Preparation method and application of citric acid-based polymer stable amorphous calcium phosphate porous scaffold for bone defect repair
A porous biomimetic mineralized scaffold was prepared by polymerizing citric acid-based polymers with amorphous calcium phosphate precursors, which solved the problems of insufficient stability and mechanical properties of ACP-based materials and achieved effective repair of bone defects and new bone formation.
Patent Information
- Application Number
- CN202511124517.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-12
- Publication Date
- 2025-12-16
AI Technical Summary
Existing ACP-based bone defect repair materials suffer from insufficient stability, poor mechanical properties, and the introduction of exogenous organic substances, making it difficult to meet the specific needs of bone defect sites.
A porous biomimetic mineralization scaffold is formed by polymerizing citric acid-based polymers with amorphous calcium phosphate precursors under specific conditions. The citric acid carboxyl groups stabilize ACP, regulate its mechanical properties and degradation characteristics, and provide a microenvironment rich in Ca2+ and PO43- to promote new bone remodeling.
The prepared porous scaffold has good biocompatibility and biodegradability, can stabilize ACP, continuously release active ions, promote osteogenic formation of mesenchymal stem cells, and effectively repair bone defects.
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Figure CN121130167A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biomedical materials, in particular to a preparation method and application of a citric acid-based polymer stabilized amorphous calcium phosphate porous scaffold for repairing bone defects. BACKGROUND
[0002] Bone defect is a global clinical challenge, which usually occurs after trauma, infection or tumor resection. Although bone transplantation has remarkable effect in repairing bone defects, its application is limited by multiple factors such as limited bone source, complications of donor site and risk of immune rejection. With the acceleration of global aging process and the rising incidence of bone diseases, the demand for ideal artificial bone repair grafts is increasingly urgent. Therefore, the development of degradable porous biomimetic mineralized scaffolds with good mechanical properties for clinical bone defect repair shows broad application prospects.
[0003] Bone mineralization process includes the formation, transport, deposition and maturation of amorphous minerals. Among them, amorphous calcium phosphate (ACP) is a key precursor of bone apatite formation, which promotes the deposition and maturation of bone minerals by continuously releasing calcium and phosphate ions. Compared with the commonly used crystalline calcium phosphate (CaP), ACP exhibits higher biological activity and biodegradability. Therefore, ACP-based biomaterials are expected to become a new choice in bone repair. However, the instability of ACP makes it easy to spontaneously transform into stable crystalline CaP, thereby reducing its inherent osteogenic properties. Therefore, how to stabilize the amorphous state of ACP and maintain its biological activity has become an important issue in the preparation of ACP-based biomaterials.
[0004] Currently, the strategies for stabilizing ACP mainly include ion substitution and surface adsorption. For example, Mg 2+ , Zn 2+ ions can substitute calcium or phosphate ions in ACP, or organic matters such as ATP and nucleic acids can be adsorbed on the surface to stabilize ACP and delay its crystallization. These methods have prolonged the stability of ACP to some extent, but still have defects such as short stabilization time, insufficient mechanical properties or introduction of exogenous organic matters. Therefore, there is an urgent need for a safe and stable amorphous calcium phosphate biomimetic mineralized scaffold with good mechanical properties and porous characteristics.
[0005] Poly(1,8-octanediol-citrate) (POC) is a new type of biodegradable elastomer with good biocompatibility, and can achieve different mechanical properties and degradation characteristics by adjusting the polymerization conditions. Studies have shown that citric acid has a natural stabilizing effect on ACP in the skeleton, and can adsorb to the surface of ACP clusters through its carboxyl group, thereby regulating the growth of crystals. POC retains a large number of citric acid carboxyl groups, but so far there has been no relevant research report on whether it can stabilize ACP and prepare a porous biomimetic mineralized scaffold with good mechanical properties. On the one hand, the scaffold stabilizes ACP to create a microenvironment rich in Ca 2+ and PO4 3- at the bone defect site, promoting the remodeling of new bone; on the other hand, by adjusting the polymerization conditions, the specific needs of the bone defect site for the mechanical properties of the implant can be met. Therefore, constructing a citric acid-based poly(1,8-octanediol-citrate) stabilized amorphous calcium phosphate porous biomimetic mineralized scaffold (POC-ACP) for bone defect repair has important clinical application value. SUMMARY
[0006] In view of the shortcomings of the existing ACP-based bone defect repair materials, the present application provides a preparation method of a citric acid-based polymer stabilized amorphous calcium phosphate porous scaffold for bone defect repair, which realizes the uniform dispersion and stable combination of ACP nanoparticles in a citric acid-based poly(1,8-octanediol-citrate) (POC) matrix, and constructs a stable ACP biomimetic mineralized bone repair scaffold with a porous structure, high mechanical strength, controllable degradability and excellent osteogenic activity.
