Bioabsorbable photocurable composition, bioabsorbable tissue regeneration inducer containing same, and transplantation method using same

Abstract

The present invention provides a bioabsorbable photocurable composition, a bioabsorbable tissue regeneration inducing material including the same, and a transplantation method using the same. The bioabsorbable photocurable composition includes a photoreduction agent and collagen, and is applied to the top of a transplantation site filled with a transplantation material, filling the empty space at the transplantation site and covering the transplantation material. The photoreduction agent is activated by irradiation with visible light to crosslink the collagen, and the collagen crosslinked by the visible light fills the minute empty space at the transplantation site and tightly bonds with the transplantation material to fix the transplantation material.

Description

[Technical field]

[0001] The present disclosure relates to a bioresorbable photocurable composition, a bioresorbable tissue regeneration-inducing material containing the same, and a transplantation method using the same. [Background technology]

[0002] In order to treat various bone losses or defects (hereinafter referred to as injuries) caused by inflammation, tumors, trauma, skeletal diseases, etc., it is necessary to fill the lost or defective area with a graft material. However, if only a graft material is filled into the damaged area, there is a disadvantage that the graft material is difficult to maintain at the graft site.

[0003] Conventionally, tissue regeneration is induced by filling the damaged area with a graft material, covering it with a masking membrane, suturing the masking membrane first, and then covering the skin again and suturing it second, as shown in Figure 1. However, when using a graft material and a masking membrane at the same time as shown in Figure 1, smooth regeneration can only occur if the graft material grafted at the bottom and the masking membrane at the top are in close contact with each other, but it is difficult to achieve perfect contact between the graft material and the masking membrane, resulting in gaps between the graft material and the masking membrane, which reduces the success rate of grafting.

[0004] Therefore, a new type of material and transplantation method that can maintain adhesion while preventing loss of the transplant material is required. Summary of the Invention [Problem to be solved by the invention]

[0005] The present disclosure provides a bioabsorbable photocurable composition that can maintain adhesion while preventing loss of the implant material.

[0006] The present disclosure provides a bioresorbable tissue regeneration-inducing material comprising a bioresorbable photocurable composition.

[0007] The present disclosure provides methods of transplantation. [Means for solving the problem]

[0008] The bioabsorbable photocurable composition according to an embodiment includes a photosensitizer and collagen, and is applied to the top of an internal transplantation site filled with a graft material to fill the empty space in the transplantation site and cover the graft material. When exposed to visible light, the photosensitizer is activated and the collagen is crosslinked, so that the collagen fills the minute empty spaces in the transplantation site and tightly bonds with the graft material to fix the graft material.

[0009] The bioabsorbable tissue regeneration-inducing material according to an embodiment includes a transplant material comprising a first agent mainly composed of collagen and a second agent containing a peptide formulated so that the first agent can be soaked in the second agent, and a bioabsorbable photocurable composition comprising a photoreduction agent and collagen, which is applied to a transplant site filled with the transplant material to cover the transplant material while filling the empty space in the transplant site, and which is activated by irradiation with visible light to crosslink the collagen, causing the collagen to fill the minute empty spaces in the transplant site and to tightly bond with the transplant material to fix the transplant material.

[0010] A transplantation method according to an embodiment includes the steps of filling an internal transplantation site with transplant material, applying a bioabsorbable photocurable composition containing a photoreduction agent and collagen to the top of the transplantation site filled with the transplant material to cover the transplant material while filling the empty space of the transplantation site, and irradiating the bioabsorbable photocurable composition with visible light to activate the photoreduction agent and crosslink the collagen, so that the collagen crosslinked by the visible light fills the tiny empty spaces of the transplantation site and tightly bonds with the transplant material to fix the transplant material.

[0011] Other specific aspects of the invention are included in the following detailed description. Effect of the Invention

[0012] According to the embodiment of the present invention, the bioabsorbable photocurable composition and the transplantation method using the same can cover the upper surface of the transplant material, fill the remaining empty space at the transplant site filled with the transplant material, and then fill the minute empty spaces at the transplant site to closely bond with the transplant material, thereby preventing the loss of the transplant material and maintaining the adhesive bond with the transplant material, thereby enabling effective tissue regeneration.

[0013] According to the embodiment of the tissue regeneration-inducing material and the method of inducing tissue regeneration using the same, a graft material having anti-inflammatory or antibacterial properties is used together with a body-absorbable photocurable composition to exert anti-inflammatory or antibacterial functions, effectively forming the microenvironment factors necessary for tissue regeneration, while preventing the loss of the graft material and maintaining the adhesive bond with the graft material, allowing tissue regeneration to occur effectively. [Brief description of the drawings]

