A strontium-doped beta-tricalcium phosphate / hydroxyapatite biphasic sintered bone

Abstract

The application provides a strontium-doped beta-tricalcium phosphate / hydroxyapatite biphasic calcined bone, and provides the strontium-doped beta-tricalcium phosphate / hydroxyapatite biphasic calcined bone, which has high porosity, a large average value of pore size, high biocompatibility, and a fast degradation rate, is used for filling and repairing of periodontal bone and maxillofacial surgical bone defects or bone insufficiency, and has a good repairing effect.

Description

Technical Field

[0001] This application relates to the field of biomedical materials technology, and in particular to a strontium-doped β-tricalcium phosphate / hydroxyapatite biphase calcined bone. Background Technology

[0002] Calcined bone is a calcium phosphate-based natural biological scaffold material, whose main component is hydroxyapatite. It has a calcium-to-phosphorus ratio and porous structure similar to human bone and is widely used for filling and repairing periodontal bone and maxillofacial bone defects or insufficient bone volume. However, it degrades slowly after implantation into the human body and is difficult to be absorbed by the body, which affects the repair effect. Therefore, developing a biological scaffold material with better repair effect for periodontal bone and maxillofacial bone defects or insufficient bone volume has become an urgent problem to be solved. Summary of the Invention

[0003] The purpose of this application is to provide a strontium-doped β-tricalcium phosphate / hydroxyapatite biphase calcined bone that has a good repair effect on periodontal bone and maxillofacial surgical bone defects or insufficient bone volume.

[0004] The first aspect of this application provides a strontium-doped β-tricalcium phosphate / hydroxyapatite biphase calcined bone, comprising strontium, β-tricalcium phosphate, and hydroxyapatite, wherein, based on the total mass of the strontium-doped β-tricalcium phosphate / hydroxyapatite biphase calcined bone, the mass percentage of strontium is 5% to 10%, the mass percentage of β-tricalcium phosphate is 20% to 50%, and the mass percentage of hydroxyapatite is 40% to 75%.

[0005] In some embodiments of this application, the porosity of the strontium-doped β-tricalcium phosphate / hydroxyapatite biphase calcined bone is 50% to 85%, and the average pore size is 100 μm to 500 μm.

[0006] In some embodiments of this application, the crystallinity of the strontium-doped β-tricalcium phosphate / hydroxyapatite biphase calcined bone is >60%.

[0007] In some embodiments of this application, the average cell viability of the strontium-doped β-tricalcium phosphate / hydroxyapatite biphasic calcined bone in the thiazolyl blue colorimetric cell assay is >85%.

[0008] A second aspect of this application provides a method for preparing strontium-doped β-tricalcium phosphate / hydroxyapatite biphase calcined bone according to any of the foregoing embodiments, comprising the following steps:

[0009] (1) Cut the beef bones into pieces with a thickness of 5mm-10mm;

[0010] (2) The bovine bone slices are mixed with a degreasing agent and shaken for 30 min to 180 min to obtain degreased bovine bone slices; wherein the mass ratio of the bovine bone slices to the degreasing agent is 1:(5-20), and the degreasing agent is selected from at least one of the following: 2wt% to 10wt% sodium dodecyl sulfate solution, 0.1wt% to 2wt% sodium hydroxide solution, 10wt% to 100wt% isopropanol solution, 20wt% to 80wt% n-propanol solution, 20wt% to 60wt% trichloromethane and 40wt% to 80wt% ethanol mixed solution, 30wt% to 50wt% dichloromethane and 50wt% to 70wt% ethanol mixed solution, or 10wt% to 65wt% petroleum ether solution;

[0011] (3) The defatted bovine bone slices described in step (2) are calcined once to obtain single-phase calcined bone. The temperature of the first calcination is 500℃-700℃ and the time is 2h-6h.

