Composition for periosteum and bone regeneration

Abstract

To provide a composition for promoting the regeneration of periosteum and bone.SOLUTION: The composition for regenerating periosteum and bone contains a culture supernatant of mesenchymal cells. Examples of the culture supernatant of mesenchymal cells include a culture supernatant of a mesenchymal cell population containing dental pulp cells, dental pulp stem cells, and the like.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition for promoting periosteal regeneration and further promoting bone regeneration. [Background technology]

[0002] The periosteum is a dense, fibrous membrane-like tissue covering the surface of bones. Its interior is rich in osteoblasts and osteoprogenitor cells, and plays an important role in bone repair. While bone marrow-derived pluripotent cells have been used in bone regenerative medicine, the use of periosteum as a source of osteoblasts and other cells is currently being explored (Non-Patent Document 1). For example, it has been suggested that cultured periosteal sheets can be used as osteoinductive biomaterials for bone regeneration (Non-Patent Document 2), and that hydrogels containing periosteal extracellular matrix components promote bone formation (Non-Patent Document 3). The function of the periosteum in bone regeneration is attracting attention.

[0003] In recent years, the importance of periosteum protection and regeneration has been highlighted in the treatment of periodontal disease. Periodontal disease encompasses various inflammatory conditions that affect the tooth-supporting structures (gums, bone, and periodontal ligament), leading not only to tooth loss but also to the spread of inflammation throughout the body and the potential for other diseases. Treatments for periodontal disease include antimicrobial therapy, host-modulating therapy, laser therapy, and tissue engineering-based regenerative therapy. It has also been reported that the application of autologous periosteum sheets to alveolar bone defects in periodontal disease, together with platelet-rich plasma (PRP) and hydroxyapatite, improved the condition (Non-Patent Document 4), and treatments using autologous periosteum sheets are also being investigated. However, the preparation and application of autologous periosteum sheets to the treatment site requires surgery, and there are risks of immune system reactions and infections after application, making less invasive treatment methods desirable. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Colnot et al., J Orthop Res., 30:1869-1878 2012 [Non-patent document 2] Kawase et al., J Tissue Eng regen Med., 3:218-229 2000 [Non-patent document 3] Qiu et al., Biomaterials 227:119552 2020 [Non-patent document 4] Yamamiya et al., J. Periodontaol., 79:811-818 2008 [Non-Patent Document 5] Yinshi et al., FASEB J. 2015, 29:2702-2711 2015 doi: 10.1096 / fj.14-265496. [Non-patent document 6] Hasegawa et al., Endocrinology. 164:bqad022. 2023 doi: 10.1210 / endocr / bqad022. Summary of the Invention [Problem to be solved by the invention]

[0005] In view of the above circumstances, an objective of the present invention is to provide a composition for promoting the regeneration of the periosteum and the bone covered by the periosteum. [Means for solving the problem]

[0006] The present inventors administered conditioned medium from dental pulp cells to the gums of a mouse model of periodontal disease, excised the maxillae of the mice, and performed contrast analysis of the alveolar bone. The results confirmed that the surface of the alveolar bone was smoother than that of a group that did not receive the conditioned medium. These results suggest that the stability of the periosteum was improved (Non-Patent Documents 5 and 6). These results suggest that the conditioned medium from dental pulp-derived stem cells, particularly the secretome (described below), promotes periosteal regeneration and promotes bone addition to the alveolar bone surface.

[0007] That is, the present invention includes the following (1) to (9). (1) A composition for periosteum and bone regeneration, comprising a culture supernatant of mesenchymal cells. (2) The composition for periosteum and bone regeneration according to (1) above, characterized in that the culture supernatant is a secretome. (3) A composition for periosteum and bone regeneration according to (1) or (2) above, wherein the mesenchymal cell culture supernatant is a culture supernatant obtained by culturing a mesenchymal cell group including mesenchymal stem cells. (4) The composition for periosteum and bone regeneration according to (1) or (2) above, wherein the mesenchymal cells are dental pulp cells and the mesenchymal stem cells are dental pulp stem cells. (5) The periosteum and bone regeneration composition according to (4) above, wherein the periosteum is the periosteum covering the alveolar bone. (6) The composition for periosteum and bone regeneration according to (1) or (2) above, wherein the composition is a pharmaceutical composition. (7) The composition for periosteum and bone regeneration according to (4) above, wherein the composition is a pharmaceutical composition. (8) A composition for periosteum and bone regeneration according to (1) or (2) above, wherein the composition is a hygiene composition. (9) The composition for periosteum and bone regeneration according to (4) above, wherein the composition is a hygiene composition. In this specification, the symbol "to" indicates a numerical range including the values ​​on either side of it. [Effects of the Invention]

