JP7186417B2 - Angiogenesis promoter - Google Patents

Description

TECHNICAL FIELD The present invention relates to an angiogenesis-promoting material that exhibits an excellent angiogenesis-inducing effect, and promotes differentiation and proliferation of transplanted stem cells by implanting the material together with a scaffold material containing stem cells. .

With the progress of an aging society, people's interest is shifting from living longer to a higher quality of life (QOL). Regenerative medicine is attracting attention under such circumstances. Regenerative medicine uses cells and tissues cultured outside the patient's body to repair and regenerate tissues and organs that have been deficient, damaged, or functionally impaired due to congenital illness, accident, aging, etc. It is complementary medicine. In contrast to conventional symptomatic therapy, regenerative medicine can be used to reconstruct missing or damaged organs, enabling radical treatment of diseases and injuries. As a result, the QOL of patients, the elderly, and the disabled will dramatically improve, and it will become possible for them to return to society and live independently. there is

At present, iPS cells (induced pluripotent stem cells) developed by Professor Shinya Yamanaka et al. of Kyoto University are attracting attention as bringing about the development of regenerative medicine. iPS cells are defined as stem cells with pluripotency, which is the ability to differentiate into many cells that constitute organs and tissues. A wide variety of somatic cells that make up the human body are differentiated from one totipotent stem cell that has complete differentiation potential. Therefore, iPS cells were produced under the assumption that totipotent stem cells and somatic cells have exactly the same nucleotide sequence of genes in the nucleus, and that this difference in differentiation ability is due to the amount of genes expressed. iPS cells are produced by introducing four genes of c-Myc, Oct3/4, Klf4 and Sox2 into somatic cells such as skin cells.

The discovery of iPS cells, which are said to have pluripotency, was revolutionary in the field of regenerative medicine, but there are still few reports of actual clinical application. For example, production of a pigment epithelial cell sheet for age-related macular degeneration patients, which requires a small number of cells, does not require a three-dimensional structure, and does not have blood vessels, has been reported. The reason for this is, of course, the dangers and ethical issues inherent in iPS cells, but also the various steps such as gene introduction, culture, purification, transplantation, and cell standardization into stem cells for industrial application and clinical use. Because it exists. Therefore, from the aspect of biomaterials, it is required to provide optimal materials and conditions for solving these various steps.

A successful example of using iPS cells for application to regenerative medicine was reported in 2007 by J.P. The report by Hanna et al. is the first. However, as described above, clinical application of regenerative medicine using iPS cells is not realistic. That is, there are almost no reports of tissue reconstruction after transplantation of cells cultured in vitro into the body, and in particular, there is no report of connection to the systemic circulation system without co-transplantation with vascular endothelial cells. Connecting to the systemic circulation system means the generation of new capillaries from existing blood vessels, that is, angiogenesis, for the purpose of taking in oxygen and nutrients essential for tissue survival and cell growth. One of the challenges in future regenerative medicine is how to establish a technique for inducing angiogenesis in cells or tissues transplanted into the body.

For example, in Patent Document 1, a bFGF/heparin-containing agarose gel is sandwiched between reservoirs of polytetramethylene glycol diacrylate or triethylene glycol dimethacrylate cured with a photopolymerization initiator for sustained release of angiogenesis-inducing factors. capsule device is disclosed. Patent Document 2 discloses a scaffold for vascular endothelial cell migration comprising genetically modified gelatin having a specific amino acid sequence. Patent Document 3 discloses a biodegradable material containing a polyhydroxysilicic acid ethyl ester compound, which is used for treatment of diseases requiring an increase in the rate of angiogenesis in the healing process. In addition, the present inventors have found that by subcutaneously implanting bFGF-encapsulated hydrogels in which polyethylene glycol chains are grafted and cross-linked with hyaluronic acid, bFGF is gradually released and angiogenesis is promoted. (Non-Patent Documents 1 and 2).

JP 2017-86313 A JP 2013-74936 A Japanese Patent Publication No. 2013-520463

T. Ooya et al., 6th Asian Biomaterials Congress. , 2017 Toru Otani et al., Japan Society for Biomaterials Symposium 2016, Hakata

As described above, the present inventors have found that a specific hydrogel containing bFGF (basic fibroblast growth factor) can enhance angiogenesis in vivo.
However, actual treatment using iPS cells has not yet been successful, and techniques for regenerating tissues and organs in vivo are extremely important. Prolonging vascularization is essential for cell proliferation, and better angiogenesis techniques are desperately needed.
Accordingly, an object of the present invention is to provide an angiogenesis-promoting material that exhibits an excellent angiogenesis-inducing effect.