[0012] (4) The single-phase calcined bone obtained in step (3) is immersed in a mixed solution of ammonium dihydrogen phosphate and strontium hydrogen phosphate for 20-26 hours and then dried to obtain dried calcined bone. The mass ratio of the single-phase calcined bone to the mixed solution is 1:(8-15), and the mass ratio of the single-phase calcined bone, ammonium dihydrogen phosphate and strontium hydrogen phosphate is 1:(8-12):(4-8).

[0013] (5) The dried calcined bone obtained in step (4) is subjected to secondary calcination to obtain the strontium-doped β-tricalcium phosphate / hydroxyapatite biphase calcined bone. The secondary calcination temperature is 800℃-1000℃ and the time is 2h-6h.

[0014] A third aspect of this application provides the use of strontium-doped β-tricalcium phosphate / hydroxyapatite biphase calcined bone, as described in any of the foregoing embodiments, as a biological scaffold material.

[0015] This application provides a strontium-doped β-tricalcium phosphate / hydroxyapatite biphase calcined bone, its preparation method, and its uses. The strontium-doped β-tricalcium phosphate / hydroxyapatite biphase calcined bone provided in this application exhibits high porosity, a large average pore size, high biocompatibility, and a rapid degradation rate. As a biological scaffold material, it demonstrates good restorative effects for the repair of periodontal bone and maxillofacial surgical bone defects or insufficient bone volume. The preparation method provided in this application can produce the aforementioned strontium-doped β-tricalcium phosphate / hydroxyapatite biphase calcined bone, and the preparation method provided in this application is simple and highly controllable.

[0016] Of course, implementing any embodiment of this application does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of this application or 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 only some embodiments of this application. For those skilled in the art, other embodiments can be obtained based on these drawings.

[0018] Figure 1 A scanning electron microscope image of single-phase calcined bone, Comparative Example 1.

[0019] Figure 2 Here is a scanning electron microscope image of biphasic calcined bone from Example 2;

[0020] Figure 3 The images show X-ray diffraction patterns of the biphase calcined bone of Example 4 and the single-phase calcined bone of Comparative Example 1. Detailed Implementation

[0021] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art based on this application are within the scope of protection of this application.

[0022] The first aspect of this application provides a strontium-doped β-tricalcium phosphate / hydroxyapatite biphasic calcined bone, comprising strontium, β-tricalcium phosphate, and hydroxyapatite. Based on the total mass of the strontium-doped β-tricalcium phosphate / hydroxyapatite biphasic calcined bone (hereinafter referred to as biphasic calcined bone), the mass percentage of strontium is 5%–10%, the mass percentage of β-tricalcium phosphate (β-TCP) is 20%–50%, and the mass percentage of hydroxyapatite is 40%–75%. The introduction of β-TCP and strontium into the biphasic calcined bone provided by this application can accelerate its degradation rate in the human body, making the biphasic calcined bone provided by this application degrade faster in the human body than single-phase hydroxyapatite. Furthermore, it can enrich a large amount of calcium and phosphorus ions on its surface in a shorter time, thereby promoting the repair of periodontal bone and maxillofacial surgical bone defects or insufficient bone volume. Meanwhile, by controlling the mass percentages of strontium, β-tricalcium phosphate, and hydroxyapatite within the aforementioned ranges, it is helpful to improve the porosity, average pore size, crystallinity, degradation rate, and biocompatibility of biphasic calcined bone, thereby promoting the repair of periodontal bone and maxillofacial surgical bone defects or insufficient bone volume.

[0023] In some embodiments of this application, the porosity of strontium-doped β-tricalcium phosphate / hydroxyapatite biphasic calcined bone is 50%–85%, with an average pore size of 100 μm–500 μm. The aforementioned porosity and average pore size indicate a high specific surface area, which facilitates the release of cytokines into the vicinity of cells, while also providing growth space for new bone and accelerating osteoblast growth. The average pore size of 100 μm–500 μm in the biphasic calcined bone meets the growth requirements of osteoblasts and osteocytes, and also supports and guides the continuous deep growth of new bone. Therefore, using the biphasic calcined bone provided in this application as a biological scaffold material for the repair of periodontal bone and maxillofacial surgical bone defects or insufficient bone volume can improve the repair effect.

