CN115784652B - Rotif bone source biphasic calcium phosphate bioactive bone repair material, and preparation method and application thereof - Google Patents
Rotif bone source biphasic calcium phosphate bioactive bone repair material, and preparation method and application thereof Download PDFInfo
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- Materials For Medical Uses (AREA)
Abstract
The invention belongs to the field of bioactive bone repair materials, and relates to a preparation method of a tilapia bone source biphasic calcium phosphate bioactive bone repair material. The invention takes waste tilapia spine vertebrae as raw materials, and prepares the bone repair material containing spongy biphase calcium phosphate bioactive bone by a gradient heating sintering method for the first time, wherein the main components of the bone repair material are beta-tricalcium phosphate (beta-TCP) and Hydroxyapatite (HAP). The invention has wide sources of raw materials, recycling of tilapia bone wastes and low cost. The waste tilapia bone is converted into the high-value biomedical bone repair material, and compared with the traditional single hydroxyapatite bone repair material, the biphasic calcium phosphate is obtained, so that the material has higher bioactivity and osteoinductive property.
Description
Technical Field
The invention belongs to the field of bioactive bone repair materials, and relates to a preparation method and application of a tilapia bone-derived biphasic calcium phosphate bioactive bone repair material.
Background
Biological materials based on calcium phosphate, particularly hydroxyapatite, are widely used for repairing defects such as bone fractures due to their good osteoinductive and osteoconductive properties. At present, synthetic hydroxyapatite is clinically used as a bone repair material, but the preparation process of the synthetic hydroxyapatite is complex, and other chemical reagents are required to be introduced. Meanwhile, the synthesized hydroxyapatite has a certain difference in physical and chemical properties with the natural hydroxyapatite in the human body, so that the biological activity of the synthesized hydroxyapatite is lower.
In recent years, natural calcium phosphate materials obtained from various natural materials such as bovine bones, fish scales, mineral materials, and the like have been attracting attention. The calcium phosphate material of natural origin has a more excellent biological activity than the synthetic hydroxyapatite.
However, the bovine bone commonly used in the present natural source calcium phosphate materials is a good source of calcium phosphate material. However, due to the risk of bovine bone with mad cow disease virus, the method threatens human life and health, and the search for an effective, safe and widely available biological material becomes particularly urgent.
The tilapia is a main cultured aquatic product in China, the fish bones of the tilapia are widely available and are rich in trace elements, and the tilapia is a natural source of active calcium phosphate materials with good prospects. However, in the process of processing the tilapia food, a large amount of fish bones are discarded as waste, so that the economic benefit is low, and the environment is adversely affected.
The invention provides a low-cost and high-feasibility method, and the spongy biphasic calcium phosphate bioactive bone repair material containing various microelements can be obtained by a gradient heating calcination method. The invention not only realizes the conversion from waste tilapia bones to high-value products, but also meets the requirements of green chemical industry and sustainable development strategy.
Disclosure of Invention
In view of the above, the invention provides a method for preparing a tilapia bone source biphasic calcium phosphate active material by a gradient heating sintering method. The main components of the material are hydroxyapatite and beta-calcium phosphate, and the converted biphasic calcium phosphate material has good application prospect in bone tissue engineering.
In order to achieve the above object, the present invention provides the following technical solutions:
a preparation method of a tilapia bone source biphasic calcium phosphate bioactive bone repair material, which specifically comprises the following steps:
(1) Removing organic matters: transferring the waste tilapia bones into a container, adding deionized water to submerge the fish bones, boiling, pouring the boiling liquid after boiling is finished, removing organic matters such as grease, washing with deionized water, draining and drying for later use;
(2) Gradient heating sintering: placing the dried tilapia bone in a muffle furnace, calcining in the first step to obtain a calcined bone containing a single-phase hydroxyapatite component, and calcining by raising a temperature gradient after the calcined bone is naturally cooled to obtain a calcined bone containing a biphasic calcium phosphate component;
(3) Grinding into powder: grinding the calcined bone containing the biphasic calcium phosphate component obtained in the step (2) into powder to obtain tilapia bone-derived biphasic calcium phosphate powder;
(4) Sterilizing: and (3) disinfecting, sterilizing, drying and preserving the tilapia bone-derived biphasic calcium phosphate powder obtained in the step (3) to obtain the tilapia bone-derived biphasic calcium phosphate bioactive bone repair material.
