Rod-like cerium-loaded hydroxyapatite, and preparation method and application thereof

Abstract

The embodiment of the application discloses rod-like cerium-loaded hydroxyapatite and a preparation method and application thereof.The method comprises the following steps: hydrothermal reaction of a water solution containing a calcium source and gelatin and a water solution containing a phosphorus source and urea is carried out in a stainless steel autoclave, then the rod-like hydroxyapatite is obtained through filtration, washing and drying; the rod-like hydroxyapatite is added into a water solution containing a cerium source, stirred uniformly, dried, and high-temperature calcination is carried out when the moisture on the surface of the material is completely removed, so that the rod-like cerium-loaded hydroxyapatite is obtained. The rod-like cerium-loaded hydroxyapatite provided by the application has good antibacterial performance, good biocompatibility and high biological activity, and has a potential application prospect in the fields of oral enamel repair, enamel mineralization regeneration and bioactive coating. The preparation process of the application is simple, convenient to operate and easy to produce.

Description

A rod-shaped cerium-supported hydroxyapatite, its preparation method and application Technical Field

[0001] This invention relates to the field of oral medical technology, specifically to a rod-shaped cerium-supported hydroxyapatite, its preparation method, and its application. Background Technology

[0002] Hydroxyapatite (HAp) is a biomedical material with great application potential, playing an important role in dental prosthetics. Hydroxyapatite shares some similarities in inorganic composition and crystal structure with human hard tissue materials (such as teeth and bone), and possesses good bioactivity and biocompatibility. After implantation, it can induce tissue growth on its surface and form a good bond with bone tissue, exhibiting osteoconductivity and osteotropy. It is inert, non-toxic, and does not cause inflammation or immune rejection. Despite its excellent biocompatibility and bioactivity, hydroxyapatite lacks antibacterial properties, which severely limits its application scope.

[0003] Ca in the crystal structure of hydroxyapatite 2+ and OH - The columnar structure formed creates an ion channel parallel to the c-axis, which easily adsorbs various metal ions and may even replace Ca in the crystal. 2+ This leads to more complex structures. Therefore, metal ion doping is a good method for modifying hydroxyapatite. By doping hydroxyapatite, the biological properties of pure-phase hydroxyapatite materials can be improved. Summary of the Invention

[0004] This application is based on the inventor's understanding as follows:

[0005] Cerium (Ce) belongs to the lanthanide group III (atomic number 58) and is the most abundant rare earth metal in the Earth's crust. Cerium exists primarily in two valence states, Ce... 4+ and Ce 3+ It can rapidly convert between these two states and has the dual properties of oxidation and reduction. In recent years, it has become a research hotspot in many fields such as alloys, fluorescence, magnetism, catalysis and biomedicine. Especially in the field of medical biomaterials, more and more studies have shown that cerium-containing composite materials have antioxidant, antitumor, antibacterial and neuroprotective effects. These effects are achieved through mechanisms such as mimicking natural enzyme activity, inducing tumor cell apoptosis, inhibiting abnormal blood vessel growth, destroying bacterial cell wall cell membranes and scavenging reactive oxygen species.

[0006] Due to the excellent biocompatibility of hydroxyapatite itself, coupled with the unique biological properties of cerium, cerium-loaded hydroxyapatite, obtained by combining hydroxyapatite and cerium, not only possesses special physicochemical properties but can also be used as a widely applicable antibacterial material. Currently, cerium-loaded hydroxyapatite is generally prepared by co-precipitation. However, the applicant has discovered certain defects in cerium-loaded hydroxyapatite prepared by co-precipitation. For example, the addition of the precipitant may lead to excessively high local concentrations, resulting in agglomeration or uneven composition, which is not conducive to cerium entering the hydroxyapatite lattice. Furthermore, during the precipitation process of calcium and cerium ions, stable cerium phosphate is formed, thereby losing the active center Ce. 4+ and Ce 3+ The reversible conversion between these components reduces the antibacterial properties of the material; in addition, the conditions for obtaining cerium-loaded hydroxyapatite with a special morphology using the co-precipitation method are quite demanding; the operation may require the use of toxic and harmful organic template agents, which is not conducive to the clinical application of the material.

