CN115321501B - Cuttlefish bone source biphasic calcium phosphate bioactive bone repair material, preparation method and application thereof - Google Patents

Cuttlefish bone source biphasic calcium phosphate bioactive bone repair material, preparation method and application thereof Download PDF

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CN115321501B
CN115321501B CN202210869794.XA CN202210869794A CN115321501B CN 115321501 B CN115321501 B CN 115321501B CN 202210869794 A CN202210869794 A CN 202210869794A CN 115321501 B CN115321501 B CN 115321501B
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calcium phosphate
cuttlefish
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CN115321501A (en
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李萌婷
冼家如
刘朝宗
尹学琼
曹夏馨
张畅泽
郑晓非
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Hainan University
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    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B25/00—Phosphorus; Compounds thereof
    • C01B25/16—Oxyacids of phosphorus; Salts thereof
    • C01B25/26—Phosphates
    • C01B25/32—Phosphates of magnesium, calcium, strontium, or barium
    • C01B25/325—Preparation by double decomposition
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    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28—Materials for coating prostheses
    • A61L27/30—Inorganic materials
    • A61L27/32—Phosphorus-containing materials, e.g. apatite
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    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
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Abstract

The invention belongs to the field of bioactive materials, and relates to a preparation method of cuttlefish bone source biphasic calcium phosphate bioactive bone repair material. The bone repairing material with bioactivity is prepared with cuttlefish bone as material and through hydrothermal sintering, and the bone repairing material HAs main components including beta-tricalcium phosphate beta-TCP and hydroxyapatite HA. The cuttlefish bone treated by hydrothermal sintering method retains microelements such as Sr for promoting bone tissue growth and inducing bone formation 2+ 、Mg 2+ 、Si 2+ 、Cu 2+ 、Zn 2+ 、Fe 2+ . The bioactive bone repair material prepared by the invention can be used as bone powder filler, bone tissue engineering bracket, implant coating and drug release carrier, and has wide application value and obvious economic benefit.

Description

Cuttlefish bone source biphasic calcium phosphate bioactive bone repair material, preparation method and application thereof
Technical Field
The invention belongs to the field of bioactive materials, and relates to a preparation method and application of cuttlefish bone source biphasic calcium phosphate bioactive bone repair material.
Background
As the main inorganic component in the bone tissue of human body is hydroxyapatite, only the hydroxyapatite is synthesized by the bone repair material commonly used in clinic. But synthetic hydroxyapatite lacks osteoinductive and osteoconductive capabilities during bone repair. In recent years, hydroxyapatite synthesized from various natural biological materials has been a hot spot, and for example, biological calcium sources using biological materials such as fish bones, corals, and bovine bones as the hydroxyapatite have been attracting attention. Coral and bovine bone are both good bone repair raw materials, but coral is used as a national protection animal, is difficult to clinically realize large-scale application, and bovine bone has the risk of carrying mad cow disease virus and has potential threat to human life health, so that the search for a biological material which is more effective, safer and more widely available is an urgent need for people in the field of bone repair.
The cuttlefish bone itself contains Sr 2+ 、Mg 2+ 、Si 2+ 、Cu 2+ 、Zn 2+ 、Fe 2+ The researches show that the trace elements are equal to Cu 2+ 、Zn 2+ Has antibacterial and endothelial cell angiogenesis promoting effects, and Sr 2+ 、Mg 2+ 、Si 2+ 、Fe 2+ The microelements are beneficial to inhibiting the generation of osteoclasts and promoting the regeneration of osteoblasts, and play a vital role in the whole physiological function and the bone combining process. Although the transformation of cuttlefish bone source into hydroxyapatite HAs been studied, the acquisition of HA/beta-TCP biphasic calcium phosphate salt containing various microelements by cuttlefish bone source HAs not been reported.
Disclosure of Invention
In view of the above, the present invention provides a method for preparing a bioactive bone repair material of cuttlefish bone source biphasic calcium phosphate by a two-step method of hydrothermal sintering. The main components of the material are beta-TCP and HA which are similar to the components of natural bone tissue of human body, and the proportion of HA/beta-TCP two phases can be regulated by the calcination of the second step, so that the ideal biodegradability, bioactivity and biological induction performance of the repairing material are obtained.
The invention aims at the patients who need to use the bone repair material clinically, so as to individually adjust the proportion of HA/beta-TCP biphasic calcium phosphate in the repair material according to the age and bone quality of the patients.
