KR20130033826A - Dental membrane - Google Patents

Abstract

PURPOSE: An antibacterial dental membrane is provided to ensure alkaline phosphatase activity in osteoblast, and to improve bond generation in a lesion. CONSTITUTION: A dental membrane contains 0.01-10 wt% of 4-hexylresorcinol, and 90-99.99 wt% of silk proteins. The silk protein is silk fibroin. A method for manufacturing the dental membrane comprises: a step of dissolving the silk fibroin in an ethanol solution containing a salt, and removing the salt and ethanol to prepare a silk fibroin solution; a step of dissolving 4-hexylresorcinol in ethanol and preparing 4-hexylresorcinol ethanol solution; a step of mixing the silk fibroin solution and the 4-hexylresorcinol ethanol solution, and comprising a dental membrane composition; and a step of pouring the dental membrane composition into a container and drying. [Reference numerals] (AA) Control group without applying a membrane; (BB) Experimental group with applying the membrane of the present invention composed of 3% hexylresorcinol and remaining amount of silk fibroin(an arrow indicates a remaining dental membrane);

Description

DENTAL MEMBRANE

TECHNICAL FIELD The present invention relates to a dental shielding membrane, and more particularly, to a dental shielding membrane that significantly improves antimicrobial activity, alkaline phosphatase activity in osteoblasts, and bone formation in bone defects.

If the tooth is lost, the gingival bone around the tooth also gradually disappears, making it difficult to reconstruct the implants later.

Shielding membranes of various materials have been developed to suppress the disappearance of the gingival bones around the teeth that occur after the loss of the teeth, or to recover some bone defects that occur during the implantation procedure.

In other words, the shielding membrane is not a bone graft but an auxiliary material to prevent the early penetration of fibroblasts or epithelial cells that proliferate in the healing step of bone defects, thereby preventing the defective part from regenerating into soft tissues and regenerating into bone.

Materials that can be used for dental shields include collagen and synthetic resin products originating from heterologous animals.

Currently, the most widely used dental shield material is collagen fiber material, but its cost is high, but the most important advantage is that it does not require a second operation for removal because it dissolves in vivo. Shielding films of chitosan material and insect origin protein materials have been developed as shielding films for other natural materials.

However, shielding membranes of these materials do not have antimicrobial properties in spite of being used in many oral cavity.

Given the fact that one of the most common reasons for tooth extraction is infection-related complications, the antimicrobial properties that shields do not have can be a serious obstacle to the application of shielding.

And the characteristic of the existing shielding membranes is that the blocking of the cell movement is most important because it is a property that can be obtained with the appropriate biodegradation rate and a suitable size of pores.

If the shielding membrane contains a component capable of activating the osteoblast activity along with cell movement shielding, it may be possible to acquire a large amount of bone formation much faster than the conventional shielding membrane.

It is an object of the present invention to provide a dental shielding film which significantly increases antimicrobial activity and alkaline phosphatase activity in osteoblast, bone defect site and bone defect site around the implant, .

Technical problems to be achieved by the present invention are not limited to the above-mentioned technical problems, and other technical problems not mentioned above may be clearly understood by those skilled in the art from the following description. There will be.

To achieve the above object, the dental shading film composition of the present invention comprises 0.01 to 10% by weight of 4-hexyl resorcinol and 90 to 99.99% by weight of silk protein.

The dental shielding film of the present invention comprises 4-hexylresorcinol and silk protein, and in particular, the 4-hexylresorcinol is contained in an amount of 0.01 to 10% by weight based on the total weight of the dental shielding film Feature.

Preferably, the dental shade film of the present invention comprises 0.01 to 10% by weight of the 4-hexyl resorcinol and 90 to 99.99% by weight of the silk protein. Preferably, the silk protein is silk fibroin.

The dental shielding film of the present invention may further comprise a biocompatible material such as collagen, gelatin, chitin, chitosan, keratin, cellulose, fibronectin, elastin, fibrinogen, pibromodulin, laminin, tenacin, (PLGA), polyglycolic acid (PGA), copolymers of polylactic acid and polyglycolic acid (PLGA), polycaprolactone (PLGA), polyglycolic acid polycaprolactone (PCL), poly {poly (ethylene oxide) terephthalate-co-butylene terephthalate} (PEOT / PBT), polyphosphoester (PPE), polyphosphazene (PPA), polyanhydride (PA), polyortho ester (polyortho ester; POE}, pluronic 127, glycerin, poly (propylene fumarate) -diacrylate; PPF-DA} and polyethylene glycol diacrylate; PEG-DA} may be further contained in an amount of 30 wt% or less.

