Magnetic response hydrogel, preparation method of magnetic response hydrogel, magnetic response hydrogel composite scaffold and preparation method and application of magnetic response hydrogel composite scaffold

Abstract

The invention provides a magnetic response hydrogel and a preparation method thereof, and a magnetic response hydrogel composite scaffold and a preparation method and application thereof, and belongs to the technical field of biological materials. According to the preparation method disclosed by the invention, SA is added to form strong coordination with ferroferric oxide, and the coordination action enables SA molecules to tightly wrap ferroferric oxide particles to form a hydrophilic protection layer, so that the dispersion uniformity of the ferroferric oxide particles is remarkably improved; by applying a magnetic field, the ferroferric oxide particles are pushed by the magnetic field to be arranged in a chain shape or a layer shape in the direction of the magnetic field, and the ferroferric oxide particles are fixed along with polymerization, so that the magnetic response hydrogel which is arranged in order is formed; when a magnetic field is applied to the magnetic response hydrogel, the ferroferric oxide particles are pushed by the magnetic field and try to rotate and arrange along the direction of the magnetic field, and the ferroferric oxide particles are in an ordered arrangement state and try to rotate in the same direction, so that resultant force is generated to initiate large change of the rigidity of the hydrogel.

Description

Technical Field

[0002] This invention belongs to the field of biomaterials technology, specifically relating to a magnetically responsive hydrogel and its preparation method, and a magnetically responsive hydrogel composite scaffold and its preparation method and application. Background Technology

[0003] Bone is a hard tissue in the human body, possessing high toughness and mechanical strength, and plays a vital role in maintaining human movement and protecting organs. Bone defects are a common orthopedic condition in clinical practice, with long treatment cycles and significant risks. Therefore, the treatment of bone defects has become a major discipline in clinical practice.

[0004] Bone is a dynamic tissue with an extremely active capacity for remodeling; small bone defects usually heal spontaneously without additional treatment. However, when large defects (usually wider than 2 cm) or bone circumference losses exceed 50% due to traumatic injury, congenital defects, surgical removal of tumors, or degenerative diseases occur, nonunion, malunion, or pathological fractures can result. These situations require surgical intervention to restore bone function and achieve healing.

[0005] Although autologous bone grafting is currently the gold standard for bone defect reconstruction, its poor availability, secondary trauma to the donor site, and long operation time are major limiting factors. Compared with autologous bone grafting, allogeneic or xenograft bone grafting, while solving the source problem, also has many limitations, such as immune rejection, disease transmission, and lack of osteogenic activity. Therefore, bone tissue engineering, which focuses on intelligent bone repair and simulates bone structure and function, has emerged.

[0006] Bone tissue engineering scaffolds, as biomaterials for bone defect repair, provide an effective method for the clinical treatment of bone defects. Scaffold-guided tissue regeneration is widely recognized as a major approach to treating bone defects. Tissue engineering scaffolds create a three-dimensional microenvironment at the bone defect site, regulating cell adhesion, proliferation, differentiation, and paracrine functions, thereby promoting effective repair of bone defects.

[0007] Mesenchymal stem cells (MSCs) are a type of stem cell with multipotent differentiation potential, capable of differentiating into adult cells such as bone, cartilage, muscle, and fat. They also secrete various growth factors and cytokines that influence the biological behavior of neighboring cells. Multiple studies have confirmed that MSCs are sensitive to mechanical stimulation, which can affect their proliferation, differentiation, and paracrine functions. In light of this, researchers have constructed scaffolds with varying stiffnesses and validated them in animal models, demonstrating that appropriate matrix stiffness is beneficial for osteogenic differentiation and bone regeneration.

[0008] However, in actual bone formation, softer callus tissue (stiffness 1-10 kPa) initially forms. As osteoid tissue forms (stiffness 25-40 kPa) and mineralization occurs (stiffness 40-100 kPa), the matrix stiffness increases, generating dynamic mechanical stimulation on cells. Therefore, static stiffness does not conform to the dynamic mechanical microenvironment during bone repair. Some studies have incorporated superparamagnetic iron oxide nanoparticles (SPION) into scaffolds, altering the particle alignment by applying a magnetic field, thereby changing the scaffold stiffness. However, since SPIONs are isotropic (macroscopically appearing non-magnetic) in the absence of a magnetic field, and the magnetic nanoparticles are randomly and uniformly aligned, the magnetization stress generated by each fixed magnetic particle is highly divergent when a magnetic field is applied, failing to work synergistically. This results in low macroscopic reinforcement efficiency of the polymer network, limited stiffness changes in the scaffold, and difficulty in meeting the dynamic requirements of bone defect repair. Summary of the Invention

