KR102262049B1 - Bone graft material for regenerating periodontal tissue and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a graft material for periodontal tissue regeneration and a method for manufacturing the same, wherein the implant material for periodontal tissue regeneration according to the present invention uses gelatin derived from natural pig skin as a main raw material to exhibit biocompatibility and to reduce manufacturing cost. have.
In addition, the graft material for periodontal tissue regeneration according to the present invention is composed of a porous hydrogel in the form of a uniform lattice structure of a certain range of diameter, so that smooth blood movement is possible, thereby promoting bone formation, and adding bioactive and biodegradable bioceramic It has the effect of facilitating the activation and proliferation of bone cells necessary for initial bone formation.
Furthermore, since the core of the synthetic bone graft material is formed so that contraction due to hydration does not occur, side effects such as periodontal tissue depression and gum volume reduction after transplantation can be prevented, and hemostatic ability is improved as well as human teeth or human periodontal tissue It is manufactured in the same form as the one with the effect of easy transplantation.

Description

Bone graft material for regenerating periodontal tissue and manufacturing method thereof

The present invention relates to a graft material for periodontal tissue regeneration and a method for manufacturing the same, and more particularly, by using a porous hydrogel and bioactive and biodegradable bioceramic to be manufactured in the same form as human periodontal tissue, rapid bone regeneration and restoration effect It relates to a graft material for periodontal tissue regeneration showing and a manufacturing method thereof.

Transplant materials for periodontal tissue regeneration are used to restore tooth defects extracted due to various causes such as dental caries, periodontal necrosis, severe periodontal disease and orthodontics. It is used as a support to prevent the need for additional alveolar bone reconstruction surgery.

Currently used products mainly use collagen as the main raw material and have a plug form.

However, existing products use collagen, which is an expensive raw material, and not only have low price competitiveness, but also heal the granulation tissue quickly, but the strength of the generated tissue is weak, making it unsuitable for implant surgery. In addition, there is a problem in that it is difficult to form perfect bone because the defective periodontal bone is biodegraded before it is completely formed.

Accordingly, there is a need for the development of a graft material for periodontal tissue regeneration that can promote rapid tissue regeneration and alveolar bone formation while maintaining the strengths of existing products, such as hemostasis, pain relief, and infection prevention, while reducing manufacturing costs.

In this regard, Korean Patent Registration No. 10-1684268 discloses an absorbable periodontal tissue and bone regeneration inducing material using bacterial cellulose through irradiation technology, and Korean Patent Publication No. 10-2015-0053371 uses gelatin nanofibers. Although a graft material for periodontal tissue regeneration has been disclosed, there is a disadvantage that a process such as irradiation or spraying of a radiation solution is required.

Republic of Korea Patent Publication No. 10-1684268 Republic of Korea Patent Publication No. 10-2015-0053371

The present invention has been devised to solve the above problems, and an object of the present invention is to provide a graft material for periodontal tissue regeneration capable of promoting rapid tissue regeneration and alveolar bone formation while reducing manufacturing cost and a method for manufacturing the same.

In order to achieve the above object, the present invention

Preparing a first hydrogel solution by mixing a hyaluronic acid solution, a gelatin solution, and beta-tricalcium phosphate (β-TCP);

injecting the first hydrogel solution into a shell mold;

Freezing after positioning the core mold in the center of the shell mold into which the first hydrogel solution is injected;

removing the core mold and separating the frozen first hydrogel solution from the shell mold to obtain a support having an internal space;

preparing a second hydrogel solution by adding dual-phase calcium phosphate (BCP) particles to the first hydrogel solution;

Freezing after injecting the second hydrogel solution into the internal space of the support;

crosslinking the frozen support; and

It provides a method for producing a graft material for periodontal tissue regeneration comprising the step of freezing the cross-linked support.

In addition, the present invention comprises the steps of preparing a first hydrogel solution by mixing a hyaluronic acid solution, a gelatin solution, and beta-tricalcium phosphate (β-TCP);

preparing a second hydrogel solution by adding dual-phase calcium phosphate (BCP) particles to the first hydrogel solution;

injecting the first hydrogel solution into a shell mold;

After freezing the second hydrogel solution by injecting it into a mold for manufacturing a core, separating from the mold to obtain a core;

Freezing after positioning the core in the center of the shell mold into which the first hydrogel solution is injected;

obtaining a support by separating the frozen first hydrogel solution from a shell mold;

crosslinking the support; and

It provides a method for producing a graft material for periodontal tissue regeneration comprising the step of freezing the cross-linked support.

Furthermore, the present invention provides a core comprising hyaluronic acid, gelatin, beta-tricalcium phosphate (β-TCP) and dual-phase calcium phosphate (BCP) particles; and

Formed on the surface of the core, hyaluronic acid, gelatin, and beta-provides a graft material for periodontal tissue regeneration comprising a shell containing tricalcium phosphate (β-TCP).

The graft material for periodontal tissue regeneration according to the present invention has the effect of not only showing biocompatibility by using gelatin derived from natural pig skin as a main raw material, but also reducing the manufacturing cost.

