JP2021116303A - Periodontal tissue formulation - Google Patents

Abstract

To provide novel periodontal tissue formulations that can be treated by simple operation and have high safety and periodontal tissue regeneration performance.SOLUTION: The present invention relates to a periodontal tissue formulation containing a culture supernatant. In particular, the periodontal tissue formulation of the present invention contains a culture supernatant capable of being obtained by a method comprising: a first culture step of culturing cells using a first medium; a second culturing step of culturing the cells using a second medium different from the first medium as a medium, after the first culturing step; and a step of obtaining a culture supernatant containing the second medium, after the second culture step, the second medium being able to contain calcium ions and a buffer, and the buffer being able to be selected from good buffers, especially HEPES.SELECTED DRAWING: None

Description

The present invention relates to a periodontal tissue preparation containing a culture supernatant and a surgical method using the preparation.

Periodontal tissue is affected by periodontal disease. As periodontal disease progresses, so-called periodontal pockets are formed between the teeth and the gingiva, and the gingiva retracts. As periodontal disease progresses further, the alveolar bone melts and the teeth move, and finally the need for tooth extraction arises. Periodontal tissue may also be affected by orthodontics.

Various methods have been studied for the regeneration of periodontal tissue affected by periodontal disease and the like. For example, bone grafting, GTR method, and periodontal tissue regeneration therapy using enamel matrix protein have been conventionally performed.

In recent years, a periodontal tissue regenerating agent called Regros (trademark) has been sold. Ligros contains a protein called fibroblast growth factor as a main component, and is said to be able to promote the proliferation and differentiation of bone, muscle, adipocytes and the like. In addition, it is said that it can regenerate periodontal tissue because it has a strong angiogenic effect. However, the procedure requires periodontal surgery and is very expensive.

Hyaluronic acid is also injected to regenerate the gingiva. Hyaluronic acid injection is effective for gingival regeneration if performed by an appropriate method, but it may not be effective depending on the physical condition and / or constitution of the patient, and it is effective for alveolar bone regeneration. There wasn't.

An object of the present invention is to provide a novel periodontal tissue preparation which can be treated by a simple operation and has high safety and periodontal tissue regeneration performance.

The present inventors have found that the above problems can be solved by the present invention having the following aspects.
<< Aspect 1 >>
A preparation for periodontal tissue containing a culture supernatant.
<< Aspect 2 >>
The culture supernatant is
The first culture step of culturing cells using the first medium and
After the first culturing step, a second culturing step of culturing the cells using a second medium different from the first medium as a medium, and a second culturing step.
The preparation for periodontal tissue according to aspect 1, which is obtained by a method including a step of obtaining a culture supernatant containing a second medium after the second culture step.
<< Aspect 3 >>
The preparation for periodontal tissue according to aspect 2, which is obtained by culturing until it becomes confluent in the first culturing step.
<< Aspect 4 >>
The preparation for periodontal tissue according to aspect 2 or 3, wherein the second medium contains calcium ions and a buffer.
<< Aspect 5 >>
The preparation for periodontal tissue according to aspect 4, wherein the buffer is selected from a good buffer.
<< Aspect 6 >>
The preparation for periodontal tissue according to aspect 5, wherein the good buffer is HEPES.
<< Aspect 7 >>
The preparation for periodontium according to any one of aspects 2 to 7, wherein the second medium is an infusion solution for injection or an infusion solution for infusion.
<< Aspect 8 >>
The periodontium according to any one of aspects 1 to 7, wherein the culture supernatant is a culture supernatant of adipose tissue-derived mesenchymal cells, epithelial-derived epithelial cells, or dental pulp-derived mesenchymal stem cells. Tissue preparation.
<< Aspect 9 >>
The preparation for periodontal tissue according to any one of aspects 1 to 8, further comprising hyaluronic acid.
<< Aspect 10 >>
The preparation for periodontal tissue according to any one of aspects 1 to 9, wherein the hyaluronic acid is a crosslinked hyaluronic acid gel.
<< Aspect 11 >>
A method for producing a periodontal tissue preparation containing a culture supernatant.
The first culture step of culturing cells using the first medium and
After the first culturing step, a second culturing step of culturing the cells using a second medium different from the first medium as a medium, and a second culturing step.
A method comprising a second culture step followed by a step of obtaining the culture supernatant containing a second medium.

FIG. 1a shows the condition of the periodontal tissue of a patient with gingival recession. The gingiva of the maxillary anterior teeth and the gingiva of the mandibular anterior teeth indicated by the arrows are retracted, resulting in a black triangle. FIG. 1b shows the state of the gingiva of the patient after multiple injections of the preparation of the present invention into the gingiva of the patient of FIG. 1a. The gingiva of the maxillary gingiva and the mandibular gingiva indicated by the arrows was restored, and the black triangle disappeared or improved. FIG. 2a shows the state of periodontal tissue in a patient with alveolar bone defect. FIG. 2b shows the state of the alveolar bone of the patient after multiple injections of the preparation of the present invention into the alveolar bone of the patient of FIG. 2a. FIG. 3a shows the state of periodontal tissue in a patient with alveolar bone defect due to severe periodontal disease. The arrow on the left in the figure indicates the alveolar bone with a defect. The right arrow in the figure indicates the apical bone of the canine. FIG. 3b shows the state of the alveolar bone of the patient after multiple injections of the preparation of the present invention into the alveolar bone of the patient of FIG. 3a. FIG. 4a shows the state of periodontal tissue in a patient whose periodontal tissue has been destroyed by severe periodontal disease. The arrow in the left figure of FIG. 4a indicates the depression resulting from the loss of the alveolar bone. The arrow in the right figure of FIG. 4a indicates the bone at the apex of the thinned canine tooth. FIG. 4b shows the condition of the alveolar bone of the patient after multiple injections of the preparation of the present invention into the gingiva of the patient of FIG. 4a. The arrow in the left figure of FIG. 4b indicates that the alveolar bone was regenerated and the depression was reduced. The arrow in the right figure of FIG. 4b indicates that the bone at the apex of the canine is regenerated. FIG. 5a shows the condition of the periodontal tissue of a patient with gingival recession. The gingiva of the maxillary anterior teeth and the gingiva of the mandibular anterior teeth indicated by the arrows are retracted, resulting in a black triangle. FIG. 5b shows the state of the gingiva of the patient after multiple injections of the preparation of the present invention containing the culture supernatant and hyaluronic acid into the gingiva of the patient of FIG. 5a. The gingiva of the maxillary gingiva and the mandibular gingiva indicated by the arrows was restored, and the black triangle disappeared or improved.

The periodontal tissue preparation of the present invention contains a culture supernatant as an active ingredient. The periodontal tissue-regenerating preparation means, for example, a periodontal tissue-regenerating preparation. The periodontal tissue includes the crown portion and the root portion. The preparation for periodontium of the present invention particularly relates to a preparation for regenerating the gingiva of the crown portion and the alveolar bone of the root portion. In one example, the periodontal tissue preparation may be a therapeutic preparation for periodontal disease or alveolar pyorrhea. In the present specification, the culture supernatant is a culture solution obtained by culturing cells, and usually contains substantially no cells. Also, as used herein, hyaluronic acid refers to all variants of hyaluronic acid, hyaluronate or hyaluronan, and all variants thereof, including various chemical modifications of various chain lengths and charged states, including cross-linking. Includes combinations of variants. Further, in the present specification, the periodontium includes teeth and surrounding tissues, for example, crown portions such as enamel, dentin, pulp and gingiva, and roots such as cementum, alveolar bone, blood vessels and nerves. Including the part.

The present inventor has found that periodontal tissue affected by periodontal disease or the like can be regenerated very effectively and easily by a preparation containing a culture supernatant. That is, while the conventional regeneration method has problems that periodontal surgery is required, the effect differs depending on the practitioner, and the safety is lowered depending on the practitioner, the present invention has a problem. According to the formulation of, it is only necessary to inject around the periodontal tissue to be regenerated, and it was found that the effect of the regeneration is very high. In particular, it was found that the alveolar bone was not substantially regenerated by the conventional regeneration method, whereas the formulation of the present invention enables the alveolar bone to be regenerated. rice field.

<Culture supernatant>
As described above, the culture supernatant is a culture solution obtained by culturing cells, and usually contains substantially no cells. The cells or stem cells used to obtain the culture supernatant may be of human or non-human mammal origin, insect origin, bird origin, or plant origin. May be good. From the viewpoint of not causing an immune reaction, the cells are preferably human-derived cells, and particularly mesenchymal or epithelial cells or stem cells are used. Preferably, the cell is an adipose tissue-derived mesenchymal cell, an epidermis-derived epithelial cell, or a dental pulp-derived mesenchymal stem cell, and particularly preferably, the cell is an adipose tissue-derived mesenchymal cell or umbilical cord. Blood stem cells.