A method for producing a dental shielding film according to the present invention comprises the steps of: preparing silk fibroin by removing sericin from a silk protein, dissolving the silk fibroin in an aqueous ethanol solution containing a salt, removing salt and ethanol to prepare a silk fibroin aqueous solution, Preparing hexane resorcinol ethanol solution by dissolving hexylresorcinol in ethanol, mixing the aqueous solution of silk fibroin and 4-hexylresorcinol ethanol solution to prepare a dental shielding film composition And pouring the dental shielding film composition into a flat container and drying the dental shielding film composition to prepare a dental shielding film.

According to the present invention, there is provided a dental shading film which remarkably improves antimicrobial activity, alkaline phosphatase activity in osteoblasts, and bone formation in bone defects.

1 is a view showing the chemical structure of 4-hexyl resorcinol.
2 is a graph showing the activity of alkaline phosphatase in the case of administration of 4-hexyl resorcinol to MG-63 cells.
3 is a graph showing the degree of expression of osteocalcin when 4-hexylresorcinol is administered to MG-63 cells
4 is a view showing the antibacterial activity against the dental shielding film composition of the present invention.
5 is a view showing the degree of new bone formation at the time of 8 weeks in the bone defect around the implant.
FIG. 6 is a micro CT scan of the state of each group at 2 weeks in the rabbit skull defect model. FIG.
FIG. 7 is a graph showing the degree of new bone formation measured in terms of volume based on the FIG. 6. FIG.
control: control group without shield,
commercials: commercialized shields installed
silk fibroin: Experimental group using a shielding film made of 4-hexyl resorcinol and silk fibroin of the present invention

The terms, techniques, and the like described in this specification are used in the meaning commonly used in the technical field to which the present invention belongs, unless otherwise specified.

Hereinafter, the present invention will be described in detail.

In order to overcome the problems of conventional dental shielding membranes, the present inventors have found that antimicrobial activity and alkaline phosphatase activity in osteoblasts are increased as compared with conventional dental shielding membranes, and bone formation in bone defects and bone defects around implants The dental shields were found to improve significantly.

The term " dental shielding film " as used herein means a shielding film to be used as an auxiliary for the purpose of restoring bone defects due to various dental diseases.

The dental shielding film of the present invention includes 4-hexylresorcinol and a silk protein.

The IUPAC name of 4-hexylresorcinol is 4-hexylbenzene-1,3-diol, which is a compound having the chemical structure shown in FIG. 1, It has been commonly used for the purpose of treatment for the purpose of taking in olive oil or recently to prevent discoloration of sea shrimp. In the present invention, 0.01 to 10% by weight is included in the total weight of the dental shielding film.

That is, when the weight of the 4-hexyl resorcinol is less than 0.01% by weight, it is difficult to expect an antimicrobial effect. When the weight of the 4-hexyl resorcinol is more than 10% by weight, The stability is not ensured and the morphological stability of the dental shielding film is not maintained.

Preferably, the dental shade film of the present invention comprises 0.01 to 10% by weight of 4-hexyl resorcinol and 90 to 99.99% by weight of the silk protein.

That is, the silk protein is a protein having the highest purity (97% or more) among many kinds of proteins present in the natural world, and contains 75% of fibroin and 25% of sericin as main constituents. These amino acids are dehydrated and condensed, When the content of the silk protein is less than 90% by weight, bone formation can be suppressed rather difficult, and it is difficult to form a membrane. When the content of the silk protein is more than 99.99% by weight, it is difficult to expect an antibacterial effect. .

Among the silk proteins, silk fibroin has 18 amino acids relatively uniformly, and glycine and alanine contents are very high, unlike keratin proteins of wool fibers.

Among the amino acids of the collagen protein, the major protein that constitutes the human skin, glycine and alanine are the most abundant in the fibroin protein.