[0007] Therefore, this invention conducts an in-depth study on the preparation method of cerium-loaded hydroxyapatite. First, rod-shaped hydroxyapatite is obtained via a hydrothermal method, and then cerium ions are loaded onto it via an impregnation method to obtain rod-shaped cerium-loaded hydroxyapatite. On the one hand, the amount of gelatin can effectively control the morphology and structure of the hydroxyapatite; on the other hand, the impregnation method can protect the morphology of the rod-shaped hydroxyapatite from damage during the cerium loading process. A special heating program is used for drying to avoid excessively rapid heating that could cause cerium salt precipitation, thus facilitating the uniform impregnation of cerium ions into the pores of the hydroxyapatite. The parallel-arranged rod-shaped cerium-loaded hydroxyapatite provided by this invention is similar to the parallel-arranged hydroxyapatite in tooth structure, possessing both excellent antibacterial properties and bioactivity, and has potential applications in oral enamel restoration, enamel mineralization regeneration, and bioactive coatings.

[0008] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0009] According to a first aspect of the present invention, the present invention provides a method for preparing rod-shaped cerium-supported hydroxyapatite, the method comprising:

[0010] An aqueous solution containing calcium source and gelatin and an aqueous solution containing phosphorus source and urea were subjected to a hydrothermal reaction in a stainless steel autoclave. After filtration, washing, and drying, rod-shaped hydroxyapatite was obtained.

[0011] Rod-shaped hydroxyapatite was added to an aqueous solution containing a cerium source, stirred until homogeneous, dried, and then calcined at high temperature when all moisture on the surface of the material was removed to obtain the rod-shaped cerium-loaded hydroxyapatite.

[0012] Furthermore, in the aqueous solution containing calcium source and gelatin, the concentration of calcium source is 10-50 mmol / L, the concentration of gelatin is 1-20 g / L, and the calcium source is selected from one or more of calcium chloride, calcium nitrate, and calcium hydroxide;

[0013] In the aqueous solution containing phosphorus source and urea, the concentration of phosphorus source is 10-50 mmol / L and the concentration of urea is 5-200 mmol / L. The phosphorus source is selected from one or more of phosphoric acid, trisodium phosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and diammonium hydrogen phosphate.

[0014] The volume ratio of the aqueous solution containing calcium source and gelatin to the aqueous solution containing phosphorus source and urea is 1:(1-3).

[0015] Furthermore, the hydrothermal reaction is carried out at a temperature of 100–300°C for a duration of 6–48 hours.

[0016] The drying temperature is 40–100°C, and the drying time is 4–12 hours.

[0017] Furthermore, in the aqueous solution containing the cerium source, the concentration of the cerium source is 2 to 200 mmol / L, and the cerium source is cerium(III) nitrate or cerium(III) chloride;

[0018] The mass-to-volume ratio of the rod-shaped hydroxyapatite to the cerium-containing aqueous solution is 1:(5-7).

[0019] Furthermore, the drying procedure is as follows: drying is carried out sequentially at temperatures of 40℃, 50℃, 60℃, 70℃, and 80℃, and each temperature is maintained for 1 to 2 hours.

[0020] Furthermore, the conditions for the high-temperature calcination are: heating to 350-550°C at a rate of 1-10°C / min and maintaining the temperature for 2-12 hours.

[0021] Furthermore, the rod-shaped cerium-supported hydroxyapatite has an overall cluster-like structure with a length of 5–50 μm, and is composed of parallel rod-shaped hydroxyapatite crystals with a diameter of 100–500 nm.

[0022] According to a second aspect of the present invention, the present invention provides rod-shaped cerium-supported hydroxyapatite, which is prepared by the preparation method described in any of the preceding claims.

[0023] According to a third aspect of the present invention, the present invention provides the application of the rod-shaped cerium-loaded hydroxyapatite as described above in oral enamel restoration, enamel mineralization regeneration, or bioactive coatings.

