A composite calcium phosphate, its preparation method and application

Abstract

The present invention discloses a complex calcium phosphate and its preparation method and application. The method comprises the following steps: (S1) reacting phytic acid with a polyhydroxy polymer to prepare a phytate polymer; (S2) mixing natural porous calcium with the phytate polymer in step (S1) to prepare a gel; and (S3) calcining the gel in step (S2) twice to prepare the complex calcium phosphate. The complex calcium phosphate of the present invention has better self-solidification properties and can be rapidly mineralized after contact with body fluids. It has a large average pore size and can serve as a bone scaffold to guide osteoblasts. In addition, micropores such as carbon dioxide and water vapor generated when organic matter in the phytate polymer is oxidized at high temperature enhance the siphoning effect of blood and body fluids.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a complex calcium phosphate and a preparation method and application thereof. Background Art

[0002] At present, animal porous cancellous bone is considered to be the best material for bone repair. Its hydroxyapatite has good biocompatibility and a loose porous structure. It not only has a high porosity but also a large pore size with an average pore size of several hundred microns. It is a good cell crawling scaffold that guides the growth of osteoblasts and promotes bone repair.

[0003] Researchers have found that synthetic hydroxyapatite cannot mimic the porous structure of natural cancellous bone, resulting in poor osteoconductivity. Furthermore, hydroxyapatite itself is non-degradable, making it difficult to form bonds with bone tissue. In recent years, various porous tricalcium phosphate scaffold materials have been developed. These materials are biodegradable in vivo, improving their degradation performance compared to hydroxyapatite. However, their porosity and pore size still fall short of the requirements for cancellous bone, resulting in poor osteoconductivity. Other natural porous materials have also been used in bone repair, with the most effective being natural coral. Its primary component is calcium carbonate, which has high porosity and an average pore size of several hundred microns. It has been directly used in bone repair materials with promising clinical therapeutic effects. However, calcium carbonate itself has poor biocompatibility and degradation. Researchers are looking to modify the surface of natural coral into calcium phosphates to improve its biodegradability and mechanical strength. However, currently modified corals are still primarily composed of calcium carbonate, which has low biodegradability, and the carbon dioxide released during degradation is detrimental to bone cell growth. Summary of the Invention

[0004] To address the problems of low porosity and poor degradation of artificially synthesized calcium phosphate, the present invention provides a complex calcium phosphate, a preparation method, and applications thereof. The method uses natural porous calcium as a base material to prepare a macroporous complex calcium phosphate, which has the characteristics of high porosity, large average pore size, good biocompatibility (relative cell survival rate can reach approximately 200%), and complete degradation (degradation can be completed in up to 3 months).

[0005] The technical solutions of the present invention are as follows:

[0006] A method for preparing complex calcium phosphate, comprising the following steps:

[0007] (S1) reacting phytic acid with a polyhydroxy polymer to prepare a phytate polymer;

[0008] (S2) mixing natural porous calcium with the phytate polymer prepared in step (S1) to prepare a gel;

[0009] (S3) calcining the gel in step (S2) twice to prepare the complex calcium phosphate.

[0010] In the method of the present invention, step (S1) is adopted to convert phytic acid into phytic acid ester polymer, which can increase the viscosity of the reaction system and reduce the acidity of the reaction system.

[0011] According to the present invention, in step (S1), the reaction of phytic acid and polyhydroxy polymer is carried out in water.

[0012] According to the present invention, in step (S1), phytic acid and polyhydroxy polymer are added in the form of aqueous solutions respectively.

[0013] According to the present invention, step (S1) specifically comprises: mixing a phytic acid aqueous solution with a polyhydroxy polymer aqueous solution to react to prepare a phytate polymer.

[0014] Specifically, the volume ratio of the phytic acid aqueous solution to the polyhydroxy polymer aqueous solution is 1:(0.8-1.5), exemplified by 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4 or 1:1.5.

[0015] Specifically, the concentration of the phytic acid aqueous solution is 2 wt% to 15 wt%, preferably 4 wt% to 10 wt%, and illustratively 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt% or 10 wt%.