[0014] [Figure 1] 1 is a schematic diagram showing a conventional bone graft procedure. FIG. [Diagram 2] 1 is a flow chart illustrating an implantation method according to one embodiment. [Diagram 3] This is a schematic diagram showing the process in which photocuring occurs in a photocurable composition. Collagen is collagen, riboflavin is riboflavin, oxygen radical is oxygen radical, and chemical link is a chemical bond. [Figure 4] 1 shows the results of analyzing the purity of a peptide having the amino acid sequence of SEQ ID NO:1 by liquid chromatography after synthesis. [Diagram 5] This shows the results of analyzing the molecular weight of a peptide having the amino acid sequence of SEQ ID NO:1 by mass spectrometry after synthesizing the peptide. [Figure 6] The results of enzyme-linked immunosorbent assay (ELISA) for IL-6 and TNF-α are shown. [Figure 7]Western blot analysis of pNFkB and plkBa is shown. [Figure 8] This shows the results of measuring the antibacterial activity of a peptide having the amino acid sequence of SEQ ID NO: 1. Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis indicate Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, respectively. The vertical axis indicates % Survial, the horizontal axis indicates Peptide, and the horizontal axis indicates minocycline. [Figure 9] This shows the results of treating a biofilm with a peptide having the amino acid sequence of SEQ ID NO: 1. On the horizontal axis, Live, Dead, and Merge indicate live, dead, and merge, respectively. [Figure 10] 1 shows the results of a confocal microscope analysis of the intracellular penetration of a peptide having the amino acid sequence of SEQ ID NO: 1. DIC stands for Dick, Nuclei stands for Nuclei, Peptide stands for Peptide, and Merge stands for Merge. [Figure 11] 1 shows the degree of intracellular penetration of a peptide having the amino acid sequence of SEQ ID NO: 1 into rabbit gingival tissue. Nuclei means nuclei, Peptide means peptide, and Merge means merge. [Figure 12] 1 shows the degree of intracellular penetration of a peptide having the amino acid sequence of SEQ ID NO: 1 into the gingival tissue of an adult dog. DIC stands for Dick, Peptide, and Merge. [Figure 13]This shows the results of measuring the antibacterial activity of a collagen sponge block containing a peptide having the amino acid sequence of SEQ ID NO: 1. Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis indicate Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, respectively. The vertical axis indicates "% Survial," the horizontal axis indicates Peptide-collage, and the horizontal axis indicates Minocycline. [Figure 14] Collagen gel containing a peptide having the amino acid sequence of SEQ ID NO: 1 was applied to an adult dog periodontitis model, and periodontal tissue regeneration was confirmed by radiography. The squares indicate the alveolar bone regenerated by the graft material. [Figure 15] The figure shows the degree of alveolar bone regeneration in a model of adult dog periodontitis when collagen gel containing a peptide having the amino acid sequence of SEQ ID NO: 1 was applied. In the figure above, the vertical axis "Regeneration / defect area (%)" indicates "regeneration / defect area (%)", the horizontal axis "Minocycline" indicates "minocycline", and the horizontal axis "Peptide" indicates "peptide". In the table below, the left column "Interdental number" indicates "interdental number", "Regeneration / defect area (%)" indicates "regeneration / defect area (%)", "Average of Regeneration / defect area (%)" indicates "average of regeneration / defect area (%)", and in the upper column "NT" indicates "group not applied", "Minocycline" indicates "minocycline", and "Peptide" indicates "peptide". [Figure 16] 1 is a photograph showing the state of gel formation in a photocurable composition according to changes in collagen content. [Figure 17] 1 is a photograph showing the state of gel formation according to changes in the riboflavin content in a photocurable composition. [Figure 18] 1 is a photograph showing the state of gel formation in a photocurable composition according to changes in the NaOH content. [Figure 19]1 is a photograph showing the change in properties of a collagen gel containing riboflavin due to light irradiation. [Figure 20] 1 is a graph showing the results of measuring the viscosity of a collagen gel containing riboflavin before and after hardening. [Figure 21] 1 is a photograph showing the measurement of stability of collagen gel containing riboflavin. [Figure 22] 1 is a photograph showing the stage of implantation using a photocurable composition (collagen gel containing riboflavin) in an adult dog with peri-implantitis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention is not limited to the specific embodiments described herein, but may be embodied in various different forms, and may be practiced by those skilled in the art.

[0016] Unless otherwise defined below, certain terms used in this disclosure, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0017] The techniques and processes described in this disclosure are generally performed according to conventional methods, which are presented throughout this application. Generally, the nomenclature and laboratory procedures in molecular biology, biochemistry, analytical chemistry and cell culture used in this disclosure are consistent with those well known and commonly used in the art.

[0018] The present disclosure relates to a bioabsorbable photocurable composition and a transplantation method using the same. The transplantation site in the body refers to a site including various bone losses or defects (hereinafter, bone damage) caused by inflammation, tumors, trauma, skeletal diseases, and the like.

[0019] The bone damage site may include both dental and non-dental damage sites. The most typical dental damage site is a damage site caused by periodontal disease. Periodontal disease includes gingivitis, which is limited to the gums, periodontitis, in which inflammation spreads to the alveolar bone surrounding the tooth root, and peri-implantitis, which is a complication after surgically embedding an artificial material in the jawbone, affecting the tissues around the implant and causing loss of supporting bone. In addition, dental damage sites may include dislocated traumatic tooth sites or tooth extraction sites.

[0020] Non-dental injury sites can include a variety of injury sites, such as congenital injury, injury due to cancer resection, acquired injury due to aging, bone degeneration and osteoporosis, and trauma-related injury such as bone fracture.

[0021] As shown in FIG. 2, a pretreatment process can be performed before filling the implant site with the implant (S1).

[0022] For example, when treating periodontal disease, which is a typical example of dental damage, a process of regenerating periodontal tissue may be performed after scaling and / or root planning, which removes the plaque and tartar that cause periodontal disease.

[0023] The implantation site is then filled with implant material (S2).

[0024] The graft material may include various types of graft materials such as skin graft materials, bone graft materials, musculoskeletal regeneration materials, nerve regeneration materials, blood vessel regeneration materials, etc. The graft material may basically include various graft material matrices such as extracellular matrix proteins, bone minerals, chitosan, biodegradable synthetic polymers, etc.

[0025] The extracellular matrix protein may be any one or more selected from the group consisting of collagen, hyaluronic acid, elastin, chondroitin sulfate, and fibroin. These extracellular matrices may be derived from humans or animals, or may be recombinant proteins produced from microorganisms.

[0026] Among extracellular matrix proteins, collagen is the most suitable. Collagen is produced by fibroblasts in the human body and is the main component of connective tissue, which is the most commonly found in the human body. It is a fibrous protein distributed throughout the human body, including bones, skin, joints, membranes of various organs, and hair. A large amount of collagen is also present around the axons of central and peripheral nerves. Collagen hydrogels present in the body have slightly different functions and roles depending on the tissue site, so there are differences in collagen concentration and degree of orientation in collagen hydrogels at different sites. For example, bone is characterized by a structure in which micro / nanocrystalline minerals are located between oriented collagen fibers. Collagen is a material that is absorbed in the body and is easily degraded, so when it is transplanted into the body, it can be gradually degraded and replaced by regenerated bone, making it suitable as a transplant matrix. In the case of collagen, it is preferable to use type 1 or type 3 isolated from pig skin.

[0027] The bone mineral component may be one or more selected from the group consisting of biologically derived bone mineral powders derived from allogeneic or xenogeneic bone, synthetic hydroxyapatite and calcium phosphate cement (CPC) powders including tetracalcium phosphate (TTCP), dicalcium phosphate anhydrous (DCPA), and monocalcium phosphate monohydrate (MCPM).

[0028] The biodegradable synthetic polymer may be at least one selected from the group consisting of polyglycolide, polylactide, polyglycolide lactide, and polycaprolactone.