[0024] The embodiments of the present invention have the following advantages:

[0025] 1) The rod-shaped cerium-loaded hydroxyapatite provided by this invention has excellent antibacterial properties, good biocompatibility, and high bioactivity, and has potential application prospects in the fields of dental enamel repair, enamel mineralization regeneration, and bioactive coatings.

[0026] 2) The preparation process of the present invention is simple, easy to operate, and easy to produce. Attached Figure Description

[0027] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0028] Figure 1 is a SEM image of rod-shaped cerium-loaded hydroxyapatite (cerium loading 5%) of Example 1 of the present invention;

[0029] Figure 2 shows the EDS mapping elemental analysis of rod-shaped cerium-loaded hydroxyapatite (cerium loading 5%) in Example 1 of the present invention;

[0030] Figure 3 shows the XRD pattern of rod-shaped cerium-loaded hydroxyapatite (cerium loading 5%) in Example 1 of the present invention. Detailed Implementation

[0031] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Unless otherwise specified, the following procedures should be performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not listed are all commercially available products.

[0033] Example 1

[0034] This embodiment provides a rod-shaped cerium-supported hydroxyapatite, the preparation method of which is as follows:

[0035] (1) Prepare 25 mL of a mixed solution containing 40 mmol / L calcium nitrate tetrahydrate and 2 g / L gelatin, and prepare 25 mL of a mixed solution containing 40 mmol / L sodium dihydrogen phosphate dihydrate and 40 mmol / L urea; mix the two solutions and stir continuously for 20 min, transfer to a 100 mL stainless steel autoclave lined with polytetrafluoroethylene, and let stand in an oven at 160 ℃ for 12 h; after natural cooling, take it out, filter it, wash it three times with deionized water and anhydrous ethanol, and dry it in an oven at 48 ℃ for 6 h to obtain rod-shaped hydroxyapatite powder.

[0036] (2) Dissolve 0.0310, 0.1549 and 0.3099 g of cerium nitrate hexahydrate in 6 mL of deionized water, add 1 g of rod-shaped hydroxyapatite powder obtained in step (1) to the solution and stir evenly; dry at 40℃, 50℃, 60℃, 70℃ and 80℃ respectively, and maintain each temperature for 1 h; finally calcine in a muffle furnace at a heating rate of 5℃ / min and maintain at 400℃ for 4 h to obtain rod-shaped cerium-loaded hydroxyapatite with cerium loading of 1%, 5% and 10% respectively.

[0037] Example 2

[0038] This embodiment provides a rod-shaped cerium-supported hydroxyapatite, the preparation method of which is as follows:

[0039] (1) Prepare 25 mL of a mixed solution containing 40 mmol / L calcium nitrate tetrahydrate and 10 g / L gelatin, and prepare 25 mL of a mixed solution containing 40 mmol / L sodium dihydrogen phosphate dihydrate and 40 mmol / L urea; mix the two solutions and stir continuously for 20 min, transfer to a 100 mL stainless steel autoclave lined with polytetrafluoroethylene, and let stand in an oven at 160 ℃ for 12 h; after natural cooling, take it out, filter it, wash it three times with deionized water and anhydrous ethanol, and dry it in an oven at 48 ℃ for 6 h to obtain rod-shaped hydroxyapatite powder.

[0040] (2) Dissolve 0.0310, 0.1549 and 0.3099 g of cerium nitrate hexahydrate in 6 mL of deionized water, add 1 g of rod-shaped hydroxyapatite powder from step (1) to the solution and stir evenly; dry at 40℃, 50℃, 60℃, 70℃ and 80℃ respectively, and maintain each temperature for 1 h; finally calcine in a muffle furnace at a heating rate of 5℃ / min and maintain at 400℃ for 4 h to obtain rod-shaped cerium-loaded hydroxyapatite with cerium loading of 1%, 5% and 10% respectively.