[0016] Specifically, the concentration of the polyhydroxy polymer aqueous solution is 5 wt% to 15 wt%, preferably 8 wt% to 12 wt%, and illustratively 8 wt%, 9 wt%, 10 wt%, 11 wt% or 12 wt%.

[0017] According to the present invention, in step (S1), the polyhydroxy polymer is selected from at least one of polyvinyl alcohol (PVA), polyethylene glycol, and the like.

[0018] According to the present invention, in step (S2), the mixing is to soak the natural porous calcium in the phytate polymer of step (S1). Specifically, the mixing is to soak the natural porous calcium in the aqueous solution of the phytate polymer of step (S1).

[0019] According to the present invention, in step (S2), the soaking is performed under vacuum conditions, which are conducive to the adsorption of the phytate polymer on the natural porous calcium and its penetration into the interior of the natural porous calcium.

[0020] According to the present invention, in step (S2), the source of the natural porous calcium is at least one of coral, shell, cuttlefish bone, etc.

[0021] According to the present invention, in step (S2), the mass volume ratio of the natural porous calcium to the phytate polymer is 1 (g): (1 to 6) (mL), illustratively 1 (g): 1 (mL), 1 (g): 2 (mL), 1 (g): 3 (mL), 1 (g): 4 (mL), 1 (g): 5 (mL) or 1 (g): 6 (mL).

[0022] According to the present invention, in step (S2), the natural porous calcium can also be washed with a phytic acid aqueous solution before being added. There is no particular limitation on the time of washing and the concentration of the phytic acid aqueous solution, as long as the surface impurities are washed away. Exemplarily, the concentration of the phytic acid aqueous solution is 2wt% to 5wt%.

[0023] According to the present invention, step (S2) specifically comprises: soaking the natural porous calcium in the phytate polymer of step (S1) for a certain period of time, taking out the natural porous calcium and letting it stand to prepare the gel.

[0024] According to the present invention, in step (S2), the soaking time is 5 min to 40 min, preferably 10 min to 30 min.

[0025] According to the present invention, in step (S2), the standing time is 1 h to 48 h, preferably 16 h to 30 h.

[0026] According to the present invention, step (S2) further comprises heating the gel after standing to prepare a xerogel. Preferably, the heating temperature is 30 to 80°C, preferably 40 to 70°C, and the heating time is 6 to 48 hours, preferably 10 to 16 hours.

[0027] In step (S2) of the present invention, the phytic acid ester polymer is mixed with the natural porous calcium, immersed, and allowed to stand. The phytic acid groups chelate with the calcium carbonate and adhere to the porous surface of the natural porous calcium, forming a gel. During continued heating, the phytic acid and the polyhydroxy polymer continue to react to form a xerogel, so that the calcium phytate generated by the reaction of the phytic acid molecules with the calcium carbonate is fixed in the xerogel network.

[0028] According to the present invention, the two calcinations in step (S3) are: a first calcination at a temperature lower than 825°C, and a second calcination at a temperature higher than 825°C.

[0029] According to the present invention, in step (S3), the temperature of the first calcination is 300-820°C, preferably 500-800°C; the time of the first calcination is 1h-6h, preferably 2h-5h.

[0030] According to the present invention, in step (S3), the temperature of the second calcination is 825-1700° C., preferably 900-1500° C.; the time of the second calcination is 1 h-10 h, preferably 2 h-8 h.

[0031] According to the present invention, step (S3) specifically comprises: calcining the gel (specifically, dry gel) in step (S2) for the first time at a temperature below 825° C., mixing the product after the first calcination with the phytate polymer in step (S1), and repeating the soaking, standing, heating, and first calcination processes multiple times, and then calcining for the second time at a temperature above 825° C. to prepare the complex calcium phosphate.