The present inventors have made intensive studies to solve the above problems. As a result, the present inventors have found that the blood vessel induction effect is remarkably improved by including a free specific polymer in a hydrogel containing a cell growth factor, and completed the present invention.
The present invention is shown below.

[1] containing a hydrogel and a cell growth factor,
An angiogenesis promoting material, wherein the hydrogel contains a crosslinked biodegradable polymer and at least one of a free water-soluble polymer and a free amphipathic polymer.
[2] The angiogenesis-promoting agent according to [1] above, wherein the crosslinked biodegradable polymer is a crosslinked acidic polysaccharide.
[3] The angiogenesis promoting agent according to [2] above, wherein the crosslinked acidic polysaccharide is crosslinked hyaluronic acid, crosslinked alginic acid or crosslinked chondroitin sulfate.
[4] The angiogenesis promoting material according to any one of [1] to [3] above, wherein the free water-soluble polymer is polyethylene glycol or polyvinyl alcohol.
[5] The angiogenesis-promoting agent according to any one of [1] to [4] above, wherein the free amphipathic polymer is a polyethylene glycol-poly(ε-caprolactone) block copolymer.
[6] The angiogenesis-promoting agent according to any one of [1] to [5] above, wherein the cell growth factor is basic fibroblast growth factor.

Blood vessels can be efficiently induced by transplanting the angiogenesis-promoting material according to the present invention together with the scaffolding material and the cells to be proliferated into the body. As a result, nutrients and the like are delivered from blood to cells, promoting cell differentiation and proliferation, and possibly regenerating tissues and organs. Therefore, the present invention is of great industrial importance as it has the potential to make regenerative medicine a reality.

FIG. 1 is a graph showing measurement results of crosslink density of various hydrogels. FIG. 2 shows a sample of a portion in which an angiogenesis-promoting material according to the present invention and a collagen gel containing P3VS cells, which are mouse vaginal stromal cell lines, and E1 cells, which are clonal cell lines of Müllerian epithelium, are implanted. is a magnified photograph stained with a CD31 antibody. FIG. 3 shows an angiogenesis-promoting material according to the present invention, a collagen gel containing E1 cells, which are epithelial clonal cell lines, and M2 cells, which are P3VS-derived clonal cell lines that induce epithelial organization. It is an enlarged photograph obtained by immunohistochemical staining of a sample of the implanted part using an antibody against HMGA2, which is a uterine epithelial marker. FIG. 4 is a graph showing the results of bFGF release studies from various hydrogels containing basic fibroblast growth factor (bFGF).

The angiogenesis promoting material according to the present invention contains a hydrogel and a cell growth factor, and the hydrogel contains a crosslinked biodegradable polymer and at least one of a free water-soluble polymer and a free amphipathic polymer. . A hydrogel is a material that is composed of a hydrophilic polymer having a three-dimensional network structure and is insoluble in water, but swells with water.

A crosslinked biodegradable polymer is a biodegradable polymer that is insolubilized by crosslinking. Since the angiogenesis promoting material of the present invention is implanted in vivo, it is preferably biodegradable. Biodegradability refers to the property of being decomposed by enzymes, microorganisms, etc. present in the living body.

Examples of biodegradable polymers include, but are not limited to, hydrophilic polymers having reactive functional groups for cross-linking, such as carboxy groups, sulfonic acid groups, amino groups, and hydroxyl groups. For example, acidic polysaccharides having carboxy groups and/or sulfonic acid groups such as hyaluronic acid, alginic acid, chondroitin sulfate; acidic peptides having side chain carboxy groups such as polyaspartic acid and polyglutamic acid; basic compounds having amino groups; polysaccharides such as chitosan; and hydroxyl-containing neutral polysaccharides such as chitin. Polysaccharides or peptides having a carboxy group and/or a sulfonic acid group are preferable because they do not excessively reduce the amount of free water-soluble polymer and amphipathic polymer based on the hydrogel production method described later. Polysaccharides with groups and/or sulfonic acid groups are more preferred.

The size of the biodegradable polymer, which is the main chain of the crosslinked biodegradable polymer, is not particularly limited and may be adjusted as appropriate. can do.

