KR20080078449A - Method for manufacturing bioactive apatite - Google Patents

Abstract

A bioactive chloride/hydroxy double structured apatite having capability of enabling a low crystalline carbonate to be formed on a surface of a hydroxy apatite by itself while the chloride apatite is dissolved during implanting of a chloride apatite into in vivo is provided, and a method for preparing the bioactive chloride/hydroxy double structured apatite is provided. A method for preparing a low crystalline carbonate/hydroxy double structured apatite comprises the steps of: (a) boiling a blood component-removed animal bone in deionized water to remove primarily fat and protein from the bone, and drying the bone; (b) crushing the dried bone, immersing the crushed bone into a volatile polar organic solvent to shake the crushed bone in the organic solvent, removing the organic solvent from the bone, and drying the bone; (c) treating the dried bone with an aqueous solution comprising chlorine ions to remove simultaneously protein from the bone and ion exchange carbonic acid ions presenting in carbonate apatite from a surface of the bone to a predetermined depth into the bone with chlorine ions; (d) heat-treating the ion exchange-completed bone at 900 to 1200 deg.C for 1 to 6 hours to simultaneously remove lipid and protein from the bone and obtain a chloride/hydroxy double structured apatite in which a chloride apatite is formed on a surface of a hydroxy apatite; and (e) immersing the obtained chloride/hydroxy double structured apatite into a solution that is supersaturated relative to the apatite to dissolve a chloride apatite layer on a surface of the chloride/hydroxy double structured apatite, and forming a low crystalline carbonate apatite crystal on the surface of the hydroxy apatite to obtain a low crystalline carbonate/hydroxy double structured apatite.

Description

Method for Manufacturing Bioactive Apatite

1 shows a scanning electron micrograph of a chlorinated / hydroxylated bistructured apatite powder according to the present invention (a: × 100 × magnification and b: × 1,000 × magnification).

Figure 2 shows the thin film XRD measurement results of the chloride / hydroxide double structure apatite powder prepared by the ion exchange process according to the present invention.

Figure 3 shows a scanning electron micrograph after immersion of the chlorinated / hydroxylated bistructured apatite powder in the pseudo-body fluid according to the present invention (a: 0 hours, b: 3 hours, c: 6 hours and d: 7 days , × 10,000 times magnified).

Figure 4 shows the results of the thin film XRD before and after the immersion of chlorine / hydroxide bi-structure apatite powder in accordance with the present invention for 7 days.

Figure 5 shows the results of FT-IR measurement before and after immersion in the physiological fluids of chlorine / hydroxide bi-structure apatite according to the present invention for 7 days.

Figure 6 shows a change in the calcium, phosphorus, pH and the ionization solubility product calculated on the basis of the chlorine / hydroxide bi-structure apatite powder in accordance with the present invention when deposited in the body fluid (a: Ca, b: P , c: pH and d: IAP).

FIG. 7 schematically shows the formation mechanism of the low crystalline apatite carbonate formed on the surface of the chlorine / hydraulic double structured apatite particles according to the present invention and deposited in the pseudo-body fluid.

Figure 8 shows a scanning electron micrograph of a chlorine / hydroxide bi-structure apatite heat treated at 1000 ° C for 3 hours after treatment of heterologous bone with aqueous sodium chloride solution according to the present invention (x1,000 times magnification).

Figure 9 shows a scanning electron micrograph after immersion of a heterogenous bone in an aqueous solution of sodium chloride in accordance with the present invention after immersing the chloride / hydroxide bi-structure apatite in a pseudo-body fluid for 7 days (x 1,000 times magnification) Picture).

Figure 10 shows a scanning electron micrograph of the powder after the xenograft treated with sodium hypochlorite according to the present invention several times using distilled water and then heat-treated at 1000 ℃ for 3 hours (x10,000 times magnified photo).

Figure 11 shows the scanning electron micrograph after immersing the xenograft with sodium hypochlorite according to the present invention after rinsing several times with distilled water several times, and immersing the powder heat-treated at 1000 ℃ for 7 days in the body fluids (× 10,000 times magnified picture).

FIG. 12 shows the XRD measurement results of the chlorine / hydraulic bistructure apatite prepared according to the present invention before washing in distilled water and after washing for 1 hour.

FIG. 13 shows a scanning electron microscope photograph of chlorine / hydroxylated bistructured apatite prepared according to the present invention in distilled water for 1 hour and then completely dried, and then immersed in pseudo fluid for 7 days (× 1,000 times magnified) ).

Figure 14 shows a scanning electron micrograph after x-ray bone was immersed in an aqueous sodium hydroxide solution and heat-treated at 1000 ℃ for 3 hours in accordance with the present invention (x100 times magnified photo).

Figure 15 shows the scanning electron micrograph after immersion of xenograft in aqueous sodium hydroxide solution for 3 hours after immersion in a sodium hydroxide aqueous solution according to the present invention, it was immersed in a pseudo-body fluid for 7 days (x10,000 times magnification).