[0032] Exemplarily, the step (S3) is specifically as follows: calcining the dry gel in step (S2) at a temperature less than 825°C for the first time, mixing and soaking the product after the first calcination with the phytate polymer in step (S1), taking out the product after the first calcination, standing, heating, preparing the dry gel, and calcining the dry gel at a temperature less than 825°C, repeating the above soaking, standing, heating, and first calcination process, and then calcining for the second time at a temperature above 825°C to prepare the complex calcium phosphate.

[0033] Preferably, the number of repetitions in step (S3) is 2-5 times.

[0034] In step (S3) of the present invention, the first calcination (i.e., calcination at a temperature lower than the decomposition temperature of calcium carbonate) causes the organic components in the phytate polymer to be oxidized and decomposed into carbon dioxide and water, causing the natural porous calcium to generate hydroxyapatite and calcium phosphate. The product after the first calcination is repeatedly subjected to the process of soaking the phytate polymer, standing, heating, and then calcining at a temperature lower than 825°C, so that the natural porous calcium reacts fully to generate hydroxyapatite and calcium phosphate. The second calcination causes the residual natural porous calcium in the reaction system to react with the hydroxyapatite to generate tetracalcium phosphate. At the same time, a small amount of β-tricalcium phosphate undergoes a phase transformation at high temperature to generate α-tricalcium phosphate, completely consuming the natural porous calcium in the reaction system to obtain a complex phase calcium phosphate, which is mainly composed of β-tricalcium phosphate and tetracalcium phosphate, mixed with a small amount of α-tricalcium phosphate and hydroxyapatite.

[0035] The present invention uses a phytate polymer prepared from phytic acid and a polyhydroxy polymer as a phosphorus source, wherein the phosphate functional groups on the phytic acid can react with natural porous calcium, gradually acid-etching and penetrating the natural porous calcium structure. Through the chelation effect of the phytic acid and the synergistic bonding effect of the polyhydroxy polymer, hydroxyapatite and calcium phosphate are formed in situ. Then, a first calcination is performed to remove organic matter to generate hydroxyapatite and calcium phosphate. The above process (i.e., soaking the first calcination product with the phytate polymer, reacting, calcining to remove organic matter, etc.) is repeated to replace the natural porous calcium with a calcium phosphate salt mainly composed of hydroxyapatite. Finally, a second calcination (i.e., at a temperature above the melting point of calcium carbonate) is performed to react the residual calcium carbonate with the hydroxyapatite and other substances to generate tetracalcium phosphate. At the temperature of the second calcination, a small amount of β-tricalcium phosphate undergoes a phase transformation to generate α-tricalcium phosphate, thereby converting the natural porous calcium into a macroporous complex phase calcium phosphate.

[0036] As an exemplary embodiment of the present invention, the preparation method of the complex calcium phosphate specifically comprises the following steps:

[0037] 1) reacting phytic acid with a polyhydroxy polymer (e.g., polyvinyl alcohol) to obtain a phytate polymer;

[0038] 2) mixing and soaking the coral particles with the phytate polymer prepared in step 1), allowing the phytate polymer to completely penetrate the porous structure of the coral particles under vacuum conditions, and removing the coral particles and allowing them to stand to obtain a gel;

[0039] 3) heating and evaporating the water in the coral particles in step 2) to form a xerogel;

[0040] 4) calcining the dry gel in step 3) at a temperature less than 825° C. for the first time;

[0041] 5) Cooling the product after the first calcination in step 4) and repeating the above steps 2) to 4) again;

[0042] 6) Rapidly raising the calcination temperature to above 825° C. for a second calcination to obtain the complex calcium phosphate.

[0043] The present invention also provides a complex calcium phosphate, which is obtained by adopting the above preparation method.

[0044] According to the present invention, the complex calcium phosphate includes at least one of β-tricalcium phosphate and tetracalcium phosphate.

[0045] According to the present invention, the complex calcium phosphate further comprises hydroxyapatite and / or α-tricalcium phosphate.

[0046] According to the present invention, the porosity of the complex calcium phosphate is 35%-88%.

[0047] According to the present invention, the average pore size of the complex calcium phosphate is 50 μm-350 μm.