[0029] The implant matrix may be in the form of a paste, powder, putty, gel, hydrogel, matrix, granules, particles, lyophilized powder, lyophilized bone, demineralized lyophilized bone, fresh frozen bone, corticocancellous bone mix, pellets, strips, plugs, membranes, lyophilized powder reconstituted with water to form a wet cake, spheres, sponges, blocks, mosels, sticks, wedges, cement, or a formulation consisting of amorphous particles.

[0030] If necessary, the implant matrix may further contain a therapeutic agent having anti-inflammatory, antibacterial or cell-penetrating properties, etc. The therapeutic agent may be a peptide having anti-inflammatory, antibacterial or cell-penetrating properties.

[0031] In one embodiment for treating dental damage, the implant can be present as a combination of a first agent based on collagen and a second agent containing a peptide formulated so that the first agent can be soaked in the second agent, which can be mixed and used prior to implantation.

[0032] The first agent, which is mainly composed of collagen, may contain collagen sponge blocks of various sizes or a complex of collagen sponge blocks and bone minerals. It is preferable to use collagen sponge blocks because collagen powder cannot form a three-dimensional structure and cannot provide an environment for easy regeneration of new tissue. When treating dental damage, it may contain collagen sponge blocks of 6X5X7mm or 8X7X9mm size, or a complex of collagen sponge blocks and bone minerals of 6X6X3mm, 6X6X6mm, 7X8X9mm, or 9X10X11mm size, depending on the size of the teeth such as premolars and molars.

[0033] The first and second agents can be mixed and used immediately before transplantation. When mixed and used immediately before transplantation, it has the advantage that collagen and peptides can be kept in a sterile state and the activity of the peptide can be maintained at a high level. Generally, proteins or peptides can be stored for a long period of time without decreasing in activity when stored in a freeze-dried state, compared to when stored in a solution state. Therefore, if the peptide is stored in a freeze-dried state and mixed with the collagen sponge block immediately before transplantation, the activity of the peptide (anti-inflammatory activity, antibacterial activity, etc.) can be maintained at a high level after transplantation.

[0034] In one aspect, the peptide formulated so that it can be soaked into the first agent constituting the implant material is a peptide having the amino acid sequence of SEQ ID NO:1 below.

[0035] SEQ ID NO:1: GKCSTRGRKSSRRKK

[0036] In one embodiment, after treatment with a peptide having the amino acid sequence of SEQ ID NO: 1, the intracellular release of inflammatory markers IL-6 and TNF-α was measured using enzyme-linked immunosorbent assay (ELISA), and it was confirmed that the peptide having the amino acid sequence of SEQ ID NO: 1 exhibits anti-inflammatory activity similar to that of IL-4, a cytokine with anti-inflammatory effects. In addition, the inhibitory effect of the peptide having the amino acid sequence of SEQ ID NO: 1 on the expression of pNF-κB, an inflammation-related enzyme, was confirmed by Western blot. As a result, it was confirmed that the peptide having the amino acid sequence of SEQ ID NO: 1 inhibits the expression of pNF-κB induced by LPS.

[0037] In another aspect, it was confirmed that the peptide having the amino acid sequence of SEQ ID NO: 1 has excellent antibacterial activity against Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, which are periodontitis-inducing bacteria, and that it exhibits the same or similar antibacterial activity as minocycline, which is often used in the treatment of periodontitis.In addition, it was confirmed that the peptide having the amino acid sequence of SEQ ID NO: 1 has antibacterial activity and also has cell-penetrating ability when it is treated with an artificial biofilm made using periodontitis-inducing bacteria.

[0038] In another aspect, the present disclosure relates to a peptide having cell penetrating ability.

[0039] In one aspect, it was confirmed through in vitro and in vivo experiments that the peptide having the amino acid sequence of SEQ ID NO: 1 penetrates into cells. Specifically, the peptide having the amino acid sequence of SEQ ID NO: 1 was treated with mouse macrophages (RAW264.7) to confirm whether it penetrates into the cells, and it was confirmed that the peptide having the amino acid sequence of SEQ ID NO: 1 penetrates into the cells. In addition, it was confirmed that the peptide having the amino acid sequence of SEQ ID NO: 1 penetrates not only at the cellular level but also at the tissue level. When the peptide having the amino acid sequence of SEQ ID NO: 1 was treated with the gingival tissue of a living rabbit and the gingival tissue of an adult dog, it was confirmed that it penetrated into the gingival tissue of the rabbit and the gingival tissue of the adult dog.

[0040] In the case of periodontitis and peri-implantitis, various types of periodontitis-inducing bacteria gather together to form a thick biofilm. Such biofilms are difficult to remove by administering antibiotics alone because they increase their resistance to antibiotics. Therefore, it is necessary to have antibacterial properties against the periodontitis-inducing bacteria that compose the biofilm while passing through such biofilms. The peptide having the amino acid sequence of SEQ ID NO: 1 is uniquely capable of penetrating cells, so there is no need to use an additional carrier. Therefore, it can penetrate the biofilm formed on the tooth to exhibit antibacterial properties and easily penetrate into the gingival tissue. In addition, by utilizing such cell / tissue penetrating properties, the peptide can be chemically conjugated to a peptide that does not have cell penetrating properties but has anti-inflammatory functions to impart cell penetrating properties, thereby increasing the desired anti-inflammatory effect.

[0041] In addition to the peptide having the amino acid sequence of SEQ ID NO: 1, peptides that can be mixed with collagen include peptides having SEQ ID NO: 2 to SEQ ID NO: 5 disclosed in applications and registrations filed by the present applicant, such as KR1249702, KR1320472, KR1329774, and KR1257228, and the specifications of these applications are incorporated herein by reference.

[0042] SEQ ID NO: 2: CPRRYKQIGTCGLPGTKCCKKP

[0043] SEQ ID NO:3: GKCSTRGRKCCRRKK

[0044] SEQ ID NO: 4: RKIEIVRKKPIFKKATVT

[0045] SEQ ID NO: 5: CKRKKKGKGLGKKRDPCLRKYK

[0046] After dissolving the peptide as the second agent in saline, the peptide solution is poured into a tube for injecting into the transplant site, and the first agent, which contains collagen as its main component, is soaked in the solution and then filled into the transplant site.