[0008] The composition of the present invention has the effect of promoting periosteum and bone regeneration, and is therefore expected to be highly effective in treating fractures and alveolar bone damage caused by periodontal disease. [Brief explanation of the drawings]

[0009] [Figure 1]Figure 1 shows the results of an investigation into the effect of secretome on the periosteum. Secretome (see Examples for details) was administered intragingivally to periodontal disease model mice, and then the maxillae were harvested and subjected to bone morphometric analysis using micro-CT. Untreated, Control, Vehicle, low-concentration, and high-concentration represent, respectively, an untreated group of Slc:ICR mice, an untreated group of periodontal disease model mice, a group of periodontal disease model mice administered 30 μL / body of saline, a group of periodontal disease model mice administered 30 μL / body of secretome, and a group of periodontal disease model mice administered 60 μL / body of secretome. [Figure 2] Figure 2 shows the results of contrast analysis of projection images from the same direction using CT3D reconstruction images. Vehicle, Low, and High represent the group of periodontal disease model mice administered 30 μL / body of saline, the group of periodontal disease model mice administered 30 μL / body of secretome, and the group of periodontal disease model mice administered 60 μL / body of secretome, respectively.

[0010] Hereinafter, embodiments of the present invention will be described. Note that, unless otherwise specified, the term "the present embodiment" refers to all embodiments described in this specification. The first embodiment is a composition for periosteum regeneration (hereinafter also referred to as "the composition according to this embodiment") containing a culture supernatant of mesenchymal cells. As described above, "periosteum" is a fibrous membrane-like tissue covering the surface of bone. It is rich in osteoblasts and osteoprogenitor cells and plays an important role in bone repair or regeneration. The composition according to this embodiment has the effect of promoting the regeneration of periosteum on the bone surface and further promoting bone regeneration. "Periosteum" and "bone" in this embodiment include "periosteum" and "bone" of any site present in a living body, and examples thereof include "periosteum covering the alveolar bone" and "alveolar bone," respectively.

[0011] In this embodiment, "mesenchymal cells" refer to cells that can differentiate into mesenchymal cells such as adipocytes and osteoblasts, and include, but are not limited to, dental pulp cells, bone marrow cells, and adipocytes. In this embodiment, "mesenchymal cells" includes "mesenchymal stem cells." "Mesenchymal stem cells" refer to stem cells or precursor cells that can differentiate into all or some mesenchymal cells, and include, for example, dental pulp stem cells, bone marrow-derived stem cells, and adipose tissue-derived stem cells. Therefore, in this embodiment, the "mesenchymal stem cell culture supernatant" (hereinafter also referred to as "culture supernatant in this embodiment") may be a culture supernatant obtained by culturing a mesenchymal stem cell population containing mesenchymal stem cells, or may be a culture supernatant obtained by culturing a cell population consisting only of mesenchymal stem cells. The culture supernatant in this embodiment may be each fraction obtained by fractionating the collected culture supernatant by filter filtration, ultrafiltration, etc. In this specification, the filtrate obtained by filtration through a 0.22 μm filter is particularly referred to as the "secretome."

