WO2012099285A1

WO2012099285A1 - Nano ceramic bone cement using animal bones and preparation method thereof

Info

Publication number: WO2012099285A1
Application number: PCT/KR2011/000422
Authority: WO
Inventors: 정종훈; 백수정; 정연훈; 정필훈
Original assignee: 서울대학교산학협력단
Priority date: 2011-01-21
Filing date: 2011-01-21
Publication date: 2012-07-26
Also published as: WO2012099285A9

Abstract

The present invention relates to nano ceramic bone cement containing animal bone powder and a preparation method thereof, and a porous bone support containing nano horse bone powder. The nano ceramic bone cement containing animal bone powder of the invention is a hydroxyapatite component similar to a real bone and has a strong strength, fast curing time, biocompatibility, and excellent characteristics for promoting bone tissue regeneration.

Description

Nanoceramic bone cement using animal bone and its manufacturing method

Tissue engineering is a multidisciplinary field of interdisciplinary life sciences, medicine, and engineering, in which the understanding of structure and function correlates in normal and pathologic abnormalities, and can repair or replace dead or damaged tissues, organs and parts of the body For the purpose of developing a biological substitute. The most common implant for treating bone defects or for union of joints or bones is known as autogenous bone and allogeneic bone. However, there is a risk that the associated complications sometimes lead to greater problems than the surgery, limited quantities to be obtained, and contamination, toxin transmission, or infection through the immune response. Tissue engineering studies have been performed extensively in search of implant materials.

Bone cement is used to fix tissues by fixing and stabilizing the fractures or bony defects that can be caused by traffic accidents, cancer, inflammation, and the like. The treatment using conventional titanium and aluminum screw or concavo-convex structure has advantages of excellent durability and strength, but metal debris that is not suitable for the living body may cause osteolysis, There is a disadvantage that the damaged bone tissue is lost. The treatment with bone cement is more biocompatible than conventional methods and maintains fluidity until it is completely cured. Therefore, it is easy to fix the tissue to the desired shape and fix the bone fragments without damaging other parts. I have.

Calcium phosphate bone cement exhibits a biocompatibility similar to that of bone tissue and has high biocompatibility. It has excellent bone conduction and induces bone formation. However, when the calcium phosphate-based bone cement is kneaded, the curing time is longer than 60 minutes, so that the curing time must be shortened to about 10 minutes in order to use it in actual practice. In addition, the calcium phosphate-based bone cement has low mechanical strength (1 to 3 MPa) compared to the actual bone mechanical strength (10 MPa or more), and various methods for improving the mechanical strength have been studied. Generally, the best material for bone cement is autogenous bone, but secondary surgery is necessary and it is difficult to obtain a large amount. In place of this, various calcium phosphate-based compounds of bone-substitute biomaterial currently in widespread use are disadvantageous in price.

In general, since bone cement is prepared as a part of the bone after being inserted into the actual patient, the physical and mechanical properties should be similar to those of the actual bone, and the bonding strength with the bone at the application site should be excellent and permanent. It should also be biocompatible so that it does not involve an immune or inflammatory reaction.

Accordingly, in the present invention, a new calcium phosphate nanoceramic material using animal bone was developed, and the mechanical strength of calcium phosphate-based nano-bone cement was improved by using chitosan and the curing time was shortened.

In the present invention, animal bone was reworked to produce hydroxyapatite similar to human bone, thereby producing a biocompatible material for bone cement free from immune rejection. Particularly, horse bone has more calcium and phosphorus than other animals. It is the only animal that stands up for 24 hours and its bone powder is also used as food for bone strengthening. In order to improve the bioabsorbability and strength of animal bone powder, they were made into nanoceramics and powder for bone cement was prepared.

In addition, chitosan solution was prepared by using natural polymer chitosan in order to compensate the long curing time (60 min) and low mechanical strength of calcium phosphate - based cement. Chitosan is a substance obtained by deacetylation of chitin, a natural polysaccharide present in the shell of crustaceans such as crabs and shrimp, and is a material that is featured in tissue engineering due to its biocompatibility, bioabsorbability and excellent bone conductivity. The chitosan solution was prepared by dissolving chitosan in a biocompatible lactic acid solution. The chitosan solution was used to shorten the curing time of the calcium phosphate bone cement made from animal bone powder, and the nanocomposite cement having similar biocompatibility, mechanical strength and elasticity Bone Cement).

In addition, a bone scaffold block was prepared by directly sintering the animal bone, especially the horse bone, at high temperature to remove all the organic material and to use the porous cancellous bone as a bone substitute.

