WO2009131323A2

WO2009131323A2 - Hyaluronic acid bone-filling complex and fabrication method thereof

Info

Publication number: WO2009131323A2
Application number: PCT/KR2009/001862
Authority: WO
Inventors: 박광범; 최석규; 양동준; 안현욱; 한세광; 염준석; 박정규; 오은주; 신홍인
Original assignee: 주식회사 메가젠임플란트; 포항공과대학교 산학협력단
Priority date: 2008-04-25
Filing date: 2009-04-10
Publication date: 2009-10-29
Also published as: KR101000491B1; KR20090112967A; WO2009131323A3

Abstract

The present invention relates to a hyaluronic acid bone-filling complex and a fabrication method thereof. The hyaluronic acid bone-filling complex according to the present invention includes a matrix containing hyaluronic acid derivatives and MGSB filling the matrix, and thus it is capable of guiding regeneration of alveolar bone in an effective manner as compared with conventional bone-filling substitutes, and maximizing guiding of bone regeneration by using a hyaluronic acid bone-filling complex containing a hyaluronic acid hydrogel in which a decomposing rate is adjusted in accordance with an in-vivo bone regeneration period.

Description

Hyaluronic Acid Bone Filling Complex and Manufacturing Method Thereof

The present invention relates to a hyaluronic acid bone filling complex and a method for producing the same. More specifically, the present invention relates to a bone filling material for artificial teeth and a method of manufacturing the same, which are considered to be the easiest commercialization among various applications of bone tissue engineering.

Recently, research on bone tissue engineering has been actively conducted worldwide. The World Health Authority (WHA) predicts the age of bones and joints from 2000 to 2010. It is expected to account for more than half of the disease among older patients.

In line with this trend, many developed countries in the United States, Europe, and Japan are intensively researching and developing bone tissue engineering in terms of welfare, socio-economics, and alternative medicine. Despite the historical necessity for the study of regeneration or reconstruction of damaged tissues, the level of bone tissue engineering research and development is still in its infancy, and the development of future bone tissue engineering research in Korea and the development of related industries are needed as a century leading technology.

When bone defects occur, there are generally two ways to treat them. Two methods are autologous bone grafts, in which bones are collected from one's own bones to fill bone defects, and heterogeneous bone grafts, which use bones or chemically synthesized bone substitutes to fill bone defects.

Autologous bone grafts are known to be the most efficient of the existing bone regeneration methods because they do not cause an immune response, but bone extraction is not easy and the amount available is very small. Necrosis of the tissue from which the bone was collected may also occur, or in severe cases, complications. In the case of xenograft transplantation, bone extraction can be performed a lot, but there is a possibility of delaying bone union due to immune response.

Recently, various kinds of bone fillers have been developed to complement the above two bone graft methods. The calcium phosphate-based compound, which is a constituent of bone, is used a lot. Even when such a bone filler is used, too much fibrous tissue is generated during the healing process, resulting in abnormal bone union and various sequelae after wound healing. . In order to solve these problems, studies are being actively conducted worldwide to allow bone regeneration to occur normally. For example, research has been conducted on how to promote bone regeneration by adding bone morphology protein (BMP) and using bone cells to actively regenerate bone regeneration.

However, since the price of BMP is very expensive and there is a risk of causing an immune response, it is not widely used. In addition, even when stem cells are used, problems such as the safety of stem cells and the need to suppress the immune response of proteins necessary for differentiating stem cells into osteoblasts should be solved. This requires a lot of effort and investment.

SUMMARY OF THE INVENTION An object of the present invention is to provide a hyaluronic acid bone filling complex and a method for producing the same, which have superior bone regeneration ability than the conventional bone filling complex without a great influence on the immune response and the human body.