As the molecule for cross-linking the cross-linked biodegradable polymer, a polymer that is hydrophilic and has reactive functional groups for cross-linking at both ends can be used. Examples of reactive functional groups for cross-linking include amino groups, hydroxyl groups, and carboxy groups. Examples of the main chain of the molecule include polyethylene glycol, polypropylene glycol, polyvinyl alcohol, and the like.

The degree of ^cross -linking of ^the biodegradable polymer is not particularly limited and may be adjusted as appropriate.

The hydrogel according to the present invention includes, in addition to the hydrogel, at least one of the free water-soluble polymer and the free amphiphilic polymer (hereinafter, at least one of the free water-soluble polymer and the free amphiphilic polymer is referred to as "free may be abbreviated as "water-soluble polymer/amphiphilic polymer").

In the hydrogel according to the present invention, the water-soluble polymer/amphiphilic polymer is present in a free state means that the water-soluble polymer/amphiphilic polymer is not covalently bonded to the crosslinked biodegradable polymer. However, it means that it exists independently of the crosslinked biodegradable polymer in the hydrogel. However, if free water-soluble polymer/amphiphilic polymer is present in the hydrogel, some of the water-soluble polymer/amphiphilic polymer is covalently bound to the crosslinked biodegradable polymer. shall be good.

Examples of water-soluble polymers include, but are not limited to, synthetic water-soluble polymers such as polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, and polyglycerol; semi-synthetic water-soluble polymers such as carboxymethylcellulose, methylcellulose, and hydroxyethylcellulose. plant-derived water-soluble polymers such as guar gum, carrageenan and alginate; microorganism-derived water-soluble polymers such as xanthan gum; animal-derived water-soluble polymers such as chondroitin sulfate and hyaluronic acid; Preferred are polyethylene glycol and polyvinyl alcohol.

Amphiphilic macromolecules are macromolecules that have both hydrophilic and hydrophobic moieties and have an affinity for both water and hydrophobic solvents, for example type AB with hydrophilic and hydrophobic moieties Mention may be made of diblock copolymers. Examples of the hydrophilic portion include the water-soluble polymers described above. Examples of hydrophobic moieties include poly(ε-caprolactone), polylactic acid, and polyglycolic acid. It is possible that such hydrophobic moieties can more stably retain the peptide cell growth factor in the hydrogel.

The molecular weight of the free water-soluble polymer/amphiphilic polymer may be appropriately selected within a range in which water solubility is maintained.

The amount of the free water-soluble polymer/amphiphilic polymer in the hydrogel may be adjusted as appropriate. The ratio of the active polymer/amphiphilic polymer can be 1% by mass or more and 80% by mass or less.

A cell growth factor generally refers to an endogenous protein that promotes the proliferation or differentiation of specific cells. In the angiogenesis-promoting material of the present invention, the cell growth factor is dissolved in the water contained in the hydrogel. Such cell growth factors include, for example, fibroblast growth factors such as basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF); VEGF-A, VEGF-B, VEGF-C , VEGF-D, VEGF-E, placental growth factor (PLGF-1), PLGF-2 and other vascular endothelial cell growth factor (VEGF) families; angiopoietin; platelet-derived growth factor (PDGF); β (TGF-β) and the like, preferably fibroblast growth factor, more preferably bGFG.

The amount of the cell growth factor is not particularly limited and may be adjusted as appropriate.

The hydrogel according to the present invention can be produced by methods known to those skilled in the art. For example, a condensing agent may be added to an aqueous solution containing a biodegradable polymer, a free water-soluble polymer/amphiphilic polymer and a cross-linking agent to cross-link the biodegradable polymer with the cross-linking agent. At this time, a portion of the free water-soluble polymer/amphiphilic polymer reacts with the biodegradable polymer, grafts onto the biodegradable polymer, or crosslinks the biodegradable polymer, resulting in a free water-soluble polymer / The amount of amphiphilic polymer may decrease. In such cases, the reactive functional groups of the biodegradable polymer, the free water-soluble polymer/amphiphilic polymer and the crosslinker can be selected such that the biodegradable polymer and the crosslinker react preferentially. , the amount of these reagents may be adjusted, and the type of condensing agent may be selected.

For example, triazine-based condensing agents such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) are used as solvents such as water and The amidation reaction between carboxylic acid and amine can be selectively allowed to proceed even in alcohol. Therefore, using such a triazine-based condensing agent, using an acidic polysaccharide having a carboxyl group and/or a sulfonic acid group as a biodegradable polymer, using a cross-linking agent having amino groups at both ends, and using a free water-soluble By using a polymer/amphiphilic polymer, the cross-linking reaction of the acidic polysaccharide with a cross-linking agent can be selectively promoted, and the reduction of the free water-soluble polymer/amphiphilic polymer can be suppressed. Alternatively, hydroxyl groups of the free water-soluble polymer/amphiphilic polymer may be protected.

