Polyhedral calcium oxalate crystal and preparation method thereof

Abstract

The invention relates to the technical field of biomedical materials, in particular to a preparation method of a polyhedral calcium oxalate crystal, which comprises the following steps: adding a calcium-containing solid compound into a water phase system, adjusting the pH value of a prepared calcium ion solution to acidity, and adjusting to a set temperature; preparing an oxalate ion-containing solution, adding the oxalate ion-containing solution into the solution, and stirring until a precipitate is separated out; transferring a product, and heating and converting in a constant-temperature oven; and washing the product, centrifugally collecting, and drying to obtain the polyhedral calcium oxalate particles with the particle size range of 12-40 microns. The preparation method of the polyhedral calcium oxalate crystal is simple in process, low in cost, short in reaction period, flexible in process control and green and environment-friendly in process, a crystal morphology regulating agent is not added, and the polyhedral calcium oxalate crystal uniform in morphology can be prepared.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials, and in particular to a polyhedral calcium oxalate crystal and a preparation method thereof. Background Art

[0002] Calcium oxalate is a widely existing biomineral in nature, a compound formed by simple dicarboxylic acids and calcium. Calcium oxalate plays a role in calcium storage, tissue support structure, plant defense, etc. in plants, and is also the main component of endogenous stones in vertebrates. This material has attracted widespread attention in the fields of biomedical engineering, drug delivery, tissue engineering, and materials science due to its non-toxicity, good biocompatibility, and charge adsorption.

[0003] Calcium oxalate crystal formation is nucleation in a supersaturated solution to form a solid-liquid interface, and further aggregates and grows on this interface. Known calcium oxalate crystal forms are divided into anhydrous calcium oxalate (COA), monohydrate calcium oxalate (COM), dihydrate calcium oxalate (COD), and trihydrate calcium oxalate (COT). Among them, COM is the most thermodynamically stable form of calcium oxalate crystals. The calcium ions on the crystal face (101) are densely distributed and have a certain positive charge. They can adhere to the negatively charged cell surface through electrostatic action, generally in the shape of hexagons, rose petals, etc. The common shapes of COD are dumbbell-shaped and quadrangular pyramidal, and the common shapes of COT are long columns, but due to thermodynamic instability, these two crystals will spontaneously transform to thermodynamically stable COM.

[0004] At present, there are mainly the following methods for preparing calcium oxalate monohydrate (COM) crystals:

[0005] Chinese patent CN201811625758.9 discloses a process for preparing calcium oxalate crystals in plants. The method is based on the fact that plant tissues contain calcium oxalate crystals, and the plant tissues are dissociated with alkali solution to separate the calcium oxalate crystals, and then the alkali solution is repeatedly washed with water, and the impurities are washed with ethanol to obtain the calcium oxalate crystals. However, this method involves a low yield of calcium oxalate, high requirements for the purification process, limited sources of raw materials, and is not suitable for industrial mass production.

[0006] According to literature reports, a document provides a method for preparing calcium oxalate with a crystal length not exceeding 10 μm, in which gelatin is used as an auxiliary template (Synthesis, characterization, adsorption properties and toxicity differences of calcium oxalate monohydrate crystals with different aspect ratios to renal epithelial cells, Journal of Inorganic Chemistry, 2022, 38(07): 1261-1271); the method disclosed in the document is difficult to prepare calcium oxalate monohydrate with a particle size greater than 10 μm, and the use of gelatin additives will bind to calcium ions in the calcium oxalate crystal nucleus and affect the crystallinity of the calcium oxalate crystal as the crystal nucleus gradually grows.

[0007] At present, the research on the synthesis and preparation method of calcium oxalate particles with small particle size (<10μm) is relatively common, but for some specific tissue engineering and regenerative medicine use scenarios, such as the application of wrinkle regeneration and repair in the field of medical beauty, the particle size of the regenerative material in the product is too small to be easily swallowed by macrophages, which significantly reduces the material's ability to stimulate its own collagen regeneration. Therefore, in the present invention, it is crucial to achieve the size of calcium oxalate particles that can be filled with materials and large-scale production by regulating the formation and growth process of calcium oxalate crystals. Summary of the invention

[0008] The technical problem to be solved by the present invention is: to overcome the deficiencies of the prior art, to provide a polyhedral calcium oxalate crystal and a preparation method thereof, and to solve the current technical problems of complex preparation process of calcium oxalate crystals, high cost, and small particle size of the obtained calcium oxalate crystals.