[0007] The technical scheme provided by the present application is as follows: a preparation method of a citric acid-based polymer stabilized amorphous calcium phosphate porous scaffold for bone defect repair, which takes amorphous calcium phosphate precursor and citric acid-based poly(1,8-octanediol-citrate) to be polymerized at 80℃ for 2-3 days or at 80℃ for 3 days and then at 120℃ for 1 day.
[0008] Citric acid-based poly(1,8-octanediol-citrate) is mainly synthesized by polycondensation reaction of citric acid and 1,8-octanediol, and its network structure provides favorable conditions for the introduction of inorganic components. It is cross-linked with inorganic ACP precursor to form a three-dimensional porous biomimetic mineralized scaffold.
[0009] In the reaction system of amorphous calcium phosphate precursor (ACP precursor) and citric acid-based poly(1,8-octanediol-citrate), the content of ACP precursor is between 40wt% and 65wt%.
[0010] In the present application, the ACP precursor is prepared by chemical precipitation method. Specifically, the calcium source and the phosphorus source are stirred uniformly to obtain a reaction solution, the reaction solution is adjusted to pH 10±0.5, and the reaction solution is washed with anhydrous ethanol, stirred and centrifuged to obtain the ACP precursor.
[0011] The molar ratio of the calcium source and the phosphorus source is 1.4-1.7:1, the calcium source is at least one of calcium nitrate and calcium chloride; and the phosphorus source is at least one of diaminium hydrogen phosphate, ammonium dihydrogen phosphate, disodium hydrogen phosphate and sodium dihydrogen phosphate.
[0012] The present application can adjust the pH of the reaction solution by using ammonia water or sodium hydroxide solution, the stirring rate is 600 r / min, and the centrifugal rate and time are 8000-10000 r / min, 3 min.
[0013] Further, the ACP precursor is added into the citric acid-based poly(1,8-octanediol-citrate) solution, stirred at 80℃ until viscous, added into a mold and then transferred into a vacuum drying oven for polymerization.
[0014] In the present application, the preparation of the citric acid-based poly(1,8-octanediol-citrate) is as follows: citric acid and 1,8-octanediol are mixed and then melted at 160℃ in an inert gas, followed by polymerization at 140℃ for 1-3 h.
[0015] Another object of the present application is to provide a citric acid-based polymer-stabilized amorphous calcium phosphate porous scaffold.
[0016] Specifically, the citric acid-based polymer-stabilized amorphous calcium phosphate porous scaffold is prepared by the above preparation method.
[0017] Another object of the present application is to provide the application of the citric acid-based polymer-stabilized amorphous calcium phosphate porous scaffold as a scaffold for repairing bone defects.
[0018] The citric acid-based polymer-stabilized amorphous calcium phosphate porous scaffold of the present application is composed of a POC organic matrix and an inorganic ACP. By adjusting the polymerization conditions of the POC, the mechanical properties of the scaffold can be adjusted to meet the specific needs of the bone defect site; a large number of citric acid carboxyl groups are retained in the POC, which can effectively stabilize the ACP to form a microenvironment rich in Ca 2+ and PO4 3- , providing a good growth basis for the remodeling of new bone; the scaffold has excellent biocompatibility and biodegradability, the released active ingredients can induce mesenchymal stem cells to osteogenesis and promote bone defect repair, and can be gradually replaced by new bone tissue in clinical application, promoting bone regeneration and repair. BRIEF DESCRIPTION OF DRAWINGS
[0019] Figure 1 POC-ACP biomimetic mineralization scaffolds prepared under different polymerization conditions and characterization;
[0020] A biomimetic mineralization scaffold structure; B XRD pattern of the phase composition of the biomimetic mineralization scaffold; C microelectronic microscope graph of the biomimetic mineralization scaffold.
[0021] Figure 2 Figure 1 is a characterization chart of physicochemical properties and ACP stability of POC-ACP biomimetic mineralization scaffolds under different polymerization conditions;
[0022] Figure 1 is a characterization chart of physicochemical properties and ACP stability of POC-ACP biomimetic mineralization scaffolds under different polymerization conditions; 2+ Figure 1 is a characterization chart of physicochemical properties and ACP stability of POC-ACP biomimetic mineralization scaffolds under different polymerization conditions;
[0023] Figure 3 Figure 1 is a characterization chart of physicochemical properties and ACP stability of POC-ACP biomimetic mineralization scaffolds under different polymerization conditions;
[0024] Figure 1 is a characterization chart of physicochemical properties and ACP stability of POC-ACP biomimetic mineralization scaffolds under different polymerization conditions;
[0025] Figure 4 Figure 1 is a characterization chart of physicochemical properties and ACP stability of POC-ACP biomimetic mineralization scaffolds under different polymerization conditions;
[0026] Figure 1 is a characterization chart of physicochemical properties and ACP stability of POC-ACP biomimetic mineralization scaffolds under different polymerization conditions;
[0027] Figure 5 Figure 1 is a characterization chart of physicochemical properties and ACP stability of POC-ACP biomimetic mineralization scaffolds under different polymerization conditions. DETAILED DESCRIPTION
[0028] In order to further understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. If the processes are not specifically described below, they can be realized or understood by those skilled in the art according to the prior art. If the reagents or instruments are not marked with the manufacturer, they are considered to be conventional products that can be purchased on the market.
