KR101438745B1 - Method for Preparing Low Crystalline Ceramics Derived from Animal Bone - Google Patents

Abstract

The present invention provides a method for producing a low crystalline ceramic material from an animal bone. The low crystalline ceramic material of the present invention is biodegradable in the body, has a very high bone conduction ability, and can be safely applied to human body. The present invention also provides a low crystalline ceramic material produced by the method of the present invention, a bone graft material comprising the same, a bone support, and a biocompatible polymer.

Description

TECHNICAL FIELD The present invention relates to a method for manufacturing a low-crystalline ceramic material derived from animal bone,

To a method for producing a low-crystalline ceramic material derived from an animal bone.

The bone is most frequently implanted next to the blood and is widely used in a wide variety of fields such as various bone reconstruction, vertebral bone fusion, nonunion fracture, severe combined fracture, delayed union, arthrodesis, limb salvage, oral maxillofacial reconstruction and dental implant surgery It is also used for filling cystic defect with small size, good extraction window of corollas, and simple fracture. However, substitute materials are needed to fill the defect size or critical sized defect. Biocompatible ceramics are the most widely used biomaterials in dentistry, orthopedics, and plastic surgery for the purpose of regenerating and treating hard tissue bone defects and swollen parts even though biocompatibility and mechanical properties are superior to polymers. Ceramics can be roughly classified into oxidized and non-oxidized ceramics. Among the most widely used ceramics are calcium phosphate, vitrified glass, alumina, zirconia, and their composites. The calcium phosphate system is composed of hydroxyapatite (HA), tricalcium phosphate (TCP), tetracalcium phosphate (TTCP), dicalcium phosphate (DCP) Include silica-based glasses, phosphate-based glasses, and glass ceramics based on glass. Ceramics can be obtained from shells of human and animal bone, fish shells, shells and squid, or corals and egg shells, and ceramics collected from human and animal bone can be used for various purposes, .

The most ideal method of bone graft to date is grafting of autologous bone taken from the patient himself. Autogenous bone is superior to other bone graft materials in bone healing and osteogenesis of bone defects. However, autogenous bone grafting has drawbacks such as general anesthesia, need for additional surgery, pain at the harvested site, limitation of bone collection, etc. At present, the use of allografts, xenografts and synthetic bone substitutes for autogenous bone There is a trend.

Allografts are obtained from allogeneic carcasses or other surviving donors. They are obtained by freezing the graft material, lyophilization, lyophilization, lyophilization, high pressure sterilization, etc. to minimize the immune response do. It is known that the method of collecting allogeneic bone from humans is obtained from the femoral head and the carcass from the hip arthroplasty. However, when it is collected from the carcass, it is known that the bacterial infection rate is about 4 times as high as that obtained from living patients. Xenografts are bone graft materials that are expected to have bone conduction capability after freezing, lyophilization, freeze drying, and radiation sterilization after bone collection in animals such as cattle and pigs. However, allogeneic bone and xenogeneic bone are not 100% safe in terms of immune response or cross-infection. To overcome these shortcomings, synthetic bone is the most popular bone graft material. Synthetic bone (alloplast) is an artificial synthesis of inorganic matter, which is the main material of human bone. It has an advantage that it is economical and easily obtainable compared with allograft or heterogeneous bone. However, these materials are classified as a bone conduction material that facilitates the formation of bone trabeculae when bone is formed. However, since there is no bone type performance unlike the autogenous bone, the period required for bone formation is longer than that of autogenous bone, , And it has a disadvantage that it is significantly lower than that of heterogeneous bone.

Among bone graft materials, heterogeneous bone grafts account for 70-80% of total implants. The heterogeneous bone is not free from the recent mad cow disease, despite the advantage of easily obtaining bone tissue by collecting and transplanting bone tissue from a cattle or a calf. In addition, it has a disadvantage of slow absorption and low bone formation inducing ability. It is known that osteoid bone, which is widely used in clinical practice, has excellent bone conduction healing as a graft material by removing organic components through special treatment process. In addition, graft materials prepared by treating pig cavernous bone with a heterogeneous bone graft method are used alone or in combination with other bone grafts or other graft materials. However, until now there is no bone graft substitute for autogenous bone.

Over the past few years, the primary focus of ceramic materials has been to develop high porosity block or particle-shaped bone substitutes for interconnected pores as a medical rather than for the development of safer ceramic materials. (Flautre et al., J Mater Sci Mater Med , 12, 679-682 (2001)), the liquid nitrogen method (Hone et al ., J Mater Sci Mater Med, 22, 349-355 (2011)), the bubble-forming method (Li et al., J Biomed Mater Res, 61, 109-120 (2002)), the template polymer sponge method (Appleford et al. a, Biomaterials, 28, 4788-4794 (2007 )), ( prototyping method ((Rumpler et al. using Computer aided desing) system, J Biomed Mater Res, 81A, 40-50 (2007)) CAD , such as a porous ceramic Materials, and in recent years, bioactive materials made of organic-inorganic hybrid materials (or hybrids) combined with biodegradable polymers have been developed.

Until recently, biomedical ceramic raw materials and bone graft materials showed high crystallinity as hydroxyapatite produced by sintering process at a high temperature of 1200 ° C or higher, and grain growth occurred during the sintering process, Which is tens of times larger than the apatite present in. When the crystallinity is high and the particle size is large, biodegradation in the body is almost impossible, the bone conduction ability is very low, and it can not be decomposed by osteoclasts. Therefore, biomedical ceramic bone graft materials, which are safer to the human body and have superior bone formation effect, are more important than the currently used various ceramics.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made efforts to develop a ceramic material having low crystallinity and high bone forming ability. As a result, the inventors of the present invention have found that after obtaining cancellous bone from pig bone, removing fat and protein from cancellous bone, drying, and then heat-treating at a temperature of 300-500 ° C and pulverizing the cancellous bone, And by confirming various applications of the ceramic material, the present invention has been completed.

Accordingly, an object of the present invention is to provide a method for producing a low-crystalline ceramic material.

Another object of the present invention is to provide a ceramic material that has been refurbished by the above method.

It is still another object of the present invention to provide a bone graft material comprising the ceramic material.

It is still another object of the present invention to provide a bone support comprising the ceramic material.

It is still another object of the present invention to provide a composite material comprising the ceramic material and the biocompatible polymer.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention and claims.

According to one aspect of the present invention, the present invention provides a method of making a low crystallinity ceramic material comprising the steps of:

(a) cutting the animal bone;

(b) removing the cortical bone and blood from the cut animal bone to obtain a cancellous bone;

(c) boiling the cancellous bone in water and performing a first removal process of lipid and protein;

(d) drying the cancellous bone;

(e) heat treating the cancellous bone at 300-500 ° C; And

(f) powdering the heat treated cane bone to obtain a low crystalline ceramic material.

The present inventors have made efforts to develop a ceramic material having low crystallinity and high bone forming ability. As a result, the inventors of the present invention have found that after obtaining cancellous bone from pig bone, removing fat and protein from cancellous bone, drying, and then heat-treating at a temperature of 300-500 ° C and pulverizing the cancellous bone, And confirmed the possibility of various applications of the recycled ceramic material.

The basic technical idea of the method of the present invention is to provide a method for manufacturing a ceramic material having low biocompatibility, capable of biodegrading in the body, .

