JP6434438B2 - Method for producing gelatin molded body - Google Patents

Description

  The present invention relates to a method for producing a gelatin molded body by discharging a gelatin solution in a droplet state and dropping it on a substrate.

  Practical application of regenerative medicine that measures the regeneration of a biological tissue or organ that has fallen into a dysfunction or dysfunction is in progress. Regenerative medicine is a medical technology that regenerates the same form and function as the original tissue using one or more of cells, scaffolds, and growth factors in biological tissues that cannot be recovered only by the natural healing ability of the living body. is there. In regenerative medicine, a molded body made of a biocompatible material may be used.

  In Patent Document 1, the substrate surface is hydrophobized and then hydrophilized, and then a natural polymer sol is poured onto the substrate surface. The natural polymer sol is cooled to form a gel. A method for producing a polymer gel, which is characterized in that it is peeled from the surface, is described. The method described in Patent Document 1 is a method for forming a gel made of a natural polymer such as gelatin into an arbitrary size and shape.

  In Patent Document 2, a plurality of coating liquids having different characteristics with respect to temperature are fed to the same coating head at different liquid feeding temperatures according to the characteristics, and a plurality of coating liquids are ejected from the coating head and run. Describes a method for simultaneous multi-layer coating. In Patent Document 2, the coating liquid having the highest liquid feeding temperature is a, the liquid feeding temperature is Tain, the discharge temperature is Taout, the coating liquid having the lowest liquid feeding temperature is b, the liquid feeding temperature is Tbin, and the discharge temperature. Multi-layer simultaneous coating, wherein a plurality of coating solutions are simultaneously coated under the conditions of 0 ° C. <Tain−Taout <10 ° C. and 0 ° C. <Tbout−Tbin <10 ° C. A method is described.

  Patent Document 3 includes a casting step of casting a polymer solution into a mold having a recess, a drying and solidifying step of drying and solidifying the polymer solution, and a peeling step of peeling the functional sheet from the mold. A method for producing a functional sheet is described in which, in the drying and solidifying step, the temperature TP of the polymer solution is adjusted to a temperature higher than the gelation temperature Tgel of the polymer solution during the constant rate drying period. .

JP 2004-263081 A JP 2002-331263 A JP 2009-241357 A

  The method of Patent Document 1 is a method that includes pouring a sol made of a natural polymer onto a hydrophilized substrate surface after hydrophobization treatment, but is not a method for producing a gelatin molded body using a gelatin solution. . The method of Patent Document 2 relates to a method of simultaneously applying a plurality of coating liquids having different characteristics with respect to temperature. The method of patent document 3 is related with the method of shape | molding using the mold which has a recessed part. As described above, there has been no report on the production of a mesh-like gelatin molded body using a gelatin solution.

  An object of the present invention is to provide a method for producing a gelatin molded body using a gelatin solution.

  As a result of intensive studies to solve the above problems, the inventors of the present invention discharged a gelatin solution in a droplet state from a nozzle portion at a temperature not lower than the gelation temperature and not higher than 32 ° C., and the discharged gelatin solution in the droplet state It was found that a gelatin molded body can be produced by dropping the solution onto a substrate having a water contact angle of 90 ° or less. The present invention has been completed based on these findings.

That is, according to the present invention, the following inventions are provided.
[1] A gelatin solution is ejected in a droplet state from a nozzle at a temperature not lower than the gelation temperature and not higher than 32 ° C., and the discharged gelatin solution is dropped onto a substrate having a water contact angle of 90 ° or less. The manufacturing method of a gelatin molded object including the process to make.
[2] The method for producing a gelatin molded product according to [1], wherein the gelatin solution is a gelatin solution containing no crosslinking agent.
[3] The method for producing a gelatin molded product according to [1] or [2], wherein the gelatin is recombinant gelatin.
[4] The method for producing a gelatin molded product according to any one of [1] to [3], wherein the gelatin molded product has a mesh shape.
[5] The method for producing a gelatin molded body according to any one of [1] to [4], wherein the water contact angle is 50 ° or more.
[6] The method for producing a gelatin molded body according to any one of [1] to [5], wherein the substrate is formed of a polymethyl methacrylate resin.
[7] The method for producing a gelatin molded body according to [5] or [6], including a peeling step of peeling the gelatin molded body from the substrate.
[8] The method for producing a gelatin molded product according to any one of [1] to [7], comprising a step of crosslinking the gelatin of the gelatin molded product.
[9] It includes a step of humidifying the gelatin molded body, a step of freezing the humidified gelatin molded body, a step of drying the frozen gelatin molded body under reduced pressure, and a step of heating the gelatin molded body dried under reduced pressure. ] The manufacturing method of the gelatin molding as described in any one of [8].

  According to the method for producing a gelatin molded product according to the present invention, a gelatin molded product can be produced using a gelatin solution.

FIG. 1 shows a state of a lattice-shaped gelatin molded body (mesh).

Hereinafter, embodiments of the present invention will be described in detail.
(1) Method for Producing Gelatin Molded Body In the method for producing a gelatin molded body according to the present invention, a gelatin solution is ejected in a droplet state from a nozzle portion at a temperature not lower than the gelation temperature and not higher than 32 ° C. The gelatin solution in a state is dropped on a substrate having a water contact angle of 90 ° or less.
Although it was possible to produce a mesh-like molded body by fiberizing a thermoplastic resin having biocompatibility such as polylactic acid, this is not the case for producing a mesh-like gelatin molded body using a gelatin solution. There was no report before. When a gelatin molded body is produced using a gelatin solution, when the gelatin solution is dropped on the substrate, the droplet of the gelatin solution is deformed (the droplet spreads), and the gelatin has a desired shape. It was difficult to mold into. In the present invention, the gelatin solution is ejected in a droplet state from the nozzle portion at a temperature not lower than the gelation temperature and not higher than 32 ° C., and the discharged gelatin solution is discharged onto the substrate having a water contact angle of 90 ° or less. By dripping, the deformation of the droplets of the gelatin solution can be suppressed and the gelatin can be formed into a desired shape. It is an unexpected finding for the first time by the present invention that gelatin can be formed into a desired shape by the above-described constitution of the present invention.