The culturing method may be appropriately adjusted according to the target cells. For example, when using mammalian cells, the culture can be carried out under any conditions suitable for culturing the mammalian cells, but generally, the cells are cultured at 37 ° C. and 5% CO ₂ for several days. The medium may be changed if necessary.

The medium is not particularly limited, and examples thereof include α-MEM and DMEM supplemented with 10 to 15% autologous serum or fetal bovine serum (FBS) and antibiotics. A medium containing no human or animal-derived components may be used. If necessary, growth factors such as fibroblast growth factor (bFGF) and adrenomeduulin may be added to the medium. Further, as the medium, those listed below can be used as the second medium.

The culture supernatant includes a first culture step of culturing cells using the first medium, and a second culture step of culturing the cells using a second medium different from the first medium after the first culture step. It can be obtained by a method including a step of obtaining a culture supernatant containing a second medium after the second culture step.

As the first culturing step, a known culturing method can be appropriately adopted. The main purpose of the first culturing step is to proliferate the cells, and the cells can be cultured until they become subconfluent or confluent.

As the first medium, any medium can be used as long as it is a medium capable of proliferating the cells to be used. As an example, α-MEM, DMEM and the like supplemented with 10 to 15% autologous serum or fetal bovine serum (FBS) and antibiotics can be mentioned. A medium containing no human or animal-derived components may be used. If necessary, growth factors such as fibroblast growth factor (bFGF) and adrenomeduulin may be added to the medium.

After the first culture step, the cells can be collected and washed as appropriate. Then, the collected cells are cultured in a second medium different from the first medium.

The second culturing step, without using the CO ₂ incubator, which does not perform a CO ₂ incubator is preferred. That is, it is preferable to culture in the range of 34 to 40 ° C., preferably around 37 ° C., using a culture vessel _{without CO 2 incubation.} The second culturing step is preferably a step of culturing cells for 5 hours or more and 5 days or less (10 hours or more and 2 days or less, or 5 hours or more and 3 days or less). The culture may be adhesive culture or suspension culture depending on the cells to be cultured, and when removing the cells, the cells may be cultured in a method that facilitates removal.

As the second medium, the medium mentioned as being usable as the first medium and other well-known media can be used, and may be prepared by further adding a calcium ion source and / or a buffer. From the viewpoint of not causing an immune reaction, it is preferable to use a serum-free medium. The second medium is preferably an electrolyte solution containing calcium ions and a buffer, and optionally containing prostaglandins. It has been found that a particularly advantageous effect can be obtained when prostaglandins are further contained. The calcium ion source is not particularly limited as long as it is a water-soluble and bioacceptable calcium salt, and examples thereof include calcium chloride.

In addition, the second medium may contain an infusion solution for injection or an infusion solution for infusion. When the second medium contains an infusion solution for injection or an infusion solution for infusion, those manufactured and sold as an infusion solution for injection or an infusion solution for infusion can be appropriately used. Examples of infusion fluids for injection are sugar fluids, extracellular fluid replacement fluids (physiological saline, Ringer's fluid, lactic acid Ringer's fluid, extracellular fluid replacement fluid, Ringer's acetate solution, Ringer's bicarbonate solution), hypotonic electrolyte solutions, amino acid preparations (high). Concentrated amino acid solution, renal failure amino acid solution, liver failure amino acid solution, pediatric amino acid solution), PPN (peripheral parenteral nutrition infusion), TPN (high calorie infusion), fat emulsion and substitute plasma bulking agent. Among these, extracellular fluid replacement solution (physiological saline solution, Ringer's solution, lactated Ringer's solution, extracellular fluid replacement solution, Ringer's acetate solution, Ringer's bicarbonate solution), and isotonic electrolyte solution are preferable. The specific infusion is Pareplus®. The above-mentioned calcium source, buffer, prostaglandin and the like may be added to these infusion solutions.

When the second medium contains calcium ions, the calcium ions are preferably 0.045 mM or more and 1.802 mM or less, 0.074 mM or more and 1.505 mM or less, 0.045 mM or more and 2 mM or less, or 0. It may be 180 mM or more and 2 mM or less, 1 mM or more and 2 mM or less, 1.3 mM or more and 1.8 mM or less, 1.2 mM or more and 1.6 mM or less, 1 mM or more and 1.6 mM or less. It may be 0.045 mM or more and 1.352 mM or less, 0.180 mM or more and 0.901 mM or less, or 20 mg / l or more and 100 mg / l or less. Examples of salts contained in the second medium are NaCl, KCl, and CaCl ₂ , which may be contained in an amount of 1 g / L or more and 30 g / L or less, or 4 g / L or more and 30 g / L or less. It may be contained in an amount of 6 g / L or more and 11 g / L or less.

As the _{buffer, MgSO 4 · 7H 2 O,} Na 2 HPO 4, KH 2 PO 4, there may be mentioned inorganic buffers such as NaHCO _3, also Good buffer (Good's buffers) organic such as A buffer can also be used. Good buffers include MES, bicine methane, ADA, PIPES, ACES, MOPSO, colamine hydrochloride, MOPS, BES, TES, HEPES, DIPSO, MOBS, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, Tricine, Tris, glycinamide, glycylglycine, HEPES, bicine, TAPS, MOPSB, CHES, AMP, AMPSO, CAPSO, CAPS, CABS, etc. can be mentioned, and among them, HEPES (hydroxyethylpiperazine ethanesulfonic acid) is particularly used. It is preferable to use it. These may be used in combination with other salts such as sodium hydrogen carbonate, sodium carbonate, pyruvate, citric acid, and salts thereof. The content of these buffers may be adjusted according to the pH range to be adjusted, for example, 1 mg / l or more and 5 g / l or less, or 2 mg / l or more and 500 mg / l or less. It may be 10 mg / l or more and 300 mg / l or less.

The acidity of the second medium is, for example, pH 5.5 or more and pH 9 or less, and may be pH 7.2 or more and pH 7.8 or less.

The second medium preferably contains little sugar, preferably contains substantially no sugar or does not contain any sugar, and preferably has a sugar (for example, glucose) content of 1 g / l or less. , 0.8 g / l or less, 0.5 g / l or less, 0.1 g / L or more and 1.5 g / L, 0.1 g / L or more and 1.2 g / L or less. It may be 0.1 g / L or more and 1 g / L or less, 0.5 g / L or more and 1.2 g / L or less, or 0.8 g / L or more and 1.1 g / L or less.

The second medium is preferably low in amino acids or substantially free of or completely free of amino acids, preferably having an amino acid content of 1 mg / ml or less, preferably 0.8 mg / l or less, and 0.5 mg / l or less. 1 or less is more preferable.

The second medium is preferably low in vitamins, substantially free of vitamins, or completely free of vitamins, and the content of vitamins is preferably 1 mg / ml or less, preferably 0.8 mg / l or less. 0.5 mg / l or less is more preferable. The second medium is preferably free of antibiotics (eg penicillin), growth factors and cytokines.

The second medium preferably contains little or no heavy metal elements such as iron, copper and lead, and trace elements. Since there are few such elements, it is possible to suppress the synthesis of metal-containing proteins and promote the synthesis of growth factors. The second medium is preferably free of carcinogens such as polyamines (eg, Putrescine 2HCl). The second medium preferably does not contain purine bases. If the second medium does not contain purine bases, it can activate the nucleic acid salvage pathway.

Since the second medium contains no or little vitamins and amino acids, it can promote autophagy and increase growth factor synthesis. The second medium can keep the environment during culturing constant in a general culturing environment (for example, it can suppress fluctuations in acidity and has a buffering capacity during culturing, so that it can be used for CO ₂ culture and the like. No need to do it.)

The second medium preferably has a larger amount of water than a normal medium. For example, when the second medium is 100% by weight, the water content is preferably 95% by weight or more and 99.99% by weight or less, 96% by weight or more and 99.9% by weight or less, and 97% by weight or more and 99.9% by weight or less. It may be as follows. The osmotic pressure can be reduced by increasing the amount of water in the medium. Then, for example, when the cells are adherently cultured, the cells are usually peeled off from the culture vessel using an animal-derived digestive enzyme such as trypsin, but when the second medium is used, it is not necessary to use the digestive enzyme. Then, when the agent containing the second medium is administered to the patient, side effects such as infection by animal-derived components can be reduced.