[0009] The technical solution adopted by the present invention to solve its technical problem is:

[0010] First aspect:

[0011] A method for preparing polyhedral calcium oxalate crystals is provided, comprising the following steps:

[0012] Step S1: adding a calcium-containing solid compound into an aqueous phase system and stirring and dispersing the compound;

[0013] Step S2: adjusting the pH value of the calcium ion solution prepared in step S1 to acidic and adjusting the temperature to a set value;

[0014] Step S3: preparing a solution containing oxalate ions and adding it to the solution in step S2, stirring until a precipitate is formed;

[0015] Step S4: transferring the product of step S3 and placing it in a constant temperature oven for heating and conversion;

[0016] Step S5: Wash the product of step S4, collect it by centrifugation and dry it to obtain polyhedral calcium oxalate particles of different particle sizes.

[0017] Further, in the calcium ion solution, the molar concentration of calcium ions is 0.1 to 1 moL / L;

[0018] In the oxalate ion-containing solution, the molar concentration of oxalate ions is 0.1 to 1 moL / L;

[0019] The molar ratio of calcium ions to oxalate ions in the solution is 1:2 to 3:1.

[0020] Furthermore, in step S2, the molar concentration of the pH regulator is 0.2-3 mol / L, and the solution temperature ranges from 0-40°C.

[0021] Furthermore, in step S3, the oxalate ion solution is added at a rate of 10 to 200 mL / min, the stirring speed ranges from 100 to 800 rpm, and the stirring time ranges from 30 to 900 s.

[0022] Furthermore, in step S4, the conversion temperature ranges from 60 to 100° C.; and the conversion time ranges from 8 to 24 hours.

[0023] Furthermore, in step S5, the centrifugal speed ranges from 500 to 4000 rpm; the centrifugal time ranges from 5 to 10 min; the drying temperature ranges from 60 to 120° C.; and the drying time ranges from 4 to 16 h.

[0024] Furthermore, the calcium-containing solid compound is one or more of calcium chloride, calcium nitrate, calcium carbonate and calcium hydroxide.

[0025] Furthermore, the oxalate ion-containing solution is prepared using one or more of oxalic acid, sodium oxalate, potassium oxalate, and ammonium hydrogen oxalate.

[0026] Furthermore, the washing solvent is one or more of deionized water, ethanol, and phosphate buffer.

[0027] Second aspect:

[0028] Provided is a polyhedral calcium oxalate crystal, which is prepared by the above-mentioned preparation method. The crystal has a three-dimensional structure and a particle size range of 12 to 40 μm.

[0029] The beneficial effects of the present invention are:

[0030] The method for preparing polyhedral calcium oxalate crystals of the present invention has the advantages of simple process, low cost, short reaction cycle, flexible process control, green and environmentally friendly process, no crystal morphology regulator is added, and polyhedral calcium oxalate crystals with uniform morphology can be prepared.

[0031] The particle size of the prepared calcium oxalate crystals can be controlled within the range of 12 to 40 μm, and the three-dimensional structure is obvious and the stability and biological safety are high. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The present invention is further described below in conjunction with the accompanying drawings.