[0024] In some embodiments of this application, the crystallinity of the strontium-doped β-tricalcium phosphate / hydroxyapatite biphase calcined bone is >60%. This indicates that the biphase calcined bone provided by this application has a high degree of crystallinity, which helps to fully utilize its space-holding performance without affecting its degradation rate in vivo. This improves the mechanical properties of the biphase calcined bone and ensures its degradation rate, biocompatibility, and repair effect on periodontal bone and maxillofacial surgical bone defects or insufficient bone volume.

[0025] In some embodiments of this application, the average cell viability of strontium-doped β-tricalcium phosphate / hydroxyapatite biphasic calcined bone is >85% in the thiazolyl blue colorimetric cell assay. Thiazolyl blue, also known as MTT, is tested according to the national standard "Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Tests" (GB / T 16886.5-2017), and biocompatibility is evaluated by the measured average cell viability. A second aspect of this application provides a method for preparing strontium-doped β-tricalcium phosphate / hydroxyapatite biphasic calcined bone, comprising the following steps:

[0026] (1) Cut the beef bones into pieces with a thickness of 5mm-10mm;

[0027] (2) Mix the bovine bone slices with the defatting agent and shake for 30 min to 180 min to obtain defatted bovine bone slices; wherein the mass ratio of bovine bone slices to defatting agent is 1:(5-20), and the defatting agent is selected from at least one of the following: 2wt% to 10wt% sodium dodecyl sulfate solution, 0.1wt% to 2wt% sodium hydroxide solution, 10wt% to 100wt% isopropanol solution, 20wt% to 80wt% n-propanol solution, 20wt% to 60wt% trichloromethane and 40wt% to 80wt% ethanol mixed solution, 30wt% to 50wt% dichloromethane and 50wt% to 70wt% ethanol mixed solution, or 10wt% to 65wt% petroleum ether solution;

[0028] (3) The defatted bovine bone slices from step (2) are calcined once to obtain single-phase calcined bone. The calcination temperature is 500℃-700℃ and the time is 2h-6h.

[0029] (4) The single-phase calcined bone obtained by step (3) is immersed in a mixed solution of ammonium dihydrogen phosphate and strontium hydrogen phosphate for 20-26 hours and then dried to obtain dried calcined bone. The mass ratio of single-phase calcined bone to mixed solution is 1:(8-15), and the mass ratio of single-phase calcined bone, ammonium dihydrogen phosphate and strontium hydrogen phosphate is 1:(8-12):(4-8).

[0030] (5) The dried calcined bone obtained in step (4) is subjected to secondary calcination to obtain strontium-doped β-tricalcium phosphate / hydroxyapatite biphase calcined bone. The secondary calcination temperature is 800℃-1000℃ and the time is 2h-6h.

[0031] The above-mentioned degreasing agent used in step (2) can effectively remove lipid substances from the surface of bovine bones without affecting the structure of the bovine bones in this application.

[0032] The initial calcination in step (3) helps eliminate the immunogenicity of bovine bone while improving the porosity and support properties of monophase calcined bone. When the initial calcination temperature is too low (e.g., below 500°C) and / or the initial calcination time is too short (e.g., less than 2 hours), organic matter in the obtained monophase calcined bone is not completely removed, resulting in a large amount of residual immunogenicity, which is not conducive to the repair of periodontal bone and maxillofacial surgical bone defects or insufficient bone volume. When the initial calcination temperature is too high (e.g., above 700°C) and / or the initial calcination time is too long (e.g., greater than 6 hours), the obtained monophase calcined bone has excessively high porosity, which reduces its support properties and is not conducive to the repair of periodontal bone and maxillofacial surgical bone defects or insufficient bone volume.