The nanoceramic bone cement containing the nano animal bone powder of the present invention is a hydroxyapatite component similar to a real bone and has excellent properties of promoting strong strength, fast curing time, biocompatibility and bone tissue regeneration. The nanoceramic bone cement of the present invention may be used in various fields in the field of tissue engineering.

1 shows SEM images and particle size analysis results of micro-pig bone powder (left) and micro-horse bone powder (right).

FIG. 2 shows SEM images and particle size analysis results of nano pig bone powder (left) and nano horse bone powder (right).

Figure 3 shows the XRD patterns of animal (pig and horse) bones and hydroxyapatite. [■: Characteristic peaks of hydroxyapatite (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ].

Figure 4 is the FTIR spectrum for micro / nano animal bone powder.

Figure 5 shows a comparison of FTIR spectra between porcine / horse and micro / nano powder.

Figure 6 shows an EDX analysis of the chemical composition of animal bone powder and hydroxyapatite.

7 shows the curing time of micro-animal bone cement (left: pork bone cement; right: horse bone cement; control means animal bone cement including distilled water, error bars indicate standard deviation, n = 5).

8 shows the hardening time of the nano animal bone cement (left: pig bone cement; right: horse bone cement; error bars show standard deviation, n = 5).

9 shows the compressive strength and Young's modulus of micro-pig bone and bone cement (control means animal bone cement including distilled water, error bars show standard deviation, n = 5).

10 shows the compressive strength and Young's modulus of nano pig bone and bone cement according to the nano powder ratio (control means animal bone cement including distilled water, error bars show standard deviation, n = 5).

FIG. 11 shows the results of an experiment to evaluate the washing resistance of an animal bone cement after immersing in simulated body fluid (SBF) for 28 days (left: pig bone cement; right: horse bone cement).

12 is an SEM image of the bone cement according to the number of days dipped in a simulated body fluid (SBF). [(A): Nano powder ratio 0%; (B): nano powder ratio 50%; (C): nano powder ratio 75%].

FIG. 13 is an SEM image of pig bone cement according to the number of days immersed in a simulated body fluid (SBF). [(A): Nano powder ratio 0%; (B): nano powder ratio 50%; (C): nano powder ratio 75%].

Fig. 14 shows the compressive strength and Young's modulus of animal bone cement according to time immersed in SBF [(A): pig bone cement; (B): bone cement (error bars represent standard deviation, n = 5).

Figure 15 shows cell viability of micro-animal bone cement to chitosan solution concentration [left: pig bone cement; Right: bone cement (error bars represent standard deviation, n = 5)].

Figure 16 shows cell viability of nano animal bone cement versus chitosan solution concentration [left: pig bone cement; Right: bone cement (error bars represent standard deviation, n = 5)].

Figure 17 shows MG63 cell proliferation of micro-animal bone cement [left: pig bone cement; Right: bone cement (error bars represent standard deviation, n = 5)].

Figure 18 shows MG63 cell proliferation of nano animal bone cement [left: pig bone cement; Right: bone cement (error bars represent standard deviation, n = 5)].

Figure 19 shows a fluorescence image of MG63 cells on the surface of bone cement [(A): Micro-pig bone cement; (B): micro / nano (50/50 (w / w)) pig bone cement; (C) micro / nano (25/75 (w / w)) pig bone cement; (D): micro-bone cement; (E): micro / nano (50/50 (w / w)) horse bone cement; (F) micro / nano (25/75 (w / w)) horse bone cement.

FIG. 20 is an SEM image after cells are divided (arrowed) into pig bone cement [(A): Micro-pig bone cement; (B): micro / nano (50% / 50% (w / w)) pig bone cement; (C) Micro / nano (25% / 75% (w / w)) pig bone cement.

FIG. 21 is an SEM image after cells are divided (arrowed) into the bone cement [(A): micro-bone cement; (B): micro / nano (50% / 50% (w / w)) horse bone cement; (C) Micro / nano (25% / 75% (w / w)) horse bone cement.

Figure 22 is a CT image of a rat skull injury site [(A): Implantation of 100% microsomal bone cement; (B): 100% nasal bone cement implants; (C) 50% Micro + 50% Nano-horse bone cement (right arrow of red: transplantation of horse bone cement; left arrow of yellow:

Figure 23 is a histological comparison of horse bone cement 3 months after transplantation at the site of the rat skull [(A): control group; (B): 100% microbial bone cement; (C): 100% nano-horse bone cement; (D): 50% micro + 50% nano-horse bone cement (left: X12.5; right: X100;

Hereinafter, the present invention will be described in more detail by way of examples. However, the scope of the present invention is not limited to the following examples.