Field of invention

The present invention relates to a bioactive apatite and a method for preparing the same, and more particularly, to treat a bone of an animal in an aqueous solution containing chlorine ions and then heat-treat it to obtain a chloride / hydroxylated bistructured apatite, wherein the chloride / hydroxylated bistructure Apatite is deposited in a solution supersaturated with apatite to dissolve the apatite chloride on the surface, and the supersaturation of the apatite in the solution is increased by the released calcium and hydroxide ions, so that the low crystalline apatite is formed on the surface of the apatite hydroxide. The present invention relates to a method for producing a bioactive apatite for obtaining a bi-hydroxylated apatite and a bioactive apatite prepared by the method.

Background of the Invention

Bone grafting substitutes (BGS) are the replacement of bone tissue defects due to various dental diseases or trauma, disease degeneration or loss of other tissues, thereby filling the space within the bone tissue and forming new bone. Implants used to promote the disease. In general, the best implants are known as autologous bones that are removed by transplanting other parts of their bones, but they require secondary surgery, are difficult to obtain as much as needed, are difficult to perform in general clinics, patients Pain and morbidity of the patient has the disadvantage that it may be serious.

Therefore, it has been used to transplant using various substitutes such as allogeneic bone, xenograft, or synthetic bone composed of hydroxide apatite, which is a component of bone. Commercially available bone graft substitutes are available in several forms, such as powders, gels, slurries / putty, tablets, chips, morsels and pellets, sticks, sheets and blocks. Possible advantages include: homogeneity, low risk of infection and disease, no pain and symptoms from harvesting bones of patients for transplantation, and small size limitations There are many differences from the structure, and there are various disadvantages such as slow tissue regeneration.

To solve these shortcomings, physicochemical treatment is applied to the bones of animals having a structure similar to that of human bones to remove organic substances, and to obtain only inorganic components that make up bones, which can be used for dental or orthopedic surgery. As a representative product, Bio-Oss ^(R) manufactured by Biomaterials Geistrich Co. ^, Ltd. may be mentioned.

In the method for producing the heterologous bone, the bone of the femur of a bovine animal is added to a solvent having a boiling point of 80 ° C. to 120 ° C. to remove fat, and then, ammonia or primary amine is added to remove proteins and organics to prepare bone minerals. Then, it is composed of a step of drying by heating for several hours at a high temperature of 250 ~ 600 ℃ (US Patent 5,167,961, US Patent 5,417,975).

On the other hand, cartilage is treated with sodium hypochlorite to selectively remove the collagen phase, but there are examples used to observe the residual cartilage structure (Broz, J. J et al., J. Mater. Sci. Mater. Med., 8: 395, 1997), the use of sodium hypochlorite to remove all proteins in bone mineral preparation has not been reported yet.

Conventional techniques related to this include 'manufacturing method of apatite hydroxide granules for biomaterials' (Korean Patent Registration No. 10-0498759), and the patent is efficient by preparing apatite hydroxide powder in an aqueous solution and filling material of bone defects. And it can be used as a drug carrier, implant surface treatment agent, there is a problem that the bioactivity is not expressed, the human compatibility is low. In addition, the preparation method of the 10-2005-0021297 bone graft substitute filed by the applicant is a low-temperature heat treatment after treating the bovine bone with sodium hypochlorite, thereby using a low crystalline apatite carbonate phase obtained from the bone without a large physical / chemical change This is a chemical inactivation of the prion protein that causes mad cow disease. However, since the prion protein is theoretically completely inactivated only after heat treatment at 900 ° C. or higher, it poses a slight risk of mad cow disease. have.

Accordingly, the present inventors have made diligent efforts to solve the shortcomings of the prior art. As a result, the bones of animals similar in structure to those of humans are ion-exchanged in an aqueous solution containing chlorine ions, and then heat-treated at a high temperature to provide a chlorine / hydroxide double structure type. When the apatite was formed and implanted in the body, the crystalline apatite layer was dissolved while the low crystalline apatite was formed on the surface of the apatite hydroxide present in the lower part of the apatite chloride layer, thereby improving bone conductivity. . In addition, by immersing the chloride / hydroxylated bistructured apatite in a supersaturated solution to the apatite in vitro, artificially coating a low crystalline apatite on the surface of the high crystalline apatite to confirm that bone conductivity is improved. The invention has been completed.

After all, the main object of the present invention is to provide a bioactive chloride / hydroxide bi-structure apatite and a method for producing a low-crystalline carbonate apatite itself on the surface of the hydroxyapatite while dissolving apatite chloride in the implant.

It is another object of the present invention to deposit peptides, proteins, fibrins, bone form-forming factors by depositing bioactive chloride / hydroxylated bistructured apatite in supersaturated solution to apatite in vitro to form low crystalline apatite on the surface of apatite hydroxide, The present invention provides a low crystalline carbonate / hydroxylated bistructure apatite and a method for preparing the same, which are easily attached to bone growth agents.

