Preparation process of sodium alginate-based micro-scaffold

Abstract

The invention belongs to the technical field of preparation of sodium alginate gel micro-scaffolds, particularly relates to a preparation process of a sodium alginate-based micro-scaffold, and aims to solve the problems that an existing traditional preparation method is low in raw material utilization rate, complex in process, difficult to consider mechanical properties and biological activity of a scaffold and the like. The raw materials are prepared in proportion; s2, spraying and shaping the prepared raw materials to obtain microspheres; s3, the microspheres are filtered, washed and freeze-dried, the SC / NCO-15 micro-scaffold is prepared by adding NaHCO3 (a pore-foaming agent) on the basis of the process of 3% of SA, 3% of CS and 6% of Ca < 2 + > (SA3 / CS-Ca6), the SC / NCO-15 micro-scaffold can be rapidly swelled for 30 min to 1 h in a culture medium and shows excellent swelling capacity (the swelling rate is 173.84%), the particle size is about 200-300 microns after swelling, and meanwhile, the size of the SC / NCO-15 micro-scaffold is greatly reduced. The SC / NCO-15 micro-scaffold can adsorb micromolecules (methylene blue) and macromolecules (protein), and provides a physicochemical basis for subsequent cell experiments.

Description

Technical Field

[0001] This invention relates to the field of sodium alginate gel microscaffold preparation technology, and in particular to a preparation process for sodium alginate-based microscaffolds. Background Technology

[0002] In the fields of tissue engineering and drug delivery, there is an urgent need for gel microscaffold materials with good biocompatibility and tunable structural properties, but current traditional preparation processes have significant shortcomings: Low raw material utilization: Traditional methods do not make full use of the main raw materials such as sodium alginate and chitosan when preparing gel microscaffolds, which leads to increased costs and low production efficiency. For example, in some precipitation or casting processes, a large amount of raw materials are discharged with waste liquid, which not only wastes resources but also increases the burden of environmental treatment. Complex processes: For example, the sol-gel method requires strict control of multiple parameters such as temperature and pH value, which is cumbersome to operate, has a long cycle, and has high requirements for equipment and operators, making it difficult to achieve large-scale stable production. Some processes that require multiple chemical cross-linking involve complex reaction condition switching and cumbersome intermediate processing steps. Performance is difficult to balance: It is difficult to simultaneously meet the requirements of mechanical properties and bioactivity. Single-material gel microscaffolds either lack sufficient mechanical strength to maintain tissue morphology, or lack bioactivity to promote cell growth and tissue repair and regeneration. For example, pure sodium alginate gel has good biocompatibility but weak mechanical strength; while gels that have been excessively cross-linked to enhance mechanical properties may destroy their bioactive components or affect cell recognition sites.

[0003] In the existing technology, traditional preparation methods have problems such as low raw material utilization, complex processes, and difficulty in balancing the mechanical properties and bioactivity of microscaffolds. To address these issues, we propose a preparation process for sodium alginate-based microscaffolds. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies, such as low raw material utilization, complex processes, and difficulty in balancing the mechanical properties and bioactivity of microscaffolds, by proposing a preparation process for sodium alginate-based microscaffolds.

[0005] The fabrication process of a sodium alginate-based microscaffold provided in this application adopts the following technical solution: A process for preparing a sodium alginate-based microscaffold includes the following steps: S1: Prepare the raw materials and mix them according to the proportions; S2: Spray and shape the prepared raw materials to obtain microspheres; S3: The microspheres are filtered, rinsed, and freeze-dried to obtain sodium alginate-based microscaffolds.

[0006] Further, in S1, a 3% sodium alginate solution (SA) (1.5g, 50mL) and a 3% chitosan solution (CS) (1.5g, 50mL) are prepared respectively, and then stirred evenly to obtain solution A (the volume ratio of SA solution to CS solution is 1:1).

[0007] Further, in step S1, 6% anhydrous calcium chloride (48g) is added to a 50% ethanol solution (800mL) at room temperature to obtain microscaffold forming solution B.

[0008] Further, in step S1, pore-forming agent NaHCO3 (450 mg, i.e., 15% of the total mass of SA and CS) is added to solution A, and the solution is placed on a stirring table and stirred at a speed of 1000 rpm for 30 min to obtain a microscaffold preparation solution. Solution B is then adjusted to pH 3 with hydrochloric acid.

[0009] Further, in step S2, solution B is added to a glass dish (20cm×10cm), which is placed directly below the nozzle of the spray freeze-drying granulation device, and the distance between the nozzle of the granulation device and the liquid surface is adjusted to 14cm.

[0010] Furthermore, in step S2, the peristaltic pump is started and the air pressure valve is opened, the pressure is adjusted to 0.08 Pa, and the preparative liquid is slowly pumped in at a rate of 5 rpm / min. The solution forms a mist of droplets through the nozzle and falls into solution B below to form microspheres. The cross-linking time is 2 hours.

[0011] Further, in step S3, after the cross-linking is completed, the microspheres are filtered through a filter screen and rinsed with deionized water. The filter screen has a pore size of 200 μm and 300 μm. The microspheres containing NaHCO3 are then soaked in deionized water at pH=1 for 24 hours to remove NaHCO3.