[Statistical analysis]

Duncan's multiple range test was used to compare the cure time, compressive strength, and biological data. The statistical treatment of the data was performed using the SAS 9.1.3 version.

[Example 1]

Manufacture of nano and micro animal bone powder

Pigs and horses were collected and immersed in distilled water for 24 hours to remove pellicles. Then, they were immersed in hydrogen peroxide (77228481, Duksan chemicals, Korea) for 48 hours to remove organic substances such as flesh on the surface of bone. The animal bone with the flesh removed from the surface was dried to remove moisture and then sintered at 1200 ° C for 2 hours using an electric sintering furnace (UP350E, Yokogawa co, Japan). The sintered bones were put into a pulverizer (A10, IKA-WERKE, Japan) to make powder, and then sintered twice at 1200 ° C for 2 hours in the same manner as described above. The sieved animal bone powder was sieved Scientific, Korea) was used to make animal bone with particle size below 100 ㎛. Nano Sizer Fine Mill (Deaga Powder Systems Co., Ltd. Korea), which is an apparatus for crushing animal bone powder by friction between zirconia particles and rotating body, was used for making animal bone powder of nanoparticles.

[Example 2]

Preparation of chitosan solution

Chitosan (molecular weight: 200,000, deacetylation degree:> 85%, Taehoon Co, Korea) was dissolved in a solution of 1-3% (v / v) lactic acid (# 50215, Duksan chemicals, % (w / v) chitosan solution.

[Example 3]

Morphological analysis

(1) Method

Scanning electron microscopy (SEM; JSM-5410LV, JEOL, Japan) was used to observe the surface of sintered animal bone and bone cement. For particle size analysis of animal bone powder made of micro and nanoparticles, a particle size analyzer (Mastersizer, Malvern Instruments Ltd, UK) was used.

(2) Results

1 and 2 are SEM images and particle size analysis results of micro / nano animal bone powder. Microparticles of animal bone powder produced by removal of organic matter and sintering showed particle sizes of 66-76 ㎛ for pig bone and 56-83 ㎛ for horse bone. As shown in FIG. 3, the particle size of the animal bone powder of nanoparticles was 150-270 nm for pig bone and 170-310 nm for horse bone, and the particle size was confirmed by SEM image.

[Example 4]

Physicochemical properties

(1) Method

X- ray diffraction to determine the crystallinity of the sintered animal bone powder was obtained an XRD graph using (# D5005, Bruker, Germany) , FTIR (Nicolet 6700, Termo Scientific, USA) for PO ₄ ^3- by using And OH ^- were observed. The chemical composition of sintered animal bone was measured using an energy dispersive X-ray spectroscope (Field Emission Scanning Electron Microscope, SUPRA 55VP, Carl Zeiss, Germany).

(2) Results

FIG. 3 shows the XRD patterns of pigs, horses and hydroxyapatite. Hydrophilic apatite (239396, Sigma Aldrich Korea, Korea) commercially available for analysis of pig and horse bone powder was used as a control. Both pig and horse bone showed similar crystallinity to hydroxyapatite. That is, the pig and horse bone powder were calcium phosphate type compounds similar to hydroxyapatite. In pigs and horse bone powder, the height of the peak increased and the width decreased, indicating a more pointed shape than that of the hydroxyapatite. This means that pig and horse bone powder have higher crystallinity than commercially available hydroxyapatite. In addition, it can be seen that the horse bone powder has a higher peak value and a sharp peak than the pig bone powder, which also shows that the horse bone powder exhibits higher crystallinity than the pig bone.

Figs. 4 and 5 show the FTIR spectra of pig and horse bone powder. As a result of the FTIR reference, the peaks of all the powders were 3571-3572 cm ^-1 , 1411-1457 cm ^-1 , and 959-962 cm ^-1 , And a peak similar to that shown in FIG. Generally FTIR spectra phosphate (PO ₄ ^3-), hydroxyl (OH ^-) and carbonate (CO ₃ ^2-) to check for the presence of ions, and the spectrum of ^{^{962cm -1, 1026-1027 cm -1, 1090}} cm ^-1 peak is derived from a phosphate bond, a peak at 1410-1450 cm- ¹ is attributed to carbonate bond, and a peak at 3571 cm- ¹ is derived from a hydroxyl bond. The results of FTIR spectra of nano- and micro-animal bone powder showed that phosphate bond, hydroxyl bond and carbonate bond were present. The results of FIG. 5 obtained by enlarging the wavelength of 1500-800 cm ^-1 showed that the bone bone, The permeability of the nanoparticle powder was lower than that of the nanoparticle powder. That is, the horseshoe and nanoparticles have more binding at the corresponding wavelengths.

