Hydroxyapatite / sodium alginate / recombinant collagen composite porous heterogeneous gel microspheres and method for preparing the same

Abstract

The application discloses a kind of hydroxyapatite / sodium alginate / recombinant collagen composite porous heterogeneous gel microspheres and a preparation method thereof.The method first mixes hydroxyapatite with sodium alginate and recombinant collagen uniformly, then injects the sodium alginate / hydroxyapatite mixture and the recombinant collagen / hydroxyapatite mixture into the pipeline of Y-type microfluidic device for mixing, drops into the oil phase to obtain heterogeneous microspheres, and obtains the composite porous heterogeneous gel microspheres after cross-linking and freeze-drying.The heterogeneous gel microspheres of the application have a more complex internal morphology, can encapsulate more components, can simulate the diversity of body tissue components, and can regulate the extracellular microenvironment through multi-component synergistic effect to realize tissue regeneration.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and relates to a hydroxyapatite / sodium alginate / recombinant collagen composite porous heterogeneous gel microsphere and its preparation method. Background Technology

[0002] The cellular microenvironment plays a crucial role in cell proliferation, differentiation, and functional expression, especially in stem cells. Subtle environmental changes can influence asymmetric differentiation of stem cells. Different cells have different microenvironments; surrounding cells, cytokines, extracellular matrix, and many other factors participate in cellular behavior, and their distribution is critical. Therefore, constructing a suitable cellular microenvironment is essential for tissue repair. The cellular environment within human tissues is a typical heterogeneous environment, containing diverse cell types and structures, as well as extracellular fluid, exhibiting high heterogeneity. The most common method for constructing in vitro cellular microenvironments is the creation of microcarriers. Microspheres are widely used for cell encapsulation, drug release, and biosensors due to their diverse manufacturing methods, controllable morphology, and ease of use.

[0003] Chinese patent CN115159483A discloses a morphology- and size-tunable hydroxyapatite microsphere and its preparation method, employing microfluidic spray drying technology. By adjusting parameters such as the type of dispersant, the mass ratio of hydroxyapatite to dispersant, and salt concentration, the geometric size and morphology of the dried microspheres can be controlled. Chinese patent CN114306737A discloses a silk fibroin-based porous microsphere and its preparation method, utilizing microfluidics combined with freeze-drying to obtain silk fibroin microspheres with tunable pore size. Chinese patent CN115429928A discloses a drug-loadable monodisperse calcium carbonate-gelatin composite embolization microsphere and drug-loaded embolization microsphere, the composition of which includes calcium carbonate nanoparticles and gelatin. However, the microspheres prepared by the above methods are all isotropic and cannot well simulate the highly heterogeneous in vitro cellular microenvironment.

[0004] Heterogeneous microspheres possess more complex and innovative internal morphologies, enabling them to encapsulate a wider range of dispersed components, including different types of cells, cytokines, and proteins. Furthermore, their complex heterogeneous structures allow for differentiated distributions of various physical parameters, such as internal morphology, composition, and mechanical strength. This provides crucial regulatory capabilities for drug release cycles, material degradation capabilities, cell migration, and proliferation behavior, resulting in a wide range of potential applications. Summary of the Invention

[0005] The present invention aims to provide a hydroxyapatite / sodium alginate / recombinant collagen composite porous heterogeneous gel microsphere and its preparation method. This composite porous heterogeneous gel microsphere has different components, which can simulate the diversity of tissue components in the body, regulate cell growth and differentiation, and achieve tissue regeneration.

[0006] The technical solution for achieving the objective of this invention is as follows:

[0007] The preparation method of hydroxyapatite / sodium alginate / recombinant collagen composite porous heterogeneous gel microspheres includes the following steps:

[0008] (1) Hydroxyapatite was mixed with sodium alginate and recombinant collagen respectively to obtain sodium alginate / hydroxyapatite and recombinant collagen / hydroxyapatite mixture.

[0009] (2) The sodium alginate / hydroxyapatite mixture and the recombinant collagen / hydroxyapatite mixture were injected into the two pipes of the Y-type microfluidic device, respectively, and then mixed and dripped into the oil phase through the Y-type microfluidic device. The oil phase was washed away to obtain different heterogeneous microspheres.

[0010] (3) The heterogeneous microspheres were cross-linked sequentially with calcium chloride solution and carbodiimide solution. After the cross-linking was completed, the microspheres were washed and freeze-dried to obtain hydroxyapatite / sodium alginate / recombinant collagen composite porous heterogeneous gel microspheres.