In addition, the graft material for periodontal tissue regeneration according to the present invention is composed of a porous hydrogel in the form of a uniform lattice structure of a certain range of diameter, so that smooth blood movement is possible, thereby promoting bone formation, and adding bioactive and biodegradable bioceramics. It has the effect of facilitating the activation and proliferation of bone cells necessary for initial bone formation.

Furthermore, since the core of the synthetic bone graft material is formed so that contraction due to hydration does not occur, side effects such as periodontal tissue depression and gum volume decrease after transplantation can be prevented, hemostatic ability is improved, and the same shape as human periodontal tissue It is manufactured with the effect of easy transplantation.

1 is a schematic diagram of the manufacturing process of Example 1, ① preparing a gelatin solution and a hyaluronic acid solution, ② mixing a gelatin solution and a hyaluronic acid solution, ③ adding β-TCP to form a first hydrogel solution Step, ④ injecting the first hydrogel solution into the shell mold, ⑤ positioning the core mold in the center of the shell mold, ⑥ freezing step, ⑦ removing the core mold and separating the support from the shell mold, ⑧ first The steps of injecting the second hydrogel with BCP particles added to the hydrogel solution into the internal space of the support, ⑨ freezing the support, ⑩ freeze drying step, ⑪ crosslinking step, ⑫ freezing and freeze drying steps are sequentially shown. .
Figure 2 is a schematic diagram of the manufacturing process of Example 2, ① preparation step of gelatin solution and hyaluronic acid solution, ② mixing step of gelatin solution and hyaluronic acid solution, ③ adding β-TCP to form a first hydrogel solution Step, ④ injecting the first hydrogel solution into the shell mold, ⑤ placing the core made of the second hydrogel to which BCP particles are added in the center of the shell mold, ⑥ freezing step, ⑦ freeze drying step, ⑧ crosslinking It shows the binding step, ⑨ freezing and freeze drying steps sequentially.
3 is a reaction scheme showing the step-by-step binding process of the EDC-NHS crosslinking reaction.
4A shows SEM images of the internal structures of Example 1 and Comparative Example 1 according to Experimental Example 1.
5 is a result of analyzing the internal characteristics of Example 1 and Comparative Example 2 according to Experimental Example 2, A is an X-ray micro-computed tomography (μ-CT) photograph, B is an energy dispersive X-ray; Spectroscopy (Energy dispersive Spectroscopy, EDS) results, C is an X-ray diffraction analysis (X-ray diffraction, XRD) results, D is a scanning electron microscope (Scanning Electron Microscope, SEM) picture.
6 is a result of measuring solubility in water of Example 1 according to Experimental Example 3, ① before submersion, ② after 1 day, ③ after 2 days, ④ after observing the change in shape.
7 is a comparison of the exploded views of the graft material for periodontal tissue regeneration according to Experimental Example 4, where A is a photograph showing the change of the implant for periodontal tissue regeneration for each elapsed day, and B is a result graph showing the mass reduction rate.
8 is an in vivo efficacy evaluation result of the graft material for periodontal tissue regeneration according to Experimental Example 5, showing the H&E staining results 3 months after transplanting the periodontal tissue regeneration graft material into the thigh of a rabbit.
9 shows an example of the form of a graft material for periodontal tissue regeneration of the present invention, A is a graft material for periodontal tissue regeneration that mimics the shape of a molar, and B is a transplant material for periodontal tissue regeneration that mimics the shape of a premolar.

Hereinafter, the present invention will be described in detail. The terms or words used in the present specification and claims should not be construed as being limited to ordinary or dictionary meanings, and the inventor may properly define the concept of the term in order to best describe his invention. It should be interpreted as meaning and concept consistent with the technical idea of the present invention based on the principle that there is.

The present invention comprises the steps of preparing a first hydrogel solution by mixing a hyaluronic acid solution, a gelatin solution, and beta-tricalcium phosphate (β-TCP);

injecting the first hydrogel solution into a shell mold;

Freezing after positioning the core mold in the center of the shell mold into which the first hydrogel solution is injected;

removing the core mold and separating the frozen first hydrogel solution from the shell mold to obtain a support having an internal space;

preparing a second hydrogel solution by adding dual-phase calcium phosphate (BCP) particles to the first hydrogel solution;

Freezing after injecting the second hydrogel solution into the internal space of the support;

crosslinking the frozen support; and

It provides a method for producing a graft material for periodontal tissue regeneration comprising the step of freezing the cross-linked support.

Hereinafter, the manufacturing method according to the present invention will be described in detail step by step.

First, a first hydrogel solution is prepared by mixing a hyaluronic acid solution, a gelatin solution, and beta-tricalcium phosphate (β-TCP).

For example, the first hydrogel solution may be prepared by mixing a hyaluronic acid solution and a gelatin solution, and then adding beta-tricalcium phosphate (β-TCP).

As the hyaluronic acid solution and the gelatin solution, an aqueous solution of hyaluronic acid and an aqueous solution of gelatin prepared by dissolving hyaluronic acid and gelatin in distilled water, respectively, may be used.

The first hydrogel solution may contain 0.1 to 50 parts by weight of hyaluronic acid based on 100 parts by weight of gelatin.