After the cross-linking reaction, it is preferable to sufficiently remove the cross-linking agent and the like by washing with water, dialysis, or the like. At this time, the free water-soluble polymer/amphiphilic polymer, which is incorporated into the three-dimensional network structure of the crosslinked biodegradable polymer and has a high affinity for the crosslinked biodegradable polymer, is difficult to remove. Small molecules such as agents are easily removed.

The method for incorporating the cell growth factor into the resulting hydrogel is not particularly limited, either, and conventional methods can be used. For example, the cell growth factor can be incorporated into the hydrogel by once drying the hydrogel by freeze-drying or the like and then adding an aqueous solution of the cell growth factor. The amount of cell growth factor can be adjusted by adjusting the amount and concentration of the aqueous cell growth factor solution.

The angiogenesis promoting material according to the present invention can promote angiogenesis in vivo. Therefore, by implanting the angiogenesis-promoting material of the present invention together with a scaffolding material containing stem cells and cells with high proliferative potential in vivo, it is possible to promote angiogenesis and promote stem cell differentiation and cell proliferation. can be possible. Cells desired to grow in vivo may be transplanted together with cells that promote their growth and organization.

However, according to the experimental findings of the present inventors, the angiogenesis-promoting agent of the present invention may exhibit toxicity to cells with high proliferative potential. In such a case, it is preferable to allow transplanted cells to exist in the scaffold material, or to implant the angiogenesis-promoting material of the present invention and the scaffold material without contacting each other. That is, if cells are present in the scaffolding material and the angiogenesis-promoting material of the present invention and the cells do not come into direct contact with each other, the angiogenesis-promoting material of the present invention and the scaffolding material are implanted in vivo while being in contact with each other. may However, the distance between the angiogenesis-promoting material of the present invention and the scaffolding material is preferably 0.1 mm or more and 1 cm or less. If the distance is 0.1 mm or more, the adverse effect of the angiogenesis-promoting material on cells to be proliferated can be sufficiently suppressed. On the other hand, if the distance is 1 cm or less, the blood vessels generated by the angiogenesis-promoting material of the present invention can more reliably reach the cells desired to proliferate. The distance is more preferably 8 mm or less, even more preferably 5 mm or less, and even more preferably 3 mm or less or 2 mm or less.

After use, that is, after the desired tissue or organ is regenerated in vivo, the angiogenesis-promoting agent of the present invention is considered to be decomposed by in vivo enzymes and excreted. Alternatively, the angiogenesis-promoting agent of the present invention may be degraded by in vivo enzymes or the like, and cell growth factors may be released over time to promote cell differentiation and proliferation.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited by the following examples, and can be modified appropriately within the scope that can conform to the gist of the above and later descriptions. It is of course possible to implement them, and all of them are included in the technical scope of the present invention.

Example 1: Preparation of hydrogel (1) Synthesis of PEG-grafted hyaluronic acid Table 1 shows the amount of each reagent used. Hyaluronic acid (HA, manufactured by JNC, M _n : 230 kDa) was dissolved in pure water in a 100 mL eggplant flask. Next, after adding α-methyl-ω-aminopropylpolyoxymethylene (PEG-NH ₂ , manufactured by NOF Corporation, M _n : 5205), which is PEG with one end aminated, a condensing agent (“DMT -MM" manufactured by Wako) was added, and the mixture was stirred at room temperature for 24 hours. After completion of the reaction, dialysis was performed for 3 days using a dialysis membrane with a molecular weight cutoff of 12,000 to 14,000 to sufficiently remove impurities. It was then lyophilized. The PEG-grafted hyaluronic acid was a white cottony material.
Differential scanning calorimetry was performed on the obtained PEG-grafted hyaluronic acid using α-alumina (5.48 mg) as a reference at a temperature elevation range of −30 to 160° C. and a temperature elevation rate of 2° C./min. The magnitude of the melting enthalpy of PEG near 60 ° C. is calculated from the area, and referring to the previous report, the melting enthalpy of PEG ΔH (mJ / mg): y and the graft ratio of PEG (wt%): y = 1 between x The graft rate was calculated from the relationship with .6113x. In addition, from previous research, the ratio of the mass of raw material PEG to the mass of raw material PEG and HA used: p and the ratio of the mass of grafted PEG to the mass of PEG-grafted hyaluronic acid: q between q = 0.678 p , the proportions of grafted PEG in the synthesized PEG-grafted hyaluronic acid were calculated to be approximately 5% by mass and 63% by mass. The yields of PEG-grafted hyaluronic acid were as high as 95% and 91%, respectively.