[0011] Preferably, in step (1), the concentration of hydroxyapatite in the sodium alginate / hydroxyapatite mixture or the recombinant collagen / hydroxyapatite mixture is 2wt%-15wt%, preferably 5wt%-10wt%; the concentration of sodium alginate in the sodium alginate / hydroxyapatite mixture is 0.1wt%-5wt%, preferably 1wt%-3wt%; and the concentration of recombinant collagen in the recombinant collagen / hydroxyapatite mixture is 0.5wt%-5wt%, preferably 1.5wt%-3wt%.

[0012] Preferably, in step (1), the recombinant collagen is produced by fermentation of Pichiapastoris Pasteurella multocida with accession number CGMCC No. 5021, which has been fully disclosed in Chinese Patent 201110327865.5.

[0013] Preferably, in step (2), the oil phase is selected from liquid paraffin oil, vegetable oil or dimethyl silicone oil, and more preferably vegetable oil.

[0014] Preferably, in step (2), the injection rate is 0.1-5 mL / h, more preferably 0.5-2 mL / h.

[0015] Preferably, in step (2), the washing method is to wash with ethanol and acetone in sequence to remove the oil phase, and the washing time for each solvent is 0.5-8h, more preferably 1-4h.

[0016] Preferably, in step (3), the concentration of calcium chloride solution is 0.5wt%-5wt%, more preferably 1.5wt%-3wt%, and the crosslinking time is 1-8h, more preferably 2-6h.

[0017] Preferably, in step (3), the concentration of the carbodiimide solution is 1wt%-5wt%, more preferably 1.5wt%-4wt%, and the crosslinking time is 1-8h, more preferably 2-4h.

[0018] Preferably, in step (3), the washing time is 1-12 hours, more preferably 2-8 hours.

[0019] Preferably, in step (3), the microsphere size of the composite porous heterogeneous gel microsphere is 100-400 μm.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] (1) The present invention obtains multi-component composite porous heterogeneous gel microspheres through Y-shaped channel design, and the preparation process is simple.

[0022] (2) The composite porous heterogeneous gel microspheres prepared in this invention have a more complex and controllable internal morphology compared to traditional gel microspheres. When encapsulating dispersed components, including various types of cytokines, they exhibit a richer spatial heterogeneous distribution. Furthermore, the complex heterogeneous structure enables differential distribution of parameters such as internal morphology, composition, and mechanical strength of the microspheres, which has important regulatory capabilities for cell migration and proliferation.

[0023] (3) The composite porous heterogeneous gel microspheres prepared in this invention, based on the fluid properties of sodium alginate, the bioactivity of hydroxyapatite, and the excellent biochemical properties of recombinant collagen, can simulate the diversity of tissue components in the body and regulate the extracellular microenvironment through the synergistic effect of multiple components to achieve tissue regeneration. Attached Figure Description

[0024] Figure 1 This is a schematic diagram illustrating the preparation of composite porous heterogeneous gel microspheres.

[0025] Figure 2 This is a SEM image of the composite porous heterogeneous gel microspheres prepared in Example 2. Detailed Implementation

[0026] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings.

[0027] Example 1

[0028] (1) Weigh 0.01g of sodium alginate and dissolve it in 10g of purified water. After it is completely dissolved by stirring, weigh 0.2g of hydroxyapatite and stir it to disperse it evenly to obtain a sodium alginate / hydroxyapatite mixture.

[0029] (2) Weigh 0.05g of recombinant collagen and dissolve it in 10g of purified water. After it is completely dissolved by stirring, weigh 0.2g of hydroxyapatite and stir it to disperse it evenly to obtain a recombinant collagen / hydroxyapatite mixture.

[0030] (3) The above sodium alginate / hydroxyapatite mixture and recombinant collagen / hydroxyapatite mixture were respectively loaded into 10mL syringes A and B, and injected into the Y-shaped tubes using a syringe pump. They were injected into the continuously stirred vegetable oil at an injection rate of 0.1mL / h to obtain heterogeneous microspheres.

[0031] (4) The heterogeneous microspheres obtained above were washed with ethanol and acetone in sequence for 0.5 h each;

[0032] (5) After cleaning, the heterogeneous microspheres are first crosslinked with 0.5wt% calcium chloride solution for 8 hours, then crosslinked with 1wt% carbodiimide solution for 8 hours, and then washed with purified water for 1 hour. After vacuum freeze-drying, porous heterogeneous gel microspheres are obtained.

