heating time is preferably 2 hours to 20 hours, more preferably 4 hours to 12 hours, and even more preferably 4.5 hours to 8 hours. preferable.
• Heating of gelatin is preferably carried out under reduced pressure, vacuum or an inert gas atmosphere, more preferably carried out under vacuum or an inert gas atmosphere, and preferably carried out under an inert gas atmosphere to prevent oxidation. is even more preferable.
• the environment is preferably 4 hPa or less. Nitrogen is preferred as the inert gas.
• a conventionally known heating device can be used to heat the gelatin, for example, DP-43 manufactured by Yamato Scientific Co., Ltd. can be used.
• the radiation used in the radiation irradiation process includes alpha rays, beta rays, gamma rays, neutron beams, electron beams, and X-rays. Among these, from the viewpoint of tissue repair speed and tissue repairability, gamma rays or Electron beams are preferred, and gamma rays are more preferred.
• the radiation dose is preferably 10 kGy to 48 kGy, more preferably 12 kGy to 45 kGy, even more preferably 12 kGy to 30 kGy.
• the preferable numerical range of the irradiation dose and irradiation time is the preferable numerical range when crosslinked gelatin is irradiated with radiation from one direction, and when irradiating from two or more directions, it is not limited to the above and may be adjusted as appropriate. Adjustment is preferred.
• a gelatin solution may be prepared by dissolving gelatin in an aqueous medium, or a prepared gelatin solution may be prepared.
• the aqueous medium that can be used is not particularly limited as long as it can dissolve gelatin and can be used for living tissues, and examples include water, physiological saline, phosphate buffer, and the like.
• the content of gelatin with respect to the total mass of the gelatin solution is not particularly limited, and is preferably 0.5% to 20% by mass, more preferably 2% to 16% by mass, and 4% to 4% by mass. More preferably, it is 12% by mass.
• the strength of the tissue repair material tends to increase, and when the gelatin content is 20% by mass or less, the tissue repair material has a highly uniform mesh. It becomes easier to form a structure, and tissue repair speed and tissue repairability tend to be improved.
• the temperature of the aqueous medium when preparing the gelatin solution can be 0°C to 60°C, preferably 3°C to 30°C.
• the gelatin solution may contain components other than gelatin, such as a crosslinking agent.
• the gelatin solution is freeze-dried to obtain a freeze-dried product.
• the method for producing a tissue repair material of the present disclosure includes an ice crystal formation step, the cooled gelatin solution is freeze-dried.
• the freezing conditions conditions normally used for protein freeze-drying may be used as they are. Freeze-drying time can be, for example, 0.5 to 300 hours. There are no particular restrictions on the freeze dryer that can be used.
• the lyophilized gelatin is pulverized to obtain a pulverized product.
• the pulverization can be carried out using a pulverizer such as a hammer mill or a screen mill. It is preferable to use a screen mill from the viewpoint that the pulverized material pulverized to a certain size can be recovered at any time and the variation in particle size is small.
• a screen mill a Cormill manufactured by Quadro, etc. can be used.
• Examples of the pulverization method include a crushing method and a cutting method.
• the pulverization step may include pulverizing the freeze-dried gelatin to obtain a pulverized product, and then classifying the pulverized product. Thereby, a pulverized product having a uniform particle size can be obtained.
• classification it is preferable to use, for example, a sieve with an opening of 300 ⁇ m to 1400 ⁇ m.
• the method for producing a tissue repair material of the present disclosure can include an ice crystal formation step before the freeze-drying step. Thereby, a gelatin-containing intermediate having ice crystals inside can be obtained.
• the formed ice crystals cause the peptide chains of gelatin to become coarser and the gelatin-containing intermediate solidifies, so that after the ice crystals disappear, a gelatin-containing intermediate having voids inside is formed.
• the disappearance of ice crystals can be achieved by drying in the freeze-drying process.
• the pore size of the voids in the gelatin-containing intermediate can be adjusted by adjusting the ice crystal temperature, cooling time, etc.
• the shape of the void there is no particular restriction on the shape of the void, and it may be a two-dimensional structure or a three-dimensional structure.
• the cross-sectional shape of the mesh is not particularly limited, and examples include polygons, circles, and ellipses.
• Examples of the three-dimensional structure of the void include columnar, spherical, and the like. From the viewpoint of tissue repair speed and tissue repairability, the shape of the void is preferably spherical.