[0012] Further, in step S3, after the microspheres are soaked, they are filtered through a filter screen with a pore size of 200 μm, and washed until the pH of the microsphere solution reaches 7. The microspheres are then collected and dried in a freeze dryer for 3 days to obtain SA / CS / NaHCO3 microscaffolds (SC / NCO).

[0013] Sodium bicarbonate (NaHCO3) is an inorganic compound, a white powder or fine crystals, odorless, with a salty taste. It is readily soluble in water, slightly soluble in ethanol (some sources say it is insoluble), and its aqueous solution is slightly alkaline. It decomposes easily upon heating, slowly in moist air to produce carbon dioxide, starting at about 50°C and completely decomposing at 270°C. It decomposes violently in the presence of acids, producing carbon dioxide. Sodium bicarbonate is widely used in chemical, pharmaceutical, food, light industry, and textile industries, as well as in daily life, playing an important role in the national economy. Sodium alginate is a byproduct of the extraction of iodine and mannitol from brown algae such as kelp or giant kelp. Its molecule is composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G) linked by (1→4) bonds. It is a natural polysaccharide possessing the stability, solubility, viscosity, and safety required for pharmaceutical excipients. First described in a patent application filed in 1881 by British chemist Edward C. Stanford, sodium alginate is defined as sodium alginate salt, with the empirical formula (C6H7O6Na)n. The physical properties of sodium alginate are that it is in powder or fibrous form, white to slightly yellow, and almost odorless. To this day, brown algae remain the primary source for extracting sodium alginate. Sodium alginate has the following properties: 1. Water solubility: Sodium alginate is easily soluble in water, forming a viscous colloidal solution; 2. Biocompatibility: It is non-toxic, has good biocompatibility, and is widely used in the biomedical field; 3. Film-forming properties: It can form a gel film with elasticity and toughness; 4. Stability: It is relatively stable under both acidic and alkaline conditions; 5. Thickening properties: It has a strong thickening effect and has a long history of wide application in the food, paper, and cosmetic industries.

[0014] In summary, this application includes at least one of the following beneficial technical effects: 1. This method, by precisely controlling the ratio and reaction conditions of sodium alginate (SA), chitosan (CS), and the pore-forming agent sodium bicarbonate (NaHCO3), can prepare sodium alginate-based microscaffolds with good swelling properties. Experimental tests show that the prepared SC / NCO-15 microscaffolds can achieve a swelling rate of up to 173.84% in the culture medium, and the particle size is uniformly distributed between 200-300 μm after swelling. This enables them to respond quickly to the biological environment, providing excellent physical conditions for cell attachment, proliferation, and nutrient transport. Thus, in tissue engineering applications, it can better simulate the extracellular matrix environment and promote tissue repair and regeneration. 2. This method utilizes spray freeze-drying granulation technology combined with specific raw material ratios and reaction conditions to obtain microscaffolds with uniform particle size and suitable porosity (17.5%). This porous structure not only facilitates deep cell growth and nutrient exchange but also enhances the interaction between the gel microscaffolds and surrounding tissues, improving the biocompatibility and integration of the microscaffolds. This allows them to better integrate into the host tissue environment in tissue engineering and regenerative medicine, promoting tissue regeneration and functional recovery.

[0015] The SC / NCO-15 microscaffold of this invention is formulated with 3% SA, 3% CS, and 6% Ca. 2It is prepared by adding NaHCO3 (porogen) to the process of (SA3 / CS-Ca6). It can swell rapidly in the culture medium (30min-1h) and exhibits excellent swelling capacity (swelling rate of 173.84%). The particle size after swelling is about 200-300μm. At the same time, the SC / NCO-15 microscaffold can adsorb small molecules (methylene blue) and large molecules (proteins), providing a physicochemical basis for subsequent cell experiments. Attached Figure Description

