Hydrogel with hollow structure as well as preparation method and application thereof

Abstract

The invention discloses hydrogel with a hollow structure and a preparation method and application thereof, the preparation method comprises the following steps: (a) adding calcium carbonate into a mixed solution of carboxymethyl chitosan and alginate, and stirring to obtain a homogeneous emulsion; (b) adding the homogeneous emulsion into an acid solution for reaction to obtain the hydrogel with the hollow structure, the hydrogel with the hollow structure can stabilize blood clots, promote the mesenchymal stem cells to gather towards the blood clots and promote the mesenchymal stem cells to differentiate towards the osteogenesis direction at the same time, and the purpose of rapidly repairing bone injuries is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogels, and particularly relates to a hydrogel with a hollow structure, a preparation method thereof, and an application thereof. Background Art

[0002] Bone injuries are caused by various reasons such as trauma, inflammatory infection, tumor resection, and congenital malformation, which pose great challenges to clinicians. For bone defects, common traditional methods include autologous grafts, allogeneic grafts, and biological fillers, but they have obvious drawbacks, such as immune rejection reactions, high costs, tissue infections, and limited donor sources. In this context, bone tissue engineering has become a promising treatment strategy. However, the osteogenic effect of current bone tissue engineering is not satisfactory. Perhaps it is more important to start from a biological perspective.

[0003] Under normal physiological conditions, bones undergo a continuous remodeling process, where old bone is resorbed and new bone is formed to adapt to mechanical loads and maintain function. Different from the characteristic of scar formation during the repair process of soft tissues such as the skin, bone defect healing forms new bone that is very similar to the original structure without forming scars. Bone regeneration is a highly complex and coordinated biological process, involving several different stages, including hematoma organization, callus formation, hard callus formation, and bone remodeling. Specifically, the coagulation cascade is first activated to form a blood clot at the bone injury site. With the formation of new blood vessels, various types of cells (such as inflammatory cells, fibroblasts, and stem cells) are recruited to the injury site. Granulation tissue forms at the fractured bone ends and is gradually replaced by fibrocartilage. At the same time, the periosteum ossifies to form an external callus. After these initial stages, the tissue is further mineralized through the deposition of hydroxyapatite to form more elastic woven bone. As the healing process progresses, the large fracture callus is replaced by secondary lamellar bone, and the blood supply in this area returns to normal, marking the completion of the bone remodeling stage.

[0004] According to the biological process, bone tissue engineering should meet the following conditions: ① promoting blood clot formation; ② recruiting relevant stem cells to the bone defect site; ③ promoting the osteogenic differentiation of stem cells.

[0005] Hydrogels are three-dimensional hydrophilic network structures formed by physical or chemical cross-linking. Since their first discovery and proposal by O. Wichterle and D. Lim in 1960, they have shown great potential in biochemical and biomedical applications and have been widely used in bone tissue engineering. Hydrogels can also play a role in recruiting cells and promoting directed cell differentiation by carrying drugs or biological factors. However, bone tissue regeneration technology based on hydrogels is still limited, and there are no related applications from laboratory to clinic. This may be because hydrogels usually occupy the entire defect site in a bulk form, minimizing the influence of blood clots. Therefore, the key to success is how to simultaneously stabilize blood clots, chemotax mesenchymal stem cells into blood clots, and promote the differentiation of mesenchymal stem cells into the osteogenic direction. Summary of the Invention

[0006] The purpose of the present invention is to provide a hydrogel with a hollow structure, its preparation method and application. The hydrogel with a hollow structure of the present invention can simultaneously stabilize blood clots, promote the aggregation of mesenchymal stem cells into blood clots, and promote the differentiation of mesenchymal stem cells into the osteogenic direction, achieving the purpose of rapid repair of bone injuries.

[0007] In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:

[0008] The first aspect of the present invention provides a preparation method of a hydrogel with a hollow structure, and the preparation method includes the following steps:

[0009] (a) Add calcium carbonate to the mixed solution of carboxymethyl chitosan and alginate and stir to obtain a homogeneous emulsion;

[0010] (b) Add the homogeneous emulsion to an acidic solution for reaction to obtain the hydrogel with a hollow structure.