According to the present invention, the hyaluronic acid bone filling complex includes a matrix containing the hyaluronic acid derivative and MGSB filled therein, which can effectively induce alveolar bone regeneration as compared to the conventional bone filling substitutes, and according to the bone regeneration period in the body The hyaluronic acid bone filling complex including hyaluronic acid hydrogel with controlled degradation rate can be maximized to induce bone regeneration.

1 is a photograph showing a scanning electron microscope (SEM) image of each bone filler ((A) is Bio-OSS®, (B) is MBCP, and (C) is MGSB).

2 is a graph analyzing the X-ray diffraction of each bone filler.

3 is a graph comparing the cell activation of each bone filler (sample without bone filler was used as a control).

4 is a schematic diagram illustrating a process of synthesizing a hydrogel using bis (sulfosuccinimidyl) suverrate of hyaluronic acid-adipicdihydrazide.

FIG. 5 is a schematic diagram illustrating a process of synthesizing hyaluronic acid-divinyl sulfone (HA-DVS) hydrogel. FIG.

FIG. 6 is a schematic diagram illustrating a process of synthesizing hyaluronic acid-citamine (HA-Cys) hydrogel. FIG.

FIG. 7 is a graph comparing the degree of degradation of hyaluronic acid-citamine (HA-Cys) hydrogel and hyaluronic acid-divinyl sulfone (HA-DVS) hydrogel according to each condition (here, (A) Hyaluronidase 90 U / ml, (B) Phosphate buffer solution at pH 6.2 containing 100 mM Glutathione (GSH)).

FIG. 8 shows the results of analysis after staining with Hematoxylin-Eosin (H & E) after 2, 4, and 8 weeks of bone regeneration when only Bio-OSS® was used as a bone filler.

9 is analyzed after staining with Hematoxylin-Eosin (H & E) after 2, 4, and 8 weeks of bone regeneration when using a hyaluronic acid bone filling complex in which Bio-OSS® is dispersed in a hyaluronic acid solution. One result.

10 is a result of analysis after staining with Hematoxylin-Eosin (H & E) after 2 weeks, 4 weeks, 8 weeks when the bone regeneration when using only MGSB as bone filler.

11 shows the results of analysis after staining with Hematoxylin-Eosin (H & E) after 2, 4, and 8 weeks of bone regeneration when the hyaluronic acid bone filling complex dispersed in MGSB in hyaluronic acid solution was used. .

Figure 12 shows the degree of bone regeneration when using the hyaluronic acid bone filling complex in which Bio-OSS® is dispersed in HA-ADH microhydrogel solution to Hematoxylin-Eosin (H & E) after 2, 4 and 8 weeks. The result is analyzed after staining.

FIG. 13 shows the degree of bone regeneration when using the hyaluronic acid bone filling complex in which Bio-OSS® is dispersed in HA-DVS microhydrogel solution with Hematoxylin-Eosin (H & E) after 2, 4, and 8 weeks. The result is analyzed after staining.

Figure 14 after staining the degree of bone regeneration when using the hyaluronic acid bone filling complex dispersed in MGSB in HA-Cys microhydrogel solution after 2 weeks, 4 weeks, 8 weeks after staining with Hematoxylin-Eosin (H & E) The result of the analysis.

The present invention provides a matrix comprising a hyaluronic acid derivative in the form of a hydrogel, and a hyaluronic acid bone filler complex including a calcium phosphate-based bone regeneration derivative filled in the matrix.

More specifically, hyaluronic acid (HA) is added to the highly compatible biocompatible calcium phosphate bone derivatives (MGSB) to promote bone regeneration by osteoconductive action of hyaluronic acid, and more. Furthermore, by using HA hydrogel with controlled degradation rate, HA can be continuously supplied from HA hydrogel decomposed during bone regeneration period, thereby providing a hyaluronic acid bone filling complex that can induce bone regeneration more effectively. .

The present invention also provides a method for preparing a hyaluronic acid bone filler complex comprising forming a matrix including a hydrogel derivative of a hyaluronic acid derivative, and filling the matrix with a calcium phosphate-based bone regeneration derivative.