  In the present invention, the gelatin solution is ejected in the form of droplets. The droplet state is a state in which the gelatin solution discharged from the nozzle part moves in the space without contacting either the nozzle part or the substrate. Regarding the discharge of the gelatin solution, there is continuous discharge as a mode other than the discharge in the droplet state. However, when the gelatin solution is continuously discharged, the gelatin solution spreads on the substrate. It becomes impossible to manufacture a mesh-shaped molded body.

In the present invention, the gelatin solution is ejected in the form of droplets from the nozzle portion at a temperature not lower than the gelation temperature and not higher than 32 ° C. By setting the temperature of the gelatin solution to be equal to or higher than the gelation temperature, the gelatin solution can be prevented from being gelled and discharged in a droplet state from the nozzle portion. By setting the temperature of the gelatin solution to 32 ° C. or lower, deformation of the droplet is suppressed when the droplet of the gelatin solution is dropped on the substrate, and it becomes possible to form the desired shape.
The temperature at which the gelatin solution is discharged in the form of droplets from the nozzle part is preferably 25 ° C. or higher and 32 ° C. or lower, more preferably 28 ° C. or higher and 32 ° C. or lower.

  The discharging of the gelatin solution from the nozzle portion in a droplet state can be performed using a dispenser. A dispenser is a device for applying a coating solution such as a polymer solution onto a substrate, and is a device capable of discharging a certain amount of coating solution. While discharging the coating liquid from the nozzle section using a discharge device such as a syringe pump, the coating liquid is uniformly applied to any location on the substrate by operating the nozzle section (discharge port) on the substrate at a constant speed. Is possible. By keeping the distance between the substrate and the nozzle portion (ejection port) constant, the coating amount can be made uniform. The dispenser may mean a system including a controller that stably supplies a predetermined amount of coating liquid and peripheral devices (such as a robot).

  As the dispenser, a pneumatic (syringe) type dispenser can be used. The air source and the syringe are connected by pneumatic piping. A plunger (piston) is preferably provided inside the syringe. When air is sent from the air source to the syringe, the plunger (piston) is pushed down, and the coating liquid inside the syringe is discharged from the nozzle portion (discharge port).

  As a specific example of the dispenser, a jet dispenser (AeroJet MJET-A manufactured by Musashi Engineering Co., Ltd., the nozzle is SNJ21-34G-SHN manufactured by Musashi Engineering Co., Ltd., and the robot is Shot mini200ΩX manufactured by Musashi Engineering Co., Ltd.). Although it can mention, it does not specifically limit.

In the present invention, the nozzle width of the nozzle provided in the dispenser is preferably 10 μm to 100 μm, and more preferably 50 μm to 80 μm.
The discharge amount of the coating liquid is controlled by air pressure × discharge time, but also depends on the viscosity of the coating liquid, the length (height) of the nozzle portion, and the nozzle width.

In the present invention, the discharged gelatin solution is dropped onto a substrate having a water contact angle of 90 ° or less.
By setting the water contact angle of the substrate to 90 ° or less, deformation of the droplet is suppressed when the droplet of the gelatin solution is dropped on the substrate, and the gelatin can be formed into a desired shape.

Although the minimum of the water contact angle of a board | substrate is not specifically limited, Generally, it is 10 degrees or more, Preferably it is 30 degrees or more, More preferably, it is 50 degrees or more. By setting the water contact angle of the substrate to 50 ° or more, it becomes easy to peel the gelatin molded body from the substrate.
The particularly preferable range of the water contact angle of the substrate is 70 ° or more and 90 ° or less, and the most preferable range is 75 ° or more and 85 ° or less. Note that 360 ° is 2π radians (rad).

  A water contact angle means the static contact angle with respect to the water measured by dripping water on the surface of a surface coating layer using the contact angle meter based on JIS-R3257. For example, the measurement can be performed using DM-500 manufactured by Kyowa Interface Science Co., Ltd. or LSE-ME1 (software 2win mini) manufactured by Nick Co., Ltd. More specifically, 2 μL of a droplet can be dropped on a substrate kept horizontal at room temperature of 20 ° C. using pure water, and the water contact angle at 20 seconds after dropping can be measured.

The material, shape, and size of the substrate are not particularly limited as long as the above water contact angle condition is satisfied, and an appropriate substrate can be used according to the purpose.
As the substrate, a substrate having a plane with a predetermined area is preferable. Although the surface area of a board | substrate is not specifically limited, Preferably it is 5-200 cm < ² >, More preferably, it is 20-100 cm < ² >.

  Examples of the material for the substrate include plastic materials such as acrylic, methacryl (polymethyl methacrylate resin, etc.), polystyrene, and polypropylene, inorganic materials such as glass, and metal materials such as copper, aluminum, and stainless steel. The material of the substrate is preferably methacryl, and particularly preferably polymethyl methacrylate resin.

  When the gelatin solution in the droplet state is dropped on the substrate, the gelatin line width is preferably 200 μm to 300 μm, and the gap between the lattice-like gelatins is 200 μm to 400 μm. By dropping the gelatin solution in a droplet state onto the substrate as described above, a gelatin molded body having a mesh shape (also referred to as a mesh) can be produced. The mesh shape (mesh) means a shape having a plurality of fine holes.