Figure 6 shows the EDX pattern of hydroxyapatite analyzed as animal bone powder and control. Both pig and horse bone powder were found to show peaks indicating calcium and phosphorus, and the EDX pattern was also similar to that of hydroxyapatite. However, as shown in Table 1, the ratio of calcium to phosphorus in horses and pig bone powder was 1.96 and higher than 1.55 in hydroxyapatite. The ratio of calcium to phosphorus is very important for the adhesion and growth of cells, and it is known that the higher the ratio of calcium to phosphorus, the better adhesion and growth of osteoblasts. Because proteins such as vibronectin, which are related to the adsorption of osteoblasts on the surface of bone cement, have a calcium binding site, the presence of calcium affects the initial protein adsorption and is effective in adhesion and growth of osteoblasts. It is also known to have a positive effect on long-term osteointegration.

Table 1

	Ca / P ratio (at%)
Hydroxyapatite	1.55
Pork bone powder	1.96
Horse bone powder	1.96

[Example 5]

Curing time measurement

(1) Method

The hardening time of animal bone bone cement was measured using a vicat needle. An appropriate amount of animal bone powder and chitosan solution was kneaded and filled into a Teflon mold having a diameter of 10 mm and a height of 5 mm. The mixture was kept in a thermostatic chamber at a temperature of 37 ° C and a humidity of 98% or more for 90 seconds, mm was placed perpendicular to the surface of the bone cement to measure the curing time. Curing time results were obtained by repeating the measurement of five samples at the time when the needles were dropped at intervals of 30 seconds and the moment when needle marks were not left on the surface of bone cement as the curing time of bone cement.

(2) Results

Fig. 7 shows the curing time according to the concentration of the chitosan solution in the micro-animal bone bone cement. The microbial bone cement was kneaded at a solution / powder ratio of 0.35, 0.40 and 0.45 ㎖ / g in a 2% (w / v) chitosan solution, respectively, with different solution and powder ratios The curing time was 11 ± 0.58 min, 15 ± 0.58 min and 21 ± 0.6 min, respectively. For the bone cement, 30 ± 0.58 min, 41 ± 1.2 min and 43 ± 0.6 min were mixed with the same concentration of chitosan solution The curing time increased as the ratio of solution to powder increased. However, when the bone cement was kneaded at a ratio of 0.35 ml / g, the water content was too small and when the ratio was 0.45 ml / g, the water was too much to be kneaded. Therefore, in the subsequent test, bone cement kneaded at a ratio of 0.40 ml / g was used. The hardening times of pig bone bone cement when kneaded with 2.0%, 3.0% and 3.5% (w / v) chitosan solution were 19 ± 0.58 min, 14 ± 0.60 min and 11 ± 0.58 min, respectively, 30 ± 0.6 min, 27 ± 0.58 min and 23 ± 0.6 min for cement, respectively. The curing time of the pig bone cement was shorter than that of the horse bone cement. This is because the crystallization degree of the horse bone powder is higher than that of the pig bone, as in the XRD analysis described above, which can be explained by ionization and crystallization, which is one of the curing processes of the calcium phosphate-based bone cement. That is, the calcium phosphate-based material dissolves in water and dissociates into Ca ²⁺ , PO ₄ ^3- , and OH ^- ions to form hydroxyapatite crystals having the structural formula Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ . Pig bone powder with low crystallinity was more soluble than horse bone powder and ion dissociation was more active. As a result of the curing time measurement, it was confirmed that the curing time was decreased with increasing concentration of chitosan solution in both types of bone cement. It was confirmed that the dissolution characteristics of chitosan dissolved in an acidic environment which is not dissolved in neutral and alkali environments, . When an alkaline bone powder and an acidic chitosan solution meet, the chitosan solution changes from a sol to a gel state due to a rapid change in pH, thereby accelerating the hardening of the bone cement. The results of Fig. 7 confirmed the effect of shortening the curing time of the chitosan solution. In the subsequent tests of the nano-bone cement, 3.5% (w / v) chitosan solution with the shortest curing time was used.