According to the present invention, bone regeneration is promoted by osteoconductive action of hyaluronic acid by adding calcium phosphate bone regeneration derivative (MGSB, Megagen Co., Ltd.) having excellent biocompatibility to a matrix containing hyaluronic acid (HA). Furthermore, hyaluronic acid bone filling is used to induce bone regeneration more effectively by continuously supplying HA from the decomposed HA hydrogel during bone regeneration using HA hydrogel with controlled degradation rate. A composite and a method for producing the same can be provided.

The matrix acts as a physical barrier to separate the tissues from each other while the hyaluronic acid bone filling complex is introduced into the living body to regenerate bone tissue, thereby covering or covering the affected area or damage that may be adherent, and after a predetermined time, Dissociated within and replaced with regenerated bone tissue, the matrix comprising hyaluronic acid derivatives in hydrogel form. The matrix can have a mesh-like structure, for example.

In more detail, the matrix is made of a material obtained by reacting hyaluronic acid with suverrate. The suverate reacted with hyaluronic acid may be, for example, Bis [sulfosuccinimmidyl] suberate.

Hyaluronic acid is a kind of glucosamide glycan consisting of a disaccharide unit in which D-glucuronic acid and N-acetylglucosamine are linked by β (1 → 3) glycosidic bonds, as shown in the following formula (1). Hyaluronic acid has no species and no chemical or physical structure, and humans have a metabolic system and are the safest biomaterials in terms of immunity and toxicity. Hyaluronic acid can be produced in large quantities, for example by fermenting microorganisms, in which case there is no risk of viral contamination, allergic reactions, and can maintain a certain level of quality.

Formula 1

The term "hyaluronic acid" in the present invention is a concept including not only hyaluronic acid itself but also salts thereof. That is, "hyaluronic acid" in the present invention includes hyaluronic acid, a hyaluronic acid salt or a mixture of hyaluronic acid and hyaluronic acid. Hyaluronic acid salts include both inorganic salts such as sodium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronic acid and the like, and organic salts such as tetrabutylammonium hyaluronic acid and the like. In some cases, at least two combinations thereof may be used. Although the molecular weight of hyaluronic acid in this invention is not specifically limited, For example, it is preferable that it is 10,000-3,000,000.

Hyaluronic acid and suverate contained in the matrix are chemically covalently bonded. Therefore, hyaluronic acid derivatives in which hyaluronic acid and suverate are combined are not separated from each other by environmental changes such as salt concentration and pH change even after administration in vivo.

Such hyaluronic acid may be bound via a linker when combined with subvertate. The linker may be introduced into the hyaluronic acid by binding to the carbonyl group of the suverate and serves to regulate the degradation rate of the hyaluronic acid derivative in vivo. Hyaluronic acid derivatives are decomposed by hyaluronic acid, a hyaluronic acid degrading enzyme present in the body, by recognizing hyaluronic acid by a carboxyl group of hyaluronic acid. Therefore, the rate of decomposition of the hyaluronic acid derivative by hyalurodinase can be controlled by binding the linker to the carboxyl group of hyaluronic acid to control the number of exposure of the carboxyl group. For example, the higher the proportion of the linker introduced into hyaluronic acid, the lower the degradation rate of the hyaluronic acid derivative in vivo. Therefore, by controlling the introduction rate of the linker introduced into the hyaluronic acid, it is possible to control the decomposition rate of the hyaluronic acid derivative in the body as desired.

Such linkers may comprise at least two amino groups. The linker may be, for example, a dihydrazide compound, a diamine compound, a hydrazine compound comprising a plurality of amino groups. The linker is for example H ₂ N- (CH ₂ ) x-NH ₂ (where x is an integer from 0 to 10), H ₂ NNHCO- (CH ₂ ) -CH _2- (O-CH ₂ -CH ₂ ) is a diamine compound represented by x-NH ₂ (wherein x is an integer of 0 to 10), and H ₂ NNHCO- (CH ₂ ) x-CONHNH ₂ (wherein x is an integer of 1 to 10). represented dihydrazide compound, or NH (R ₁₎ -NH (R ₂₎ may be a hydrazine compound represented by (wherein, R ₁ and R ₂ are each a hydrogen atom or a C ₁ _-6 alkyl group).