  The size of the gelatin molded product is not particularly limited, but when the gelatin molded product is approximated to a rectangular parallelepiped, the vertical, horizontal, and height of the rectangular parallelepiped are each preferably 200 mm to 1 mm, more preferably 200 mm to 0.1 mm. It is.

  In the present invention, the gelatin molded body can be peeled off from the substrate after the gelatin solution in a droplet state is dropped on the substrate to produce the gelatin molded body. That is, the method of the present invention may include a peeling step of peeling the gelatin molded body from the substrate. According to the present invention, the gelatin molded body can be peeled from the substrate. In particular, the gelatin molded body can be easily peeled from the substrate by using a substrate having a water contact angle of 50 ° or more.

  In the present invention, when the gelatin crosslinking step described later is performed, the gelatin molded product is peeled off from the substrate before the gelatin crosslinking step, during the gelatin crosslinking step, or during the gelatin crosslinking step. It can be done at any later stage.

(2) Gelatin solution (2-1) Gelatin Gelatin may be natural gelatin (animal gelatin or the like), recombinant gelatin, or chemically synthesized gelatin.
Natural gelatin means gelatin made from naturally derived collagen. The organism from which gelatin is derived is not particularly limited, and for example, gelatin derived from animals (mammals, fish, etc.) can be used.

  Chemically synthesized gelatin means artificially synthesized gelatin. The peptide such as gelatin may be synthesized by solid phase synthesis or liquid phase synthesis, but is preferably solid phase synthesis. Solid-phase synthesis of peptides is known to those skilled in the art. For example, Fmoc group synthesis method using Fmoc group (Fluorenyl-Methoxy-Carbonyl group) as amino group protection, and Boc group (tert-Butyl group) as amino group protection Boc group synthesis method using Oxy Carbonyl group).

  The preferred embodiment of the natural gelatin and the chemically synthesized gelatin can be applied to the contents described in the recombinant gelatin described later in this specification.

(2-2) Recombinant gelatin The gelatin used in the present invention is preferably recombinant gelatin.
Recombinant gelatin means a polypeptide or protein-like substance having an amino acid sequence similar to gelatin produced by a genetic recombination technique. The recombinant gelatin that can be used in the present invention preferably has a repeating sequence represented by Gly-X-Y, which is characteristic of collagen (X and Y each independently represents an amino acid). Here, the plurality of Gly-XY may be the same or different. Preferably, two or more cell adhesion signals are contained in one molecule. As the recombinant gelatin used in the present invention, recombinant gelatin having an amino acid sequence derived from a partial amino acid sequence of collagen can be used. For example, EP1014176, US Pat. No. 6,992,172, International Publication WO2004 / 85473, International Publication WO2008 / 103041, and the like can be used, but are not limited thereto. A preferable example of the recombinant gelatin used in the present invention is the recombinant gelatin of the following embodiment.

  Recombinant gelatin has excellent biocompatibility due to the inherent performance of natural gelatin, and is not naturally derived, so there is no concern about bovine spongiform encephalopathy (BSE) and excellent non-infectivity. Recombinant gelatin is more uniform than natural gelatin and its sequence is determined, so that strength and degradability can be precisely designed with less blur due to crosslinking or the like.

  The molecular weight of the recombinant gelatin is not particularly limited, but is preferably 2000 or more and 100000 or less (2 kDa (kilo dalton) or more and 100 kDa or less), more preferably 2500 or more and 95000 or less (2.5 kDa or more and 95 kDa or less), and further preferably. Is from 5,000 to 90,000 (5 kDa to 90 kDa), and most preferably from 10,000 to 90,000 (10 kDa to 90 kDa).

  Recombinant gelatin preferably has a repeating sequence represented by Gly-XY characteristic of collagen. Here, the plurality of Gly-XY may be the same or different. In Gly-XY, Gly represents glycine, and X and Y represent any amino acid (preferably any amino acid other than glycine). The sequence represented by Gly-XY, which is characteristic of collagen, is a very specific partial structure in the amino acid composition and sequence of gelatin and collagen compared to other proteins. In this part, glycine accounts for about one third of the whole, and in the amino acid sequence, it is one in three repeats. Glycine is the simplest amino acid, has few constraints on the arrangement of molecular chains, and greatly contributes to the regeneration of the helix structure upon gelation. The amino acids represented by X and Y are rich in imino acids (proline, oxyproline) and preferably account for 10% to 45% of the total. Preferably, 80% or more, more preferably 95% or more, and most preferably 99% or more of the amino acid sequence of the recombinant gelatin is a Gly-XY repeating structure.

  In general gelatin, polar amino acids have a charged amino acid and an uncharged one in a ratio of 1: 1. Here, the polar amino acid specifically refers to cysteine, aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine, serine, threonine, tyrosine and arginine, and among these polar uncharged amino acids are cysteine, asparagine, glutamine, serine. Refers to threonine and tyrosine. In the recombinant gelatin used in the present invention, the proportion of polar amino acids is 10 to 40%, preferably 20 to 30%, of all the constituting amino acids. The proportion of uncharged amino acids in the polar amino acid is preferably 5% or more and less than 20%, more preferably 5% or more and less than 10%. Furthermore, it is preferable that any one amino acid of serine, threonine, asparagine, tyrosine and cysteine is not included in the sequence, and that two or more amino acids of serine, threonine, asparagine, tyrosine and cysteine are not included in the sequence. More preferred.