(2) Preparation of hydrogel Add HA to pure water (2.0 mL) on a 35 mm dish, and further polyethylene glycol (PEG, manufactured by Nacalai Tesque, M _n : 7400 to 10200) or ethylene glycol-ε-caprolactone block. A copolymer (PEG-PCL, available from Kansai University, M _n : 10300) was added and stirred with a spatula until a homogeneous solution was obtained. Then, α-aminopropyl-ω-aminopropylpolyoxymethylene (PEG-BA, manufactured by NOF Corporation, M _n : 2121), which is a PEG having both ends aminated, was added and stirred until completely dissolved. A condensing agent (“DMT-MM” manufactured by Wako) was added. After confirming that all the reagents were completely dissolved, the mixture was allowed to stand at room temperature for 24 hours. After 24 hours, each hydrogel was die-cut to a diameter of 1 cm and a thickness of 2 mm using the tip of an Eppendorf tube. After that, it was immersed in pure water for two days to remove unreacted substances. During this time, pure water was exchanged three times. The washing water was stored for calculation of the cross-linking agent introduction rate. After washing, the dried hydrogel was recovered by freeze-drying. The resulting dry hydrogel was a white solid. Also, the PEG-grafted hyaluron obtained in (1) above was crosslinked in the same manner to obtain a dry hydrogel.
Table 2 shows the amounts of reagents used to prepare each dry hydrogel. Hydrogels obtained from PEG-grafted hyaluronic acid grafted with about 5% by mass and 63% by mass of PEG are referred to as "PEG5-g-HA" and "PEG63-g-HA", respectively. In addition, the numbers in front of “PEG/HA” and “PEG-PCL/HA”, which are hydrogels in which a water-soluble polymer or an amphipathic polymer is not grafted to HA but are simply mixed and crosslinked, are PEG Mass percentage of PEG derivatives relative to the sum of derivatives and HA.

In "PEG5-g-HA" and "PEG63-g-HA", all PEGs are amide-bonded to HA, and free PEG is removed by dialysis and substantially does not exist in the hydrogel. Not likely. In "PEG/HA" and "PEG-PCL/HA", the condensing agent DMT-MM is highly reactive to amines, and even when alcohols and amines coexist, the resulting esters are amides. Since it is 1% or less (M. Kunishima et al., Tetrahedron, 57, 1551 (2001)), almost all PEG and PEG-PCL are not covalently bound to HA and exist free in the hydrogel. It is thought that

(3) Water Content Measurement Each dry hydrogel (10 mg) prepared was immersed in water for 9 days, and then the mass of the swollen hydrogel was measured. From the weight of the swollen hydrogel (W _wet ) and the weight of the dry hydrogel (W _dry ), the water content was calculated according to the following formula. Table 3 shows the results.
Water content (%) = [(W _wet -W _dry )/W _wet ] x 100

The water content of each hydrogel was 90-94%, with a maximum difference of 4% among the hydrogels. This difference is considered to be due to the measurement error due to the operation of draining the water when measuring the mass of the swollen hydrogel, and it was judged that the hydrogels prepared this time had almost the same water content.
The hydrogels other than the hydrogel containing PEG-PCL became colorless and transparent by containing water, but the swollen hydrogel containing PGE-PCL was white. Therefore, it was suggested that PEG-PCL forms aggregates in the gel.

(4) Measurement of cross-linking agent introduction rate The cross-linking agent (PEG-BA) contained in the washing water used during the preparation of each hydrogel was reacted with 2,4,6-trinitrobenzenesulfonic acid (TNBS), and measured using a spectrophotometer. was used to measure the absorbance at 345 nm to determine the amount of the cross-linking agent contained in the washing water. The cross-linking agent reaction rate of each hydrogel was calculated from the amount and the amount of the raw material cross-linking agent used. Table 4 shows the results.

The cross-linking agent introduction rate was 89 to 96%, and a maximum difference of 7% was observed between the hydrogels. The carboxylic acid of HA is used for grafting in the graft body, and in PEG63-g-HA, only about 50% of the carboxylic acid that can react with the cross-linking agent exists compared to other gels. However, since the graft ratio was 91%, it was judged that grafting had no effect on the introduction ratio of the cross-linking agent.

