WO2012099285A9 - Nanociment osseux céramique utilisant des os d'animaux et son procédé de préparation - Google Patents

Nanociment osseux céramique utilisant des os d'animaux et son procédé de préparation Download PDF

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WO2012099285A9
WO2012099285A9 PCT/KR2011/000422 KR2011000422W WO2012099285A9 WO 2012099285 A9 WO2012099285 A9 WO 2012099285A9 KR 2011000422 W KR2011000422 W KR 2011000422W WO 2012099285 A9 WO2012099285 A9 WO 2012099285A9
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bone
bone cement
powder
nano
animal
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WO2012099285A1 (fr
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정종훈
백수정
정연훈
정필훈
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서울대학교산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/722—Chitin, chitosan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A61L24/0036—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/02—Surgical adhesives or cements; Adhesives for colostomy devices containing inorganic materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic materials
    • A61L27/12—Phosphorus-containing materials, e.g. apatite
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • animal bone was reworked to produce hydroxyapatite similar to human bone, thereby producing a biocompatible material for bone cement free from immune rejection.
  • 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.
  • 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).
  • 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.
  • FIG 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.
  • 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).
  • SBF simulated body fluid
  • FIG. 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%].
  • 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;
  • 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.
  • a pulverizer A10, IKA-WERKE, Japan
  • the sieved animal bone powder was sieved Scientific, Korea) was used to make animal bone with particle size below 100 ⁇ m.
  • 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.
  • 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.
  • Scanning electron microscopy (SEM; JSM-5410LV, JEOL, Japan) was used to observe the surface of sintered animal bone and bone cement.
  • SEM Scanning electron microscopy
  • a particle size analyzer (Mastersizer, Malvern Instruments Ltd, UK) was used.
  • 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 ⁇ m for pig bone and 56-83 ⁇ m for horse bone.
  • 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.
  • 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).
  • 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.
  • the height of the peak increased and the width decreased, indicating a more pointed shape than that of the hydroxyapatite.
  • pig and horse bone powder have higher crystallinity than commercially available hydroxyapatite.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ml / 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.
  • 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.
  • bone cement 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.
  • 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.
  • 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.
  • pig bone cement showed faster curing than that of horse bone cement.
  • 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.
  • 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, .
  • 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.
  • 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.
  • FIG. 1 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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 °C, humidity of 100% and of 5% CO 2 concentration of the environment Respectively.
  • DMEM Dulbeco's modified eagle's minimum essential medium
  • FBS fetal bovine serum
  • 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).
  • a kit Cell titer 96 non radioactive cell proliferation assay, Promega.
  • 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.
  • 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 ⁇ l of MTT solution was added to each well and allowed to react for 4 hours.
  • the culture medium was removed for a certain period of time, washed three times with PBS, and then immobilized for SEM imaging.
  • 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.
  • 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.
  • HMDS hexamethyldisilazane
  • 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.
  • 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.
  • 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.
  • Figure 23 shows the results of histological analysis through H & E staining.
  • no change was observed in the bone defect site where no bone cement was inserted, such as formation of new tissue.
  • 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).
  • more dense tissue was formed in the bone tissue grafted with bone cement using nanoparticles than the bone cement using microparticles.
  • 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 .

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  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Materials For Medical Uses (AREA)

Abstract

La présente invention porte sur un nanociment osseux céramique qui contient de la poudre d'os d'animaux et sur un procédé de préparation de celui-ci, et sur un support osseux poreux qui contient de la nanopoudre d'os de cheval. Le nanociment osseux céramique contenant de la poudre d'os d'animaux de l'invention est un composant à base d'hydroxyapatite similaire à un vrai os et présente une forte résistance, un temps de durcissement court, une biocompatibilité et d'excellentes caractéristiques pour favoriser la régénération de tissu osseux.
PCT/KR2011/000422 2011-01-21 2011-01-21 Nanociment osseux céramique utilisant des os d'animaux et son procédé de préparation WO2012099285A1 (fr)

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WO2012099285A9 true WO2012099285A9 (fr) 2013-05-23

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US6696073B2 (en) * 1999-02-23 2004-02-24 Osteotech, Inc. Shaped load-bearing osteoimplant and methods of making same
DK1366774T3 (da) * 2002-05-29 2007-10-08 Heraeus Kulzer Gmbh Knoglecementblanding og röntgenkontrastmiddel

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