[0033] Figure 1 This is a morphology picture of the calcium oxalate particles prepared in Example 1, with a scale bar of 50 μm;

[0034] Figure 2 This is a morphology picture of the calcium oxalate particles prepared in Example 2, with a scale bar of 50 μm;

[0035] Figure 3 This is a morphology picture of the calcium oxalate particles prepared in Example 3, with a scale bar of 50 μm;

[0036] Figure 4 XRD of calcium oxalate particles prepared in Example 3;

[0037] Figure 5 This is a morphology picture of the calcium oxalate particles prepared in Example 4, with a scale bar of 50 μm;

[0038] Figure 6 This is a morphology picture of the calcium oxalate particles prepared in Example 5, with a scale bar of 50 μm;

[0039] Figure 7 This is a SEM image of the calcium oxalate particles prepared in Example 5, with a scale bar of 2 μm;

[0040] Figure 8 The results of evaluating the cell activity of L929 cells after treating them with calcium oxalate particles prepared in Example 3 at different concentrations;

[0041] Fig. 9 The results of evaluating the cell activity of Raw264.7 cells after the calcium oxalate particles prepared in Example 3 were treated with different concentrations;

[0042] Fig.10 Western Blot and Sirius red staining results of L929 cells treated with different concentrations of calcium oxalate particles prepared in Example 2 to stimulate intracellular collagen production. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] The present application provides a method for preparing polyhedral calcium oxalate crystals, comprising the following steps:

[0045] Step S1: adding a calcium-containing solid compound into an aqueous phase system and stirring and dispersing the compound;

[0046] Step S2: adjusting the pH value of the calcium ion solution prepared in step S1 to acidic and adjusting the temperature to a set value;

[0047] Step S3: preparing a solution containing oxalate ions and adding it to the solution in step S2, stirring until a precipitate is formed;

[0048] Step S4: transferring the product of step S3 and placing it in a constant temperature oven for heating and conversion;

[0049] Step S5: Wash the product of step S4, collect it by centrifugation and dry it to obtain polyhedral calcium oxalate particles of different particle sizes.

[0050] In this embodiment, the molar concentration of calcium ions in the calcium ion solution is 0.1 to 1 moL / L;

[0051] In the oxalate ion-containing solution, the molar concentration of oxalate ions is 0.1 to 1 moL / L;

[0052] The molar ratio of calcium ions to oxalate ions in the solution is 1:2 to 3:1.

[0053] In this embodiment, in step S2, the molar concentration of the pH regulator is 0.2-3 mol / L, and the solution temperature ranges from 0-40°C.

[0054] In this embodiment, in step S3, the oxalate ion solution is added at a rate of 10 to 200 mL / min, the stirring speed ranges from 100 to 800 rpm, and the stirring time ranges from 30 to 900 s.

[0055] In this embodiment, in step S4, the conversion temperature ranges from 60 to 100° C.; and the conversion time ranges from 8 to 24 hours.

[0056] In this embodiment, in step S5, the centrifugal speed ranges from 500 to 4000 rpm; the centrifugal time ranges from 5 to 10 min; the drying temperature ranges from 60 to 120° C.; and the drying time ranges from 4 to 16 h.

[0057] In this embodiment, the calcium-containing solid compound is one or more of calcium chloride, calcium nitrate, calcium carbonate, and calcium hydroxide.

[0058] In this embodiment, the oxalate ion-containing solution is prepared by one or more of oxalic acid, sodium oxalate, potassium oxalate, and ammonium hydrogen oxalate.

[0059] In this embodiment, the washing solvent is one or more of deionized water, ethanol, and phosphate buffer.

[0060] The crystals prepared by the preparation method are three-dimensional structures with a particle size range of 12 to 40 μm.

[0061] The present invention is described below through several embodiments: It should be noted that the description order of the following embodiments is not intended to limit the preferred order of the embodiments of the present application. In the following embodiments, the description of each embodiment has its own emphasis. For parts that are not described in detail in a certain embodiment, please refer to the relevant description of other embodiments.

[0062] Example 1

[0063] A method for preparing polyhedral calcium oxalate crystals comprises the following steps:

[0064] S1: Weigh 0.74g calcium hydroxide and add it into 10mL deionized water, stirring thoroughly until completely dispersed;

[0065] S2: Adjust the pH of 1 mol / L calcium hydroxide dispersion with an equal volume of 1 M hydrochloric acid and adjust the solution temperature to 25°C;

[0066] S3: Weigh 2.68 g of sodium oxalate to prepare a 1 mol / L sodium oxalate solution, add it to the stirred calcium hydroxide solution at a speed of 30 mL / min, the molar ratio of calcium ions to oxalate ions in the solution is 1:2, the stirring speed is 100 rpm, and the stirring time is 30 s;

[0067] S4: transfer the above reaction system to a 75°C constant temperature oven for conversion for 12 hours;

[0068] S5: Wash the calcium oxalate particles with deionized water for several times, centrifuge at 3000 rpm for 10 min, collect the product and dry it at 105°C for 12 h, and finally obtain the following: Figure 1 The polyhedral calcium oxalate crystals shown have a particle size of about 13.8±1.2 μm.