The position at which the suverate or linker is introduced into the hyaluronic acid can be, for example, a carboxyl group of hyaluronic acid. When introduced to a carboxyl group, the subvertate or linker moiety can be introduced to the hyaluronic acid by forming an amide group, hydrazide group, diacylhydrazide group, carboxylic acid ester group. In the hyaluronic acid derivative formed by the introduction of a submerate or a linker to the hyaluronic acid by forming a carboxylic acid ester with the hyaluronic acid, the desorption of the suverate may occur in a relatively short time due to hydrolysis in the administration solution and in vivo. Therefore, it is preferable to introduce submerate into hyaluronic acid via a linker containing an amide group, a hydrazide group, or the like having a slow hydrolysis rate.

The matrix of the hyaluronic acid bone filling complex according to an embodiment of the present invention as described above may include a repeating structure of a unit represented by the following formula (2) in the molecule.

Formula 2

Wherein, R ₁ and R ₂ are each selected from hydrogen atoms, C ₁ _-6 alkyl group, R ₃ represents a single _{bond, - (CH 2) x-,} -CH 2 -CH 2 - (O-CH 2 -CH 2 ) x- or -NHCO- (CH ₂ ) y-CONH-, x is an integer from 1 to 10, and y is an integer from 0 to 10. In this case, preferably, R ₁ and R ₂ are each a hydrogen atom, and R ₃ may be -NHCO- (CH ₂ ) y-CONH-.

This matrix is filled with bone regeneration derivatives. The matrix may have, for example, a mesh-like network structure, and bone regeneration derivatives may be filled between the inside of the network structure and between the networks.

The bone regeneration derivative is a calcium phosphate-based bone regeneration derivative, may be a calcium phosphate-based compound derived from nature, or may be an artificially synthesized calcium phosphate-based compound, and is not particularly limited as long as it induces regeneration of bone tissue. Although, it is preferable to use MGSB (Megagen Synthetic Bone, Megagen Co., Ltd.).

The MGSB is biphasic calcium phosphate or calcium nitrate tetrahydrate and tribasic ammonium including tricalcium phosphate and hydroxyapatite. It is preferred that it is a chemical precipitate of phosphate (ammonium phosphate), and the formation of the matrix and the filling of bone regeneration derivatives can be carried out substantially in-situ.

The production method of the hyaluronic acid bone filling complex as described above will be described.

First, the carboxyl group of hyaluronic acid is activated. For example N, N'-carbonyldiimidazole (CDI), N, N'-dicyclohexylcarbonylimide (DCC), N-ethoxycarbonyl-2-ethoxy-1,2-di Hydroxynoline (EEDQ), 4- (4,6-dimethoxy-1,3,5-triazine) -4-methylmorpholium (DMT-MM), 2-benzotriazole-1,1,3, 3-tetramethyluronium tetrafluoroborate (TBTU), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HODhbt), benzotriazole-1-oxy-tris Pyrrolidino-phosphonium hexafluorophosphate (PyBOP), benzotriazol-1-yl-oxy-tris (dimethylamino) phosphonium hexafluorophosphate (BOP) or 1-ethyl-3- (3-dimethylamino Condensation agents, such as propyl) carbodiimide (EDC) or N-hydroxysuccinimide (NHS), are used alone or in combination as appropriate to active esterify the carboxyl group of hyaluronic acid.