Preferably, based on 100 parts by weight of gelatin, 1 to 30 parts by weight of hyaluronic acid may be included, and more preferably, 5 to 20 parts by weight of hyaluronic acid may be included with respect to 100 parts by weight of gelatin.

If the content of hyaluronic acid based on the gelatin is less than the above range, there is a problem in that the lattice-shaped pore structure is not implemented when the hydrogel is formed.

In addition, due to the increase in the content of gelatin, the shape of the graft material may be contracted and deformed, and it is difficult to hydrate the hydrogel, and there is a problem in that the effect of relieving pain is reduced because the sponge properties after hydration are reduced.

If the content of hyaluronic acid based on the gelatin exceeds the above range, hygroscopicity may increase and hydration may occur after transplantation or hemostatic ability may be reduced.

In addition, due to the decrease in the content of gelatin, it is difficult to implement a plug-type graft material, the durability of the product itself is very low, and the cross-linking effect is reduced, so that it is rapidly decomposed after hydration.

The first hydrogel solution may be characterized in that 0.1 to 30 wt% of beta-tricalcium phosphate (β-TCP) is included.

Preferably 0.5 to 20 wt% may be included, and more preferably 1 to 10 wt% may be included.

If the beta-tricalcium phosphate (β-TCP) content is included below the above range, the osteogenesis effect may be reduced, and if it exceeds the above range, it is difficult to uniformly mix with hydrogel and gelatin, as well as periodontal The internal pores of the shell for tissue regeneration may become non-uniform.

Therefore, the content range of the beta-tricalcium phosphate (β-TCP) corresponds to a content range in which new bone can be induced as much as possible and mixing can be facilitated.

The beta-tricalcium phosphate (β-Tricalcium phosphate, β-TCP) is a bioceramic and has a chemical composition similar to that of natural bone, and has excellent osteoconductivity, biodegradability and biocompatibility.

The beta-tricalcium phosphate (β-TCP) may be used in powder form.

Then, the prepared first hydrogel solution is injected into the shell mold.

The shell mold is for manufacturing a shell region of a graft material for periodontal tissue regeneration, and a human periodontal shape, a tooth shape, a bullet shape, a plug shape, etc. may be used in an intaglio shape.

For example, when using a shell mold in which the human periodontal shape (premolars) is engraved, those having a height of 5 to 20 mm and 10 to 30 mm in upper diameter may be used, preferably 10 to 15 mm in upper diameter and 20 Those having a height of ~ 30 mm can be used.

This is to manufacture the implant material for periodontal tissue regeneration in the same shape as the actual human periodontium, and the implantation material for periodontal tissue regeneration in the same shape as the human periodontium is easy to transplant, so that commercial convenience can be increased.

In consideration of the volume of the human periodontal tissue, the first hydrogel may be injected into the shell mold in a volume of 1 to 10 ml, preferably in a volume of 2 to 5 ml.

A core mold is placed in the center of the shell mold into which the first hydrogel solution is injected and frozen.

The core mold is for forming a core region of a graft material for periodontal tissue regeneration, and a mold having a embossed core shape or a structure having a core shape may be used.

The core shape may be a columnar shape such as a cylinder or a polygonal column, and preferably may be a columnar shape having a convex lower portion.

The ratio of the upper diameter of the core mold to the upper diameter of the shell mold may be 1: 2 to 5, preferably 1: 2 to 3.

A ratio of the upper diagonal length of the core mold to the upper diagonal length of the shell mold may be 1: 2 to 5, preferably 1: 2 to 3.

For example, a structure having a cylindrical shape with a convex lower portion may be used as the core mold, and a core mold having an upper diameter of 1 to 10 mm and a height of 5 to 10 mm may be used in consideration of the size of the core region.

The core mold may be made of the same material as the shell mold.

The core mold is frozen by being positioned at the center of the shell mold in which the first hydrogel solution is injected, specifically, the core mold is injected into the center of the upper surface of the shell mold in which the first hydrogel is injected, so that the lower part of the core mold is the shell mold It is placed so that it does not come into contact with the lower part of the

In this case, the upper portions of the shell mold and the core mold mean a portion having a maximum diameter (diagonal length).

In addition, the shell mold and the lower part of the core mold mean a portion formed to be concave (shell mold) or convex (core mold) due to a decrease in diameter (diagonal length).

The freezing may be made at a temperature of 0 ~ 30 ℃ for 0.1 ~ 5 hours, preferably at a temperature of 0 ~ 20 ℃ may be made for 0.5 ~ 3 hours, more preferably at a temperature of 0 ~ 10 ℃ 1 It can be done for ~2 hours.

The freezing temperature and time range correspond to the range required to remove the core mold located in the center of the shell mold after freezing while maintaining the shape to the extent that it does not collapse after freezing.

Then, the core mold positioned at the center of the shell mold is removed, and the frozen first hydrogel solution is separated from the shell mold to obtain a support having an internal space.

When the core mold is removed, an inner space is formed in a shape in which the shape of the core mold is depressed.