[0016] Figure 1 This is a flowchart of the preparation process of a sodium alginate-based microscaffold proposed in this invention; Figure 2 This is a complete view of the spray freeze-drying granulation device for the preparation process of sodium alginate-based microscaffolds proposed in this invention. Figure 2 a); Sample preparation area ( Figure 2 b); Transformer ( Figure 2 c); Feeding area ( Figure 2 d); Pressure regulating zone ( Figure 2 e); air pressure pump (2f); Figure 3 The preparation process of the sodium alginate-based microscaffold proposed in this invention is based on SA3 / CS-Ca4 ( Figure 3 a) SA3 / CS-Ca6 ( Figure 3 b), SA3 / CS-Ca8 ( Figure 3 c) and SA3 / CS-Ca10 ( Figure 3 d) Microscaffolds in Ca 2+ Photographs of cross-linked components in solution; Figure 4 This invention provides a fabrication process for an SA3 / CS-Ca6 microscaffold based on sodium alginate. Figure 4 a, d), SA4 / CS-Ca6 micro-scaffold ( Figure 4 b, e) and SA4.5 / CS-Ca6 ( Figure 4 c, f) Photographs of the microscaffolds after swelling in deionized water and phenol red medium; Figure 5 This invention provides a fabrication process for an SA3 / CS-Ca6 microscaffold based on sodium alginate. Figure 5 a, d), SA4 / CS-Ca6 micro-scaffold ( Figure 5 b, e) and SA4.5 / CS-Ca6 micro-scaffold ( Figure 5 c, f) Optical micrographs of the swollen medium in deionized water and phenol red; Figure 6 This invention provides a fabrication process for an SA3 / CS-Ca6 microscaffold based on sodium alginate. Figure 6a, d), SA4 / CS-Ca6 micro-scaffold ( Figure 6 b, e) and SA4.5 / CS-Ca6 micro-scaffold ( Figure 6 Scanning electron microscope (SEM) images of (c) and (f); Figure 7 The image shows the BET adsorption data of the SA3 / CS-Ca6 microscaffold prepared according to the sodium alginate-based microscaffold proposed in this invention. Figure 8 This is a schematic diagram of the adsorption isotherm of the microscaffold according to the preparation process of the sodium alginate-based microscaffold proposed in this invention. Figure 9 Photographs of different batches of SA3 / CS-Ca6 microscaffold lyophilized powder, representing the preparation process of a sodium alginate-based microscaffold proposed in this invention. Figure 9 ac); Figure 10 The SC / NCO-10 microscaffold is a fabrication process for a sodium alginate-based microscaffold proposed in this invention. Figure 10 a, g, m), SC / NCO-15 micro-scaffold ( Figure 10 b, h, n), SC / NCO-20 micro-scaffold ( Figure 10 c, i, o), SC / NCO-30 micro-scaffold ( Figure 10 d, j, p), SC / NCO-40 micro-scaffold ( Figure 10 e, k, q) and SC / NCO-50 micro-scaffold ( Figure 10 SEM images of different magnifications (f, l, r); Figure 11 The SC / NCO-10 microscaffold is a fabrication process for a sodium alginate-based microscaffold proposed in this invention. Figure 11 a, g), SC / NCO-15 micro-scaffold ( Figure 11 b, h), SC / NCO-20 micro-scaffold ( Figure 11 c, i), SC / NCO-30 micro-scaffold ( Figure 11 d, j), SC / NCO-40 micro-scaffold ( Figure 11 e, k) and SC / NCO-50 micro-scaffold ( Figure 11 f, l) Optical micrographs of the swollen medium after swelling in phenol red-free medium; Figure 12 Photograph of SC / NCO-15 microscaffold freeze-dried powder, which is a preparation process of sodium alginate-based microscaffold proposed in this invention. Figure 13 The SC / NCO-15 microscaffold is a fabrication process for a sodium alginate-based microscaffold proposed in this invention. Figure 13 a, b) Optical microscope images and swelling ratios after swelling in the culture medium. Figure 13 c) Figure 14 Absorption curves of small molecules adsorbed by SC / NCO-15 microscaffolds in accordance with the preparation process of sodium alginate-based microscaffolds proposed in this invention ( Figure 14 a) and adsorption photographs ( Figure 14 b) Figure 15 The SC / NCO-15 microscaffold is a fabrication process for a sodium alginate-based microscaffold proposed in this invention. Figure 15 (ac) Laser confocal image of fluorescent bovine serum albumin (green) adsorbed; Figure 16 The present invention proposes a process for preparing a sodium alginate-based microscaffold by adding SC / NCO-15 (which swells and adsorbs in methylene blue culture medium) to a basic hydrogel. Figure 16 a); Hydrogel cultured without phenol red ( Figure 16 b) and phenol red medium ( Figure 16 c) Photographs of the 2D paving and 3D dome formed; the seepage of methylene blue from the hydrogel at different times: 3h ( Figure 16 d), 24h Figure 16 e). Detailed Implementation

[0017] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0018] Example 1 Reference Figure 1 A process for preparing a sodium alginate-based microscaffold includes the following steps: S1: Prepare 3% sodium alginate solution (SA) (1.5g, 50mL) and 3% chitosan solution (CS) (1.5g, 50mL) respectively, and then stir them evenly to obtain solution A (the volume ratio of SA solution to CS solution is 1:1). At room temperature, add 6% anhydrous calcium chloride (48g) to 50% ethanol solution (800mL) to obtain microscaffold forming solution (solution B). Add porogen NaHCO3 (450mg, i.e., 15% of the total mass of SA and CS) to solution A, place it on a stirring table and stir at a stirring speed of 1000rpm for 30min to obtain microscaffold preparation solution. Adjust the pH of solution B to 3 with hydrochloric acid. S2: Add solution B to a glass dish (20cm×10cm) and place it directly below the nozzle of the spray freeze-drying granulation device. Adjust the distance between the nozzle and the liquid surface of the granulation device to 14cm. Start the peristaltic pump and open the air pressure valve. Adjust the pressure to 0.08Pa and slowly pump in the preparative solution at a rate of 5rpm / min. The solution forms a mist droplet through the nozzle and falls into solution B below to form microspheres. The cross-linking time is 2h. S3: After cross-linking, the microspheres were filtered through a filter screen with pore sizes of 200 μm and 300 μm and rinsed with deionized water. The microspheres (NaHCO3) were then soaked in deionized water at pH 1 for 24 hours to remove NaHCO3. 3， After soaking, the microspheres were filtered through a 200 μm sieve and washed until the pH of the microsphere solution reached 7. The microspheres were then collected and dried in a freeze dryer for 3 days to obtain SA / CS / NaHCO3 microscaffolds (SC / NCO).