[0047] The peptide was added in an amount of 100 parts by weight of the graft. -4 0.5 to 10 parts by weight, more preferably 10 -3 ~10 -1 This means that the peptide can contain 10 parts by weight. -3 If it is contained in an amount less than 0.5 parts by weight, the regenerative effect is not significant, and if it is contained in an amount exceeding 0.5 parts by weight, it is difficult to include it in the transplant material.

[0048] Next, a bioabsorbable photocurable composition is applied to the upper part of the implantation site filled with the implant material (S3).

[0049] The bioabsorbable photocurable composition includes collagen, a photosensitizer, a pH adjuster, and an isotonicity agent. The bioabsorbable photocurable composition is applied in a gel state when applied to the upper part of the transplant site. Therefore, the viscosity of the bioabsorbable photocurable composition can be such that it is easy to administer to the transplant site and does not run off. The viscosity of the bioabsorbable photocurable composition can be in the range of 25 to 270 cP. Therefore, the bioabsorbable photocurable composition can cover the transplant material while efficiently filling the vacant space at the transplant site filled with the transplant material.

[0050] Collagen may be a type 1, 2, 3 or 4 collagen solution. Among them, type 1 collagen may be effectively applied. This is because type 1 collagen is derived from most connective tissues and is less likely to cause a negative immune reaction with the tissue at the implantation site. Collagen may be included in the range of 0.75-3 w / v%. If the content is less than 0.75 w / v% or more than 3 w / v%, gel formation may be insufficient.

[0051] The photosensitizer may be a substance capable of inducing collagen gelation by irradiation with light. For example, riboflavin is suitable as the photosensitizer. Riboflavin may be contained in the range of 0.0008 w / v% to 0.008 w / v%. Outside this range, gel formation with an appropriate viscosity may not be achieved. Preferably, riboflavin may be contained in the range of 0.0008 w / v% to 0.004 w / v%.

[0052] Therefore, the weight ratio of collagen:riboflavin can be 1:0.00056 to 1:0.0533, and within this range a gel of the desired state can be formed.

[0053] As the pH adjusting agent, NaOH or the like can be used. The pH adjusting agent can be contained in the range of 26 mM to 30 mM. Outside this range, gel formation with an appropriate viscosity may not be achieved.

[0054] As an isotonicity agent, a combination of NaCl, KCl, Na2HPO4, and KH2PO4 can be used.

[0055] Next, light curing is carried out (S4).

[0056] As shown in Figure 3, when visible light is applied, it stimulates the photoactivator riboflavin, causing a chemical reaction, and the oxygen radicals released from this reaction form chemical bonding points on the collagen, hardening it.

[0057] As a visible light source, an LED light source in the blue wavelength range is suitable. Although light curing is also possible with UV, it can cause damage to cells, so a light source in the blue wavelength range is more appropriate. The light source can irradiate a 450 nm wavelength light source with sufficient power and time to cure the collagen. For example, the power can be up to 2 W / cm 2 The transplant site can be irradiated for 20 to 60 seconds so that the patient does not feel any heat, and the photocurable composition can be sufficiently cured to perform its shielding function without flowing.

[0058] If necessary, the LED light irradiator can penetrate into the body-absorbed photosensitive composition to more effectively transmit the light source and allow photocuring to occur uniformly throughout the body-absorbed photosensitive composition. For example, in the case of treating periodontitis, the LED light irradiator can penetrate into the photocurable composition filled between the teeth and gums to allow the light to diffuse uniformly within the photocurable composition, allowing photocuring to occur more efficiently.

[0059] When the photocurable composition (e.g., collagen gel containing a photoreduction agent) is crosslinked by visible light, the collagen crosslinked by visible light fills the minute empty spaces at the transplant site and bonds closely with the graft material to fix the graft material. The collagen gel can also bond with natural collagen present at the transplant site. Therefore, regardless of the various patterns and sizes of the transplant site, the minute empty spaces at the transplant site can be filled and the graft material can be fixed by bonding with strong adhesion. Therefore, the problems that occur when applying the conventional shielding film can be solved, and the troublesome process of suturing the shielding film is also unnecessary. When initially applied, the viscosity of the photocurable composition increases from 25 cP to 270 cP to 110 cP to 410 cP by about 1.5 to 4.5 times due to photocuring, and the viscosity is tightly bonded to the underlying graft material to prevent the graft material from coming off.

[0060] In particular, when the graft material is composed of a collagen block and a peptide having antibacterial or anti-inflammatory properties, not only is the adhesion between the collagen block and the collagen sol strengthened by bonding, but it is also expected that they will act as a scaffold that allows the peptide having antibacterial or anti-inflammatory properties to act in a sustained release form. In the case of ointments containing minocycline as the main ingredient, which are sold as conventional periodontitis treatments, minocycline mainly exhibits antibacterial effects, and is therefore insufficient in inducing regeneration of the transplant site accompanied by inflammation, but when a peptide exhibiting antibacterial and anti-inflammatory properties is applied, it has the advantage of being able to effectively induce regeneration of the transplant site.

[0061] In particular, in the case of periodontal disease, it can induce an environment suitable for regeneration by reducing inflammation such as various peri- or periodontitis along with antibacterial activity against periodontitis-inducing bacteria. Collagen also provides space that can be replaced by regenerated bone by being absorbent and decomposed over time while supporting natural bone until it is fully regenerated, thereby increasing the efficiency of periodontal tissue regeneration.

[0062] In the following, preferred experimental examples are presented to aid in understanding the present invention. However, the following experimental examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following experimental examples.

[0063] Peptide synthesis

[0064] The peptide of sequence number 1 was synthesized from the C-terminus by F-moc solid-phase chemical synthesis using a synthesis apparatus. That is, it was synthesized using Rink resin (0.075mmol / g, 100-200mesh, 1% DVB crosslinking) to which Fmoc-(9-Fluorenylmethoxycarbonyl) was bound as a blocking group, and 50mg of Rink Amide MBHA resin was placed in the synthesis apparatus, and the resin was swelled with DMF, and then 20% piperidine / DMF solution was used to remove the Fmoc-group. From the C-terminus, 5, 10, and 5 equivalents of 0.5M amino acid solution (solvent: DMF), 1.0M DIPEA (solvent: DMF and NMP), and 0.5M HBTU (solvent: DMF) were placed in the sequence, respectively, and reacted under a nitrogen stream for 1-2 hours. After each of the deprotection and coupling steps, the product was washed twice with DMF and methanol. After coupling of the last amino acid, deprotection was also performed to remove the Fmoc group.