Specifically, the invention obtains the calcium-deficient hydroxyapatite d-HA and CaPO by controlling the components and the temperature of the hydrothermal reaction system in the first stage 3 A precursor of (OH); and through the second stage of calcination, d-HA and CaPO are calcined under the high temperature condition 3 (OH) is decomposed to form beta-TCP, and d-HA and CaPO are generated with the increase of the calcination temperature 3 (OH) is decomposed more and the content of beta-TCP is increased, so that the ratio of HA to beta-TCP in the double phases can be controlled, and the bone tissue composition and structure required for repair can be obtained; in addition, the cuttlefish bone treated by hydrothermal sintering two-step method retains microelements such as Sr for promoting bone tissue growth and inducing bone formation 2+ 、Mg 2+ 、Si 2+ 、Cu 2+ 、Zn 2+ 、Fe 2+ 。
In order to achieve the above object, the present invention provides the following technical solutions:
a preparation method of cuttlefish bone source biphasic calcium phosphate bioactive bone repair material, which specifically comprises the following steps:
(1) Cleaning the bones of the black carp, oven drying, grinding into powder, sieving, and mixing with 30% H 2 O 2 After soaking in the solution for 24 hours, carrying out solid-liquid separation on the product, and drying and calcining the sample for later use;
(2) Weighing a proper amount of sample, dissolving in deionized water, performing ultrasonic dispersion, slowly dripping phosphoric acid into the deionized water, adjusting the ratio of calcium to phosphorus, stirring after titration is completed, and performing full hydrothermal reaction under high-temperature and high-pressure conditions to obtain a product for later use;
(3) And (3) performing solid-liquid separation, drying and calcining on the product obtained after the hydrothermal treatment in the step (2) to obtain the cuttlefish bone source biphasic calcium phosphate bioactive bone repair material.
Optionally, in the step (1), the centrifugal speed is 2500-6000r/min, the centrifugal time is 10-20min, and the calcination temperature is 600-1000 ℃.
Optionally, the ratio of calcium to phosphorus in the step (2) is 1-1.7, the hydrothermal reaction temperature in the step (3) is 100-200 ℃, and the reaction time is 6-15 hours; the calcination temperature in the step (3) is 600-1200 ℃, the heating rate is 10 ℃/min, and the temperature is kept for 2-5 hours.
The calcium carbonate may be converted into calcium oxide by calcination;
and the content of hydroxyapatite is increased when the hydrothermal temperature is 100-200 ℃, and HA/beta-TCP biphasic calcium phosphate can be obtained by calcining at 600-1200 ℃.
The invention also requests a biphasic calcium phosphate material prepared by the method, which contains round beta-calcium phosphate particles beta-TCP and short rod-shaped hydroxyapatite whisker HA, wherein the mass percentage of the beta-TCP is changed from 5% to 100%.
Wherein the biphasic calcium phosphate material has a micro-nano pore structure, and the pore radius is 100-1000nm.
The invention also claims the application of the biphasic calcium phosphate material prepared by the method or the biphasic calcium phosphate material as claimed in claim 5 in preparing bone defect bone repair materials and bone tissue engineering.
Further comprises: the application of the biphasic calcium phosphate material in preparing bone implant coating materials and the application of the biphasic calcium phosphate material in preparing materials of drug carriers.
Compared with the prior art, the cuttlefish 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:
the invention can adjust the proportion of beta-TCP and HA in the material by adjusting the calcining temperature after hydrothermal treatment, thereby adjusting the biodegradation rate, bioactivity and the like of the bone repair material; wherein, beta-TCP has a faster dissolution rate, so that the beta-TCP has good biodegradability and bioactivity; compared with beta-TCP, HA HAs higher mechanical strength and lower degradation rate, and can adjust the mechanical properties of the repair material. Aiming at the bone injury position and bone quality of a patient, the HA/beta-TCP composite proportion is comprehensively considered, the personalized setting is realized, the physical, chemical, mechanical and biological properties of the bone repair material are improved, and the repair function of the bone repair material is heightened.
In addition, trace elements in the cuttlefish bone for promoting bone repair are still reserved after the two-step method of hydrothermal calcination, and the trace elements exist in the form of ions and play an important role in the aspects of proliferation and mineralization of bone tissues, bone strength improvement and the like. Therefore, the invention obtains a high-value bone repair product of cuttlefish bone source with low cost.