[0019] Example 2 The difference between this embodiment and Embodiment 1 is that: (Refer to...) Figure 1 A process for preparing a sodium alginate-based microscaffold includes the following steps: S1: Prepare 3% basic SA solution (1.5g, 50mL) and 3% CS solution (1.5g, 50mL) respectively, and then stir evenly to obtain solution A (the volume ratio of SA solution to chitosan solution is 1:1). At room temperature, add 6% anhydrous calcium chloride (48g) to 50% ethanol solution (800mL) to obtain solution B. Add different amounts of NaHCO3 to solution A (10%, 15%, and 20% of the total mass of SA and CS respectively), place on a stirring table and stir at 1000rpm for 30min to obtain microscaffold preparation solution. Adjust the pH of solution B to 3 with hydrochloric acid. S2: Add solution B to a glass dish (20cm×10cm) and place it directly below the nozzle of the spray freeze-drying granulation device. Adjust the distance between the nozzle and the liquid surface of the granulation device to 14cm. Start the peristaltic pump and open the air pressure valve to a pressure of 0.08Pa. Slowly pump in the preparative solution at a rate of 5rpm / min. The solution forms a mist droplet through the nozzle and falls into solution B below to form microspheres. The cross-linking time is 2h. S3: After cross-linking, the microspheres were filtered through a filter screen with pore sizes of 200 μm and 300 μm and rinsed with deionized water. The microspheres (NaHCO3) were then soaked in deionized water at pH 1 for 24 hours to remove NaHCO3. 3， After soaking the microspheres, they were filtered through a 200 μm sieve and washed until the pH of the microsphere solution reached 7. The microspheres were then collected and dried in a freeze dryer for 3 days to obtain SA / CS / NaHCO3 microscaffolds (SC / NCO). With increasing NaHCO3 dosage, the particle size of the microscaffold slightly increased, and its morphology gradually became flatter, with a more pronounced pore structure. The microscaffold exhibited optimal swelling performance with a NaHCO3 dosage of 15%, reaching a swelling rate of 173.84%. When the dosage was below 15%, the swelling rate was lower; when the dosage was above 15%, the swelling rate decreased, but the larger particle size after swelling may lead to a decrease in the mechanical strength of the scaffold. Microscaffolds with different amounts of porogen exhibit varying adsorption capacities for small molecules (methylene blue) and large molecules (fluorescent bovine serum albumin). The microscaffold with 15% NaHCO3 content shows more balanced adsorption performance, achieving an adsorption capacity of 0.540 g / g for methylene blue, while also effectively adsorbing fluorescent bovine serum albumin. Based on these results, the SC / NCO microscaffold prepared with 15% NaHCO3 content demonstrates superior overall performance.

[0020] Example 3 The difference between this embodiment and Embodiment 1 is that: (Refer to...) Figure 1 A process for preparing a sodium alginate-based microscaffold includes the following steps: S1: Prepare 3% basic SA solution (1.5g, 50mL) and 3% CS solution (1.5g, 50mL) respectively, and then stir evenly to obtain solution A (the volume ratio of SA solution to chitosan solution is 1:1). Prepare CaCl2 solutions of different concentrations (4%, 6%, 8%, 10%). Add the CaCl2 solutions of different concentrations to 50% ethanol solution (800mL) at room temperature to obtain solution B. Add porogen NaHCO3 (450mg, i.e., 15% of the total mass of SA and CS) to solution A, place it on a stirring table and stir at 1000rpm for 30min to obtain microscaffold preparation solution. Adjust the pH of solution B to 3 with hydrochloric acid. S2: Add solution B to a glass dish (20cm×10cm) and place it directly below the nozzle of the spray freeze-drying granulation device. Adjust the distance between the nozzle and the liquid surface of the granulation device to 14cm. Start the peristaltic pump and open the air pressure valve to a pressure of 0.08Pa. Slowly pump in the preparative solution at a rate of 5rpm / min. The solution forms a mist droplet through the nozzle and falls into the CaCl2 solution below to form microspheres. The cross-linking time is 2h. S3: After cross-linking, the microspheres were filtered through a filter screen with pore sizes of 200 μm and 300 μm and rinsed with deionized water. The microspheres (NaHCO3) were then soaked in deionized water at pH 1 for 24 hours to remove NaHCO3. 3，After soaking the microspheres, they were filtered through a 200 μm sieve and washed until the pH of the microsphere solution reached 7. The microspheres were then collected and dried in a freeze dryer for 3 days to obtain SA / CS / NaHCO3 microscaffolds (SC / NCO). As the concentration of solution B increases, the swelling rate of the microscaffold gradually decreases. When the CaCl2 concentration is 6%, the microscaffold swells the fastest in phenol red-free medium (10-30 min), and the particle size after swelling is moderate. When the CaCl2 concentration is higher than 6%, the swelling rate slows down significantly, and the particle size after swelling is smaller, which may lead to an overly dense internal structure of the scaffold, which is not conducive to cell growth and nutrient transport. The microscaffold with a solution B concentration of 6% has good mechanical properties and can maintain a certain shape and structural integrity under simulated physiological conditions, without breaking or deforming due to excessive swelling. However, when the solution B concentration is low, the mechanical strength of the microscaffold is weak, and it is prone to breakage during the swelling process. When the solution B concentration is too high, although the mechanical strength is improved, the swelling performance decreases, which may affect its biocompatibility and cell viability. Scanning electron microscopy (SEM) revealed that the microscaffold with a solution B concentration of 6% had a relatively uniform pore structure and a moderate pore size, which was conducive to cell attachment and growth. The pore structure of microscaffolds with other concentrations was either not obvious enough or the pore size was too large or too small, which was not conducive to cell growth and material exchange. In summary, the SC / NCO microscaffolds prepared with a solution B concentration of 6% exhibited the best overall performance in terms of swelling properties, mechanical properties, and microstructure.