In order to achieve the above object, the present invention comprises the steps of (a) boiling the animal bone from which the blood component is removed in deionized water to remove fat and protein first, and drying; (b) pulverizing the dried bone, dipping the ground bone in a volatile polar organic solvent and shaking the mixture, and then removing and drying the organic solvent; (c) treating the dried bone with an aqueous solution containing chlorine ions to remove proteins and ion-exchanging carbonate ions present in the apatite to a predetermined thickness from the bone surface with chlorine ions; And (d) heat-treating the ion-exchanged bone at 900 to 1200 ° C. for 1 to 6 hours to remove lipids and proteins, and at the same time to obtain a chloride / hydroxylated bistructured apatite having an apatite chloride layer formed on the surface of the apatite hydroxide. It provides a method for producing a bioactive chloride / hydroxide bi-structure apatite comprising.

The present invention also comprises the steps of (a) boiling animal bones from which blood components have been removed in deionized water to first remove fats and proteins, and drying them; (b) pulverizing the dried bone, dipping the ground bone in a volatile polar organic solvent and shaking the mixture, and then removing and drying the organic solvent; (c) treating the dried bone with an aqueous solution containing chlorine ions to remove proteins and ion-exchanging carbonate ions present in the carbonate apatite up to a predetermined thickness from the bone surface with chlorine ions; (d) heat treating the ion-treated bone at 900 to 1200 ° C. for 1 to 6 hours to remove lipids and proteins, and at the same time to obtain a chloride / hydroxylated bistructured apatite having an apatite chloride layer formed on the surface of the apatite hydroxide; And (e) dipping the obtained chlorinated / hydroxylated bistructured apatite into a supersaturated solution for the apatite to dissolve the apatite chloride layer on the chlorinated / hydroxylated bistructured apatite surface and to form low crystalline apatite crystals on the apatite hydroxide surface. It provides a method for producing a low crystalline carbonate / hydroxide bi-structure apatite by forming to obtain a low crystalline carbonate / hydroxide bi-structure apatite.

In the present invention, the organic solvent may be characterized in that the chloroform / methanol mixed solvent.

In the present invention, the aqueous solution containing chlorine ions may be used sodium hypochlorite aqueous solution or sodium chloride aqueous solution, the concentration of the aqueous solution containing chlorine ion may be characterized in that 30 to 80% by weight.

In the present invention, the supersaturated solution for the apatite may be characterized in that the solubility product of the apatite is 5.5 × 10 ^-118 to 10 ^-90 .

The present invention also provides a bioactive chloride / hydroxide bistructured apatite prepared by the above method, wherein the interior is apatite hydroxide, and the apatite chloride is formed on the surface of the apatite, and the interior is apatite. Provided is a low crystalline carbonate / hydroxylated bistructured apatite in which low crystalline apatite is formed.

The present invention also provides a bone graft substitute containing the bioactive chloride / hydroxylated bistructured apatite and a bone graft substitute containing the low crystalline carbonate / hydroxylated bistructured apatite.

In the present invention, growth factors for promoting bone growth, peptides and proteins for promoting bone tissue formation, fibrin, bone morphogenesis factors, bone growth agents, chemotherapeutic agents, antibiotics, analgesics, bisphosphonates, strontium salts, fire It may be characterized in that it is formulated by adding one or more biologically active substances selected from the group consisting of anti-inflammatory, magnesium salt and sodium salt.

In the present invention, it is formulated by adding one or more chemical compounds selected from the group consisting of hyaluronic acid (hyaluronic acid), chondroitin sulfate, alginic acid, chitosan, collagen, hydroxyapatite, calcium carbonate, calcium phosphate, calcium sulfate and ceramics You can do

Hereinafter, the present invention will be described in detail.

One aspect of the present invention relates to a method for preparing a bioactive chloride / hydroxylated bistructured apatite and a bioactive chloride / hydroxylated bistructured apatite prepared by the above method.

In order to manufacture the bioactive chloride / hydroxylated bistructured apatite, first, the animal bones from which blood components have been removed are boiled in deionized water to first remove fat and protein and then dry. The dried bone is immersed in a volatile polar organic solvent and shaken, and the organic solvent is removed and dried. The organic solvent may be characterized by using a chloroform / methanol mixed solvent.

The bone is immersed in an aqueous solution containing chlorine ions for 1 hour to remove the remaining protein in the bone, and the primary ion exchange of carbonate ions of apatite carbonate present in the bone with chlorine ions. The aqueous solution containing chlorine ions can be prepared by dissolving all inorganic salts containing chlorine ions in water, it is preferable to use an aqueous sodium hypochlorite solution or an aqueous sodium chloride solution. The concentration of the aqueous solution containing chlorine ions is preferably 30 to 80% by weight. When the aqueous solution containing the chlorine ion is less than 30% by weight, the protein removal efficiency is lowered, when the weight is more than 80% by weight the yield is low. After several times, the process was ion exchanged for 12 hours to completely replace the carbonate ions in the apatite carbonate present from the bone surface to a certain thickness with chlorine ions, and dried at room temperature. At this time, the thickness of the layer in which carbonate ions are replaced with chlorine ions from the bone surface may be characterized in that less than 100㎛. After the ion exchange treatment is completed, the bone is heat-treated at 900 to 1200 ° C. for 1 to 6 hours to completely remove lipids and proteins, thereby obtaining a chloride / hydroxylated bistructured apatite having a constant thickness of apatite chloride layer formed on the surface of the apatite hydroxide. The form of the chlorinated / hydroxylated bistructured apatite may be selected from the group consisting of powder, tablets, chips, morsels, pellets, sticks, sheets, blocks and scaffolds. Can be.

