Device, method and application for preparing ultrafine nano-hydroxyapatite particles from animal bones

Abstract

The present invention belongs to the technical field of synthesis of biomedical materials, and particularly relates to a device, method and application for preparing hydroxyapatite ultrafine nanoparticles using animal bone as a raw material. The device includes: a quartz tube with both ends open, one end of the quartz tube is an inert gas inlet, the other end is an inert gas outlet, a collection device is arranged at the inert gas outlet, the pretreated animal bone is placed in the quartz tube, and an ultrasonic transducer is fixedly connected to the quartz tube surface opposite to the pretreated animal bone; the device also includes a laser, and the emission port of the laser is opposite to the pretreated animal bone. In the present invention, the pretreated animal bone is processed by using an ultrasonic field-assisted laser direct writing technique to obtain hydroxyapatite ultrafine nanoparticles with regular morphology, uniform pore size and less impurities.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical material synthesis, and particularly relates to a device, method and application for preparing hydroxyapatite ultrafine nanoparticles using animal bone as a raw material. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Hydroxyapatite (HA for short) is the main inorganic component of the bones and teeth of humans and vertebrates. In bone mass, the content of hydroxyapatite is approximately 69 wt%. Its basic unit is a nanosized needle-like apatite crystal, and its composition and structure are similar to natural bone. It is non-toxic, harmless and non-carcinogenic to the human body, has good biocompatibility, bioactivity, bioaffinity and osteoconductive effect. Its surface can undergo selective chemical reactions with the physiological environment, induce and promote bone tissue growth, and can form a strong chemical bond with bone at the interface. Therefore, it can be widely used in the repair of human hard tissue defects and is the most ideal alternative material for bones.

[0004] Currently, the research on new hydroxyapatite preparation technologies aims at better and more effective biomedical applications to make their properties closer to living bone, such as nano and monolithic structures. Nano-phase hydroxyapatite has a small particle size and a high specific surface free energy, can control the interaction of proteins, and enhance the adhesion function of osteoblasts.

[0005] Multiple methods for preparing hydroxyapatite nanoparticles are disclosed in the prior art, including wet chemical deposition method, biomimetic deposition method, sol-gel method and electrodeposition method. However, these methods have relatively high requirements for production equipment, and the finally synthesized hydroxyapatite nanoparticles have irregular morphologies, uneven pore sizes, and it is difficult to achieve the controllable preparation of hydroxyapatite ultrafine nanoparticles. Summary of the Invention

[0006] In order to overcome the above problems, the present invention provides a device, method and application for preparing hydroxyapatite ultrafine nanoparticles using animal bone as a raw material. In the present invention, the pretreated animal bone is processed by an ultrasonic field-assisted laser direct writing technique to obtain hydroxyapatite ultrafine nanoparticles with regular morphologies, uniform pore sizes and fewer impurities.

[0007] To achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] In the first aspect of the present invention, there is provided a device for preparing hydroxyapatite ultrafine nanoparticles using animal bone as a raw material, comprising:

[0009] A quartz tube with both ends open, one end of the quartz tube is an inert gas inlet, and the other end is an inert gas outlet. A collection device is provided at the inert gas outlet. Pretreated animal bone is placed inside the quartz tube, and an ultrasonic transducer is fixedly connected to the quartz tube surface opposite to the pretreated animal bone;

[0010] The device further includes a laser, and the emission port of the laser faces the pretreated animal bone.

[0011] In a second aspect of the present invention, there is provided a method for preparing hydroxyapatite ultrafine nanoparticles by using the device for preparing hydroxyapatite ultrafine nanoparticles with animal bone as a raw material, including:

[0012] (1) Using an ultrasonic field-assisted laser direct writing technique to process the pretreated animal bone to generate oxyapatite nanoparticles. Introduce an inert gas through the inert gas inlet, and with the aid of the flow of the inert gas, collect the oxyapatite nanoparticles at the collection device;

[0013] (2) Disperse the collected oxyapatite nanoparticles in water to obtain an oxyapatite nanoparticle suspension; add an alkali solution to the suspension for reaction. After the reaction is completed, perform gradient centrifugation and drying to obtain hydroxyapatite ultrafine nanoparticles.

[0014] In a third aspect of the present invention, there is provided the application of the hydroxyapatite ultrafine nanoparticles prepared by the above method in the preparation of bone repair materials.