[0021] Example 4 The difference between this embodiment and Embodiment 1 is that: (Refer to...) Figure 1 A process for preparing a sodium alginate-based microscaffold includes the following steps: S1: Prepare 3% sodium alginate solution (SA) (1.5g, 50mL) and 3% chitosan solution (CS) (1.5g, 50mL) respectively, and then stir them evenly to obtain solution A (the volume ratio of SA solution to CS solution is 1:1). At room temperature, add 6% anhydrous calcium chloride (48g) to 50% ethanol solution (800mL) to obtain solution B. Add porogen NaHCO3 (450mg, i.e., 15% of the total mass of SA and CS) to solution A, place it on a stirring table and stir at a stirring speed of 1000rpm for 30min to obtain microscaffold preparation solution. Adjust the pH of solution B to 3 with hydrochloric acid. S2: Add solution B to a glass dish (20cm×10cm) and place it directly below the nozzle of the spray freeze-drying granulation device. Adjust the distance between the nozzle and the liquid surface of the granulation device, start the peristaltic pump and open the air pressure valve. Adjust the distance between the nozzle and the liquid surface to 12cm, 14cm and 16cm respectively, and the air pressure to 0.05Pa, 0.08Pa and 0.10Pa respectively. Slowly pump in the preparative solution at a rate of 5rpm / min. The solution forms a mist droplet through the nozzle and falls into solution B below to form microspheres. The cross-linking time is 2h. S3: After cross-linking, the microspheres were filtered through a filter screen with pore sizes of 200 μm and 300 μm and rinsed with deionized water. The microspheres (NaHCO3) were then soaked in deionized water at pH 1 for 24 hours to remove NaHCO3. 3， After soaking the microspheres, they were filtered through a 200 μm sieve and washed until the pH of the microsphere solution reached 7. The microspheres were then collected and dried in a freeze dryer for 3 days to obtain SA / CS / NaHCO3 microscaffolds (SC / NCO). As the distance between the nozzle and the liquid surface increases, the particle size of the microspheres decreases slightly, and the particle size is relatively uniform when the distance is 14 cm. The higher the gas pressure, the smaller the particle size of the microspheres. However, when the gas pressure is too high (such as 0.10 Pa), the microspheres are prone to breakage, resulting in uneven particle size distribution. When the distance between the nozzle and the liquid surface is 14 cm and the gas pressure is 0.08 Pa, the swelling performance of the microscaffold is optimal, with a high swelling rate and a moderate particle size after swelling, which remains in the range of 200-300 μm. SEM observation revealed that when the distance between the nozzle and the liquid surface was 14 cm and the air pressure was 0.08 Pa, the surface pores of the microscaffold were evenly distributed and the structure was relatively complete, which was conducive to cell attachment and growth.

