Preparation method and application of high-stability bioactive amorphous calcium magnesium phosphate

Abstract

The present application relates to a kind of preparation method and application of high stability bioactive amorphous calcium magnesium phosphate.The preparation method includes the following steps: (1) preparation pyrophosphate aqueous solution;(2) soluble magnesium salt is dispersed into pyrophosphate aqueous solution, fully stirred, and pyrophosphate magnesium dispersion is prepared;(3) inorganic calcium salt is dispersed into pyrophosphate magnesium dispersion, and pH is adjusted to 10 or more, fully stirred, and colloidal substance is obtained, and high stability bioactive amorphous calcium magnesium phosphate is prepared by separating colloidal substance.The application of high stability bioactive amorphous calcium magnesium phosphate prepared is used for the preparation of bone defect support and the repair of bone defect.The beneficial effect is that pyrophosphate with complexing property realizes efficient loading and controllable release of calcium and magnesium ions.Pyrophosphate can be hydrolyzed into phosphate in vivo, and is an important substance involved in in vivo metabolism.The raw materials added in the whole preparation process are cheap and easy to obtain, simple and non-toxic, simple and green process, and simple operation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological materials, in particular to a preparation method and application of high-stability bioactive amorphous calcium magnesium phosphate. BACKGROUND

[0002] Calcium phosphate has a composition similar to human bone and good biocompatibility, and is the most widely studied bone repair material. Magnesium ions are essential nutrients for the human body, which are mainly distributed in human bones and have excellent activity in promoting osteogenic proliferation, differentiation and blood vessel growth. Pyrophosphate has excellent metal ion complexing ability and ion regulation ability, and is an active substance involved in cell metabolism.

[0003] However, the existing calcium phosphate material still has the problems of slow degradation rate and low bioactivity. In addition, in the preparation method of improving the activity of calcium phosphate by doping magnesium ions, it is difficult to increase the magnesium loading content and to obtain stable calcium and magnesium ion release, which reduces the osteogenic ability of the material. SUMMARY

[0004] The technical problem to be solved by the present application is that the existing calcium phosphate has a slow degradation rate, the magnesium loading content of magnesium-doped calcium phosphate is low, and the release of calcium and magnesium ions is difficult to control.

[0005] The technical solution adopted by the present application to solve the technical problem is: a preparation method of high-stability bioactive amorphous calcium magnesium phosphate, comprising the following steps:

[0006] (1) preparing a pyrophosphate aqueous solution;

[0007] (2) preparation of a magnesium pyrophosphate dispersion:

[0008] The soluble magnesium salt is dispersed into the pyrophosphate aqueous solution prepared in step 1, and is fully stirred to prepare a magnesium pyrophosphate dispersion;

[0009] (3) preparation of high-stability bioactive amorphous calcium magnesium phosphate: the inorganic calcium salt is dispersed into the magnesium pyrophosphate dispersion prepared in step 2, and the pH is adjusted to above 10, and the colloidal substance is fully stirred, separated, and high-stability bioactive amorphous calcium magnesium phosphate is prepared.

[0010] Further limitation, the pyrophosphate is one of potassium pyrophosphate and sodium pyrophosphate, and the purity of the pyrophosphate is > 98%.

[0011] Further limited, in step 1, pyrophosphate is dissolved in water, ultrasonic stirring, preparation of pyrophosphate aqueous solution, ultrasonic power is 20-40W, stirring rate is 40-60r / min, ultrasonic stirring time is 1-3min; in step 2, by ultrasonic stirring is fully stirred, ultrasonic power is 60-80W, stirring speed is 60-80r / min, ultrasonic stirring time is 5-10min; in step 3, by ultrasonic stirring is fully stirred, ultrasonic power is 80-100W, stirring speed is 80-100r / min, ultrasonic stirring time is 2-4h.

[0012] Further limited, the soluble magnesium salt is one of magnesium chloride, magnesium nitrate and magnesium sulfate.

[0013] Further limited, the molar ratio of the soluble magnesium salt to the pyrophosphate is 0

[0014] Further limited, the molar ratio of the sum of the amount of substance of the soluble magnesium salt and the inorganic calcium salt to the pyrophosphate is 2.

[0015] Further limited, the inorganic calcium salt is one of calcium chloride, calcium nitrate, calcium hydroxide and calcium carbonate.