8 shows the result of measuring the curing time of the nano-bone cement. In case of pig bone cement, the curing time of bone cement without any nano powder was 15 ± 0.96 min, 12 ± 0.57 min with 25% (w / w) nanopowder, 50% (w / w) , 10 ± 0.5 minutes, 8 ± 0.5 minutes for 75% (w / w), and 7 ± 0.58 minutes for pig bone cement made only with nano powder. In the case of the bone cement, it was 30 ± 0.58 min, 22 ± 1.53 min, 17 ± 1.15 min, 7 ± 0.5 min and 2 ± 0.1 min, respectively, depending on the ratio of nano powder. At 25 and 50% level, pig bone cement showed faster curing than that of horse bone cement. However, at 75 and 100% level of nano, bone cement hardened faster than pig bone cement. It has been confirmed that the hardening time is shortened as the ratio of the nano powder increases. This phenomenon is due to the fact that the animal bone molecules having nanoparticles have a larger surface area and the curing action of the calcium phosphate-based bone cement which forms the hydroxyapatite crystal It is thought that it is actively done.

This experiment showed that the hardening time of bone cement can be controlled by the concentration of animal bone powder and chitosan in nano and microparticles. In other words, it has been shown that bone cement with desired curing time can be manufactured according to the situation in clinical application. Among the bone cements of various conditions developed in this experiment, curing time was too fast when 100% (w / w) nano powder was used and the content of nano powder was 50% (w / w) and 75% (w / w) ), It was presumed that it has proper curing time. Therefore, only 50% (w / w) and 75% (w / w) of nanoparticles were selected for the preparation of bone cement.

[Example 6]

Compressive strength

(1) Method

The compressive strength of bone cement was measured using a texture analyzer (TAXT2i, Stable Microsystems Co, US) and the crosshead speed was set at 1 mm / min.

(2) Results

FIG. 9 is a graph showing compressive strength and Young's modulus of an animal bone bone cement having a diameter of 10 mm and a height of 5 mm. The compressive strength of pig bone cement was 1.47 ± 0.24 MPa, 2.0%, 3.0%, and 3.5% (w / v) for the control treated with water, and 1.66 ± 0.32 and 2.33 ± 0.28 and 3.10 ± 0.13 MPa, respectively. The Young's modulus was 6.89 ± 1.27 MPa for the control group and 8.03 ± 1.57, 11.81 ± 1.32, and 12.60 ± 0.66 MPa for the chitosan solution of each concentration, respectively. The compressive strength of the control group was 2.75 ± 0.31 MPa and 4.06 ± 0.34, 4.21 ± 0.18 and 9.25 ± 1.30 MPa, respectively, for bone cement using 2.0%, 3.0% and 3.5% (w / v) chitosan solution , The Young's modulus of the control group was 18.64 ± 2.29 MPa, and the concentration of chitosan solution was 22.58 ± 8.01, 29.15 ± 6.94, and 41.54 ± 9.63 MPa, respectively.

Both of the pig bone and the bone bone cement showed higher compressive strength than the control group treated with distilled water and the compressive strength was increased with increasing concentration of chitosan solution (p <0.05). The increase in the compressive strength of the bone cement using the chitosan solution is due to the fact that the curing time of the bone cement in the curing process is shorter than that of the alkaline bone powder and the acidic chitosan solution according to the solubility characteristics of the chitosan, . In addition, the compressive strength of the horse bone cement is higher than that of the pig bone cement. This is because the crystallinity of the horse bone powder is higher as confirmed by the XRD analysis. In general, bone cement requires a value of 10 MPa or more, which is the compressive strength of bone. Pig bone cement was initially inferior to expected value, but in case of bone cement 3.5% (w / v) chitosan solution Cement was 9.25 ± 1.30 MPa, confirming the compressive strength approaching the required value.

The graph of compressive strength and Young's modulus of bone cement including nano powder is shown in FIG. Pig bone cement showed compressive strength of 8.62 ± 1.38 MPa and 16.52 ± 1.30 MPa, respectively, compared to 4.13 ± 1.04 MPa of micro-bone cement with 50% and 75% (w / w) appear. The bone cement showed the compressive strength of 8.18 ± 2.78 MPa containing 50% (w / w) of nano powder and 24.88 ± 1.97 MPa containing 75% (w / w) It was confirmed that the compressive strength of bone cement including nano powder increased more than 2.56 MPa (p <0.05). It is thought that the nanopowder has a larger surface area than the micropowder and actively dissociates and crystallizes.