[0033] Example 2

[0034] (1) Weigh 0.15g of sodium alginate and dissolve it in 10g of purified water. After it is completely dissolved by stirring, weigh 1g of hydroxyapatite and stir it to disperse it evenly to obtain a sodium alginate / hydroxyapatite mixture.

[0035] (2) Weigh 0.15g of recombinant collagen and dissolve it in 10g of purified water. After it is completely dissolved by stirring, weigh 1g of hydroxyapatite and stir it to disperse it evenly to obtain a recombinant collagen / hydroxyapatite mixture.

[0036] (3) The above sodium alginate / hydroxyapatite mixture and recombinant collagen / hydroxyapatite mixture were respectively loaded into 10mL syringes A and B, and injected into the Y-shaped tubes using a syringe pump. They were injected into the continuously stirred vegetable oil at an injection rate of 1mL / h to obtain heterogeneous microspheres.

[0037] (4) The heterogeneous microspheres obtained above were washed with ethanol and acetone in sequence for 3 hours each;

[0038] (5) After cleaning, the heterogeneous microspheres are first crosslinked with 2wt% calcium chloride solution for 4 hours, then crosslinked with 2wt% carbodiimide solution for 4 hours, and then washed with purified water for 4 hours. After vacuum freeze-drying, porous heterogeneous gel microspheres are obtained.

[0039] Example 3

[0040] (1) Weigh 0.5g of sodium alginate and dissolve it in 10g of purified water. After it is completely dissolved by stirring, weigh 1g of hydroxyapatite and stir it to disperse it evenly to obtain a sodium alginate / hydroxyapatite mixture.

[0041] (2) Weigh 0.2g of recombinant collagen and dissolve it in 10g of purified water. After it is completely dissolved by stirring, weigh 2g of hydroxyapatite and stir it to disperse it evenly to obtain a recombinant collagen / hydroxyapatite mixture.

[0042] (3) The above sodium alginate / hydroxyapatite mixture and recombinant collagen / hydroxyapatite mixture were respectively loaded into 10mL syringes A and B, and injected into the Y-shaped tubes using a syringe pump. They were injected into the continuously stirred vegetable oil at an injection rate of 0.5mL / h to obtain heterogeneous microspheres.

[0043] (4) The heterogeneous microspheres obtained above were washed with ethanol and acetone in sequence for 4 h and 1 h, respectively;

[0044] (5) After cleaning, the heterogeneous microspheres are first cross-linked with 1.5wt% calcium chloride solution for 6 hours, then cross-linked with 5wt% carbodiimide for 2 hours, and then washed with purified water for 8 hours. After vacuum freeze-drying, porous heterogeneous gel microspheres are obtained.

[0045] Example 4

[0046] (1) Weigh 0.3g of sodium alginate and dissolve it in 10g of purified water. After it is completely dissolved by stirring, weigh 1g of hydroxyapatite and stir it to disperse it evenly to obtain a sodium alginate / hydroxyapatite mixture.

[0047] (2) Weigh 0.5g of recombinant collagen and dissolve it in 10g of purified water. After it is completely dissolved by stirring, weigh 1.5g of hydroxyapatite and stir it to disperse it evenly to obtain a recombinant collagen / hydroxyapatite mixture.

[0048] (3) The above sodium alginate / hydroxyapatite mixture and recombinant collagen / hydroxyapatite mixture were respectively loaded into 10mL syringes A and B, and injected into the Y-shaped tubes using a syringe pump. They were injected into the continuously stirred vegetable oil at an injection rate of 2mL / h to obtain heterogeneous microspheres.

[0049] (4) The heterogeneous microspheres obtained above were washed with ethanol and acetone for 1 h and 5 h respectively.

[0050] (5) After cleaning, the heterogeneous microspheres are first crosslinked with 5wt% calcium chloride solution for 1h, then crosslinked with 1.5wt% carbodiimide for 6h, and then washed with purified water for 12h. After vacuum freeze-drying, porous heterogeneous gel microspheres are obtained.

[0051] Example 5

[0052] (1) Weigh 0.1g of sodium alginate and dissolve it in 10g of purified water. After it is completely dissolved by stirring, weigh 0.8g of hydroxyapatite and stir it to disperse it evenly to obtain a sodium alginate / hydroxyapatite mixture.