[0016] Further limited, in step 3, the colloidal substance is separated through the steps of filtration, washing and drying.

[0017] Further limited, in step 3, the drying temperature is 40-80 DEG C.

[0018] Further limited, in step 3, NaOH is used to adjust pH.

[0019] The application of the high-stability bioactive amorphous calcium magnesium phosphate prepared by the above method.

[0020] The application has the following advantages: pyrophosphate is used as the phosphorus source, which has metal ion complexing ability, can increase the solubility of metal ions, and can realize the regulation of active ions; and pyrophosphate can be hydrolyzed into phosphate in the body, which is an important substance involved in metabolism. By introducing calcium and magnesium ions as active ions, high-stability bioactive amorphous calcium magnesium phosphate is synthesized at room temperature. Pyrophosphate with complexing performance realizes efficient loading and controllable release of calcium and magnesium ions. Calcium ions have the effect of promoting bone mineralization, magnesium ions can promote the proliferation and differentiation of osteoblasts, and can promote the growth of blood vessels. Therefore, the regulation of calcium and magnesium ions has the potential to continuously stimulate bone repair.

[0021] The raw materials added in the preparation process of the application are cheap and easy to obtain, simple, non-toxic, simple, green, and easy to operate, and the obtained high-stability bioactive amorphous calcium magnesium phosphate is in a colloidal morphology and has an irregular flaky shape, uniform morphology, and excellent ion regulation capacity.

[0022] The application overcomes the difficulties of low degradation of calcium phosphate, low magnesium content, and difficulty in regulating the release speed of calcium and magnesium ions. By introducing pyrophosphate as a phosphorus source, using the strong complexing property of pyrophosphate, introducing calcium and magnesium ions, and synthesizing colloidal amorphous calcium magnesium phosphate at room temperature, the release of calcium and magnesium ions can be regulated according to the performance of the product.

[0023] The application uses a liquid deposition method to prepare high-stability bioactive amorphous calcium magnesium phosphate, which has the advantages of simple required equipment, easy-to-obtain raw materials, low reaction energy consumption, and fast and large-scale preparation, and the product has uniform morphology and is in an irregular colloidal flaky shape, which is a convenient and fast preparation method of high-magnesium-loaded amorphous calcium magnesium phosphate.

[0024] In the preparation of the application, the pyrophosphate, calcium ions, and magnesium ions have important mutual coordination effects. As the main framework of the reaction, pyrophosphate has strong electronegativity and metal ion complexing ability, and can load more magnesium ions through electrostatic and covalent interactions to obtain a magnesium pyrophosphate dispersion. On this basis, calcium salt is introduced to regulate the reaction pH to above 10 to improve the stability of the reaction, and through covalent interaction, an irregular flaky aggregate with excellent thermodynamic stability and performance is spontaneously formed.

[0025] The high-stability bioactive amorphous calcium magnesium phosphate prepared by the application retains the pyrophosphate framework and is an irregular flaky nanometer aggregate. The aggregate has the characteristics of high surface activity, porous structure, high stability, and multiple modification sites, and the retained pyrophosphate framework has long-term ion dynamic loading capacity, which shows application potential in the fields of drug delivery, tumor diagnosis and treatment, stent engineering, fuel cells, and environmental governance.

[0026] The high-stability bioactive amorphous calcium magnesium phosphate prepared by the application also shows good osteogenesis and angiogenesis capacity on the basis of excellent ion regulation capacity. BRIEF DESCRIPTION OF DRAWINGS

[0027] The application will be further described in detail below in combination with the drawings and specific embodiments.

[0028] Figure 1 SEM image of the amorphous calcium magnesium phosphate obtained in Example 1;

[0029] Figure 2 SEM image of the amorphous calcium magnesium phosphate obtained in Example 2;

[0030] Figure 3SEM image of amorphous magnesium phosphate obtained from Comparative Example 1;

[0031] Figure 4 SEM image of amorphous magnesium phosphate obtained from Comparative Example 1;

[0032] Figure 5 SEM image of amorphous calcium phosphate obtained from Comparative Example 2;

[0033] Figure 6 XRD image of amorphous calcium magnesium phosphate obtained from Example 3;

[0034] Figure 7 XRD image of amorphous calcium magnesium phosphate obtained from Example 3 after calcination at 400℃;