[0041] Example 3

[0042] This embodiment provides a rod-shaped cerium-supported hydroxyapatite, the preparation method of which is as follows:

[0043] (1) Prepare 25 mL of a mixed solution containing 40 mmol / L calcium nitrate tetrahydrate and 20 g / L gelatin; prepare 25 mL of a mixed solution containing 40 mmol / L sodium dihydrogen phosphate dihydrate and 40 mmol / L urea; mix the two solutions and stir continuously for 20 min, transfer to a 100 mL stainless steel autoclave lined with polytetrafluoroethylene, and let stand in an oven at 160 ℃ for 12 h; after natural cooling, remove and filter, wash three times with deionized water and anhydrous ethanol, and dry in an oven at 48 ℃ for 6 h to obtain rod-shaped hydroxyapatite powder.

[0044] (2) Dissolve 0.0310, 0.1549 and 0.3099 g of cerium nitrate hexahydrate in 6 mL of deionized water, add 1 g of rod-shaped hydroxyapatite powder from step (1) to the solution, and then stir evenly; dry at 40℃, 50℃, 60℃, 70℃ and 80℃ respectively, and maintain each temperature for 1 h; finally calcine in a muffle furnace at a heating rate of 5℃ / min and maintain at 400℃ for 4 h to obtain rod-shaped cerium-loaded hydroxyapatite with cerium loading of 1%, 5% and 10% respectively.

[0045] Comparative Example 1

[0046] This comparative example provides a rod-shaped hydroxyapatite without cerium loading, which is prepared by the following method:

[0047] Prepare 25 mL of a mixed solution containing 40 mmol / L calcium nitrate tetrahydrate and 2 g / L gelatin; prepare 25 mL of a mixed solution containing 40 mmol / L sodium dihydrogen phosphate dihydrate and 40 mmol / L urea; mix the two solutions and stir continuously for 20 min, then transfer to a 100 mL stainless steel autoclave lined with polytetrafluoroethylene, and let stand in an oven at 160 °C for 12 h; after natural cooling, remove and filter, wash three times with deionized water and anhydrous ethanol, dry in an oven at 48 °C for 6 h, and then calcine in a muffle furnace at a heating rate of 5 °C / min, and maintain at 400 °C for 4 h to obtain rod-shaped hydroxyapatite.

[0048] Comparative Example 2

[0049] This comparative example provides a cerium-loaded hydroxyapatite prepared by a co-precipitation method, the preparation method of which is as follows:

[0050] Prepare 25 mL of a mixed solution containing 40 mmol / L calcium nitrate tetrahydrate and 2.86 mmol / L cerium nitrate hexahydrate; prepare 25 mL of a solution containing 40 mmol / L sodium dihydrogen phosphate dihydrate; mix the two solutions, adjust the pH to 9 with sodium hydroxide, and stir continuously at 80 °C for 6 h; after natural cooling, remove the mixture, filter it, and wash it three times with deionized water and anhydrous ethanol; dry it in an oven at 80 °C for 2 h, calcine it in a muffle furnace at a heating rate of 5 °C / min, and maintain it at 400 °C for 4 h to obtain cerium-loaded hydroxyapatite with a cerium loading of 1%.

[0051] Comparative Example 3

[0052] This comparative example provides a cerium-loaded hydroxyapatite, the preparation method of which is as follows:

[0053] (1) Same as step (1) in Example 1;

[0054] (2) Dissolve 0.0310 g of cerium nitrate hexahydrate in 6 mL of deionized water, add 1 g of rod-shaped hydroxyapatite powder from step (1) to the solution, stir evenly; dry at 80 °C for 5 h; finally calcine in a muffle furnace at a heating rate of 5 °C / min and maintain at 400 °C for 4 h to obtain rod-shaped cerium-loaded hydroxyapatite with a cerium loading of 1%.

[0055] Comparative Example 4

[0056] This comparative example provides a cerium-loaded hydroxyapatite, the preparation method of which is as follows:

[0057] (1) Same as step (1) in Example 1;

[0058] (2) Dissolve 0.0310g of cerium nitrate hexahydrate in 6mL of deionized water, add 1g of rod-shaped hydroxyapatite powder from step (1) to the solution, stir evenly; dry at 40℃ for 5h; finally calcine in a muffle furnace at a heating rate of 5℃ / min, and maintain at 400℃ for 4h to obtain rod-shaped cerium-loaded hydroxyapatite with a cerium loading of 1%.