[0069] Example 2

[0070] A method for preparing polyhedral calcium oxalate crystals comprises the following steps:

[0071] S1: Weigh 4.45 g of calcium hydroxide and add it to 200 mL of deionized water, stirring thoroughly until it is completely dispersed;

[0072] S: Adjust the pH of 0.3 mol / L calcium hydroxide dispersion with an equal volume of 2 M nitric acid and adjust the solution temperature to 8°C;

[0073] S3: Weigh 3.6 g of oxalic acid to prepare a 0.1 mol / L oxalic acid solution, add it to the stirred calcium hydroxide solution at a speed of 60 mL / min, the molar ratio of calcium ions to oxalate ions in the solution is 3:2, the stirring speed is 800 rpm, and the stirring time is 240 s;

[0074] S4: transfer the above reaction system to a constant temperature oven at 80°C for conversion for 8h;

[0075] S5: Wash the calcium oxalate particles with ethanol several times, centrifuge at 1000 rpm for 5 min, collect the product and dry it at 60°C for 16 h, and finally obtain the following: Figure 2 The polyhedral calcium oxalate crystals shown have a particle size of about 18.4±2.6 μm.

[0076] Example 3

[0077] A method for preparing polyhedral calcium oxalate crystals comprises the following steps:

[0078] S1: Weigh 8.2 g of calcium nitrate and add it to 500 mL of deionized water, stirring thoroughly until it is completely dispersed;

[0079] S2: The pH value of 0.1 mol / L calcium nitrate dispersion was adjusted with an equal volume of 0.2 M nitric acid, and the solution temperature was adjusted to 40°C;

[0080] S3: Weigh 10.7 g of ammonium hydrogen oxalate to prepare a 0.1 mol / L ammonium hydrogen oxalate solution, and add it to the stirred calcium nitrate solution at a speed of 200 mL / min. The molar ratio of calcium ions to oxalate ions in the solution is 1:2. The stirring speed is 350 rpm and the stirring time is 480 s.

[0081] S4: transfer the above reaction system to a constant temperature oven at 100°C for conversion for 8 hours;

[0082] S5: Wash the calcium oxalate particles with ethanol several times, centrifuge at 1000 rpm for 5 min, collect the product and dry it at 120°C for 4 h, and finally obtain the following: Figure 3 The polyhedral calcium oxalate crystals shown have a particle size of about 24.5±3.2 μm. Figure 4 The XRD results shown show that it is a standard calcium oxalate monohydrate crystal form.

[0083] Example 4

[0084] A method for preparing polyhedral calcium oxalate crystals comprises the following steps:

[0085] S1: Weigh 0.67g calcium chloride and add it to 10mL deionized water, stir thoroughly until it is completely dissolved;

[0086] S2: Adjust the pH of 0.6 mol / L calcium chloride solution with an equal volume of 0.2 M hydrochloric acid and adjust the solution temperature to 4 °C;

[0087] S3: Weigh 0.54 g of oxalic acid to prepare a 0.3 mol / L oxalic acid solution, add it to the stirred calcium chloride solution at a speed of 10 mL / min, the molar ratio of calcium ions to oxalate ions in the solution is 1:1, the stirring speed is 300 rpm, and the stirring time is 600 s;

[0088] S4: transfer the above reaction system to a 60°C constant temperature oven for conversion for 24 hours;

[0089] S5: Wash the calcium oxalate particles with deionized water for several times, centrifuge at 4000 rpm for 10 min, collect the product and dry it at 65°C for 16 h, and finally obtain the following: Figure 5 The polyhedral calcium oxalate crystals shown have an average particle size of 30.2±2.4 μm.