[0035] Figure 8 ICP image of calcium magnesium ion release of amorphous calcium magnesium phosphate obtained from Example 3 after 21 days of PBS immersion;

[0036] Figure 9 Cell migration results of osteoblast MC3T3-E1 treated with complete medium;

[0037] Figure 10 Cell migration results of endothelial cell HUVECs treated with complete medium;

[0038] Figure 11 Cell migration results of osteoblast MC3T3-E1 treated with amorphous calcium magnesium phosphate obtained from Example 3;

[0039] Figure 12 Cell migration image of endothelial cell HUVECs treated with amorphous calcium magnesium phosphate obtained from Example 3. DETAILED DESCRIPTION

[0040] Example 1

[0041] A method for preparing a high-stability bioactive amorphous calcium magnesium phosphate, comprising the following steps:

[0042] (1) preparing a pyrophosphate aqueous solution: dissolving pyrophosphate in water and ultrasonic stirring to obtain a pyrophosphate solution with a substance concentration of 50 mM;

[0043] wherein the pyrophosphate is one of potassium pyrophosphate and sodium pyrophosphate, and the purity of the pyrophosphate is >98%. The ultrasonic power is 20 W, the stirring rate is 40 r / min, and the ultrasonic stirring time is 1 min.

[0044] (2) Preparation of magnesium pyrophosphate dispersion liquid: magnesium chloride was weighed and dispersed into the pyrophosphate solution prepared in step 1, and was fully stirred by ultrasonic stirring, and after the reaction was completed, the magnesium pyrophosphate dispersion liquid was prepared;

[0045] The molar ratio of magnesium chloride to pyrophosphate was 2:3. The ultrasonic power was 60 W, the stirring rate was 60 r / min, and the ultrasonic stirring time was 5 min.

[0046] (3) Preparation of high-stability bioactive amorphous calcium magnesium phosphate: calcium hydroxide was weighed and dispersed into the magnesium pyrophosphate dispersion liquid prepared in step 2, the pH was above 10, and there was no need to adjust the pH again, and the reaction was fully stirred by ultrasonic stirring, and after the reaction was completed, the colloidal material was obtained, and the colloidal material was separated after the steps of filtration, washing and drying, and the high-stability bioactive amorphous calcium magnesium phosphate was prepared;

[0047] The molar ratio of calcium hydroxide to pyrophosphate was 4:3. The ultrasonic power was 100 W, the stirring rate was 100 r / min, and the ultrasonic stirring time was 4 h. The drying temperature was 80°C, and the time was 18 h.

[0048] Example 2

[0049] A preparation method of a high-stability bioactive amorphous calcium magnesium phosphate, comprising the following steps:

[0050] (1) Preparation of pyrophosphate aqueous solution: pyrophosphate was dissolved in water and stirred by ultrasonic to obtain a pyrophosphate solution with a concentration of 50 mM;

[0051] The pyrophosphate is one of potassium pyrophosphate and sodium pyrophosphate, and the purity of the pyrophosphate is greater than 98%. The ultrasonic power is 30 W, the stirring rate is 50 r / min, and the ultrasonic stirring time is 2 min.

[0052] (2) Preparation of magnesium pyrophosphate dispersion liquid: magnesium chloride was weighed and dispersed into the pyrophosphate solution prepared in step 1, and was fully stirred by ultrasonic stirring, and after the reaction was completed, the magnesium pyrophosphate dispersion liquid was prepared;

[0053] The molar ratio of magnesium chloride to pyrophosphate was 1:1. The ultrasonic power was 70 W, the stirring rate was 70 r / min, and the ultrasonic stirring time was 7 min.

[0054] (3) Preparation of high-stability bioactive amorphous calcium magnesium phosphate: calcium hydroxide was weighed and dispersed into the magnesium pyrophosphate dispersion liquid prepared in step 2, the pH was above 10, and there was no need to adjust the pH again, and the reaction was fully stirred by ultrasonic stirring, and after the reaction was completed, the colloidal material was obtained, and the colloidal material was separated after the steps of filtration, washing and drying, and the high-stability bioactive amorphous calcium magnesium phosphate was prepared;

[0055] The molar ratio of calcium hydroxide to pyrophosphate is 1:1. The ultrasonic power is 90 W, the stirring rate is 90 r / min, and the ultrasonic stirring time is 3 h. The drying temperature is 60℃, and the time is 16 h.