• the gelatin-containing intermediate may have communicating pores in which voids are continuously formed. Since the communicating holes have been described above, their description will be omitted here.
• the pore diameter of the voids is preferably 10 ⁇ m or more, more preferably 50 ⁇ m or more, and even more preferably 100 ⁇ m or more, from the viewpoint of tissue repair speed and tissue repair properties.
• the pore diameter of the void is the average diameter in the major axis direction (major axis), and is measured as follows.
• a test piece obtained by cutting the dried intermediate obtained after drying the gelatin-containing intermediate in the horizontal direction and a test piece cut in the vertical direction are prepared.
• the horizontal direction of the dry intermediate means a direction that is horizontal to a flat surface when the dry intermediate is left standing. Note that the dry intermediate is left standing so that the area in contact with a flat surface is maximized.
• the cross section of each test piece is stained by bringing it into close contact with a stamp stand, and a 2.0 mm x 2.0 mm area is observed using an optical microscope.
• the circumscribed rectangle in which the distance between two opposing sides of the rectangle is the largest is selected.
• the length of the long side of the circumscribed rectangle where the distance between the two opposing sides is maximum is 50 within the observation area of each of the cross section of the test piece cut in the horizontal direction and the cross section of the test piece cut in the vertical direction.
• the measurement is performed one by one, and the average value is taken as the average value of the long diameter of the voids in the gelatin-containing intermediate.
• the aspect ratio of the void is between 1 and 3, it is defined as “spherical", and when it is outside this range, it is defined as “columnar”.
• the aspect ratio is preferably 4 or 5 from the viewpoint of tissue repair speed and tissue repairability.
• the porosity of the gelatin-containing intermediate is preferably 80% to 99.99%, more preferably 95.01% to 99.9%.
• the porosity of the gelatin-containing intermediate is determined using the bulk density ( ⁇ 1) of the gelatin-containing intermediate, the true density ( ⁇ c1) of crosslinked gelatin, and the following formula.
• the bulk density ( ⁇ 1) is calculated from the dry mass and volume of the gelatin-containing intermediate, and the true density ( ⁇ c1) of the gelatin-containing intermediate is determined by the pycnometer method using a Gay-Lussac type pycnometer.
• Porosity P1 (%) (1- ⁇ 1/ ⁇ c1) x 100
• Ice crystal formation temperature means the temperature at which at least a portion of the gelatin solution freezes.
• the ice crystal formation temperature varies depending on the solid content concentration of the gelatin solution, but can generally be set to -10°C or lower.
• the cooling temperature of the gelatin solution is preferably -100°C to -10°C, more preferably -80°C to -20°C, even more preferably -40°C to -60°C.
• the cooling time is preferably 1 to 6 hours from the viewpoint of uniform ice crystal formation.
• tissue repair material of the present disclosure includes applying the tissue repair material to a defective or damaged site in a target tissue.
• the target tissue is preferably a hard tissue such as a tooth or bone, and the tissue repair material is preferably used for bone regeneration.
• Use of the tissue repair material of the present disclosure includes applying at least one of transplanted cells and an osteoinductive agent to the site to which the tissue repair material is applied, as necessary, before or after application of the tissue repair material. You can stay there.
• the use of tissue repair materials includes treatment methods or restorative methods such as periodontal defects in the maxillofacial region, implant defects, etc.; GBR method as a preliminary treatment for implant placement, gingival augmentation, etc. It can be preferably applied to ridge augmentation method, sinus lift method, socket reservation method, etc.
• CBE3 described in WO2008/103041A1 was prepared as a recombinant gelatin.
• the details of CBE3 are as follows. ⁇ Molecular weight: 51.6kDa ⁇ Number of amino acid residues: 571 ⁇ Number of RGD sequences: 12 ⁇ Amino acid sequence: SEQ ID NO: 1 GAP(GAPGLQGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGPAGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGPAGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGPAGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGPA
• An aqueous solution containing 7.5% by mass of CBE3 was prepared.
• the aqueous solution was poured into a cylindrical container and then placed in a freeze dryer.
• the aqueous solution was frozen at ⁇ 60° C. for 1 hour or more, and primary drying was performed at ⁇ 15° C. for 38 hours under vacuum and secondary drying was performed at 23° C. for 2 hours to obtain sponge-like gelatin.
• Sponge-like gelatin was pulverized using a screen pulverizer (Cormil U10, manufactured by Quadro) using a 0.079-inch screen and then a 0.040-inch screen to obtain a pulverized product.