[0048] The present invention also provides application of the complex calcium phosphate in bone scaffolds.

[0049] Beneficial effects of the present invention:

[0050] The invention uses phytate polymer formed by the reaction of phytic acid and polyhydroxy polymer as a phosphorus source, acid-leaches natural porous calcium, causes the phosphate functional groups in the phytic acid to react with part of the calcium carbonate and form a chelate reaction, and adheres a layer of phytate polymer to the surface of the natural porous calcium. The natural porous calcium is then decomposed through a first calcination and a second calcination to simultaneously generate a composite calcium phosphate mixed with tetracalcium phosphate, α-tricalcium phosphate, β-tricalcium phosphate and hydroxyapatite. The macroporous composite calcium phosphate has the characteristics of high porosity (35% to 88%) and large average pore diameter (50 to 350 μm), contains four components of hydroxyapatite, α-tricalcium phosphate, β-tricalcium phosphate and tetracalcium phosphate, and has excellent biocompatibility, osteoconductivity and degradability.

[0051] Unlike traditional tricalcium phosphate and hydroxyapatite, the complex calcium phosphate of the present invention has better self-solidification properties and can be rapidly mineralized after contact with body fluids. Its average pore size is large, which can serve as a bone scaffold to guide osteoblasts. In addition, the micropores such as carbon dioxide and water vapor generated when organic matter in the phytate polymer is oxidized at high temperature enhance the siphoning effect of blood and body fluids.

[0052] The invention is suitable for preparing macroporous complex phase calcium phosphate using coral, shell, cuttlefish bone and the like as raw material sources of natural porous calcium, wherein the main component of cuttlefish bone is hydroxyapatite. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is the XRD pattern of the complex calcium phosphate in Example 1.

[0054] Figure 2 This is the SEM image of the complex calcium phosphate in Example 1.

[0055] Figure 3 This is the XRD pattern of the complex calcium phosphate in Example 2.

[0056] Figure 4 This is the XRD pattern of the complex calcium phosphate in Example 3.

[0057] Figure 5 This is the XRD pattern of the complex calcium phosphate in Example 4.

[0058] Figure 6 This is the XRD pattern of the sample of the complex calcium phosphate in Test Example 1 after being deposited in SBF solution for 5 hours.

[0059] Figure 7 This is the FTIR graph of the sample after the complex calcium phosphate in Test Example 1 was deposited in SBF solution for 5 hours.

[0060] Figure 8 This is a test diagram of the compatibility of complex calcium phosphate and osteoblasts in Test Example 3.

[0061] Figure 9 This is a histological section of the rabbit femoral condyle implanted with the complex calcium phosphate in Test Example 3 for 3 months. DETAILED DESCRIPTION

[0062] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0063] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0064] Example 1

[0065] First, 5 g of crushed coral particles were placed in a 5 wt% phytic acid solution and washed for 1 minute, and then taken out for use; then 10 mL of a 10 wt% phytic acid aqueous solution and 10 mL of a 10 wt% polyvinyl alcohol (PVA) aqueous solution were mixed and stirred evenly to obtain a phytate polymer, and the washed coral particles were added and vacuum-soaked for 10 minutes (soaking). The coral particles were taken out and placed in a culture dish and allowed to stand for 24 hours (standing) to obtain a gel, which was then placed in a 60°C oven for drying for 12 hours (drying) to obtain a dry gel, which was then placed in a muffle furnace and calcined at 800°C for 2 hours (calcining). The gel was taken out and the soaking-standing-drying-calcining process was repeated twice, and finally calcined at 1500°C for 4 hours to obtain a complex calcium phosphate, which includes hydroxyapatite, α-tricalcium phosphate, β-tricalcium phosphate and tetracalcium phosphate; its XRD is as follows: Figure 1 As shown, the surface morphology is Figure 2 shown.