Herein, a linker as described above containing a functional group reactive with active esters such as hydrazide groups and amino groups is introduced. In this case, the linker may be introduced into the carboxyl group of hyaluronic acid in a solution containing distilled water or at least one organic solvent. The organic solvent may be, for example, ethanol, and the proportion of the organic solvent in the total solution may be about 60% by volume or less, and preferably about 50% by volume.

Alternatively, the HA-DVS hydrogel can be synthesized by reacting hyaluronic acid with HA at pH 13 and 37 ℃ and then reacting with DVS (Divinylsulfone), or the carboxyl group of hyaluronic acid with EDC and 1-Hydroxybenzotriazole monohydrate (HOBt). ), And then HA-Cys hydrogel can be synthesized using Cystamine dihydrochloride.

Subsequently, a bone regenerated derivative, for example, a calcium phosphate compound is dispersed in a hyaluronic acid solution to which the linker is introduced, and then bis (sulfosuccinimidyl) suvrate is added to form a network structure including a hyaluronic acid derivative in the form of hydrogel. The hyaluronic acid bone filling complex in which the bone derivative is filled in the matrix can be completed.

The hyaluronic acid bone filling complex may be freeze-dried to make various formulations suitable for the purpose of use such as block, granule, or injection.

The hyaluronic acid bone filling complex according to the embodiment of the present invention as described above may be used, for example, as a dental bone filling material. That is, it is possible to induce the regeneration of the alveolar bone by filling in the damaged alveolar bone defect site.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the following examples are merely illustrative for the understanding of the present invention, and the protection scope of the present invention is not limited to the following examples.

EXAMPLE

Example 1: MGSB Synthesis

Calcium nitrate tetrahydrate and ammonium phosphate dibasic were reacted in distilled water at 80 ° C. for 1 hour and then filtered through a 0.2 μm filter. The filtered solution is dried in an environment of 80 ° C. for 12 hours. Finally the powder terminates the synthesis of MGSB by calcifying at 900 ° C. The produced MGSB was characterized by SEM and X-ray diffraction analysis, and the degree of cell activation was compared with other materials (MBCP, Bio-OSS®) using MC3T3-E1 extracted from the cranial canal of rats.

Experimental Example 1: Characterization of the prepared MGSB

The surface of the MGSB prepared in Example 1 was analyzed using SEM and X-ray diffraction. The results are shown in Figure 1 (SEM picture) and Figure 2 (X-ray diffraction pattern). The prepared MGSB powder, as shown in Figure 1 Bio-OSS®, compared to MBCP surface is very porous (porous) is easy to adsorb the cells, it can be seen that the uniform powder of 500 ㎛. In addition, it can be seen that the MGSB synthesized in FIG. 2 is closer to MBCP, which is a synthetic bone, than Bio-OSS®, which is a living bone (right bone). MGSB confirmed that Hydroxyapatite and β-TCP are the major constituents and are similar to Bio-OSS® biopsy bone.

Experimental Example 2: Analysis of Cell Activation of MGSB

MC3T3-E1 extracted from the cranial canal of rats was used to analyze the degree of activation of cells compared to other materials (MBCP, Bio-OSS®) (Fig. 3). The number of cells was counted after treatment by trypsin-EDTA after hemocytometer treatment. As a control, bone filler-free samples, Bio-OSS®, MBCP, and MGSB samples were prepared and cell cultures were performed on them for 3, 4 and 5 days. Cell activation in MGSB was much better than previously used Bio-OSS® and MBCP, and was similar to the control without bone filler.

Example 2: Hyaluronic Acid Hydrogel Synthesis

Experimental Example 3: HA-ADH Hydrogel Synthesis

A hyaluronic acid solution having a concentration of 5 mg / ml of hyaluronic acid (molecular weight 235,000, Lifecore, USA) in distilled water was prepared. Herein, 1-ethyl-3- [3- (dimethylamino) propyl] carbodiimide (EDC) (Tokyo Chemical Industry, TCI, Japan) was used to activate the carboxyl group of hyaluronic acid, followed by adipic acid dihydrazide. , ADH) (TCI, Japan) was added, and the mixture was reacted at room temperature for about 2 hours while maintaining the pH at about 4.8 with 1N hydrochloric acid. At this time, the addition amount of EDC and ADH was 4 times and 40 times in molar ratio with respect to the carboxyl group of hyaluronic acid, respectively.