[Example 7]

Simulated Body Fluid (SBF) Experiment

(1) Method

Of distilled water to measure the biological activity of the bone cement manufactured using a sintering animal bones and chitosan solution _{NaCl, NaHCO 3, KCl, K} 2 HPO 4 · 3H 2 O, MgCl 2 · 6H 2 O, Na 2 SO 4 . The surface of the bone cement was changed, and the compressive strength was measured at 37 ℃ for 7, 14, 21, 28 days.

(2) Results

Porcine and horse bone cements using chitosan solution were found to be much less washed out than bone cements prepared using distilled water (FIG. 11). Bone cement kneaded with distilled water did not maintain its shape when placed in a similar solution, but the bone cement prepared with chitosan solution had the shape even after 28 days. It is known that this is related to the dissolution characteristics of chitosan which can shorten the curing time by pH change. It rapidly changed into a hard form due to the pH change. It could not be dissolved in the environment of pH 7 or more and could maintain its original shape.

Figs. 12 and 13 are SEM images showing changes in surface when the horse and pig bone cements were immersed in a simulated body fluid for 7, 14, 21, and 28 days, respectively. The appearance of the surface of the bone cement before immersing it in the simulated fluid was rough and the shape of the hole could not be found, but after observing the surface of the bone cement after immersing it in the similar fluid, holes were found in various places, And the shape of the surface. These crystals were formed in the image of high magnification (X5,000), and the crystal layer appeared. The chemical composition of these crystals (points A to C) was confirmed using EDX and the ratio of calcium and phosphorus thereof was 1.61-1.65, confirming that it was a hydroxyapatite crystal (Table 2). This can be interpreted in the same context as the hardening action of calcium phosphate bone cement, which is a calcium phosphate compound, an animal bone powder dissociated into Ca ²⁺ , PO ₄ ^3- , and OH ^- ions to form hydroxyapatite crystals.

SEM images showed that the surface of the bone cement made with micropowder had a rougher surface compared to the bone cement containing 50% and 75% of the nano powder, but the surface of the pig bone cement and the bone cement showed a big difference I did.

Table 2

	Point A	Point B	Point C
Ca / P ratio (at%)	1.65	1.60	1.61

FIG. 14 shows changes in compressive strength and Young's modulus with time in immersing the bone cement in a simulated body fluid. The compressive strength and Young's modulus of micro-pig bone cement were 4.13 ± 1.04, 8.14 ± 1.80 MPa and 8.18 ± 2.41 and 33.99 ± 2.94 MPa respectively. The compressive strength and Young's modulus of the pig bone cement after immersion in the simulated body fluid for 28 days were 41.53 ± 0.60 and 53.04 ± 3.42 MPa, respectively. The bone cement showed 27.41 ± 1.12 and 99.61 ± 3.27 MPa, respectively. (P <0.05). It was also observed that the compressive strength of the cement mortar was increased before immersion.

As described above, calcium phosphate-based bone cement is known to dissociate into ions and form hydroxyapatite crystals. The increase in the compressive strength and Young's modulus of the bone cement after being immersed in a similar solution can be explained by the fact that the Ca ²⁺ , PO ₄ ^3- , and OH ^- It can be concluded that the hydroxyapatite crystal layer is formed on the surface of the bone cement and the compressive strength is increased.

[Example 8]

In vitro test

(1) Cell culture

Osteogenic cells derived from human; incubation at (Human osteoblast-like cells MG63, KCLB 21427, Korean Cell Line Bank, Seoul national university college of medicine, Korea) to a temperature 37 ℃, humidity of 100% and of 5% CO ₂ concentration of the environment Respectively. Were cultured in Dulbeco's modified eagle's minimum essential medium (DMEM; Hyclone, Atlanta, GA) containing 10% fetal bovine serum (FBS) and 1% antibiotics.

(2) Toxicity test

MTT assay for analyzing the toxicity of the new bone cement of the present invention was performed using a kit (Cell titer 96 non radioactive cell proliferation assay, Promega). First, bone cement specimens, 10 mm in diameter and 5 mm in height, were placed in DMEM medium for extraction for 1, 3, and 5 days. 5 × 10 ⁴ cells were seeded on a 24-micro plate and incubated for 4 hours. Then, the medium was replaced with an extraction medium and incubated for 24 hours at 37 ° C. in an atmosphere of 100% humidity and 5% CO ₂ Lt; / RTI > After incubation, 150 μl of MTT solution was added to each well and allowed to react for 4 hours. Then, 1 ml of solubilization solution / stop mix was added and reacted for another 1 hour. After the reaction was completed, absorbance was measured at 570 nm using a microplate spectrophotometer (Sunrise, Tecan, Australia).