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 reaction scheme of a two-step hydrothermal calcination process.
Fig. 2 is a scanning electron microscope image of a cuttlefish bone source biphasic calcium phosphate active bone repair material prepared by a hydrothermal calcination two-step method.
FIG. 3 XRD patterns of different proportions of HA/beta-TCP composites were obtained at a calcination temperature of 700 ℃, 800 ℃, 900 ℃, 1000 ℃.
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 cuttlefish 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) Cleaning the black fish bone, oven drying, grinding, sieving, and mixing with 30% H 2 O 2 After soaking in the solution for 24 hours, carrying out solid-liquid separation on the product, and drying and calcining the sample for later use;
(2) Weighing a proper amount of sample, dissolving in deionized water, performing ultrasonic dispersion, slowly dripping phosphoric acid into the suspension, and adjusting the ratio of calcium to phosphorus;
(3) After titration is completed, stirring for 3 hours, then placing the mixture into a hydrothermal reaction kettle, carrying out hydrothermal treatment at 180 ℃ for 10 hours, carrying out solid-liquid separation on a product obtained after the hydrothermal treatment, drying the product, and then placing the product into a medium-temperature furnace for calcining at 900 ℃ for 2 hours to obtain the cuttlefish bone source biphasic calcium phosphate bioactive bone repair material.
Example 2
(1) Cleaning the black fish bone, oven drying, grinding, sieving, and mixing with 30% H 2 O 2 After soaking in the solution for 24 hours, carrying out solid-liquid separation on the product, and drying and calcining the sample for later use;
(2) Weighing a proper amount of sample, dissolving in deionized water, performing ultrasonic dispersion, slowly dripping phosphoric acid into the suspension, and adjusting the ratio of calcium to phosphorus;
(3) After titration is finished, stirring is continued for 3 hours at 30 ℃, then the obtained mixture is placed in a hydrothermal reaction kettle, hydrothermal treatment is carried out for 10 hours at 180 ℃, and the obtained product after the hydrothermal treatment is subjected to solid-liquid separation, dried and then placed in a medium temperature furnace for calcination at 1000 ℃ for 2 hours, so that the cuttlefish bone source biphasic calcium phosphate bioactive bone repair material is obtained.
Example 3
(1) Cleaning the black fish bone, oven drying, grinding, sieving, and mixing with 30% H 2 O 2 After soaking in the solution for 24 hours, carrying out solid-liquid separation on the product, and drying and calcining the sample for later use;
(2) Weighing a proper amount of sample, dissolving in deionized water, performing ultrasonic dispersion, slowly dripping phosphoric acid into the suspension, and adjusting the ratio of calcium to phosphorus;
(3) After titration is completed, stirring for 3 hours, then placing the mixture into a hydrothermal reaction kettle, carrying out hydrothermal treatment at 110 ℃ for 10 hours, carrying out solid-liquid separation on a product obtained after the hydrothermal treatment, drying the product, and then placing the product into a medium-temperature furnace for calcining at 900 ℃ for 2 hours to obtain the cuttlefish bone source biphasic calcium phosphate bioactive bone repair material.
Example 4
(1) Cleaning the black fish bone, oven drying, grinding, sieving, and mixing with 30% H 2 O 2 After soaking in the solution for 24 hours, carrying out solid-liquid separation on the product, and drying and calcining the sample for later use;
(2) Weighing a proper amount of sample, dissolving in deionized water, performing ultrasonic dispersion, slowly dripping phosphoric acid into the suspension, and adjusting the ratio of calcium to phosphorus;
(3) After titration is completed, stirring for 3 hours, then placing the mixture into a hydrothermal reaction kettle, carrying out hydrothermal treatment at 150 ℃ for 10 hours, carrying out solid-liquid separation on a product obtained after the hydrothermal treatment, drying the product, and then placing the product into a medium-temperature furnace for calcining at 900 ℃ for 2 hours to obtain the cuttlefish bone source biphasic calcium phosphate bioactive bone repair material.
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 figure 1, the precursor required for preparing the biphasic calcium phosphate is obtained by the hydrothermal reaction of the cuttlefish bone and the phosphoric acid in the first stage, the beta-TCP phase is gradually increased along with the increase of the calcination temperature in the second stage, HA/beta-TCP with different proportions can be obtained, and when the temperature is increased to a certain temperature, all the precursor obtained in the hydrothermal process is converted into the beta-TCP.