[0033] In step (4), soaking the single-phase calcined bone in the mixed solution helps to enhance the adsorption of hydrogen phosphate ions and strontium ions by the single-phase calcined bone. Furthermore, the mass ratio of the single-phase calcined bone to the mixed solution within the above range can ensure that the single-phase calcined bone is completely immersed in the mixed solution, so that hydrogen phosphate ions and strontium ions can be fully adsorbed. When the soaking time is too short (e.g., less than 20 hours), the single-phase calcined bone adsorbs fewer hydrogen phosphate and strontium ions, resulting in less β-tricalcium phosphate generated during the secondary calcination process. Simultaneously, the strontium incorporation is low, which is detrimental to the repair of periodontal bone and maxillofacial surgical bone defects or insufficient bone volume. Conversely, when the soaking time is too long (e.g., more than 26 hours), the single-phase calcined bone adsorbs excessive amounts of hydrogen phosphate and strontium ions, resulting in more β-tricalcium phosphate generated during the secondary calcination process. Furthermore, the strontium incorporation is too high. When used as a biological scaffold material for the repair of periodontal bone and maxillofacial surgical bone defects or insufficient bone volume, excessive strontium can have adverse effects on the human body. For example, it can exhibit dose-dependency, reducing its therapeutic effect with long-term use. High doses of strontium can also have harmful effects on bone mineralization by reducing calcium absorption and potentially altering bone mineral properties.

[0034] The secondary calcination in step (5) helps to transform monophase calcined bone into strontium-doped β-tricalcium phosphate / hydroxyapatite biphase calcined bone. When the secondary calcination temperature is too low (e.g., below 800℃) and / or the secondary calcination time is too short (e.g., less than 2h), the resulting biphase calcined bone contains too little β-tricalcium phosphate and too little strontium, which is not conducive to the repair of periodontal bone and maxillofacial surgical bone defects or insufficient bone volume. When the secondary calcination temperature is too high (e.g., above 1000℃) and / or the secondary calcination time is too long (e.g., greater than 6h), the resulting biphase calcined bone contains too much β-tricalcium phosphate and too much strontium, which reduces the space retention performance of the biphase calcined bone and causes side effects on the human body, thus being unfavorable to the repair of periodontal bone and maxillofacial surgical bone defects or insufficient bone volume.

[0035] Biphasic calcined bone was prepared by the above preparation method. The thickness of the bovine bone slices, the amount of each substance added, the soaking time of the single-phase calcined bone, the calcination temperature and time in steps (1) to (5) were controlled within the above range. The resulting biphasic calcined bone has high porosity, large average pore size and high biocompatibility, as well as fast degradation rate. As a biological scaffold material, it can be used for the repair of periodontal bone and maxillofacial bone defects or insufficient bone volume, and has a good repair effect. Moreover, the above preparation method is simple to operate and highly controllable.

[0036] The third aspect of this application provides the use of strontium-doped β-tricalcium phosphate / hydroxyapatite biphase calcined bone as a biological scaffold material. For example, the biphase calcined bone provided in this application is used for the repair of periodontal bone and maxillofacial surgical bone defects or insufficient bone volume, and has a good repair effect.

[0037] Example

[0038] The embodiments and comparative examples provided below illustrate the implementation of this application in more detail. Various tests and evaluations were conducted according to the methods described below. Furthermore, unless otherwise specified, "parts" and "%" are quality standards.

[0039] Test methods and instruments:

[0040] Scanning electron microscopy (SEM) test:

[0041] Measurements were taken using a solar-powered scanning electron microscope (SU8010).

[0042] X-ray diffraction (XRD) test:

[0043] Measurements were taken using a Rigaku X-ray diffractometer (Smartlab) from Japan.