[0015] The beneficial effects of the present invention are as follows:

[0016] (1) The laser direct writing technique uses the high energy of the laser to perform surface micro-region processing on the pretreated animal bone, so that the hydroxyapatite in the animal bone generates oxyapatite under the treatment of the high energy of the laser, and at the same time evaporates and forms a gas phase; under the action of the ultrasonic wave generated by the ultrasonic transducer, the oxyapatite crystallizes into uniform nanoparticles, and the nanoparticles vibrate and reduce the adsorption between each other. With the aid of the flow of the inert gas, the oxyapatite nanoparticles are collected at the collection device, and the oxyapatite nanoparticles are processed to form hydroxyapatite ultrafine nanoparticles.

[0017] (2) The ultrasonic wave makes the oxyapatite crystallize into uniform nanoparticles, and the nanoparticles vibrate and reduce the adsorption between each other, thereby improving the yield of the oxyapatite nanoparticles, and further improving the yield of the hydroxyapatite ultrafine nanoparticles.

[0018] (3) Use the ultrasonic field-assisted laser direct writing technique to process the pretreated animal bone to obtain oxyapatite nanoparticles with regular morphology, uniform pore size, and fewer impurities, and then generate hydroxyapatite ultrafine nanoparticles with regular morphology, uniform pore size, and fewer impurities through subsequent processing. Brief Description of the Drawings

[0019] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0020] Figure 1 It is a schematic structural diagram of the device for preparing hydroxyapatite ultrafine nanoparticles using animal bone as the raw material in Example 1; wherein 1 - quartz tube, 2 - pretreated animal bone, 3 - inert gas inlet, 4 - inert gas outlet, 5 - ultrasonic transducer, 6 - laser, 7 - collection device;

[0021] Figure 2 In it, a is untreated bovine bone, and b is cut bovine bone;

[0022] Figure 3 It is a TEM image of hydroxyapatite ultrafine nanoparticles;

[0023] Figure 4 It is an XRD image of hydroxyapatite ultrafine nanoparticles;

[0024] Figure 5 It is a Raman image of hydroxyapatite ultrafine nanoparticles. Detailed Description of the Specific Embodiments

[0025] It should be noted that the following detailed descriptions are all exemplary and are intended to provide further explanations of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0026] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0027] The first typical embodiment of the present invention provides a device for preparing hydroxyapatite ultrafine nanoparticles using animal bone as the raw material, including:

[0028] A quartz tube with both ends open, one end of the quartz tube is an inert gas inlet, the other end is an inert gas outlet, a collection device is arranged at the inert gas outlet, pre-treated animal bone is placed in the quartz tube, and an ultrasonic transducer is fixedly connected to the quartz tube surface opposite to the pre-treated animal bone;

[0029] The device further includes a laser, and the emission port of the laser is opposite to the pre-treated animal bone.

[0030] In one or more embodiments, the collection device is a filter membrane, and the filter membrane has good air permeability and can allow inert gas to escape while collecting hydroxyapatite nanoparticles.

[0031] In one or more embodiments, the laser is a nanosecond pulsed laser.

[0032] A second typical embodiment of the present invention provides a method for preparing hydroxyapatite ultrafine nanoparticles by using the device for preparing hydroxyapatite ultrafine nanoparticles with animal bone as a raw material, including:

[0033] (1) Using ultrasonic field-assisted laser direct writing technology to process the pre-treated animal bone to generate hydroxyapatite nanoparticles, introducing inert gas from the inert gas inlet, and collecting the hydroxyapatite nanoparticles at the collection device by means of the flow of the inert gas;

[0034] (2) Dispersing the collected hydroxyapatite nanoparticles in water to obtain a hydroxyapatite nanoparticle suspension; adding an alkali solution to the suspension for reaction, and performing gradient centrifugation and drying after the reaction is completed to obtain hydroxyapatite ultrafine nanoparticles.

[0035] The particle size of the hydroxyapatite ultrafine nanoparticles is 5nm - 20nm.

[0036] In one or more embodiments, the animal bone includes bovine bone, porcine bone or ovine bone.

[0037] In one or more embodiments, the pre-treatment method of the animal bone includes:

[0038] (1) Layer by layer removing the soft tissue, fascia layer, periosteum and bone marrow in the medullary cavity attached to the surface of the animal bone, and rinsing with sterile distilled water;

[0039] (2) Performing freeze-thaw cycles, then performing decellularization, removing organic matter and dehydration to obtain the pre-treated animal bone.

[0040] Preferably, in the freeze-thaw cycle, the freezing temperature is -90°C to -70°C, preferably -80°C, and the freezing time is at least 6h; the thawing temperature is 35 - 38°C, preferably 37°C.

[0041] Preferably, TritonX-100 is used for decellularization, and the mass fraction of TritonX-100 is 0.8-1.2%, preferably 1%.

[0042] Preferably, organic matter is removed by soaking in ether for 20-30 h.

[0043] Preferably, dehydration is carried out by soaking in absolute ethanol for 20-30 h.