Another aspect of the present invention relates to a method for preparing a low crystalline carbonate / hydroxylated bistructured apatite and a low crystalline carbonate / hydroxylated bistructured apatite prepared by the above method.

In order to prepare the low crystalline carbonate / hydroxylated bistructure apatite, first, animal bones from which blood components have been removed are boiled in deionized water to first remove fat and protein and then dry. The dried bone is immersed in a volatile polar organic solvent and shaken, and the organic solvent is removed and dried. The organic solvent may be characterized by using a chloroform / methanol mixed solvent.

The bone is immersed in an aqueous solution containing chlorine ions for 1 hour to remove the remaining protein in the bone, and the primary ion exchange of carbonate ions of apatite carbonate with chlorine ions. The aqueous solution containing chlorine ions can be prepared by dissolving all inorganic salts containing chlorine ions in water, it is preferable to use an aqueous sodium hypochlorite solution or an aqueous sodium chloride solution. The concentration of the aqueous solution containing chlorine ions is preferably 30 to 80% by weight. When the aqueous solution containing the chlorine ion is less than 30% by weight, the protein removal efficiency is lowered, when the weight is more than 80% by weight the yield is low. After several times, the process was ion exchanged for 12 hours to completely replace the carbonate ions in the apatite carbonate present from the bone surface to a certain thickness with chlorine ions, and dried at room temperature. At this time, the thickness of the layer in which carbonate ions are replaced with chlorine ions from the bone surface may be characterized in that less than 100㎛. After the ion exchange treatment is completed, the bone is heat-treated at 900 to 1200 ° C. for 1 to 6 hours to completely remove lipids and proteins, thereby obtaining a chloride / hydroxylated bistructured apatite having a constant thickness of apatite chloride layer formed on the surface of the apatite hydroxide. The chlorine / hydroxide bistructured apatite may be selected from the group consisting of powder, tablets, chips, morsels, pellets, sticks, sheets, blocks and scaffolds. have. When the heat-treated bone is immersed in a supersaturated solution of apatite, calcium and hydroxide ions are eluted while the apatite chloride layer on the surface of the chlorinated / hydroxylated bistructured apatite is dissolved, thereby increasing the ionization solubility of the apatite in the aqueous solution. Low crystalline apatite carbonate crystals are formed on the surface of the apatite hydroxide to obtain a low crystalline carbonate / hydroxylated bistructured apatite.

Another aspect of the invention relates to bone graft substitutes containing bioactive chloride / hydroxylated bistructured apatite and bone graft substitutes containing low crystalline carbonate / hydroxylated bistructured apatite.

Compositions for bone graft replacement are commonly used with the term "bone grafting substitute" and are materials for filling the space within bone tissue. Bone graft substitutes can be used in the form of putty, paste, moldable strips, blocks, chips, etc., using compression, compression, pressure contact, packing, compression, hardening, etc. It can be used in the form of powder, paste, tablets, pellets and the like, and can also be used as it is in powder form.

When formulated and used as a bone graft substitute as described above, it is preferable to use a biologically active substance, and the growth factor for promoting bone growth as the biologically active substance, peptides and proteins for inducing bone tissue formation, fibrin, Bone morphogenesis factors, bone growth agents, chemotherapeutic agents, antibiotics, analgesics, bisphosphonates, strontum salts, fluoride salts, magnesium salts, sodium salts and the like can be used.

The growth factors include bone morphogenic protein (BMP), platelet-derived growth factor (PDGF), transgenic growth factor (TGF-beta), insulin-like growth factor (IGF-I), IGF-II, and fibroblast growth factor (FGF). ) And BGDF-II (beta-2-microglobulin) can be used. As the bone tissue formation enhancing peptides and proteins, various peptides including RGD sequences and various proteins such as collagen and fibronogen may be used. As the bone morphogenesis factor, osteocalcin, headquarter alloprotein, bonesialo protein, osteogenin, BMP and the like can be used. The bone growth agent may be used without limitation as long as it is harmless to the human body and promotes bone growth, and may use nucleic acids that promote bone formation, antagonists of substances that inhibit bone formation, and the like.

Chemical additives used to formulate bone graft substitutes in the present invention include hyaluronic acid, chondroitin sulfate, alginic acid, chitosan, collagen, hydroxyapatite, calcium carbonate, calcium phosphate, calcium sulfate, ceramics, and the like. Depending on the type, it can be formulated in the form of gel, strip, powder, chip, pellet, tablet, paste, and the like.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Method for Producing Low Crystalline Carbonic Acid / Hydroxy Bistructure Apatite Powder Using Sodium Hypochlorite Aqueous Solution According to the Present Invention

[Pretreatment Process]

Bones obtained at the femur site of the animal were cut to a size of about 5 cm ³ using a bone cutter. The cut bone pieces were immersed in deionized water for 24 hours to remove blood components present in the bone. The bone flakes washed with deionized water were boiled for 72 hours while exchanging deionized water every 12 hours to remove lipids and proteins present in the bone. The bone fragment from which the fat and protein were first removed was completely dried in an oven at 60 ° C. for 24 hours, and then ground to a size of 1.5 mm or less using a grinder.