  In general, the minimum amino acid sequence that acts as a cell adhesion signal in a polypeptide is known (for example, “Pathophysiology” published by Nagai Publishing Co., Ltd., Vol. 9, No. 7 (1990), page 527). The recombinant gelatin used in the present invention preferably has two or more of these cell adhesion signals in one molecule. Specific sequences include RGD sequences, LDV sequences, REDV sequences, YIGSR sequences, PDSGR sequences, RYVVLPR sequences, LGITIPG sequences, RNIAEIIKDI sequences, which are expressed in one-letter amino acid notation in that many types of cells adhere. , IKVAV sequences, LRE sequences, DGEA sequences, and HAV sequences are preferred. More preferred are RGD sequence, YIGSR sequence, PDSGR sequence, LGTIPG sequence, IKVAV sequence and HAV sequence, and particularly preferred is RGD sequence. Of the RGD sequences, an ERGD sequence is preferred. By using recombinant gelatin having a cell adhesion signal, the amount of cell substrate produced can be improved.

The arrangement of RGD sequences in the recombinant gelatin used in the present invention is preferably such that the number of amino acids between RGDs is not uniform between 0 and 100, and more preferably the number of amino acids between RGDs is not uniform between 25 and 60. .
The content of the minimum amino acid sequence is preferably 3 to 50, more preferably 4 to 30, and particularly preferably 5 to 20 in one protein molecule from the viewpoint of cell adhesion and proliferation. Most preferably, it is 12.

  In the recombinant gelatin used in the present invention, the ratio of the RGD motif to the total number of amino acids is preferably at least 0.4%. When the recombinant gelatin contains 350 or more amino acids, it is preferred that each stretch of 350 amino acids contains at least one RGD motif. The ratio of the RGD motif to the total number of amino acids is more preferably at least 0.6%, even more preferably at least 0.8%, even more preferably at least 1.0%, particularly preferably at least 1.2%. %, Most preferably at least 1.5%. The number of RGD motifs in the recombinant peptide is preferably at least 4, more preferably 6, more preferably 8, even more preferably 12 or more and 16 or less per 250 amino acids. A ratio of 0.4% of the RGD motif corresponds to at least one RGD sequence per 250 amino acids. Since the number of RGD motifs is an integer, a gelatin of 251 amino acids must contain at least two RGD sequences to meet the 0.4% feature. Preferably, the recombinant gelatin of the present invention comprises at least 2 RGD sequences per 250 amino acids, more preferably comprises at least 3 RGD sequences per 250 amino acids, more preferably at least 4 per 250 amino acids. Contains the RGD sequence. As a further aspect of the recombinant gelatin of the present invention, it contains at least 4 RGD motifs, preferably 6, more preferably 8, more preferably 12 or more and 16 or less.

  Recombinant gelatin may be partially hydrolyzed.

Preferably, the recombinant gelatin used in the present invention is represented by A-[(Gly-XY) _n ] _m -B. Each of n Xs independently represents any of amino acids, and each of n Ys independently represents any of amino acids. m preferably represents an integer of 2 to 10, more preferably an integer of 3 to 5. n is preferably an integer of 3 to 100, more preferably an integer of 15 to 70, and most preferably an integer of 50 to 65. A represents any amino acid or amino acid sequence, and B represents any amino acid or amino acid sequence. Note that the n Gly-XY may be the same or different.

More preferably, the recombinant gelatin used in the present invention is Gly-Ala-Pro-[(Gly-XY) ₆₃ ] ₃ -Gly (wherein 63 X independently represent any of amino acids, 63 Y's each independently represent any amino acid, and 63 Gly-X-Y's may be the same or different.

  It is preferable to bind a plurality of naturally occurring collagen sequence units to the repeating unit. The naturally occurring collagen referred to here may be any naturally occurring collagen, but is preferably type I, type II, type III, type IV, or type V collagen. More preferred is type I, type II, or type III collagen. According to another form, the collagen origin is preferably human, bovine, porcine, mouse or rat, more preferably human.

  The isoelectric point of the recombinant gelatin used in the present invention is preferably 5 to 10, more preferably 6 to 10, and further preferably 7 to 9.5. The measurement of the isoelectric point of the recombinant gelatin was described in isoelectric focusing (Maxey, CR (1976; Phitogr. Gelatin 2, Editor Cox, P. J. Academic, London, Engl.). As described above, it can be carried out by measuring the pH after passing a 1% by weight gelatin solution through a mixed crystal column of cation and anion exchange resin.

Preferably, the recombinant gelatin is not deaminated.
Preferably, the recombinant gelatin has no telopeptide.
Preferably, the recombinant gelatin is a substantially pure polypeptide prepared with a nucleic acid encoding an amino acid sequence.

Particularly preferably as the recombinant gelatin used in the present invention,
(1) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 1;
(2) A peptide having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in SEQ ID NO: 1 and having biocompatibility; or (3) described in SEQ ID NO: 1 A peptide consisting of an amino acid sequence having a sequence identity of 80% or more (more preferably 90% or more, particularly preferably 95% or more, most preferably 98% or more) with the amino acid sequence and having biocompatibility;
Any of them.

The sequence identity in the present invention refers to a value calculated by the following formula.
% Sequence identity = [(number of identical residues) / (alignment length)] × 100
Sequence identity between two amino acid sequences can be determined by any method known to those skilled in the art, and the BLAST ((Basic Local Alignment Search Tool)) program (J. Mol. Biol. 215: 403-410, 1990) Etc. can be used to determine.

  The “one or several” in the “amino acid sequence in which one or several amino acids have been deleted, substituted or added” is preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5. Means, particularly preferably 1 to 3.