The reaction product was dialyzed with 100 mM sodium chloride solution, 25% ethanol solution, distilled water, and lyophilized to obtain HA-ADH into which a hydrazide group was introduced. The introduction rate of hyaluronic acid was quantified by 1H-NMR (DPX300, Bruker, Germany) to confirm that about 70 mol% was introduced.

The HA-ADH thus obtained was prepared in a HA-ADH solution having a concentration of 40 mg / ml against Phosphate Buffered Saline (PBS, pH 7.4, Sigma-aldrich, USA). Bis (sulfosuccinimidyl) suverate was added here and mixed uniformly so that it might be about 20 mol% of the amine group of HA-ADH. The precursor solution was incubated at 37 ° C. for one hour to complete the crosslinking reaction to obtain a HA-ADH hydrogel as shown in FIG. 4.

Experimental Example 4 HA-DVS Hydrogel Synthesis

A hyaluronic acid solution having a concentration of hyaluronic acid (molecular weight 235,000, Lifecore, USA) to 0.2 M hydroxy sodium (pH 13.0) at 40 mg / ml was prepared. Divinylsulfone (DVS) was added and mixed uniformly while the hydroxyl group of hyaluronic acid was activated in an environment of pH 13.0. The precursor solution was incubated at 37 ° C. for one hour to complete the crosslinking reaction, resulting in a hydrogel as shown in FIG. 5. However, since the glucosidine bond of hyaluronic acid is broken at a high pH of pH 13, as soon as the hydrogel is made, dialysis was performed with PBS solution of pH 7.4 to stabilize the pH to 7.4 to obtain HA-DVS hydrogel.

Experimental Example 5 HA-Cys Hydrogel Synthesis

A hyaluronic acid solution having a concentration of hyaluronic acid (molecular weight 235,000, Lifecore, USA) with a concentration of 0.2 M Sodium Phosphate Dibasic (pH 8.5) of 40 mg / ml was prepared. EDC and HOBt were added to activate the carboxyl group of hyaluronic acid at pH 8.5, and then mixed with Cystamine (TCI, Japan). When the precursor solution is incubated at 37 ° C. for one hour to complete the crosslinking reaction, hydrogel is synthesized through the process as shown in FIG. 6. At this time, the addition amount of EDC, HOBt, and Cystamine was added one by one in molar ratio with respect to the carboxyl group of hyaluronic acid.

The reaction product was dialyzed with PBS solution of pH 7.4 to remove EDC and HOBt remaining after the reaction to obtain HA-Cys hydrogel.

Experimental Example 6: Preparation of Microhydrogel

In order to use hyaluronic acid hydrogel as a hyaluronic acid bone filling complex, each hyaluronic acid was pulverized by using a homogenizer (T-18 basic, IKA, Japan) at 8000 rpm for a hydrogel: PBS = 1: 3 ratio for 5 minutes. A microhydrogel of lonic acid derivative was obtained.

Experimental Example 7 Degradation of Hyaluronic Acid Hydrogel

In the study on the decomposition of HA-ADH hydrogel and HA-DVS hydrogel, the degradation rate of HA-ADH was very long (more than 6 months), so it would not be suitable for bone regeneration. HADVS hydrogel and HA-Cys hydro The degree of decomposition of the gel was compared. In the same manner as in Experimental Example 4 and Experimental Example 5, a cylindrical 100ul dose of hyaluronic acid hydrogel was synthesized in a 1 ml insulin syringe. The degree of degradation by a solution containing 90 U of Hyaluronidase and the degree of degradation by a solution containing 100 mM Glutathione (GSH) in 0.2 molar sodium phosphate buffer (pH 6.2) was determined by measuring the mass of the hydrogel over time. saw.