[0011] Preferably, the concentration of carboxymethyl chitosan in the mixed solution of carboxymethyl chitosan and alginate is 0.1% - 10%, and the concentration of alginate is 0.1% - 10%.

[0012] Preferably, the final added concentration of calcium carbonate is 1% - 5%.

[0013] Preferably, the alginate is selected from at least one of sodium alginate, potassium alginate, calcium alginate, ammonium alginate, and magnesium alginate.

[0014] Preferably, the pH value of the acidic solution is 0.1 - 6.5.

[0015] Preferably, the acidic solution is selected from at least one of acetic acid solution, hydrochloric acid solution, sulfuric acid solution, and nitric acid solution.

[0016] Preferably, the volume ratio of the homogeneous emulsion to the acidic solution is 1∶(4 - 10).

[0017] Preferably, the reaction temperature is room temperature and the reaction time is 8 - 24 h.

[0018] In the second aspect of the present invention, there is provided a hydrogel with a hollow structure prepared by the above preparation method.

[0019] In the third aspect of the present invention, there is provided an application of a hydrogel with a hollow structure prepared by the above preparation method in the preparation of bone injury repair products.

[0020] Compared with the prior art, the beneficial effects of the present invention at least include:

[0021] The hydrogel with a hollow structure of the present invention can simultaneously stabilize blood clots, promote the aggregation of mesenchymal stem cells towards blood clots and the differentiation of mesenchymal stem cells towards the osteogenic direction. Specifically, by fully mixing sodium alginate, chitosan and CaCO3, and then reacting with an acid, CaCO3 is dissolved, releasing carbon dioxide and Ca 2+ , Ca 2+ can undergo ionic cross - linking with sodium alginate to form a hydrogel. Among them, positively charged chitosan and negatively charged sodium alginate undergo physical entanglement, which can further enhance the mechanical properties of the hydrogel; in addition, due to the differences in cross - linking time and cross - linking density between the surface polymer and the internal polymer, the polymer gradually aggregates towards the gel surface layer, forming a hollow structure, which provides a good space for the stabilization of blood clots; after introducing the hollow hydrogel of the present invention into the bone defect site, blood forms blood clots in the hydrogel, showing a good stabilizing effect. In the early stage, Ca 2+ is released, aggregating mesenchymal stem cells onto the blood clots and providing nutrients for mesenchymal stem cells. Subsequently, Ca 2+ cross - linked with sodium alginate will also be gradually released as the gel gradually degrades, promoting the osteogenic differentiation of mesenchymal stem cells and ultimately realizing tissue regeneration; it can be seen that the hydrogel material with a hollow structure of the present invention follows the physiological process, achieving the stabilization of blood clots, chemotaxis of stem cells and promotion of stem cell osteogenic differentiation in sequence. Compared with tissue engineering scaffolds for clinical applications, the indications are relatively wide, and the amino groups carried by chitosan can also achieve an antibacterial effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts do not necessarily draw according to the actual scale.

[0023] Figure 1 Morphological structures of different hydrogels under electron microscope in Experimental Example 1 of the present invention;

[0024] Figure 2 SEM image of the hydrogel-blood composition in Experimental Example 2 of the present invention;

[0025] Figure 3 Sample section image of the hydrogel-blood composition in Experimental Example 2 of the present invention;

[0026] Figure 4 Results of CCK8 experiment for measuring the toxicity of the hydrogel in Experimental Example 3 of the present invention;

[0027] Figure 5 Cell ALP staining image on the 7th day in Experimental Example 4 of the present invention;

[0028] Figure 6 Cell alizarin red staining image on the 21st day in Experimental Example 4 of the present invention;

[0029] Figure 7 Results of qRT-PCR analysis in the experimental examples of the present invention. Detailed implementation manners

[0030] The embodiments of the technical solution of the present invention will be described in detail below in conjunction with the embodiments. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, so they are only examples and cannot be used to limit the protection scope of the present invention.

[0031] It should be noted that unless otherwise specified, the technical terms or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art to which the present invention belongs.