The “1 / IOB” value, which is the hydrophilicity value of gelatin used in the present invention, is preferably from 0 to 1.0. More preferably, it is 0 to 0.6, and still more preferably 0 to 0.4. IOB is an index of hydrophilicity / hydrophobicity based on an organic conceptual diagram representing the polarity / non-polarity of an organic compound proposed by Satoshi Fujita. Details thereof can be found in, for example, “Pharmaceutical Bulletin”, vol.2, 2, pp .163-173 (1954), “Area of Chemistry” vol.11, 10, pp.719-725 (1957), “Fragrance Journal”, vol.50, pp.79-82 (1981), etc. Yes. To put it simply, methane (CH ₄ ) is the source of all organic compounds, and all the other compounds are all methane derivatives, with certain numbers set for their carbon number, substituents, transformations, rings, etc. Then, the score is added to obtain an organic value (OV) and an inorganic value (IV), and these values are plotted on a diagram with the organic value on the X axis and the inorganic value on the Y axis. It is going. 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, that is, “inorganic value (IV) / organic value (OV)”. For details of the organic conceptual diagram, please refer to “New Organic Conceptual Diagram: Basics and Applications” (Yoshio Koda et al., Sankyo Publishing, 2008). In the present specification, hydrophilicity / hydrophobicity is represented by a “1 / IOB” value obtained by taking the reciprocal of IOB. The smaller the “1 / IOB” value (closer to 0), the more hydrophilic it is.

  By setting the “1 / IOB” value of the gelatin used in the present invention within the above range, the hydrophilicity is high and the water absorption is high.

The gelatin used in the present invention preferably has a hydrophilicity / hydrophobicity index represented by the Grand average of hydropathicity (GRAVY) value of 0.3 or less and minus 9.0 or more, 0.0 or less, minus 7.0 or more. More preferably. Grand average of hydropathicity (GRAVY) values are based on Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins MR, Appel RD, 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 RD, Bairoch A .; ExPASy: the proteomics server for in-depth protein knowledge and analysis .; Nucleic Acids Res. 31: 3784-3788 (2003).
By setting the GRAVY value of the gelatin used in the present invention within the above range, the hydrophilicity is high and the water absorption is high.

  The recombinant gelatin used in the present invention can be produced by a genetic recombination technique known to those skilled in the art. For example, in EP1014176A2, US Pat. No. 6,992,172, International Publication WO2004 / 85473, International Publication WO2008 / 103041, etc. It can be produced according to the method described. Specifically, a gene encoding the amino acid sequence of a predetermined recombinant gelatin is obtained, and this is incorporated into an expression vector to produce a recombinant expression vector, which is introduced into an appropriate host to produce a transformant. . Recombinant gelatin is produced by culturing the obtained transformant in an appropriate medium. Therefore, the recombinant gelatin used in the present invention can be prepared by recovering the recombinant gelatin produced from the culture. .

(2-3) Gelatin solution In the present invention, a gelatin solution is used. The solvent used for the gelatin solution is not particularly limited as long as it is a solvent that can dissolve gelatin. Generally, it is water, an organic solvent, or a mixture of water and an organic solvent, preferably water, or water and an organic solvent. An aqueous medium such as a mixture of solvents. Examples of the organic solvent include acetone, ethanol and the like, and the surface tension and liquid viscosity can be adjusted by adding “very small amount” to water. A gelatin aqueous solution can be preferably used as the gelatin solution.

  The gelatin concentration in the gelatin solution is not particularly limited, but is generally 1% by mass to 50% by mass, preferably 2 to 40% by mass, more preferably 3 to 30% by mass, and further preferably 5%. It is -20 mass%, Most preferably, it is 10-20 mass%. In order to produce a gelatin molded body having a desired shape, it is advantageous that the nozzle width is narrow. However, when the nozzle width is narrow, when the viscosity of the gelatin solution is too high, it becomes difficult to discharge. There is a case.

  When the use of the gelatin molded product is for medical purposes (regeneration of skin defect, etc.), it is desirable to produce the gelatin molded product without using a crosslinking agent. Therefore, in the present invention, the gelatin solution is preferably a gelatin solution containing no crosslinking agent. Examples of the crosslinking agent include aldehydes (for example, formaldehyde, glutaraldehyde, etc.) and condensing agents (carbodiimide, cyanamide, etc.).

(3) Step of Crosslinking Gelatin of Gelatin Molded Body In the present invention, after the gelatin molded body is produced, a step of crosslinking gelatin of the gelatin molded body (hereinafter also referred to as gelatin crosslinking step) may be performed. However, the gelatin crosslinking step is not essential, and the method of the present invention includes the case where the gelatin crosslinking step is not performed.

  The gelatin crosslinking step preferably includes a step of heating and crosslinking the gelatin molded body. More preferably, the gelatin crosslinking step is a step of humidifying the gelatin molded body, a step of freezing the humidified gelatin molded body, a step of drying the frozen gelatin molded body under reduced pressure, and heating the gelatin molded body dried under reduced pressure. Process. By performing the gelatin crosslinking step, a gelatin molded body having high strength can be produced.

  By discharging the gelatin solution in a droplet state from the nozzle portion at a temperature not lower than the gelation temperature and not higher than 32 ° C., and dropping the discharged gelatin solution onto a substrate having a water contact angle of 90 ° or less. The manufactured gelatin molded body is presumed to be a mixture of gel and sol. By humidifying the gelatin molded body, it is assumed that the gelatin molded body obtains a solvent (water), fluidizes the molecules, and forms a stable gelled structure. By performing the humidification step, the strength of the gelatin crosslinked in the heating step described later can be further improved. The conditions of the humidification step are not particularly limited as long as the conditions allow water to be applied to the gelatin molded body as described above, but in general, the relative humidity is 40% to 60%, and the temperature is 20 ° C to 25 ° C ( For example, it can be performed for about 1 to 3 hours under the conditions of room temperature).

