A macroporous hydrogel with oriented structure, its preparation method and application

Abstract

This invention provides a macroporous hydrogel with a directional structure, its preparation method, and its applications. The preparation method includes: adding a photoinitiator to a mixed solution of sodium alginate and hyaluronic acid methacrylate, stirring in the dark to form a homogeneous solution; cross-linking the homogeneous solution with a cross-linking agent to form hydrogel microfilaments, and washing away excess cross-linking agent; implanting the hydrogel microfilaments into a mold groove, and combing them in the same direction using a fine-tooth comb until a directional structure is macroscopically observed; irradiating the hydrogel with a UV lamp to promote UV cross-linking, and then removing the hydrogel microfilaments from the mold to form a macroporous SA / HA hydrogel with a directional structure. This invention utilizes a mild and simple method to prepare a macroporous hydrogel with a directional structure. This hydrogel has good biocompatibility, significantly promotes cell ingrowth and tissue integration, and promotes tendon stem cell differentiation into tendons, thus facilitating tendon repair.

Description

Technical Field

[0001] This invention relates to the field of medical materials, specifically to a macroporous hydrogel with a directional structure, its preparation method, and its applications. Background Technology

[0002] Due to their ability to mimic the extracellular matrix and their excellent biocompatibility, hydrogels have shown great promise in tissue engineering. The composition, pore size, and structure of hydrogels all influence their biological functions. Compared to hydrogels with nanoscale pores, hydrogels with micron-scale pore structures can promote cell ingrowth and the exchange of nutrients and oxygen. Simultaneously, macroporous hydrogels can improve the survival rate of transplanted cells and promote the integration of hydrogels with tissues, demonstrating good effects in various tissue repair and regeneration processes.

[0003] However, for tissues with oriented structures, such as tendons, scaffolds with oriented structures are more conducive to their repair. Compared to non-oriented scaffolds, oriented scaffolds can significantly promote tendon stem cell differentiation into tendons, thus facilitating tendon repair. Currently, commonly used methods for synthesizing macroporous oriented hydrogels include the template method, the method of preparing nanoporous hydrogels by pressing a mold grid, and the rotational receiver method.

[0004] One study used a template method to remove micron-sized oriented sodium acetate trihydrate crystals from agarose hydrogels to prepare macroporous oriented hydrogels. The principle is that supersaturated sodium acetate solution can crystallize to form micron-sized oriented sodium acetate trihydrate crystals. After removing the sodium acetate trihydrate crystals from the hydrogel, the remaining hydrogel forms a macroporous oriented structure.

[0005] The method of preparing nanoporous hydrogels by pressing a mold mesh involves pressing a nanoporous hydrogel through a mold mesh with micron-sized pores, thereby giving the nanoporous hydrogel an oriented structure. The gaps next to the pores of the mold mesh form pores in the hydrogel, thus forming a macroporous oriented hydrogel overall.

[0006] The rotating receiver method involves synthesizing hydrogels using wet spinning and then using a rotating receiver to receive hydrogel microfibers, forming a macroporous hydrogel with an oriented structure on a rotating platform.

[0007] However, the template-based hydrogel synthesis process involves steps reaching 90℃, which is detrimental to the loading of bioactive substances onto the hydrogel. The mold-grid pressing method for preparing nanoporous hydrogels has very high requirements for mold preparation and requires the hydrogel to be gelled before pressing, which is not feasible for hydrogels with high viscosity. The rotational receiver method used in this study involves organic solvents such as methanol and ethanol, which can easily destroy the bioactive substances in the hydrogel. Therefore, a simple method with mild synthesis conditions is needed to prepare macroporous hydrogels with oriented structures. Summary of the Invention

[0008] To overcome the shortcomings of the prior art, the present invention provides a macroporous hydrogel with a directional structure, its preparation method and application, which is simple to prepare and has mild synthesis conditions.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] The first aspect of this invention is to provide a method for preparing a macroporous hydrogel with a directional structure, comprising the following steps:

[0011] Step 1: Add a photoinitiator to the mixed solution of sodium alginate and hyaluronic acid methacrylate, and stir in the dark to form a homogeneous solution;