The inner space is a space for forming the core of the graft material for periodontal tissue regeneration, and the second hydrogel solution constituting the core is injected.

The second hydrogel solution is prepared by adding dual-phase calcium phosphate (BCP) particles to the first hydrogel solution.

The dual-phase calcium phosphate (BCP) particles are bioceramics in which hydroxyapatite (HAp) and tricalcium phosphate (TCP) are mixed, and have a chemical composition similar to that of natural bone, and have excellent biodegradability and biocompatibility. . In addition, it has bone conductivity and has the effect of promoting bone formation.

The second hydrogel solution may be characterized in that 0.1 to 30 wt% of dual-phase calcium phosphate (BCP) particles are included.

Preferably 1 to 20 wt% may be included, and more preferably 5 to 15 wt% may be included.

If the double-phase calcium phosphate (BCP) particles are less than the above range, there may be a problem in that the bone formation promoting effect and shape-retaining power are lowered, and if it exceeds the above range, the particles are distributed on the surface of the graft material and the gum tissue at the graft site is There may be a problem that it is not smoothly restored.

If it is out of the above range, problems such as reduced bone formation effect and reduced shape retention may occur.

Then, the second hydrogel solution is injected into the internal space of the support and then frozen, and the frozen support is cross-linked.

The second hydrogel may be injected into the internal space in a volume of 0.1 to 10 ml, preferably in a volume of 0.5 to 5 ml, in consideration of the volume ratio of the human periodontal tissue and the volume ratio of the shell region.

The injected second hydrogel forms a core, and may descend from the upper surface of the shell due to a weight difference according to the double-phase calcium phosphate (BCP) particles.

Accordingly, the finally prepared graft material for periodontal tissue regeneration is composed of a core and a shell surrounding it, and the shell may be formed to surround the surface of the core as a whole or the rest of the surface except for the upper surface of the core.

The upper surface of the core refers to a surface adjacent to the upper surface of the shell, and the upper portion of the shell refers to a portion having a maximum diameter or maximum diagonal length in a graft material for periodontal tissue regeneration.

The freezing may be made at a temperature of -10 ~ -100 ℃ for 1 to 36 hours, preferably at a temperature of -20 ~ -50 ℃ for 6 to 12 hours.

After the freezing process, it may be freeze-dried at a temperature of -50 ~ -200 ℃ for 12 to 72 hours to remove moisture, preferably freeze-dried at a temperature of -100 ~ -120 ℃ for 24 ~ 60 hours. have.

Since the frozen support itself is easily soluble in water, a reaction for crosslinking is required to maintain the shape of the support.

For the cross-linking, a cross-linking solution capable of cross-linking hyaluronic acid and gelatin may be treated.

For example, the cross-linking may be carried out by treating the frozen support with a 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) solution and cross-linking.

The treatment of the crosslinking solution is preferably performed at 1 to 10° C. for 12 to 36 hours.

Since the crosslinking solution such as EDC is a substance harmful to the human body, a washing process may be performed to remove it.

The washing process may be repeated 1 to 10 times for 5 to 30 minutes using distilled water (DI-Water), and preferably, the washing process for 10 to 20 minutes may be repeated 3 to 7 times. .

Finally, the cross-linked scaffold is frozen to obtain a final graft material for periodontal tissue regeneration.

The freezing may be made at a temperature of -10 ~ -100 ℃ for 1 to 36 hours, preferably at a temperature of -20 ~ -50 ℃ for 6 to 12 hours.

After the freezing process, it may be freeze-dried at a temperature of -50 ~ -200 ℃ for 12 to 72 hours to remove moisture, preferably freeze-dried at a temperature of -100 ~ -120 ℃ for 24 ~ 60 hours. have.

The final graft material for periodontal tissue regeneration consists of a core and a shell, and the shell is formed on the surface of the core, and may be formed to surround the entire surface of the core or to surround the rest of the surface except for the upper surface of the core.

The upper surface of the core refers to a surface adjacent to the upper surface of the shell, and the upper surface of the shell refers to a portion having a maximum diameter in a graft material for periodontal tissue regeneration.

In addition, the present invention comprises the steps of preparing a first hydrogel solution by mixing a hyaluronic acid solution, a gelatin solution, and beta-tricalcium phosphate (β-TCP);

preparing a second hydrogel solution by adding dual-phase calcium phosphate (BCP) particles to the first hydrogel solution;

injecting the first hydrogel solution into a shell mold;

After freezing the second hydrogel solution by injecting it into a mold for manufacturing a core, separating from the mold to obtain a core;

Freezing after positioning the core in the center of the shell mold into which the first hydrogel solution is injected;

obtaining a support by separating the frozen first hydrogel solution from a shell mold;

crosslinking the support; and

It provides a method for producing a graft material for periodontal tissue regeneration comprising the step of freezing the cross-linked support.

Here, the process of preparing the first hydrogel solution, the process of preparing the second hydrogel solution, and the process of injecting the first hydrogel solution into the shell mold are the same as described above.

Then, the second hydrogel solution is injected into a mold for core production and frozen, and then separated from the mold to obtain a core.