[Degreasing treatment process]

A solvent in which 20 ml of chloroform and methanol were mixed in a 1: 1 volume ratio was added per 1 g of the pulverized bone powder, followed by shaking for 24 hours at a rotational speed of 120 rpm to perform degreasing treatment. Deionized water was added at a rate of 50 g per 1 g of bone powder to remove the remaining solvent in the deionized bone powder, followed by shaking at 120 rpm for 12 hours to remove the remaining solvent in the powder. The flushing efficiency was improved by exchanging with ionized water. The washed bone powder was completely dried in an oven at 60 ℃.

[Ion exchange process]

40 g of dried bone powder degreased in 100 g of aqueous sodium hypochlorite solution is mixed and shaken at a rotational speed of 120 rpm for 1 hour to remove the protein present in the bone powder and at the same time chlorine ion of the apatite carbonate present in the bone. Primary ion exchange was performed. Thereafter, the same process was performed once more, and finally, 12 hours of ion exchange was performed under the same conditions to completely replace carbonate ions present in the carbonate apatite from the surface of the powder to chlorine ions and completely dry at room temperature.

[Heat treatment process]

After the ion-exchanged powder was heated to 2 ° C / min in an electric furnace, heat-treated at 1000 ° C or higher for 3 hours, and after the furnace cooling process, the remaining lipids and proteins in the powder were completely removed, and the apatite chloride layer was formed on the surface of the apatite hydroxide. Hydroxyl bistructured apatite powder was prepared.

[Sieving process]

The heat-treated powder is sieved using a sieve of 212 ~ 1000㎛ size, and use only those coming in 212 ~ 1000㎛ size.

[Low Crystalline Apatite Coating Process]

The chlorinated / hydroxylated bistructured apatite powder with sieving is obtained from pseudo fluids (Kokubo T, Kushitani H, Sakka S, Kitsugi T, Yamamuro T. Solutions able to reproduce in vivo surface-structure changes in bioactive glass-ceramic AW.J Biomed Mater Res 1990; 24 (6): 721-34) in other words, the ionization solubility enemy (ionic activity product) for the deposition of apatite in the solution is ^10-95 chloride apatite layer is calcium and hydroxide ions are released as soluble, the apatite As the ionization solubility was increased, a low crystalline apatite layer was formed on the surface of the apatite hydroxide powder to obtain a low crystalline carbonate / hydroxylated bistructured apatite.

As a result of analyzing the chlorinated / hydroxylated bistructured apatite powder prepared by the ion exchange process through a scanning electron microscope, abnormal grain growth of the apatite particles occurred, resulting in the development of hexagonal columnar particles having a size of 50 μm or more (FIG. One).

In addition, as a result of analyzing the surface layer of the chlorinated / hydraulic double structured apatite powder prepared by the ion exchange process by the thin film XRD, it was confirmed that the surface of the powder was composed mostly of the apatite chloride phase, and some of the apatite hydroxide phase. (FIG. 2).

The chlorine / hydroxylated bistructured apatite powder was deposited in pseudo fluid, and analyzed by scanning electron microscopy according to the deposition time. As a result, the particles present in the chlorinated / hydroxide bistructured apatite powder had a smooth surface before deposition (FIG. 3A). ), As the deposition time passes, the surface layer dissolves into irregularities (FIG. 3B), and after further time, small lobed crystals start to form in the surface layer (FIG. 3C), and the deposition occurs. On day 7, all particles were covered with nanometer-sized small apatite particles (Fig. 3d).

Before and after the deposition of the chlorinated / hydroxylated bistructured apatite powder in the pseudo fluids, the apatite chloride phase was observed to be converted to the apatite hydroxide phase after deposition. It can be seen that the particles are apatite hydroxide (FIG. 4).

The FT-IR was measured before and after immersion of the chlorinated / hydroxylated bistructured apatite in pseudo-body fluid for 7 days, and it was found that the carbonate group was not detected before deposition, and that after the deposition, the carbonate group was detected to form apatite. (Figure 5).

In combination with the thin film XRD results of FIG. 4 and the FT-IR results of FIG. 5, it was confirmed that the small particles formed in the body fluid were low crystalline apatite carbonate phases having chemical and physical properties similar to those of human bone.

When the chlorine / hydraulic double structured apatite powder was deposited in pseudo fluids, the results of analysis of the amount of calcium and phosphorus present in the pseudo fluids by time using ICP-AES are shown in FIGS. 6A and 6B. It is a result of measuring pH value, and FIG. 6D is a result of calculating the ionization solubility product with respect to the apatite in a pseudobody liquid based on these data. As shown in FIG. 6, when the chlorinated / hydroxylated bistructured apatite is deposited in pseudo fluid, the surface of the apatite chloride is dissolved to dissolve calcium and hydroxide ions, and phosphorus does not dissolve. It can be seen that the ionization solubility of apatite increases due to the elution of ions, resulting in the formation of low crystalline apatite carbonate on the surface.