[0012] Step 2: The above homogeneous solution is cross-linked with a cross-linking agent to form hydrogel microfilaments, and excess cross-linking agent is washed away;

[0013] Step 3: Insert the above-mentioned hydrogel microfilaments into the mold groove, and comb them in the same direction with a fine-tooth comb until they present a directional structure in general appearance;

[0014] Step four: Use a UV lamp to irradiate the hydrogel to promote UV cross-linking of the hydrogel, and then remove the hydrogel microfilaments from the mold to form a macroporous SA / HA hydrogel with a directional structure.

[0015] Further, in step one, the mixed solution of sodium alginate and hyaluronic acid methacrylate is formed by dissolving sodium alginate (≥2,000 cP, 2% (25°C) (lit.)) and hyaluronic acid methacrylate in double-distilled water; the concentration range of sodium alginate and hyaluronic acid methacrylate in the mixed solution is both in the range of 0.5-2% (w / v), preferably 0.8-1.2% (w / v); the concentration ratio of the two is preferably 1:1; more preferably, the concentration of sodium alginate and hyaluronic acid methacrylate in the mixed solution is both 1% (w / v).

[0016] Furthermore, the above photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate or 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylphenylacetone, preferably lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

[0017] Furthermore, the volume ratio of the above mixed solution to the photoinitiator is 6-14:1, preferably 6-10:1, and more preferably 9:1.

[0018] Furthermore, the crosslinking agent in step two is a solution containing calcium ions, preferably a calcium chloride solution, with a concentration preferably of 0.05-0.3 mol / L, more preferably of 0.15-0.24 mol / L, and even more preferably of 0.2 mol / L.

[0019] Further, the specific steps of step two are as follows: using a propulsion pump to push the above homogeneous solution into a 0.2 mol / L calcium chloride solution through a steel needle, sodium alginate is cross-linked by calcium ions to form SA / HA hydrogel microfilaments.

[0020] Furthermore, the propulsion rate is 3-8 ml / min; preferably 5 ml / min.

[0021] Furthermore, in step three, after each combing, the elongated hydrogel microfilaments are folded into the mold groove and combed again until they generally present an oriented structure.

[0022] Furthermore, in step four, the ultraviolet light wavelength range of the ultraviolet lamp is 200-400nm, and the irradiation time is 10-30s; preferably, the ultraviolet light wavelength of the ultraviolet lamp is 365nm, and the irradiation time is 20s.

[0023] A second aspect of the present invention is to provide a macroporous hydrogel prepared by the above-described preparation method.

[0024] A third aspect of the present invention is to provide the application of the above-described macroporous hydrogel in the preparation of tissue repair products with oriented structures.

[0025] Furthermore, the aforementioned tissues with oriented structures include, but are not limited to, tendons.

[0026] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0027] This invention utilizes a mild and simple method to prepare macroporous hydrogels with directional structures. These hydrogels exhibit good biocompatibility, significantly promote cell ingrowth and tissue integration, and facilitate tendon stem cell differentiation into tendons, thus enhancing tendon repair. Attached Figure Description

[0028] Figure 1This is the process of synthesizing macroporous hydrogels with aligned microfibers (MHA) in one embodiment of the present invention; for easy observation, the microfibers are doped with fluorescein isothiocyanate isomer I (FITC) dye; wherein, (A) the SA / HA solution is cross-linked with calcium ions to form hydrogel microfibers, (B) the hydrogel microfibers are washed with double-distilled water to remove excess calcium ions, (C) the hydrogel microfibers are placed on a mold, (D) ultraviolet cross-linking, (E) the MHA preparation is completed;

[0029] Figure 2 The image shows a scanning electron microscope image of a hydrogel in one embodiment of the present invention, wherein NH represents nanoporous hydrogels and MHR represents macroporous hydrogels with random microfibers.

[0030] Figure 3 The results of the in vitro biosafety assessment of MHA in one embodiment of the present invention are shown; wherein, Figure A shows the results of live and dead cell staining, with the skeleton stained green as live cells and stained red as dead cells, and Figure B shows the results of Cell Counting 8 (CCK-8) detection.