  By performing the step of freezing the humidified gelatin molded body, the strength of the gelatin crosslinked in the heating step described later can be further improved. The freezing conditions are not particularly limited, but generally the freezing temperature is in the range of -10 ° C to -25 ° C, and the freezing time is about 1 hour to 3 hours.

  In the step of drying the frozen gelatin molded body under reduced pressure, drying is carried out while removing the water inside the gelatin molded body and maintaining the gelled structure. The conditions for reducing the pressure are generally -0.01 MPa to -1.0 MPa, preferably -0.01 MPa to -0.5 MPa. The drying under reduced pressure can be generally performed at a temperature of 20 ° C. to 25 ° C. (for example, room temperature) for about 1 hour to 3 hours.

  Gelatin can be crosslinked by the step of heating the gelatin molded product. The heating temperature is not particularly limited as long as crosslinking is possible, but is preferably 0 ° C to 300 ° C, more preferably 50 ° C to 300 ° C, still more preferably 100 ° C to 250 ° C, and particularly preferably 120 ° C. ° C to 200 ° C. However, T [Kelvin: K] = t [degree of Celsius: ° C.] + 273.15.

  The crosslinking time is not particularly limited, but is generally 1 hour to 120 hours, preferably 2 hours to 72 hours, more preferably 4 hours to 60 hours, and even more preferably 8 hours to 48 hours. It is.

(4) Use of gelatin molded body The use of the gelatin molded body produced by the method of the present invention is not particularly limited, but preferably a scaffold for regenerative medicine, a product such as regenerative medicine (a tissue cultured outside the body) or tissue Can be used as a restoration material.

  The tissue repair material in the present invention is a material that contributes to the formation of tissue in the implanted site by being implanted in a living body, and may or may not contain cells. Good. Moreover, it does not need to contain the component which accelerates | stimulates the reaction of a biological body like a growth factor or a chemical | medical agent. Further, it may be mixed with an inorganic material such as hydroxyapatite, or a composite may be produced and applied. The tissue repair material in the present invention includes not only a material that contributes to the formation of normal tissue normally present at the site of implantation, but also a material that promotes the formation of non-normal tissue including scar tissue and the like.

  Specific examples of the tissue repair material include, but are not limited to, a cartilage, meniscus, skin or bone repair material. That is, the tissue repair material in the present invention can be used as a therapeutic agent for regeneration of cartilage, meniscus, skin or bone. As long as the above regeneration is necessary, the disease is not limited, but as an example, diseases involving cartilage defects include osteoarthritis, osteochondral defect, severe osteochondritis, traumatic cartilage damage , Osteoarthritis, relapsing polychondritis, achondroplasia, intervertebral disc injury, disc herniation and the like.

  The tissue repair material can also be used as a bone regeneration treatment agent in combination with transplanted cells or osteoinductive agents. Examples of the osteoinductive agent include BMP (bone forming factor) and bFGF (basic fibroblast growth factor), but are not particularly limited.

Since the gelatin molded product of the present invention can be used as a tissue repair material, a tissue repair method and a treatment method for diseases associated with tissue damage are also included in the present invention. The tissue repair method in the present invention includes applying a tissue repair material, which is a gelatin molded body, to a site where the target tissue is deficient or damaged, and may include other steps as necessary. As another process, for example, the transplanted cell and / or the osteoinductive agent may be applied to a site to which the tissue repair material is applied before, or after, the application of the tissue repair material.
Incision, injection, arthroscope, endoscope, etc. can be used as a method of applying the gelatin molded body to a site where the target tissue is deficient or damaged.

  The following examples further illustrate the present invention, but the present invention is not limited to the examples.

[Recombinant gelatin]
The following CBE3 was prepared as a recombinant gelatin (described in International Publication No. WO2008 / 103041).
CBE3:
Molecular weight: 51.6 kD
Structure: GAP [(GXY) ₆₃ ] ₃ G
Number of amino acids: 571 RGD sequence: 12 Imino acid content: 33%
Almost 100% of amino acids are GXY repeating structures. The amino acid sequence of CBE3 does not include serine, threonine, asparagine, tyrosine and cysteine. CBE3 has an ERGD sequence.
Isoelectric point: 9.34
GRAVY value: -0.682
1 / IOB value: 0.323
Amino acid sequence (SEQ ID NO: 1 in the sequence listing) (same as SEQ ID NO: 3 of International Publication WO2008 / 103041; however, the suffix X is corrected to “P”)
GAP (GAPGLQGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGPAGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPP) ₃ G

[dispenser]
In the following examples and comparative examples, the discharge of dot-shaped droplets was performed using a jet dispenser (Musashi Engineering Co., Ltd., AeroJet MJET-A. The nozzle part was made by Musashi Engineering Co., Ltd., SNJ21-34G. -The diameter of the nozzle part was 0.07 mm, and the robot used was Musi Engineering Co., Ltd., Shot mini 200ΩX).

[Example 1]
Powdered recombinant gelatin (CBE3) was dissolved in distilled water to prepare a 20% by mass gelatin aqueous solution. Droplets of the above-mentioned gelatin aqueous solution on a 10 cm × 10 cm acrylic substrate (water contact angle is 70 to 90 °) (substrate made of polymethyl methacrylate resin: the same is used in the following examples and comparative examples) Were ejected in the form of dots, and these dots were overlapped to form a line, thereby producing a 30 mm × 30 mm lattice-shaped gelatin molded body (mesh). The temperature at the time of discharging the gelatin aqueous solution was 30 ° C. A state of a lattice-shaped gelatin molded body (mesh) is shown in FIG. The line width of the gelatin was 200 μm to 300 μm, and the gap between the lattice-like gelatins was about 300 μm.