[0065] The synthesis was confirmed using the ninhydrin test method, and the resin that had been synthesized after the test was dried and then Reagent K cleavage cocktail was added at a ratio of 20 ml per 1 g of resin and shaken for 3 hours, after which the resin and the cocktail in which the peptide was dissolved were separated through filtering. The filtered solution was added with cold ether to crystallize the peptide in the solid phase, and this was separated by centrifugation. At this time, the Reagent K cleavage cocktail was completely removed through several washings with ether and centrifugation processes. The crude thus obtained was dissolved in distilled water and separated and purified using liquid chromatography. The purified peptide was freeze-dried. The purity was analyzed by liquid chromatography and the results of mass spectrometry are shown in Figures 4 and 5.

[0066] 4 and 5, it can be confirmed that the synthesized peptide has a purity of 98.2%, a molecular weight of 1733.99, and a 15-mer amino acid sequence.

[0067] Anti-inflammatory activity evaluation

[0068] 1) Measurement of the production of proinflammatory cytokines (IL-6 and TNF-α)

[0069] Anti-inflammatory activity was examined using a human mononuclear cell line (THP-1, ATCC, TIB-202). In the case of the mononuclear cell line, PMA (Phorbol 12-myristate 13-acetate, Merck, P1585) was inoculated to differentiate the mononuclear cells into macrophages, and then 1μg / ml LPS (lipopolysaccharide, Merck, L3024), an inflammation inducer, was treated. After 16 hours, 200μM of a peptide having the amino acid sequence of SEQ ID NO:1 and 20ng / ml of IL-4 (R&D system, 204-IL-010 / CF) as a positive control were inoculated, and the cell culture medium was obtained 24 hours later. The amount of IL-6 (R&D system, D6050) and TNF-α (R&D system, DTA00D), which are inflammation-inducing cytokines secreted by the cells present in the medium, was confirmed by ELISA (enzyme-linked immunosorbent assay).

[0070] As a result, as shown in Figure 6, it was confirmed that when the peptide having the amino acid sequence of SEQ ID NO: 1 was treated (LPS + S3), the IL-6 and TNF-α levels were reduced to a level similar to that when IL-4 was treated. When only LPS was treated, the IL-6 and TNF-α levels increased. Therefore, it was found that the peptide having the amino acid sequence of SEQ ID NO: 1 has an anti-inflammatory effect.

[0071] 2) Western blot of pNFkB and plkBa

[0072] Raw264.7 cells were used in the experiment, provided by ATCC (American Type Culture Collection). Raw264.7 cells were cultured in a 37℃ CO2 incubator with 5% CO2 and 95% air, using DMEM (Dulbecco's modified Eagle medium) medium supplemented with 10% FBS and 100 units / ml antibiotics (penicillin-streptomycin).

[0073] Raw264.7 cells were treated with 200μM of peptide having the amino acid sequence of SEQ ID NO:1 for 30 minutes, and then treated with 1μg / ml LPS (lipopolysaccharide, Merck, L3024). After 8 hours, the treated cells were collected, and RIPA buffer (Radioimmunoprecipitation assay buffer, Thermo Fisher, 89900) was added with a protease inhibitor cocktail and a phosphatase inhibitor cocktail, and the cells were treated to lyse the cells. The cell lysate was centrifuged, the supernatant was collected, and the concentration of the cell lysate was measured using a BCA assay (Bicinchoninic acid assay, Thermo Fisher, 23227), and RIPA buffer was added to prepare a cell lysate at a final concentration of 5mg / ml. The mixture was then mixed with a buffer solution consisting of 1M Tris-HCl (pH 6.8), 50% glycerol, and 10% SDS in a 4:1 ratio, and subjected to SDS-polyacrylamide gel electrophoresis (sodium dodecyl sulfate-poly acrylamide gel electrophoresis, SDS-PAGE). The polyacrylamide gel was then transferred to a nitrocellulose membrane, and the membrane was blocked with a blocking solution (5% skim milk in Tris-buffered saline with 0.1% Tween® 20), and then reacted with the blocking solution and primary antibodies (phosphorylated NF-κB (Signaling Technology, 3033), NF-κB (Cell Signaling Technology, 8242), phosphorylated IκBα (Cell Signaling Technology, 9246), IκBα (Cell Signaling Technology, 4812), and β-actin (Santa Cruz Biotechnology, sc-47778) at a ratio of 1:1000 overnight in a refrigerator.Then, the secondary antibodies for each primary antibody were reacted at room temperature for 1 hour in blocking solution 1:2000, and then reacted with ECL (enhanced chemiluminescence, Thermo Fisher, 34094) solution, and the membrane was developed using ChemiDoc Imaging Systems (Bio-Rad). Phosphorylated NF-κB and phosphorylated plkBa are indicators that can confirm the degree of inflammatory response at the cellular level, and the higher the inflammatory response, the more phosphorylated NF-κB and phosphorylated plkBa expression can be confirmed.

[0074] As a result, as shown in Figure 7, it was confirmed that the expression of pNFκB and plkBa was increased when only LPS was treated, but the expression of pNFκB and plkBa was suppressed when LPS was treated after treatment with the peptide having the amino acid sequence of SEQ ID NO: 1. Therefore, it was found that the peptide having the amino acid sequence of SEQ ID NO: 1 has an anti-inflammatory effect.

[0075] However, treatment with only the peptide having the amino acid sequence of SEQ ID NO: 1 did not affect the expression of pNFκB and plkBa in the cells, which indirectly proves that the peptide is safe.

[0076] Antibacterial activity evaluation

[0077] 1) Inhibition of the growth of periodontitis-causing bacteria

[0078] To evaluate the antibacterial activity, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, which are representative periodontitis-causing bacteria, were used. Each bacterium was inoculated into BHI (brain heart infusion) liquid medium and then cultured in an anaerobic incubator at 37°C and 120 rpm for 48 hours. After the culture, the peptide having the amino acid sequence of SEQ ID NO: 1 was treated at 100μM, 250μM, and 500μM. As a positive control, minocycline (Merck, M9511) was inoculated at 500ng / ml, and the degree of growth of the bacteria was confirmed by measuring the absorbance at 600nm wavelength using a spectrophotometer after 24 hours.