[0090] Example 5

[0091] A method for preparing polyhedral calcium oxalate crystals comprises the following steps:

[0092] S1: Weigh 9g of calcium carbonate and add it into 150mL of deionized water, stirring thoroughly until it is completely dissolved;

[0093] S2: Adjust the pH value of 0.6 mol / L calcium carbonate solution with an equal volume of 3 M hydrochloric acid and adjust the solution temperature to 0°C;

[0094] S3: Weigh 4.99 g potassium oxalate to prepare a 0.1 mol / L potassium oxalate solution, add it to the stirred calcium carbonate solution at a speed of 15 mL / min, the molar ratio of calcium ions to oxalate ions in the solution is 3:1, the stirring speed is 600 rpm, and the stirring time is 900 s;

[0095] S4: transfer the above reaction system to a constant temperature oven at 80°C for conversion for 8h;

[0096] S5: Wash the calcium oxalate particles with deionized water for several times, centrifuge at 500 rpm for 10 min, collect the product and dry it at 100 °C for 8 h, and finally obtain the following Figure 6 and Figure 7 The particle size shown is about 36.4±

[0097] 3.6μm polyhedral calcium oxalate crystals.

[0098] Comparative Example 1

[0099] The difference from Example 1 is that the calcium oxalate precipitate precipitated in S3 is freeze-dried to obtain calcium oxalate dihydrate crystalline particles.

[0100] Comparative Example 2

[0101] The difference from Example 2 is that in S2, the solution temperature is adjusted to 60° C., and after the reaction is completed, irregular calcium oxalate crystals with a particle size of about 8 to 12 μm are obtained.

[0102] Comparative Example 3

[0103] The difference from Example 3 is that the prepared calcium oxalate monohydrate crystals are dry-baked at 250° C. for 60 min, and the calcium oxalate monohydrate crystals are transformed into anhydrous calcium oxalate structures.

[0104] Comparative Example 4

[0105] The difference from Example 4 is that the obtained polyhedral calcium oxalate crystals were placed in a pH-neutral aqueous environment and microscopically observed 30 days later. The morphology and size did not change, and the material had good stability.

[0106] After the micron-sized calcium oxalate particles prepared in Examples 1-5 were co-cultured with mouse fibroblast L929 for 1 day, Example 3 was preferred. Figure 8 As shown, the presence of calcium oxalate particles did not affect the normal growth and survival of cells, and the cell morphology did not change and still stretched in a spindle shape.

[0107] After the micron-sized calcium oxalate particles prepared in Examples 1-5 were co-cultured with mouse mononuclear macrophage Raw264.7 for 1 day, Example 3 was preferred. Fig. 9 As shown, calcium oxalate particles aggregate a large number of macrophages, promoting the proliferation and differentiation of macrophages to a certain extent. However, since there is no cell phagocytic behavior, the inflammatory effect induced by them is relatively mild.

[0108] After the micron-sized calcium oxalate particles prepared in Examples 1-5 were co-cultured with mouse fibroblast L929 for 3 days, Example 2 was preferred. Fig.10 As shown, calcium oxalate particles can effectively stimulate the production of collagen, especially the content of type I collagen is significantly increased, thereby providing good structural support and tensile strength for the tissue.

[0109] In summary, the present invention provides a method for preparing polyhedral calcium oxalate crystals through the exploration of the preparation process. Compared with the methods in the existing patents, the present invention has a simple process, low cost and short reaction cycle; the process has no sewage and waste gas emissions, and is green and environmentally friendly; secondly, the process control is flexible, and polyhedral calcium oxalate crystals with stable morphology can be obtained without adding crystal morphology regulators, and the particle size can be controlled within the range of 12 to 40 μm; it is suitable for industrial mass production.

[0110] The polyhedral calcium oxalate particles prepared by the present invention have uniform morphology, adjustable particle size, biological safety and structural stability. Since calcium oxalate crystals are endogenous substances of the body, they can achieve low immunogenicity, be metabolized by the body, and have non-toxic metabolites, and can be used as an injectable implant material in the fields of tissue engineering and medical beauty filling.