The fibroin protein having an amino acid structure similar to that of human skin has excellent biocompatibility and exhibits no inflammatory effect. Therefore, in the present invention, it is preferable to use silk fibroin as the silk protein.

The dental shielding film constructed as described above can be produced so that the total amount of the 4-hexyl resorcinol and the silk protein is 100% by weight. A biocompatible material for biocompatible tissue engineering, which does not impair the transparency and physical properties of the shielding film, By weight based on the total weight of the shielding film.

If it exceeds 30% by weight, transparency and the like may be deteriorated.

In the present invention, the terms "transparent" and "transparency" are used to mean a transparent state in which the inside of the film can be observed in addition to the completely transparent state.

The biocompatible material may be any of a biocompatible material, but it may be any of a collagen, gelatin, chitin, chitosan, keratin, cellulosic, fibronectin, elastin, fibrinogen, pibromodulin, laminin, tenacin, Polylactic acid (PLA), polyglycolic acid (PGA), copolymer of polylactic acid and polyglycolic acid (PLGA), polycaprolactone (polycaprolactone), polylactic acid PCL), poly {poly (ethylene oxide) terephthalate-co-butylene terephthalate} (PEOT / PBT), polyphosphoester (PPE), polyphosphazene (PPA), polyanhydride PA), polyortho ester {polyortho ester; POE}, pluronic, glycerin, poly (propylene fumarate) -diacrylate; PPF-DA} and polyethylene glycol diacrylate; PEG-DA} is preferably added.

In addition, various additives such as antibiotics, antiviral agents, antimicrobial agents, nucleic acids, peptides, and proteins may be added to the dental shade membrane of the present invention, so long as they do not adversely affect the structure and function of the dental shielding membrane.

The antibiotics, antiviral agents, antibacterial agents, and the like serve to prevent the infection of the dental shielding membrane.

The protein is preferably selected from the group consisting of a hormone, a cytokine, an enzyme, an antibody, a growth factor, a transcription factor, a vaccine, a structural protein, a ligand protein, a receptor, a cell surface antigen and a receptor antagonist.

The dental shielding film of the present invention thus formed is treated on the surface of the bone defect, and then the implant is implanted.

The term 'implant' refers to the replacement of a missing natural tooth, or a screw-like implant fixture is fastened to the alveolar bone to be fused with the bone for a certain period of time, and then an abutment Refers to a dental procedure for restoring the original function of a tooth by fixing a prosthesis such as an artificial tooth, a crown, etc. However, in the present specification, it means a replacement of a lost natural tooth.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples.

Example 1 Preparation of Dental Shielding Layer 1 of the Present Invention

Cocoon cocoa was used as a sample, and marlyilles soap and sodium carbonate solution were refined at 100 ° C for 1 hour to remove sericin.

The silk fibroin obtained by refining was dissolved in a calcium chloride-alcohol-aqueous solution prepared at a ratio of 1: 2: 8 (molar ratio) of calcium chloride, ethanol and water at 80 ° C for 20 minutes. The resulting silk fibroin was dialyzed against distilled water for 4 days The salt and ethanol were removed to prepare a silk fibroin aqueous solution.

4-Hexylresorcinol ethanol solution was prepared by dissolving 4-hexylresorcinol with ethanol.

To the aqueous silk fibroin solution prepared above, the 4-hexyl resorcinol ethanol solution prepared above was added and mixed, and 3% by weight of the total weight of the solution was made up of 4-hexyl resorcinol ethanol solution and the remaining silk fibroin aqueous solution To prepare a dental shielding film composition.

The dental shielding film composition was poured into a flat polystyrene container and dried at 60 DEG C to prepare a dental shielding film 1 of the present invention.

Example 2 Preparation of Dental Shielding Layer 2 of the Present Invention

The composition was prepared in the same manner as in Example 1, except that 5 wt% of 4-hexylresorcinol ethanol solution and the remaining silk fibroin aqueous solution were mixed in an amount of 5 wt% based on the total weight of the dental shading film composition. The shielding film composition was poured into a flat polystyrene container and dried at 60 DEG C to prepare a dental shielding film 2 of the present invention.

Example 3 Preparation of Dental Shielding Layer 3 of the Present Invention

Cocoon cocoa was used as a sample, and marlyilles soap and sodium carbonate solution were refined at 100 ° C for 1 hour to remove sericin.