  Under the above conditions, droplets of the gelatin aqueous solution could be stably discharged. Further, the deformability during production of the gelatin molded body was suppressed, and the moldability was good. Furthermore, the gelatin molded product could be easily peeled from the acrylic substrate.

[Example 2]
Powdered recombinant gelatin (CBE3) was dissolved in distilled water to prepare a 20% by mass gelatin aqueous solution. On a 10 cm × 10 cm acrylic substrate (water contact angle is 70 to 90 °), the droplets of the gelatin aqueous solution are ejected in the form of dots, and the dots are overlapped to form a line to form a 30 mm × 30 mm grid. -Shaped gelatin molded body (mesh) was produced. The temperature at the time of discharging the gelatin aqueous solution was 32 ° C.

  Under the above conditions, droplets of the gelatin aqueous solution could be stably discharged. Further, the deformability during production of the gelatin molded body was suppressed, and the moldability was good. Furthermore, the gelatin molded product could be easily peeled from the acrylic substrate.

[Example 3]
Powdered animal (fish) gelatin (Nitta Gelatin “Ikuos” series: FGL100SP: average molecular weight of about 50,000) was dissolved in distilled water to prepare a 20 mass% gelatin aqueous solution. On a 10 cm × 10 cm acrylic substrate (water contact angle is 70 to 90 °), the droplets of the gelatin aqueous solution are ejected in the form of dots, and the dots are overlapped to form a line to form a 30 mm × 30 mm grid. -Shaped gelatin molded body (mesh) was produced. The temperature at the time of discharging the gelatin aqueous solution was 30 ° C.

  Under the above conditions, droplets of the gelatin aqueous solution could be stably discharged. Further, the deformability during production of the gelatin molded body was suppressed, and the moldability was good. Furthermore, the gelatin molded product could be easily peeled from the acrylic substrate.

[Example 4]
Powdered recombinant gelatin (CBE3) was dissolved in distilled water to prepare a 20% by mass gelatin aqueous solution. On a 10 cm x 10 cm glass substrate (with a water contact angle of 30 ° or less), the droplets of the gelatin aqueous solution are ejected in the form of dots, and these dots are overlapped to form a line, thereby forming a 30 mm x 30 mm grid. A gelatin molded body (mesh) was prepared. The temperature at the time of discharging the gelatin aqueous solution was 30 ° C.

  Under the above conditions, droplets of the gelatin aqueous solution could be stably discharged. Further, the deformability during production of the gelatin molded body was suppressed, and the moldability was good. The gelatin molded product was in close contact with the glass substrate, and peeling was not easy, but it was possible to peel off.

[Comparative Example 1]
Powdered recombinant gelatin (CBE3) was dissolved in distilled water to prepare a 20% by mass gelatin aqueous solution. On a 10 cm × 10 cm acrylic substrate (water contact angle is 70 to 90 °), the droplets of the gelatin aqueous solution are ejected in the form of dots, and the dots are overlapped to form a line to form a 30 mm × 30 mm grid. -Shaped gelatin molded body (mesh) was produced. The temperature at the time of discharging the gelatin aqueous solution was set to 25 ° C.

  Under the above conditions, it was possible to discharge droplets of an aqueous gelatin solution at first, but the gelatin aqueous solution gradually became a gel and could not be stably discharged due to an increase in viscosity. As described above, in Comparative Example 1, since stable ejection could not be performed, the moldability of the gelatin molded body and the peelability from the acrylic substrate could not be evaluated.

[Comparative Example 2]
Powdered recombinant gelatin (CBE3) was dissolved in distilled water to prepare a 20% by mass gelatin aqueous solution. On a 10 cm × 10 cm acrylic substrate (water contact angle is 70 to 90 °), the droplets of the gelatin aqueous solution are ejected in the form of dots, and the dots are overlapped to form a line to form a 30 mm × 30 mm grid. -Shaped gelatin molded body (mesh) was produced. The temperature at the time of discharging the gelatin aqueous solution was set to 36 ° C.

  Under the above conditions, the droplets of the gelatin aqueous solution did not solidify and diffused to the periphery, and the gaps in the lattice of the gelatin molded body (mesh) were filled with the droplets of the gelatin aqueous solution. In Comparative Example 2, the moldability of the gelatin molded body and the peelability from the acrylic substrate could not be evaluated.

[Comparative Example 3]
Powdered recombinant gelatin (CBE3) was dissolved in distilled water to prepare a 20% by mass gelatin aqueous solution. On a 10 cm × 10 cm Teflon (registered trademark) substrate (water contact angle is 100 ° or more), the droplets of the gelatin aqueous solution are ejected in the form of dots, and the dots are overlapped to form a line. A 30 mm lattice-shaped gelatin molded body (mesh) was prepared. The temperature at the time of discharging the gelatin aqueous solution was 30 ° C.

  Under the above conditions, droplets of the gelatin aqueous solution could be stably discharged. However, the droplet dropped on the substrate is repelled, the position of the droplet on the substrate is not fixed, the droplets are not easily connected to each other, it is difficult to form a line, and it is difficult to produce a gelatin molded body . In Comparative Example 3, the peelability of the gelatin molded product from the Teflon (registered trademark) substrate could not be evaluated.

[Example 11]
In a container having a relative humidity of 95% or more and a temperature of 20 ° C., the acrylic substrate having the gelatin molded product produced in Example 1 was left for 3 hours. Thereby, the gelatin molded body was humidified.
The acrylic substrate and the gelatin molded body were taken out of the container, and immediately put in a freezer (−20 ° C.) and left for 3 hours. Thereby, the humidified gelatin molded body was frozen. In this case, a vacuum dryer is not necessary because a vacuum is not used and normal pressure is sufficient.