(3) Cell adhesion and proliferation experiments

Cells, 5 X 10 ⁴ to dispense the bone cement, respectively 1, 3, 5 being the incubation after live for cell adhesion and proliferation check stained cells in green fluorescence by the knife-old acetoxymethyl ester (calcein AM) principles for Live / Dead Viability / Cytotoxicity kit (L3224, Molecular Probes, Eugene, Ore) was used. After culturing on the surface of bone cement for a fixed period, bone cement samples were fixed in 4% paraformaldehyde (PFA) solution and washed three times with PBS. 200 μl of the staining sample was added to each well and allowed to react in a dark room for 45 minutes. Then, the adsorption pattern of the cells was observed using a fluorescence microscope (Axipot microscope, Zeiss, Oberkochen, Germany).

In order to observe the morphological shape of the cells adhered to the bone cement, the culture medium was removed for a certain period of time, washed three times with PBS, and then immobilized for SEM imaging. For initial fixation, immersed in 2 ml of modified Karnovsky's fixative, reacted at 4 ° C for two hours, and washed three times with 0.05 M sodium cacodylate buffer. For late fixation, bone cement samples are immersed in 2 ml of 1% osium tetroxide solution and reacted at 4 ° C for 2 hours. After removing the fixing solution, the sample was washed with distilled water, and the solution was immersed in 30, 50, 70, 80, 90, and 100% ethanol for 10 minutes. Finally, 15 ml of hexamethyldisilazane (HMDS) (Zeiss, Supra 55VP) was used to observe the growth of cells on the surface of bone cement.

(4) Results

15 shows the results of MTT analysis of micro-bone cement using MG63, an osteoblast. As a result, the cell viability of pig and horse bone cement prepared with 2.0, 3.0 and 3.5% (w / v) chitosan solution, respectively, was not statistically different when compared with the survival rate of the control group cultured in the normal medium p > 0.05).

FIG. 16 shows the results of MTT analysis of nano-bone cement, and it was confirmed that the cell viability of bone cement containing microparticle and micro-bone cement was not statistically different. In conclusion, bone cement prepared from chitosan solution, pig bone and horse bone powder is not toxic and can minimize the damage such as necrosis of surrounding cells due to toxicity when injected into living body. It can also be concluded that porcine and horse bone cements, including nanoparticles powder, are also not toxic.

FIG. 17 shows the cell growth curve in the micro-bone cement, and it was observed that the cells not only maintained the initial growth but also showed an increase in the OD value. It was found that bone cement prepared with 2.0, 3.0 and 3.5% (w / v) chitosan solution was suitable for osteoblast growth (p <0.05). The growth curve of MG 63 cells according to the content of nanopowder of pig and horse bone cement is shown in FIG. It was confirmed that the OD value was increased due to the osteoblast growth even in the bone cement containing 50% and 75% of the nano powder, and the growth of the cells was not observed in both the pig and the bone cement.

FIG. 19 is an image of MG63 cells grown on the surface of pig and horse bone cement and observed by fluorescence staining. Live cells were stained green by calcein AM. After 1, 3, and 5 days of incubation, live cells were found to be increased after 5 days. This means that cells are not only attached to the surface of the bone cement at the initial stage of culture but also grow continuously.

20 and 21 show the result of SEM observation of MG63 cells immobilized on pig and horse bone cement surface. As in the previous fluorescence image, it was confirmed that the number of cells after culturing for 1, 3, and 5 days after cell division increased. After 1 day of culture, the cells were attached to the surface of bone cement and the polygonal shape of MG63 osteoclast was maintained. After 5 days of culture, osteoblast was grown on the surface of bone cement as monolayer culture.

In vitro analysis such as toxicity test and cell adhesion experiment showed that bone cement prepared with chitosan solution, pig and horse bone powder had no toxicity and played a positive role in cell adhesion and growth.

[Example 9]

In vivo testing

(1) Surgical procedure

Eighteen healthy rats (Wistar) weighing approximately 250 g were anesthetized with tiletamine-zolazepam (10 mg / kg, Zolethyl 50, Virbac Laboratory, Carros, France) It was cleanly removed. The skull of the rat's skull was lengthened to reveal the skull. Two holes were made using a surgical drill to create an artificial bone defect site, and the right side was filled with bone cement. The left side was left as a control.

(2) Computed tomography (CT) analysis

Three months after insertion of the bone cement, new bone tissue formation was observed in the rat skull deficient area through analysis of CT (64-slice multidetector CT scanner, Brillance 64, Phillips Medical Systems).