[0022] Example 5 The difference between this embodiment and Embodiment 1 is that: (Refer to...) Figure 1 A process for preparing a sodium alginate-based microscaffold includes the following steps: S1: Prepare 3% sodium alginate solution (SA) (1.5g, 50mL) and 3% chitosan solution (CS) (1.5g, 50mL) respectively, and then stir them evenly to obtain solution A (the volume ratio of SA solution to CS solution is 1:1). At room temperature, add 6% anhydrous calcium chloride (48g) to 50% ethanol solution (800mL) to obtain solution B. Add porogen NaHCO3 (450mg, i.e., 15% of the total mass of SA and CS) to solution A, place it on a stirring table and stir at a stirring speed of 1000rpm for 30min to obtain microscaffold preparation solution. Adjust the pH of solution B to 3 with hydrochloric acid. S2: Add solution B to a glass dish (20cm×10cm) and place it directly below the nozzle of the spray freeze-drying granulation device. Adjust the distance between the nozzle and the liquid surface of the granulation device to 14cm. Start the peristaltic pump and open the air pressure valve to a pressure of 0.08Pa. Slowly pump in the preparative solution at a rate of 5rpm / min. The solution forms a mist droplet through the nozzle and falls into solution B below to form microspheres. The cross-linking time is 2h. S3: After cross-linking, the microspheres were filtered through a filter screen with pore sizes of 200 μm and 300 μm and rinsed with deionized water. The microspheres (NaHCO3) were then immersed in deionized water at pH=1 for different times (12 h, 24 h, 36 h) to remove NaHCO3. 3， After soaking the microspheres, they were filtered through a 200 μm sieve and washed until the pH of the microsphere solution reached 7. The microspheres were then collected and dried in a freeze dryer for different times (2 days, 3 days, and 4 days) to obtain SA / CS / NaHCO3 microscaffolds (SC / NCO). When the soaking time is 24 hours, NaHCO3 is basically completely removed, and the surface of the microscaffold is smooth with no obvious residue. When the soaking time is too short (12 hours), a small amount of NaHCO3 remains on the surface of the microscaffold. When the soaking time is too long (36 hours), there is no significant improvement in the removal effect. When the drying time is 3 days, the microscaffold is fully dried and its shape is stable. When the drying time is too short (2 days), the moisture inside the microscaffold is not completely removed, and it is easy to agglomerate. When the drying time is too long (4 days), although there is no significant negative impact on the performance of the microscaffold, it will increase the production cost. The microscaffolds soaked for 24 hours and dried for 3 days have the best overall performance, with moderate swelling rate, good mechanical properties, and uniform pore structure, which can meet the requirements of tissue engineering and drug delivery.