[0009] The purpose of this invention is to provide a magnetically responsive hydrogel and its preparation method, as well as a magnetically responsive hydrogel composite scaffold and its preparation method and application. The hydrogel prepared by the method provided by this invention can exhibit significant stiffness changes under the influence of a magnetic field, meeting the dynamic requirements of bone defect repair.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing a magnetically responsive hydrogel, comprising the following steps: Acrylamide, crosslinking agent, sodium alginate, ferric oxide, and water are mixed to obtain a monomer solution; the mass fraction of ferric oxide in the monomer solution is 2-8%. The monomer solution was mixed with an initiator and polymerized in a static magnetic field to obtain a magnetically responsive hydrogel.

[0011] Preferably, the mass fraction of sodium alginate in the monomer solution is 0.5-1.5%.

[0012] Preferably, the strength of the static magnetic field is 0.1~0.5T.

[0013] Preferably, the initiator is persulfate, and the volume ratio of the initiator to the monomer solution is (0.5~1.5):200.

[0014] Preferably, the mass ratio of acrylamide to crosslinking agent is (8~12):(0.05~0.15).

[0015] The present invention also provides a magnetically responsive hydrogel prepared by the preparation method described above, wherein the iron(III) oxide in the magnetically responsive hydrogel is arranged in an orderly manner.

[0016] The present invention also provides a magnetically responsive hydrogel composite scaffold, comprising a porous PCL scaffold and a magnetically responsive hydrogel filling the pores of the porous PCL scaffold; the magnetically responsive hydrogel is the magnetically responsive hydrogel described in the above technical solution.

[0017] Preferably, the porous PCL scaffold is made of polycaprolactone and nano-hydroxyapatite, and the mass ratio of polycaprolactone to nano-hydroxyapatite is (7~9):(1~3).

[0018] The present invention also provides a method for preparing the magnetically responsive hydrogel composite scaffold described above, comprising the following steps: Porous PCL scaffolds were fabricated using 3D printing. A magnetically responsive hydrogel precursor is polymerized in the porous PCL scaffold in a static magnetic field to obtain a magnetically responsive hydrogel composite scaffold. The magnetically responsive hydrogel precursor is a solution obtained by mixing the monomer solution and the initiator described in the above technical solution.

[0019] The present invention also provides the application of the magnetically responsive hydrogel composite scaffold described above in bone injury repair, including: applying a dynamic magnetic field to the magnetically responsive hydrogel composite scaffold.

[0020] This invention provides a method for preparing a magnetically responsive hydrogel, comprising the following steps: mixing acrylamide, a crosslinking agent, sodium alginate, iron(III) oxide, and water to obtain a monomer solution; wherein the mass fraction of iron(III) oxide in the monomer solution is 2-8%; and then polymerizing the monomer solution with an initiator in a static magnetic field to obtain the magnetically responsive hydrogel. This invention utilizes the carboxyl groups in sodium alginate (SA) and the Fe groups in iron(III) oxide... 2+ / Fe 3+Empty orbitals form strong coordination bonds, which cause SA molecules to tightly encapsulate the iron oxide particles, forming a hydrophilic protective layer and significantly improving the dispersion uniformity of iron oxide particles in the monomer solution. Furthermore, the high viscosity of the SA aqueous solution also prevents the iron oxide particles from settling. By applying a static magnetic field during polymerization, the iron oxide particles are pushed along the magnetic field direction to align into chains or layers. As polymerization proceeds, the iron oxide particles are fixed, forming a magnetically responsive hydrogel with ordered iron oxide particle arrangement. By limiting the mass fraction of iron oxide in the monomer solution, magnetic responsiveness can be imparted to the hydrogel without affecting its swelling capacity, and excessive iron oxide agglomeration is avoided. When a magnetic field is applied to this magnetically responsive hydrogel, the iron oxide particles attempt to rotate and align along the magnetic field direction. Since the iron oxide particles are all in an ordered state, their attempted rotation directions are consistent, generating a resultant force that causes a significant change in the hydrogel's stiffness. The results of the examples show that the elastic modulus of the magnetically responsive hydrogel prepared by the method provided by the present invention is increased from 30 kPa to 82 kPa under a magnetic field strength of 0.4 T. Attached Figure Description