FIG. 7 shows a schematic diagram of a chlorinated / hydroxylated bistructured apatite particle and a low crystalline apatite carbonate formed on the surface when it is immersed in the body fluid, and (a) shows carbonic acid present in the bone of a degreased animal. When apatite (CO ₃ Ap) is treated with sodium hypochlorite and ion exchanged and subjected to a high temperature heat treatment, apatite chloride (ClAp) is formed on the surface of apatite hydroxide (HAp) as shown in (b). Subsequently, it is again deposited in a supersaturated solution of apatite, and as a result of dissolving the apatite chloride layer on the surface, calcium and hydroxide ions are released, and calcium, phosphorus and carbonate ions present in the aqueous solution as shown in (d). Together they form new low crystalline apatite carbonate (CO ₃ Ap) on the surface of the apatite hydroxide (HAp).

Example 2 Preparation of Low Crystalline Carbonic Acid / Hydraulic Double Structure Apatite Powder Using Aqueous Sodium Chloride Solution

The same process as in Example 1 was carried out by replacing the aqueous sodium hypochlorite solution with an aqueous sodium chloride solution. The concentration of the aqueous sodium chloride solution was completely dissolved by adding 10 g of sodium chloride per 100 g of water, and after degreasing treatment, 20 g of bone powder having been washed with water was added to a mixed solution of sodium chloride, mixed at a speed of 120 rpm, and ion exchanged for 12 hours. Next, heat treatment at 1000 ° C. stably fixed the chloride ions in the apatite structure.

After analysis with a mixed solution of sodium chloride, the chlorinated / hydroxylated bistructured apatite heat-treated at 1000 ° C. was analyzed by scanning electron microscopy. As in the case of using sodium hypochlorite as a source of chlorine ion, apatite particles showed abnormal grain growth, resulting in 50 μm. It was observed that the above size (Fig. 8).

The chlorine / hydraulic bistructured apatite treated with the aqueous sodium chloride solution and then heat treated at 1000 ° C. was immersed in pseudo fluid for 7 days, and analyzed by scanning electron microscopy. It was confirmed that this was generated (Fig. 9).

Comparative Example 1 Apatite Powder Prepared by Sodium Hypochlorite Treatment and Water Treatment to Completely Remove Sodium Hypochlorite Components and Heat Treatment

[Pretreatment Process]

Bones obtained at the femur site of the animal were cut to a size of about 5 cm ³ using a bone cutter. The cut bone pieces were immersed in deionized water for 24 hours to remove blood components present in the bone. The bone flakes washed with deionized water were boiled for 72 hours while exchanging deionized water every 12 hours to remove lipids and proteins present in the bone. The bone fragment from which the fat and protein were first removed was completely dried in an oven at 60 ° C. for 24 hours, and then ground to a size of 1.5 mm or less using a grinder.

[Degreasing treatment process]

A solvent in which 20 ml of chloroform and methanol were mixed in a 1: 1 volume ratio was added per 1 g of the pulverized bone powder, followed by shaking for 24 hours at a rotational speed of 120 rpm to perform degreasing treatment. Deionized water was added at a rate of 50 g per 1 g of bone powder in order to remove the remaining solvent in the bone powder completed degreasing, and then shaken at 120 rpm for 12 hours to remove the solvent remaining in the powder. At this time, the washing efficiency was improved by replacing with fresh deionized water every two hours. The washed bone powder was completely dried in an oven at 60 ℃.

[Ion exchange process]

40 g of dried bone powder degreased in 100 g of aqueous sodium hypochlorite solution is mixed, shaken for 1 hour at a rotational speed of 120 rpm to remove the protein present in the bone powder, and the carbonate ions of the apatite carbonate are firstly converted into chlorine ions. Ion exchange. Thereafter, the same process was performed once more, followed by ion exchange under the same conditions for 12 hours to completely replace carbonate ions in the carbonate apatite from the surface of the powder to a predetermined thickness. Next, 50 g of deionized water was added per 1 g of bone powder to remove the sodium hypochlorite component present on the surface, and the remaining sodium hypochlorite was removed by shaking at 120 rpm for 72 hours, and every 2 hours for the first 12 hours. It was exchanged with fresh deionized water, after which it was replaced with fresh deionized water every 12 hours. The washed bone powder was completely dried in an oven at 60 ℃.

[Heat treatment process]

The dried bone powder after degreasing and deproteinization was heated to 2 ° C. per minute and heat-treated at 1000 ° C. for 3 hours.

As in the above process, after the sodium hypochlorite treatment was not directly heat-treated and removed by washing with water, and analyzed by a scanning electron microscope, the hexagonal columnar particles shown in Figure 1 was not found, the angle of about 1㎛ Only particles were observed (FIG. 10).

The hydroxyapatite granules shown in FIG. 10 were immersed in the pseudo-body fluid for one week and analyzed by scanning electron microscopy. As a result, low crystalline apatite carbonate crystals were not produced at all (FIG. 11).

Comparative Example 2 Production of Low Crystalline Apatite by Chlorinated Apatite Confirmation Experiment 1

The chlorinated / hydroxylated bistructured apatite prepared in Example 1 was washed with deionized water to dissolve the apatite chloride layer, and then it was confirmed that low crystalline apatite was produced even when deposited in pseudo fluid.