[0066] Example 2

[0067] First, 4 g of crushed shell particles were placed in a 3 wt % phytic acid solution and washed for 1 minute, and then taken out for use; then 10 mL of a 6 wt % phytic acid aqueous solution and 10 mL of a 10 wt % polyvinyl alcohol (PVA) aqueous solution were mixed and stirred evenly to obtain a phytate polymer, and the cleaned coral particles were added and vacuum-soaked for 20 minutes (soaking), taken out and placed in a culture dish, and allowed to stand for 24 hours (standing) to obtain a gel, which was placed in a 60° C. oven for drying for 12 hours (drying) to obtain a dry gel, which was then placed in a muffle furnace and calcined at 800° C. for 3 hours (calcining), taken out, and the soaking-standing-drying-calcining process was repeated 3 times, and finally calcined at 1500° C. for 2 hours to obtain a complex calcium phosphate, which includes hydroxyapatite (HA), α-tricalcium phosphate, β-tricalcium phosphate and tetracalcium phosphate; its porosity is 51%, the pore size is 55-200 μm, and the XRD is as follows: Figure 3 shown.

[0068] Example 3

[0069] First, 3 g of crushed cuttlebone particles were placed in a 2 wt % phytic acid solution and washed for 1 minute, and then taken out for use; then 10 mL of a 4 wt % phytic acid aqueous solution and 10 mL of a 10 wt % polyvinyl alcohol (PVA) aqueous solution were mixed and stirred uniformly to obtain a phytate polymer, and the washed cuttlebone particles were added and vacuum-soaked for 15 minutes (soaking), taken out and placed in a culture dish, and allowed to stand for 24 hours (standing) to obtain a gel, which was then placed in a 60° C. oven for drying for 12 hours (drying) to obtain a dry gel, which was then placed in a muffle furnace and calcined at 800° C. for 2 hours (calcining), taken out, and the soaking-standing-drying-calcining process was repeated 3 times, and finally calcined at 1500° C. for 4 hours to obtain a complex calcium phosphate, which includes hydroxyapatite, α-tricalcium phosphate and β-tricalcium phosphate; its porosity is 60%, the pore size is 60 to 230 μm, and the XRD results are as follows: Figure 4 shown.

[0070] Example 4

[0071] First, 3 g of crushed cuttlebone particles were placed in a 2 wt % phytic acid solution and washed for 1 minute, and then taken out for use; then 10 mL of a 4 wt % phytic acid aqueous solution and 10 mL of a 10 wt % polyvinyl alcohol (PVA) aqueous solution were mixed and stirred evenly to obtain a phytate polymer, and the washed coral particles were added and vacuum-soaked for 15 minutes (soaking), taken out and placed in a culture dish, and allowed to stand for 24 hours (standing) to obtain a gel, which was placed in a 60° C. oven for drying for 12 hours (drying) to obtain a dry gel, which was then placed in a muffle furnace and calcined at 700° C. for 2 hours (calcining), taken out, and the soaking-standing-drying-calcining process was repeated 3 times, and finally calcined at 1200° C. for 3 hours to obtain a complex calcium phosphate, which includes hydroxyapatite (HA) and β-tricalcium phosphate; its XRD is as follows Figure 5shown.

[0072] Test Example 1

[0073] The complex calcium phosphate in Example 1 was immersed in a simulated body fluid SBF solution for 5 hours, and a deposition test was performed. The results showed that after 5 hours of deposition, a hydroxyapatite layer was formed on the surface of the complex calcium phosphate. The XRD characteristics of the sample after 5 hours of deposition of the complex calcium phosphate in the SBF solution were as follows: Figure 6 As shown, FTIR Figure 7 As shown (i.e., SBF-5h); Figure 7 In the present invention, HA is hydroxyapatite.

[0074] Test Example 2

[0075] The porosity of the complex calcium phosphate in Example 1 was tested by density method, and it was found that its porosity was 81%, which is within the range of cancellous bone (30% to 90%); the pore size was observed by SEM to be 60 to 350 μm.