[0021] Figure 1 This is a graph showing the stability changes of the magnetically responsive hydrogel precursors in Example 1 and Comparative Example 4 of the present invention. Figure 2 The figures show the hydrogel adsorption performance of Examples 1-2 and Comparative Examples 1-3 of this invention. Figure 3 This is a diagram showing the mechanical properties of the magnetically responsive hydrogel in Embodiment 1 of the present invention; Figure 4 This is a diagram showing the mechanical properties of the magnetically responsive hydrogel in Embodiment 2 of the present invention. Figure 5 This is a diagram showing the mechanical properties of the magnetically responsive hydrogel in Comparative Example 1 of the present invention. Figure 6 This is a diagram showing the mechanical properties of the magnetically responsive hydrogel in Comparative Example 2 of the present invention. Figure 7 The figures show the bioactivity of the hydrogels from Examples 1-2, Comparative Examples 1-3, and Comparative Example 5 of this invention. Figure 8 This is a cell activity diagram of the magnetically responsive hydrogel under different magnetic stimuli in Example 2 of the present invention; Figure 9 These are SEM images of the porous PCL support of Comparative Example 6 of the present invention at different magnifications; Figure 10 These are SEM images of the porous PCL support in Embodiment 4 of the present invention at different magnifications; Figure 11 These are SEM images of the porous PCL support in Embodiment 3 of the present invention at different magnifications; Figure 12 These are SEM images of the porous PCL support in Embodiment 5 of the present invention at different magnifications; Figure 13 These are compression performance diagrams of the porous PCL stents in Embodiments 3-5 and Comparative Example 6 of the present invention; Figure 14 The diagram shows the bioactivity of the porous PCL scaffolds in Examples 3-5 and Comparative Example 6 of this invention. Figure 15 This is an ALP activity diagram of the magnetically responsive hydrogel composite scaffold in Embodiment 3 of the present invention under different magnetic stimuli. Detailed Implementation

[0022] All raw materials used in this invention are not particularly limited in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0023] There are no particular restrictions on the purity of any raw materials used in this invention. However, it is preferred to use analytical grade or conventional purity in the field of biological materials.

[0024] This invention provides a method for preparing a magnetically responsive hydrogel, comprising the following steps: Acrylamide, crosslinking agent, sodium alginate, ferric oxide, and water are mixed to obtain a monomer solution; the mass fraction of ferric oxide in the monomer solution is 2-8%. The monomer solution was mixed with an initiator and polymerized in a static magnetic field to obtain a magnetically responsive hydrogel.

[0025] This invention involves mixing acrylamide, a crosslinking agent, sodium alginate, ferric oxide, and water to obtain a monomer solution.

[0026] As one embodiment of the present invention, the particle size of the iron oxide can be 1~3μm; iron oxide with a particle size within the above range has excellent magnetic responsiveness and low cost.

[0027] In one embodiment of the present invention, the crosslinking agent may be N,N'-methylenebisacrylamide; N,N'-methylenebisacrylamide can enable polyacrylamide to form a three-dimensional network structure, which is beneficial to improving the adsorption performance of the hydrogel.

[0028] In this invention, the preferred mass ratio of acrylamide to crosslinking agent is (8~12):(0.05~0.15), more preferably 10:(0.05~0.15); as one embodiment of this invention, the mass ratio of acrylamide to crosslinking agent can be 10:0.06, 10:0.08, 10:0.1, 10:0.12, or 10:0.14. Acrylamide is a polymeric monomer, and a mass ratio of acrylamide to crosslinking agent within the above range is beneficial for improving the mechanical stability of the hydrogel.

[0029] In one embodiment of the present invention, the mass fraction of acrylamide in the monomer solution may be 10%, and the mass fraction of N,N'-methylenebisacrylamide may be 0.1%.

[0030] In this invention, the mass fraction of sodium alginate in the monomer solution is preferably 0.5-1.5%; more preferably 1%. A mass fraction of sodium alginate within the above range in the monomer solution is beneficial for coordination with ferric oxide, further improving the dispersion uniformity of ferric oxide.