Chloride / hydraulic bistructured apatite was washed with deionized water for 1 hour and then washed with deionized water for thin film XRD. After washing in deionized water for 1 hour, no apatite chloride phase was found and only pure apatite phase was observed. . Therefore, it can be seen that the chlorinated / hydroxylated bistructured apatite prepared by the above method has only a surface layer and a double structure of hydroxide apatite (FIG. 12).

Chlorinated / hydroxylated bistructured apatite was immersed in distilled water for 1 hour, then completely dried, and again immersed in pseudobody fluid for 7 days, and then observed by scanning electron microscopy. No low crystalline apatite was produced on the surface. It can be seen from this that the ability to produce low crystalline apatite occurs from the dissolution of the apatite chloride layer (FIG. 13).

Comparative Example 3 Production of Low Crystalline Apatite by Chlorinated Apatite Confirmation Experiment 2

Comparative Example 3 to demonstrate that low crystalline apatite was not produced even when deposited in a supersaturated solution for apatite when treated as in Example 1 using a salt containing sodium ions but no chlorine ion. The experiment was performed. After dissolving 10 g of sodium hydroxide (NaOH) in 100 g of water and adding 20 g of bone powder, the microstructure did not generate abnormal grain growth as shown in FIG. In addition, low crystalline apatite carbonate was not produced at all even in the microstructure after immersion in the body fluid for 7 days (FIG. 15).

As shown in FIG. 14 and FIG. 15, it can be seen that in Comparative Example 3, not only apatite chloride was produced but also low crystalline apatite was not produced even in a solution supersaturated with the apatite.

Test Example 1 Evaluation of Specific Surface Area and Peptide Adsorption Efficiency

The specific surface area was measured using a BET tester using a low bone carbonate / hydroxylated bistructured apatite powder according to the present invention and a heterologous bone (Korean Patent No. 10-0635385) consisting of pure hydroxyapatite and low crystalline apatite. The adsorption amount of RGD peptide was measured and compared using a flurocytometer. The measurement results are as shown in Table 1 below.

Specific surface area and RGD peptide adsorption results Specific surface area RGD peptide adsorption amount Example 1 versus versus Example 2 versus versus Comparative Example 1 small small Comparative Example 2 small small Comparative Example 3 small small Low crystalline apatite carbonate bone (registered patent 10-0635385) medium medium

For quantitative determination of RGD peptides, a fluorescent material was attached to the RGD peptides and then nonspecifically adsorbed onto the apatite surface in PBS solution for 24 hours, and then separated by using a sonicator, and a predetermined amount of the supernatant of PBS solution was taken. Quantification was carried out using a flurocytometer.

As a result, it can be seen that the low crystalline carbonic acid / hydroxyl bistructured apatite powder according to the present invention has a specific surface area increased compared to the heterologous bone consisting of pure hydroxyapatite and low crystalline apatite carbonate, thereby increasing the adsorption efficiency of the peptide. .

As described above, when the bones of animals are ion-exchanged in an aqueous solution containing chlorine ions such as an aqueous sodium hypochlorite solution or an aqueous sodium chloride solution and subjected to high temperature heat treatment, the chloride / hydroxide double structured apatite layer is formed on the surface of the hydroxide apatite layer. Is obtained. When this is deposited in a solution supersaturated with apatite, that is, a pseudo-body fluid, the surface of the apatite chloride layer dissolves and calcium and hydroxide ions are eluted, and the ionization solubility of the apatite increases, resulting in the formation of low crystalline apatite on the surface of the apatite hydroxide. Low crystalline carbonate / hydroxylated bistructured apatite can be obtained.

However, it can be seen that low crystalline apatite is not produced in the body fluid when washed after heat treatment without direct heat treatment after sodium hypochlorite treatment. In addition, after washing the chlorinated / hydroxylated double structured apatite in distilled water, all of the surface apatite chloride phase was dissolved and not found at all, and only pure apatite phase was observed. It was not created either. It can be seen from this that the formation of low crystalline apatite carbonate is caused by the dissolution of the apatite chloride layer.

Having described the specific parts of the present invention in detail, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

As described in detail above, the present invention provides a bioactive apatite and a method for preparing the same, which facilitate the adsorption of peptides and proteins that induce bone tissue regeneration, increase bone conductivity, and improve human suitability. According to the present invention, carbonic acid ions of carbonate apatite existing in human bones and animal bones similar in structure and composition are ion-exchanged with chlorine ions in an aqueous solution containing chlorine ions, and then heat treated at high temperature to remove harmful fats from the human body. Effective removal of organics results in bioactive chloride / hydroxylated bistructured apatite that completely eliminates the risk of mad cow disease. Even when the chlorinated / hydroxylated bistructured apatite is directly implanted into the body without a low crystalline apatite carbonate, the apatite chloride layer dissolves in the blood and the supersaturation of the apatite increases, resulting in the formation of low crystalline apatite on the surface of the apatite. Has the effect of improving. In addition, depositing the chlorinated / hydroxylated bistructured apatite in a supersaturated solution to the apatite coats nanocrystalline low crystalline apatite carbonate particles with excellent bone conductivity to induce bone tissue formation enhancement, and provides a low crystalline apatite layer. Through this, there is an effect of increasing the adhesion efficiency of various peptides and proteins actively inducing bone tissue regeneration and improving bone conductivity.