[0032] The embodiment of the present invention provides a preparation method of a hydrogel with a hollow structure, and the preparation method includes the following steps:

[0033] (a) Add calcium carbonate to the mixed solution of carboxymethyl chitosan and alginate and stir to obtain a homogeneous emulsion;

[0034] (b) Add the homogeneous emulsion to an acidic solution for reaction to obtain the hydrogel with a hollow structure.

[0035] The present invention does not specifically limit the concentrations of carboxymethyl chitosan and alginate. In some embodiments, the concentration of carboxymethyl chitosan in the mixed solution of carboxymethyl chitosan and alginate can be any value from 0.1% to 10%, and the concentration of alginate can be any value from 0.1% to 10%.

[0036] In some embodiments, the final added concentration of the calcium carbonate can be 1% to 5%, specifically 1%, 2.5% or 5%.

[0037] The present invention does not specifically limit the type of alginate, and conventional alginates in the art can be used. In one embodiment, the alginate is selected from one of sodium alginate, potassium alginate, calcium alginate, ammonium alginate, and magnesium alginate.

[0038] In one embodiment, the pH value of the acidic solution can be any value in the range of 0.1 to 6.5.

[0039] In one embodiment, the acidic solution is selected from at least one of acetic acid solution, hydrochloric acid solution, sulfuric acid solution, and nitric acid solution.

[0040] In one embodiment, the volume ratio of the homogeneous emulsion to the acidic solution can be any value in the range of 1∶(4 - 10).

[0041] In one embodiment, the reaction temperature can be room temperature, and the reaction time can be any value in the range of 8 to 24 h.

[0042] Another embodiment of the present invention provides a hydrogel with a hollow structure prepared by the above - mentioned preparation method.

[0043] Another embodiment of the present invention provides an application of a hydrogel with a hollow structure prepared by the above - mentioned preparation method in the preparation of bone injury repair products.

[0044] The technical solution of the present invention will be further described in detail through specific examples below.

[0045] The raw materials used in the following examples are as follows:

[0046] Preparation and Characterization of Calcium Carbonate

[0047] In the presence of N - hexadecyltrimethylammonium chloride (CTAC), calcium carbonate (CaCO3) was prepared by a chemical precipitation reaction between sodium carbonate and calcium chloride at room temperature. Specifically, 100 g / L CTAC was added to a 60 mM Na2CO3 solution and mixed thoroughly. Then, an equal volume of 60 mM CaCl2 was slowly added dropwise to the mixed solution under vigorous stirring for 30 min. The precipitate was separated by centrifugation and washed three times with deionized water. Finally, white powdery CaCO3 was obtained by vacuum drying.

[0048] Example 1

[0049] This example provides a preparation method of a hydrogel with a hollow structure, and the preparation method includes the following steps:

[0050] (a) Calcium carbonate was added to the mixed solution of carboxymethyl chitosan and sodium alginate and stirred to obtain a homogeneous emulsion. Among them, the concentration of carboxymethyl chitosan in the mixed solution of carboxymethyl chitosan and alginate was 5%, and the concentration of alginate was 5%; the final added concentration of calcium carbonate was 1%.

[0051] (b) According to the volume ratio of the homogeneous emulsion to the acetic acid solution being 1:5, the homogeneous emulsion was added to the acetic acid solution with a pH value of 0.1 and reacted at room temperature for 12 h to obtain the hydrogel with a hollow structure (denoted as CACC@1% hydrogel).

[0052] Example 2

[0053] This example provides a preparation method of a hydrogel with a hollow structure. The preparation method includes the following steps:

[0054] (a) Calcium carbonate was added to the mixed solution of carboxymethyl chitosan and sodium alginate and stirred to obtain a homogeneous emulsion. Among them, the concentration of carboxymethyl chitosan in the mixed solution of carboxymethyl chitosan and alginate was 5%, and the concentration of alginate was 5%; the final added concentration of calcium carbonate was 2.5%.

[0055] (b) According to the volume ratio of the homogeneous emulsion to the acetic acid solution being 1:5, the homogeneous emulsion was added to the acetic acid solution with a pH value of 0.1 and reacted at room temperature for 12 h to obtain the hydrogel with a hollow structure (denoted as CACC@2.5% hydrogel).