Optionally, in the step (1), the cooking is performed by an electromagnetic oven with a heating power of 240-300w and a cooking time of 15-30min, the cooking liquid is poured, and then the deionized water is washed 3-5 times and dried in a constant temperature oven at 60 ℃.
Optionally, in the step (2), the first step is calcining at 600-800 ℃ for 1h, and then raising the temperature gradient to 900-1200 ℃ for 1-4h; wherein, the temperature rising rate of the muffle furnace is 5-10 ℃/min, and the temperature reducing rate is 10 ℃/min.
Optionally, in the step (4), the sterilization mode is gamma rays, and the irradiation dose of the gamma rays is controlled to be 25-30 kGy; and after disinfection and sterilization, drying in a constant temperature oven at 60 ℃ and preserving.
It should be noted that, as the temperature of the high-temperature calcination increases, the content of β -TCP increases to some extent. However, too high a temperature may cause an increase in crystallinity of the material and a change in grain size, thereby affecting the degradation rate of the corresponding material.
Further, the single-phase hydroxyapatite bioactive material can be obtained by calcining for 1h at the temperature gradient of 600-800 ℃ in the first step, and the biphasic calcium phosphate bioactive bone repair material with HAP and beta-TCP as main components can be obtained by calcining for 1-4h at the temperature gradient of 900-1200 ℃ in the second step, as shown in fig. 4.
The invention also claims a tilapia bone-derived biphasic calcium phosphate bioactive bone repair material prepared by the method, wherein the tilapia bone-derived biphasic calcium phosphate bioactive bone repair material contains a spongy structure, and main components of the tilapia bone-derived biphasic calcium phosphate bioactive bone repair material comprise Hydroxyapatite (HAP) and beta-tricalcium phosphate (beta-TCP).
Wherein the particle size of the tilapia bone source biphasic calcium phosphate bioactive bone repair material particles is 100nm-2mm, the particle size of the tilapia bone source biphasic calcium phosphate bioactive bone repair material particles contains a plurality of microelements which are beneficial to bone tissue growth, and the mass percentage of the microelements is 0.04-1%.
The invention also claims the application of the tilapia bone source biphasic calcium phosphate bioactive bone repair material prepared by the method in preparing bone defect bone repair materials and bone tissue engineering.
Further comprises: the application of the tilapia bone source biphasic calcium phosphate active material in preparing a bone implant coating material and the application of the tilapia bone source biphasic calcium phosphate bioactive bone repair material in preparing a material of a drug carrier.
Compared with the prior art, the tilapia bone source biphasic calcium phosphate bioactive bone repair material, the preparation method and the application thereof provided by the invention have the following excellent effects:
1) Tilapia bone is a natural bone repair material source, has wide material sources and contains various microelements capable of promoting bone repair. Meanwhile, the waste tilapia bones are used as sources to obtain the natural bioactive calcium phosphate material, so that the economic benefit can be improved, and the effect of protecting the environment can be achieved.
2) In the gradient heating sintering process, the tilapia bone can retain various microelements contained in the material, and the sintered material contains a spongy porous structure, so that bone tissue regeneration is promoted more effectively, and the operation method is simple, convenient and feasible.
Among them, beta-TCP has superior biological performance, better biocompatibility and biodegradability. The biphasic calcium phosphate material obtained by the gradient calcination method has the main components of HAP and beta-TCP, and can comprehensively utilize the advantages of the two and the actions of various microelements in promoting the bone repair material, thereby remarkably improving various biological properties of the bone repair material.
In conclusion, the tilapia bone source high-valued bioactive calcium phosphate material with low cost and high feasibility is obtained.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
Fig. 1 is a diagram showing a two-phase ICP data analysis of tilapia bone.
Fig. 2 is an SEM image of a "spongy" tilapia bone-derived biphasic calcium phosphate bioactive bone repair material.
FIG. 3 is an XRD pattern of the phase obtained in the first step of gradient temperature-rising sintering.
Fig. 4 is an XRD pattern of a biphasic calcium phosphate bioactive bone repair material obtained by gradient temperature-rising sintering.
Detailed Description
The following description of embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The embodiment of the invention discloses a preparation method of a tilapia bone source biphasic calcium phosphate active bone repair material.