The present invention relates to a method for producing a low crystalline ceramic material and its use.

As used herein, the term " low crystallinity " means that the broader the width of the peak in the X-ray diffraction analysis, the lower the crystallinity. On the other hand, the narrower the width of the peak, the higher the crystallinity.

As a result of X-ray diffraction analysis, the total area of all peaks was small, and the width of each peak (diffraction line index) was broad as a whole (Table 2).

The method of the present invention will be described in detail in each step as follows:

(a) Cutting of animal bone

According to the present invention, animal bone is first cut.

The animal bone used in the present invention is preferably a pig bone, a bones or a horse bone, more preferably a pig bone.

The pig bone used in the present invention is preferably a hair bone, an arm bone, a leg bone, a rib, a dress bone, a vertebra bone, a sacral bone, a caudal bone or a tooth of a pig, more preferably an arm bone, a leg bone or a rib, More preferably an arm or leg bone, and most preferably a femur.

Animal bone cleavage can be accomplished through a variety of methods known in the art.

According to a preferred embodiment of the invention, the animal bone has a size of 0.5-5.0 cm ³ , more preferably 0.5-3.0 cm ³ , even more preferably 0.5-2.0 cm ³ , most preferably 1.0-1.5 cm ³ Cut it.

(b) obtaining cancellous bone from animal bone

Then, the cortical bone is removed from the cut animal bone. Cortical bone removal can be accomplished through a variety of methods known in the art.

The cortical bone is removed by immersing the cortical bone piece in the water. Preferably 12-72 hours, more preferably 12-48 hours, even more preferably 12-24 hours, most preferably 24 hours, in water to remove the blood components.

The amount of water to impregnate (impregnate) a piece of cancellous bone from which cortical bone has been removed is not particularly limited.

A cancellous bone from which cortical bone and blood have been removed from the cut animal bone can be obtained.

(c) Primary removal of fat and protein from cancellous bone

The resultant sponge bone of step (b) is put into water (impregnated) and boiled. In the present invention, boiling of the cancellous bone by impregnating the cancellous bone with water is performed in order to primarily remove the lipid and protein present in the cancellous bone.

According to a preferred embodiment of the present invention, the process of boiling cancellous bone is preferably carried out for 24-96 hours, more preferably 48-84 hours, even more preferably 60-72 hours, most preferably 72 hours .

(d) Drying of cancellous bone with primary removal of fat and protein

Next, the cancellous bone is first dried with the fat and protein removed.

The drying may be carried out by various methods known in the art, and most preferably by using an oven.

According to a preferred embodiment of the invention, drying is carried out in an oven at 60 DEG C for 24 hours.

According to a preferred embodiment of the present invention, a step of chemically removing lipids and proteins from the dried cancellous bone between steps (d) and (e) may be further included.

Removal of lipids and proteins can be accomplished through a variety of chemical removal methods known in the art.

According to a preferred embodiment of the present invention, removal of fat from dried cancellous bone is performed by treating the dried cancellous bone with a solvent (e.g., toluene, methylcyclohexane or chloroform and methanol solution) containing hydrocarbon. Preferably, the fat is removed by treatment with toluene or a solution of chloroform and methanol, and then the cancellous bone is dried in an oven.

According to a preferred embodiment of the present invention, the removal of the protein from the dried cancellous bone can be accomplished by removing a water soluble amine solvent (e.g., cyclohexylamine, ethanolamine, ethylenediamine) or sodium hypochlorite . The solvent may be used in admixture with water to a maximum extent of 50%. Preferably, the cancellous bone is treated with ethylene diamine or sodium hypochlorite to remove the protein, and then washed to remove residual solvent. The solution used for washing is water, methanol, ethanol, acetone and / or a mixed solution thereof, more preferably water. An ultrasonic cleaner can be used to enhance the cleaning effect.

Preferably, the washing is carried out by placing the cancellous bone in a container capable of continuously inflowing water and introducing water, more preferably from 1 to 8 days, even more preferably from 3 to 8 days, still more preferably from 5 to 6 Day. Washing is carried out at a water temperature of 10-60 ° C to prevent recrystallization of bone mineral.

Ethylene diamine solution is added to the spongy bone after washing (primary), and the protein present in the spongy bone is once again removed by heating. The process of adding and heating the ethylenediamine solution is preferably carried out at a temperature of 80 to 200 DEG C for 30 to 60 hours. The cancellous bone that has undergone the protein removal process is secondly washed with water to remove residual solvent. The second washing is preferably carried out for 5-20 days, more preferably 10-20 days, even more preferably 15-17 days. After the second washing, the piece of cancellous bone is dried in the oven.

(e) Heat treatment of dried cancellous bone

Heat dried cancellous bone.

According to a preferred embodiment of the present invention, the dried cancellous bone is heat-treated under dry heat at 300-500 ° C. More preferably 300-450 占 폚, even more preferably 350-450 占 폚, and most preferably 350-400 占 폚. The temperature is increased by 10 ° C per minute to reach the temperature used for the heat treatment. Preferably, the heat treatment is carried out for 10-30 hours, more preferably 15-30 hours, even more preferably 15-25 hours.

If no further chemical removal of lipids and proteins is carried out between steps (d) and (e), the heat treatment is carried out at 100-400 ° C for 10-48 hours, more preferably at 200-400 ° C for 20-48 hours , Still more preferably at 300-400 DEG C for 30-48 hours.

In the present invention, the heat treatment is performed at a temperature lower than a conventionally known heat treatment temperature (1200 ° C). The apatite present in the bone tissue has a high crystallinity while being subjected to a heat treatment at a high temperature. The ceramic material produced by the method of the present invention exhibits a low crystallinity due to a low temperature heat treatment (FIGS. Ceramic material with high crystallinity is almost impossible to biodegrade in the body, has very low bone conduction ability, and can not be degraded by osteoclasts, making it difficult to use it as bone graft material and bone support.

(f) pulverizing cancellous bone to obtain a low-crystalline ceramic material

The heat treated cancellous bone is pulverized to finally obtain a low crystalline ceramic material. The pulverization may be carried out through various pulverization methods known in the art.

The prepared low crystalline ceramic material has a Ca / P ratio of 1.6-1.8 At%, a specific surface area of 105-130 m ² / g and a BJH desorption dV / dlog (D) of 0.8-0.95 cm ³ / And has a pupil volume value.

According to the present invention, in addition to the low-crystalline ceramic material produced by the method of the present invention, an antibiotic / antimicrobial treatment or radiation (gamma ray) can be irradiated.

In the present invention, various antibiotics and antibacterial agents known in the art can be used as antibiotics and antibacterial agents. Preferred examples of the antioxidant include phencyclene, cephem, vancomycin, streptomycin, bacitracin, gramicidin, flavopolpol, polymnic, polyether, nitroimidazole, nitrofuran, quinolone, And may be selected from the group consisting of rifampicin, sulfomimide, trimethoprim, aminoglycoside, pennikol, rinkosamid, macrolide, streptoglamine, flurohmutilin and tetracycline.

According to the present invention, demineralization can be performed in addition to the low-crystalline ceramic material produced by the method of the present invention. The demineralization process of the present invention may use nitric acid, sulfuric acid, hydrochloric acid, phosphoric acid, or a mixture thereof, and may be used by immersing in the range of 5 to 80% by volume for 1 to 24 hours.

