Hemostatic crystal glue microspheres, and preparation method and application thereof

Abstract

This invention discloses a hemostatic crystalloid microsphere, its preparation method, and its application, relating to the field of biomedical materials. The hemostatic crystalloid microsphere of this invention has the following characteristics: the microsphere has interconnected through-pores internally; the porosity of the microsphere is 50-98%; the average particle size of the microsphere ranges from 10 to 1000 μm; the pore size ranges from 5 to 400 μm; the surface charge of the microsphere is -100 to 0 mV; and the density of the microsphere is 0.01-1.5 g / cm³. 3 This invention utilizes a low-temperature crosslinking process with carboxyl and amino groups to obtain microspheres via an emulsion method, thus preparing crystalloid microspheres that integrate hemostasis and repair functions. The highly interconnected macropores within the hemostatic crystalloid microspheres prepared by this method facilitate rapid blood absorption, accumulation of blood cells, support for cell adhesion, proliferation, and migration, and promote the repair of the oral mucosa and alveolar bone.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, and more specifically, to a hemostatic crystalloid microsphere, its preparation method, and its application. Background Technology

[0002] Periapical abscesses, tooth defects, and fractures caused by external forces often require surgical repair. After tooth extraction, bleeding is common in the extraction socket, and the bone around the socket tends to undergo irreversible resorption and atrophy. Bone regeneration surgery can reduce bone atrophy around the extraction socket, providing a good foundation for later implantation and restoration.

[0003] Existing alveolar bone repair techniques utilize bone powder and collagen membranes that lack hemostatic capabilities and have low osteoinductive activity, limiting the effectiveness of bone repair. Existing techniques, as described in *International Journal of Biological Macromolecules* 260(2024)129454, show that hydrogel materials have weaker mechanical properties compared to other scaffold materials and degrade rapidly. Existing techniques, as described in *Bioactive Materials* 27(2023)231–256, utilize 3D-printed scaffold materials, offering higher precision and personalization, but significantly increasing material and time costs. Therefore, based on the entire dental restoration process, including extraction and implantation, there is a particular need for a restorative material that can cover hemostasis, repair, and bone growth induction processes after tooth extraction. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a hemostatic crystalloid microsphere, its preparation method, and its applications. This invention utilizes a low-temperature crosslinking process with carboxyl and amino-containing polymer compounds to obtain microsphere morphology based on an emulsion method, thus preparing crystalloid microspheres with both hemostatic and repair functions. The highly interconnected macropores within the hemostatic crystalloid microspheres prepared by this invention facilitate rapid blood absorption, enrichment of blood cells, support for cell adhesion, proliferation, and migration, and promote the repair of the oral mucosa and alveolar bone, thereby solving the problem of poor bone repair effects of current artificial bone powder. The raw materials used in this invention are inexpensive, the process is simple, it does not require the introduction of small molecules such as photoinitiators and crosslinking agents, and it exhibits good biocompatibility. Compared with existing artificial bone powders, the hemostatic and repair effects of these crystalloid microspheres are significantly improved.

[0005] One of the objectives of this invention is to provide a hemostatic crystalloid microsphere.

[0006] The hemostatic crystalloid microspheres of this invention:

[0007] The interior of the crystalline microspheres has interconnected through-holes;

[0008] The porosity of the crystalline microspheres is 50-98%;

[0009] The average particle size of the crystalline microspheres ranges from 10 to 1000 μm;

[0010] The diameter of the through hole ranges from 5 to 400 μm;

[0011] The surface charge of the crystalline microspheres is -100 to 0 mV;

[0012] The density of the crystalline microspheres is 0.01–1.5 g / cm³. 3 .

[0013] In a preferred embodiment of the present invention:

[0014] The porosity of the crystalline microspheres is 70-95%;

[0015] The average particle size of the crystalline microspheres ranges from 100 to 500 μm.

[0016] The diameter of the through hole ranges from 10 to 100 μm;

[0017] The surface charge of the crystalline microspheres is -60 to -20 mV;

[0018] The density of the crystalline microspheres is 0.1–0.5 g / cm³. 3 .

[0019] The second objective of this invention is to provide a method for preparing the hemostatic crystalloid microspheres described in the first objective of this invention.

[0020] The method for preparing hemostatic crystalloid microspheres according to the present invention includes:

[0021] Microsphere emulsions were prepared by emulsion method using carboxyl and amino polymers, and then the crystalline microspheres were obtained by freeze polymerization.