[0059] Test Example 1

[0060] Structural analysis and characterization

[0061] Figure 1 shows a SEM image of cerium-loaded hydroxyapatite (cerium loading 5%) from Example 1. It can be seen that rod-shaped cerium-loaded hydroxyapatite was successfully synthesized. The overall structure is a cluster structure composed of parallel rod-shaped cerium-loaded hydroxyapatite crystals. The diameter of the rod-shaped cerium-loaded hydroxyapatite crystals is about 200 nm, and the length of the cluster structure is about 50 μm.

[0062] Figure 2 shows the EDS mapping image of cerium-loaded hydroxyapatite (cerium loading 5%) in Example 1. It can be seen that cerium is uniformly distributed on the surface of hydroxyapatite, indicating that cerium has been successfully loaded onto hydroxyapatite.

[0063] Figure 3 shows the XRD pattern of cerium-loaded hydroxyapatite (5% cerium loading) from Example 1. It can be observed that the cerium-loaded hydroxyapatite is mainly composed of the hydroxyapatite (JCPDS#09-0432) phase, and no diffraction peaks related to cerium were found. Combined with Figure 2, this indicates that cerium was successfully loaded onto the hydroxyapatite, exhibiting a very high degree of dispersion, and that cerium is integrated into the hydroxyapatite crystals.

[0064] Test Example 2

[0065] Antibacterial properties

[0066] Materials preparation: Yeast extract peptone glucose medium (YPD), PBS buffer, materials, and the experimental equipment and instruments mentioned below were all autoclaved (121℃, 20min) for later use; the yeast extract peptone glucose medium was cooled to 60℃, poured into plates, and allowed to solidify to form solid culture dishes.

[0067] Antibacterial experiment: Candida albicans was inoculated into YPD solid medium for activation for 24 h; the activated cells were inoculated into 10 mL of YPD liquid medium and cultured overnight at 37 °C and 200 rpm in a constant temperature shaker; 1-2% of the bacterial suspension was inoculated into 50 mL of YPD liquid medium and cultured at 37 °C and 200 rpm in a constant temperature shaker, with the OD value measured by UV spectrophotometer between 0.4 and 0.5; then 1 mL of the bacterial suspension was centrifuged (5000 rpm, 10 min) to remove the supernatant; then 1.5 mL of PBS was added to the precipitate and vortexed; 1.5 mL of the well-mixed bacterial suspension was transferred to a 5 mL EP tube containing 50 mg of rod-shaped cerium hydroxyapatite material; the tube was placed in a rotating shaker and cultured for 72 h at 200 rpm; the EP tube was removed, allowed to stand for 2-3 min, and 50 μL was spread onto a plate. After incubating at 37℃ for 24 hours, the colony count on each plate was determined. Three experiments were repeated, and the colony count was calculated as the average.

[0068] The formula for calculating the antibacterial ratio is as follows:

[0069]

[0070] Wherein, A is the average colony count (CFU / sample) of the un-cerium-loaded hydroxyapatite provided in Comparative Example 1, and B is the average colony count (CFU / sample) of the cerium-loaded hydroxyapatite provided in Examples 1-3 and Comparative Examples 2-4.

[0071] The results of the antibacterial performance test are shown in Table 1.

[0072] Table 1

[0073]

[0074]

[0075] The results showed that the rod-shaped cerium-loaded hydroxyapatite provided in the embodiments of the present invention exhibits excellent antibacterial properties against Candida albicans. Among them, under the same cerium loading, Example 2 showed better antibacterial properties, indicating that its corresponding morphology possesses higher antibacterial performance.