[0029] Embodiment 1
[0030] 0.1 mol of citric acid and 0.1 mol of 1,8-octanediol were weighed into a three-necked flask, nitrogen was introduced, and the mixture was fully stirred at 160°C. After melting, the POC prepolymer was obtained by polymerization at 140°C for 1 h. The POC prepolymer was dissolved in anhydrous ethanol to obtain a prepolymer solution, i.e., a citric acid-based poly(1,8-octanediol-citrate) solution.
[0031] Take 100 mL of 15 mmol / L (NH4)2HPO4 solution into 100 mL of 22.5 mmol / L Ca(NO3)2·4H2O solution, stir with stirrer at 600 r / min, adjust the pH of the mixed solution to 10±0.5 with 25% ammonia water, and take the precipitate by low-temperature centrifuge at 10000 r / min for 2 min. Wash with a large amount of anhydrous ethanol, and then centrifuge at 10000 r / min for 3 min to obtain the ACP precursor.
[0032] Add the ACP precursor to the prepolymer solution, and the content of the ACP precursor is 60 wt%. Stir at 80°C until it becomes viscous, then transfer it to a vacuum drying box for polymerization after adding a mold. The amorphous calcium phosphate precursor is crosslinked with citric acid-based poly(1,8-octanediol-citrate) to obtain the porous biomimetic mineralized scaffold POC-ACP1-3, as shown in Figure 1 A.
[0033] Among them, POC-ACP1 is obtained by polymerization at 80°C for 2 days; POC-ACP2 is obtained by polymerization at 80°C for 3 days and then at 120°C for 1 day; and POC-ACP3 is obtained by polymerization at 80°C for 3 days.
[0034] XRD detection of the phase characteristics of the scaffold shows that no crystal characteristic peak appears, indicating that the preparation process of the scaffold does not affect the amorphous characteristics of ACP, which provides favorable conditions for the biological activity of the scaffold Figure 1 B). SEM detection of the microstructure of the cross section of the scaffold shows that the scaffolds POC-ACP1-3 all have a porous structure, and the pore sizes are in the range of 100-300 μm, which provides favorable conditions for the growth of new bone tissue and the exchange of nutrient components Figure 1 C).
[0035] Example 2
[0036] The mechanical properties, ion release capacity and ACP stability of the biomimetic mineralized scaffolds POC-ACP1-3 are characterized, as shown in Figure 2 .
[0037] The results show that the pore sizes of POC-ACP1, PC-ACP2 and POC-ACP3 are 298.06, 135.1 and 266.44 μm, respectively Figure 2 A). Excellent mechanical properties are a key characteristic of bone repair materials. The mechanical characteristics of the scaffolds are detected by a universal mechanical testing machine, and the results show that the compressive strengths of the scaffolds are 9.90 MPa, 24.62 MPa and 16.22 MPa, respectively Figure 2 B). This is comparable to the compressive strength of cancellous bone, which can meet the needs of the support of the scaffold after implantation. In vitro degradation experiments show that the scaffold can be degraded in a simulated body fluid environment Figure 2C). Simultaneously, the stent continuously releases Ca through a stable ACP. 2+ For at least 14 days, during which POC-ACP3 releases Ca 2+ Its stronger ability indirectly proves that its stable ACP effect is better than the other two groups. Figure 2 D). To verify the ACP stability of the scaffold under different polymerization conditions, XRD analysis was performed on the surface of the scaffold mineralized in simulated body fluid. It was found that the characteristic crystal peaks in the POC-ACP3 group appeared only after 3 days, indicating better ACP stability and a delayed crystallization effect compared to the other two groups. Figure 2 E).
[0038] Example 3
[0039] The constructed porous biomimetic mineralized scaffold POC-ACP1-3 was co-cultured with bone marrow mesenchymal stem cells for 5 days. CCK-8 staining was used to measure absorbance, and scanning electron microscopy was used to detect cell surface adhesion and proliferation. These results showed that POC-ACP1-3 exhibited good cell compatibility. Figure 3 As shown. CC-8 assay revealed that cells could proliferate normally on the scaffold surface. Figure 3 A), and SEM analysis revealed that the cells adhered and spread well on the scaffold surface, and the cell density gradually increased with increasing culture time. Figure 3 B). This indicates that POC-ACP1-3 has good biocompatibility and will not produce toxicity after implantation in the body.