[0056] Example 3

[0057] A preparation method of a high-stability bioactive amorphous calcium magnesium phosphate, comprising the following steps:

[0058] (1) Preparation of pyrophosphate aqueous solution: dissolve pyrophosphate in water and ultrasonically stir to obtain a pyrophosphate solution with a substance concentration of 50 mM.

[0059] The pyrophosphate is one of potassium pyrophosphate and sodium pyrophosphate, and the purity of the pyrophosphate is > 98%. The ultrasonic power is 40 W, the stirring rate is 60 r / min, and the ultrasonic stirring time is 3 min.

[0060] (2) Preparation of magnesium pyrophosphate dispersion: weigh magnesium chloride, disperse the magnesium chloride into the pyrophosphate solution prepared in step 1, and fully stir by ultrasonic stirring. After the reaction is completed, a magnesium pyrophosphate dispersion is obtained.

[0061] The molar ratio of magnesium chloride to pyrophosphate is 4:3. The ultrasonic power is 80 W, the stirring rate is 80 r / min, and the ultrasonic stirring time is 10 min.

[0062] (3) Preparation of high-stability bioactive amorphous calcium magnesium phosphate: weigh calcium hydroxide, disperse the calcium hydroxide into the magnesium pyrophosphate dispersion prepared in step 2, and adjust the pH to be above 10. No further pH adjustment is needed. Fully stir by ultrasonic stirring. After the reaction is completed, a colloidal substance is obtained. The colloidal substance is separated by filtration, washing, and drying steps to obtain high-stability bioactive amorphous calcium magnesium phosphate.

[0063] The molar ratio of calcium hydroxide to pyrophosphate is 2:3. The ultrasonic power is 80 W, the stirring rate is 80 r / min, and the ultrasonic stirring time is 2 h. The drying temperature is 40℃, and the time is 14 h.

[0064] Comparative Example 1

[0065] A preparation method of amorphous magnesium pyrophosphate, comprising the following steps:

[0066] (1) Dissolve pyrophosphate (purity > 98%) in water and ultrasonically stir to obtain a pyrophosphate solution with a substance concentration of 50 mM. The ultrasonic power is 40 W, the stirring rate is 60 r / min, and the ultrasonic stirring time is 3 min.

[0067] (2) Weigh magnesium chloride and ultrasonically stir it evenly into the pyrophosphate solution prepared in step 1. The molar ratio of magnesium chloride to pyrophosphate is 2:1, the ultrasonic power is 80W, the stirring rate is 80r / min, and the ultrasonic stirring time is 2h. After the reaction is completed, the mixture is washed, filtered, and dried to obtain amorphous magnesium pyrophosphate. The drying temperature is 40℃ and the time is 12h.

[0068] Comparative Example 2

[0069] A method for preparing amorphous calcium pyrophosphate includes the following steps:

[0070] (1) Dissolve pyrophosphate (purity > 98%) in water, and stir ultrasonically. The ultrasonic power is 20W, the stirring rate is 40r / min, and the ultrasonic stirring time is 1min to obtain a pyrophosphate solution with a molar concentration of 50mM.

[0071] (2) Weigh calcium hydroxide and ultrasonically stir it evenly into the pyrophosphate solution prepared in step 1. The molar ratio of calcium hydroxide to pyrophosphate is 2:1, the ultrasonic power is 100W, the stirring rate is 100r / min, and the ultrasonic stirring time is 5h. After the reaction is completed, the solution is washed, filtered, and dried to obtain amorphous calcium pyrophosphate. The drying temperature is 80℃ and the time is 24h.