[0111] The present invention regulates the nucleation and growth process of calcium oxalate crystals by optimizing the reaction temperature, stirring speed and system pH, thereby avoiding the addition and use of a crystal form controller; at the same time, the calcium oxalate crystals are regulated to transform from thermodynamically unstable calcium oxalate dihydrate to calcium oxalate monohydrate through a temperature-raising drying process, and the morphology is a polyhedral three-dimensional structure. The calcium oxalate prepared by the present invention has high crystallinity, good dispersibility, good thermal stability, low cytotoxicity and a certain ability to stimulate collagen regeneration, providing a new direction and idea for the selection of filling materials in tissue engineering and regenerative medicine.

[0112] In addition, the preparation method of the present invention is suitable for mass production of micron-sized polyhedral calcium oxalate particles because of the advantages of readily available raw materials, simple process, green environmental protection, uniform morphology, and controllable particle size.

[0113] Based on the above ideal embodiments of the present invention, the relevant staff can make various changes and modifications without departing from the technical concept of the present invention through the above description. The technical scope of the present invention is not limited to the contents of the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A method for preparing polyhedral calcium oxalate crystals, characterized in that: The steps include: Step S1: adding a calcium-containing solid compound into an aqueous phase system and stirring and dispersing the compound; Step S2: adjusting the pH value of the calcium ion solution prepared in step S1 to acidic and adjusting the temperature to a set value; Step S3: preparing a solution containing oxalate ions and adding it to the solution in step S2, stirring until a precipitate is formed; Step S4: transferring the product of step S3 and placing it in a constant temperature oven for heating and conversion; Step S5: Wash the product of step S4, collect it by centrifugation and dry it to obtain polyhedral calcium oxalate particles of different particle sizes.

2. The method for preparing polyhedral calcium oxalate crystals according to claim 1, wherein: In the calcium ion solution, the molar concentration of calcium ions is 0.1 to 1 moL / L; In the oxalate ion-containing solution, the molar concentration of oxalate ions is 0.1 to 1 moL / L; The molar ratio of calcium ions to oxalate ions in the solution is 1:2 to 3:

1.

3. The method for preparing polyhedral calcium oxalate crystals according to claim 1, wherein: In step S2, the molar concentration of the pH regulator is 0.2-3 mol / L, and the solution temperature ranges from 0-40°C.

4. The method for preparing polyhedral calcium oxalate crystals according to claim 1, wherein: In step S3, the oxalate ion solution is added at a rate of 10 to 200 mL / min, the stirring speed ranges from 100 to 800 rpm, and the stirring time ranges from 30 to 900 s.

5. The method for preparing polyhedral calcium oxalate crystals according to claim 1, wherein: In step S4, the conversion temperature ranges from 60 to 100° C.; the conversion time ranges from 8 to 24 hours.

6. The method for preparing polyhedral calcium oxalate crystals according to claim 1, characterized in that: In step S5, the centrifugal speed ranges from 500 to 4000 rpm; the centrifugal time ranges from 5 to 10 min; the drying temperature ranges from 60 to 120° C.; and the drying time ranges from 4 to 16 h.

7. The method for preparing polyhedral calcium oxalate crystals according to claim 1, characterized in that: The calcium-containing solid compound is one or more of calcium chloride, calcium nitrate, calcium carbonate and calcium hydroxide.

8. The method for preparing polyhedral calcium oxalate crystals according to claim 1, characterized in that: The oxalate ion-containing solution is prepared by using one or more of oxalic acid, sodium oxalate, potassium oxalate and ammonium hydrogen oxalate.

9. The method for preparing polyhedral calcium oxalate crystals according to claim 1, characterized in that: The washing solvent is one or more of deionized water, ethanol, and phosphate buffer.

10. A polyhedral calcium oxalate crystal, characterized in that: The crystal is prepared by the preparation method according to any one of claims 1 to 9, and has a three-dimensional structure and a particle size range of 12 to 40 μm.

Images