The second medium, carbohydrates (e.g., glucose), (including the source of calcium ions, for example NaCl, those containing them as one major salts composed only KCl and CaCl ₂₎ salts and buffers (e.g. MgSO ₄ · _{H 2 O, Na 2 HPO 4} · 2H 2 O, KH 2 PO 4, NaHCO 3, and Good's buffer agent (particularly HEPES) those containing them as made or main buffer only) and the balance solvent (e.g. water ) Only. With such a composition, as its effectiveness was confirmed in Examples described later, it highly expresses a specific gene or protein and functions as a medium and an infusion solution having excellent biocompatibility.

Since the second medium has a low amino acid content, it avoids interaction with synthetically secreted growth factors (mainly oxidation / reduction) and polymerization reaction, prevents alteration of components, and absorbs moisture by mixed amino acids. And can prevent alteration. In addition, since the second medium has a low amino acid content, the risk of bacterial growth when used as a raw material for cosmetics is reduced, so that the stability of the product can be ensured without adding preservatives or antioxidants. .. When the second medium does not contain amino acids, the de-amino acid step for use in the affected area becomes unnecessary, and it does not serve as a nutrient for the growth of bacteria and the like resident in the affected area. This means that when used on the wound surface, it does not cause a foul odor. In addition, since it does not cause dysgeusia or body odor when an agent containing a second medium is administered by injection or the like after being formulated, it is highly convenient when used as a medicine. When the second medium does not contain amino acids, desalting is easy. Therefore, when a large amount of cells are cultured, a high-concentration growth factor dried product can be easily obtained by freeze-drying. In the case of normal medium, the medium must be changed in 48-72 hours in any case. When the second medium is not an amino acid-containing medium, the viable environment can be maintained for a long period of time (for example, about 7 days) by suppressing the metabolic activity of the cultured cells, and then the medium is replaced with a medium. By doing so, the cells can proliferate again. The second medium is preferably a medium containing calcium ions and a buffer, and preferably has a simple composition, so that it is highly versatile and contains all animal cells (including ES cells, iPS cells / stem cells) and It can be applied to plant cells (particularly plant callus culture and maintenance of plant stem cells).

The step of obtaining the culture supernatant containing the second medium is performed after the second culture step. This step preferably further includes a step of adding trehalose. The preparation containing the culture supernatant preferably contains 50% by weight or more and 100% by weight or less of the second medium after the second culture step. The preparation containing the culture supernatant may contain 60% by weight or more and 100% by weight or less of the second medium (including the culture supernatant) after the second culture step, or 70% by weight or more and 99% by weight or less, 70% by weight. It is preferable to include 90% by weight or more, 80% by weight or more and 99% by weight or less, 90% by weight or more and 100% by weight or less, and 90% by weight or more and 95% by weight or less. Usually, as the culture supernatant of stem cells or the like, a supernatant component obtained by solid-liquid separation of the culture supernatant by centrifugation is usually used. Since the method described in this specification can positively include the second medium in the preparation, a filtered medium after the second culture step may be simply used.

The pharmaceutical product is a treated product obtained by removing water from the culture supernatant obtained as described above by freeze-drying, a treated product obtained by concentrating the culture supernatant under reduced pressure using an evaporator or the like, and ultrafiltration. The processed product obtained by concentrating the culture supernatant using a membrane or the like, the treated product obtained by solid-liquid separation of the culture supernatant using a filter, or the culture supernatant before the above-mentioned treatment. It may be undiluted solution. In addition, for example, the supernatant obtained by culturing cells is centrifuged (for example, 1,000 × g, 10 minutes) and then fractionated with ammonium sulfate (for example, 65% saturated ammonium sulfate), and the precipitate is subjected to an appropriate buffer solution. After suspension, dialysis treatment may be performed and filtered through a syringe filter (for example, 0.2 μm) to obtain a sterile culture supernatant. The collected culture supernatant can be used as it is, or it can be cryopreserved and thawed at the time of use. Further, a pharmaceutically acceptable carrier may be added and dispensed into a sterilization container so that the amount of liquid is easy to handle, for example, 0.2 ml or 0.5 ml. Furthermore, as a countermeasure against the risk of infectious pathogens, the culture supernatant may be treated with a virus clearance filter or γ-ray irradiation.

As described above, a freezing step of freezing the collected culture supernatant may be included after the second culture step and the step of obtaining the culture supernatant. In order to freeze the culture supernatant, for example, the culture supernatant may be adjusted to −200 ° C. or higher and 0 ° C. or lower, or −100 ° C. or higher and −5 ° C. or lower. The preparation containing the culture supernatant may be a product obtained by disrupting cells after the second culture step, centrifuging and then filtering using a filter, or a product obtained by further freezing and drying the filtered product. good.

<hyaluronic acid>
The present inventors have found that a preparation containing hyaluronic acid in addition to the culture supernatant has a remarkable effect. Although the formulation containing only the culture supernatant has an effect on the alveolar bone, it takes time to realize the effect, and the formulation using the culture supernatant tends to be expensive. In some patients, the treatment was stopped before the effect appeared, but the combined use of hyaluronic acid in the formulation of the present invention made it possible to realize the effect at an early stage. The fact that the effect can be realized at an early stage is very important for the continuation of treatment, and the formulation further containing hyaluronic acid has become a highly satisfying formulation for the patient.

Without being bound by theory, hyaluronic acid can be used to eliminate the black triangle caused by the thinning of the gums due to the swelling of the hyaluronic acid injection site. Hyaluronic acid is absorbed over time, but does not return to its original position after absorption. This is because the administration of hyaluronic acid can swell the gingiva to give a stretching stimulus to the gingival cells and enhance the activity of fibroblasts, for example, collagen synthesis ability and proliferative ability to promote gingival regeneration. However, continuous administration is required for such effects to be exhibited. On the other hand, although the culture supernatant has no immediate effect, it acts on the gingiva and alveolar bone to promote the regeneration of periodontal tissue, and the tissue regeneration ability of the culture supernatant around the tissue stretched by hyaluronic acid. Was effectively exhibited, and it is considered that a synergistic effect was obtained.

The term "hyaluronic acid" is a type of glycosaminoglycan composed mainly of glucuronic acid and N-acetylglucosamine. As mentioned above, "hyaluronic acid" is all variants of hyaluronic acid, hyaluronate or hyaluronan, and modifications thereof, including various chemical modifications of various chain lengths and charged states, including cross-linking. Including body combinations. That is, the term also includes various hyaluronates of hyaluronic acid with different counterions, such as sodium hyaluronate. Also, various modifications of hyaluronic acid include oxidation (eg, oxidation of -CH ₂ OH to -CHO and / or -COOH); periodate oxidation of nearby hydroxyl groups, and optionally subsequent reduction (eg,-. Reduction of CHO to -CH ₂ OH), or coupling with amines to form imines and reduction to secondary amines; sulfated; deaminated acid, optionally followed by deaminated with a new acid or Amide formation; esterification; cross-linking; substitution with various compounds, such as using cross-linking agents or carbodiimide-supported couplings; terms involving coupling of different molecules such as proteins, peptides and active agent components to hyaluronic acid, etc. Included by. Other examples of modifications are isourea, hydrazide, bromoyan, monoepoxide and monosulfone couplings.

Hyaluronic acid can be obtained from various sources of animal and non-animal sources. Sources of non-animal sources include yeast, preferably bacteria. The molecular weight of a single hyaluronic acid molecule is typically in the range 0.1-10 MDa, but other molecular weights are possible.

In certain embodiments, the concentration of the hyaluronic acid is in the range of 1-100 mg / ml. In some embodiments, the concentration of the hyaluronic acid is in the range of 2-50 mg / ml. In certain embodiments, the concentration of the hyaluronic acid is in the range of 5-30 mg / ml, or in the range of 10-30 mg / ml. In certain embodiments, hyaluronic acid is crosslinked. The cross-linked hyaluronic acid is a hyaluronic acid molecule held together by covalent cross-linking, physical entrainment of hyaluronic acid chains, and various interactions such as electrostatic interactions, hydrogen bonds and van der Waals forces. Includes cross-linking between hyaluronic acid chains to create a continuous network.

Cross-linking of hyaluronic acid may be achieved by modification with a chemical cross-linking agent. The chemical cross-linking agent may be selected, for example, from the group consisting of divinyl sulfone, multiepoxides and diepoxides. According to embodiments, the chemical cross-linking agent is selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE) and diepoxy octane. According to a preferred embodiment, the chemical cross-linking agent is 1,4-butanediol diglycidyl ether (BDDE).

The crosslinked hyaluronic acid product is preferably biocompatible. This implies that no or very mild immune response occurs in treated individuals. That is, it occurs in individuals treated with no or very mild unwanted local or systemic effects.