• a test sieve of ISO3310 standard the pulverized material was sieved in accordance with the method described in Method 2 of Section 3.04 of the 16th revised Japanese Pharmacopoeia. Specifically, the fraction that passed through a sieve with an opening of 1400 ⁇ m and remained on a sieve with an opening of 300 ⁇ m was collected, and about 0.09 g was placed in a 10 mL glass vial ( ⁇ 24.3 mm, height 46.5 mm). Filled.
• the crushed product filled in a glass vial was placed in a clean oven (NCO-500A600L-WS, manufactured by Nitto Rika Kogyo Co., Ltd.), and heated at a heating temperature of 135°C to 140°C for 4.75 hours in a nitrogen atmosphere.
• the pulverized material was heated to obtain particulate crosslinked gelatin.
• the glass vial was capped with a rubber stopper (rubber stopper 2A) and sealed with an aluminum cap.
• the glass vial filled with the crosslinked gelatin was placed in a cardboard box, a polymethyl methacrylate dosimeter was attached to the side of the cardboard box, and a dose of 18 kGy was irradiated to obtain a tissue repair material.
• Example 2 A tissue repair material was obtained in the same manner as in Example 1, except that the dose of irradiated gamma rays was changed to 28 kGy.
• Example 3 A tissue repair material was obtained in the same manner as in Example 1, except that the dose of irradiated gamma rays was changed to 13 kGy and the heating time of the crushed material was changed to 6.25 hours.
• Example 4 A tissue repair material was obtained in the same manner as in Example 1, except that the dose of irradiated gamma rays was changed to 28 kGy and the heating time of the crushed material was changed to 6.25 hours.
• Example 5 A tissue repair material was obtained in the same manner as in Example 1, except that the dose of irradiated gamma rays was changed to 28 kGy and the heating time of the crushed material was changed to 8.00 hours.
• Example 1 A tissue repair material was obtained in the same manner as in Example 1 except that ⁇ -ray irradiation was not performed.
• the elution curves obtained from the tissue repair materials of Examples 1 to 7 have molecular weights in the range of 43 kDa or more and 51 kDa or less, molecular weights in the range of 28 kDa or more and less than 43 kDa, and molecular weights in the range of 14 kDa or more and less than 28 kDa. At least two absorption peaks with an absorbance of 0.025 or more (described as absorption peak number in Table 1) were observed.
• the elution curves obtained from the tissue repair materials of Comparative Examples 1 and 2 have molecular weights in the range of 43 kDa or more and 51 kDa or less, molecular weights in the range of 28 kDa or more and less than 43 kDa, and molecular weights in the range of 14 kDa or more and less than 28 kDa. No absorption peak with an absorbance of 0.025 or more was observed in any range.
• a microtube (hereinafter referred to as tube) was prepared, and its mass (A) was measured. 15.0 ( ⁇ 0.2) mg of the tissue repair material produced in the Examples and Comparative Examples was weighed (mass: B) and filled into a tube. 1.7 mL of 1 mol/L hydrochloric acid was added to the tube containing the tissue repair material, and heated for 3 hours using a heat block set at 37°C. After heating, the tube was placed on ice to stop the acid decomposition reaction, and centrifuged at 10,000 xg for 1 minute using a centrifuge preset at 4°C.
• the supernatant was sucked out, 1 mL of ultrapure water previously chilled on ice was added, and the tube was centrifuged again under the same conditions as above. After centrifugation, the supernatant was sucked out, ultrapure water was added again, and centrifugation was performed again under the same conditions as above. After centrifugation, the supernatant is sucked out, and the tube filled with the tissue repair agent is placed in a -80°C freezer (Ultra-low temperature freezer CLN-31UW, manufactured by Nippon Freezer Co., Ltd.) for at least 1 hour to obtain the frozen tissue repair agent. I got it.
• a -80°C freezer Ultra-low temperature freezer CLN-31UW, manufactured by Nippon Freezer Co., Ltd.
• tissue repair materials obtained in the Examples have excellent tissue repair properties compared to the tissue repair materials obtained in the Comparative Examples. Moreover, from Table 1, it can be seen that the tissue repair materials obtained in the examples have excellent tissue repair speed.

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• 2022
  • 2022-10-25 JP JP2024511185A patent/JPWO2023188492A1/ja active Pending
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• 2024
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