[0056] Example 3

[0057] This example provides a preparation method of a hydrogel with a hollow structure. The preparation method includes the following steps:

[0058] (a) Calcium carbonate was added to the mixed solution of carboxymethyl chitosan and sodium alginate and stirred to obtain a homogeneous emulsion. Among them, the concentration of carboxymethyl chitosan in the mixed solution of carboxymethyl chitosan and alginate was 5%, and the concentration of alginate was 5%; the final added concentration of calcium carbonate was 5%.

[0059] (b) According to the volume ratio of the homogeneous emulsion to the acetic acid solution being 1:5, the homogeneous emulsion was added to the acetic acid solution with a pH value of 0.1 and reacted at room temperature for 12 h to obtain the hydrogel with a hollow structure (denoted as CACC@5% hydrogel).

[0060] Comparative Example 1

[0061] This comparative example provides a preparation method of a hydrogel with a hollow structure. The preparation method includes the following steps:

[0062] (a) Stir the mixed solution of carboxymethyl chitosan and sodium alginate to obtain a homogeneous emulsion. Among them, the concentration of carboxymethyl chitosan in the mixed solution of carboxymethyl chitosan and alginate is 5%, and the concentration of alginate is 5%.

[0063] (b) According to the volume ratio of the homogeneous emulsion to the calcium chloride solution being 1:5, add the homogeneous emulsion to a 5% calcium chloride solution and react at room temperature for 12 h to obtain the hydrogel without a hollow structure (denoted as CAC hydrogel).

[0064] Experimental Example 1

[0065] This experiment is for observing the hydrogel structures of the examples and comparative examples.

[0066] Use a freeze dryer to freeze the hydrogel overnight at -80 °C, and then freeze it at -54 °C for 24 hours to obtain the freeze-dried hydrogel. After sputtering gold plating for 6 minutes at an acceleration voltage of 20 kV using a sputtering gold plating machine, observe the morphological characteristics of the hydrogel with a scanning electron microscope. The observation results are as Figure 1 shown.

[0067] It can be seen from Figure 1 that: CACC@1% hydrogel, CACC@2.5% hydrogel, and CACC@5% hydrogel have a pore structure with a hollow core and tube wall, while the CAC hydrogel does not contain the corresponding structure.

[0068] Experimental Example 2

[0069] This experimental example is for the stable blood clot experiment:

[0070] Mix CACC@2.5% hydrogel and CAC hydrogel with fresh animal whole blood respectively until the whole blood coagulates to form a blood clot. Use a freeze dryer to freeze the hydrogel-blood clot composition overnight at -80 °C, and then freeze it at -54 °C for 24 hours to obtain the freeze-dried hydrogel-blood composition. After sputtering gold plating for 6 minutes at an acceleration voltage of 20 kV using a sputtering gold plating machine, observe the morphological characteristics of the blood clot in the hydrogel with a scanning electron microscope. The observation results are as Figure 2 shown, Figure 2 The left figure in the middle is CACC@2.5% hydrogel, and the right figure is CAC hydrogel.

[0071] Embed the hydrogel-blood composition in the optimal cutting temperature compound (OCT). Cut the sample into slices with a thickness of 80 μm, and observe and take pictures with an optical microscope. The observation results are as Figure 3 shown, Figure 3 in which the left figure is CACC@2.5% hydrogel, and the right figure is CAC hydrogel.

[0072] It can be seen from Figure 2 andFigure 3 It can be known that:

[0073] Blood can successfully enter the hollow core of the CACC@2.5% hydrogel and the pore structure within the wall, and a hydrogel-blood clot complex is formed.

[0074] Experimental Example 3

[0075] This experimental example is for in vitro biocompatibility determination:

[0076] CCK-8 cytotoxicity determination:

[0077] L929 cells were seeded in a 96-well plate at a density of 5000 cells per well. After incubation for 24 h, culture media containing CACC@1% hydrogel, CACC@2.5% hydrogel, and CACC@5% hydrogel were added to the experimental groups respectively, and incubated with L929 for 24 h and 72 h. According to the instructions of the CCK-8 kit, the OD value at 450 nm was measured, and the relative cytotoxicity was evaluated using the following formula:

[0078] Cell survival rate (%) = (A - B) / (C - B) × 100%;

[0079] In this formula, A is the absorbance of the experimental group (including L929, culture medium, hydrogel, CCK-8 solution); B is the absorbance of the blank group (containing culture medium and CCK-8 solution); C is the absorbance of the control group (containing L929, culture medium, CCK-8 solution).