The crosslinked hyaluronic acid product according to the present invention may be a gel or a hydrogel. That is, it can be considered water insoluble, but can substantially dilute the cross-linking system of hyaluronic acid molecules when subjected to a liquid, typically an aqueous liquid.

The gel contains almost liquid by weight and can contain, for example, 90-99.9% water and behaves like a solid due to the three-dimensional crosslinked hyaluronic acid network in the liquid. Due to its important liquid content, the gel is structurally flexible and resembles a natural tissue that makes it very useful for tissue augmentation as a scaffold in tissue engineering.

As described above, cross-linking of hyaluronic acid to form a cross-linked hyaluronic acid gel can be achieved by modification with a chemical cross-linking agent such as BDDE (such as 1,4-butanediol diglycidyl ether). The hyaluronic acid concentration and the degree of cross-linking affect the mechanical properties, such as the elastic modulus G'and stability of the gel.

Cross-linked hyaluronic acid gels are often characterized in terms of "degree of modification". The degree of modification of hyaluronic acid gel is generally in the range of 0.1 to 15 mol%. The degree of modification of the hyaluronic acid gel is preferably 2 mol% or less, for example 1.5 mol% or less, for example 1.25 mol% or less, for example 0. The range is 1 to 2 mol% or less, for example, 0.2 to 1.5 mol%, for example, 0.3 mol to 1.25 mol%. The degree of modification (mol%) is the amount of the cross-linking agent (s) bound to HA, that is, the amount of the cross-linking agent (s) bound to the total molar amount of repeating the disaccharide unit of HA. Is shown. The degree of modification reflects how much HA was chemically modified by the chemical cross-linking agent. All reaction conditions for cross-linking techniques and suitable analytical techniques for determining the degree of modification are well known to those skilled in the art who can readily adjust for these and other related factors, thereby providing the appropriate conditions. , The degree of modification in the range of 0.1 to 2% can be obtained to modify the obtained product characteristics with respect to the degree of modification. BDDE (1,4-butanediol diglycidyl ether) crosslinked hyaluronic acid gels may be prepared, for example, according to the methods described in Examples 1 and 2 of WO97 / 04012A.

In a preferred embodiment, the hyaluronic acid is present in the form of a cross-linked hyaluronic acid gel cross-linked by a chemical cross-linking agent, wherein the concentration of the hyaluronic acid is in the range of 10-30 mg / ml and the chemical cross-linking. The degree of modification with the agent is in the range of 0.1 to 2 mol%.

Hyaluronic acid gels can also contain moieties of hyaluronic acid that are not cross-linked, i.e., not attached to a three-dimensional cross-linked hyaluronic acid network. However, preferably at least 50% by weight, preferably at least 60% by weight, more preferably 70% by weight, and most preferably at least 80% by weight of hyaluronic acid forms part of the crosslinked hyaluronic acid network.

The type of hyaluronic acid used can be changed depending on the site of the periodontal tissue in which the preparation of the present invention is used. For example, when injecting into the gingiva, the hyaluronic acid gel particles are relatively small and soft. For example, it is preferable to use Restyrene Vital Skin Boosters (registered trademark).

<others>
The preparation for periodontal tissue of the present invention can be produced by using a known method, and can be administered by using a known administration method such as injection or application into periodontal tissue. An example is intragingival or intraosseous alveolar administration, which may be administered to one or more sites. It is preferable to administer the preparation of the present invention as one administration unit in an amount of 0.1 mL or more and 3.0 mL or less, more preferably 0.2 mL or more and 2.0 mL or less, 0.3 mL or more and 1.5 mL or less, or 0.5 mL or more. It is administered in 1.2 mL or less.

When the preparation is used as a liquid preparation, the liquid preparation can be produced by a known method. For example, lyophilized culture supernatant powder is mixed with a pharmaceutically acceptable solvent and filled in a sterilized liquid preparation container. Then, it can be produced by optionally further adding hyaluronic acid to the container. Examples of pharmaceutically acceptable solvents are water for injection, distilled water, physiological saline, electrolyte solution, or a solution having a composition similar to that of a culture solution, and it is preferable to use a sterilized solvent. Examples of containers for sterile liquids are ampoules, vials, syringes, and bags. As these containers, known containers such as those made of glass or plastic can be used. Specifically, examples of plastic containers are those using materials such as polyvinyl chloride, polyethylene, polypropylene, and ethylene-vinyl acetate copolymer. Examples of sterilization methods for these containers and solvents are heating methods (flame method, drying method, high temperature steam method, circulating steam method, boiling method, etc.), filtration methods, irradiation methods (radiation method, ultraviolet method, high frequency method, etc.), The gas method and the chemical method. Such a sterilization method can be appropriately selected and used by those skilled in the art according to the material of the container and the properties of the solvent.

In one embodiment, the culture supernatant may contain cultured cells. When cells are used therapeutically as a liquid, intravenous injection may be the most frequently used transplantation method. As an example, in the case of intravenous injection, it is preferable to adjust the solution as a solution at ^{1 × 10 5} cells / mL or more and 5 × 10 ^{7 cells / mL or less, and 1 × 10 6} cells / mL or more and 1 × 10 ⁷ cells / mL. mL is more preferred. Further, as the mesenchymal stem cell agent adjusted as one administration unit of intravenous injection in humans, 1 × 10 ⁵ cells or more and 1 × 10 ⁹ cells are preferable, and 2 × 10 ⁷ cells or more and 2 × 10 ⁸ cells are more preferable. Is. For other administration routes, it can be used within the range of the amount of liquid that can be transplanted into tissues and the maximum number of cells that can be suspended in the amount of liquid.

The formulation may be prepared with a pharmaceutically acceptable carrier or vehicle. Pharmaceutically acceptable carriers or vehicles include, for example, pharmaceutically acceptable substances such as stabilizers, solubilizers, suspending agents, buffers, isotonic agents, antioxidants, or preservatives. can give. Examples of isotonic agents are sodium chloride and glucose. Examples of buffers are citrates, acetates, boric acid, and phosphates. As the aqueous medium for suspending the cells, for example, an aqueous solution for injection in which the osmotic pressure and pH are adjusted to near the blood value and the salts concentration and the like are adjusted may be appropriately used. Ringer's solution such as acetic acid Ringer's solution and other infusion solutions, physiological saline, glucose solution and the like can be used, but the present invention is not limited thereto. For example, when an infusion Ringer's solution is used, an acceptable amount of dimethyl sulfoxide (DMSO) or human serum albumin (HSA) may be added thereto. Examples of antioxidants are ascorbic acid, sodium bisulfite, and sodium metabisulfite.

It has been found that the pharmaceutical product of the present invention is particularly effective for women and men in their 20s to 50s, and is particularly effective for women in their 20s to 40s.

The present specification also discloses a treatment method for the periodontal tissue of a subject (human or non-human mammal) using the periodontal tissue preparation containing the culture supernatant as described above. When treatment is performed using this, it can be administered to the affected area by injection or the like.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

<< Manufacturing Examples 1 and 2 >>
<First culture step>
Adipose tissue-derived stromal cells extracted by enzymatically treating normal human adipose tissue are subjected to a non-coated culture flask (FALCON) using DME medium containing 20% FBS (fetal bovine serum) (Dalbeco modified Eagle's medium manufactured by Gibco, high glucose). It was cultivated in (manufactured by the company) and used as the primary culture. The primary cultured cells immediately before the confluence were collected by enzyme treatment, seeded on a 12-well plate for non-coated culture (manufactured by Sumitomo Bakelite Co., Ltd.) in the same medium, and cultured until they became confluent.

After confirming that the cells had become confluent, the medium was removed, and the cell surface was washed with PBS (Dulbecoline buffer manufactured by DS Pharma Biomedical).

<Second culture step>
The washed cells were replaced with FBS-free DME medium in Production Example 1 and HBSS (Hanks Balanced Salt Solution manufactured by Sigma Aldrich) -HEPES in Production Example 2. After that, 12 wells were divided into 4 groups of 3 wells each, and in Production Example 1 using FBS-free DME medium _{, the cells were cultured in a 5% CO 2} incubator, and in Production Example 2 using HBSS-HEPES medium, 5 The cells were cultured in an incubator without using _{% CO 2.}

<Recovery of culture supernatant>
Immediately after the replacement (0 hours), 3 hours, 6 hours, 24 hours, and 48 hours after each of Production Examples 1 and 2, the culture supernatant in each well was collected.

The FBS-free DME medium is Dulbecco's modified Eagle's medium that does not contain FBS. The DME medium contains various components such as glucose and L-glutamine.