[0012] In this embodiment, particularly preferred mesenchymal cells and mesenchymal stem cells are dental pulp cells or dental pulp stem cells. Here, "dental pulp cells" refer to cells isolated from dental pulp tissue. Dental pulp cells may be cells recovered by treating dental pulp tissue collected from a tooth with an enzyme such as collagenase. Furthermore, "dental pulp stem cells" are stem cells present in dental pulp tissue (Gronthos et al., Proc Natl Acad Sci USA, 97:13625-13630, 2000) and can be obtained by known methods (see, for example, Yamaza et al., Stem Cell Res Ther. 1:5, 2010). Dental pulp stem cells can be cultured, selected using surface antigen markers of dental pulp stem cells, and finally isolated. Although the cell population collected from dental pulp contains a mixture of dental pulp stem cells and other cells, dental pulp stem cells have a higher proliferation rate than other cells, resulting in a higher proportion of dental pulp stem cells at the end of the culture. Therefore, by repeatedly culturing a cell population collected from dental pulp, the proportion of dental pulp stem cells present increases, and cells containing almost only dental pulp stem cells can be obtained. Furthermore, the mesenchymal stem cells used in this embodiment may be either primary cultured cells or immortalized cells.

[0013] The animal species from which the mesenchymal cells in this embodiment are derived is not particularly limited, and may include humans, pet animals such as dogs, cats, and rabbits, and livestock animals such as cows, pigs, sheep, and horses, among others, with humans being a particularly preferred animal.

[0014] In this embodiment, the culture supernatant preferably does not contain animal serum to enhance safety. Serum and other substances can be easily removed from the culture supernatant by dialysis, solvent substitution, or the like. The culture supernatant in this embodiment may be in the form of a frozen product or a lyophilized product, or may be a solution obtained by dissolving the lyophilized product in an appropriate solvent. As described above, the "mesenchymal cell culture supernatant" in this embodiment is preferably a culture supernatant obtained by culturing dental pulp cells (population) or dental pulp stem cells (population).

[0015] The culture medium used to produce the culture supernatant in this embodiment may be any culture medium that can be used to culture mesenchymal cells, and is not particularly limited. Examples of such culture medium include a basal culture medium such as DMEM (Dulbecco's Modified Eagle Medium), αMEM (alpha Modified Eagle Minimum Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), Ham's F-12, RPMI (Roswell Park Memorial Institute)-1640, or a mixture thereof, and a serum such as fetal bovine serum (FBS) or Knockout Reagents (KSR). TM The culture medium may be a culture medium to which serum substitutes such as erythrocyte serum replacement (ESR), glucose, amino acids, vitamins, antibiotics, etc. have been appropriately added. However, it is preferable that the culture supernatant finally obtained does not contain animal-derived serum, and a culture supernatant from which serum has been removed by the above-mentioned method is particularly preferable.

[0016] The composition according to this embodiment has the effect of promoting the regeneration of periosteum and bone. Therefore, it can be used as a pharmaceutical composition for promoting the regeneration of periosteum and / or bone, as well as for treating fractures, and for preventing or treating bone resorption due to autoimmune diseases such as osteoarthritis and rheumatism, and alveolar bone damage due to periodontal disease. Furthermore, the composition according to this embodiment can be provided as, for example, a food or drink composition, a feed composition, and a hygiene composition in addition to pharmaceutical compositions, but is not limited to these compositions.

[0017] When the composition according to this embodiment is a pharmaceutical composition, its dosage form is not particularly limited and may be an oral or parenteral dosage form, such as tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, or injections. These preparations are prepared according to conventional methods. Liquid preparations may be dissolved or suspended in water or other suitable solvents before use. Tablets and granules may also be coated by known methods. In the case of injections, for example, a lyophilized product of a mesenchymal cell culture supernatant may be dissolved in physiological saline or a glucose solution as needed to prepare the preparation.

[0018] Those skilled in the art can appropriately select the type of formulation additive used in the production of the pharmaceutical composition of this embodiment, the ratio of the formulation additive to the culture supernatant or its lyophilized product as the active ingredient, the production method, etc. The formulation additive can be an inorganic or organic substance, or a solid or liquid substance, and can generally be blended in an amount of 1 to 90% by weight based on the weight of the active ingredient. Specific examples of pharmaceutical additives include lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminometasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, calcium carboxymethylcellulose, ion exchange resins, methylcellulose, gelatin, gum arabic, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, Veegum, titanium oxide, sorbitan fatty acid esters, sodium lauryl sulfate, glycerin, fatty acid glycerin esters, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oils, wax, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants, propylene glycol, and water.