The mold for manufacturing the core is for forming the core to be injected into the implant for periodontal tissue regeneration, and a mold in which the shape of the core is formed in an intaglio may be used.

The core shape may be a columnar shape such as a cylinder or a polygonal column, and preferably may be a columnar shape having a convex lower portion.

The ratio of the upper diameter of the core to the upper diameter of the shell mold may be 1: 2 to 5, preferably 1: 2 to 3.

A ratio of the upper diagonal length of the core to the upper diagonal length of the shell mold may be 1:2-5, preferably 1:2-3.

As an example, when using a mold for manufacturing a core in which a cylindrical shape with a convex lower portion is formed in an intaglio, the core shape having an upper diameter of 1 to 10 mm and a height of 5 to 10 mm in consideration of the size of the core region is formed by intaglio Can be used.

The second hydrogel may be injected into the mold for core manufacturing in a volume of 0.1 to 10 ml, preferably in a volume of 0.5 to 5 ml, in consideration of the volume ratio of the human periodontal tissue and the volume ratio of the shell region.

The freezing may be made at a temperature of -10 ~ -100 ℃ for 1 to 36 hours, preferably at a temperature of -20 ~ -50 ℃ for 6 to 12 hours.

After the freezing process, the core obtained by separating from the mold may be freeze-dried for 12 to 72 hours at a temperature of -50 to -200 ℃ to remove moisture, preferably 24 to at a temperature of -100 to -120 ℃ It can be freeze-dried for 60 hours.

Next, the core is placed in the center of the shell mold into which the first hydrogel solution is injected and then frozen.

Specifically, the core is put into the center of the upper surface of the shell mold in which the first hydrogel is injected, the lower part of the core is positioned so as not to contact the lower part of the shell mold, and then is frozen.

In this case, the upper portions of the shell mold and the core mold mean a portion having a maximum diameter (diagonal length).

In addition, the shell mold and the lower part of the core mold mean a portion formed to be concave (shell mold) or convex (core mold) due to a decrease in diameter (diagonal length).

The core may descend from the upper surface of the shell due to the weight difference according to the dual-phase calcium phosphate (BCP) particles.

Accordingly, the finally prepared graft material for periodontal tissue regeneration is composed of a core and a shell surrounding it, and the shell may be formed to surround the entire surface of the core or the rest of the surface except for the upper surface of the core.

The upper surface of the core refers to a surface adjacent to the upper surface of the shell, and the upper portion of the shell refers to a portion having a maximum diameter or maximum diagonal length in a graft material for periodontal tissue regeneration.

The freezing may be made at a temperature of -10 ~ -100 ℃ for 1 to 36 hours, preferably at a temperature of -20 ~ -50 ℃ for 6 to 12 hours.

After the freezing process, it can be freeze-dried for 12 to 72 hours at a temperature of -50 to -200 ℃ to remove moisture, preferably freeze-dried at a temperature of -100 to -120 ℃ for 24 to 60 hours. have.

Then, the frozen first hydrogel solution is separated from the shell mold to obtain a support.

Since the frozen support itself is easily soluble in water, a reaction for cross-linking is required to induce the shape of the support, and the cross-linking reaction is the same as described above.

Finally, the cross-linked scaffold is frozen to obtain a final graft material for periodontal tissue regeneration.

The freezing may be made at a temperature of -10 ~ -100 ℃ for 1 to 36 hours, preferably at a temperature of -20 ~ -50 ℃ for 6 to 12 hours.

After the freezing process, it may be freeze-dried at a temperature of -50 ~ -200 ℃ for 12 to 72 hours to remove moisture, preferably freeze-dried at a temperature of -100 ~ -120 ℃ for 24 ~ 60 hours. have.

Furthermore, the present invention provides a core comprising hyaluronic acid, gelatin, beta-tricalcium phosphate (β-TCP) and dual-phase calcium phosphate (BCP) particles; and

Formed on the surface of the core, hyaluronic acid, gelatin, and beta-provides a graft material for periodontal tissue regeneration comprising a shell containing tricalcium phosphate (β-TCP).

The core is composed of a synthetic bone graft material to promote alveolar bone induction, and to prevent contraction due to hydration, thereby preventing side effects such as periodontal tissue depression and gum volume reduction after transplantation.

The core is formed inside the implant for periodontal tissue regeneration, and may be formed in a plug-shaped bullet shape, a flat surface on one side and a convex column shape on the other surface.

For example, when one side is flat and the other side is a convex cylindrical shape, it may have a diameter of 1 to 10 mm and a height of 5 to 10 mm, preferably a diameter of 2 to 8 mm and a height of 6 to 8 mm. can have

As another example, when one side is flat and the other side has a rectangular prism shape with convex corners rounded, the width of the square forming the flat side may be 1 to 10 mm, preferably 2 to 8 mm.

In addition, the height of the square pillar shape may be 5 to 10 mm, preferably 6 to 8 mm.

The shell is formed on the surface of the core, and may be formed to surround the entire surface of the core or to surround the rest of the surface except for the upper surface of the core.

The upper surface of the core refers to a surface adjacent to the upper surface of the shell, and the upper surface of the shell refers to a portion having a maximum diameter in a graft material for periodontal tissue regeneration.