Claims (15)

A method for preparing a bioactive chloride / hydroxylated bistructured apatite comprising the following steps: (a) boiling animal bones from which blood components have been removed in deionized water to first remove fats and proteins and to dry them; (b) pulverizing the dried bone, dipping the ground bone in a volatile polar organic solvent and shaking the mixture, and then removing and drying the organic solvent; (c) treating the dried bone with an aqueous solution containing chlorine ions to remove proteins and ion-exchanging carbonate ions present in the apatite to a predetermined thickness from the bone surface with chlorine ions; And (d) heat treating the ion-treated bone at 900 to 1200 ° C. for 1 to 6 hours to remove lipids and proteins, and at the same time to obtain a chloride / hydroxylated bistructured apatite in which an apatite chloride layer is formed on the surface of the apatite hydroxide. The method of claim 1, wherein the organic solvent is a chloroform / methanol mixed solvent. The method of claim 1, wherein the aqueous solution containing chlorine ions may use sodium hypochlorite or sodium chloride, and the concentration of the aqueous solution containing chlorine ions is 30 to 80% by weight. A bioactive chloride / hydroxylated double structured apatite prepared by the method of any one of claims 1 to 3, wherein the inside is apatite hydroxide, and the apatite chloride is formed on the surface of the apatite hydroxide. A bone graft substitute comprising the bioactive chloride / hydroxylated bistructured apatite of claim 4. According to claim 5, The bioactive chloride / hydroxide bi-structure apatite growth factor for promoting bone growth, peptides and proteins for promoting bone tissue formation, fibrin, bone morphogenesis factor, bone growth agent, chemotherapeutic agent, A bone graft substitute, which is formulated by adding at least one biologically active substance selected from the group consisting of antibiotics, analgesics, bisphosphonates, strontium salts, fluoride salts, magnesium salts and sodium salts. The method of claim 5, wherein the bioactive chloride / hydroxyl bistructured apatite is selected from the group consisting of hyaluronic acid, chondroitin sulfate, alginic acid, chitosan, collagen, hydroxyapatite, calcium carbonate, calcium phosphate, calcium sulfate, and ceramics. A bone graft substitute characterized in that it is formulated with the addition of one or more chemical compounds. A process for preparing a low crystalline carbonate / hydroxylated bistructure apatite comprising the following steps: (a) boiling animal bones from which blood components have been removed in deionized water to first remove fats and proteins and to dry them; (b) pulverizing the dried bone, dipping the ground bone in a volatile polar organic solvent and shaking the mixture, and then removing and drying the organic solvent; (c) treating the dried bone with an aqueous solution containing chlorine ions to remove proteins and ion-exchanging carbonate ions present in the apatite to a predetermined thickness from the bone surface with chlorine ions; (d) heat treating the ion-treated bone at 900 to 1200 ° C. for 1 to 6 hours to remove lipids and proteins, and at the same time to obtain a chloride / hydroxylated bistructured apatite having an apatite chloride layer formed on the surface of the apatite hydroxide; And (e) The obtained chloride / hydroxylated bistructured apatite is deposited in a supersaturated solution of apatite to dissolve the apatite chloride layer on the surface of the chloride / hydroxylated bistructured apatite to form low crystalline apatite carbonate crystals on the hydroxide apatite surface. To obtain a low crystalline carbonate / hydroxylated bistructured apatite. The method of claim 8, wherein the organic solvent is a chloroform / methanol mixed solvent. The method of claim 8, wherein the aqueous solution containing chlorine ions may use sodium hypochlorite or sodium chloride, and the concentration of the aqueous solution containing chlorine ions is 30 to 80% by weight. The method of claim 8, wherein the supersaturated solution for the apatite has a solubility product of 5.5 × 10 ⁻¹¹⁸ to 10 ⁻⁹⁰ for the apatite. A low crystalline carbonate / hydroxylated double structured apatite prepared by the method according to any one of claims 8 to 11, wherein the inside is apatite hydroxide, and low crystalline apatite is formed on the surface of the apatite hydroxide. A bone graft substitute comprising the low crystalline carbonate / hydroxylated bistructured apatite of claim 12. 15. The method of claim 13, wherein the low crystalline carbonic acid / hydroxide bi-structure apatite growth factor for promoting bone growth, peptides and proteins for promoting bone tissue formation, fibrin, bone morphogenesis factor, bone growth agent, chemotherapy agent Bone graft substitute, characterized in that the formulation of the addition of one or more biologically active substances selected from the group consisting of antibiotics, analgesics, bisphosphonates, strontium salts, fluoride salts, magnesium salts and sodium salts. The method of claim 13, wherein the low crystalline carbonate / hydroxylated bistructured apatite is selected from the group consisting of hyaluronic acid, chondroitin sulfate, alginic acid, chitosan, collagen, hydroxyapatite, calcium carbonate, calcium phosphate, calcium sulfate, and ceramics. A bone graft substitute characterized in that it is formulated by adding one or more selected chemical compounds.

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