The present invention will be further specifically illustrated by the following examples, which are not to be construed as limiting the invention, but rather as falling within the scope of the present invention, for some non-essential modifications and adaptations of the invention that are apparent to those skilled in the art based on the foregoing disclosure.
The technical scheme of the invention will be further described below with reference to specific embodiments.
Example 1
(1) Taking waste tilapia bones, transferring the waste tilapia bones into a container, adopting an electromagnetic oven to cook, pouring cooking liquid, flushing with deionized water, draining, and drying in an oven at 60 ℃ for later use;
(2) A proper amount of fishbone is taken and placed in a muffle furnace for calcination at 800 ℃ for 1h, and after the fishbone is naturally cooled, single-phase hydroxyapatite calcined bone is obtained;
(3) Placing the single-phase hydroxyapatite calcined bone in a muffle furnace for calcining for 4 hours at 1200 ℃, and naturally cooling to obtain a biphasic calcium phosphate calcined bone;
(4) Grinding the biphasic calcium phosphate calcined bone into powder in an agate mortar;
(5) Sterilizing the obtained powder with gamma rays, controlling irradiation dose at 25-30 kGy, and then at 60deg.C
Oven drying and storing.
Example 2
(1) Taking waste tilapia bones, transferring the waste tilapia bones into a container, adopting an electromagnetic oven to cook, pouring cooking liquid, flushing with deionized water, draining, and drying in an oven at 60 ℃ for later use;
(2) A proper amount of fishbone is taken and placed in a muffle furnace for calcination at 800 ℃ for 1h, and after the fishbone is naturally cooled, single-phase hydroxyapatite calcined bone is obtained;
(3) Placing the single-phase hydroxyapatite calcined bone in a muffle furnace for calcining for 4 hours at 1100 ℃, and naturally cooling to obtain a biphasic calcium phosphate calcined bone;
(4) Grinding the biphasic calcium phosphate calcined bone into powder in an agate mortar;
(5) The obtained powder is sterilized by gamma rays, the irradiation dose is controlled to be 25-30 kGy, and then the powder is dried in a baking oven at 60 ℃ and stored.
Example 3
(1) Taking waste tilapia bones, transferring the waste tilapia bones into a container, adopting an electromagnetic oven to cook, pouring cooking liquid, flushing with deionized water, draining, and drying in an oven at 60 ℃ for later use;
(2) A proper amount of fishbone is taken and placed in a muffle furnace for calcination at 800 ℃ for 1h, and after the fishbone is naturally cooled, single-phase hydroxyapatite calcined bone is obtained;
(3) Placing the single-phase hydroxyapatite calcined bone in a muffle furnace for calcining for 4 hours at 900 ℃, and obtaining the biphasic calcium phosphate calcined bone after the single-phase hydroxyapatite calcined bone is naturally cooled;
(4) Grinding the biphasic calcium phosphate calcined bone into powder in an agate mortar;
(5) The obtained powder is sterilized by gamma rays, the irradiation dose is controlled to be 25-30 kGy, and then the powder is dried in a baking oven at 60 ℃ and stored.
Example 4
(1) Taking waste tilapia bones, transferring the waste tilapia bones into a container, adopting an electromagnetic oven to cook, pouring cooking liquid, flushing with deionized water, draining, and drying in an oven at 60 ℃ for later use;
(2) A proper amount of fishbone is taken and placed in a muffle furnace to be calcined for 1h at 700 ℃, and after the fishbone is naturally cooled, single-phase hydroxyapatite calcined bone is obtained;
(3) Placing the single-phase hydroxyapatite calcined bone in a muffle furnace for calcining for 4 hours at 1000 ℃, and naturally cooling to obtain a biphasic calcium phosphate calcined bone;
(4) Grinding the biphasic calcium phosphate calcined bone into powder in an agate mortar;
(5) The obtained powder is sterilized by gamma rays, the irradiation dose is controlled to be 25-30 kGy, and then the powder is dried in a baking oven at 60 ℃ and stored.