HBSS (Hank's balanced salt solution) containing (1) sodium bicarbonate solution isotonic _{solution, (2) NaCl, KCl,} MgSO 4 · 7H 2 O, Na 2 HPO 4, glucose, and _KH 2 PO ₄ It is a solution in which a buffer solution and (3) CaCl _{2 solution are mixed.}

HEPES is a hydroxyethylpiperazine ethanesulfonic acid, which is a buffer or pH regulator.

The amount of sugar contained in the FBS-free DME medium was 4.5 g / L, the total amount of amino acids (L-glutamic acid, etc.) was 1.6 g / L, and the amount of calcium ions was 1.8 mM.

On the other hand, the amount of sugar contained in the HBSS-HEEPS medium (hanks-HEEPES) was 1.0 g / L, NaCl was 8.0 g / L, KCl was 0.4 g / L, and CaCl ₂ was 0. _{14g / L, MgSO 4 · H} 2 O _{is, 0.2g / L, Na 2 HPO} 4 · 2H 2 O _{is, 0.06g / L, KH 2 PO} 4 is 0.06 g / L, NaHCO ₃ is It was 0.35 g / L, 20 mM HEPES pH 7.4, the total amount of amino acids (L-glutamic acid, etc.) was 0 g / L, and the calcium ion was 1.5 mM.

<Measurement method of gene expression level>
After collecting the culture supernatant as shown above, 400 microliters of RNA extract ISOGEN (Nippon Gene No. 319-90211) was added to each well, and total RNA was extracted from the cells according to a conventional method, and etatin mate was used. The total RNA precipitate obtained using (Nippon Gene No. 312-01791) was dissolved in nuclease-free water. The total RNA concentration was measured by nanodrop (Thermoscientific) using 1.5 microliters of this. Complementary DNA was synthesized from 100 nanograms of the obtained total RNA using the eye script cDNA synthesis kit (BIO RAD No. 1708891), and used for quantification of the expression level of each gene by real-time PCR (Rotor Gene Q manufactured by QIAGEN). ..

<Quantification of gene expression by real-time PCR method>
The synthesized DNA, the primer specific to the gene to be detected and quantified (Quantitect Primer Assay, QIAGEN), and the real-time PCR reagent (RotorGene SYBR Green, QIAGEN) are mixed, and real-time PCR is performed by the RotorGeneQ system (QIAGEN). The gene fragment to be detected and quantified was amplified. At this time, β-Actin-specific primers, which are housekeeping genes, were similarly amplified, and the quantitative values of the genes to be detected and quantified were relatively calculated using the amplification curve as an index. The following primer mixture from QIAGEN was used as a primer specific to each gene.

Human β-Actin
Hs_ACTB_1_SG Catalog number QT00095431
Human FGF2
Hs_FGF2_1_SG Catalog number QT000457579
Human VEGFA
Hs_VEGFA_1_SG Catalog number QT01010184

<Method for quantifying proteins contained in culture supernatant>
The collected culture supernatant was passed through a filter having a diameter of 0.22 micrometers to prepare a sample for protein quantification. A QUANTIKINE ELISA (Human VEGF, FGF-2) kit manufactured by R & D SYSTEMS was used for protein quantification of the sample, and quantified values of each protein were obtained according to the protocol.

The results are shown in Table 1.

<Results of real-time PCR>
The expression level of the VEGF gene in the cells was higher in Production Example 2 (Hanks-HEPES medium). In addition, the expression level of the FGF-2 gene in the cells was also high in Production Example 2 (Hanks-HEPES medium).

<Results of protein quantification>
The amount of VEGF in the culture supernatant was large in Production Example 2 (Hanks-HEPES medium). The amount of FGF-2 in the culture supernatant was large in Production Example 1 (DME medium).

The gene expression level and the amount of protein in the culture supernatant all tended to increase over time for 0, 6, 24, and 48 hours. The expression level of the VEGF gene and the amount of protein in the culture supernatant were higher in Production Example 2. The gene expression level of FGF-2 was higher in Production Example 2, but the amount of protein in the culture supernatant was higher in Production Example 1.

<< Manufacturing Examples 3 and 4 >>
<First culture step>
Epidermal keratinized cells extracted by enzymatically treating normal human skin tissue are subjected to a non-coated culture flask (FALCON) using a serum-free cell medium (Gibco No. MEPI500CA) containing supplement S7 (Gibco No. S0175). ) Was used as the primary culture. The primary cultured cells immediately before the confluence were collected by enzyme treatment, seeded on a 12-well plate for non-coated culture (manufactured by Sumitomo Bakelite Co., Ltd.) in the same medium, and cultured until they became confluent.

After confirming that the cells had become confluent, the medium was removed, and the cell surface was washed with PBS (Dulbecoline buffer manufactured by DS Pharma Biomedical).

<Second culture step>
The washed cells were replaced with a supplement 7-free serum-free cell medium (EpiLife) in Production Example 3 and a solution of HBSS-HEPES pH 7.4 in Production Example 4.

<Recovery of culture supernatant>
After that, 12 wells were divided into 4 groups of 3 wells each, and the culture supernatant in each well was collected immediately after replacement (0 hours), 3 hours, 6 hours, and 24 hours.

The amount of sugar contained in the supplement 7-free serum-free cell medium (EpiLife) of Production Example 3 was 1.0 g / L, the total amount of amino acids (L-glutamic acid, etc.) was 1.5 g / L, and calcium ions. Was 8.4 mg / L.

<Quantification of gene expression by real-time PCR method>
As described in Production Examples 1 and 2, gene expression and protein were quantified by the real-time PCR method. However, the following primer mixture from QIAGEN was used as a primer specific to human EGF.

Human EGF
Hs_EGF_1_SG Catalog number QT00051646

The results are shown in Table 2.

The intracellular expression level of the EGF gene was higher in Production Example 4 (Hanks-HEPES medium) than in Production Example 3 (EpiLife medium). The intracellular FGF-2 gene expression level was also higher in Production Example 4 than in Production Example 3. The intracellular VEGF gene expression level was also higher in Production Example 3 and Production Example 4.

<< Manufacturing Examples 5 to 7 >>
The culture solution of adipose tissue-derived stromal cells that became confluent by culturing in the same manner as in the first culturing step of Production Example 1 was replaced with three kinds of solutions and cultured without using _{5% CO 2 for 48 hours.} The expression level of each intracellular mRNA was quantified later. In Production Example 5, it was replaced with FBS-free DME medium, in Production Example 6, it was replaced with HBSS-HEPES pH 7.4 medium, and in Production Example 7, it was replaced with Preplus infusion.

Pareplus means Pareplus infusion, glucose, sodium chloride, L-sodium lactate, calcium chloride hydrate, magnesium sulfate hydrate, zinc sulfate hydrate, thiamine chloride hydrochloride, pyridoxin hydrochloride, cyanocobalamine, bread Includes tenol, glacial acetic acid, amino acids, electrolytes, stabilizers, and pH adjusters.

The results of quantifying the expression level of mRNA are shown in Table 3.

From Table 3, it can be seen that the gene expression level of the electrolyte solution medium (Hanks-HEPES and Pareplus) of Production Example 6 is higher than that of the DME medium of Production Example 5.

<< Manufacturing Examples 8 to 12 >>
The culture solution of adipose tissue-derived stromal cells that became confluent by culturing in the same manner as in the first culturing step of Production Example 1 was replaced with the solution shown in Table 4 below, and each of the intracellular cells 48 hours after culturing. The expression level of mRNA was quantified. In addition, only Production Example 8 was cultured using _{5% CO 2.}

KN2 indicates KN2 infusion. KN2 infusion contains sodium chloride, potassium chloride, L-sodium lactate, magnesium chloride, sodium dihydrogen phosphate hydrate, dipotassium phosphate and glucose.

Further, "+ Ca" means that calcium chloride is additionally added so that the calcium ion becomes 1.0 mM.

Comparing Production Example 9 and Production Example 10, it can be seen that the addition of HEPES (pH 7.4) slows down the rate at which PH becomes acidic, thereby promoting gene expression.

<< Example of freeze-drying method >>
After 48 hours have passed in the second culture step, the culture supernatant is collected in a 50 mL centrifuge tube (Sumitomo Bakelite Co., Ltd.) and then centrifuged (740 G, 5 minutes). It was passed through a filtration filter (Kurabo Industries Ltd.) and dispensed into a syringe (Termo Co., Ltd.). The syringe was sealed in a sterilization bag, frozen in a freezer at −80 ° C., transferred to a freeze-drying apparatus (Yamato Scientific Co., Ltd.), and freeze-dried. After freeze-drying, it was sealed with a syringe lure cap (Terumo Corporation).