[0023] Example I. SC / NCO R&D Process Data 1. Spray freeze-drying granulation device Reference Figure 2 A complete view of the Tokyo Rika SF-1100 spray freeze-drying granulation unit (Japan) Figure 2 a); Sample preparation area (nozzle, stirring table, glassware) Figure 2 b); Transformer ( Figure 2 c); Feeding area (peristaltic pump, feed pipe) Figure 2 d); Pressure regulating zone ( Figure 2 e); air pump ( Figure 2 f); The basic process for microscaffold preparation is as follows: First, adjust the distance between the nozzle and the solution surface B in the sample preparation area ( Figure 2 b); then turn on the transformer power supply ( Figure 2 c), then adjust the peristaltic pump to adjust the feed rate ( Figure 2 d); After feeding is complete, open the air pressure valve in the air pressure regulating zone and adjust its rate ( Figure 2 e); Finally, turn on the air pump ( Figure 2 f) Once a clear mist of liquid droplets is ejected from the nozzle, preparation can begin; 2. Solubility of sodium alginate SA (intrinsic viscosity 86 mPa·s) Table 1. Solubility of SA at different concentrations Table 1 shows the solubility of SA at different concentrations. As the concentration of SA increases, the solubility gradually decreases. Among them, 1.5% and 2% SA dissolve the fastest (about 30 min), followed by 3% SA (about 1-2 h), 4% SA takes 5 h to dissolve, and 4.5% SA dissolves the slowest (about 12 h). 5%-6% SA is difficult to dissolve. Therefore, SA at intermediate concentrations of 3%, 4%, and 4.5% were selected for subsequent experiments. 3. SA / CS micro-scaffolds (different Ca 2+ Concentration) Swelling rate Preparation of different Ca 2+ Microscaffolds with crosslinking concentrations (4%, 6%, 8% and 10%), where the SA concentration is 3%, are abbreviated as SA3 / CS-Ca4, SA3 / CS-Ca6, SA3 / CS-Ca8 and SA3 / CS-Ca10, respectively; like Figure 3 As shown, SA3 / CS-Ca4 exhibits severe agglomeration after freeze-drying and cannot be dispersed. Figure 2 a); SA3 / CS-Ca6, SA3 / CS-Ca8, and SA3 / CS-Ca10 all swelled in phenol red-free medium ( Figure 3 bd); with Ca 2+ With increasing concentration, the swelling rate of the microscaffold gradually decreased; among them, SA3 / CS-Ca6 swelled the fastest (10-30 min), while SA3 / CS-Ca8 and SA3 / CS-Ca10 swelled slowly (more than 1 h); therefore, the Ca used for crosslinking... 2+ The optimal concentration of the solution was determined to be 6%, and the concentration of SA was further screened based on this Ca2+ solution concentration. 4. Swelling properties of SA / CS microscaffolds (different SA concentrations) in water and culture media Microscaffolds with SA concentrations of 3%, 4%, and 4.5% were prepared, with a Ca2+ concentration of 6%, and the corresponding sample abbreviations were SA3 / CS-Ca6, SA4 / CS-Ca6, and SA4.5 / CS-Ca6, respectively. like Figure 4As shown in af, all microscaffolds swelled significantly in deionized water and phenol red medium, with no significant difference. Therefore, the effect of SA concentration on the swelling of microscaffolds could not be evaluated by ordinary visual observation. Therefore, optical microscopy was used to further observe the swelling. like Figure 5 As shown in Figure af, the morphology of SA3 / CS-Ca6, SA4 / CS-Ca6, and SA4.5 / CS-Ca6 after swelling was further observed using an optical microscope; with the increase of SA concentration, the swelling capacity of the microscaffolds gradually decreased; SA3 / CS-Ca6, after swelling in deionized water and phenol red medium, had smooth spherical surfaces without wrinkles, indicating its excellent swelling properties, and the particle size after swelling was approximately 200-300 μm. Figure 5 a, d); SA4 / CS-Ca6 and SA4.5 / CS-Ca6 showed obvious surface wrinkles after swelling in deionized water and phenol red medium. Figure 5 (b, c, e, f) indicates that it has not completely swollen; 5. Micro / nanostructures of SA / CS microscaffolds (with different SA concentrations) As shown in Figures (6a-c, e, f), all microscaffolds exhibit a wrinkled, seed-like shape, with a particle size of approximately 100-200 μm. This indicates that SA concentration has no effect on the morphology of the microscaffolds. Furthermore, magnified images of SA3 / CS-Ca6 are also shown. Figure 6 d) No obvious micro / nanoporous structures were observed; In summary, SA3 / CS-Ca6 exhibits no agglomeration after freeze-drying, swells rapidly, and results in a smooth, wrinkle-free surface after swelling, making it the optimal group. 6. BET adsorption test of SA3 / CS-Ca6 microscaffold Table 2 BET adsorption data of SA3 / CS-Ca6 microscaffolds BET adsorption testing can be used to obtain data related to pore structure; Figure 7 The mass, density, pore volume, and pore size of the microcarriers and SA3 / CS-Ca6 are compiled in Table 2. The porosity can be calculated using the following formula: Porosity = Pore volume / Sample volume = Pore volume / (mass / density) * 100% ---------① Figure 8 As shown, the SA3 / CS-Ca6 adsorption isotherm does not show a clear saturation adsorption plateau, belonging to a composite hysteresis loop of type H1 and H3, indicating that the pore size distribution is non-uniform; the porosity of SA3 / CS-Ca6 is calculated to be 17.5% using BET data (Table 2) and formula ①. 7. Dispersibility and stability of SA3 / CS-Ca6 dry powder preparation process Figure 9 As shown in a, SA3 / CS-Ca6 was granular after freeze-drying without agglomeration; and, different batches of microscaffold freeze-dried powder ( Figure 9 The morphology of the ac is basically the same, indicating that the current microscaffold fabrication process is relatively stable; SC / NCO characterization and analysis During the preparation of the microscaffold, different amounts of NaHCO3 (10wt%, 15wt%, 20wt%, 30wt%, 40wt%, 50wt%) were added, and the corresponding samples were simply referred to as SC / NCO-10, SC / NCO-15, SC / NCO-20, SC / NCO-30, SC / NCO-40 and SC / NCO-50. 1. SEM of SC / NCO microscaffolds like( Figure 10 (ac, bf) showed that all SC / NCO microscaffolds were uniformly dispersed with a particle size of 100-200 μm; when the NaHCO3 content was low, the SC / NCO-10 and SC / NCO-15 microscaffolds were seed-shaped. Figure 10 g, h), and a uniform pore structure can be observed ( Figure 10 m, n); When the NaHCO3 content is high, SC / NCO-20 ( Figure 10 c,i,o), SC / NCO-30 ( Figure 10 d,j,p), SC / NCO-40 ( Figure 10 e,k,q) and SC / NCO-50 ( Figure 10 f,l,r) The microscaffolds are flat and no obvious pore structure can be observed; 2. Swelling properties of SC / NCO Figure 11 As shown in af, all SC / NCO microscaffolds can swell; Figure 11 gl showed that the particle size of the SC / NCO microscaffolds after swelling was 200-300 μm. With increasing NaHCO3 content, the swelling capacity of the SC / NCO microscaffolds first increased and then decreased, with SC / NCO-15 showing the best swelling capacity and the highest uniformity after swelling. Considering the SEM morphology, pore structure, and swelling performance, SC / NCO-15 was the superior group. 3. Macroscopic morphology Figure 12 As shown, the SC / NCO-15 microscaffold did not show obvious agglomeration after freeze-drying, and the granular texture was obvious; 4. Swelling and swelling rate Figure 13As shown in ab, after swelling in the culture medium, the SC / NCO-15 microscaffolds exhibited high uniformity, with a particle size distribution of 250-300 μm and a swelling rate of 173.84%. 5. Adsorption performance of SC / NCO-15 microscaffold (small molecule-methylene blue) The adsorption curves show that the absorbance of the methylene blue solution was highest at 0.687 before adsorption; as the adsorption time increased, the highest absorbance value decreased, indicating that the adsorption capacity of the SC / NCO-15 microscaffold for methylene blue increased. Figure 14 a), and the adsorption reaches its maximum value in 3 hours; at this time, the highest value of the curve is 0.147 (the adsorption amount is 0.540). 6. Adsorption performance of macromolecules (fluorescent bovine serum albumin) Figure 15 The ac results show that the surface of the SC / NCO-15 microscaffold has green fluorescence, indicating that it can adsorb bovine serum albumin. 7. SC / NCO-15 microscaffolds can be used in combination with basic hydrogel. Figure 16 a indicates that the SC / NCO-15 microscaffold was immersed in a methylene blue solution for adsorption; Figure 16 As shown in b, after the basic hydrogel (sodium alginate-based hydrogel) is uniformly mixed with the SC / NCO-15 microscaffold adsorbed with methylene blue, it can be spread to a certain thickness in a cell culture well plate and form a three-dimensional dome-shaped gel; then, as... Figure 16 c. Add a culture medium containing phenol red to cover the mixture of the above hydrogel and the SC / NCO-15 microscaffold adsorbed with methylene blue, and observe the color change in the wells; Figure 16 As shown in Figure d, with the increase of the standing time to 3 hours, the color of the phenol red-containing medium in the above wells changed from red to purple, indicating that the methylene blue adsorbed in the SC / NCO-15 microscaffold can be released into the medium through the hydrogel, and the microscaffold has the ability to release small molecules; Figure 16 As shown in Figure e, after standing for 24 hours, the color of the SC / NCO-15 microscaffold changed from blue to red, indicating that the small molecule (phenol red) can penetrate into SC / NCO-15. The above results show that SC / NCO-15 can be mixed with the basic hydrogel and can exchange substances with the culture medium in the hydrogel, and can be used for cell culture. Summarize In summary, the currently developed SC / NCO-15 micro-scaffold is based on 3% SA, 3% CS, and 6% Ca. 2+The SC / NCO-15 microscaffold is prepared by adding NaHCO3 (a porogen) to the (SA3 / CS-Ca6) process. It is a porous hydrogel microsphere in the shape of a melon seed, with a particle size of about 100-200 μm after freeze-drying. Its freeze-dried powder can swell rapidly in the culture medium (30 min-1 h) and exhibits excellent swelling ability (swelling rate of 173.84%), with a particle size of about 200-300 μm after swelling. At the same time, the SC / NCO-15 microscaffold can adsorb small molecules (methylene blue) and large molecules (proteins), providing a physicochemical basis for subsequent cell experiments.