[0044] In one or more embodiments, the wavelength of the laser is 532-1064 nm, and the pulse width is 0.4-20 ns.

[0045] In one or more embodiments, the laser power density is 0.5-15.0 J / cm 2 , the area of the pretreated animal bone to be treated is 2-6 cm 2 , and the treatment time is 30-50 min.

[0046] In one or more embodiments, the ultrasonic frequency of the ultrasonic transducer is 20-50 kHz, and the power is 20-1000 W.

[0047] In one or more embodiments, the inert gas is high-purity argon, and the ventilation rate is 100-300 mL / min.

[0048] In one or more embodiments, the alkali solution is sodium hydroxide or potassium hydroxide solution, and the concentration of sodium hydroxide or potassium hydroxide solution in the mixed solution is 0.1-0.5 mol / L.

[0049] The third typical embodiment of the present invention provides the application of the hydroxyapatite ultrafine nanoparticles prepared by the above method in the preparation of bone repair materials.

[0050] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with specific examples.

[0051] Example 1

[0052] See Figure 1 , a device for preparing hydroxyapatite ultrafine nanoparticles from animal bones, comprising:

[0053] A quartz tube 1 with both ends open, one end of the quartz tube 1 is an inert gas inlet 3, and the other end is an inert gas outlet 4. The inert gas enters the quartz tube 1 from the inert gas inlet 3 and escapes from the inert gas outlet 4; a filter membrane is provided at the inert gas outlet 4 as a collection device 7, and the pretreated animal bone 2 is placed in the quartz tube 1, and the ultrasonic transducer 5 is fixedly connected to the surface of the quartz tube 1 opposite to the pretreated animal bone 2;

[0054] The device further includes a nanosecond pulsed laser 6. The emission port of the nanosecond pulsed laser 6 faces the pretreated animal bone 2, and the laser beam emitted by the nanosecond pulsed laser 6 is perpendicularly incident on the pretreated animal bone 2.

[0055] Example 2

[0056] Provide hydroxyapatite ultrafine nanoparticles prepared by using the device for preparing hydroxyapatite ultrafine nanoparticles with animal bone as the raw material in Example 1.

[0057] In this example, the animal bone used is bovine bone ( Figure 2 as shown in a). The soft tissues, fascia layer, periosteum and bone marrow in the medullary cavity attached to the surface of the bovine bone are removed layer by layer, and the articular cartilage around the joint is removed as much as possible. It is rinsed 3 times with sterile distilled water and processed into bone blocks of 4 cm × 4 cm size with a steel saw (as Figure 2 shown in b). The bone blocks are placed in a -80 °C freezer for 24 h, and then thawed in a 37 °C water bath, and this is repeated 3 cycles. The bone blocks are subjected to decellularization treatment: the bone blocks are placed in 1% TritonX-100 and treated on a shaker at a constant speed (100 rpm) for 12 h. The decellularized bone blocks are thoroughly rinsed with sterile distilled water. The bovine bone blocks are put into a beaker, first soaked in ether for 24 h to remove grease. After pouring out the ether, they are soaked in absolute ethanol for 24 h for dehydration, and finally freeze-dried in a vacuum freeze dryer for standby.

[0058] Put the pretreated bovine bone blocks into the device in Example 1, turn on the ultrasonic transducer 5 and the laser 6, and at the same time introduce high-purity argon gas from the inert gas inlet 3. The ultrasonic frequency of the ultrasonic transducer 5 is 50 kHz and the power is 400 W. The wavelength of the nanosecond pulsed laser 6 is 532 nm, and the laser power density is 4 J / cm 2 , the area of the pretreated animal bone to be processed is 2 cm 2 , and the processing time is 45 min. The flow rate of the high-purity argon gas is 200 mL / min.

[0059] Use the ultrasonic field-assisted laser direct writing technology to process the pretreated animal bone to generate oxyapatite nanoparticles. Introduce inert gas from the inert gas inlet, and collect the oxyapatite nanoparticles at the filter membrane with the help of the inert gas flow.

[0060] Put the filter membrane with oxyapatite nanoparticles into a centrifuge tube, add pure water and ultrasonicate for 15 min to obtain a nanoparticle suspension;

[0061] Sodium hydroxide solution is added to the suspension, and the concentration of the sodium hydroxide solution in the final mixed solution is 0.1 mol / L. After sufficient mixing and reaction, gradient centrifugation is performed at 3000r and 9000r, and vacuum drying is performed for 6 hours to obtain hydroxyapatite ultrafine nanoparticles.

[0062] The hydroxyapatite ultrafine nanoparticles obtained in this example were characterized.