According to another aspect of the present invention, the present invention provides a process for the preparation of a composition comprising a Ca / P ratio of 1.6-1.8 At%, a specific surface area of 105-130 m ² / g and a BJH desorption dV / dlog of 0.8-0.95 cm ³ / (D) a low crystalline ceramic material having a pore volume value.

The low crystalline ceramic material of the present invention has a high calcium / phosphorus ratio compared to conventional ceramic materials and raw bone particles (Table 1). The calcium / phosphorus ratio is an important factor in cell adhesion and growth, and the high calcium / phosphorus ratio has a positive effect on bone formation.

The low crystalline ceramic material of the present invention has a higher specific surface area than the conventional ceramic material and raw bone particles (Fig. 5). The specific surface area is an area per unit volume, and as the specific surface area is larger, cell attachment is facilitated and osseointegration is promoted.

The low crystalline ceramic material of the present invention exhibits a BJH desorption dV / dlog (D) pore volume value (FIG. 6) higher than conventional ceramic materials and raw bone particles, Pore size and distribution, and is expected to promote osteogenesis as a result.

According to another aspect of the present invention, there is provided a bone graft material comprising the ceramic material of the present invention.

As used herein, the term " bone graft material " refers to a material that acts as a bridge for filling the spaces between the bones, and plays an important role in bone healing by immobilizing and stabilizing the bone by filling fractured portions or bone defects. . The bone graft material is used in orthopedics, neurosurgery and dentistry, and is used, for example, in bone defect sites during disc surgery to induce bone regeneration, and can be used for implant treatment and oral bone defect repair.

According to another aspect of the present invention, there is provided a bone support comprising the ceramic material of the present invention.

As used herein, the term " bone scaffold " refers to a biomaterial that provides a support for the attachment, differentiation, and proliferation and differentiation of cells that are sown in and out of the structure.

According to the present invention, the bone support of the present invention includes a bone shaft, a wedge, a ring, a bone strip, a bone block, a bone chip, But not limited to, bone particles, bone powder, and the like.

The ocular lipid of the present invention can be produced as a biodegradable support for tissue regeneration and wound or treatment of various diseases. The bone support of the present invention can be safely used in the human body than other heterogeneous bone and allogeneic bone-derived ceramics, and is more easily supplied than the autogenous bone-derived ceramics, and is more biocompatible than artificial synthetic ceramics.

According to another aspect of the present invention, there is provided a composite material comprising the ceramic material and the biocompatible polymer of the present invention.

As used herein, the term " biocompatible "means administered to a living body to not induce undesirable long-term effects.

The biocompatible polymer to be used in the present invention may be at least one selected from the group consisting of polydioxanone, polyglycolic acid, polylactic acid, polycaprolactone, lactic acid-glycolic acid copolymer, glycolic acid-trimethyl carbonate, glycolic acid-epsilon -caprolactone, polyglyconate , Polyglyactins, polyamino acids, polyanhydrides, polyorthoesters, mixtures thereof, and copolymers thereof; Protein derivatives such as collagen, gelatin, chitin / chitosan, alginate, albumin, hyaluronic acid, heparin, fibrinogen, celluloses, dextran, pectin, polylysine, polyethyleneimine, dexamethasone, chondroitin sulfate, lysozyme, DNA, RNA and RGD ; Lipids, growth factors, growth hormones, peptide drugs, protein drugs, antiinflammatory agents, anticancer agents, antiviral agents, sex hormones, antibiotics, antibacterial agents and compounds thereof; Collagen, albumin, cellulose, agarose, alginate, heparin, hyaluronic acid, chitosan.

The composite material of the present invention may include not only the ceramic material of the present invention, but also ceramic materials conventionally known in the art. The ceramic material of the present invention can control the size of the particles in various ways during the manufacturing process and can be used not only with the ceramic material derived from the pig bone, but also with the mixed bone, allogeneic bone, autogenous bone and artificial synthetic ceramics. The artificial synthetic ceramics may be calcium phosphate-based ceramics, life-like glass, alumina, zirconia, or a composite thereof.

The composite material of the present invention can be used as a support, joint, bone for hard tissue regeneration including artificial bone, artificial joint, bone cement, jaw bone, small bones of bone, limb bones, implants, abutments, fillers, ceramics, brackets, Fixed devices, spinal fixation devices, and composite shielding membranes.

When the composite material of the present invention is used as a biomedical material, it can be applied in any form, and examples thereof include a block, a film, a filament, a fiber, a mesh, a woven / nonwoven fabric, a knit, And the like, or a combination of two or more forms is possible.

The features and advantages of the present invention are summarized as follows:

(I) The present invention provides a method for producing a low crystallinity ceramic material from animal bone.

(Ii) The low crystalline ceramic material of the present invention is biodegradable in the body, has a high bone conduction ability, and can be safely applied to human body.

(Iii) The present invention provides a low crystalline ceramic material produced by the method of the present invention.

(Iv) The present invention provides a bone graft material and a bone support comprising the low-crystalline ceramic material of the present invention, the ceramic material of the present invention, and a composite material comprising the biocompatible polymer.

Figure 1 shows the results of a representative bone-derived product BioOss, ceramic particles (P350C) prepared from porcine bone according to the method 1 of the present invention and ceramic particles (B350C) prepared using bovine bone, The morphological characteristics of the ceramic particles (P350N) were observed with a scanning electron microscope (SEM).
FIG. 2 shows results of energy dispersive spectrometry (EDS) analysis of BioOss, P350C, B350C, P350N and calcium / phosphorus ratios on the surface of live bone without heat treatment.
Fig. 3 shows XRD (X-ray diffraction) results of BioOss, P350C, B350C, P350N and untreated porcine bone particles.
FIG. 4 is a TGA (thermogravimetry analyzer) result of BioOss, P350C, B350C, P350N and untreated porcine raw bone particles.
Figure 5 shows the results of the BET (Brunauer-Emmett-Teller) test of BioOss, P350C, B350C and P350N.
6 shows the results of measurement of pore size and distribution of BioOss, P350C, B350C, and P350N by the BJH (Barret-Joyner-Halenda) method.
7 shows XRD (X-ray diffraction) measurement results of porcelain bone-derived ceramic particles (P1200N) prepared at P350C, P350N and high temperature (1200 DEG C).
Figure 8 is a plain radiograph of the rat skull defect model at 4 and 8 weeks after bone grafting.
B350C: cow sponge graft heat treated at 350 ℃; P350C: Porcine sponge bone implant heat treated at 350 ° C.
FIG. 9 is a micro CT 3D photograph taken at 4 weeks and 8 weeks after bone graft in a rat skull defect model. FIG.
B350C: cow sponge graft heat treated at 350 ℃; P350C: Porcine sponge bone implant heat treated at 350 ° C.
10 is a median cross-sectional view of a micro CT 3D photograph taken at 4 and 8 weeks after bone graft in a rat skull defect model.
B350C: cow sponge graft heat treated at 350 ℃; P350C: Porcine sponge bone implant heat treated at 350 ° C.
11 is an SEM photograph of a porcine sponge bone piece heat-treated at 400 ° C.
a1-3: P400, b1-3: P1200. a1 and b1: x30, a2 and b2: x2,000, a3 and b3: x20,000
12 is X-ray diffraction data of a porcine spongy bone fragment heat-treated at 400 ° C.
13 is pore size distribution data of a porcine spongy bone fragment heat-treated at 400 ° C.
a: P400, b: P1200.
14 is a plain radiograph of the rat skull defect model at 4 and 8 weeks after bone graft.
b1: P400 group at the 8th week, c1: P1200 group at the 4th week (P400 group at the 4th week after the heat treatment at 1200 占 폚) Transplantation), c2: P1200 group at 8 weeks.
15 is a micro CT 3D photograph taken at 4 and 8 weeks after bone graft in a rat skull defect model.
b1: P400 group at the 4th week, b2: P400 group at the 8th week, c1: P1200 group at the 4th week, c2: P1200 group at the 8th week, a1: control group at the 4th week, a2: control group at the 8th week.
FIG. 16 is a photograph of a rat skull defect model in which a skull defect area is stained at 4 weeks after bone grafting.
a1-3: Control group, b1-3: P400 group, c1-3: P1200 group, Arrow: Edge of defected area, asterisk: Suture material.
The samples were stained with H & E (a1, b1 and c1; × 10, a2-3, b2-3 and c2-3; × 100, a2, b2 and c2; marginal edge, a3, b3 and c3;
FIG. 17 is a photograph of a skull defect model at the eighth week after bone graft in a rat skull defect model. FIG.
a1-3: Control group, b1-3: P400 group, c1-3: P1200 group, Arrow: Edge of defected area, asterisk: Suture material.
The samples were stained with H & E (a1, b1 and c1; × 10, a2-3, b2-3 and c2-3; × 100, a2, b2 and c2; marginal edge, a3, b3 and c3;