The silk fibroin obtained by refining was dissolved in a calcium chloride-alcohol-aqueous solution prepared at a ratio of 1: 2: 8 (molar ratio) of calcium chloride, ethanol and water at 80 ° C for 20 minutes. The resulting silk fibroin was dialyzed against distilled water for 4 days The salt and ethanol were removed to prepare a silk fibroin aqueous solution.

4-Hexylresorcinol ethanol solution was prepared by dissolving 4-hexylresorcinol with ethanol.

To the aqueous silk fibroin solution prepared above, the 4-hexyl resorcinol ethanol solution prepared above was added and mixed, and 3% by weight of the total weight of the solution was made up of 4-hexyl resorcinol ethanol solution and the remaining silk fibroin aqueous solution To prepare a dental shielding film composition.

10% by weight of PEG 20,000 (polyethylene glycol) as a biomaterial was added to the dental shielding film composition and concentrated. Thereafter, it was poured into a flat polystyrene container and dried at 60 DEG C to prepare a dental shielding film 3 of the present invention.

Experimental Examples 1 and 2. Measurement of alkaline phosphatase activity and expression level of osteocalcin

1) Experimental methods and materials

MG63 cells (ATCC, US; osteoblasts (osteoblast like cell)) in vitro by using the (In in vitro test.

(Dulbecco Modified Eagles Medium) high glucose (PAA Laboratories, Austria) supplemented with 10% fetal bovine serum (PAA, Canada) and containing 1% penicillin / streptomycin It was to proliferation of the cells, and stored at 5% CO ₂ and 99% humidity under 37 ℃. The medium was changed three times a day.

Then, the protein concentration of the lysate was measured by a colorimetric assay.

At this time, the activity of alkaline phosphatase was measured by using an alkaline phosphatase assay kit. It was found that the conversion of p-nitrophenyl phosphate to p-nitrophenol Was measured by measuring the absorbance.

The amount of osteocalcin expression was measured according to the manufacturer's protocol at 72 ° C using an ELISA kit.

2) Test result

A. Results of alkaline phosphatase activity measurement

As shown in FIG. 2, the activity of the alkaline phosphatase was determined by adding 4-hexylresorcinol to the plate cultured on MG-63. As a result, the alkaline phosphoric acid decomposition was continuously performed up to 10 μg / It was confirmed that the activity of the enzyme was increased.

N. Results of osteocalcin expression

As shown in FIG. 3, the expression level of osteocalcin was determined by adding 4-hexylresorcinol to the MG-63 culture dish. As a result, the expression of osteocalcin was continuously increased to 10 ㎍ / .

Experimental Example 3. Antibacterial Experiment

1) Experimental method

A. Bacteria

Two periodontal bacteria ( Porphyromonas gingivalis , ATCC 49417 And Prevotella intermedia , ATCC 25611) were purchased from KCTC (Korean Collection for Type Cultures).

Staphylococcus aureus , ATCC 25923) was purchased from the American Type Culture Collection (ATCC).

The Porphyromonas jinx valis And The Porothella Intermedia was incubated under anaerobic conditions with an anaerobic brain infusion broth containing 1.0 g / ml Vit K1 and 5 g / ml Hemin.

Staphylococcus longus was cultured on nutrient agar under aerobic conditions.

B. Screening of Antimicrobial Activity

Each of the bacteria was cultured and diluted with sterile medium at 0.5 absorbance at 450 nm.

Porphyromonas jinx valis (ATCC49417) And 200 [mu] l of the bacterial solution was added onto an agar plate agar plate supplemented with 5% sheep blood, 1.0 g / ml Vit K1 and 5 g / ml hemine for the Freibereller Intermedia (ATCC 25611) Lt; / RTI >

Staphylococcus longus (ATCC 25923) was distributed in the same manner on a muller hinton agar medium.

Each of the strains was coated with a shielding film composition (the shielding film composition prepared in Example 1) containing a general shielding film, 0.1, 1, 10 μg / ml, 0.1, 1 and 10 mg / ml of 4-hexylresorcinol Respectively.