The acrylic substrate and the frozen gelatin molded body were placed in a desiccator capable of reducing pressure, and the frozen gelatin molded body was dried under reduced pressure at room temperature for about 1 hour. The degree of vacuum was -0.1 MPa.
After removing the acrylic substrate and the gelatin molded product dried under reduced pressure from the desiccator, the gelatin molded product was gently peeled from the acrylic substrate using tweezers. At this time, the gelatin molded product could be easily peeled from the acrylic substrate.

The gelatin molded body peeled from the acrylic substrate was heated in an oven at 140 ° C. for 24 hours. During the heating, in order to drive out oxygen, an oven VAC-201P (manufactured by ESPEC CORP.) Capable of reducing the pressure by introducing N ₂ gas was used. Moreover, N2 purge was performed before the start of heating.
As a result, a gelatin molded product in which gelatin was crosslinked was obtained.

  The gelatin molded product obtained by crosslinking the gelatin produced above swelled when immersed in water. The ratio of the length of one side of the gelatin molded body after swelling to the length of one side of the gelatin molded body before being immersed in water was determined as the swelling ratio. As a result, the swelling rate was 1.2.

[Example 12]
It peeled from the acrylic board | substrate which has the gelatin molded object manufactured in Example 1, and heated at 140 degreeC for 24 hours in oven. During the heating, in order to drive out oxygen, an oven VAC-201P (manufactured by ESPEC CORP.) Capable of reducing pressure was introduced by introducing N ₂ gas. Moreover, N2 purge was performed before the start of heating. As a result, a gelatin molded product in which gelatin was crosslinked was obtained.

  The gelatin molded product obtained by crosslinking the gelatin produced above swelled when immersed in water. As a result of obtaining the swelling ratio in the same manner as in Example 11, the swelling ratio was 1.5.

[Example 13]
In a container having a relative humidity of 95% or more and a temperature of 20 ° C., the acrylic substrate having the gelatin molded product produced in Example 1 was left for 3 hours. Thereby, the gelatin molded body was humidified.

The humidified gelatin molded body was placed in a desiccator capable of reducing pressure, and the humidified gelatin molded body was dried under reduced pressure at room temperature for about 1 hour. The degree of vacuum was -0.1 MPa.
After removing the acrylic substrate and the gelatin molded product dried under reduced pressure from the desiccator, the gelatin molded product was gently peeled from the acrylic substrate using tweezers. At this time, the gelatin molded product could be easily peeled from the acrylic substrate.

The gelatin molded body peeled from the acrylic substrate was heated in an oven at 140 ° C. for 24 hours. During the heating, in order to drive out oxygen, an oven VAC-201P (manufactured by ESPEC CORP.) Capable of reducing the pressure by introducing N ₂ gas was used. Moreover, N2 purge was performed before the start of heating.
As a result, a gelatin molded product in which gelatin was crosslinked was obtained.

  The gelatin molded product obtained by crosslinking the gelatin produced above swelled when immersed in water. As a result of obtaining the swelling rate in the same manner as in Example 11, the swelling rate was 1.4.

The results of the above-described Examples and Comparative Examples are summarized in Table 1 and Table 2 below.
The evaluation criteria for the suitability of discharge are as follows.
A: When stable and good discharge is possible.
B: When the liquid cannot be stably ejected due to an increase in viscosity, or when the droplets of the gelatin aqueous solution diffuse around without being hardened.

Evaluation criteria for deformation suppression (formability) at the time of forming a molded body are as follows.
A: Deformability during production of a gelatin molded body is suppressed, and moldability is good.
B: When the droplet dropped on the substrate is repelled, the position of the droplet on the substrate is not fixed, and the droplets are not easily connected to each other and are not easily formed into a line.

The evaluation criteria for peelability are as follows.
A: The gelatin molded product can be easily peeled from the substrate.
B: The gelatin molded product is in close contact with the substrate but can be peeled off.

  According to the methods of Examples 1 to 4 having the constitution of the present invention, it was shown that a gelatin molded body can be produced using a gelatin solution. Further, by carrying out the step of crosslinking the gelatin of the gelatin molded product, a strong gelatin molded product having a reduced swelling ratio when immersed in water can be produced.

Claims (9)

A step of discharging a gelatin solution in a droplet state from a nozzle portion at a temperature not lower than the gelation temperature and not higher than 32 ° C., and dropping the discharged gelatin solution onto a substrate having a water contact angle of 90 ° or less. A method for producing a gelatin molded product. The method for producing a gelatin molded product according to claim 1, wherein the gelatin solution is a gelatin solution containing no crosslinking agent. The method for producing a gelatin molded product according to claim 1 or 2, wherein the gelatin is recombinant gelatin. The manufacturing method of the gelatin molding as described in any one of Claims 1-3 whose shape of the said gelatin molding is mesh shape. The manufacturing method of the gelatin molding as described in any one of Claims 1-4 whose said water contact angle is 50 degrees or more. The manufacturing method of the gelatin molding as described in any one of Claims 1-5 with which the said board | substrate is formed with the polymethylmethacrylate resin. The manufacturing method of the gelatin molding of Claim 5 or 6 including the peeling process which peels a gelatin molding from a board | substrate. The manufacturing method of the gelatin molding as described in any one of Claims 1-7 including the process of bridge | crosslinking the gelatin of a gelatin molding. The method includes the steps of humidifying a gelatin molded body, freezing the humidified gelatin molded body, drying the frozen gelatin molded body under reduced pressure, and heating the vacuum-dried gelatin molded body. The manufacturing method of the gelatin molded object as described in any one of these.