[0022] In a preferred embodiment of the present invention, the method includes:

[0023] (1) Dissolve carboxyl polymers and amino polymers in deionized water, add water-soluble surfactants, activators and stabilizing intermediates, and mix well to obtain the first solution;

[0024] (2) Add an oil-soluble surfactant to the oil phase and mix well to obtain a second solution;

[0025] (3) The first solution is added to the second solution to obtain a microsphere emulsion; the microsphere emulsion is a water-in-oil emulsion, which can be prepared by methods commonly used in the art, such as stirring, electrostatic spraying, 3D printing or microfluidic methods;

[0026] (4) The microsphere emulsion is cooled and then freeze-polymerized, and the crystal microspheres are obtained after post-treatment.

[0027] In a preferred embodiment of the present invention:

[0028] The carboxyl polymer is at least one of sodium alginate, hyaluronic acid, and gelatin; and / or

[0029] The amino polymer compound is at least one of carboxymethyl chitosan, gelatin, and polylysine; and / or,

[0030] The activator is 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and / or N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide methyl p-toluenesulfonate (CMC); and / or,

[0031] The stable intermediate is N-hydroxysuccinimide (NHS) and / or sodium N-hydroxythiosuccinimide (Slufo-NHS); and / or,

[0032] The oil phase is at least one of vegetable oil, mineral oil, and synthetic oil. Preferably, the vegetable oil is at least one of soybean oil, peanut oil, rapeseed oil, corn oil, and sesame oil, and / or the mineral oil is at least one of alkanes, aromatic hydrocarbons, and cycloalkanes, and / or the synthetic oil is at least one of ester oils, polyethers, chlorofluorocarbon oils, fluorinated oils, and silicone oils.

[0033] In a preferred embodiment of the present invention:

[0034] The total weight fraction of carboxyl polymers, amino polymers, activators and stabilizing intermediates in the first solution is 0.1-20 wt%, preferably 1-5 wt%.

[0035] The weight ratio of the carboxyl polymer, amino polymer, activator and stabilizing intermediate is 1:(0.5-1.5):(0.01-0.2):(0.01-0.2), preferably 1:(0.8-1.2):(0.05-0.15):(0.05-0.15).

[0036] In a preferred embodiment of the present invention:

[0037] The water-soluble surfactant is at least one of anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants. Preferably, the anionic surfactant is at least one of stearic acid, sodium dodecylbenzene sulfonate, and sodium fatty acid methyl ester sulfonate, and / or the cationic surfactant is a quaternary ammonium compound, and / or the amphoteric surfactant is at least one of lecithin, amino acid-type surfactants, and betaine-type surfactants, and / or the nonionic surfactant is fatty acid glycerides and / or polysorbates (such as Tween 60); and / or...

[0038] The oil-soluble surfactant is at least one of petroleum sulfonate, amine surfactant, and polyol ester surfactant, such as Span 80, glycerol, and N-dodecylglucosamine surfactant.

[0039] In a preferred embodiment of the present invention:

[0040] The concentration of the water-soluble surfactant in the first solution is 0.1-20 mg / mL, preferably 1-5 mg / mL; and / or,

[0041] The concentration of the oil-soluble surfactant in the second solution is 0.1-20 mg / mL, preferably 1-5 mg / mL; and / or,

[0042] The volume ratio of the first solution to the second solution is 1:(1-20), preferably 1:(2-6).

[0043] In a preferred embodiment of the present invention:

[0044] The polymerization temperature of the freeze polymerization is -30 to -10°C, and / or the polymerization time is 4-48 hours;

[0045] In a preferred embodiment of the present invention:

[0046] The post-processing includes:

[0047] Collect by centrifugation, wash 3-5 times with solvent, wash 3-5 times with deionized water, and freeze-dry at -30 to -10°C for 24-48 hours. Specifically, after centrifugation, wash 3-5 times with acetone and 3-5 times with deionized water; then freeze-dry in a freeze dryer for 24-48 hours.

[0048] A third objective of this invention is to provide a hemostatic crystalloid microsphere prepared by the method described in the second objective of this invention.

[0049] like Figure 1As shown, this invention provides hemostatic crystalloid microspheres based on amide bonds. The crystalloid microspheres are obtained through crosslinking of carboxyl-containing and amino-containing polymeric compounds under the catalysis of an activator and a stabilizing intermediate, followed by low-temperature freeze polymerization. The product of this invention is a white powder.

[0050] The fourth objective of this invention is to provide an application of hemostatic crystalloid microspheres as described in the first objective of this invention or as described in the third objective of this invention in promoting alveolar bone regeneration.