Example 5
(1) Taking waste tilapia bones, transferring the waste tilapia bones into a container, adopting an electromagnetic oven to cook, pouring cooking liquid, flushing with deionized water, draining, and drying in an oven at 60 ℃ for later use;
(2) A proper amount of fishbone is taken and placed in a muffle furnace to be calcined for 1h at 700 ℃, and after the fishbone is naturally cooled, single-phase hydroxyapatite calcined bone is obtained;
(3) Placing the single-phase hydroxyapatite calcined bone in a muffle furnace for calcining for 4 hours at 1200 ℃, and naturally cooling to obtain a biphasic calcium phosphate calcined bone;
(4) Grinding the biphasic calcium phosphate calcined bone into powder in an agate mortar;
(5) The obtained powder is sterilized by gamma rays, the irradiation dose is controlled to be 25-30 kGy, and then the powder is dried in a baking oven at 60 ℃ and stored.
In addition, to further illustrate the technical scheme of the invention, the inventors respectively measure the morphology and the crystal phase of the materials prepared in the examples, and specifically:
as shown in fig. 1, the bioactive material after tilapia bone transformation contains Sr, mg, si, cu, zn, fe, mn microelements, and the microelements have good effect in the bone repair process. For example, mg can improve the degradability and biological inducibility of the bioactive material, zn can activate bone metabolism related enzymes, sr and Mn can promote proliferation and differentiation of osteoblasts, and the coordination of various microelements is more beneficial to bone repair. The material obtained by the technical scheme of the invention is in a sponge shape, and the local structure is in a porous structure as shown in figure 2. The phase of the first step of sintering of tilapia bone is shown in fig. 3 and is HAP.
And as shown in fig. 4, the main components of the bioactive material obtained by gradient heating sintering conversion of tilapia bone are beta-TCP and HAP, and compared with HAP, the beta-TCP has more excellent biodegradability, so that the problem of excessively slow degradation speed of single HAP can be better improved, and the material has excellent biological properties. Therefore, the tilapia bone-converted calcium phosphate material has good application prospect in bone tissue engineering.
And, in order to further describe the specific application of the prepared tilapia bone source biphasic calcium phosphate bioactive material, the inventor also carries out the following application experiments, and the specific contents are as follows:
experimental example 1: preparation of bionic bone repair stent material
(1) Taking waste tilapia bones, transferring the waste tilapia bones into a container, adopting an electromagnetic oven to cook, pouring cooking liquid, flushing with deionized water, draining, and drying in an oven at 60 ℃ for later use;
(2) A proper amount of fishbone is taken and placed in a muffle furnace for calcination at 800 ℃ for 1h, and after the fishbone is naturally cooled, single-phase hydroxyapatite calcined bone is obtained;
(3) Placing the single-phase hydroxyapatite calcined bone in a muffle furnace for calcining for 4 hours at 1100 ℃, and naturally cooling to obtain a biphasic calcium phosphate calcined bone;
(4) Grinding the biphasic calcium phosphate calcined bone into powder in an agate mortar;
(5) Sterilizing the obtained powder by gamma rays, controlling the irradiation dose to be 25-30 kGy, then drying in a baking oven at 60 ℃, and preserving;
(6) Firstly, pre-drying a polylactic acid and tilapia bone-derived biphasic calcium phosphate material for 12 hours, taking 1g of the polylactic acid and biphasic calcium phosphate bioactive material as a solute according to the mass ratio of the polylactic acid to the biphasic calcium phosphate bioactive material of 85:15, weighing, vibrating and uniformly mixing, dissolving in 8ml of 1, 4-dioxane organic solvent, heating and stirring until the polylactic acid is completely dissolved, placing the mixed solution into a refrigerator at the temperature of minus 20 ℃ for pre-freezing for 5 hours; taking out from the refrigerator, and freeze-drying in a vacuum freeze dryer with the initial temperature of-60 ℃ for 24 hours to obtain the n-HA/PLA composite three-dimensional porous scaffold material; and finally, putting the dried test piece into absolute ethyl alcohol to be soaked for 24 hours, repeatedly washing with deionized water, drying in a constant-temperature blast drying oven for 12 hours, and preserving.