As can be seen from fig. 2, the powder particles are in an ellipsoidal shape after high-temperature calcination, a large number of sintering necks appear, and have a micro-nano pore structure; and FIG. 3 shows XRD patterns of the powder after hydrothermal calcination and at different calcination temperatures in the examples, and as can be seen from FIG. 3, the main components of the powder are HA and beta-TCP, and as the calcination temperature increases, the beta-TCP ratio in the two phases increases.
And, in order to further describe the specific application of the prepared cuttlefish 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 method of bone meal filler by using cuttlefish bone
(1) Cleaning cuttlefish bone, cutting into small pieces, and mixing with 30% H 2 O 2 After soaking in the solution for 24 hours, the product is subjected to solid-liquid separation, and the sample is dried and calcined for standby.
(2) Soaking the processed cuttlefish bone pieces in distilled water, and adding appropriate amount of phosphoric acid after full reaction.
(3) Soaking for 3 hours, then placing the mixture into a hydrothermal reaction kettle, carrying out hydrothermal treatment at 180 ℃ for 10 hours, and carrying out solid-liquid separation on the product after the hydrothermal treatment.
(4) Drying and calcining for 2 hours at 1000 ℃ in a medium temperature furnace.
(5) Grinding, sieving with 20 mesh sieve and 10 mesh sieve to obtain cuttlefish bone powder of 1-2 mm.
(6) And (5) compositing PEEK, PLA, PLLA, PLGA, PVA or PCL with the cuttlefish bone powder obtained in the step (5) to obtain the composite bone powder filler.
Experiment 2: preparation method of warm spraying HA/beta-TCP coating
(1) Cleaning the black fish bone, oven drying, grinding, sieving, and mixing with 30% H 2 O 2 After soaking in the solution for 24 hours, the product is subjected to solid-liquid separation, and the sample is dried and calcined for standby.
(2) Weighing a proper amount of sample, dissolving in deionized water, performing ultrasonic dispersion, slowly dripping phosphoric acid into the suspension, and adjusting the ratio of calcium to phosphorus.
(3) After titration is completed, stirring for 3 hours, then placing the mixture into a hydrothermal reaction kettle, carrying out hydrothermal treatment at 180 ℃ for 10 hours, and carrying out solid-liquid separation on the product after the hydrothermal treatment.
(4) Drying and calcining for 2 hours at 900 ℃ in a medium temperature furnace.
(5) Adopting a CH-2000 type supersonic flame spraying system, sampling, adjusting the spraying parameter of propane pressure to be 0.4MPa, the flow to be 15slpm, the oxygen pressure to be 0.5MPa, the flow to be 60slpm, the nitrogen pressure to be 0.6MPa respectively, the flow to be 40slpm, the moving speed of a spray gun to be 1500mm/s, and the spraying distance to be 30mm, thereby obtaining the HA/beta-TCP/Ti matrix bone repair material.
Experiment 3: preparation method of HA/beta-TCP material loaded baicalin
(1) Cleaning the black fish bone, oven drying, grinding, sieving, and mixing with 30% H 2 O 2 After soaking in the solution for 24 hours, the product is subjected to solid-liquid separation, and the sample is dried and calcined for standby.
(2) Weighing a proper amount of sample, dissolving in deionized water, performing ultrasonic dispersion, slowly dripping phosphoric acid into the suspension, and adjusting the ratio of calcium to phosphorus.
(3) After titration is completed, stirring for 3 hours, then placing the mixture into a hydrothermal reaction kettle, carrying out hydrothermal treatment at 180 ℃ for 10 hours, and carrying out solid-liquid separation on the product after the hydrothermal treatment.
(4) Drying and calcining for 2 hours at 900 ℃ in a medium temperature furnace.
(5) Preparing 100-1000 mug/mL of BA solution by using PBS, putting 3mL of prepared baicalin solution into a plastic EP tube, weighing 5mg of powder obtained in the step (4), adding the powder into the solution, sealing a container, placing the container in a constant temperature culture oscillator for shaking for 24 hours, centrifuging, taking precipitate, and freeze-drying to obtain the BA-loaded HA/beta-TCP material.
Experiment 4: preparation of bone repair 3D printing stent material
(1) Cleaning the black fish bone, oven drying, grinding, sieving, and mixing with 30% H 2 O 2 After soaking in the solution for 24 hours, the product is subjected to solid-liquid separation, and the sample is dried and calcined for standby.