The plates were allowed to react for 2 days under anaerobic conditions at < RTI ID = 0.0 > 37 C. < / RTI >

In the case of Staphylococcus longovis, the plate was reacted under aerobic conditions at 37 ° C for 1 day. The diameter of the inhibition zone was measured.

2) Experimental results

Fig. 4 shows the result of applying the antimicrobial agent to be tested to a bacterial medium after wetting the antimicrobial agent test disk with the antimicrobial agent to be tested.

That is, the antimicrobial activity against Porphyromonas junzi varis (ATCC 49417) according to the effective content of 4-hexyl resorcinol was examined. As a result, it was found that when 0.1 μg / ml of 4-hexyl resorcinol and 1 μg / It was observed that the inhibitory ring was observed up to 10mg / ㎖ from 1mg / ㎖, but it showed higher antimicrobial activity than the disc containing the vancomycin used as the control.

In addition, it exhibited antibacterial activity against Fibotella Intermedia (ATCC25611) and Staphylococcus longus (ATCC25923).

That is, when the effective content of 4-hexyl resorcinol was 0.1 mg / ml, 1 mg / ml, and 10 mg / ml, a marked inhibitory ring was observed. In particular, the effective content of 4-hexyl resorcinol was 1 mg / 10 mg / ml, the antimicrobial activity was higher than that of the disc containing the vancomycin used as a control.

Therefore, it was confirmed that the content of 4-hexylresorcinol in the present invention exhibits the antimicrobial activity when it is contained in an amount of 0.1 to 10% by weight based on the total weight of the shielding film.

EXPERIMENTAL EXAMPLE 4. Bone Regeneration Experiments around Bone Defects around the Implant

1) Animals and experimental materials

Ten quadrants of New Zealand rape with an average weight of 2.7 kg (2.5 to 3.0 kg) were used in the experiments of the present invention. This experiment was approved by the Animal Experimental Ethics Committee (No. 2009-NCT-006) of Wonju National University, Gangneung, Korea.

2) Operation method

An intramuscular injection of 0.4 ml of a combination of ketamine (100 mg / ml) (Ketara and 0.3 ml of xylazine (10 mg / kg body weight; Rompun; Bayer Korea) (general anesthesia).

The tibia area was shaved and disinfected with povidine-iodine.

Then, an incision was made in the periosteum. Sharp sub-periosteal dissection was performed to expose the tibial area.

At the tibia region, a bone defect of 3.0 mm in width and 5.0 mm in length was formed on each unilateral cortical bone.

The distance between the bone defect portions was 10.0 mm. Implant (diameter: 3.0 mm, length: 10.0 mm: MSP30103R, Osstem) was placed in each bone defect portion.

The periosteum and skin then filled the layer with 3-0 silk.

Gentamicin 1 mg / kg (Kookje Inc.) was injected intramuscularly three times a day for three days after surgery.

Each rabbit was individually housed and fed with feed and water. At the 8th week, ten rabbits were sacrificed and used for this experiment. For accurate identification of these rabbits, villanueva bone dyes were stained for 7 days.

After the dehydration process, the sample was imbedded in methylmethacylate resin.

The slice was then sliced along the long axis of the implant using a low speed diamond wheel saw.

Finally, the slice was ground to a thickness of 30 m. Then, a digital image of the section was photographed using a digital camera.

3) Experiment result

As shown in FIG. 5, when the shielding film containing 3% 4-hexylresorcinol and the remaining silk fibroin of the present invention was provided (Example 1), the amount of formation of lamellar bones of good quality Excellent results can be observed.

EXPERIMENTAL EXAMPLE 5. Comparison with general shielding film in skull defect model of rabbit

1) Animals and experimental materials

Ten 10-month old New Zealand razors weighing 2.7 kg (2.5-3.0 kg) were used in the experiments of the present invention.

The experiment of the present invention was approved by the Animal Experimental Ethics Committee (No. 2009-NCT-006) of Wonju National University, Gangneung National University.

2) Operation method

An intramuscular injection of 0.4 ml of a combination of ketamine (100 mg / ml) (Ketara and 0.3 ml of xylazine (10 mg / kg body weight; Rompun; Bayer Korea) (general anesthesia).

The cranial bone was shaved and disinfected with povidine-iodine. Then, an incision was made in the periosteum.

A sharp pericosteal dissection was performed to expose the parietal bone area.