The shell may have the same shape as human teeth or human periodontal tissue.

Here, the shell may have a diameter or a width length of 5 to 20 mm, preferably it may have a diameter or a width length of 10 to 20 mm.

In addition, it may have a height of 10 ~ 30 mm, preferably may have a height of 20 ~ 30 mm.

Furthermore, the shell is characterized in that it has a lattice structure consisting of pores having a diameter of 100 ~ 500um.

Specifically, the shell may have a lattice structure in which pores having a diameter of 100 to 500 μm are uniformly formed, preferably a lattice structure in which pores having a diameter of 100 to 400 μm are uniformly formed, and more preferably a lattice structure in which pores having a diameter of 100 to 400 μm are uniformly formed. It may have a lattice structure in which pores are uniformly formed.

The shell of the lattice structure has a structure similar to an extracellular matrix (ECM), and exhibits excellent performance in early osteoblast activation and differentiation by facilitating the movement of blood.

The maximum diameter of the core and the shell may have a ratio of 1: 2 to 5, preferably, a ratio of 1: 3 to 3.

In addition, the ratio of the diagonal length of the shell of the core may be 1: 2 to 5, preferably 1: 2-3.

The ratio has an effect that tissue restoration through the shell region and bone tissue regeneration effect and shape retention through the core region can be optimized.

The graft material for periodontal tissue regeneration is composed of a core and a shell surrounding it, and the shell may be formed to surround the entire surface of the core or to surround the rest of the surfaces except for the upper surface of the core.

The upper surface of the core refers to a surface adjacent to the upper surface of the shell, and the upper portion of the shell refers to a portion having a maximum diameter or a diagonal length in a graft material for periodontal tissue regeneration.

The graft material for periodontal tissue regeneration is formed in the same shape as human teeth or human periodontal tissue, thereby facilitating transplantation and increasing commercial convenience.

In addition, it exhibits an effect on periodontal tissue volume maintenance and bone tissue reconstruction as well as a rapid regeneration effect of periodontal tissue, and exhibits excellent hemostatic properties.

Furthermore, compared with the conventional graft material for periodontal tissue regeneration composed of collagen, there is an effect of improving the strength of periodontal tissue by significantly increasing bone cells after transplantation.

Hereinafter, examples and experimental examples will be described in detail to describe the present invention in detail. However, the embodiments according to the present invention may be modified in various other forms, and the scope of the present invention should not be construed as being limited to the embodiments described below. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Example 1> Preparation of a core-formed periodontal tissue regeneration graft material 1

Gelatin and hyaluronic acid were dissolved in water to prepare 7.5 wt% and 0.75 wt%, respectively, and the prepared gelatin solution and hyaluronic acid solution were mixed in a 1:1 volume ratio to prepare a hydrogel solution.

A first hydrogel solution was prepared by adding β-TCP to the hydrogel solution so as to be 4 wt%, and 2.836 ml of the prepared first hydrogel solution was molded in a shell mold.

As the shell mold, a mold having the same shape as the human periodontium (premolars) having an upper diameter of 15 mm and a height of 25 mm was formed in an engraved form.

After binding a convex cylindrical core mold with a diameter of 7.5 mm and a height of 16.5 mm to the center of the molded first hydrogel solution, it was frozen at a temperature of 8° C. for 1 hour.

Thereafter, the core mold was removed, and the frozen first hydrogel solution was separated from the mold to obtain a support having an internal space.

Then, a second hydrogel solution was prepared by adding BCP particles to the β-TCP-added hydrogel solution at a ratio of 12.5 wt%. 0.963 ml of the prepared second hydrogel solution was added to the inner space of the support and frozen at a temperature of -25°C for 8 hours, and then freeze-dried at a temperature of -110°C for 48 hours.

Next, a crosslinking solution was prepared by mixing 4.79 g of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) and 1.15 g of Nhydroxysulfosuccinimide (NHS) in 500 ml of 80 wt% ethanol.

The freeze-dried support was crosslinked by treating the EDC (1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide)/NHS (N-hydroxysuccinimide) crosslinking solution at a temperature of 4°C for 24 hours. Then, it was washed 5 times with distilled water (DI-Water) for 10 to 15 minutes.

Then, the cross-linked tissue was frozen at a temperature of -25°C for 8 hours, and then freeze-dried at a temperature of -110°C for 48 hours to prepare a graft material for periodontal tissue regeneration (see FIG. 1).

<Example 2> Preparation of a core-formed periodontal tissue regeneration graft material 2

Gelatin and hyaluronic acid were dissolved in water to prepare 7.5 wt% and 0.75 wt%, respectively, and the prepared gelatin solution and hyaluronic acid solution were mixed in a 1:1 volume ratio to prepare a hydrogel solution.

A first hydrogel solution was prepared by adding β-TCP to the hydrogel solution so as to be 4 wt%, and 2.836 ml of the prepared first hydrogel solution was molded in a shell mold.

As the shell mold, a mold having the same shape as the human periodontium (premolars) having an upper diameter of 15 mm and a height of 25 mm was formed in an engraved form.