[0053] (2) Weigh 0.3g of recombinant collagen and dissolve it in 10g of purified water. After it is completely dissolved by stirring, weigh 0.5g of hydroxyapatite and stir it to disperse it evenly to obtain a recombinant collagen / hydroxyapatite mixture.

[0054] (3) The above sodium alginate / hydroxyapatite mixture and recombinant collagen / hydroxyapatite mixture were respectively loaded into 10mL syringes A and B, and injected into the Y-shaped tubes using a syringe pump. They were injected into the continuously stirred vegetable oil at an injection rate of 5mL / h to obtain heterogeneous microspheres.

[0055] (4) The heterogeneous microspheres obtained above were washed with ethanol and acetone in sequence for 8 h and 4 h, respectively;

[0056] (5) After cleaning, the heterogeneous microspheres are first crosslinked with 4wt% calcium chloride solution for 2h, then crosslinked with 4wt% carbodiimide for 3h, and then washed with purified water for 6h. After vacuum freeze-drying, porous heterogeneous gel microspheres are obtained.

[0057] Example 6

[0058] (1) Weigh 0.12g of sodium alginate and dissolve it in 10g of purified water. After it is completely dissolved by stirring, weigh 1g of hydroxyapatite and stir it to disperse it evenly to obtain a sodium alginate / hydroxyapatite mixture.

[0059] (2) Weigh 0.3g of recombinant collagen and dissolve it in 10g of purified water. After it is completely dissolved by stirring, weigh 1g of hydroxyapatite and stir it to disperse it evenly to obtain a recombinant collagen / hydroxyapatite mixture.

[0060] (3) The above sodium alginate / hydroxyapatite mixture and recombinant collagen / hydroxyapatite mixture were respectively loaded into 10mL syringes A and B, and injected into the Y-shaped tubes using a syringe pump. They were injected into the continuously stirred vegetable oil at an injection rate of 1.5mL / h to obtain heterogeneous microspheres.

[0061] (4) The heterogeneous microspheres obtained above were washed with ethanol and acetone in sequence for 6 h and 2 h, respectively;

[0062] (5) After cleaning, the heterogeneous microspheres are first crosslinked with 3wt% calcium chloride solution for 4h, then crosslinked with 5wt% carbodiimide for 1h, and then washed with purified water for 2h. After vacuum freeze-drying, porous heterogeneous gel microspheres are obtained.

[0063] Comparative Example 1

[0064] Hydroxyapatite / sodium alginate / recombinant collagen composite porous heterogeneous gel microspheres were prepared according to the method in Example 2, except that sodium alginate was not added.

[0065] Comparative Example 2

[0066] Hydroxyapatite / sodium alginate / recombinant collagen composite porous heterogeneous gel microspheres were prepared according to the method in Example 2, except that recombinant collagen was not added.

[0067] Comparative Example 3

[0068] Hydroxyapatite / sodium alginate / recombinant collagen composite porous heterogeneous gel microspheres were prepared according to the method in Example 2, except that hydroxyapatite was not added.

[0069] Comparative Example 4

[0070] Hydroxyapatite / sodium alginate / recombinant collagen composite porous heterogeneous gel microspheres were prepared according to the method in Example 2, except that the injection rate was 0.01 ml / h.

[0071] Comparative Example 5

[0072] Hydroxyapatite / sodium alginate / recombinant collagen composite porous heterogeneous gel microspheres were prepared according to the method in Example 2, except that the injection rate was 10 ml / h.

[0073] Comparative Example 6

[0074] Hydroxyapatite / sodium alginate / recombinant collagen composite porous heterogeneous gel microspheres were prepared according to the method in Example 2, except that they were not washed.

[0075] Comparative Example 7

[0076] Hydroxyapatite / sodium alginate / recombinant collagen composite porous heterogeneous gel microspheres were prepared according to the method in Example 2, except that calcium chloride solution was not used.

[0077] Comparative Example 8

[0078] Hydroxyapatite / sodium alginate / recombinant collagen composite porous heterogeneous gel microspheres were prepared according to the method in Example 2, except that carbodiimide was not used.