[0024] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A process for preparing a sodium alginate-based microscaffold, characterized in that: Includes the following steps: S1: Prepare the raw materials and mix them according to the proportions; S2: Spray and shape the prepared raw materials to obtain microspheres; S3: The microspheres are filtered, rinsed, and freeze-dried to obtain sodium alginate-based microscaffolds.

2. The preparation process of a sodium alginate-based microscaffold according to claim 1, characterized in that: In step S1, a 3% sodium alginate solution (1.5g, 50mL) and a 3% chitosan solution (1.5g, 50mL) are prepared and then stirred until homogeneous to obtain solution A.

3. The preparation process of a sodium alginate-based microscaffold according to claim 2, characterized in that: In step S1, 6% anhydrous calcium chloride is added to a 50% ethanol solution at room temperature to obtain microscaffold forming solution B.

4. The preparation process of a sodium alginate-based microscaffold according to claim 3, characterized in that: In step S1, pore-forming agent NaHCO3 is added to solution A and stirred on a stirring table.

5. The preparation process of a sodium alginate-based microscaffold according to claim 4, characterized in that: In step S1, the stirring speed is 1000 rpm and the stirring time is 30 min to obtain the microscaffold preparation solution; Adjust the pH of solution B to 3 using hydrochloric acid.

6. The preparation process of a sodium alginate-based microscaffold according to claim 5, characterized in that: In step S2, solution B is added to a glass dish and placed directly below the nozzle of the spray freeze-drying granulation device. The distance between the nozzle of the granulation device and the liquid surface is adjusted to 14 cm.

7. The preparation process of a sodium alginate-based microscaffold according to claim 6, characterized in that: In step S2, the peristaltic pump is started and the air pressure valve is opened. The pressure is adjusted to 0.08 Pa, and the preparative liquid is slowly pumped in at a rate of 5 rpm / min. The solution forms a mist of droplets through the nozzle and falls into solution B below to form microspheres. The cross-linking time is 2 hours.

8. The preparation process of a sodium alginate-based microscaffold according to claim 7, characterized in that: In step S3, after cross-linking is completed, the microspheres are filtered through a filter screen and rinsed with deionized water. The filter screen has a pore size of 200 μm and 300 μm.

9. The preparation process of a sodium alginate-based microscaffold according to claim 8, characterized in that: In step S3, the microspheres are soaked in deionized water with pH=1 for 24 hours to remove NaHCO3. After soaking, the microspheres are filtered through a filter screen with a pore size of 200μm.

10. The preparation process of a sodium alginate-based microscaffold according to claim 9, characterized in that: In step S3, the microspheres are washed until the pH of the microsphere solution reaches 7, then collected and dried in a freeze dryer for 3 days to obtain SA / CS / NaHCO3 microscaffolds.

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