[0079] As a result, as shown in Figure 8, when treated with a peptide having the amino acid sequence of SEQ ID NO: 1, the survival rate of Aa (Aggregatibacter actinomycetemcomitans) was 50% or less at 250 μM or more, and the survival rate of Pg (Porphyromonas gingivalis) was 50% or less at 100 μM or more.

[0080] Meanwhile, when compared with the effect of minocycline, the survival rate was higher when treated with 500μM concentration of Aa than when treated with 500ng / ml of minocycline, but the effect was similar. While minocycline only has antibacterial activity, the peptide having the amino acid sequence of SEQ ID NO:1 has antibacterial and anti-inflammatory activity, so even if there is a slight difference in antibacterial activity, this part can be complemented. For Pg, it showed a better effect than minocycline at all concentration ranges.

[0081] 2) Evaluation of antibacterial activity in biofilms

[0082] Periodontitis-inducing bacteria exist in the oral cavity in the form of a biofilm, and to mimic this, an artificial biofilm was created on the surface of a titanium disk using Aa (Aggregatibacr actinomycetemcomitans) and Pg (Porphyromonas gingivalis). Saliva and bacteria were mixed with the titanium disk, then placed in BHI liquid medium and cultured in an anaerobic incubator at 37°C. After 72 hours, 50μg / ml (=29μM), 100μg / ml (=58μM), 250μg / ml (=144μM), and 500μg / ml (=288μM) peptides and 1μg / ml minocycline as a positive control were inoculated. 24 hours after inoculation, titanium disks with biofilms formed on their surfaces were obtained and stained using the Filmtracer™ LIVE / DEAD™ Biofilm Viability Kit (Thermo Fisher, L10316). Live bacteria were stained green, and dead bacteria were stained red. The titanium disks were observed using a confocal scanning fluorescence microscope.

[0083] As a result, as shown in FIG. 9, in the case of minocycline used as a positive control, almost no dead bacteria (red) were observed because it could not penetrate the biofilm to kill the bacteria. In contrast, in the case of treatment with the peptide having the amino acid sequence of SEQ ID NO: 1, dead bacteria (red) were observed from a concentration of 50 μg / ml (= 29 μM). Therefore, it can be confirmed that existing antibiotics cannot kill bacteria that exist in the form of a biofilm, but the peptide having the amino acid sequence of SEQ ID NO: 1 penetrates the biofilm and has antibacterial properties.

[0084] Cell / tissue penetration assessment

[0085] 1) Evaluation of cell permeability at the cellular level

[0086] The N-terminus of the peptide having the amino acid sequence of SEQ ID NO:1 prepared in Example 1 was fluorescently labeled with rhodamine B (Tokyo Chemical Industry Co., LTD., A5102). Raw264.7 cells were dispensed into a 4-well chamber at 3X104 cells each and cultured in DMEM medium for 24 hours to stabilize the cells. After 24 hours, the fluorescently labeled peptide was added to the medium at concentrations of 50, 100, and 200 μM. One hour after peptide treatment, the degree of intracellular peptide permeation was observed using a confocal scanning fluorescence microscope. The cell nuclei were stained using DAPI (4',6-diamidino-2-phenylindole, Thermo Fisher, D1306). DIC indicates observations made using a general microscope, Nuclei indicates the cell nucleus, Peptide indicates the degree of permeation of the peptide bound to rhodamine B, and MERGE indicates an image obtained by overlapping the images of DIC, Nuclei, and the permeated peptide. As a result, as shown in Figure 10, the fluorescence of rhodamine B (red) was observed in the cells treated with the peptide having the amino acid sequence of SEQ ID NO: 1, but the red color was not observed in the untreated experimental group. It was also found that the intensity of the fluorescence increased with increasing peptide concentration.

[0087] 2) Evaluation of tissue penetration in animal tissues

[0088] The amine group of the peptide having the amino acid sequence of SEQ ID NO: 1 was fluorescently labeled using Alexa Fluor (trademark) 680 NHS Ester (Thermo Fisher, A20008). The fluorescent labeling method was according to the method provided by the manufacturer. The fluorescently labeled peptide was dissolved in water for injection at a concentration of 250 mg / ml, and 10 μL was injected into the gingival cleft of the premolar of an adult dog (beagle dog) and the gingival cleft of the anterior tooth of a rabbit. After 2 hours, the animals were sacrificed and the gingival tissue was collected and embedded in Tissue-Tek (registered trademark) OCT Compound (Sakura Finetek, 4583) to confirm the penetration of the peptide in the tissue. The embedded tissue was sectioned to a thickness of 10 μm, and the fluorescently labeled peptide was confirmed by a confocal scanning fluorescence microscope. The nuclei of the tissue cells were stained with DAPI.

[0089] As a result, it was confirmed that the fluorescently labeled peptide penetrated the gingival tissue cells and reached a depth of approximately 500 (440-459) μm, as shown in Figure 11 (the gingival tissue of a rabbit) and Figure 12 (the gingival tissue of an adult dog).

[0090] Therefore, it was confirmed that the peptide having the amino acid sequence of SEQ ID NO: 1 penetrates not only at the cellular level but also into tissues.

[0091] Evaluation of the antibacterial activity of implant materials

[0092] A collagen sponge block of 6x5x7 mm size was immersed in 500 μg / 0.5 ml of a peptide having the amino acid sequence of SEQ ID NO: 1, left at 4° C. for 8 hours, and then freeze-dried. As a positive control, a collagen sponge block of 6x5x7 mm size was immersed in 250 ng / 0.5 ml of minocycline (Merck, M9511), left at 4° C. for 8 hours, and then freeze-dried.

[0093] To evaluate the antibacterial activity of the implant containing the peptide having the amino acid sequence of SEQ ID NO: 1, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, which are representative periodontitis-causing bacteria, were used. Each bacterium was inoculated into a BHI (brain heart infusion) liquid medium and then cultured in an anaerobic incubator at 37°C and 120 rpm for 48 hours. After the culture, a collagen sponge block containing the peptide having the amino acid sequence of SEQ ID NO: 1 of Example 2 was added to the medium. After 24 hours, the absorbance was measured at a wavelength of 600 nm to confirm the degree of bacterial growth.