(2) Weighing a proper amount of sample, dissolving in deionized water, performing ultrasonic dispersion, slowly dripping phosphoric acid into the suspension, and adjusting the ratio of calcium to phosphorus.
(3) After titration is completed, stirring for 3 hours, then placing the mixture into a hydrothermal reaction kettle, carrying out hydrothermal treatment at 180 ℃ for 10 hours, and carrying out solid-liquid separation on the product after the hydrothermal treatment.
(4) Drying and calcining for 2 hours at 900 ℃ in a medium temperature furnace.
(5) Respectively soaking PEEK, PLA, PLLA, PLGA, PVA or PCL and the powder obtained in the step (4) in ethanol solution, respectively magnetically stirring for 30min, performing ultrasonic treatment for 30min, and then mixing the obtained mixed solution with a solution of 1:1, and after stirring by magnetic force for 1h and sonicating for 1h, the mixture powder was collected by filtration, washed with deionized water, and dried at 60 ℃. Fabricating a stent using Selective Laser Sintering (SLS); the laser sintering of the powder adopts the spot diameter of 500 mu m, the scanning speed of 120mm s < -1 >, the scanning line interval of 950 mu m and the layer thickness of 0.1 mm-0.2 mm; after sintering is completed, the scaffold is removed and the unsintered powder is removed with blown compressed air.
Experiment 5: porous HA/beta-TCP ceramic material prepared by sponge impregnation method
(1) Cleaning blockDrying cuttlefish bone, grinding, sieving, and mixing with 30% H 2 O 2 After soaking in the solution for 24 hours, the product is subjected to solid-liquid separation, and the sample is dried and calcined for standby.
(2) Weighing a proper amount of sample, dissolving in deionized water, performing ultrasonic dispersion, slowly dripping phosphoric acid into the suspension, and adjusting the ratio of calcium to phosphorus.
(3) After titration is completed, stirring for 3 hours, then placing the mixture into a hydrothermal reaction kettle, carrying out hydrothermal treatment at 180 ℃ for 10 hours, and carrying out solid-liquid separation on the product after the hydrothermal treatment.
(4) Drying and calcining for 5 hours at 900 ℃ in a medium temperature furnace.
(5) Mixing the powder obtained in the step (4) with a high-temperature binder (P 2 O 5 、Na 2 O、MgO、AlPO 4 CaO) according to 85:15 weight percent, adding proper distilled water, ball milling for 10 hours to prepare slurry, adding H 3 PO 4 The pH value is adjusted to be weak acid. And then impregnating the slurry with the cut high-elasticity polyurethane sponge, drying at 80 ℃, and finally, preserving heat at 880 ℃ and sintering to obtain the porous HA/beta-TCP ceramic material.
Experiment 6: in vitro cytotoxicity experiment of bone meal filler, HA/beta-TCP coating material, bone repair 3D printing stent material and HA/beta-TCP as drug release carrier
The mouse preosteoblast MC3T3-E1 used in this study was cultured in MEM medium (Hyclone, logan, UT) containing 10% fetal bovine serum (Hyclone), 100U/mL penicillin (Amresco), 100. Mu.g/mL streptomycin (Amresco, cleveland, ohio) at 37℃in a humid environment of 5% carbon dioxide.
The toxicity of bone meal filler, HA/β -TCP coating material, bone repair 3D printing scaffold material, HA/β -TCP as drug delivery vehicle to MC3T3-E1 cells was evaluated using the MTT test, the specific experimental procedure is as follows:
cytotoxicity of the material: MC3T3-E1 cells were seeded in 24-well plates at a density of 0.5X10 4 After 24 hours, the cells were washed twice with phosphate buffered saline; bone powder filler, HA/beta-TCP coating material, bone repair 3D printing stent material and HA/beta-TCP as drug release carrier sample with a proportion of 100150, 200 μg/mL were placed in 24 well plates, 1 sample per group; after incubation at 37℃for 1 day, the medium was removed, MTT at a concentration of 5mg/mL was added, and further incubation was carried out in an incubator at 37℃for 4 hours; sucking out MTT working solution, adding a proper amount of DMSO into each well, incubating for 10min on a shaker, and dividing the DMSO of each well into 3 96-well plates; absorbance was measured at 490nm using an enzyme-labeled instrument.