[0019] When the pharmaceutical composition of this embodiment is produced as an injection, the culture supernatant or a freeze-dried product thereof, which is the active ingredient, is mixed with distilled water for injection, optionally together with a pH adjuster such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate, sodium dihydrogen phosphate, etc., and an isotonicity adjuster such as sodium chloride or glucose, and the mixture is sterile filtered and filled into ampoules; alternatively, mannitol, dextrin, cyclodextrin, gelatin, etc. may be added, followed by vacuum freeze-drying to produce an injection that is dissolved just before use.

[0020] When the pharmaceutical composition according to this embodiment is prepared as an ointment or cream, it can be produced by kneading and mixing the culture supernatant or its lyophilized product, which is the active ingredient, with a base and additives. An oleaginous ointment can be produced, for example, by warming and melting an oleaginous base such as oils, waxes, or hydrocarbons such as paraffin, adding the active ingredient, mixing to dissolve or disperse the active ingredient, and kneading the mixture until homogeneous. A water-soluble ointment can be produced, for example, by warming and melting a water-soluble base such as macrogol, adding the active ingredient, and kneading the mixture until homogeneous.

[0021] The pharmaceutical composition according to this embodiment can be administered by known methods such as injection (infusion) or application to the tissues surrounding the bone to be treated. The dosage of the pharmaceutical composition can be easily determined by a specialist such as a physician, dentist, or veterinarian, but preferably, for example, an amount in the range of approximately 0.1 mL to 5.0 mL of culture supernatant per administration.

[0022] The pharmaceutical compositions of this embodiment can be prepared as sustained-release formulations, such as implants or microencapsulated delivery systems, using carriers that can prevent immediate elimination from the body. Such carriers can include biodegradable and biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such materials can be readily prepared by those skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. Liposomes can be prepared using a lipid composition containing, but not limited to, phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanol (PEG-PE), filtered through an appropriate pore size filter to obtain a suitable size, and purified by reverse-phase evaporation.

[0023] When the composition according to this embodiment is provided as a hygiene composition, examples of the hygiene composition include, but are not limited to, compositions for preventing alveolar bone damage associated with periodontal disease. The form of the composition according to this embodiment when provided as a hygiene composition is also not particularly limited, and may be, for example, a liquid (including foam and mist), solid, gel, or paste. More specific examples include toothpaste, mouthwash, denture cleaner, and spray. The hygiene composition may be for human or non-human use.

[0024] A second embodiment is a method for regenerating periosteum and / or bone, comprising administering to a subject a composition containing a mesenchymal cell culture supernatant. The second embodiment also includes a method for treating or preventing injuries or diseases for which therapeutic or preventive effects are expected through periosteum regeneration or bone regeneration, such as fractures and periodontal disease, comprising administering to a subject a composition containing a mesenchymal cell culture supernatant. Here, "treatment" means preventing or alleviating the progression or worsening of the pathological condition of the target disease, etc., and "prevention" means treatment aimed at preventing the onset of the target disease, etc. The subjects of the treatment and prevention methods according to the present embodiment are not particularly limited, and may be any animal classified as a mammal, including, in addition to humans, pet animals such as dogs, cats, and rabbits, and livestock animals such as cows, pigs, sheep, and horses. A particularly preferred "mammal" is a human.

[0025] When this specification is translated into English and includes the singular words "a," "an," and "the," it is intended to include the plural as well as the singular, unless the context clearly indicates otherwise. Also, in this specification, "about" or "to the extent" means a numerical range of ±10%. The present invention will be further explained below by showing examples, but these examples are merely illustrative of embodiments of the present invention and do not limit the scope of the present invention. [Example]

[0026] 1. Materials and Experimental Methods 1-1.Culture supernatant During the culture process, the liquid containing secretions from the mesenchymal stem cells was used as the culture supernatant. The culture supernatant was then filtered and purified using a filter with a pore size of 0.22 μM (220 nM). This purified product, containing substances with a particle size of 220 nM or less, was used as the secretome. Furthermore, the secretome was ultrafiltered, and only the fraction containing substances with a particle size of approximately 100 nM was collected and used as the exosome. In this example, the "secretome (including exosomes)" that had been filtered and purified using a filter with a pore size of 220 nM was used.