Experiment 2: preparation method of tilapia bone as bone filling material
(1) Taking waste tilapia bones, transferring the waste tilapia bones into a container, adopting an electromagnetic oven to cook, pouring cooking liquid, flushing with deionized water, draining, and drying in an oven at 60 ℃ for later use;
(2) A proper amount of fishbone is taken and placed in a muffle furnace for calcination at 800 ℃ for 1h, and after the fishbone is naturally cooled, single-phase hydroxyapatite calcined bone is obtained;
(3) Placing the single-phase hydroxyapatite calcined bone in a muffle furnace for calcining for 4 hours at 1100 ℃, and naturally cooling to obtain a biphasic calcium phosphate calcined bone;
(4) Grinding the biphasic calcium phosphate calcined bone into powder in an agate mortar;
(5) Sterilizing the obtained powder by gamma rays, controlling the irradiation dose to be 25-30 kGy, then drying in a baking oven at 60 ℃, and preserving;
(6) And (3) compounding the powder obtained in the step (5) with PLA, PLLA, PLGA, PVA, PEEK or PCL to prepare the compound bone powder filler.
Experiment 3: preparation method of HAP/beta-TCP coating
(1) Taking waste tilapia bones, transferring the waste tilapia bones into a container, adopting an electromagnetic oven to cook, pouring cooking liquid, flushing with deionized water, draining, and drying in an oven at 60 ℃ for later use;
(2) A proper amount of fishbone is taken and placed in a muffle furnace for calcination at 800 ℃ for 1h, and after the fishbone is naturally cooled, single-phase hydroxyapatite calcined bone is obtained;
(3) Placing the single-phase hydroxyapatite calcined bone in a muffle furnace for calcining for 4 hours at 1000 ℃, and naturally cooling to obtain a biphasic calcium phosphate calcined bone;
(4) Grinding the biphasic calcium phosphate calcined bone into powder in an agate mortar;
(5) Sterilizing the obtained powder by gamma rays, controlling the irradiation dose to be 25-30 kGy, then drying in a baking oven at 60 ℃, and preserving;
(6) 7700 type plasma spraying equipment adopting SG-100 type spray gun is used, and the spraying parameters are set as follows: main air (N) 2 ) The flow rate is 90L.min -1 Auxiliary gas (H) 2 ) The flow rate was 1L.min -1 The power was 81kw, the current 530A, the feed rate was 32g.min -1 The spraying distance is 90mm, the spraying times are 2, and the HAP/beta-TCP coating material is obtained.
Experiment 4: preparation method of HAP/beta-TCP sodium alginate drug-loaded stent
(1) Taking waste tilapia bones, transferring the waste tilapia bones into a container, adopting an electromagnetic oven to cook, pouring cooking liquid, flushing with deionized water, draining, and drying in an oven at 60 ℃ for later use;
(2) A proper amount of fishbone is taken and placed in a muffle furnace for calcination at 800 ℃ for 1h, and after the fishbone is naturally cooled, single-phase hydroxyapatite calcined bone is obtained;
(3) Placing the single-phase hydroxyapatite calcined bone in a muffle furnace for calcining for 4 hours at 1200 ℃, and naturally cooling to obtain a biphasic calcium phosphate calcined bone;
(4) Grinding the biphasic calcium phosphate calcined bone into powder in an agate mortar;
(5) Sterilizing the obtained powder by gamma rays, controlling the irradiation dose to be 25-30 kGy, then drying in a baking oven at 60 ℃, and preserving;
(6) Adding 10ml of deionized water into a 30ml glass beaker, slowly heating to 37 ℃ under the water bath condition, setting the rotating speed of a mechanical stirrer to 1000r/min for stirring, sequentially adding 3.5g of the double-tilapia bone source biphasic hydroxyapatite active material, 0.0058gNG (naringin) (maintained for 10 min) and 1gSA (sodium alginate), and uniformly stirring; after using an ultrasonic vibration instrument, placing the biological ink into a 50ml centrifuge tube, setting the rotating speed of a low-temperature centrifuge to 3000r/min for centrifugation, and carefully placing the biological ink into a special charging barrel of a printer to remove redundant bubbles in the material; using a needle with a specification of 0.2mm, setting the air pressure value to 0.52MPa, setting the moving speed of the spray head to 28mm/s, setting the spacing between the bracket pores to 1.2mm, and using a 3D modeling bracket program in advance in software: circular 8mm x 2mm; immediately after printing, the stent is soaked and immersed in 10% CaCl at 4 ℃ entirely 2 Crosslinking (about 10 minutes) was performed, and distilled water was washed 3 times; and finally, placing the bracket into an ultralow temperature refrigerator for defrosting, and then placing the bracket into a vacuum freeze dryer for freeze drying for later use.