A second hydrogel solution was prepared by adding dual-phase calcium phosphate (BCP) particles to the first hydrogel solution at a ratio of 12.5 wt%, and this was injected into a mold for core production and frozen at a temperature of -25°C for 8 hours. , separated from the mold to obtain a core.

As the mold for manufacturing the core, a mold in which a convex cylindrical shape having a diameter of 7.5 mm and a height of 16.5 mm was formed in a concave shape was used.

After placing the prepared core in the center of the shell mold in which the first hydrogel solution was molded, it was frozen at a temperature of -25° C. for 8 hours, and the frozen first hydrogel solution was separated from the shell mold to obtain a support.

The support was freeze-dried at a temperature of -110 °C for 48 hours.

Then, a crosslinking solution was prepared by mixing 4.79 g of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) and 1.15 g of Nhydroxysulfosuccinimide (NHS) in 500 ml of 80 wt% ethanol.

The freeze-dried support was cross-linked by treating the cross-linking solution at a temperature of 4° C. for 24 hours. Then, it was washed 5 times with distilled water (DI-Water) for 10 to 15 minutes.

Thereafter, the cross-linked tissue was frozen at a temperature of -25°C for 8 hours, and then freeze-dried at a temperature of -110°C for 48 hours to prepare a graft material for periodontal tissue regeneration (see FIG. 2 ).

<Comparative Example 1> Preparation of graft material for periodontal tissue regeneration composed of collagen

A commercially available porcine dermis extracted collagen component was molded in the same shell mold as in Example 1, and then frozen and freeze-dried under the same conditions to prepare a graft material for periodontal tissue regeneration.

<Comparative Example 2> Preparation of graft material for periodontal tissue regeneration without core

Gelatin and hyaluronic acid were dissolved in water to prepare 7.5 wt% and 0.75 wt%, respectively, and the prepared gelatin solution and hyaluronic acid solution were mixed in a 1:1 volume ratio to prepare a hydrogel solution.

A first hydrogel solution was prepared by adding β-TCP to the hydrogel solution so as to be 4 wt%, and 2.836 ml of the prepared first hydrogel solution was molded in a shell mold.

As the shell mold, a mold having the same shape as the human periodontium (premolars) having an upper diameter of 15 mm and a height of 25 mm was formed in an engraved form.

Thereafter, the shell mold was frozen at a temperature of -25°C for 8 hours, and then freeze-dried at a temperature of -110°C for 48 hours.

Next, a crosslinking solution was prepared by mixing 4.79 g of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) and 1.15 g of Nhydroxysulfosuccinimide (NHS) in 500 ml of 80 wt% ethanol.

The freeze-dried support was cross-linked by treating the cross-linking solution at a temperature of 4° C. for 24 hours.

Then, it was washed alternately 6 times with distilled water (DI-Water) at 37° C. and room temperature for 15 to 20 minutes. The washing effect is good at high temperature, but if the washing is continued at high temperature, the shape of the product collapses. Therefore, when washing, washing was performed alternately with distilled water at 37°C and distilled water at room temperature once.

The cross-linked paper body was frozen at a temperature of -25°C for 8 hours, and then freeze-dried at a temperature of -110°C for 48 hours.

<Experimental Example 1> Comparison of internal structure of graft material for periodontal tissue regeneration

(1) Experimental method

The internal structures of Example 1 and Comparative Example 1 were observed using a scanning electron microscope (SEM).

(2) Experimental results

The results of the above experiments are shown in FIG. 4 .

As shown, in the case of Example 1, it was confirmed that the internal structure of the shell region had a honeycomb-shaped structure as a whole, and pores having a diameter of 200 to 300 um were formed.

In particular, it was confirmed that the β-TCP particles were evenly distributed in the internal structure.

In comparison, Comparative Example 1 showed a form in which collagen was tangled.

Therefore, the graft material for periodontal tissue regeneration of Example 1 was thought to be effective for bone formation because it enables smooth blood movement through pores having a relatively uniform 200-300 μm diameter, and β-TCP particles are evenly distributed.

<Experimental Example 2> Analysis of internal characteristics of graft material for periodontal tissue regeneration

(1) Experimental method

In order to specifically analyze the internal properties of implants for periodontal tissue regeneration, X-ray micro-computed tomography (μ-CT), Energy dispersive Spectroscopy (EDS), X-ray diffraction Analysis (X-ray diffraction, XRD) and scanning electron microscope analysis (Scanning Electron Microscope, SEM) were performed for Examples 1 and 2.

(2) Experimental results

The results of the above experiments are shown in FIG. 5 .

As shown, in the case of Example 1, an inner core layer was formed, the results of component analysis were different from those of the outer hydrogel layer, and BCP particles distributed in the inner core layer were confirmed.

<Experimental Example 3> Measurement of solubility of graft material for periodontal tissue regeneration

(1) Experimental method

After the implant material for periodontal tissue regeneration prepared in Example 1 was immersed in 10 ml of water, it was placed in an oven at 37° C. and the shape change of the product was observed. The solution was replaced every 24 hours, and morphological changes were observed before immersion, after 1 day, after 2 days, and after 6 days, respectively.