[0027] 1-2. Experimental method 1-2-1. Preparation of human dental pulp stem cells Human dental pulp stem cells (hDPSCs) were prepared using extracted teeth provided by donors at the University of Tokyo Hospital or affiliated hospitals, with approval from the University of Tokyo Hospital Ethics Committee.

[0028] 1-2-2. Isolation of hDPSCs All extracted teeth used in this example were placed in a 50 mL conical tube (IWAKI) containing DMEM high glucose (GlutaMax) containing antibiotics (1% penicillin, streptomycin, amphotericin, 200 μg / mL meropene, 20 μg / mL minocycline). TM The cells were quickly stored at 4°C in a refrigerator (Thermo Fisher Scientific, MA, US) and transported from the donor (a collaborating hospital) to the laboratory at 4°C within 24 hours of starting hDPSC isolation. The isolation of hDPSCs was carried out under xeno-free conditions. The extracted teeth were excised to remove the dental pulp tissue, which was then minced with a scalpel. The tissue was then placed in a sterile Ca 2+ and Mg 2+ Tissue digestion reagent diluted in the above medium without 0.3% Collagenase TM The cells were digested in 20 mL of PBS (Thermo Fisher Scientific, MA, US) at 130 rpm at 37°C for 45 minutes. After centrifugation at 1500 rpm for 5 minutes, the supernatant was removed. The number of cells recovered in the pellet was counted, and the cell density was determined to be 0.5-1.0 x 10 6 The cells were seeded onto a 100 mm polystyrene dish at 100 cells / dish. The medium was changed twice a week, and the cells were passaged when they reached confluence (P0: Passage 0 (primary cells), the same applies below).

[0029] 1-2-3. hDPSC cell passage and proliferation The culture medium was removed, and the cells on the culture dish were washed twice with 1 mL of PBS (Nacalai Tesque). Then, hDPSCs were treated with the phenol red-free cell removal reagent TrypLE. TM 1 mL of Thermo Fisher Scientific select (Thermo Fisher Scientific) was added and the mixture was left to stand at 37°C for 5 minutes to allow the reagent to penetrate the cells and remove cell adhesion. The detached hDPSCs were then centrifuged at 1,500 rpm for 5 minutes to recover the pellet, and the supernatant was removed. From passages P1 to P6, the cells were collected at a cell density of 0.5 × 106 The cells were seeded at 10 cm per dish and cultured in DMEM containing a non-animal serum substitute. The medium was changed twice a week, and the cells were subcultured after 10 days. The same procedure was repeated up to the P6 subculture.

[0030] 1-2-4. Quality control of hDPSC culture supernatant Five days after the P7 passage, the entire culture supernatant was collected without changing the medium and passed through a 0.22 μm filter. At each passage, the culture supernatant was tested for viruses (HIV1, HIV2, HTLV1, HTLV2, HCV, HBV, CMV, ParvoB19, and WNV). Endotoxin, mycoplasma, and sterility tests were also performed, confirming negative results.

[0031] 1-2-5. Lyophilization of hDPSC culture supernatant The culture supernatant was collected from each dish and placed in a 5 mL vial. The collected culture supernatant was placed in a freeze-dryer for 24 hours to remove excess water and produce a dried product. The freeze-dried product weighed 0.05 g per vial.

[0032] 1-2-6. Test substance and solvent The test substance was prepared by dissolving the lyophilized product prepared in 1-2-5 above in Otsuka distilled water (Japanese Pharmacopoeia, Water for Injection; Otsuka Pharmaceutical Factory, Inc.) (hereinafter referred to as "water for injection"). For the high-dose secretome, 0.1 mL of water for injection was added to one vial of dried secretome to dissolve it. For the low-dose secretome, 0.5 mL of water for injection was added to one vial of dried secretome to dissolve it. The test substance was stored frozen at -20°C until use. Furthermore, Otsuka saline injection (Japanese Pharmacopoeia, Physiological Saline; Otsuka Pharmaceutical Factory, Inc.) (hereinafter referred to as "physiological saline") was used as a control substance. The volume of the administered solution was 30 μL / body for the vehicle group, 30 μL / body for the low dose group, and 60 μL / body for the high dose group.