[0094] As a result, as shown in Figure 13, when a collagen sponge block containing a peptide having the amino acid sequence of SEQ ID NO: 1 was treated, the survival rate of Aa (Aggregatibacter actinomycetemcomitans) and Pg (Porphyromonas gingivalis) was 40% or less. Therefore, it was proven that the peptide having the amino acid sequence of SEQ ID NO: 1 has an antibacterial effect when applied to a collagen sponge block.

[0095] Evaluation of periodontal tissue regeneration ability of implant materials

[0096] Collagen gel was dissolved in 0.1M PBS at a concentration of 4%. 125 mg of a peptide having the amino acid sequence of SEQ ID NO: 1 was dissolved in 1 g of collagen gel. As a positive control, 20 mg of minocycline was dissolved in the same 1 g of collagen gel.

[0097] To evaluate the periodontal tissue regeneration ability of a graft material containing a peptide having the amino acid sequence of SEQ ID NO:1, a collagen sponge block containing the peptide was grafted after inducing periodontitis in adult beagle dogs. Periodontitis was induced by wrapping a dental wire around the cervical area of ​​the mandibular premolars no. 2, 3, and 4 several times. Three months after the wire was wrapped, the wire was removed and scaling and root planning were performed. 15 μl of collagen gel containing a peptide having the amino acid sequence of SEQ ID NO:1 and collagen gel containing minocycline were applied to the gingival cleft twice a week for five weeks. The periodontal condition was observed for 12 weeks, and x-ray photography was performed to observe alveolar bone regeneration.

[0098] As a result, as shown in Figures 14 and 15, the group to which nothing was applied (NT) did not generate alveolar bone after 12 weeks. The group to which the collagen gel containing minocycline was implanted showed a similar degree of alveolar bone regeneration as the NT group. However, when the collagen gel to which the peptide having the amino acid sequence of SEQ ID NO: 1 was applied was implanted, the regeneration of alveolar bone (white part in the square) increased by more than 10 times compared to the group to which the collagen gel containing minocycline was implanted. Therefore, it was proven that the collagen gel to which the peptide having the amino acid sequence of SEQ ID NO: 1 was applied has excellent periodontal tissue regeneration effects.

[0099] Measurement of gel formation as a function of collagen content in photocurable compositions

[0100] A collagen gel containing riboflavin (riboflavin sodium phosphate, Sigma, 77623-50G-F) was prepared using porcine type 1 collagen. As shown in Table 1, gels were prepared by varying the collagen concentration to 0.375w / v%, 0.75w / v%, 1.125w / v%, 1.5w / v%, and 3w / v%, and the riboflavin, NaCl, KCl, Na2HPO4, KH2PO4, and NaOH were fixed to prepare collagen gels. Purified water was used as the solvent.

[0101] [Table 1]

[0102] As a result, as shown in FIG. 16, it was confirmed that a gel was formed from 0.75% collagen, and the hardest gel was formed at 3%. Gel formation measurement table according to the change in riboflavin content in photocurable compositions As shown in 2, gels were produced by changing the concentration of riboflavin to 0.0008w / v%, 0.0016w / v%, 0.004w / v%, and 0.008w / v%, and collagen, NaCl, KCl, Na2HPO4, KH2PO4, and NaOH were fixed to produce collagen gels. Purified water was used as the solvent.

[0103] [Table 2]

[0104] As a result, as shown in FIG. 17, gels with appropriate viscosity were formed in the range of 0.0008 w / v % to 0.008 w / v %, and gels with more preferable viscosity were formed in the range of 0.0008 w / v % to 0.004 w / v %. Gel formation measurement table according to NaOH content change in photocurable composition As shown in 3, gels were produced by varying the NaOH concentration to 20 mM, 26 mM, and 30 mM, and collagen, riboflavin, NaCl, KCl, Na2HPO4, and KH2PO4 were fixed to produce collagen gels. Purified water was used as the solvent.

[0105] [Table 3]

[0106] As a result, as shown in FIG. 18, no gel was formed with 20 mM NaOH, but gels were formed with 26 mM and 30 mM NaOH. Changes in properties of a photocurable composition (collagen gel containing riboflavin) due to light irradiation 300 μl of collagen gel of composition No. 4 in Table 1 was irradiated with light of 450 nm wavelength at 2 W / cm 2 The collagen gel was irradiated for 10, 20, 30, 50, and 60 seconds at an intensity of 100 nm, and the shape of the collagen gel was observed.

[0107] The results are shown in Figure 19. Referring to Figure 19, no phase change occurred when the material was irradiated with light for 10 seconds. When the material was irradiated with light for 20 seconds, it began to change to a solid state as it hardened, and after 30 seconds, it changed to a solid state.

[0108] Viscosity measurement of photocurable compositions

[0109] The gels of composition numbers 2 to 5 in Table 1 were measured using a viscometer (DV-II Proviscosity Meter, Brookfield). The spindle used for the measurement was No. 42, and the viscosity was measured before and after curing at 90 rpm.

[0110] The results are shown in Figure 20. Referring to Figure 20, the viscosity of the gels of compositions 2 to 5 before curing was measured to be between 25 and 270 cP, and the viscosity after curing increased to 110 to 410 cP. In other words, although it varies depending on the composition, the viscosity after curing can increase by about 1.5 to 4 times compared to before curing.

[0111] Stability of collagen gel containing riboflavin

[0112] To confirm the stability of collagen gel containing riboflavin, collagen gel of composition number 4 in Table 1 was irradiated with light for 30 seconds, placed in 500 μl of purified water and stored at 37° C. for 7, 14 and 30 days. As a control, collagen gel of composition number 4 was not irradiated with light, placed in 500 μl of purified water and stored at 37° C. for 7, 14 and 30 days.

[0113] The results are shown in Figure 21. Referring to Figure 21, after irradiating light for 30 seconds, the gel containing purified water maintained its shape, while the gel not irradiated with light could not maintain its shape and was mixed with purified water. Therefore, it was confirmed that the collagen gel containing riboflavin of composition number 4 irradiated with light for 30 seconds formed a stable solid gel for up to 30 days.