[0063] Figure 3 TEM image of hydroxyapatite ultrafine nanoparticles, such as Figure 3 As shown, the particle size of the hydroxyapatite ultrafine nanoparticles is in the range of 5 to 20 nm, and the particles are evenly distributed.

[0064] Figure 4 is the XRD image of hydroxyapatite ultrafine nanoparticles. Figure 4 As shown, the diffraction peaks of hydroxyapatite ultrafine nanoparticles observed at 18.17°, 21.82°, 22.90°, 25.34°, 25.87°, 31.81°, 32.91°, 34.20°, 47.15°, 49.30°, 50.83°, 52.97°, 53.10°, 54.32°, 64.26°, and 71.87° correspond to Ca 10 (PO 4 ) 6 (OH) 2 (PDF#09-0432) has the (110), (002), (201), (200), (211), (122), (300), (222), (213), (321), (402), (004), (104), (304), and (431) crystal planes. Therefore, the composition of the hydroxyapatite ultrafine nanoparticle powder obtained in this embodiment conforms to the hydroxyapatite phase.

[0065] Figure 5 Figure 5 shows the Raman image of ultrafine hydroxyapatite nanoparticles. Two obvious lattice vibration characteristic peaks of hydroxyapatite can be observed, at 244 cm-1 and 960 cm-1 respectively. -1 The Raman spectrum of hydroxyapatite nanocrystals is consistent with that of hydroxyapatite nanocrystals.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing ultrafine nanoparticles of hydroxyapatite, characterized in that: include: (1) Using ultrasonic field assisted laser direct writing technology to process pretreated animal bones to generate oxyapatite nanoparticles, the device used includes: a quartz tube with two ends open, one end of the quartz tube is an inert gas inlet, and the other end is an inert gas outlet, a collecting device is arranged at the inert gas outlet, the pretreated animal bones are placed in the quartz tube, and an ultrasonic transducer is fixedly connected to the quartz tube surface opposite to the pretreated animal bones; the device also includes a laser, and the emission port of the laser is opposite to the pretreated animal bones; the ultrasonic transducer and the laser are turned on, and inert gas is introduced from the inert gas inlet, and the oxyapatite nanoparticles are collected at the collecting device with the help of the inert gas flow; (2) dispersing the collected oxyapatite nanoparticles in water to obtain an oxyapatite nanoparticle suspension; adding an alkaline solution to the suspension to react, and after the reaction is completed, performing gradient centrifugation and drying to obtain hydroxyapatite ultrafine nanoparticles.

2. The method according to claim 1, characterized in that The collecting device is a filter membrane.

3. The method according to claim 1, characterized in that The laser is a nanosecond pulse laser.

4. The method according to claim 1, characterized in that The animal bones include cattle bones, pig bones or sheep bones.

5. The method according to claim 1, characterized in that Animal bone pretreatment methods include: (1) Remove the soft tissue, fascia, periosteum and bone marrow in the medullary cavity attached to the surface of the animal bone layer by layer, and rinse with sterile distilled water; (2) performing a freeze-thaw cycle, followed by decellularization, removal of organic matter, and dehydration to obtain pretreated animal bones.

6. The method according to claim 5, characterized in that In the freeze-thaw cycle, the freezing temperature is -90°C to -70°C, the freezing time is at least 6 hours, and the thawing temperature is 35°C to 38°C.

7. The method according to claim 6, characterized in that In the freeze-thaw cycle, the freezing temperature is -80°C and the thawing temperature is 37°C.

8. The method according to claim 5, characterized in that TritonX-100 was used for decellularization, wherein the mass fraction of TritonX-100 was 0.8~1.2%.

9. The method according to claim 8, characterized in that The mass fraction of TritonX-100 is 1%.

10. The method according to claim 5, characterized in that The organic matter was removed by ether immersion treatment, and the immersion time was 20~30 h.

11. The method according to claim 5, characterized in that Dehydration was carried out by immersion in anhydrous ethanol for 20 to 30 h.

12. The method according to claim 1, characterized in that The wavelength of the laser is 532~1064 nm and the pulse width is 0.4~20ns.

13. The method according to claim 1, characterized in that Laser power density is 0.5-15.0 J / cm 2 The area of ​​animal bones after pretreatment is 2~6 cm 2 , the processing time is 30~50 min.

14. The method according to claim 1, wherein: The ultrasonic frequency of the ultrasonic transducer is 20~50 kHz and the power is 20~1000 W.

15. The method according to claim 1, wherein: The alkali solution is a sodium hydroxide or potassium hydroxide solution, and the concentration of the sodium hydroxide or potassium hydroxide solution in the mixed solution is 0.1-0.5 mol / L.

16. Use of ultrafine nano-particles of hydroxyapatite prepared by the method according to any one of claims 1 to 15 in preparing bone repair materials.

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