Experiment 5: preparation method of HAP/beta-TCP grafted folic acid
(1) Taking waste tilapia bones, transferring the waste tilapia bones into a container, adopting an electromagnetic oven to cook, pouring cooking liquid, flushing with deionized water, draining, and drying in an oven at 60 ℃ for later use;
(2) A proper amount of fishbone is taken and placed in a muffle furnace for calcination at 800 ℃ for 1h, and after the fishbone is naturally cooled, single-phase hydroxyapatite calcined bone is obtained;
(3) Placing the single-phase hydroxyapatite calcined bone in a muffle furnace for calcining for 4 hours at 900 ℃, and obtaining the biphasic calcium phosphate calcined bone after the single-phase hydroxyapatite calcined bone is naturally cooled;
(4) Grinding the biphasic calcium phosphate calcined bone into powder in an agate mortar;
(5) Sterilizing the obtained powder by gamma rays, controlling the irradiation dose to be 25-30 kGy, then drying in a baking oven at 60 ℃, and preserving;
(6) 0.3g of tilapia bone-derived biphasic calcium phosphate powder was dissolved in 100ml of ethanol: adding a certain amount of 3-aminopropyl triethoxy into a solution of water (volume ratio is 1:1) under the protection of nitrogen and stirring at 500r/min to enable the molar ratio of the tilapia bone source biphasic calcium phosphate to the silane to be 1:4, reacting the silane for 2h, separating, treating and drying;
(7) Folic Acid (FA) 0.6g was dissolved in 28ml of N, N-dimethylimide: adding 0.186g Dicyclohexylcarbodiimide (DCC) and 0.154g hydroxysuccinimide (NHS) into a solvent with the volume ratio of dimethyl sulfoxide (DMF: DMSO) of 3:1, stirring for 12h at a medium rotating speed under the conditions of shading and nitrogen protection to fully activate FA, and removing byproducts by suction filtration to obtain folic acid activated ester solution;
(8) Adding 0.3g of reacted tilapia bone source biphase calcium phosphate powder into folic acid activated ester (molar ratio is 1:3) solution, and magnetically stirring for 24 hours under the protection of light and nitrogen; and (3) centrifugally separating the obtained product, and drying the sediment in a 60 ℃ oven for 12 hours to finally obtain HAP/beta-TCP grafted with folic acid.
Experiment 6: in vitro cytotoxicity experiments of biomimetic bone repair scaffold material, bone filler material, HAP/β -TCP coating material, HAP/β -TCP as drug carrier:
preparing a material leaching solution: firstly, the prepared bionic bone repair material, bone filling material, HAP/beta-TCP coating material and HAP-beta-TCP serving as drug carriers are immersed in a sterile DMEM culture medium according to the standard of 20mg/ml, centrifuged at 120rpm, placed in an oscillating box for incubation at 37 ℃ for 24 hours, and the supernatant is taken as leaching liquor after centrifugation. MC3T3-E1 cells were cultured with fresh DMEM medium for 24 hours, after which the medium was removed by pipetting, and the stock solution of the leaching solution and the gradient dilution (1/2, 1/4,1/8,1/16,1/32, 1/64) were added for further culture for 22 hours. The extract was diluted, a culture medium containing 10% CCK-8 (V/V) was added, incubated at 37℃for 2 hours, and absorbance at OD450nm was measured by a multifunctional microplate reader. Three replicates per group were examined for the effect of the material on cell proliferation.
Staining of live dead cells: the bionic bone repair scaffold material, bone filling material, HAP/beta-TCP coating material and HAP/beta-TCP film slide used as medicine carrier are prepared, 6 parallel samples are prepared for each group, and the slide is placed in 24 pore plates, and each pore is seeded with 2X 10 4 And (3) cells. Placing the well plate inoculated with cells and growth factors into a constant temperature cell incubator at 37deg.C and 5% CO 2 Is cultured for 1 day and 3 days respectively. The medium was aspirated and discarded after one wash with PBS. 1ml of paraformaldehyde solution was added to each well for fixation, and the mixture was left at room temperature for 30 minutes. The paraformaldehyde is sucked and discarded after PBS is washed once. 200 mu L of the prepared calcein is added to each well, the mixture is stained for 5 minutes and then is sucked off, and PBS is washed once and then is sucked off. 200 μLPI was added, and after ten minutes incubation at 37℃the cells were removed, blotted off PI, washed once with PBS and visualized using an inverted fluorescence microscope.