[0076] Test Example 3

[0077] Cell activity

[0078] The activity of rod-shaped cerium-loaded hydroxyapatite with human primary gingival fibroblasts was evaluated using the CCK-8 assay. In short, human primary gingival fibroblasts (8 × 10⁶) were used to... 3 Cells / well were co-cultured with different concentrations of samples at 37°C for 24 h. CCK-8 (10 μL) was added to the cell culture medium. After 2 h, the absorbance was read at 450 nm using a microplate reader. The cell viability of the control group (without added material) was considered 100%. Cell viability is expressed as a percentage and calculated using the following formula:

[0079]

[0080] Among them, OD T The absorbance of the sample group; OD C The absorbance of the blank group is shown.

[0081] The results of the cell viability test are shown in Table 2.

[0082] Table 2

[0083]

[0084]

[0085] The activity test results of the rod-shaped cerium-loaded hydroxyapatite materials provided in Examples 1 and 3 on human primary gingival fibroblasts showed no significant difference from those in Example 2. The results indicate that the rod-shaped cerium-loaded hydroxyapatite provided in these embodiments of the present invention possesses excellent cell compatibility and cell activity.

[0086] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing rod-shaped cerium-supported hydroxyapatite, characterized in that, The method includes: a hydrothermal reaction of an aqueous solution containing a calcium source and gelatin with an aqueous solution containing a phosphorus source and urea in a stainless steel autoclave; followed by filtration, washing, and drying to obtain rod-shaped hydroxyapatite; adding the rod-shaped hydroxyapatite to an aqueous solution containing a cerium source, stirring evenly, drying, and calcining at high temperature when the surface moisture of the material is completely removed to obtain the rod-shaped cerium-supported hydroxyapatite; the drying procedure is as follows: the temperature is successively 40℃, 50℃, 60℃, 70℃, and 80℃, and each temperature is maintained for 1 to 2 hours.

2. The method for preparing rod-shaped cerium-supported hydroxyapatite according to claim 1, characterized in that, In the aqueous solution containing calcium source and gelatin, the concentration of calcium source is 10-50 mmol / L, and the concentration of gelatin is 1-20 g / L. The calcium source is selected from one or more of calcium chloride, calcium nitrate, and calcium hydroxide. In the aqueous solution containing phosphorus source and urea, the concentration of phosphorus source is 10-50 mmol / L, and the concentration of urea is 5-200 mmol / L. The phosphorus source is selected from one or more of phosphoric acid, trisodium phosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and diammonium hydrogen phosphate. The volume ratio of the aqueous solution containing calcium source and gelatin to the aqueous solution containing phosphorus source and urea is 1:(1-3).

3. The method for preparing rod-shaped cerium-supported hydroxyapatite according to claim 1, characterized in that, The hydrothermal reaction is carried out at a temperature of 100–300°C for 6–48 hours; the drying process is carried out at a temperature of 40–100°C for 4–12 hours.

4. The method for preparing rod-shaped cerium-supported hydroxyapatite according to claim 1, characterized in that, The concentration of the cerium source in the aqueous solution is 2-200 mmol / L, and the cerium source is cerium(III) nitrate or cerium(III) chloride; the mass-to-volume ratio of the rod-shaped hydroxyapatite to the aqueous solution containing the cerium source is 1:(5-7).

5. The method for preparing rod-shaped cerium-supported hydroxyapatite according to claim 1, characterized in that, The high-temperature calcination conditions are as follows: the temperature is increased to 350-550℃ at a rate of 1-10℃ / min and maintained for 2-12 hours.

6. The method for preparing rod-shaped cerium-supported hydroxyapatite according to claim 1, characterized in that, The rod-shaped cerium-supported hydroxyapatite has an overall cluster-like structure with a length of 5–50 μm, and is composed of parallel rod-shaped hydroxyapatite crystals with a diameter of 100–500 nm.

7. A rod-shaped cerium-supported hydroxyapatite, characterized in that, It is prepared by any one of claims 1 to 6.

8. The application of the rod-shaped cerium-loaded hydroxyapatite according to claim 7 in oral enamel restoration, enamel mineralization regeneration, or bioactive coating.

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