[0051] This invention utilizes a low-temperature crosslinking process with carboxyl and amino-containing polymers to obtain microspheres via an emulsion method, thus preparing crystalloid microspheres that integrate hemostasis and repair functions. The hemostatic crystalloid microspheres of this invention exhibit high structural order, with polymer chains forming a continuous network structure and highly interconnected macropores within the material matrix, enabling them to maintain a specific shape and structure while possessing reversible deformation capabilities. These hemostatic crystalloid microspheres possess a large specific surface area, combining the characteristics of crystalloid materials with the morphology and size of microspheres, making them suitable for use as drug delivery carriers, microreactors, and cell culture substrates. The hemostatic crystalloid microspheres of this invention can effectively fill deep wounds and achieve rapid hemostasis, while also serving as microporous scaffolds for tissue engineering, supporting cell adhesion, proliferation, and migration. Attached Figure Description

[0052] Figure 1 This is a schematic flowchart of the preparation method of the crystalloid microspheres of the present invention;

[0053] Figure 2 A scanning electron microscope image of the hemostatic crystalloid microspheres prepared in Example 1;

[0054] Figure 3 Here is a magnified scanning electron microscope image of a portion of the hemostatic crystalloid microspheres prepared in Example 1;

[0055] Figure 4 A scanning electron microscope image of the hemostatic crystalloid microspheres prepared in Example 2;

[0056] Figure 5 This is a magnified scanning electron microscope image of a portion of the hemostatic crystalloid microspheres prepared in Example 2;

[0057] Figure 6 This is a scanning electron microscope image of cells cultured with hemostatic crystalloid microspheres prepared in Example 3;

[0058] Figure 7 This is a magnified scanning electron microscope image of a portion of the hemostatic crystalloid microspheres prepared in Example 3 after culturing cells.

[0059] Figure 8 This is a scanning electron microscope image of the crystal microspheres prepared in Comparative Example 1 after culturing cells. Detailed Implementation

[0060] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the content of the present invention are still within the scope of protection of the present invention.

[0061] The raw materials used in the embodiments and comparative examples of this invention are all commercially available products.

[0062] Example 1

[0063] Preparation of hemostatic crystalloid microspheres:

[0064] Step 1: Dissolve 0.4g hyaluronic acid, 0.4g carboxymethyl chitosan, 8mg EDC, 8mg NHS, and 40mg Tween 60 in 9.2mL of deionized water to obtain the first solution. Dissolve 40mg Span 80 in 10mL of liquid paraffin to obtain the second solution.

[0065] Step 2: Add the first solution to the second solution to obtain a water-in-oil emulsion (microsphere emulsion) of hyaluronic acid, carboxymethyl chitosan and oil.

[0066] Step 3: Freeze the water-in-oil emulsion at -80℃ for 1 hour, then freeze it at -20℃ for 24 hours to obtain crystal gel microspheres through freeze polymerization;

[0067] Step 4: Centrifuge to collect the crystalloid microspheres, wash with acetone 3 times, wash with deionized water 3 times, and freeze-dry at -20℃ for 48h to obtain hemostatic crystalloid microspheres.

[0068] Example 2

[0069] Preparation of hemostatic crystalloid microspheres:

[0070] Step 1: Dissolve 0.1g sodium alginate, 0.08g polylysine, 15mg EDC, 15mg Slufo-NHS, and 5mg sodium dodecylbenzenesulfonate in 9.85mL of deionized water to obtain the first solution. Dissolve 5mg glycerol in 50mL of soybean oil to obtain the second solution.

[0071] Step 2: Add the first solution to the second solution to obtain a water-in-oil emulsion (microsphere emulsion) of sodium alginate, carboxymethyl chitosan and oil.

[0072] Step 3: Freeze the water-in-oil emulsion at -60℃ for 1 hour, then freeze it at -20℃ for 12 hours to obtain crystal gel microspheres through freeze polymerization;

[0073] Step 4: Centrifuge to collect the crystalloid microspheres, wash with acetone 3 times, wash with deionized water 3 times, and freeze-dry at -20℃ for 48h to obtain hemostatic crystalloid microspheres.

[0074] Example 3

[0075] Preparation of hemostatic crystalloid microspheres:

[0076] Step 1: Dissolve 1.8g of gelatin (0.9g each as a carboxyl polymer and an amino polymer), 5mg of CMC, 5mg of NHS, and 0.19g of lauramide propyl hydroxysulfonate betaine in 9.78mL of deionized water to obtain the first solution. Dissolve 15mg of N-dodecyl-N-methylglucamide in 190mL of petroleum ether to obtain the second solution.