<< Manufacturing Example 13 >>
After confirming that the adipose tissue-derived stromal cells became confluent in the same manner as in Production Example 1, the medium was removed and the cell surface was washed with PBS.

The washed cells were replaced with HBSS-HEPES pH 7.4 medium. Then, 12 wells were divided into 6 groups of 2 wells each, and these were cultured in an incubator (Production Example 13). Immediately after replacement (0 hours), 1 day, 2 days, 3 days, 4 days, 5 days, 6 days later, total RNA was extracted from the cells of each well using ISOGEN (manufactured by Nippon Gene), and the VEGF gene was extracted by real-time PCR. Relative quantification of the expression of.

The results are shown in Table 5.

As shown in Table 5, it was revealed that gene expression was maintained until 5 days later.

<< Manufacturing Example 14 >>
After confirming that the adipose tissue-derived stromal cells became confluent in the same manner as in Production Example 1, the medium was removed and the cell surface was washed with PBS.

The washed cells were replaced with HBSS-HEPES pH 6.5, HBSS-HEPES pH 7.0, HBSS-HEPES pH 7.4, HBSS-HEPES pH 7.8, and HBSS-HEPES pH 9.0. Immediately after the replacement (0 hours) and 48 hours later, total RNA was extracted from the cells of each well using ISOGEN (manufactured by Nippon Gene), and the expression of the VEGF gene was relatively quantified by real-time PCR.

The results are shown in Table 6.

As shown in Table 6, it was revealed that gene expression was maintained from HBSS-HEPES pH 7.0 to HBSS-HEPES pH 7.8.

<< Manufacturing Example 15 >>
In the same manner as in Production Example 1, adipose tissue-derived stromal cells were cultured in a 12-well plate, and after confirming that they became confluent, the medium was removed and the cell surface was washed with PBS.
The washed cells were divided into 4 groups of 3 wells each, and HBSS-HEEPS added with PGE1 (Prostandin Maruishi Pharmaceutical Co., Ltd.) at concentrations of 0 ng / mL, 4 ng / mL, 40 ng / mL, and 400 ng / mL, respectively. Was replaced with, and the cells were cultured in an incubator. Forty-eight hours after the replacement, the culture supernatant was collected from each well and protein quantified by QUANTIKINE ELISA Human VEGF (manufactured by R & D SYSTEMS). In addition, total RNA is extracted from each well after collecting the culture supernatant using ISOGEN (manufactured by Nippon Gene), and complementary DNA is synthesized using the eye script cDNA synthesis kit (manufactured by BIO RAD), which is then subjected to real-time PCR. It was used for relative quantification of VEGF gene expression level by (Rotor Gene Q, manufactured by QIAGEN).

The results are shown in Table 7.

As shown in Table 7, the relative quantitative values of the mRNA expression levels were almost the same regardless of the amount of PGE1 added. It was clarified that the amount of protein tends to increase according to the amount of PGE1 added. From this, it was found that by substituting the medium with HBSS-HEPES containing PGE1 and culturing for 48 hours, a culture supernatant containing a higher concentration of VEGF can be obtained.

<< Manufacturing Examples 16-18 >>
<First culture step>
Adipose tissue-derived stromal cells (ASC) extracted by enzymatically treating normal human adipose tissue are cultured in DME medium containing 20% FBS (fetal bovine serum) (Dalveco modified Eagle's medium manufactured by Gibco, high glucose). The cells were cultured in a flask T75 (manufactured by BD FALCON) to prepare a primary culture. The primary cultured cells immediately before the confluence were collected by enzymatic treatment, diluted appropriately, and the appropriate number of cells was seeded on one 6-well plate for culture (manufactured by Sumitomo Bakelite Co., Ltd.) in the same medium and cultured until the confluence was reached.

The cells of each confluent well were divided into two groups of 3 wells, the medium of each was removed, and the cell surface of each well was washed with PBS (DS Pharma Biomedical Co., Ltd. d'Albeccholine buffer).

<Second culture step>
After washing, Production Example 16 was prepared with a DME high glucose medium, Production Example 17 was prepared with HBSS (Hanks Balanced Salt Solution manufactured by Sigma-Aldrich) having a final concentration of 10 mM HEPES pH 7.4, and Production Example 18 was prepared with a final concentration of 20 mM HEPES pH 7. HBSS prepared to be 4 was added.

After culturing in the incubator for 48 hours, the culture supernatant was collected in a tube (manufactured by BD FALCON) having a volume of 5 mL each, and the VEGF protein was quantified by QUANTIKINE ELISA Human VEGF (manufactured by R & D SYSTEMS). Further, total RNA was extracted and concentrated from the cells of each well using ISOGEN (manufactured by Nippon Gene), and complementary DNA was obtained using an eye script cDNA synthesis kit (manufactured by BIO RAD). Then, using it, the relative quantification of each expression level of each of the VEGF, FGF2basic, MMP1, EFNA3, BMP1 and WNT5A genes was performed by real-time PCR (Rotorgene Q, manufactured by QIAGEN).

The results are shown in Table 8. The relative quantitative value was calculated using the value in the DME high glucose medium as the reference value after correcting between the samples using beta actin as the internal standard.

As shown in Table 8, each intracellular mRNA tends to be more expressed in cells cultured at HBSS-10 mM HEPES pH 7.4 than in DME medium, and the concentration of HEPES is higher at 20 mM. I understood.

<< Manufacturing Examples 19 to 20 >>
<First culture step>
In the same manner as in Production Examples 8 to 12, adipose tissue-derived stromal cells (ASC) were seeded on 5 6-well plates for culture (manufactured by Sumitomo Bakelite Co., Ltd.) and cultured until they became confluent (Production Example 19).

Similarly, human dental pulp stem cells (SHED) were seeded on 5 6-well plates and cultured until confluent (Production Example 20).

For ASC and SHED that became confluent, the cells in each well were divided into 3 groups of 2 wells each, the medium was removed from each well, and the cell surface of each well was subjected to PBS (DS Pharma Biomedical Co., Ltd. dalbecolinic acid buffer). Washed with.

<Second culture step>
After washing, 3 mL, 2 mL, and 1 mL of HBSS (Hanks Balanced Salt Solution manufactured by Sigma-Aldrich) -HEPES pH 7.4 were added to each of the three groups, and the cells were cultured in an incubator for 48 hours.

Then, each of the culture supernatants was collected in a tube (manufactured by BD FALCON) having a volume of 5 mL, and the VEGF protein was quantified by QUANTIKINE ELISA Human VEGF (manufactured by R & D SYSTEMS). Further, total RNA was extracted and concentrated from the cells of each well using ISOGEN (manufactured by Nippon Gene), and complementary DNA was obtained using an eye script cDNA synthesis kit (manufactured by BIO RAD). Then, using it, the relative quantification of the VEGF gene expression level was performed by real-time PCR (Rotorgene Q, manufactured by QIAGEN).

The results are shown in Table 9. The relative quantification value was calculated using beta actin as an internal standard, correction between samples, and a normal medium volume of 2 mL as a reference value.

The expression level of mRNA in the cells did not change depending on the amount of the culture supernatant. The amount of VEGF protein contained in the culture supernatant was smaller when the volume of the culture supernatant was larger and larger when the volume of the culture supernatant was smaller. From this, it was found that a culture supernatant containing more growth factors can be obtained by using a medium in an amount smaller than the volume of the medium normally used.

<< Manufacturing Examples 21 to 22 >>
In the same manner as in the above example of the freeze-drying method, the culture supernatant after 48 hours had passed in the second culture step was collected, and the culture supernatant obtained by centrifugation (740 G, 5 minutes) was added without addition. (Production Example 21) and 250 mM trehalose (manufactured by Hayashibara Co., Ltd.) were added (Production Example 22). Then, it was passed through a filtration filter (Kurabo Industries Ltd.) having a membrane pore size of 0.2 μm and dispensed into a syringe (Terumo Corporation). The syringe was sealed in a sterilization bag, frozen in a freezer at −80 ° C., transferred to a freeze-drying apparatus (Yamato Scientific Co., Ltd.), and freeze-dried. After freeze-drying, it was sealed with a syringe lure cap (Terumo Corporation). In a tightly closed state, each was stored for 3 weeks under three temperature conditions of −80 ° C., 4 ° C., and 25 ° C. After 3 weeks, the lyophilized sample was resuspended in water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.), and protein was quantified by QUANTIKINE ELISA Human VEGF (manufactured by R & D SYSTEMS). The quantitative value was calculated based on the quantitative value of VEGF protein contained in the culture supernatant after storage at −80 ° C. for 3 weeks, and the relative quantitative value of VEGF protein at each storage temperature was calculated.