Proliferation experiment, preparing coating slide with bionic bone repair stent material, bone filler material, HAP/beta-TCP coating material, HAP/beta-TCP as drug carrier, preparing 6 parallel samples, placing slide in 24-well plate, and seeding 2×10 per well 4 And (3) cells. Placing the well plate after inoculating cells into a constant temperature cell incubator at 37deg.C and 5% CO 2 On average 48 hours, and is ready to be assayed for cell proliferation with CCK-8 at 1 day, 3 days and 7 days, respectively.
Experimental results show that the bionic bone repair stent material, the bone filling material, the HAP/beta-TCP coating material and the HAP/beta-TCP are non-cytotoxic when used as drug carriers.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (8)
1. The preparation method of the tilapia bone source biphasic calcium phosphate bioactive bone repair material is characterized by comprising the following steps of:
(1) Removing organic matters: transferring the waste tilapia bones into a container, adding deionized water to submerge the fish bones, boiling, pouring the boiling liquid after boiling is finished, removing organic matters such as grease, washing with deionized water, draining and drying for later use;
(2) Gradient heating sintering: placing the dried tilapia bone in a muffle furnace, calcining in the first step to obtain a calcined bone containing a single-phase hydroxyapatite component, and calcining by raising a temperature gradient after the calcined bone is naturally cooled to obtain a calcined bone containing a biphasic calcium phosphate component;
the first step of calcination is carried out at the temperature gradient of 600-800 ℃ for 1h, and then the temperature gradient is raised to 900-1200 ℃ for 1-4h; wherein, the temperature rising rate of the muffle furnace is 5-10 ℃/min, and the temperature reducing rate is 10 ℃/min;
(3) Grinding into powder: grinding the calcined bone containing the biphasic calcium phosphate component obtained in the step (2) into powder to obtain tilapia bone-derived biphasic calcium phosphate powder;
(4) Sterilizing: sterilizing the tilapia bone source biphasic calcium phosphate powder obtained in the step (3), drying and preserving to obtain the tilapia bone source biphasic calcium phosphate bioactive bone repair material;
the tilapia bone-derived biphasic calcium phosphate bioactive bone repair material has a spongy structure, and the main components of the tilapia bone-derived biphasic calcium phosphate bioactive bone repair material comprise hydroxyapatite and beta-tricalcium phosphate.
2. The method for preparing the tilapia bone source biphasic calcium phosphate bioactive bone repair material according to claim 1, wherein in the step (1), the cooking is performed by an electromagnetic oven, the heating power is 240-300w, the cooking time is 15-30min, the deionized water is washed 3-5 times after the cooking liquid is poured, and the tilapia bone source biphasic calcium phosphate bioactive bone repair material is dried in a constant-temperature oven at 60 ℃.
3. The method for preparing the tilapia bone source biphasic calcium phosphate bioactive bone repair material according to claim 1, wherein in the step (4), a sterilization mode is adopted, and the irradiation dose of gamma rays is controlled to be 25-30 kGy; and after disinfection and sterilization, drying in a constant temperature oven at 60 ℃ and preserving.
4. A tilapia bone-derived biphasic calcium phosphate bioactive bone repair material prepared according to the method of claim 1.
5. The tilapia bone-derived biphasic calcium phosphate bioactive bone repair material according to claim 4, wherein the particle size of the tilapia bone-derived biphasic calcium phosphate bioactive bone repair material particles is 100nm-2mm, and the tilapia bone-derived biphasic calcium phosphate bioactive bone repair material contains a plurality of microelements which are beneficial to bone tissue growth, and the microelements are 0.04-1% by mass.
6. Use of the tilapia bone-derived biphasic calcium phosphate bioactive bone repair material of claim 4 in bone tissue engineering.
7. The use according to claim 6, further comprising: the application of the tilapia bone source biphasic calcium phosphate bioactive bone repair material in preparing a bone implant coating material.
8. The use according to claim 6, further comprising: the application of the tilapia bone source biphasic calcium phosphate bioactive bone repair material in preparing a drug carrier material.
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| Sintering behavior and mechanical properties of hydroxyapatite ceramics prepared from Nile Tilapia (Oreochromis niloticus) bone and commercial powder for biomedical applications;Atchara Khamkongkaeo等;《Ceramics International》;第47卷(第24期);第34575-34584页 * |
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