[0077] Step 2: Add the first solution to the second solution to obtain a water-in-oil emulsion (microsphere emulsion) of gelatin and oil.

[0078] Step 3: Freeze the water-in-oil emulsion at -40℃ for 1 hour, then freeze it at -20℃ for 48 hours to obtain crystal gel microspheres through freeze polymerization;

[0079] Step 4: Centrifuge to collect the crystalloid microspheres, wash with acetone 3 times, wash with deionized water 3 times, and freeze-dry at -20℃ for 48h to obtain hemostatic crystalloid microspheres.

[0080] Comparative Example 1

[0081] Preparation of crystalline microspheres:

[0082] Step 1: Dissolve 1.8g of gelatin (0.9g as a carboxyl polymer and 0.9g as an amino polymer), 10mg of glutaraldehyde, and 0.19g of lauramide propyl hydroxysulfonate betaine in 9.78mL of deionized water to obtain the first solution. Dissolve 15mg of N-dodecyl-N-methylglucamide in 190mL of petroleum ether to obtain the second solution.

[0083] Step 2: Add the first solution to the second solution to obtain a water-in-oil emulsion (microsphere emulsion) of gelatin and oil.

[0084] Step 3: Freeze the water-in-oil emulsion at -40℃ for 1 hour, then freeze it at -20℃ for 48 hours to obtain crystal gel microspheres through freeze polymerization;

[0085] Step 4: Centrifuge to collect the crystalline microspheres, wash with acetone 3 times, wash with deionized water 3 times, and freeze-dry at -20℃ for 48h to obtain the crystalline microspheres.

[0086] The hemostatic crystalloid microspheres prepared in Examples 1-3 and the crystalloid microspheres prepared in Comparative Example 1 were tested as follows:

[0087] (1) The average particle size and pore size of the crystalline microspheres were observed by scanning electron microscopy.

[0088] (2) Surface charge test: The test was performed in accordance with the method in the prior art Colloids and Surfaces B:Biointerfaces 216(2022)112596;

[0089] (3) Porosity and density testing: The tests were conducted in accordance with the methods described in the existing technology Carbohydrate Polymers 316(2023)121058;

[0090] The test results are shown in Table 1 below.

[0091] Table 1

[0092]

[0093] As shown in Table 1, the difference between Comparative Example 1 and Example 3 lies in the use of different crosslinking agents. The crosslinking principle of glutaraldehyde mainly involves the reaction of the two aldehyde groups of glutaraldehyde with the amino groups in other molecules to form a Schiff base, which then forms a stable CN bond through a reduction reaction. Glutaraldehyde has a high crosslinking efficiency and a large degree of crosslinking, thus affecting the porosity. The porosity of the embodiments of the present invention is large, thus absorbing more liquid and achieving a better hemostatic effect.

[0094] Cells were cultured using the hemostatic crystalloid microspheres prepared in Example 3 and the crystalloid microspheres prepared in Comparative Example 1, respectively.

[0095] 2×10 5 L929 cells were slowly seeded into 96-well plates, with 10 mg of sample microspheres placed in each well. After culturing the cells in complete culture medium under standard cell culture conditions for 3 days, the rinsed sample microspheres were immersed in a 4% paraformaldehyde solution at 4°C overnight. Then, they were dehydrated using a gradient of ethanol. Finally, the sample microspheres were dried in an oven at 40°C. After drying, the surface of the material was vacuum-coated with uranium-gold, and the cell morphology on the microspheres was observed under a 10 kV accelerating voltage. The results are as follows: Figure 6-8 As shown.

[0096] Depend on Figure 6 and 7 It can be seen that the hemostatic crystalloid microspheres of Example 3 show obvious cell adhesion, indicating that the hemostatic crystalloid microspheres prepared in the embodiments of the present invention can serve as microporous scaffolds for tissue engineering, supporting cell adhesion, proliferation, and migration; Figure 8 and Figure 6 and 7The comparison shows that there are almost no cells attached to the surface of the crystalloid microspheres in Comparative Example 1, while the surface of the hemostatic crystalloid microspheres in Example 3 shows more cells adhering to the surface of the microspheres, indicating that the hemostatic crystalloid microspheres of the present invention have better biocompatibility.