The results are shown in Table 10.

As shown in Table 10, in the freeze-dried culture supernatant without additives, a remarkable decrease in VEGF protein was confirmed by storage in an environment of 25 ° C. This suggests the degradation of proteins associated with moisture absorption and photooxidation. On the other hand, it was found that the addition of trehalose at a final concentration of 250 mM suppressed the decrease in VEGF protein.

<< Manufacturing Examples 23 to 24 >>
Adipose tissue-derived stromal cells extracted by enzymatically treating normal human adipose tissue are cultured in a culture flask (FALCON) using DME medium containing 20% FBS (fetal bovine serum) (Dalbeco modified Eagle's medium manufactured by Gibco, high glucose). It was cultivated in (manufactured by the company) and used as the primary culture. After collecting the primary cultured cells immediately before the confluence by enzyme treatment, the cells were seeded in 5 culture flasks T75 (manufactured by BD FALCON) in the same medium and cultured until they became confluent.

After confirming that the cells had become confluent, the medium was removed, and the cell surface was washed with PBS (Dulbecoline buffer manufactured by DS Pharma Biomedical).

After washing, the cells were replaced with HBSS (Hanks Balanced Salt Solution manufactured by Sigma Aldrich) -HEPES and cultured in an incubator for 48 hours, and then 15 mL of each culture supernatant was placed in a 20 mL syringe (Terumo). Recovered. The collected culture supernatant was lyophilized.

Two culture supernatants lyophilized with a 20 mL syringe were suspended in 15 mL of water for injection, and concentrated to 0.6 mL using VIVASPIN20, VS2041 (manufactured by Sartorius) and a centrifuge KUBOTA2800 (manufactured by Kubota). The operation was performed.

After the concentration operation, Exosome in the culture supernatant was isolated and extracted according to a conventional method using Exosome Isolation Kit (manufactured by Fuji Film Wako). The extracted Exosome extract was 0.1 mL. The remaining one was stored as it was in a 1.5 mL tube (manufactured by Eppendorf) (Production Example 24).

Exosome ELISA Kit (manufactured by Fuji Film Wako Co., Ltd.) was used for sample 1 in which exosomes were isolated and extracted by performing a concentration operation, and sample 2 in which the culture supernatant was only subjected to a concentration operation. Quantified. For the quantitative value, the difference between the absorbance at 450 nm and the absorbance at 650 nm was determined, corrected with a blank value, and expressed as an integrated value of the absorbance contained in 15 mL of the culture supernatant based on the absorbance value.

The results of exosome quantitative values were 116.94 and 150.06, respectively, in Production Examples 23 and 24.

As shown in this result, from the comparison of the absorbance values of both, it was found that the culture supernatant by this production method contains exosomes, and a considerable amount of exosomes can be utilized without intentionally isolating and extracting them. rice field.

<< Manufacturing Examples 25 to 26 >>
<First culture step>
Adipose tissue-derived stromal cells (ASC) extracted by enzymatically treating normal human adipose tissue are cultured in DME medium containing 20% FBS (fetal bovine serum) (Dalveco modified Eagle's medium manufactured by Gibco, high glucose). The cells were cultured in a flask T75 (manufactured by BD FALCON) to prepare a primary culture. After collecting the primary cultured cells immediately before the confluence by enzyme treatment, they were seeded on two 6-well plates for culture (manufactured by Sumitomo Bakelite Co., Ltd.) in the same medium and cultured until they became confluent.

The cells of each confluent well were divided into two groups of 3 wells, the medium of each was removed, and the cell surface of each well was washed with PBS (DS Pharma Biomedical Co., Ltd. d'Albeccholine buffer).

<Second culture step>
After washing, 2 mL of HBSS (Hanks Balanced Salt Solution manufactured by Sigma-Aldrich) -HEPES pH 7.4 was added to the two groups, and the cells were cultured in an incubator for 24 hours or 48 hours. After 24 hours or 48 hours, the culture supernatant of Production Example 25 was collected in a 5 mL volume tube (manufactured by BD FALCON) as usual. Regarding Production Example 26, a cell scraper (manufactured by Asahi Glass Co., Ltd.) was used to collect the adherent cells together with the culture supernatant in a tube having a capacity of 5 mL, and the cells were crushed and measured using Hiscotron (manufactured by Nichion Medical Science Instruments Mfg. Co., Ltd.). It was used as a sample. Each sample was quantified for VEGF protein using QUANTIKINE ELISA Human VEGF (manufactured by R & D SYSTEMS). The relative quantitative value of VEGF protein contained in the cell-containing culture supernatant was calculated based on the quantitative value of VEGF protein obtained from the first group (culture supernatant).

The results are shown in Table 11.

In Table 11, "* (relative quantification value)" is a relative quantification of VEGF protein contained in each sample after culturing for 48 hours, based on the quantification value of VEGF protein obtained from the culture supernatant after culturing for 24 hours. The value is calculated. As shown in Table 11, it was revealed that more growth factors can be utilized by using the cell-containing culture supernatant.

<< Manufacturing Examples 27 to 29 >>
Fibroblasts were selectively cultured from epidermal cells derived from healthy human skin tissue by a conventional method. The obtained primary culture was maintained and cultured, and the 4th passage was seeded on 5 6-well plates (manufactured by Sumitomo Bakelite) and cultured until it became confluent. The cells of each confluent well were divided into two groups of 3 wells, the medium of each was removed, and the cell surface of each well was washed with PBS (DS Pharma Biomedical Co., Ltd. d'Albeccholine buffer).

After washing, in Production Example 27, HBSS (Hanks Balanced Salt Solution manufactured by Sigma-Aldrich) -HEPES pH 7.4 was replaced with 2 mL each, and in Production Examples 28 and 29, the same amount of HBSS-HEPES pH 7.4 was added to Production Example 1 and The adipose tissue stem cell-derived culture supernatant (ASCsup.) Shown in 2 was replaced with a solution added so as to have a final concentration of 10% and 50%, respectively. Then, the cells were cultured in an incubator for 3, 6, 24 or 48 hours.

After each time elapses, total RNA is extracted from each well using ISOGEN (manufactured by Nippon Gene), complementary DNA is synthesized using the Iscript cDNA synthesis kit (manufactured by BIO RAD), and the complementary DNA is synthesized by real-time PCR (rotor). -Used for relative quantification of FGFbasic and VEGF gene expression levels by Gene Q (manufactured by QIAGEN). The relative quantification value was calculated using beta actin as an internal standard, correction between samples, and the value of each gene expression level in cells at the time of confluence as a reference value.

The results are shown in Table 12.

As shown in Table 12, with respect to the FGF-basic gene, ASC-sup. In the additive-free group (Production Example 27), the expression tended to increase with the passage of time up to 24 hours after the medium replacement, but stagnated after 48 hours. On the other hand, in the addition group (Production Examples 28 to 29), the expression increased with the passage of time up to 48 hours after the replacement, and the rate of increase increased depending on the amount of addition.

Regarding the VEGF gene, the expression of the VEGF gene in both the non-addition group and the addition group tended to increase with the passage of time up to 48 hours after the replacement, and ASC-sup. The rate of increase was significantly higher depending on the amount of addition. It was suggested that the addition of adipose tissue-derived stromal cell culture supernatant to fibroblasts promoted the expression of growth factors of the cells themselves and was effective for tissue regeneration.

<< Manufacturing Examples 30 to 31 >>
Epidermal cells derived from healthy human skin tissue were extracted by a conventional method and seeded on a 6-well plate. At that time, the cells were divided into two groups, EpiLife medium (Production Example 30) and EpiLife medium supplemented with the adipose tissue stem cell-derived culture supernatant shown in Production Example 1 (Production Example 31), and cultured until they became confluent. After confirming that the cells became confluent, L-DOPA staining was performed according to a conventional method, and the color tone of the pigment stem cells contained in the epidermal cells was quantified. Quantitative values were obtained from the proportion of the area of the black portion of L-DOPA-stained pigment stem cells using image software ImageJ.

The results were 1884 and 1174 for the original supplement-containing EpiLife medium (Production Example 30) and the ASC culture supernatant and the original supplement-containing EpiLife medium (Production Example 31), respectively.

Since the quantification value of the color tone of the pigment stem cells present in the epidermal cells by L-DOPA staining is lower in the medium to which the culture supernatant derived from adipose tissue stem cells is added, the culture supernatant derived from adipose tissue stem cells is dyed. It was suggested that it has a function of suppressing the proliferation of stem cells and the differentiation into pigment cells.