(5) Measurement of crosslink density The crosslink density of the prepared hydrogel was calculated by a compression test using an autograph. Specifically, the prepared hydrogel was cut into a cylindrical shape, and the thickness t ₀ and volume V ₀ before swelling were measured. Furthermore, after the hydrogel was swollen with pure water until it reached swelling equilibrium, the thickness t _s and the volume V _s were measured. After lightly wiping off surface moisture and inserting the hydrogel between fixed thick plates, an autograph test was performed at a compression rate of 1.0 mm/min to measure the stress of the hydrogel. The crosslink density was calculated using the following theoretical formula for rubber elasticity.
τ=RT×(ν _e /V ₀ )×ν ₂ ^1/3 ×(α−1/α ² )
[In the formula, τ represents stress (N/m ² ), R represents gas constant (J/Kmol), T represents absolute temperature (K), and ν _e /V ₀ represents crosslink density (mol/m ³ ), ν ₂ is the volume ratio before and after swelling of the hydrogel (no units), α is the elongation rate (Δl/l ₀ ), and l is the thickness of the hydrogel. ]
The results between each hydrogel were subjected to one way ANOVA Fisher's test using data analysis and graphing software ("ORIGIN 8.03" Lightstone). The results are shown in FIG. In FIG. 1, "*" indicates that there is a significant difference at p<0.05.
63PEG/HA had a crosslink density of about 51%, 63PEG-PCL/HA had a crosslink density of about 31%, and PEG63-g-HA had a crosslink density of about 10%, which were significantly lower than those of HA. Furthermore, a significant difference was confirmed between 63PEG/HA and PEG63-g-HA. This is probably because the PEG derivative present in the hydrogel exhibits a steric inhibition effect and reduces the introduction rate of the cross-linking agent. This is proved by the above experiments. Therefore, this result suggests that there exists a cross-linking agent that reacts with only one functional group of a bifunctional cross-linking agent and has a structure as if it were grafted onto HA. Therefore, it was considered that the introduction ratio of the cross-linking agent was the same as that of other hydrogels, and that a hydrogel with a low cross-linking density was prepared. In addition, PEG63-g-HA showed the lowest cross-linking density because the amount of carboxylic acid that can react with the cross-linking agent due to grafting is only half that of other hydrogels, and the cross-linking density decreased. Conceivable.

(6) Observation of Surface Properties Each freeze-dried hydrogel was cut into pieces of about 3 mm square and adhered to an aluminum plate using a carbon tape. Furthermore, platinum deposition was performed and observed using a scanning electron microscope (SEM).
The HA hydrogel was confirmed to have a porous structure with a large number of pores of several tens to 100 μm. Regarding hydrogels containing free PEG, a porous structure was confirmed in which pores from several tens of μm to about 200 μm in size were observed in 5PEG/HA, but there were a few pores of approximately 100 μm in 63PEG/HA. I just couldn't confirm. Hydrogels containing free PEG-PCL were observed to have surface roughness compared to other hydrogels. This is thought to be related to the crystal structure of PEG-PCL. Regarding pores, only a few pores of several tens of μm can be confirmed in both 5PEG-PCL/HA and 63PEG-PCL/HA, and it was determined that the weight ratio of PEG-PCL has little effect on the number and size of pores. can. Regarding PEG-g-HA hydrogel, a few pores of about 100 μm were confirmed in PEG5-g-HA, but PEG63-g-HA had a very few pores of about 1000 μm in a cracked structure. It could be confirmed. PEG63-g-HA was a brittle hydrogel exhibiting the lowest crosslink density, and the huge pores in the hydrogel were considered to qualitatively support the low crosslink density.