[0114] Adaptation of riboflavin-containing collagen gel as a transplant material

[0115] Collagen gel containing riboflavin can be used when fixation is required at a local site in the body, like a collagen block. Collagen gel of composition number 4 in Table 1 was applied to artificially induced peri-implantitis in adult dogs. As shown in Figure 22, the gums at the site where peri-implantitis appeared were incised, and the area around the implant was washed with saline as much as possible. After washing was completed, the collagen block containing the peptide was transplanted into the site of peri-implantitis (Figure 22a). After transplantation, 0.5 ml of collagen gel containing riboflavin was applied to fix the collagen block (Figure 22b), and then a 450 nm wavelength, 2 W / cm 2 The tissue was then exposed to light for 30 seconds at an intensity of 100 nm (Figure 22c). After confirming that the riboflavin-containing collagen gel had gelled, the incised gums were sutured (Figure 22d). It was confirmed that the implant site was well maintained without any side effects even 4 weeks after implantation.

[0116] Although the above describes experimental examples, the scope of the invention is not limited thereto. The embodied forms can be modified in various ways within the scope of the detailed description of the invention and the attached drawings, and these also naturally fall within the scope of the invention. [Industrial Applicability]

[0117] It can be used as a graft material to treat various bone losses or defects caused by inflammation, tumors, trauma, and skeletal diseases. [Sequence List Free Text]

[0118] Sequence Catalog No. 1: GKCSTRGRKSSRRKK Sequence Catalog No. 2: CPRRYKQIGTCGLPGTKCCKKP Sequence Catalog No. 3: GKCSTRGRKCCRRKK Sequence Catalog No. 4:RKIEIVRKKPIFKKATVT Sequence Catalog No. 5: CKRKKKGKGLGKKRDPCLRKYK

Claims

1. Contains a photosensitizer and collagen, Apply the composition to the top of the implantation site filled with implant material to cover the implant material while filling the empty space of the implantation site; The photoactivator is activated by irradiation with visible light, crosslinking the collagen, and the collagen crosslinked by the visible light fills minute empty spaces at the transplant site and tightly bonds with the transplant material to fix the transplant material.

2. 2. The photocurable composition according to claim 1, wherein the viscosity of the photocurable composition after irradiation with visible light is increased by 1.5 to 4.5 times compared to before irradiation with visible light.

3. 2. The bioresorbable photocurable composition of claim 1, wherein the photosensitizer is riboflavin.

4. 4. The bioabsorbable photocurable composition according to claim 3, wherein the weight ratio of riboflavin to collagen is 1:0.00056 to 1:0533.

5. The visible light is a light source in the blue wavelength range and has a maximum intensity of 2 W / cm ２ The photocurable composition of claim 3, which is a light source having an output.

6. A transplant material comprising a first agent mainly composed of collagen and a second agent containing a peptide formulated so that the first agent can be immersed therein; and A bioabsorbable tissue regeneration inducing material comprising a bioabsorbable photocurable composition which comprises a photoreduction agent and collagen, which is applied to a transplant site filled with the transplant material to fill the empty space in the transplant site and cover the transplant material, and which is activated by irradiation with visible light to crosslink the collagen, so that the collagen crosslinked by the visible light fills the minute empty spaces in the transplant site and tightly bonds with the transplant material to fix the transplant material.

7. The bioresorbable tissue regeneration inducing material according to claim 6 , wherein the peptide is a peptide having antibacterial activity, anti-inflammatory activity, antibacterial activity and anti-inflammatory activity, or antibacterial activity, anti-inflammatory activity and cell-penetrating activity.

8. 8. The bioresorbable tissue regeneration inducing material according to claim 7, wherein the peptide is a peptide represented by an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5.

9. The implant contains the peptide at 10 －４ The tissue regeneration-inducing material absorbable in the body according to claim 7, containing from 0.5 parts by weight to 0.5 parts by weight.

10. The tissue regeneration inducing material absorbable in the body according to claim 6, wherein the viscosity of the photocurable composition after irradiation with visible light is increased by 1.5 to 4.5 times compared to before irradiation with visible light.

11. The bioresorbable tissue regeneration inducing material according to claim 6 , wherein the photosensitizer is riboflavin.

12. The bioresorbable photocurable composition according to claim 11, wherein the weight ratio of riboflavin to collagen is 1:0.00056 to 1:0.0533.

13. The visible light is a light source in the blue wavelength range and has a maximum intensity of 2 W / cm ２ The photocurable composition of claim 6, which is a light source having an output.

14. filling the implantation site with implant material; applying a bioabsorbable photocurable composition containing a photosensitizer and collagen to the upper portion of the transplant site filled with the transplant material to fill the empty space of the transplant site and cover the transplant material; and The transplantation method includes a step of irradiating the body-absorbable photocurable composition with visible light to activate the photoactivator and crosslink the collagen, and the collagen crosslinked by the visible light fills minute empty spaces at the transplantation site and tightly bonds with the transplant material to fix the transplant material.

15. Prior to the step of filling the implant, The method of claim 14, further comprising the step of preparing the implantation site.

16. The transplant site is a site of periodontal disease, The implantation method of claim 15, wherein the preparation step includes scaling or scaling and root planing.

17. The transplantation method according to claim 14, wherein the viscosity of the photocurable composition after irradiation with visible light is increased by 1.5 to 4.5 times compared to before irradiation with visible light.

18. The method of claim 14, wherein the photosensitizer is riboflavin.

19. The transplantation method according to claim 18, wherein the weight ratio of riboflavin:collagen is 1:0.00056 to 1:0.0533.

20. The visible light is a light source in the blue wavelength range and has a maximum intensity of 2 W / cm ２ 15. The method of claim 14, further comprising an output.

21. The implantation method according to claim 14, wherein the light irradiator for irradiating the visible light transmits a light source by penetrating into the internally absorbable photosensitive composition.

22. The transplant method according to claim 14 , wherein the transplant material comprises a first agent mainly composed of collagen and a second agent containing a peptide formulated so that the first agent can be soaked therein.

23. The transplantation method according to claim 22, wherein the peptide is a peptide having antibacterial activity, anti-inflammatory activity, antibacterial activity and anti-inflammatory activity, or antibacterial activity, anti-inflammatory activity and cell penetrating activity.

24. The peptide transplantation method according to claim 23, wherein the peptide is represented by an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5.

25. The implant contains the peptide at 10 －４ The implantation method of claim 23, comprising from about 0.5 parts by weight.

Images