[0072] Material morphology testing

[0073] To investigate the effect of different calcium-magnesium ratios on the morphology of the product. Figures 1-3 The images show SEM images of amorphous calcium magnesium phosphate obtained in Examples 1-3, with calcium-to-magnesium ratios of 2:1, 1:1, and 1:2, respectively. Figure 4 SEM image of amorphous magnesium phosphate obtained from the reaction of magnesium chloride with pyrophosphate in Comparative Example 1 without the addition of calcium hydroxide. Figure 5 SEM image of amorphous calcium phosphate obtained from the reaction of calcium hydroxide with pyrophosphate in Comparative Example 2 without the addition of magnesium chloride. Figure 6 The image shows the XRD pattern of amorphous calcium magnesium phosphate obtained from the reaction in Example 3, where the calcium-to-magnesium ratio was 1:2. Figure 7 The image shows the XRD pattern of amorphous calcium magnesium phosphate obtained from the reaction in Example 3 (calcium-magnesium ratio 1:2) after calcination at 400°C. Figure 8 The image shows the ion release diagram of amorphous calcium magnesium phosphate obtained from the reaction in Example 3 with a calcium-to-magnesium ratio of 1:2.

[0074] Depend on Figures 1-5 It can be seen that as the magnesium ion content increases, the morphology tends towards colloidal formation, and the particle size decreases. The particle size is smallest when the calcium-to-magnesium ratio is 1:2. The product obtained in Comparative Example 1 without the addition of calcium hydroxide is a cross-linked bulk. The product obtained in Comparative Example 2 without the addition of magnesium chloride has poor cross-linking properties and contains unknown bulk particles. Figure 6As can be seen, the XRD results of the amorphous calcium magnesium phosphate obtained in Example 3 show typical peaks resembling steamed buns, indicating that it is an amorphous product. From Figure 7 It can be seen that the XRD results of the amorphous calcium magnesium phosphate obtained in Example 3 after calcination at 400℃ still show typical amorphous peaks, indicating that the amorphous calcium magnesium phosphate obtained in Example 3 has high stability. Figure 8 As can be seen from the ICP results of the amorphous calcium magnesium phosphate obtained in Example 3 after long-term immersion in PBS solution (also known as PBS buffer), it is evident that the obtained amorphous calcium magnesium phosphate possesses a stable calcium and magnesium ion release capability. Figures 1-8 It can be seen that Example 3 is the optimal example, and the structure of the amorphous calcium magnesium phosphate prepared under the given conditions in this example is significantly better than that of other examples and comparative examples.

[0075] Materials application testing

[0076] The amorphous calcium magnesium phosphate material prepared in Example 3 was used to evaluate its osteogenic and angiogenic properties.

[0077] The specific testing process is as follows:

[0078] 1. Preparation of material culture medium:

[0079] The amorphous calcium magnesium pyrophosphate prepared in Example 3 was sterilized and then soaked in DMEM medium to prepare a material extraction solution of 0.5 mg / mL.

[0080] 2. Cell culture:

[0081] MC3T3-E1 and HUVECs were cultured in a complete medium prepared with DMEM, 10% FBS and 1% double antibiotics. When the cell confluence reached 80%, the cells were washed, digested, centrifuged, resuspended in the medium, and seeded into plates.

[0082] 3. Cell migration:

[0083] Cells were seeded at a rate of 10,000 per well in a six-well plate. When the cell confluence reached 80%, the cells were scratched with a 1 mL pipette tip and the extraction medium was changed for cell culture. The complete culture medium group served as a blank control. The cells were observed and photographed at the same location under a microscope on days 0, 1, and 2 (denoted as D0, D1, and D2).

[0084] The test results are as follows: Figures 9-12 As shown, in Figures 9-12 The dashed lines in the diagram represent the locations of cell migration, and the gaps between the dashed lines represent areas not covered by the cells. Figure 9 Cell migration results of osteoblast MC3T3-E1 cells treated with complete culture medium. Figure 10 Cell migration results of HUVECs endothelial cells treated with complete culture medium. Figure 11The cell migration results of the osteoblast MC3T3-E1 treated by the amorphous calcium magnesium phosphate obtained in Example 3. Figure 12 The cell migration results of the endothelial cell HUVECs treated by the amorphous calcium magnesium phosphate obtained in Example 3. Figures 9-12 It can be seen that the amorphous calcium magnesium phosphate obtained in Example 3 has the effect of promoting the migration of MC3T3-E1 and HUVECs, and is presumed to have the potential of promoting the vascularized bone repair.

[0085] The above experiments show that the high-stability bioactive amorphous calcium magnesium phosphate synthesized in the application has the ability to promote the migration of osteoblasts and endothelial cells.

[0086] The application of the high-stability bioactive amorphous calcium magnesium phosphate prepared by the preparation method of the high-stability bioactive amorphous calcium magnesium phosphate, for the preparation of bone defect scaffolds and the repair of bone defects.