[0079] Performance Test 1

[0080] Weigh 10 mg of microsphere sample and disperse it in 1 mL of phosphate buffer. Mix thoroughly using a pipette. Transfer 20 μL of the liquid onto a glass slide, cover with a coverslip, and observe under an optical microscope. Take five random photographs of the samples. Process the images using ImageJ software. Measure the diameter of each microsphere sample. Perform Gaussian distribution analysis on the obtained data. The statistical results are shown in Table 1. The experimental results of the examples show that when the injection rate is 0.1-5 ml / h, the diameter of the microspheres obtained is within the range of 100-400 μm. The microsphere diameters of Comparative Examples 1 and 2 are not significantly different from those of the examples, indicating that the microsphere diameter is not related to the changes in components. Comparative Example 3 did not form microspheres, and the microsphere diameter could not be measured, indicating that hydroxyapatite is the core factor affecting the microspheres. The microsphere diameters of Comparative Examples 4 and 5 are significantly different from those of the examples, indicating that the microsphere diameter is related to the injection rate; the higher the injection rate, the smaller the microsphere diameter, and vice versa. The microsphere diameters of Comparative Examples 6, 7, and 8 are not significantly different from those of the examples, indicating that the microsphere diameter is not related to cleaning or cross-linking.

[0081] Table 1 Average diameter of microspheres

[0082]

[0083]

[0084] Performance Test 2

[0085] The morphology of the sample in Example 2 was observed using a scanning electron microscope, and the specific results can be seen in [the image below]. Figure 2 The results showed that the prepared sample was in the form of microspheres.

Claims

1. A method for preparing a hydroxyapatite / sodium alginate / recombinant collagen composite porous heterogeneous gel microsphere, characterized by, The method comprises the following steps: (1) stirring and mixing hydroxyapatite with sodium alginate and recombinant collagen respectively to obtain a sodium alginate / hydroxyapatite mixture and a recombinant collagen / hydroxyapatite mixture; the concentration of hydroxyapatite in the sodium alginate / hydroxyapatite mixture or the recombinant collagen / hydroxyapatite mixture is 2wt%-15wt%; the concentration of sodium alginate in the sodium alginate / hydroxyapatite mixture is 0.1wt%-5wt%; the concentration of recombinant collagen in the recombinant collagen / hydroxyapatite mixture is 0.5wt%-5wt%; (2) injecting the sodium alginate / hydroxyapatite mixture and the recombinant collagen / hydroxyapatite mixture into two channels of a Y-shaped microfluidic device respectively, mixing and dropping the mixture into an oil phase through the Y-shaped microfluidic device, and washing and removing the oil phase to obtain heterogeneous microspheres; (3) crosslinking the heterogeneous microspheres with a calcium chloride solution and a carbodiimide solution in sequence, washing after crosslinking, and freeze-drying to obtain hydroxyapatite / sodium alginate / recombinant collagen composite porous heterogeneous gel microspheres.

2. The production method according to claim 1, characterized by, In step (1), the concentration of hydroxyapatite in the sodium alginate / hydroxyapatite mixture or the recombinant collagen / hydroxyapatite mixture is 5wt%-10wt%; the concentration of sodium alginate in the sodium alginate / hydroxyapatite mixture is 1wt%-3wt%; and the concentration of recombinant collagen in the recombinant collagen / hydroxyapatite mixture is 1.5wt%-3wt%.

3. The production method according to claim 1, characterized by, In step (1), the recombinant collagen is produced by fermentation of Pichia pastoris with a preservation number of CGMCC No. 5021; and in step (2), the oil phase is selected from liquid paraffin oil, vegetable oil or dimethyl silicone oil.

4. The method of claim 1, wherein, In step (2), the injection rate is 0.1-5mL / h, and the washing method is to sequentially wash and remove the oil phase with ethanol and acetone, and the washing time of each solvent is 0.5-8h.

5. The preparation method according to claim 4, characterized in that, In step (2), the injection rate is 0.5-2mL / h, and the washing time of each solvent is 1-4h.

6. The method of claim 1, wherein, In step (3), the concentration of the calcium chloride solution is 0.5wt%-5wt%, the crosslinking time is 1-8h, the concentration of the carbodiimide solution is 1wt%-5wt%, the crosslinking time is 1-8h, and the washing time is 1-12h.

7. The preparation method according to claim 1, characterized in that, In step (3), the concentration of the calcium chloride solution is 1.5wt%-3wt%, the crosslinking time is 2-6h, the concentration of the carbodiimide solution is 1.5wt%-4wt%, the crosslinking time is 2-4h, and the washing time is 2-8h.

8. The production method according to claim 1, characterized by, In step (3), the size of the composite porous heterogeneous gel microspheres is 100-400 μm.

9. The hydroxyapatite / sodium alginate / recombinant collagen composite porous heterogeneous gel microspheres according to any one of claims 1-8.

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