As shown in FIG. 7, the hydrogel decomposition by Hyaluronidase showed that HA-DVS and HA-Cys had almost the same pattern. However, glutathione (GSH), which serves to break disulfone bond (SS) groups, showed that HA-Cys hydrogels containing cystamine with disulfone groups were almost completely decomposed about 30 hours. DVS did not dissolve as expected because there was no disulfone. This is because HA-Cys hydrogel is degraded faster by hyaluronidase and GSH than other hydrogels in the body. Therefore, it was expected that bone regeneration could be performed by supplying hyaluronic acid necessary for bone regeneration.

Example 3: Bone Regeneration Experiment

Experimental Example 8: Evaluation of Hyaluronic Acid and Newly Prepared MGSB on Bone Regeneration

New Zealand rabbits (2 kg, male) were anesthetized intramuscularly with zoletil and lump (v / v = 1/1, 0.1 cc / kg), and then 9 mm in diameter with trephine bur (8 mm diameter) in the rabbit's skull. Drilled two by two. Each hole was filled with Bio-OSS®, MGSB with hyaluronic acid solution (1%, molecular weight 3,000,000, LG Life Sciences) added to Bio-OSS®, PBS. After 2, 4, and 8 weeks, respectively, rabbits were sacrificed to assess bone regeneration. The portion of the rabbit's skull used for bone regeneration was dissected and fixed in 10% formalin solution. After removing ash from 10% EDTA solution for 2 to 3 weeks, staining with Hematoxylin-Eosin (H & E) was performed for tissue analysis.

First, when the degree of bone regeneration of Bio-OSS® and MGSB was evaluated, in FIG. 8, in the case of Bio-OSS®, the thickness of the recovered bone tissue was thin due to the weak generation of new bone tissue, and the area covered by the bone tissue was narrow. In FIG. 10, in the case of MGSB, mature bone tissue was formed around the bone filler, and it was confirmed that osteogenic recovery proceeded effectively. In addition, when evaluating the effect of hyaluronic acid on bone regeneration, FIG. 9 confirms that filling with a mixture of Bio-OSS® and hyaluronic acid induces a significant osteogenic recovery by forming a thick bone plate compared with only Bio-OSS®. As shown in FIG. 11, the filling of MGSB with hyaluronic acid was confirmed that osteogenic recovery was better than that of MGSB. Therefore, it was concluded that the newly synthesized MGSB had superior bone regeneration effect than Bio-OSS®, and that bone regeneration was more active when hyaluronic acid was added.

Experimental Example 9 Evaluation of Bone Regeneration by Hyaluronic Acid Supply and Degradation Rate

Bone-damage tissue is made in the same manner with rabbits of the same kind as in Experimental Example 8. This hole was filled with hyaluronic acid microhydrogel (HA-DVS, HA-ADH) prepared in Experimental Example 6 and mixed with Bio-OSS®, and the degree of bone regeneration was evaluated.

In the case of Bio-OSS® containing HA-ADH hydrogel, it was confirmed that the osteogenic recovery was weak in FIG. 12, and in the case of using Bio-OSS® containing HA-DVS hydrogel, partial osteogenic recovery was observed. What is happening can be confirmed by FIG. However, Bio-OSS® containing hyaluronic acid solution shows excellent effect on osteogenic recovery, which means that the decomposition rate of hyaluronic acid and hydrogel is more than 6 months for HA-ADH hydrogel and HA-DVS. HA-ADH and HA because the supply of hyaluronic acid rarely occurred so that bone regeneration occurred because it was very slow under 6 months, and because there was little space for bone regeneration due to the adsorption of bone cells to the bone filler due to little decomposition. It was concluded that DVS hydrogel had little effect on bone regeneration.