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

1: Preparation of Low Crystalline Ceramic Material Removing Organics from Porcine Bone (350 ℃ Heat Treatment)

1-1. Pretreatment process

The bone obtained from the femoral region of the pig was cut to a size of about 1-1.5 cm ³ using a short-term fracture, and the cortical bone was removed. Since the cortical bone in the thigh of the pig is distinguishable from the cancellous bone and the cancellous bone, it is simply removed using a short-term fracture. The piece of cancellous bone obtained by cutting was immersed in distilled water for 24 hours to remove blood components present in the cancellous bone. The pieces of cancellous bone washed with the distilled water were replaced with distilled water every 24 hours and boiled for 72 hours to primarily remove the lipids and proteins present in the cancellous bone. At this time, the amount of distilled water used for washing and primary removal of fat and protein was 4 times the volume of bone used. The cancellous bone fragments, from which the fat and protein were removed, were completely dried in an oven at 60 DEG C for 24 hours.

1-2. Degreasing process

The pretreated cancellous bone pieces were degreased for 72 hours using a toluene solvent and a Soxhlet apparatus designed for degreasing treatment. The degreased bone fragments were completely dried in an oven at 80 ° C.

1-3. Deproteinization process

1,000 ml of a 99% ethylene diamine solution and 50 ml distilled water were added to 600 g of the cancellous bone after the degreasing treatment (80 ml of ethylenediamine solution and 50 ml of distilled water were added) The preferred temperature was 115 [deg.] C, and the upper temperature limit was 200 [deg.] C) to remove proteins present in the cancellous bone fragments for 50 hours. In order to remove the solvent remaining on the decolorized cancellous bone pieces, the cancellous bone pieces were placed in a cylindrical glass container with grooves at the bottom and continuously washed with distilled water for 6 days. 1,000 ml of a 99% ethylene diamine solution was added per 600 g of the cancellous bone piece subjected to the first washing, and the protein present in the cancellous bone piece was removed by heating at 115 ° C or higher for 50 hours. To remove the solvent remaining in the bone powder after deproteinization, a piece of cancellous bone was placed in a cylindrical glass container, and distilled water was continuously infused and washed for 17 days. After the second washing, the cancellous bone pieces were completely dried in an oven at 160 ° C.

1-4. Heat treatment process

Dehydrated and deproteinized pieces of dried cancellous bone were heat - treated to remove residual lipids and proteins. The cancellous bone pieces were heated at a rate of 10 ° C per minute and then heat-treated at 350 ° C for 20 hours and then cooled.

1-5. Crushing and sieving processes

After the heat treatment was completed, the mixture was pulverized using a cancellous bone fragments, and the mixture was sieved using a sieve having a size of 300-1,000 mu m and used as a bone graft material.

2: Manufacture of Low-Crystalline Ceramic Material Containing Organic Material Using Porcine Bone

2-1. Pretreatment process

Bones taken from the thigh region of the pigs were cut to about 1-1.5 cm ³ using a short-term fracture and the cortical bone was removed. The piece of cancellous bone obtained by cutting was immersed in distilled water for 24 hours to remove blood components present in the cancellous bone. The pieces of cancellous bone washed with the distilled water were replaced with distilled water every 24 hours and boiled for 72 hours to primarily remove the lipids and proteins present in the cancellous bone. The cancellous bone fragments, from which the fat and protein were removed, were completely dried in an oven at 60 DEG C for 24 hours.

2-2. Heat treatment process

The electric furnace used for the heat treatment was heated to 10 ° C per minute, and the cancellous bone pieces were heat treated at 350 ° C for 20 hours and then cooled.

2-3. Crushing and sieving processes

After the heat treatment was completed, the mixture was pulverized using a cancellous bone fragments, and the mixture was sieved using a sieve having a size of 300-1000 μm and used as a bone graft material.

3: Manufacture of Bone Ceramic Particles / PLGA Polymer Granules

The bone ceramic particles were prepared in the same manner as in Method 1. 1 g of PLGA (poly D, L-lactic-co-glycolic acid) polymer was dissolved in 5 mL of dichloromethane, 500 mg of porcine bone ceramic particles were added thereto, and the mixture was uniformly dispersed with a stirrer. Subsequently, the granules were immersed in liquid nitrogen to solidify them, and then lyophilized to prepare porous PLGA composite granules containing porcine bone ceramic particles.

4: Pig bone ceramics particle / PGA polymer composite granule manufacture

The bone ceramic particles were prepared in the same manner as in Method 1. 1 g of PGA (Poly-Glycolic Acid) polymer was dissolved in 5 mL of dichloromethane, 500 mg of porcine bone ceramic particles were added thereto, and the mixture was homogeneously dispersed with a stirrer. Then immersed in liquid nitrogen to solidify, and then lyophilized to prepare a porous PGA composite granule containing porcine bone ceramic particles.

5: Manufacture of Porcine Bone Ceramic Particles / PCL Polymer Granules

The bone ceramic particles were prepared in the same manner as in Method 1. PCL polycaprolactone) polymer was dissolved in 5 mL of dichloromethane, 500 mg of porcine bone ceramic particles were added thereto, and the mixture was homogeneously dispersed with a stirrer. Subsequently, the granules were immersed in liquid nitrogen to solidify them and then lyophilized to prepare porous PCL granules containing porcine bone ceramic particles.

6: Manufacture of Porcine Bone Ceramic Particles / PLA Composite Filaments

Porcine bone ceramic particles were prepared in the same manner as in Method 1. Then, PLA (poly lactic acid) polymer was put into an extruder of a fiber emitter and heated at 250 ° C. to melt. Then, the prepared porcine bone ceramic particles were uniformly mixed and then spun with an extrusion nozzle of 1.0? Equipped in an extruder to prepare a PLA composite filament containing the final transformed porcine bone ceramic particles.