[0076] Test Example 3

[0077] The biocompatibility of the composite calcium phosphate in Example 1 was tested using osteoblasts (MC-3T3). The test results are as follows: Figure 8 As shown (where Control represents a blank sample and Scaffold represents a complex calcium phosphate), Figure 8 As can be seen from the figure, the relative cell viability (OD scaffold / OD control *100%) can reach almost 200%, and can promote cell proliferation. Figure 9 The histological sections of the composite calcium phosphate implanted into the rabbit femoral condyle for 3 months are shown in FIG. Figure 9 As can be seen from the figure, the composite calcium phosphate in Example 1 was implanted into the rabbit femoral condyle for 3 months, and was found to be able to promote the formation of new bone tissue, and was completely degraded after 3 months.

[0078] The above is an exemplary description of the embodiments of the present invention. However, the scope of protection of the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc. made by those skilled in the art within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A method for preparing a complex calcium phosphate, characterized in that: The method comprises the following steps: (S1) reacting phytic acid with a polyhydroxy polymer to prepare a phytate polymer; (S2) mixing natural porous calcium with the phytate polymer prepared in step (S1) to prepare a gel; (S3) calcining the gel in step (S2) twice to prepare the complex calcium phosphate; In step (S2), the source of the natural porous calcium is at least one of coral, shell, and cuttlefish bone; The two calcinations in step (S3) are: a first calcination at a temperature lower than 825°C, and a second calcination at a temperature higher than 825°C.

2. The method according to claim 1, characterized in that In step (S1), the reaction of phytic acid and polyhydroxy polymer is carried out in water.

3. The method according to claim 1, characterized in that Step (S1) specifically comprises: mixing a phytic acid aqueous solution with a polyhydroxy polymer aqueous solution to react and prepare a phytate polymer.

4. The method according to claim 3, characterized in that The volume ratio of the phytic acid aqueous solution to the polyhydroxy polymer aqueous solution is 1:(0.8-1.5).

5. The method according to any one of claims 1 to 4, characterized in that In step (S1), the polyhydroxy polymer is selected from at least one of polyvinyl alcohol and polyethylene glycol.

6. The method according to claim 1, characterized in that In step (S2), the mass volume ratio of the natural porous calcium to the phytate polymer is 1 g:(1-6) mL.

7. The method according to claim 1, characterized in that Step (S2) specifically comprises: soaking the natural porous calcium in the phytate polymer of step (S1) for a certain period of time, taking out the natural porous calcium and letting it stand to prepare the gel.

8. The method according to claim 7, characterized in that Step (S2) further includes heating the gel after standing to prepare a dry gel; The heating temperature is 30-80°C; the heating time is 6-48h.

9. The method according to claim 1, characterized in that In step (S3), the temperature of the first calcination is 300-820° C.; and the time of the first calcination is 1-6 hours.

10. The method according to claim 1, characterized in that In step (S3), the temperature of the second calcination is 825-1700° C.; and the time of the second calcination is 1-10 hours.

11. The method according to claim 1, wherein Step (S3) further includes calcining the dry gel in step (S2) at a temperature below 825° C. for the first time, mixing the product after the first calcination with the phytate polymer in step (S1), and repeating the soaking, standing, heating and first calcination processes multiple times, and then calcining for the second time at a temperature above 825° C. to prepare the complex calcium phosphate.

12. A complex calcium phosphate, characterized in that: The complex calcium phosphate is prepared by the preparation method according to any one of claims 1 to 11.

13. The complex calcium phosphate according to claim 12, characterized in that The complex calcium phosphate includes at least one of β-tricalcium phosphate and tetracalcium phosphate.

14. The complex calcium phosphate according to claim 13, characterized in that The complex calcium phosphate further comprises hydroxyapatite and / or α-tricalcium phosphate.

15. The complex calcium phosphate according to claim 12, characterized in that The porosity of the complex calcium phosphate is 35% to 88%.

16. The complex calcium phosphate according to claim 12, characterized in that The average pore size of the complex calcium phosphate is 50 to 350 μm.

17. Use of the complex calcium phosphate according to any one of claims 12 to 16 in a bone scaffold.

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