Two bony defects (diameter: 8.0 mm) were formed in one. Twelve bone defects were obtained in six rabbits, which were divided into three groups. The first group consisted of a control group with no material attached, the second group received a shielding film as a commodity, (Example 2) consisting of 5% 4-hexyl resorcinol and the remaining silk fibroin.

Gentamicin 1 mg / kg (Kookje Inc.) was injected intramuscularly three times a day for 3 days after surgery. Each rabbit was individually housed and fed with feed and water. At week 2, the rabbits were sacrificed and analyzed by micro CT scans.

3) Experiment result

As can be seen from FIG. 6, in the case of the shielding film made of 5% 4-hexyl resorcinol and the rest silk fibroin of the present invention (Example 2), it was observed that the shielding film was not applied, Faster bone regeneration can be seen than in the group with shielding.

As can be seen from Fig. In the results of micro CT analysis, the volume of the new bone was 8.90 1.51 mm ^{3 in} the case of the control group to which no shielding film was applied, whereas in the case of the shielding film made of 5% 4-hexyl resorcinol and the remaining silk fibroin of the present invention 23.27 5.80 mm < ^{3 &} gt ;. The difference between the two groups was statistically significant (p = 0.014).

On the contrary, the volume of new bone was 11.43 2.46 mm ³ in the case of the commercialized shields, but there was no statistically significant difference (p> 0.05).

However, the difference between the two groups was statistically significant (p = 0.031) when comparing the shielding film of the present invention with the shielding film of 5% 4-hexylresorcinol and the remaining silk fibroin of the present invention (Example 2).

Accordingly, the present invention provides a dental shielding membrane that significantly improves antimicrobial activity, alkaline phosphatase activity in osteoblasts, and bone formation in bone defects.

The present invention has been described with reference to preferred embodiments and experimental examples, and those skilled in the art to which the present invention pertains within the essential technical scope of the present invention and other embodiments of the detailed description of the present invention Examples and experimental examples may be implemented. The scope of the present invention is defined by the appended claims, and all differences within the scope of the claims are to be construed as being included in the present invention.

Claims (7)

Consisting of 0.01 to 10% by weight of 4-hexyl resorcinol and 90 to 99.99% by weight of silk protein,
Dental shielding membrane composition. Including 4-hexylresorcinol and silk protein,
Dental shields. The method of claim 2,
The 4-hexyl resorcinol contains 0.01 to 10% by weight based on the total weight of the dental shielding film,
Dental shields. The method of claim 2,
0.01 to 10% by weight of the 4-hexyl resorcinol and 90 to 99.99% by weight of the silk protein,
Dental shields. The method according to any one of claims 2 to 4,
The silk protein is characterized in that the silk fibroin,
Dental shields. The method according to claim 2 or 3,
Biomaterials collagen, gelatin, chitin, chitosan, keratin, cellulose, fibronectin, elastin, fibrinogen, fibromodulin, laminin, tenacin, vitronectin, alginate, hyaluronic acid, silk based on the total weight of the dental shielding membrane Proteins and derivatives thereof, agarose, polylactic acid (PLA), polyglycolic acid (PGA), copolymers of polylactic acid and polyglycolic acid (PLGA), polycaprolactone (PCL), poly {Poly (ethylene oxide) terephthalate-co-butylene terephthalate} (PEOT / PBT), polyphosphoester (PPE), polyphosphazene (PPA), polyanhydride (PA), poly Ortho esters; POE}, pluronic 127, glycerin, poly (propylene fumarate) -diacrylate; PPF-DA} and polyethylene glycol diacrylate; PEG-DA} further comprises at least 30% by weight,
Dental shields. Removing sericin from silk protein to obtain silk fibroin;
Preparing a silk fibroin aqueous solution by dissolving the silk fibroin with an ethanol aqueous solution containing a salt and then removing the salt and ethanol;
Dissolving 4-hexyl resorcinol with ethanol to prepare a 4-hexyl resorcinol ethanol solution;
Preparing a dental shielding membrane composition by mixing the silk fibroin aqueous solution and 4-hexyl resorcinol ethanol solution; And
Pouring the dental shielding film composition in a flat container and dried to prepare a dental shielding film; comprising,
A method for manufacturing a dental shielding film.

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