[0044] Porosity and pore size testing:

[0045] The results were obtained using a mercury porosimeter in accordance with the national standard "Determination of Pore Size Distribution and Porosity of Solid Materials by Mercury Porosimetry and Gas Adsorption Methods - Part 1: Mercury Porosimetry" (GB / T 21650.1-2008).

[0046] Crystallinity test:

[0047] The XRD patterns were obtained using Jade software.

[0048] Density test:

[0049] The density was measured using a density tester.

[0050] Biocompatibility testing:

[0051] According to the national standard "Biological evaluation of medical devices - Part 5: In vitro cytotoxicity test" (GB / T16886.5-2017), the average cell viability was measured by the MTT assay to evaluate biocompatibility.

[0052] Example 1

[0053] <Preparation of Strontium-doped β-tricalcium phosphate / hydroxyapatite biphase calcined bone>

[0054] (1) Cut the beef bone into pieces with a thickness of 7.5±2.5mm and crush them.

[0055] (2) Mix the bovine bone slices with the defatting agent and shake for 120 min to obtain defatted bovine bone slices; wherein the mass ratio of bovine bone slices to defatting agent is 1:10 and the defatting agent is 2wt% sodium hydroxide solution.

[0056] (3) The defatted bovine bone slices from step (2) are calcined once to obtain single-phase calcined bone. The calcination temperature T0 is 500℃ and the time t0 is 3h.

[0057] (4) The single-phase calcined bone obtained by step (3) is immersed in a mixed solution of ammonium dihydrogen phosphate and strontium hydrogen phosphate for 24 hours, and then dried to obtain the dried calcined bone. The mass ratio A of single-phase calcined bone to mixed solution is 1:10, and the mass ratio B of single-phase calcined bone, ammonium dihydrogen phosphate and strontium hydrogen phosphate is 1:8:4.

[0058] (5) The dried calcined bone obtained in step (4) is subjected to secondary calcination to obtain strontium-doped β-tricalcium phosphate / hydroxyapatite biphase calcined bone. The temperature T2 of the secondary calcination is 900℃ and the time t2 is 4h.

[0059] Examples 2 to 11

[0060] Except for adjusting the relevant preparation parameters according to Table 1, everything else is the same as in Example 1.

[0061] Comparative Example 1

[0062] (1) Cut the beef bone into pieces with a thickness of 7.5±2.5mm;

[0063] (2) Mix the bovine bone slices with the defatting agent and shake for 120 min; wherein the mass ratio of bovine bone slices to defatting agent is 1:10, and the defatting agent is a 2wt% sodium hydroxide solution;

[0064] (3) The defatted bovine bone slices from step (2) are calcined once to obtain single-phase calcined bone. The calcination temperature T0 is 500℃ and the time t0 is 3h.

[0065] The preparation parameters and performance tests for each embodiment are shown in Table 1.

[0066]

[0067] As can be seen from Examples 1 to 4 and Comparative Example 1, when the mass percentages of strontium, β-tricalcium phosphate, and hydroxyapatite are within the range of this application, the biphasic calcined bone has a high porosity and a large average pore size, as well as high crystallinity and density. In addition, it degrades quickly and has high biocompatibility.

[0068] Figure 1 This is a scanning electron microscope image of single-phase calcined bone from Comparative Example 1. Figure 2The image shows a scanning electron microscope image of the biphase calcined bone of Example 2. As can be seen from the image, the biphase calcined bone of Example 2 of this application has a larger pore size compared with the single-phase calcined bone of Comparative Example 1.

[0069] Figure 3 The images show X-ray diffraction patterns of the biphase calcined bone of Example 4 and the single-phase calcined bone of Comparative Example 1. As can be seen from the images, the single-phase calcined bone in Comparative Example 1 contains hydroxyapatite, while the biphase calcined bone of Example 4 of this application contains β-tricalcium phosphate and hydroxyapatite, and has a high degree of crystallinity.