(3) Histological analysis

After removing the surrounding tissues from the bone cement, they were fixed with 4% formalin solution, and then dehydrated using the ethanol concentration stepwise, and then filled with methyl methacrylate. Embedded specimens were cut into 10 ㎛ thick by freezing incision, stained with hematoxylin and eosin (H & E), and the appearance and extent of new bone tissue were observed using an optical microscope.

(4) Results

Three bone marrow cements of 100% micro cement, 100% nano cement and 50% micro and 50% nano were implanted into the skull of the rats and bone tissue was detected three months later. No infection or inflammation was found. FIG. 22 shows a CT image of a rat skull, in which (R) is the site where the animal bone bone cement is inserted, and (L) is the control where the bone damage site is left empty. After 3 months of bone cement implantation, CT images showed that the bone defect site was reduced or completely disappeared, whereas the control site was empty of the original defect site. It was confirmed that 5 out of the 6 mice implanted with 100% micro-chitosan and chitosan solution had decreased or completely disappeared, and 100% cement with nano and 50% + nano 50% Six cage-treated rats treated with cement were observed to have reduced or completely disappeared bone defects in all 6 rats. In particular, as shown in FIG. 22 (A), when 100% micro cement was implanted, the defect was formed to have a thicker structure than the surrounding area, and when the nano 100% cement was implanted as in (B) , And (C), 50% and 50% of micro-bone cement were implanted in the same manner, and it was found that 50% + 50% nano-mixed bone cement was most effective .

Figure 23 shows the results of histological analysis through H & E staining. As shown in FIG. 22, no change was observed in the bone defect site where no bone cement was inserted, such as formation of new tissue. However, if we look at the image of the bone grafted bone tissue in the skull deficient area, it is confirmed that the original bone graft is well adhered to the grafted bone cement and the traces of osteoid tissue formation on the surface of the original bone graft were found (Arrow point). It was also observed that more dense tissue was formed in the bone tissue grafted with bone cement using nanoparticles than the bone cement using microparticles. In conclusion, in the present invention, bone cement prepared by sintering animal bone has the ability to form new bone tissue, and it is confirmed that the bone formation ability of bone cement using nanoparticles is superior to that of microcrystalline bone cement .

Claims

A nanoceramic bone cement characterized by containing a nano animal bone powder.
The nano-ceramic bone cement according to claim 1, wherein the chitosan solution is a liquid.
The nanoceramic bone cement according to claim 2, wherein the chitosan solution is a chitosan solution prepared by dissolving at least 85% and at most 99% of deacetylated chitosan having a molecular weight of 100,000 or more and 900,000 or less in a solvent.
4. The nanoceramic bone cement according to claim 3, wherein the solvent is a solution prepared by mixing 1 to 5% (v / v) of lactic acid or acetic acid with distilled water.
The nanoceramic bone cement according to claim 1 or 2, wherein the nano animal bone powder exhibits a particle size distribution of 150 nm or more and 500 nm or less.
The nanoceramic bone cement according to claim 1 or 2, wherein the animal bone is a horse bone.
A porous osteoporosis and a sintered bone block support comprising the nano-horse bone powder.
A method for manufacturing a nanoceramic bone cement, comprising the steps of:

(1) removing blood and organic matter from animal bone;

(2) drying the animal bone from which blood and organic matter have been removed;

(3) sintering the dried animal bone;

(4) pulverizing the sintered animal bone to produce micro animal bone powder;

(5) sintering the crushed animal bone powder at least once more than 5 times;

(6) pulverizing the sintered animal bone powder to prepare nano animal bone powder; And

(7) mixing nano animal bone powder with a chitosan solution.
9. The method of claim 8, wherein the sintering is performed at 1200 DEG C for 2 hours in step (3).
The method according to claim 8 or 9, wherein the micro-animal bone powder produced in step (4) has a particle diameter of 100 μm or less.
The method of claim 8 or 9, wherein the nano animal bone powder produced in step (6) exhibits a particle size distribution of 150 nm or more and 500 nm or less.
The chitosan solution according to claim 8 or 9, wherein the chitosan solution of step (7) is a chitosan solution prepared by dissolving not less than 99% and not more than 99% of deacetylated chitosan having a molecular weight of not less than 100,000 and not more than 900,000 in a solvent A method for manufacturing a nanoceramic bone cement.
[Claim 9] The method according to claim 8 or 9, wherein the animal bone is a horse bone.