[0031] Figure 4 Figure A shows the performance verification results of MHA subcutaneous implantation in rats in one embodiment of the present invention; Figure B shows the HE staining image of MHA promoting cell infiltration and tissue integration, and Figure B shows the semi-quantitative analysis results of the number of cells infiltrating in the hydrogel. "**" represents P<0.01 compared with the NH group, and "***" represents P<0.001 compared with the NH group.

[0032] Figure 5 The images show the immunofluorescence results of MHA promoting tendon stem cell differentiation into tendons in one embodiment of the present invention. Figures A and D show the immunofluorescence staining and semi-quantitative analysis results of type I collagen (COL I), Figures B and E show the immunofluorescence staining and semi-quantitative analysis results of Scleraxis (Scx), and Figures C and F show the immunofluorescence staining and semi-quantitative analysis results of Tenomodulin (Tnmd). "**" indicates P < 0.01 compared to the MHR group, and "***" indicates P < 0.001 compared to the MHR group. Detailed Implementation

[0033] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings to enable a better understanding of the invention. However, the following embodiments do not limit the scope of the invention.

[0034] Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared according to conventional methods, unless otherwise specified.

[0035] Example 1

[0036] This embodiment provides a macroporous hydrogel with a directional structure, and its preparation method includes the following steps (see...). Figure 1 ):

[0037] 1. Dissolve medium-viscosity sodium alginate (SA) and methacrylated hyaluronic acid (HA) in double-distilled water to prepare a mixed solution with a concentration of (1%) / 1%. Add a sterile magnetic stir bar and stir on a magnetic stirrer until completely dissolved. Then add the photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl) phosphate at a volume ratio of (9:1) and stir in the dark until a homogeneous solution is formed.

[0038] 2. Using a propulsion pump (5 mL / min), the SA / HA solution is propelled into a 0.2 mol / L calcium chloride solution through a steel needle. The reaction is carried out at room temperature for about 10 seconds. After the SA is cross-linked by calcium ions, it forms SA / HA hydrogel microfilaments.

[0039] 3. Use a thin iron rod to scoop the hydrogel filaments into double-distilled water to wash away excess calcium chloride solution.

[0040] 4. Then place the hydrogel microfilaments into the mold groove (groove size: 25mm*10mm*1mm).

[0041] 5. Use a fine-toothed comb to comb in the same direction. After each combing, fold the elongated hydrogel filaments into the mold groove and comb again until a generally oriented structure is achieved.

[0042] 6. Irradiate the hydrogel with a UV lamp (365nm) for 20s to promote UV cross-linking. Then, remove the hydrogel microfilaments from the mold to form a macroporous SA / HA hydrogel with a oriented structure. Its surface morphology is as follows: Figure 2 As shown.

[0043] Verification Implementation Examples

[0044] This embodiment verifies the performance of the MHA provided in Example 1. The specific experimental steps and results are as follows:

[0045] 1. In vitro biosafety assessment

[0046] MHA was soaked in culture medium to prepare a hydrogel extract, which was then filtered through a 0.22 μm filter and used for bone marrow mesenchymal stem cell (BMSC) culture. BMSCs were seeded in 96-well plates, and after intervention with the extract for 1, 3, and 5 days, live / dead staining and CCK8 assay were performed. For live / dead staining, the culture medium was aspirated, and the cells were washed with phosphate-buffered saline (PBS). 100 μl of Calcein AM / PI assay working solution was added; the cells were incubated at 37°C in the dark for 30 min, and then photographed under a fluorescence microscope. For CCK8 assay, the culture medium was removed, the cells were washed with PBS, and then incubated in complete culture medium containing 10% CCK8 reagent in the dark for 2 hours. The absorbance at 450 nm was then measured using a microplate reader.

[0047] like Figure 3 As shown, live / dead staining and CCK8 assays revealed that MHA exhibited good biocompatibility with BMSCs, and had no inhibitory effect on BMSC growth after 1, 3, and 5 days of intervention.