[0087] The above is only the preferred specific implementation of the application, but the protection scope of the application is not limited to this, any person skilled in the art can make equivalent replacement or change according to the technical solution and the inventive concept of the application within the technical range disclosed by the application, which should be covered in the protection scope of the application.

[0088] For example, the soluble magnesium salt of the application can also be magnesium nitrate or magnesium sulfate. The inorganic calcium salt can also be one of calcium chloride, calcium nitrate and calcium carbonate. When the inorganic calcium salt is one of calcium chloride, calcium nitrate and calcium carbonate, the pH is adjusted to above 10 by adding NaOH in step 3.

Claims

1. A method for preparing high-stability biologically active amorphous calcium magnesium phosphate, characterized by comprising the steps of: It comprises the following steps: ​ (1) preparing a pyrophosphate aqueous solution; (2) preparing a magnesium pyrophosphate dispersion: dispersing a soluble magnesium salt into the pyrophosphate aqueous solution prepared in step 1, and fully stirring to prepare a magnesium pyrophosphate dispersion; (3) preparing a high-stability bioactive amorphous calcium magnesium phosphate: dispersing an inorganic calcium salt into the magnesium pyrophosphate dispersion prepared in step 2, adjusting the pH to above 10, fully stirring to obtain a colloidal substance, and separating the colloidal substance to prepare a high-stability bioactive amorphous calcium magnesium phosphate; The molar ratio of the soluble magnesium salt to the pyrophosphate is 0 < molar ratio < 2. The molar ratio of the inorganic calcium salt to the pyrophosphate is 0 < molar ratio < 2.

2. The method of claim 1, wherein the high-stability bioactive amorphous calcium magnesium phosphate is prepared by the following steps of: The pyrophosphate is one of potassium pyrophosphate and sodium pyrophosphate, and the purity of the pyrophosphate is > 98%. ​ 3. The method of claim 1, wherein the high-stability bioactive amorphous calcium magnesium phosphate is prepared by the process of: In step 1, the pyrophosphate is dissolved in water, and ultrasonic stirring is performed to prepare the pyrophosphate aqueous solution, the ultrasonic power is 20-40 W, the stirring speed is 40-60 r / min, and the ultrasonic stirring time is 1-3 min. ​ In step 2, the fully stirring is performed by ultrasonic stirring, the ultrasonic power is 60-80 W, the stirring speed is 60-80 r / min, and the ultrasonic stirring time is 5-10 min. In step 3, the fully stirring is performed by ultrasonic stirring, the ultrasonic power is 80-100 W, the stirring speed is 80-100 r / min, and the ultrasonic stirring time is 2-4 h.

4. The method of claim 1, wherein the high-stability bioactive amorphous calcium magnesium phosphate is prepared by the following steps of: The soluble magnesium salt is one of magnesium chloride, magnesium nitrate and magnesium sulfate. ​ 5. The method of claim 1, wherein the high-stability bioactive amorphous calcium magnesium phosphate is prepared by the following steps of: The molar ratio of the sum of the amounts of substance of the soluble magnesium salt and the inorganic calcium salt to the pyrophosphate is 2. ​ 6. The method for preparing highly stable bioactive amorphous calcium magnesium phosphate according to claim 1, characterized in that: The inorganic calcium salt is one of calcium chloride, calcium nitrate, calcium hydroxide and calcium carbonate.

7. The method for preparing highly stable bioactive amorphous calcium magnesium phosphate according to claim 1, characterized in that: In step 3, the colloidal substance is separated through the steps of filtration, washing and drying.

8. The method of claim 7, wherein the high-stability bioactive amorphous calcium magnesium phosphate is prepared by the following steps of: In step 3, the drying temperature is 40-80℃. ​ 9. The method of claim 1, wherein the high-stable bioactive amorphous calcium magnesium phosphate is prepared by the following steps of: In step 3, NaOH is used to adjust the pH. ​ 10. Use of the highly stable bioactive amorphous calcium magnesium phosphate according to any one of claims 1 to 9, wherein the highly stable bioactive amorphous calcium magnesium phosphate is prepared by the method according to any one of claims 1 to 9. The preparation of a scaffold for bone defect and the repair of bone defect.

Images