Example 2: Vascular Inductivity Measurement Mouse tails were subjected to extraction with 0.1% acetic acid to obtain a 3 mg/mL Type I collagen solution. 10× Dulbecco's Modified Eagle's Medium/Nutrient Mixture Ham's F- containing the collagen solution and P3VS cells (2.6×10 ⁵ cells/well), which are vaginal stromal cells derived from p53 knockout mice 12 (“DMEM/F12” manufactured by Sigma) and 200 mM HEPES buffer containing 262 mM NaHCO ₃ and 0.05 N NaOH were mixed at a ratio of 8:1:1 so that the total volume was 500 μL, and PET culture inserts ( A collagen gel was obtained by pouring it onto a Falcon, pore size: 0.4 μm, and hardening it by treating it at 37° C. for 20 minutes. E1 cells (1.8×10 ⁵ cells/well), which are clonal strain cells of Mullerian epithelium, were seeded on the collagen gel and placed in a 24-well plate. DMEM/F12 medium supplemented with 10% Equa FETAL (Atlas Biological), 10 μg/mL insulin (Wako), 10 μg/mL transferrin (BBI Solutions), and 50 μg/mL ascorbic acid (Wako) in each well was added and cultured at 37°C for 1 day. After confirming adhesion of the epithelial cells, the culture medium was aspirated from the culture insert and co-cultured while exchanging the medium every day for a week to obtain a collagen gel containing E1 cells and P3VS cells.
Separately, dried hydrogels of HA, 5PEG/HA, 5PEG-PCL/HA, PEG5-g-HA, and PEG63-g-HA were cut to 2.5 mm ³ . Basic fibroblast growth factor (bFGF) at a concentration of 25 μg/mL was dissolved in phosphate-buffered saline containing 0.1% bovine serum albumin, and 0.1 mL of the resulting solution was applied to each dry hydrogel. A hydrogel containing a certain amount of bFGF was prepared by dripping and allowing to stand for 24 hours.
The above collagen gel was subcutaneously implanted into C57BL/6J mice (Sankyo Labo Service) together with each hydrogel, and the mice were bred for 2 weeks with free access to food and water.
Then, the transplanted specimen was taken out, fixed in a 4% paraformaldehyde aqueous solution at 4° C. overnight, and dehydrated using a tissue dehydration solution (Fujifilm Wako Pure Chemical Industries, Ltd.). Samples were embedded in paraffin and cut into 6 μm sections. Sections were deparaffinized with xylene, rehydrated, rinsed with PBS, and treated in 0.5% aqueous sodium citraconate solution at 95° C. for 45 minutes to expose the antigen. Non-specific binding was blocked for 30-60 minutes at room temperature in PBS containing 5% goat serum, 1% bovine serum albumin (BSA, Sigma), and 0.3% Triton X-100. A rabbit anti-mouse CD31 antibody (1/200, Cell Signaling Technology), which is an endothelial cell marker, was used as a primary antibody. Sections were incubated overnight at 4°C with primary antibody. After washing several times with PBS, the sections were incubated with Alexa Fluor 488 goat anti-rabbit antibody (1/300, Jackson ImmunoResearch Laboratories) for 2 hours at room temperature to stain vascular endothelial cells. A photograph of a sample using 5PEG/HA hydrogel is shown in FIG.
The subcutaneously implanted collagen adhered to the host tissue. In FIG. 2, the dotted line is the boundary between the collagen gel and the adhesion host tissue, with the host tissue on the left and the collagen gel on the right. In addition, green fluorescence indicates cell membranes of vascular endothelial cells, and blue fluorescence indicates cell nuclei. Since vascular endothelial cells were not included in the implanted collagen gel, it was confirmed that blood vessels were generated from the host tissue side to the collagen gel side. Thus, 5PEG/HA hydrogel was found to have a clear blood vessel-inducing ability, and similar results were confirmed for 5PEG-PCL/HA hydrogel. When the same experiment was repeated three times, the collagen gel was maintained all three times only when 5PEG/HA hydrogel and 5PEG-PCL/HA hydrogel were used. Experiments with /HA hydrogels were repeated six times.
On the other hand, when hydrogels of HA, PEG5-g-HA, and PEG63-g-HA were used, the collagen gel was retracted, and the collagen gel was maintained only once. The experiment could not be repeated enough to calculate . As a reason for this, it is conceivable that blood vessels were not formed up to the collagen gel and the transplanted cells died, causing an inflammatory reaction or the like and accelerating the decomposition of the collagen gel. On the other hand, when 5PEG/HA hydrogel and 5PEG-PCL/HA hydrogel were used, blood vessels were generated up to the collagen gel, and the transplanted cells were maintained, suggesting that the collagen gel was also maintained.
The obtained images were analyzed, and the ratio of the CD31 antibody-stained portion, that is, the blood vessel portion in each image was calculated. Table 5 shows the results.

As shown in Table 5, the 5PEG/HA hydrogel and 5PEG-PCL/HA hydrogel according to the present invention were clearly superior in angiogenic activity to other hydrogels.