Experimental Example 10 Evaluation of Bone Regeneration of MGSB Bone Filling Complex Including HA-Cys Hydrogel

After bone damage tissue was made in the same manner as in Experimental Example 8, the HA-Cys hydrogel prepared in Experimental Example 6 was filled with MGSB in the damaged area, and after 4 weeks of bone regeneration, the same method as in Experimental Example 8 was performed. Evaluation was made.

The bone filling complex including HA-Cys hydrogel was only seen after 4 weeks, but it can be seen in FIG. 14 that more effective bone recovery appeared than the MGSB bone filling complex including the hyaluronic acid solution of Experimental Example 6. Therefore, HA-Cys hydrogel can be concluded in agreement with Experimental Example 7 that the bone regeneration was secured in the gel state and then bone regeneration was effectively performed due to the rapid decomposition rate and continuous supply of hyaluronic acid. there was.

Claims

A matrix comprising a hyaluronic acid derivative in the form of a hydrogel, and a hyaluronic acid bone filler complex comprising a calcium phosphate-based bone regeneration derivative filled in the matrix.
The hyaluronic acid bone filler complex according to claim 1, wherein the bone regeneration derivative is a megagen synthetic bone filler (MGSB, manufactured by Megagen).
The method of claim 2, wherein the MGSB is biphasic calcium phosphate or calcium nitrate tetrahydrate including tricalcium phosphate and hydroxyapatite. Hyaluronic acid bone filler complex, a chemical precipitate of dibasic ammonium phosphate.
The hyaluronic acid bone filler complex according to claim 1, wherein the hyaluronic acid derivative has a molecular weight of 10,000 to 3,000,000.
The hyaluronic acid bone filler complex according to claim 1, wherein the hydrogel derivative of the hyaluronic acid is in the form of a micro hydrogel milled with a homogenizer.
The hyaluronic acid bone filler complex according to claim 1, wherein the hyaluronic acid derivative is crosslinked with divinyl sulfone (DVS) or cystamine.
The hyaluronic acid bone filler complex according to claim 1, wherein the hyaluronic acid in the hydrogel form is supplied with hyaluronic acid continuously over the bone regeneration period by controlling the rate of decomposition by changing a crosslinking agent, a degree of crosslinking and a molecular weight of hyaluronic acid.
The hyaluronic acid bone filler complex according to claim 1, wherein the matrix has a network structure.
Forming a matrix comprising a hyaluronic acid derivative in the form of a hydrogel; And

Method of producing a hyaluronic acid bone filler complex comprising the step of filling a bone regeneration derivative in the matrix.
The method for producing a hyaluronic acid bone filler complex according to claim 9, wherein the bone regeneration derivative is a megagen synthetic bone filler (MGSB, manufactured by Megagen).
11. The method of claim 10, wherein the MGSB is biphasic calcium phosphate or calcium nitrate tetrahydrate (Tricalcium phosphate) and hydroxyapatite (Hydroxyapatite) and calcium nitrate tetrahydrate (Calcium nitrate tetrahydrate) and A method for preparing a hyaluronic acid bone filler complex, which is a chemical precipitate of dibasic ammonium phosphate.
The method of claim 9, wherein the hyaluronic acid derivative has a molecular weight of 10,000 to 3,000,000.
The method of claim 9, wherein the hydrogel derivative of the hyaluronic acid is in the form of a micro hydrogel pulverized with a homogenizer.
The method of claim 9, wherein the hyaluronic acid derivative is cross-linked with divinyl sulfone (DVS) or cystamine.
10. The method of claim 9, wherein the hyaluronic acid in the form of hydrogel is a hyaluronic acid bone filler complex which is continuously supplied over the bone regeneration period by controlling the rate of decomposition by changing the crosslinking agent, the degree of crosslinking and the molecular weight of hyaluronic acid. .
10. The method of claim 9, wherein the matrix has a network structure.