[0040] Example 4
[0041] The constructed porous biomimetic mineralization scaffold POC-ACP1-3 was co-cultured with bone marrow mesenchymal stem cells for 14 days. The scaffold's ability to promote osteogenic differentiation of mesenchymal stem cells in vitro was assessed using ALP / ARS staining, qPCR, and Western blot. Figure 4 As shown. The results showed that bone marrow mesenchymal stem cells (rBMSCs) were co-cultured on the scaffold in vitro. ALP / ARS staining indicated that the POC-ACP3 group had the highest positive staining intensity on the scaffold surface. Figure 4 AB). The expression of osteogenic-related genes and proteins in rBMSCs was detected, and the study found that the expression of these genes and proteins was highest in the POC-ACP3 group. Figure 4 (CD). This result indirectly proves that POC-ACP3 has better ACP stability, thereby promoting osteogenic differentiation of rBMSCs.
[0042] Example 5
[0043] A bone defect with a diameter of 3.5 mm and a depth of 4 mm was constructed in the lateral femoral condyle of SD rats. A porous biomimetic mineralization scaffold (POC-ACP1-3) was implanted into the defect site. The scaffold perfectly filled the defect, provided mechanical support, closed the wound, and allowed for free movement post-surgery. Eight weeks post-surgery, micro-CT was used to assess new bone growth at the defect site, and three-dimensional reconstruction of the new bone was performed. Figure 5 As shown in the figure. The results showed that the scaffold significantly promoted bone regeneration, with the POC-ACP3 group exhibiting better bone defect repair effects. Figure 5 This indicates that the scaffold can promote bone regeneration in vivo by stabilizing ACP.
Claims
1. A method for preparing a citrate-based polymer-stabilized amorphous calcium phosphate porous scaffold for bone defect repair, characterized in that, Amorphous calcium phosphate precursor was polymerized with citrate-based poly(1,8-octanediol-citrate) at 80°C for 2-3 days, or polymerized at 80°C for 3 days followed by polymerization at 120°C for 1 day.
2. The method for preparing a citrate-based polymer-stabilized amorphous calcium phosphate porous scaffold for bone defect repair according to claim 1, characterized in that, in In the reaction system of the amorphous calcium phosphate precursor and citrate-based poly(1,8-octanediol-citrate), the content of the amorphous calcium phosphate precursor is between 40 wt% and 65 wt%.
3. The method for preparing a citrate-based polymer-stabilized amorphous calcium phosphate porous scaffold for bone defect repair according to claim 1, characterized in that, The amorphous calcium phosphate precursor was prepared by chemical precipitation.
4. The method for preparing a citrate-based polymer-stabilized amorphous calcium phosphate porous scaffold for bone defect repair according to claim 3, characterized in that, The calcium and phosphorus sources were stirred evenly to obtain a reaction solution. The pH of the reaction solution was adjusted to 10±0.
5. The reaction solution was washed with anhydrous ethanol, stirred, and centrifuged to obtain the amorphous calcium phosphate precursor.
5. The method for preparing a citrate-based polymer-stabilized amorphous calcium phosphate porous scaffold for bone defect repair according to claim 4, characterized in that, The molar ratio of the calcium source to the phosphorus source is 1.4-1.7:1, and the calcium source is at least one of calcium nitrate and calcium chloride; the phosphorus source is at least one of diamine hydrogen phosphate, ammonium dihydrogen phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate.
6. The method for preparing a citrate-based polymer-stabilized amorphous calcium phosphate porous scaffold for bone defect repair according to claim 4, characterized in that, The pH of the reaction solution is adjusted by using ammonia or sodium hydroxide solution. The stirring rate is 600 r / min, the centrifugation rate is 8000-10000 r / min, and the time is 3 min.
7. The method for preparing a citrate-based polymer-stabilized amorphous calcium phosphate porous scaffold for bone defect repair according to claim 4, characterized in that, The amorphous calcium phosphate precursor was added to the citrate-based poly(1,8-octanediol-citrate) solution and stirred at 80°C until it became viscous. The solution was then poured into a mold and transferred to a vacuum drying oven for polymerization.
8. The method for preparing a citrate-based polymer-stabilized amorphous calcium phosphate porous scaffold for bone defect repair according to claim 4, characterized in that, The citric acid-based poly(1,8-octanediol-citric acid ester) prepolymer is prepared by mixing citric acid and 1,8-octanediol, melting them in an inert gas at 160°C, and then polymerizing them at 140°C for 1-3 hours.
9. The citric acid-based polymer-stabilized amorphous calcium phosphate porous scaffold is prepared by the preparation method described in any one of claims 1-8.
10. The application of the citric acid-based polymer-stabilized amorphous calcium phosphate porous scaffold according to claim 9 as a bone defect repair scaffold.