Claims

1. A hemostatic crystalloid microsphere, characterized in that: The interior of the crystalline microspheres has interconnected through-holes; The porosity of the crystalline microspheres is 50-98%; The average particle size of the crystalline microspheres ranges from 10 to 1000 μm; The diameter of the through hole ranges from 5 to 400 μm; The surface charge of the crystalline microspheres is -100 to 0 mV; The density of the crystalline microspheres is 0.01–1.5 g / cm³. 3 .

2. The crystalline microspheres according to claim 1, characterized in that: The porosity of the crystalline microspheres is 70-95%; The average particle size of the crystalline microspheres ranges from 100 to 500 μm. The diameter of the through hole ranges from 10 to 100 μm; The surface charge of the crystalline microspheres is -60 to -20 mV; The density of the crystalline microspheres is 0.1–0.5 g / cm³. 3 .

3. A method for preparing hemostatic crystalloid microspheres as described in claim 1 or 2, the method comprising: Microsphere emulsions were prepared by emulsion method using carboxyl and amino polymers, and then the hemostatic crystalloid microspheres were obtained by freeze polymerization.

4. The method according to claim 3, characterized in that... The method includes: (1) Dissolve carboxyl polymers and amino polymers in deionized water, add water-soluble surfactants, activators and stabilizing intermediates, and mix well to obtain the first solution; (2) Add an oil-soluble surfactant to the oil phase and mix well to obtain a second solution; (3) Add the first solution to the second solution to obtain a microsphere emulsion; (4) The microsphere emulsion is cooled and then freeze-polymerized, and the crystal microspheres are obtained after post-treatment.

5. The method according to claim 4, characterized in that: The carboxyl polymer is at least one of sodium alginate, hyaluronic acid, and gelatin; and / or The amino polymer compound is at least one of carboxymethyl chitosan, gelatin, and polylysine; and / or, The activator is 1-ethyl-(3-dimethylaminopropyl)carbodiimide and / or N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide methyl p-toluenesulfonate; and / or, The stable intermediate is N-hydroxysuccinimide and / or sodium salt of N-hydroxythiosuccinimide; and / or, The oil phase is at least one of vegetable oil, mineral oil, and synthetic oil. Preferably, the vegetable oil is at least one of soybean oil, peanut oil, rapeseed oil, corn oil, and sesame oil, and / or the mineral oil is at least one of alkanes, aromatic hydrocarbons, and cycloalkanes, and / or the synthetic oil is at least one of ester oils, polyethers, chlorofluorocarbon oils, fluorinated oils, and silicone oils.

6. The method according to claim 4, characterized in that: The total weight fraction of carboxyl polymers, amino polymers, activators and stabilizing intermediates in the first solution is 0.1-20 wt%, preferably 1-5 wt%. The weight ratio of the carboxyl polymer, amino polymer, activator and stabilizing intermediate is 1:(0.5-1.5):(0.01-0.2):(0.01-0.2), preferably 1:(0.8-1.2):(0.05-0.15):(0.05-0.15).

7. The method according to claim 4, characterized in that: The water-soluble surfactant is at least one of anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants. Preferably, the anionic surfactant is at least one of stearic acid, sodium dodecylbenzene sulfonate, and sodium fatty acid methyl ester sulfonate, and / or the cationic surfactant is a quaternary ammonium compound, and / or the amphoteric surfactant is at least one of lecithin, amino acid-type surfactants, and betaine-type surfactants, and / or the nonionic surfactant is fatty acid glycerides and / or polysorbates; and / or... The oil-soluble surfactant is at least one of petroleum sulfonate, amine surfactant, and polyol ester surfactant.

8. The method according to claim 4, characterized in that: The concentration of the water-soluble surfactant in the first solution is 0.1-20 mg / mL, preferably 1-5 mg / mL; and / or, The concentration of the oil-soluble surfactant in the second solution is 0.1-20 mg / mL, preferably 1-5 mg / mL; and / or, The volume ratio of the first solution to the second solution is 1:(1-20), preferably 1:(2-6).

9. The method according to claim 4, characterized in that: The freezing polymerization temperature is -30 to -10°C, and / or the polymerization time is 4-48 hours.

10. The method according to claim 4, characterized in that: The post-processing includes: Centrifuge to collect, wash, and freeze dry at -30 to -10℃ for 24-48 hours.

11. A hemostatic crystalloid microsphere prepared by the method according to any one of claims 3-10.

12. The application of a hemostatic crystalloid microsphere as described in claim 1 or 2, or as described in claim 11, in promoting alveolar bone regeneration.

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