<< Manufacturing Example 32 >>
A freeze-dried product of the culture supernatant of the example of the above freeze-drying method was produced. The freeze-dried culture supernatant prepared from 1 ml of the culture supernatant was thawed in 1 ml of two types of hyaluronic acid preparations, lestyrene lid (manufactured by GALDETRAMA) and belotello (manufactured by MERZ). It was revealed that the lyophilized culture supernatant was thawed in hyaluronic acid. Therefore, when hyaluronic acid is administered to the periodontal tissue, the freeze-dried culture supernatant can be administered together with hyaluronic acid.

<< Pharmacological effect of culture supernatant on periodontal tissue >>
The lyophilized culture supernatant prepared from 1 ml of the culture supernatant of Production Example 2 was dissolved in 1 ml of physiological saline to prepare a preparation for injection.

<Subject 1>
A 26-year-old woman with gingival recession after orthodontics was injected with hyaluronic acid three times, once every three to four weeks, by the usual method. Although there was severe pain during the procedure, the gingiva was regenerated to some extent and the subject was satisfied.

<Subject 2>
FIG. 1a is a photograph of the periodontal tissue of a 29-year-old woman with gingival recession after orthodontics. Gingival recession is seen in the anterior teeth of the mandible and the anterior teeth of the maxilla. There is an interdental space in the anterior teeth of the maxilla, resulting in a black triangle.

The patient's mandibular anterior teeth and maxillary anterior teeth were injected with the above 1 ml of the above-mentioned preparation three times at a rate of once every 3 to 4 weeks.

FIG. 1b is a photograph of the periodontal tissue after three injections. It can be clearly seen that the gingiva of the maxillary anterior teeth is thicker and thicker than before the treatment, and the black triangle is less noticeable. As for the anterior teeth of the mandible, the gingiva increased and the nipple became thicker. The subject was very satisfied. However, the subject could not feel the result after the first injection and felt that the result was difficult to understand even after the second injection.

<Subject 3>
FIG. 2a is a photograph of the periodontal tissue of a 28-year-old woman with gingival recession after periodontal disease and orthodontics. Gingival recession and reduction of attached gingiva are observed in the anterior teeth of the mandible and the anterior teeth of the maxilla, and the gingiva is thinned. In the anterior teeth of the mandible, a decrease in the interdental papilla due to gingival recession and a black triangle occur.

The patient's mandibular anterior teeth and maxillary anterior teeth were injected with the above 1 ml of the above-mentioned preparation 9 times at a rate of once every 3 to 4 weeks.

FIG. 2b is a photograph of the periodontal tissue after 9 injections. It can be seen that the gingiva of the maxillary anterior teeth and the mandibular anterior teeth is increased and thickened as compared with before the treatment. In particular, in the anterior teeth of the mandible, it can be clearly seen that the interdental space is clearly reduced and the black triangle is inconspicuous due to the increase in the attached gingiva and the thickness of the papilla. As for the anterior teeth of the mandible, the black triangle became smaller due to the increased gingiva and the thicker nipple. The subject was very satisfied. However, the subject could not feel the result after the first injection and felt that the result was difficult to understand even after the second injection.

<Subject 4>
FIG. 3a is a panoramic X-ray image of the periodontal tissue of a 35-year-old man whose alveolar bone is missing due to severe periodontal disease. At the first visit, due to severe periodontal disease, unevenness in the depressed part of the tooth was noticeable, and the bone at the apex of the canine was thin.

The patient's alveolar bone was administered by injecting the above 1 ml preparation four times at a rate of once every 3 to 4 weeks.

FIG. 3b is a panoramic X-ray image of the periodontal tissue after four injections. It can be seen that the alveolar bone has increased in the missing recessed part and the bone has been regenerated as compared with before the treatment. In addition, it can be seen that the bone is also regenerated at the apex of the canine tooth, and the area of the white part is increased. The subject was very satisfied. However, the subject could not feel the result after the first injection and felt that the result was difficult to understand even after the second injection.

<Subject 5>
FIG. 4a is a photograph of the periodontal tissue of a man whose alveolar bone is missing due to severe periodontal disease. At the first visit, due to severe periodontal disease, the gingiva in the depressed part of the tooth and the gingiva behind the canine were reduced. As a result, when I touched the canines, it was in a state of fluttering.

The patient's alveolar bone was administered by injecting the above 1 ml preparation three times at a rate of once every 3 to 4 weeks.

FIG. 4b is a photograph of the periodontal tissue after three injections. Compared to before the procedure, the gingiva at the depressed part was regenerated and the thickness of the gingiva behind the canines was increased. As a result, the glaring of the canines subsided to a considerable extent. The subject was very satisfied. However, the subject could not feel the result after the first injection and felt that the result was difficult to understand even after the second injection.

<Subject 6>
The lyophilized culture supernatant prepared from 1 ml of the culture supernatant of Production Example 2 is dissolved in 1 ml of physiological saline, and 1 ml of hyaluronic acid (Restyrene Vital Skin Boosters (registered trademark)) is further mixed. It was prepared for injection.

FIG. 1a is a photograph of the periodontal tissue of a 30-year-old woman with gingival recession due to periodontal disease. Gingival recession is seen in the anterior teeth of the mandible and the anterior teeth of the maxilla. There is an interdental space in the anterior teeth of the maxilla, resulting in a black triangle.

The patient's mandibular anterior teeth and maxillary anterior teeth were injected with 2 ml of the above-mentioned pharmaceutical product of the present invention six times at a rate of once every 1 to 2 months.

FIG. 1b is a photograph of the periodontal tissue after 6 injections. It can be clearly seen that the gingiva of the maxillary anterior teeth has increased and thickened compared to before the treatment, and the black triangle has disappeared. As for the anterior teeth of the mandible, the gingiva increased and the nipple became thicker. The subject was very satisfied. The six injections gave such very good results, but the subjects were already aware of the results after the first and second injections.

<summary>
Although there are individual differences for each patient, the effect of periodontal tissue regeneration using conventional hyaluronic acid, the effect of periodontal tissue regeneration when using a preparation containing a culture supernatant, and patient satisfaction are summarized. And, it can be summarized as shown in Table 13 below. The effect of regeneration performance was judged by the dentist. Here, "0" means no effect, "1" means effective or slightly satisfied, "2" means highly effective or satisfied, and "3" means very effective or very satisfied.

That is, when the preparation containing the culture supernatant was used, the alveolar bone could be regenerated, which was not possible with the conventional technique, and the gingival regeneration was also more effective than the conventional technique. In addition, the use of the culture supernatant and the preparation containing hyaluronic acid enabled the regeneration of the alveolar bone, which was not possible with the prior art, and a high effect on the regeneration of the gingiva was obtained from the early stage of treatment. .. Therefore, according to the pharmaceutical product of the present invention, in addition to obtaining a high regenerative effect, the patient's satisfaction is very high, and the treatment can be continuously performed without interruption.

Claims (11)

A preparation for periodontal tissue containing a culture supernatant. The culture supernatant is
The first culture step of culturing cells using the first medium and
After the first culturing step, a second culturing step of culturing the cells using a second medium different from the first medium as a medium, and a second culturing step.
The preparation for periodontium according to claim 1, which is obtained by a method including a step of obtaining a culture supernatant containing a second medium after the second culture step. The preparation for periodontal tissue according to claim 2, which is obtained by culturing until it becomes confluent in the first culturing step. The preparation for periodontal tissue according to claim 2 or 3, wherein the second medium contains calcium ions and a buffer. The preparation for periodontal tissue according to claim 4, wherein the buffer is selected from a good buffer. The preparation for periodontal tissue according to claim 5, wherein the good buffer is HEPES. The preparation for periodontium according to any one of claims 2 to 7, wherein the second medium is an infusion solution for injection or an infusion solution for infusion. The tooth according to any one of claims 1 to 7, wherein the culture supernatant is a culture supernatant of adipose tissue-derived mesenchymal cells, epithelial-derived epithelial cells, or dental pulp-derived mesenchymal stem cells. Peri-tissue preparation. The preparation for periodontal tissue according to any one of claims 1 to 8, further comprising hyaluronic acid. The preparation for periodontal tissue according to any one of claims 1 to 9, wherein the hyaluronic acid is a crosslinked hyaluronic acid gel. A method for producing a periodontal tissue preparation containing a culture supernatant.
The first culture step of culturing cells using the first medium and
After the first culturing step, a second culturing step of culturing the cells using a second medium different from the first medium as a medium, and a second culturing step.
A method comprising a second culture step followed by a step of obtaining the culture supernatant containing a second medium.

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