[0048] 2. Performance Verification

[0049] 2.1 Subcutaneous Implantation Detection of the Role of Macroporous Structure of Hydrogel in Cell Growth

[0050] Rats were anesthetized with sodium pentobarbital. After shaving the hair on the back of the rats, a 1.5 cm incision was made, and the appropriate hydrogel was implanted. Samples were collected 2 and 4 weeks postoperatively. Rats were euthanized by over-anesthesia. Skin specimens were fixed in fixative for 24 hours, then dehydrated in a gradient, embedded in paraffin, sectioned, and stained with hematoxylin and eosin.

[0051] like Figure 4 As shown, after 2 and 4 weeks of subcutaneous implantation in rats, MHA significantly promoted cell ingrowth and integration of the hydrogel with the host, compared to NH.

[0052] 2.2 Detection of the effect of hydrogel orientation structure on tendon stem cell differentiation

[0053] Hydrogels were placed in 6-well plates, and tendon stem cells (TDSCs) were then seeded onto the hydrogels. After intervention, samples were fixed with paraformaldehyde and washed with PBS. Quickblock was used for further analysis. TM (Beyotime, China) After blocking for 15 minutes, the cells were incubated overnight with primary antibodies (type I collagen, Scleraxis, and Tenomodulin). After incubation with secondary antibodies, the cells were photographed using a fluorescence microscope.

[0054] like Figure 5As shown, compared to MHR, MHA can significantly promote the expression of tendon-related genes (type I collagen, Scleraxis and Tenomodulin) in tendon stem cells.

[0055] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A method for preparing a macroporous hydrogel with a directional structure, characterized in that, Includes the following steps: Step 1: Add a photoinitiator to the mixed solution of sodium alginate and hyaluronic acid methacrylate, and stir in the dark to form a homogeneous solution; the concentration range of sodium alginate and hyaluronic acid methacrylate in the mixed solution is 0.8-1.2% (w / v), and the concentration ratio of the two is 1:1; Step 2: The homogeneous solution is cross-linked with a cross-linking agent to form hydrogel microfilaments, and excess cross-linking agent is washed away; the cross-linking agent is a calcium chloride solution with a concentration of 0.15~0.24 mol / L. Step 3: Insert the hydrogel microfilaments into the mold groove and comb them in the same direction with a fine-tooth comb until the microfilaments show an oriented structure along the combing direction; Step four: Use a UV lamp to irradiate the hydrogel to promote UV cross-linking of the hydrogel, and then remove the hydrogel microfilaments from the mold to form a macroporous SA / HAMA hydrogel with a directional structure.

2. The preparation method according to claim 1, characterized in that, In step one, the mixed solution of sodium alginate and hyaluronic acid methacrylate is formed by dissolving sodium alginate and hyaluronic acid methacrylate in double-distilled water with medium viscosity; the concentration of sodium alginate and hyaluronic acid methacrylate in the mixed solution is 1% (w / v).

3. The preparation method according to claim 1, characterized in that, The photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate or 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylphenylacetone.

4. The preparation method according to claim 3, characterized in that, The volume ratio of the mixed solution to the photoinitiator is 9:

1.

5. The preparation method according to claim 1, characterized in that, In step two, the concentration of the calcium chloride solution is 0.2 mol / L.

6. The preparation method according to claim 5, characterized in that, The specific steps of step two are as follows: using a propulsion pump to push the homogeneous solution into a 0.2 mol / L calcium chloride solution through a steel needle, sodium alginate is cross-linked by calcium ions to form SA / HAMA hydrogel microfilaments.

7. The preparation method according to claim 6, characterized in that, The propulsion rate is 3-8 ml / min.

8. The preparation method according to claim 1, characterized in that, In step four, the ultraviolet light wavelength range of the ultraviolet lamp is 200-400nm, and the irradiation time is 10-30s.

9. The macroporous hydrogel prepared by the preparation method according to any one of claims 1-8.

10. The application of the macroporous hydrogel as described in claim 9 in the preparation of tissue repair products with oriented structures.

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