7: Manufacture porcine bone ceramic scaffold

Polymers such as PLGA (Poly D, L-lactic-co-glycolic acid), PGA (Poly-Glycolic Acid), PCL (polycaprolactone) and PLA (poly lactic acid) were melted at 100-150 ° C. Then, the bone ceramic powder prepared by the same method as that of Method 1 was mixed with a high-booster, and a three-dimensional scaffold (including a bone scraping support and a shielding film) was prepared using a microstereolithography system.

8: Preparation of heat-treated porcine sponge bone fragments (400 ℃ heat treatment)

To obtain pig spongy tissue, the pig femur was surgically removed and soft tissue and cartilage were removed. The cancellous bone was separated from the femur and cut to a size of 2 cm ³ or less. To remove blood and bleach, the cancellous bone was immersed in distilled water for 24 hours, then immersed in 10% H ₂ O ₂ for 12 hours, and the cancellous bone was washed by replacing the distilled water every 2 hours.

The carpal bone fragments washed with distilled water were boiled for 72 hours after replacing distilled water every 12 hours to remove primarily the lipids and proteins present in the cancellous bone. The carpal bone fragments, from which the fat and protein were removed, were first completely dried in an oven at 60 ° C for 24 hours and then processed into pieces of 1 mm or less using a grinder (MM400, Retsch, Germany).

Protein was removed by treating the bone fragments with chloroform / methanol solution (1: 1 mixed solution, 130 rpm, 24 hours) to remove lipids and treating with sodium hypochlorite (4%, 130 rpm, 72 hours). In each step, purified water was added to remove the solvent from the bone fragments and shaken at 130 rpm for 24 hours or 72 hours. Purified water was exchanged every two hours to increase cleaning efficiency.

The degreased and deproteinized cancellous bone pieces were heat-treated at 400 ° C. (5 hours) and 1,200 ° C. (3 hours) using a muffle kiln (MF-21G, JeioTech, Korea). The heat - treated cancellous bone pieces were immersed in acetone and washed with an ultrasonic washing machine for 30 minutes. To completely remove moisture, the bone fragments were lyophilized and stored at -70 ° C.

9. Characterization of heat treated pork sponge bone fragments

The morphological characteristics of porcine sponge bone fragments heat-treated using a scanning electron microscope (SEM, S-4300, Hitachi, Japan) were analyzed. Prior to testing, a gold sputter-coating was applied to impart electrical conductivity to the sample.

Calcium and phosphorus contents of bone fragments were analyzed by energy dispersive spectrometer (EDS). The EDS profile was analyzed using a field emission scanning electron microscope (FE-SEM) system (Quanta 200 FEG, FEI company, USA) under conditions of 15 kV voltage, spot size 3 and random 5 points.

Crystallinity of heat-treated porcine cancellous bone fragments was analyzed at 40 kV, 200 mA, 2 ° / min (2θ, 5 - 60 °) using an X-ray diffractometer (Rigaku, Max-2500, Japan) , And the crystallite size was measured semiquantitatively using the Scherrer equation (9).

D is the average domain size, λ is the incident X-ray wavelength, β1 / 2 is the peak width at 2θ (maximum width in half the maximum), and θ is the diffraction angle of the corresponding reflection.

The specific surface area of the sample was determined by nitrogen adsorption-desorption isotherms at liquid nitrogen temperature using Micromeritics (ASAP 2420, Norcross, USA) at 300 ° C and 1.333 Pa outgas conditions . The surface area was calculated by BET (Bruaauer-Emmett-Teller) method and the pore size distribution (pore diameter and volume) was calculated by BJH (Barrett-Joyner-Halenda) method (7).

10. Cranial defect model and bone graft

Thirty healthy 7-week-old male Sprague-Dawley rats (Samtaco, Osan, Korea; weight, 250-280 g) were used in the experiment. The experimental animals were housed in a room maintained at 23 ± 2 ° C, 12 hours night-day cycle, and 60 ± 10% relative humidity. Tap water and rodent feed (Samyang feed, Korea) were supplied at random. Experiments and breeding protocols were approved by Chonnam National University Animal Experimental Ethics Committee. Experimental animals were randomly assigned to three groups (control, severe defect; transplantation of P400 group, heat treated pork sponge bone pieces at 400 ° C; P1200 group, heat treated pork sponge bone pieces at 1200 ° C).

11. Surgical procedure

The animals were anesthetized by intraperitoneal injection of 40 mg / kg of ketamine (Ketamine 50, Yuhan Co., Korea) and 10 mg / kg of xylazine (Rompun, Bayerkorea, Korea). The skin was disinfected and then incised, exposing the periosteum of the skull. The skull was injured (diameter: 8 mm) using a surgical micromotor (NSK, Kanuma, Japan) and bone marrow pieces of P400 and P1200 were implanted in the defect sites of P400 and P1200 groups. I did not implant anything. The periosteum was then sutured using a 4-0 sorbent (Surgisorb, Samyang Co., Korea). The skin was sutured using 3-0 non-absorbable cotton (Black Silk, Ailee, Korea). The animals were sacrificed by CO ₂ choking at 4 or 8 weeks postoperatively.

12. Evaluation of bone formation ability

At 4 weeks and 8 weeks after bone grafting, a sample was obtained using a diamond saw in a rat skull defect model and radiographs were performed at 70 kVp, 7 mA, 0.031 s and 15 cm FFD (focal film distance) conditions.

Samples were analyzed by micro-computed tomography (CT) using a Skyscan 1172 desktop X-ray micro-tomograph (Skyscan, Aartselaar, Belgium) at 48 kVp and 201 μA. Bone volume and bone density were analyzed by micro CT at 4 and 8 weeks after bone grafting.

Samples were fixed in 10% formalin and then decalcified using Calci-ClearTM Rapid (National diagnostics, Atlanta, USA). Samples were dehydrated by washing with ascending series of alcohol and embedded in para-plast (Sherwood Medical Industries, St. Louis, USA). The embedded sample was cut to a thickness of 5 using a microtome (Reichet-Jung 820). Slides were stained with hematoxylene and eosin (H & E) and then observed under a microscope.

13. Statistical analysis

Experimental results were expressed as ± SD, and a one-way ANOVA (SPSS version 12) analysis was used to assess bone volume and bone mineral density by the feroney assay. P values <0.05 are considered to be statistically significant in all experimental results.

Experiment result

1. Scanning electron microscope observation

(P350C) prepared from porcine bone and ceramic particles (B350C) prepared from bovine bone according to Method 1, a ceramic particle (P350N) prepared from porcine bone according to Method 2 As a result of observing the morphological characteristics with a scanning electron microscope (SEM), it was confirmed that porcine bone particles prepared by the method 1 are superior in porosity to other particles (Fig. 1).

2. Energy dispersive spectrometer (EDS) analysis

The calcium / phosphorus ratio of P350C, P350C, B350C, P350N and untreated porcine live bone surfaces was analyzed. As shown in Table 1, the calcium / phosphorus ratio of P350C was 1.70, 2a-e). The ratio of calcium to phosphorus is very important for cell adhesion and growth and has a positive effect on bone formation.