Live-read staining analysis: the sterilized bone powder filler, HA/beta-TCP coating material, bone repair 3D printing stent material and HA/beta-TCP are used as drug release carriers to be soaked in PBS buffer solution for 24 hours, taken out and then further soaked in a complete culture medium, and soaked in a cell incubator for 24 hours. After BMSCs are inoculated on bone powder filler, HA/beta-TCP coating material, bone repair 3D printing bracket material and HA/beta-TCP serving as a drug release carrier for 3, 5 and 7 days of surface culture, the BMSCs are subjected to Live/read staining, and the survival condition of cells is observed by adopting a Calcein/propidium iodide (Calcein-AM/PI) double-fluorescence staining method. The dyeing steps are as follows: 1) Taking 10 mu L of Calcein-AM (1 mg/mL) and 15 mu L of PI (1 mg/mL) in 5mLPBS, and preparing a dyeing working solution for light-shielding for later use; 2) Taking out bone powder filler, HA/beta-TCP coating material, bone repair 3D printing stent material and HA/beta-TCP as drug release carrier, discarding culture solution, flushing with PBS for 3 times, and adding appropriate amount of staining solution to immerse cells. After incubation for 20min in dark, flushing with PBS for 3 times again; 3) The excitation light wavelengths were set to 488nm and 561nm, the cells were observed for their death state on the scaffold surface under LCSM, red fluorescence was apoptotic cells, and green fluorescence was spread viable cells.
Experimental results show that the bone powder filler, the HA/beta-TCP coating material, the bone repair 3D printing stent material and the HA/beta-TCP are not cytotoxic when used as drug release 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 (2)

1. The preparation method of the cuttlefish bone source biphasic calcium phosphate bioactive bone repair material is characterized by comprising the following steps of:
(1) Cleaning the bones of the black carp, oven drying, grinding into powder, sieving, and mixing with 30% H 2 O 2 After soaking in the solution for 24 hours, carrying out solid-liquid separation on the product, and drying and calcining the sample for later use;
(2) Weighing a proper amount of sample, dissolving in deionized water, performing ultrasonic dispersion, slowly dripping phosphoric acid into the deionized water, adjusting the ratio of calcium to phosphorus, stirring after titration is completed, and performing full hydrothermal reaction under high-temperature and high-pressure conditions to obtain a product for later use;
(3) Performing solid-liquid separation, drying and calcination on the product obtained after the hydrothermal treatment in the step (2) to obtain the cuttlefish bone source biphasic calcium phosphate bioactive bone repair material;
in the step (1), the centrifugal speed is 2500-6000r/min, the centrifugal time is 10-20min, and the calcining temperature is 600-1000 ℃;
the ratio of calcium to phosphorus in the step (2) is 1-1.7, the hydrothermal reaction temperature in the step (3) is 100-200 ℃, and the reaction time is 6-15 hours; the calcination temperature in the step (3) is 600-1200 ℃, the heating rate is 10 ℃/min, and the temperature is kept for 2-5 hours;
the cuttlefish bone source biphasic calcium phosphate bioactive bone repair material contains round beta-calcium phosphate particles beta-TCP and short rod-shaped hydroxyapatite whisker HA, wherein the mass percentage of the beta-TCP is changed from 5% to 100%.
2. The method for preparing the cuttlefish bone-derived biphasic calcium phosphate bioactive bone repair material according to claim 1, wherein the cuttlefish bone-derived biphasic calcium phosphate bioactive bone repair material has a micro-nano pore structure, and the pore radius is 100-1000nm.
CN202210869794.XA 2022-07-22 2022-07-22 Cuttlefish bone source biphasic calcium phosphate bioactive bone repair material, preparation method and application thereof Active CN115321501B (en)

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CN102764450A (en) * 2011-07-19 2012-11-07 李亚屏 Cuttlebone transformation series porous composite bio-ceramic, its preparation method and application
WO2015034307A1 (en) * 2013-09-09 2015-03-12 주식회사 본셀바이오텍 Bone graft material using cuttlefish bones and method for preparing same

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CN102764450A (en) * 2011-07-19 2012-11-07 李亚屏 Cuttlebone transformation series porous composite bio-ceramic, its preparation method and application
WO2015034307A1 (en) * 2013-09-09 2015-03-12 주식회사 본셀바이오텍 Bone graft material using cuttlefish bones and method for preparing same

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