Example 3: Evaluation of Epithelial Induction In the same manner as in Example 2 above, E1 cells, a clonal cell line of Müllerian epithelium, and M2 cells, a P3VS-derived clonal stromal cell that induces epithelial organization, are included. A collagen gel was prepared and subcutaneously implanted into mice either alone or together with the 5PEG/HA hydrogel and 5PEG-PCL/HA hydrogel whose effects were observed in Example 2 above, and the mice were bred for 2 weeks to obtain samples. . It was observed whether epithelial tissue was present in the obtained sample and whether collagen gel containing surviving stromal cells (M2 cells) remained. Samples were categorized as having no epithelial tissue and no collagen gel containing viable stromal cells, or both. Table 6 shows the results.

As the results shown in Table 6, when 5PEG/HA hydrogel and 5PEG-PCL/HA hydrogel were co-implanted, mice were bred for 2 weeks, and the samples obtained were composed of epithelial cells. Tissue and/or collagen gel containing viable stromal cells was included. When these results were subjected to the χ ² -test, a significant difference was recognized at p<0.05.
Fig. 3 shows a photograph of a 5PEG/HA hydrogel-collagen gel co-implanted sample in which the nucleus of the uterine epithelium was immunohistochemically stained with green fluorescence using an antibody against HMGA2, a transcription factor specifically expressed in the uterine epithelium. shown in As shown in FIG. 3, when 5PEG/HA hydrogel and collagen gel were co-implanted, a single epithelial layer was formed near the boundary between the collagen gel and the host tissue, and HMGA2, a uterine epithelial marker, was also expressed. Therefore, it was suggested that a uterine-like epithelial tissue was induced.
These results are considered to be due to the formation of blood vessels by the hydrogel of the present invention, which supplied oxygen and nutrients to epithelial cells in collagen.

Example 4 Evaluation of bFGF Release Behavior Basic fibroblast growth factor (bFGF) labeled with a fluorescent molecule (FITC) was synthesized and a 12 mM PBS solution with a concentration of 1000 μg/mL was prepared. 0.1 mL of the solution was added dropwise to each dried hydrogel and allowed to stand for 42 hours to prepare a hydrogel containing 100 μg of FITC-bFGF per gel.
The above hydrogel was wrapped in a tea pack, placed in 12 mM PBS (pH 7.5) containing 0.1 mass % NaN ₃ and incubated at 37°C. After 5, 10, 20, 30, 40, 50, 60, 120, 180, 360, 540, 720, 1440 minutes after the start of incubation, and every 24 hours thereafter, solution samples were obtained and measured by fluorescence. The concentration of FITC-bFGF released into PBS was determined. The measurement was performed 6 times for each hydrogel, and the average value was obtained from the 4 data excluding the maximum and minimum values. The results are shown in FIG.
The results between each of the two groups obtained were tested for significance by a t-test. No significant difference was observed except for the significant difference at p<0.05.
5PEG/HA hydrogel and 5PEG-PCL/HA hydrogel were found to have angiogenic and tissue formation promoting effects, whereas HA hydrogel, PEG5-g-HA hydrogel and PEG63-g-HA hydrogel had Considering the results of Examples 2 and 3 that no effect was observed, it was found that the angiogenic effect and tissue formation promoting effect of the hydrogel are not related to the sustained release action of cell growth factors. rice field.

Claims (5)

containing hydrogels and cell growth factors,
An angiogenesis-promoting material, wherein the hydrogel contains hyaluronic acid crosslinked with polyethylene glycol, and at least one of polyethylene glycol and polyethylene glycol-poly(ε-caprolactone) block copolymer . The angiogenesis-promoting agent according to claim 1, wherein the polyethylene glycol and the polyethylene glycol-poly(ε-caprolactone) block copolymer have a number average molecular weight of 1,000 or more and 20,000 or less. The polyethylene glycol and the polyethylene glycol-poly(ε-caprolactone) block copolymer for the sum of the polyethylene glycol cross-linked hyaluronic acid, the polyethylene glycol and the polyethylene glycol-poly(ε-caprolactone) block copolymer The angiogenesis-promoting material according to claim 1 or 2 , wherein the ratio of is 1% by mass or more and 80% by mass or less . The angiogenesis-promoting material according to any one of claims 1 to 3 , wherein the amount of cell growth factor in said hydrogel is 1 µg/g or more and 3000 µg/g or less . The angiogenesis promoting material according to any one of claims 1 to 4 , wherein the cell growth factor is basic fibroblast growth factor.

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