Types of bone particles Calcium / phosphorus (unit: At%) Porcine bone bone (live bone) 1.57 Products BioOss Bovine bone particle 1.55 Bone bone particles (B350C) prepared according to Method 1 1.58 Pig bone particles (P350C) prepared according to Method 1 1.70 Porcine bone particles (P350N) prepared according to Method 2 1.51

3. X-ray diffraction (XRD)

When X-rays strike the crystal, some of them cause diffraction, and its diffraction angle and intensity are inherent in the material structure. Using this diffraction X-ray, information related to the kind and amount of the crystalline material contained in the sample can be obtained . XRD analysis of BioOss, P350C, B350C, P350N and untreated porcine raw bone particles showed that the XRD patterns of BioOss (FIG. 3A), P350C (FIG. 3B), B350C (FIG. 3C) and P350N Shows characteristics of a low-crystalline ceramic similar to untreated raw bone (Fig. 3E). In the analysis of peak area and height for XRD analysis, the broader the width of the XRD peak is, the lower the crystallinity is, and the narrower the XRD peak is, the higher the crystallinity. As shown in Table 2, P350C and P350N have a smaller overall peak area than BioOss and B350C, and the width of each peak (index: 002, 210, 211, 310, 222, 213, 004) It can be seen that the particles made with bone are relatively low crystalline compared to those made with bovine bone.

Diffraction Index Total area 002 210 211 310 222 213 004 FWHM area (%) FWHM area (%) FWHM area (%) FWHM area (%) FWHM area (%) FWHM area (%) FWHM area (%) BioOss 1155363 0.272 18 0.59 13.9 0.706 100 0.483 8.8 0.508 18.2 0.47 19.1 0.324 9.3 B350C 1207423 0.048 0.2 0.607 7.2 0.857 100 0.375 2.9 0.638 19.3 0.55 17.3 0.386 6.7 P350C 984662 0.322 23.4 0.712 17 0.974 100 1.088 22 0.65 21 0.58 22.3 0.355 9.8 P350N 1078132 0.392 14.6 0.779 9.1 1.126 100 1.09 13.3 0.662 10.9 0.56 7.7 0.468 4.2 Live Bone 348670 0.45 6 3.399 22.4 1.915 100 1.25 12.5 1.197 16.6 0.972 10.2 1.766 8.5

4. Thermogravimetry analysis (TGA)

The TGA shows the result of measuring the change of temperature with temperature (0-100 ℃ moisture, 200-400 ℃ organic matter, 600-800 ℃ carbonate removal) and time change with heating at given temperature condition. TGA analysis of BioOss, P350C, B350C, P350N and untreated porcine bone grains showed the largest weight change of sample according to temperature and time of porcine raw bone particles without heat treatment (Fig. 4e) P350C ceramic particles showed the smallest weight change (Fig. 4B).

5. Analysis of BET (Brunauer-Emmett-Teller)

BET analysis of BioOss, P350C, B350C, P350N and untreated porcine bone grains revealed that P350N (127.3825 m ² / g) and P350C (110.6533 m ² / g) were B350C (98.3558 m ² / g) and BioOss 93.7732 m &lt; ² &gt; / g) (Fig. 5). The larger the specific surface area, the more favorable the adhesion of cells and the positive effect on osteosynthesis and osteogenesis.

6. Analysis by BJH (Barret-Joyner-Halenda) method

The pore size and distribution of BioOss, P350C, B350C, and P350N ceramic particles were measured by the BJH method, and the pore size distribution of all the particles was mostly in the range of 20-500 (2-50 nm) (FIG. 6). Micropores promote cell adhesion and metabolism. BJH desorption of Figure 6 (desorption) dV / dlog ( D) pore When the volume value, P350C is 0.9 cm ³ / g ·, P350N is 0.88 cm ³ / g ·, B350C is 0.69 cm ³ / g ·, BioOss 0.49 cm ³ / g · the bar, P350N and P350C is to have fine pore size and distribution superior to BioOss and B350C is expected that a positive effect have the goal new ^{[P350C (0.9 cm 3 / g} ·)> P350N ( 0.88 cm ³ / g B350 C 0.69 cm ³ / g BioOss 0.49 cm ³ / g.

7. Crystallinity analysis

XRD measurements of porcelain bone-derived ceramic particles (P1200N) prepared at P350C, P350N and high temperature (1200 ° C) show that the P350C and P350N exhibit less ceramic characteristics than the ceramic particles produced at high temperature (FIG.

8. Estimation of bone regeneration ability of porcine sponge bone pieces heat treated at 350 ℃

8-1. General radiograph

In the critical defect, which formed only the critical defect, the bone was formed from the edge of the defect at 4th week, and the new bone formed from the border was gradually formed in the middle of the defect at 8th week. In the Bio-Oss group, new bone formation occurred at the 4th week after fusion with the original bone at the transplant margin. At 8th week, the new bone formation from the margin of the defect was more increased compared to the 4th week. Bone mineral density of the transplanted bone at 4 weeks and 8 weeks in B350C group was almost similar, and it was confirmed that the degree of new bone formation from the edge was increased at 8 weeks after 4 weeks. In the P350C group, fusion between the graft from the edge of the graft site and the original bone was confirmed at 4 and 8 weeks, and at the 8th week, the osseointegration and new bone formation from the marginal zone were further increased compared with the other groups 8).

8-2. Micro CT pictures

In both the 4th and 8th weeks of micro CT imaging, the graft material remained in the bone defect area and the density of the graft material did not change between 4 and 8 weeks. Critical defect was observed at 4 and 8 weeks after formation of new bone at the border of bone defect and at 8th week of new bone formation. In the Bio-Oss group, B350C group and P350C group, new bone was formed at the border of bone defect at 4th week compared to the control group. At 8 weeks, fusion between the graft material and original bone was further increased at the micro- , And new bone formation between the implant materials was also confirmed (Figs. 9 and 10). In the P350C group derived from the pig bone, new bone formation was observed between the border of the bone defect and the graft material in the micro CT 3D median plane, compared with the other groups, and it was confirmed that new bone was formed from the dura of the skull. Especially, at 8 weeks, a large amount of new bone was formed at the center of the bone defect and the defect, compared with the Bio-Oss group and the B350C group derived from bovine bone.

9. Characterization of Pork Sponge Carving by Heat Treatment at 400 ℃

9-1. SEM and EDS analysis

In SEM images of porcine bone pieces (P400) heat treated at 400 ° C, the bone fragments had rough surfaces and were found to have various sizes of several hundred micrometers to several hundreds of millimeters. The bone fragments have a porous structure on the surface by a heat treatment process (Fig. 11, a1-3). Pig bone fragments (P1200) heat treated at 1200 ℃ showed similar characteristics to P400 bone fragments in SEM, but the surface was rougher than P400 and showed higher bone density than P400 (Fig. 11, b1-3). In the EDS profile of P400, the calcium / phosphorus ratio was 1.61 at P400 and 1.73 at P1200 (Table 3).

Calcium / phosphorus ratio in the EDS profile Experimental group ingredient(%) Calcium / phosphorus ratio (At%) P400 sign 12.39 1.61 calcium 19.95 P1200 sign 14.73 1.73 calcium 25.56

9-2. X-ray diffraction method

The chemical composition (presence of crystal phase) shown in the X-ray diffraction analysis results is shown in FIG. 12 and Table 2. The D-spacing value was 2.81, and the 2-theta included in each spectrum was enlarged to 30-35, and the peak was calculated to be 31.8 (FIG. 12). A hydroxyapatite crystal peak was identified in all samples, consistent with the JCPDS data (No. 090432). In other words, all samples consist of hydroxyapatite particles. The domain size of P400 was 3.580, the P1200 was 16.793, and the crystal size was further increased by heat treatment at 1,200 ° C (Table 4). The relatively low crystallinity of P400 is explained by the broad diffraction peaks, and the high crystallinity of P1200 is explained by the relatively narrow diffraction peaks (FIG. 12).