(2) Experimental results

The results of the experiment are shown in FIG. 6 .

As shown, it was found that the graft material for periodontal tissue regeneration of Example 1 did not undergo morphological changes such as softening or spreading even after 6 days had elapsed.

<Experimental Example 4> Evaluation of decomposition of graft material for periodontal tissue regeneration

(1) Experimental method

For the evaluation of the decomposition degree of Example 1, Comparative Example 1 and Comparative Example 2, the resulting graft material was immersed in water, and then the mass reduction rate for 14 days was measured.

(2) Experimental results

The results of the above experiments are shown in FIG. 7 .

As shown, in the case of Comparative Example 1, the mass decreased by 100% after 7 days, but in the case of Examples 1 and 2, it was found that the mass reduction rate was less than 50% even after 14 days.

Therefore, it was confirmed that the graft material for periodontal tissue regeneration of Example 1 had a low degree of decomposition by moisture, so that it was possible to sufficiently secure time for bone formation.

<Experimental Example 5> Evaluation of the in vivo efficacy of a graft material for periodontal tissue regeneration

(1) Experimental method

The graft materials corresponding to Example 1, Comparative Example 1 and Comparative Example 2 were transplanted into the thigh of a rabbit for 3 months, and then the progress was analyzed through Hematoxyline and eosin (H&E) staining.

(2) Experimental results

The results of the above experiments are shown in FIG. 8 .

As shown, in the case of Comparative Example 1, it was confirmed that the inside was composed of chondrocytes, not osteocytes, and thus the strength of the tissue was significantly reduced.

Also, in Comparative Example 2, new bone formation was excellent, but it was confirmed that the volume of the graft site was reduced because the inner core, a component of the bone graft material, was not included. It was difficult to do.

In comparison, in the case of Example 1, the inner core of the bone graft material was formed along with excellent new bone formation, so volume maintenance was very excellent, and additional bone regeneration could be expected because the bone graft material existing in the inner core remained. there was.

Claims (10)

Preparing a first hydrogel solution by mixing a hyaluronic acid solution, a gelatin solution, and beta-tricalcium phosphate (β-TCP);
injecting the first hydrogel solution into a shell mold;
Freezing after positioning the core mold in the center of the shell mold into which the first hydrogel solution is injected;
removing the core mold and separating the frozen first hydrogel solution from the shell mold to obtain a support having an internal space;
preparing a second hydrogel solution by adding dual-phase calcium phosphate (BCP) particles to the first hydrogel solution;
Freezing after injecting the second hydrogel solution into the internal space of the support;
crosslinking the frozen support; and
freezing the cross-linked support;
The first hydrogel solution contains 1 to 10 wt% of beta-tricalcium phosphate (β-TCP),
The second hydrogel solution is a method of manufacturing a graft material for periodontal tissue regeneration, characterized in that the double-phase calcium phosphate (BCP) particles are included in 5 to 20 wt%.
According to claim 1,
The first hydrogel solution is a method of manufacturing a graft material for periodontal tissue regeneration, characterized in that 0.1 to 50 parts by weight of hyaluronic acid is included with respect to 100 parts by weight of gelatin.
delete delete Preparing a first hydrogel solution by mixing a hyaluronic acid solution, a gelatin solution, and beta-tricalcium phosphate (β-TCP);
preparing a second hydrogel solution by adding dual-phase calcium phosphate (BCP) particles to the first hydrogel solution;
injecting the first hydrogel solution into a shell mold;
After freezing the second hydrogel solution by injecting it into a mold for manufacturing a core, separating from the mold to obtain a core;
Freezing after positioning the core in the center of the shell mold into which the first hydrogel solution is injected;
obtaining a support by separating the frozen first hydrogel solution from a shell mold;
crosslinking the support; and
freezing the cross-linked support;
The first hydrogel solution contains 1 to 10 wt% of beta-tricalcium phosphate (β-TCP),
The second hydrogel solution is a method of manufacturing a graft material for periodontal tissue regeneration, characterized in that the double-phase calcium phosphate (BCP) particles are included in 5 to 20 wt%.
6. The method of claim 5,
The first hydrogel solution is a method of manufacturing a graft material for periodontal tissue regeneration, characterized in that 0.1 to 50 parts by weight of hyaluronic acid is included with respect to 100 parts by weight of gelatin.
delete delete a core comprising particles of hyaluronic acid, gelatin, beta-tricalcium phosphate (β-TCP) and dual phase calcium phosphate (BCP); and
It is formed on the surface of the core, and includes a shell comprising hyaluronic acid, gelatin, and beta-tricalcium phosphate (β-TCP),
The shell is formed by freezing a hydrogel solution containing 1 to 10 wt% of beta-tricalcium phosphate (β-TCP),
The core is a graft material for periodontal tissue regeneration, characterized in that the second hydrogel solution containing 5 to 20 wt% of dual phase calcium phosphate (BCP) particles is frozen and formed.
10. The method of claim 9,
The shell is a graft material for periodontal tissue regeneration, characterized in that the lattice structure consisting of pores with a diameter of 100 ~ 500um.

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