[0033] 1-2-7.Statistical analysis All data are presented as mean ± SE (standard error). Data were plotted and statistically analyzed using MATLAB® R2023a (MathWorks). The Lilliefors test was used to confirm normal distribution of the data. Significant differences between data groups were also examined using the Kruskal-Wallis test and the Steel-Dwass test.

[0034] 2.Results 2-1. Safety evaluation of secretome The safety of secretome was evaluated by administering a single dose to Slc:ICR mice. Slc:ICR mice (Japan SLC, Inc., Shizuoka, Japan) of both sexes were delivered at 5 weeks of age and, after acclimation, were used for the study at 6 weeks of age. Male and female mice were divided into seven groups of three so that their weights were approximately equal. Four groups of each sex were used for intravenous administration, and three groups of each sex were used for gingival administration. The intravenous administration groups received saline, a low-dose secretome, or a high-dose secretome, while the gingival administration groups received saline, a low-dose secretome, or a high-dose secretome administered gingivally. After two weeks of administration, the mice were observed for general condition, followed by blood sampling and necropsy. No notable changes were observed in general condition, body weight, or necropsy results. Although statistically significant differences were observed in blood tests for MCV, reticulocytes, and eosinophils, and in blood biochemistry tests for AST, albumin, BUN, creatinine, and Cl, these were not toxicologically significant changes. Based on the above, it was considered that the culture supernatant, the test substance, had no obvious toxicity when administered intravenously or gingivally to mice.

[0035] 2-2. Examination of the effect of secretome on the periosteum Male Slc:ICR mice were delivered at 4 weeks of age and, after acclimation, were used to create a periodontal disease model at 5 weeks of age. The periodontal disease model was created according to a previous study (Abe et al., J Immunol Methods. 394:49-54 2013. doi: 10.1016 / j.jim.2013.05.002). One week after model creation, the mice were divided into eight groups of three, with six groups used for gingival administration. Treatment consisted of saline, low-dose Secretome, or high-dose Secretome administered gingivally. After seven days of observation, the mice were then observed for general condition. The maxillae were then excised for bone morphometric analysis using micro-CT (Figure 1). No significant differences were observed in the maximum distance from the cementoenamel junction (CEJ) to the alveolar crest in the periodontal disease model (mandibular second molar) (Figure 1).

[0036] Next, the gingival region, mesial, and distal alveolar bone of the mandibular second molar were excised, and the contrast of projection images from the same direction as the 3D reconstructed CT images was analyzed using MATLAB® R2023a (MathWorks). On day 7 of administration, the low-concentration group (low-dose group) and the high-concentration group (high-dose group) showed significantly lower contrast than the control group, indicating a smoother alveolar bone surface. This result suggests that bone addition occurred on the alveolar bone surface due to periosteal regeneration caused by Secretome administration (Figure 2). [Industrial Applicability]

[0037] The present invention is expected to be used in the fields of medicine, dentistry, veterinary medicine, and the like.

Claims

1. A composition for periosteum and bone regeneration, comprising a culture supernatant of mesenchymal cells.

2. The composition for periosteum and bone regeneration according to claim 1, wherein the culture supernatant is a secretome.

3. 3. The composition for regenerating periosteum and bone according to claim 1, wherein the mesenchymal cell culture supernatant is a culture supernatant obtained by culturing a mesenchymal cell population containing mesenchymal stem cells.

4. The composition for periosteum and bone regeneration according to claim 1 or 2, wherein the mesenchymal cells are dental pulp cells and the mesenchymal stem cells are dental pulp stem cells.

5. The periosteum and bone regeneration composition according to claim 4, wherein the periosteum is the periosteum covering the alveolar bone.

6. The composition for periosteum and bone regeneration according to claim 1 or 2, wherein the composition is a pharmaceutical composition.

7. The composition for periosteum and bone regeneration according to claim 4, wherein the composition is a pharmaceutical composition.

8. The composition for periosteum and bone regeneration according to claim 1 or 2, wherein the composition is a hygiene composition.

9. The periosteum and bone regeneration composition according to claim 4, wherein the composition is a hygiene composition.

Images