[0070] As can be seen from Examples 5 to 7, by controlling the mass percentage content of strontium, β-tricalcium phosphate and hydroxyapatite within the range of this application and using the preparation method provided in this application, the porosity of the obtained biphasic calcined bone is also within the range of this application, and its porosity is high.

[0071] As can be seen from Examples 8 to 11, by controlling the temperature and time of the secondary calcination within the range of this application, the resulting biphase calcined bone has high porosity and large average pore size, as well as high crystallinity and density. In addition, it degrades quickly and has high biocompatibility.

[0072] It should be noted that, in this document, the term "comprising" or any other variation thereof is intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0073] The various embodiments in this specification are described in a related manner. For the same or similar parts between the various embodiments, refer to each other. Each embodiment focuses on describing the differences from other embodiments.

[0074] The above description is merely a preferred embodiment of this application and is not intended to limit the scope of protection of this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application are included within the scope of protection of this application.

Claims

1. A strontium-doped β-tricalcium phosphate / hydroxyapatite biphase calcined bone, comprising strontium, β-tricalcium phosphate, and hydroxyapatite, wherein, Based on the total mass of the strontium-doped β-tricalcium phosphate / hydroxyapatite biphase calcined bone, the mass percentage of strontium is 6%–10%, the mass percentage of β-tricalcium phosphate is 20%–50%, and the mass percentage of hydroxyapatite is 40%–75%; the crystallinity of the strontium-doped β-tricalcium phosphate / hydroxyapatite biphase calcined bone is >60%.

2. The strontium-doped β-tricalcium phosphate / hydroxyapatite biphase calcined bone according to claim 1 has a porosity of 50% to 85% and an average pore size of 100 μm to 500 μm.

3. The strontium-doped β-tricalcium phosphate / hydroxyapatite biphasic calcined bone according to claim 1 has an average cell viability >85% in the thiazolyl blue colorimetric cell assay.

4. A method for preparing strontium-doped β-tricalcium phosphate / hydroxyapatite biphase calcined bone according to any one of claims 1-3, comprising the following steps: (1) Cut the beef bones into pieces with a thickness of 5mm-10mm; (2) The bovine bone slices are mixed with a degreasing agent and shaken for 30 min to 180 min to obtain degreased bovine bone slices; wherein the mass ratio of the bovine bone slices to the degreasing agent is 1:(5-20), and the degreasing agent is selected from at least one of the following: 2wt% to 10wt% sodium dodecyl sulfate solution, 0.1wt% to 2wt% sodium hydroxide solution, 10wt% to 100wt% isopropanol solution, 20wt% to 80wt% n-propanol solution, 20wt% to 60wt% trichloromethane and 40wt% to 80wt% ethanol mixed solution, 30wt% to 50wt% dichloromethane and 50wt% to 70wt% ethanol mixed solution, or 10wt% to 65wt% petroleum ether solution; (3) The defatted bovine bone slices described in step (2) are calcined once to obtain single-phase calcined bone. The temperature of the first calcination is 500℃-700℃ and the time is 2h-6h. (4) The single-phase calcined bone obtained in step (3) is immersed in a mixed solution of ammonium dihydrogen phosphate and strontium hydrogen phosphate for 20-26 hours and then dried to obtain dried calcined bone. The mass ratio of the single-phase calcined bone to the mixed solution is 1:(8-15), and the mass ratio of the single-phase calcined bone, ammonium dihydrogen phosphate and strontium hydrogen phosphate is 1:(8-12):(4-8). (5) The dried calcined bone obtained in step (4) is subjected to secondary calcination to obtain the strontium-doped β-tricalcium phosphate / hydroxyapatite biphase calcined bone. The secondary calcination temperature is 800℃-1000℃ and the time is 2h-6h.

5. Use of strontium-doped β-tricalcium phosphate / hydroxyapatite biphase calcined bone according to any one of claims 1-3 for use as a biological scaffold material.

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