[0080] The measurement results are as Figure 4 shown;

[0081] It can be known from Figure 4 that:

[0082] Neither the CACC@1% hydrogel, the CACC@2.5% hydrogel nor the CACC@5% hydrogel showed significant inhibitory effects on the growth of L929 on the 1st day and the 3rd day, indicating that the hydrogel has good biocompatibility.

[0083] Experimental Example 4

[0084] This experimental example is for osteogenic ability determination:

[0085] Cells at 2×10 4 cells / cm 2Inoculate into the well plate at a density of. When the cell confluence almost reaches 100%, replace the complete medium with osteogenic induction medium containing CACC@2.5% hydrogel (denoted as hollow hydrogel), CAC hydrogel (denoted as non-hollow hydrogel), and no CACC@2.5% hydrogel (denoted as control). Perform ALP staining on the cells on the 7th day, and alizarin red staining on the cells on the 21st day. The staining results are as Figure 5 , Figure 6 shown;

[0086] Cells are inoculated into the six-well plate at a density of 2×10 4 cells / cm 2 . When the cell confluence almost reaches 100%, replace the complete medium with osteogenic induction medium containing CACC@2.5% hydrogel (denoted as hollow hydrogel), containing CAC hydrogel (denoted as non-hollow hydrogel), and no CACC@2.5% hydrogel (denoted as control). Extract RNA on the 7th day and perform qRT-PCR analysis, as Figure 7 shown;

[0087] It can be seen from Figures 5 to 7 that:

[0088] The results of ALP staining and semi-quantitative analysis show that the CACC@2.5% hydrogel increases the blue-purple precipitate of the cells on the 7th day; the results of alizarin red staining and semi-quantitative analysis show that the CACC@2.5% hydrogel significantly promotes the formation of calcium nodules of the cells on the 21st day; as shown by the qRT-PCR results, the CACC@2.5% hydrogel significantly promotes the expression of osteogenic genes on the 7th day, while the expression of osteogenic genes in the CAC hydrogel treatment group does not increase significantly compared to the control. This indicates that the CACC@2.5% hydrogel has the ability to promote osteogenic differentiation of cells.

[0089] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered by the scope of the claims and the description of the present invention.

Claims

1. A method for preparing a hydrogel having a hollow structure, characterized in that: The preparation method comprises the following steps: (a) adding calcium carbonate to a mixed solution of carboxymethyl chitosan and alginate and stirring to obtain a homogeneous emulsion; (b) adding the homogeneous emulsion into an acidic solution for reaction to obtain the hydrogel having a hollow structure.

2. The preparation method according to claim 1, characterized in that: In the mixed solution of carboxymethyl chitosan and alginate, the concentration of carboxymethyl chitosan is 0.1% to 10%, and the concentration of alginate is 0.1% to 10%.

3. The preparation method according to claim 1, characterized in that: The final concentration of calcium carbonate added is 1% to 5%.

4. The preparation method according to claim 1, characterized in that: The alginate is selected from at least one of sodium alginate, potassium alginate, calcium alginate, ammonium alginate and magnesium alginate.

5. The preparation method according to claim 1, characterized in that: The pH value of the acidic solution is 0.1-6.

5.

6. The preparation method according to claim 1, characterized in that: The acidic solution is selected from at least one of acetic acid solution, hydrochloric acid solution, sulfuric acid solution and nitric acid solution.

7. The preparation method according to claim 1, characterized in that: The volume ratio of the homogeneous emulsion to the acidic solution is 1:(4-10).

8. The preparation method according to claim 1, characterized in that: The reaction temperature is room temperature, and the reaction time is 8 to 24 hours.

9. A hydrogel with a hollow structure prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the hydrogel with a hollow structure prepared by the preparation method according to any one of claims 1 to 8 in the preparation of bone damage repair products.

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