Evaluation of domain size from diffraction peaks of bone fragments Experimental group Maximum width in half the maximum value
(Full width at half maximum) Domain Size P400 0.731 3.580 P1200 0.156 16.793

9-3. Surface area analysis of sample using BET method

Detailed parameters for the porous structure of P400 and P1200 bone fragments were determined using the N2 adsorption-desorption isotherms method (Table 5). The BET surface area of P400 and P1200 was 61.02 And 0.41 m ² / g, the surface area of P400 was remarkably wider than that of P1200. This means that the pore volume and size of P400 is greater than P1200. In addition, the pore size distributions of P400 and P1200 were 76.01-571.47 nm and 17.60-409.44 nm, respectively, indicating that P400 contains macropores and P1200 mainly contains mesopores and macropores (Fig. 13).

Analysis of porous structure of sample using BET method Sample P400 P1200 BET surface area (m ² / g) 61.02 0.41 Pore volume (cm ³ / g) 0.32 0.01 Pore size (nm) 206.69 105.00

10. Evaluation of bone formation ability in rat skull defect model

10-1. Plain radiographs

During the fourth week, the bone fragments of the P400 and P1200 groups were well maintained during the experiment. In the control group, new bone formation was observed at the edge of the defect, and soft tissue was observed at the center of the defect (Fig. 15 a1). In P400 and P1200, new bone formation was observed at the edge of the defect, and the density in the defect was also increased compared to the control (Fig. 15 b1 and c1). At 8 weeks, new bone formation was clearly observed in the parietal region of the P400 and P1200 defects. On the other hand, in the control group, most of the central portion of the defect position was composed of a soft tissue (Fig. 15 a2). On the other hand, new bone formation was observed in the central region and the edge of the P400 and P1200 group defective positions (Fig. 15 b2 and c2).

10-2. Micro CT 3D analysis

Analysis of micro CT 3D images showed that new bone formation was observed at the edge of all the experimental groups and bone fragments were well maintained in the P400 and P1200 groups. At 8 weeks, bone density was higher than P1200 at P400 (Fig. 16).

Bone volume (BV) was significantly higher in the P400 and P1200 groups than in the control group. At 8 weeks, the bone volume of the P400 group was 45.527 ± 7.033%, which was slightly higher than that of the P1200 group (42.879 ± 8.108%). There was no significant difference in the bone volume of the P400 and P1200 groups. Bone density of the P400 and P1200 groups was significantly higher than that of the control group, and bone density of the P400 group was significantly higher than that of the P1200 group (Table 6).

Bone volume and bone density Experimental group BV (%) BMD (g / mm ³ ) 4 weeks 8 weeks 4 weeks 8 weeks Control group 2.106 ± 1.987 10.771 + - 4.471 0.445 + 0.021 0.491 + 0.044 P400 43.617 ± 7.102 ^** 45.527 ± 7.033 ^** 0.686 ± 0.010 ^{** (a)} 0.692 + - 0.014 ^{** (a)} P1200 42.879 ± 8.635 ^** 42.879 ± 8.108 ^** 0.585 ± 0.010 ^** 0.585 ± 0.006 ^**

BV (bone volume): bone volume; BMD (bone mineral density): Bone mineral density.

^** P <0.01 compared to the control group.

^(a) P < 0.01 compared to the 1200 & lt ^{; 0 &gt;} C group.

Measured values are expressed as ± standard deviation (n = 5).

11. Histological evaluation

At 4 weeks, the new bone formation in the control group is attributed to the existing bone, and the defect position is filled with fibrous connective tissue (Fig. 16 a1-3). In the P400 group, the space between the xenografts was filled with fibrous connective tissue, and the osteogenic cells were observed to penetrate into the defect site (Fig. 16, b1 and b3). In the P400 and P1200 groups, (Fig. 16 (b2) and (c2)). Fibrous connective tissue was observed between the bone fragments in the P400 and P1200 groups, and more fibrous connective tissue was found in the P400 group (Fig. 17, c1 and c3).

At week 8, there was more new bone formation at the margins of the defect than at the 4th week (Figs. 17a1, b1, c1 and a2, b2, c2). In the control group, osteogenesis was more frequent at 8 weeks than at 4 weeks, and the defect site was filled with fibrous connective tissue, but there was almost no osteocyte infiltration (Fig. 17 a1-3). In the P400 group, the space between the bone fragments was filled with fibrous connective tissue, and more bone formation was observed compared to the P1200 group. P400 and P1200 groups showed bone formation around the bone graft, especially in the P400 group, more bone formation was observed in the bone graft material as well as around the bone graft material. It was observed that the osteogenic cells were attached to the surface of the heat-treated bone graft material and were active in bone formation. In comparison with P1200 (Fig. 17C3), new bone formation was further increased around the bone graft site of P400 (Fig. 17B3). Immune responses did not appear in any of the experimental groups.

Argument

Autotransplantation is the most ideal bone grafting method in that it has bone formation and bone conduction / bone inducing activity. However, synthetic bone grafts and bone xenografts are increasingly being used as an alternative to autologous grafting for a variety of reasons (10, 11). Human apatite has low crystallinity, but commercially available synthetic bone materials have high crystallinity because they are exposed to high temperatures during manufacturing. For example, hydroxyapatite is highly crystalline due to the sintering process and has a larger particle size than bone apatite due to grain growth. These large particles exhibit high resistance to biodegradation and very low bone conduction in the human body and are not degraded by osteoclasts (12). Thus, it is believed that the heating temperature and crystallinity will affect the bone remodeling rate around bone graft particles, and the present inventors have found that the porogen-derived xenografts produced under different temperature conditions / Physical characteristics were analyzed.

Bone volume and bone density were significantly higher in the P400 and P1200 groups than in the control group. This means that the bone fragments are well maintained at the defect site and new bone is formed in the space of the bone fragments. In particular, bone density measurements due to increased mineralization were significantly higher in the P400 group than in the P1200 group (13, 14). Histological evaluation showed that the P400 group showed higher osteogenic effect than the P1200 group at 8 weeks, and new bone formation started from the edge of the defect area. The gaps of the heat treated bone pieces were mainly filled with osteogenic cells.

XRD is a method of determining the crystal structure and constituent materials of a sample by analyzing a diffraction pattern. Although the two materials in the chemical composition are the same, the crystal structure may show a large difference. In XRD, a broad peak means low crystallinity and a narrow peak means high crystallinity (12, 15-17). In the present invention, P400 and P1200 exhibit hydroxyapatite peaks, P400 exhibits higher peaks and exhibits lower crystallinity than P1200, which is similar to low crystalline carbonic apatite, a synthetic bone material known to have bone conduction (18).

The heat - treated porcine cancellous bone fragments, which could be prepared by various methods, were confirmed to maintain the surface pore structure of cancellous bone as a result of SEM imaging. In the EDS profile, P400 and P1200 were mainly composed of calcium and phosphorus, and their calcium: phosphorus ratio was similar to human bone.

In the present invention, the porous structure analysis was performed using adsorption-desorption N ₂ gas analysis. N ₂ is readily absorbed on solid surfaces, and these properties are well known. The BET value, pore volume, and pore size of the sample measured by this method are shown in Table 3. In bone regeneration, the micropore size is 0.3-2 nm, the mesopore size is 2-20 nm, and the macropore size is 50-105 nm (19). As shown in FIG. 3, the porosity of the sample was changed from macropore to mesopore as the heat treatment temperature of the porcine sponge bone was increased. Analysis of the porous structure revealed that P400 has a larger surface area and larger pore volume than P1200. These results indicate that the densification size of the apatite particles in the bone fragments is increased by the high temperature heat treatment and the densification is the connected or non-connected micropore generated by the removal of organic components in the porcine bone fragment (20).

According to previous studies, human bone crystals change under various sintering conditions, and crystallinity increases with increasing firing temperature (21). According to other experimental results based on cortical bone, the crystallinity of bone increases with increasing heat treatment temperature and time (22). Materials with low crystallinity have a larger surface area and can easily combine with organic materials. These materials have high affinity and high biodegradability for cells in In vivo. In other words, low-crystallinity materials can be directly degraded by keel cells and provide a more favorable environment for bone material remodeling (23). In addition, P400 and P1200 represent the morphological characteristics of a low crystalline structure characterized by increased porosity compared to P1200, although P400 and P1200 are derived from the same material. Based on these results, the inventors of the present invention could predict that the P400 bone structure prepared with low crystallinity had remarkable bone conduction and bone inducing ability than other graft materials prepared with high crystallinity.

In this respect, porcine cancellous bone can be used to produce bone fragments of various sizes and various crystal structures, and can also be used to develop low crystalline bone graft materials suitable for human and animal bone regeneration.

references

1. Culpan P et al., J Bone Joint Surg Br 89: 11781183 (2007).

2. Haidukewych GJ et al., J Arthroplasty 20: 344349 (2005).

3. Millis DL et al., In: Textbook of Small Animal Surgery, 3rd ed. Philadelphia: Saunders; 18491861 (2003).

4. Yoo D, Giulivi A. Can J Vet Res 64 (4): 193-203 (2000).

5. Puga YG, Schneider MK, Seebach JD. Curr Opin Organ Transplant 14 (2): 154-60 (2009).

6. Ladavos AK et al., Micropor Mesopor Mat 151: 126-133 (2012).

7. Singh TP, Singh H, Singh H. Journal of Thermal Spray Technology 21 (5): 917-927 (2012).

8. Onoki T et al., Applied Surface Science 262: 263-266 (2012).

9. Allen WB, Kenneth O, Thomas R, Ignatius YC. Micropor Mesopor Mat 117 (1-2): 75-90 (2009).

10. Ilan DI, Ladd AL. Oper Tech Plast Reconstr Surg 9: 151160 (2003).

11. Giannoudis PV, Dinopoulos H, Tsiridis E. Bone substitutes: an update. Injury 36 (1): S20S27 (2005).

12. Balasundaram G, Sato M, Webster TJ. Biomaterials 27: 2798-2805 (2006).

13. Compston J. Arq Bras Endocrinol Metabol 50 (4): 579-585 (2006).

14. Stauber M et al., J Bone Miner Res 21 (4): 586-595 (2006).

15. Klug HP, Alexander LE. X-ray diffraction procedures for polycrystalline and a morphous materials. 2nd ed. New York: Wiley-Interscience; 1974. Chapter 9.

16. Tadic D, Epple M. Biomaterials 25: 987-994 (2004).

17. Kirik SD et al., Acta Crystallogr B 56: 419-425 (2000).

18. Lee SH. J Korean Dental Assoc 44: 524-33 (2006).

19. Jozef L et al., Ceramics Silik 50 (1): 22-26 (2006).

20. Bouler JM et al., J Biomed Mater Res 32 (4): 603-609 (1996).

21. Rogers KD, Daniels P. Biomaterials 23: 25772585 (2002).

22. Hiller JC et al., Biomaterials 24: 50915097 (2003).

23. Wopenka B, Pasteris JD. Mater Sci Eng C 25: 131-143 (2005).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (16)

A method of making a ceramic material comprising the steps of:
(a) cutting the pig bone;
(b) removing the cortical bone and blood from the severed pig bone to obtain a cancellous bone;
(c) boiling the cancellous bone in water and performing a first removal process of lipid and protein;
(d) drying the cancellous bone;
(e) heat treating the cancellous bone at 300-500 ° C; And
(f) powdering the heat treated cane bone to obtain a low crystalline ceramic material, wherein the full width at half maximum (FWHM) value for the X-ray diffraction peak of the low crystalline ceramic material is (i) 0.322 in the X-ray diffraction index having; (Ii) 0.974 at the X-ray diffraction index with the index 210 and 0.712 (iii) at the X-ray diffraction index with the index 211; (Iv) 1.088 at the X-ray diffraction index with 310 index; (V) 0.65 at the X-ray diffraction index with index 222; (Vi) 0.58 at the X-ray diffraction index with index of 213; And an X-ray diffraction index having a (004) index of 0.355.
2. The method of claim 1, wherein the cutting of the pig bone in step (a) is performed at a size of 0.5-5.0 cm &lt; ^{3 &} gt ;.
2. The method of claim 1, wherein the pig bone is selected from the group consisting of a head bone, an arm bone, a leg bone, a rib, a dress bone, a vertebra bone, a sac bone, a caudal bone and a tooth.
4. The method of claim 3, wherein the pig bone is a femur.
The method of claim 1, further comprising the step of chemically removing lipids and proteins from the dried cancellous bone between steps (d) and (e).
6. The method of claim 5, wherein the lipid removal is performed by treating toluene or a solution of chloroform and methanol.
6. The method of claim 5, wherein the protein removal is performed by treating ethylene diamine or sodium hypochlorite.
The method of claim 1, wherein the heat treatment of step (e) is performed for 5-20 hours.
The method of claim 1, wherein the ceramic material has a calcium / phosphorus ratio of 1.6-1.8 At%.
The method according to claim 1, wherein the ceramic material has a specific surface area of 105-130 m ² / g.
The method of claim 1, wherein the ceramic material produced has a BJH desorption dV / dlog (D) pore volume value of 0.8-0.95 cm ³ / g.
(D) pore volume value and X-ray diffraction (XRD) values of 1.6-1.8 At% calcium / phosphorus, 105-130 m ² / g specific surface area and 0.8-0.95 cm ³ / g BJH desorption dV / (FWHM) value for the peak (i) 0.322 at the X-ray diffraction index with index 002; (Ii) 0.974 at the X-ray diffraction index with the index 210 and 0.712 (iii) at the X-ray diffraction index with the index 211; (Iv) 1.088 at the X-ray diffraction index with 310 index; (V) 0.65 at the X-ray diffraction index with index 222; (Vi) 0.58 at the X-ray diffraction index with index of 213; And a low crystalline ceramic material having an X-ray diffraction index of 0.355 with a (004) index.
13. The ceramic material according to claim 12, wherein the ceramic material is produced by the method according to any one of claims 1 to 11.
A bone graft material comprising the ceramic material of claim 12.
A bone support comprising the ceramic material of claim 12.
A composite material comprising the ceramic material of claim 12 and a biocompatible polymer.

Images