[0031] In this invention, the mass fraction of ferric oxide in the monomer solution is 2-8%, preferably 3-7%. As one embodiment of this invention, the mass fraction of ferric oxide in the monomer solution can be 2.5%, 3%, 4%, 5%, 6%, 7%, or 8%. Ferric oxide has magnetic responsiveness, and a mass fraction of ferric oxide within the above range can impart magnetic responsiveness to the hydrogel without affecting its swelling capacity, and also avoids excessive ferric oxide from forming agglomerates.

[0032] In one embodiment of the present invention, the mixing can be as follows: first, acrylamide and N,N'-methylenebisacrylamide are dissolved in water to obtain an AM-MBA solution; SA is added to water and dissolved at 60°C to obtain an SA solution; then the AM-MBA solution and the SA solution are mixed and magnetically stirred for 1 hour to obtain an AM-SA solution; iron(III) oxide is added to the AM-SA solution, and stirring is continued for 1 hour, followed by ultrasonic dispersion for 2 hours to obtain a monomer solution.

[0033] After obtaining the monomer solution, the present invention mixes the monomer solution with an initiator and polymerizes it in a static magnetic field to obtain a magnetically responsive hydrogel.

[0034] In this invention, the initiator is preferably a persulfate, specifically ammonium persulfate. Persulfates can initiate acrylamide polymerization via free radicals, and byproducts can be removed by washing with water, which helps improve the purity of the hydrogel.

[0035] In this invention, the preferred volume ratio of the initiator to the monomer solution is (0.5~1.5):200, more preferably 1:200. A volume ratio of initiator to monomer solution within this range is beneficial for polymerization and further increases the reaction rate.

[0036] In one embodiment of the present invention, tetramethylethylenediamine may be added to the polymerization system before polymerization; the volume ratio of tetramethylethylenediamine to monomer solution may be 1:2000; the tetramethylethylenediamine may promote polymerization.

[0037] In this invention, the strength of the static magnetic field is preferably 0.1~0.5T, more preferably 0.2~0.4T; as one embodiment of this invention, the strength of the static magnetic field can be 0.1T, 0.2T, 0.3T, 0.4T, or 0.5T. A static magnetic field strength within the above range is beneficial for guiding the regular arrangement of iron(III) oxide particles, further improving the magnetic responsiveness of the hydrogel.

[0038] In one embodiment of the present invention, the container for polymerization can be a predetermined mold.

[0039] This invention utilizes the carboxyl group in SA and the Fe in iron(III) oxide. 2+ / Fe 3+ Empty orbitals form strong coordination bonds, which cause SA molecules to tightly encapsulate the iron oxide particles, forming a hydrophilic protective layer and significantly improving the dispersion uniformity of iron oxide particles in the monomer solution. Furthermore, the high viscosity of the SA aqueous solution also prevents the iron oxide particles from settling. By applying a magnetic field during polymerization, the iron oxide particles are pushed along the magnetic field direction to align into chains or layers. As polymerization progresses, the iron oxide particles are fixed, forming a magnetically responsive hydrogel with ordered iron oxide particle arrangement. By limiting the mass fraction of iron oxide in the monomer solution, magnetic responsiveness can be imparted to the hydrogel without affecting its swelling capacity, and excessive iron oxide agglomeration is avoided. When a magnetic field is applied to this magnetically responsive hydrogel, the iron oxide particles attempt to rotate and align along the magnetic field direction. Since the iron oxide particles are all in an ordered state, their attempted rotation directions are consistent, generating a resultant force that causes a significant change in the hydrogel's stiffness.

[0040] The present invention also provides a magnetically responsive hydrogel prepared by the preparation method described in the above technical solution.

[0041] In this invention, the iron(III) oxide in the magnetically responsive hydrogel is arranged in an orderly manner.

[0042] The present invention also provides a magnetically responsive hydrogel composite scaffold, the magnetically responsive hydrogel composite scaffold comprising a porous PCL scaffold and a magnetically responsive hydrogel filling the pores of the porous PCL scaffold; the magnetically responsive hydrogel is the magnetically responsive hydrogel described in the above technical solution.

[0043] In this invention, the porous PCL scaffold is preferably made of polycaprolactone and nano-hydroxyapatite. Adding nano-hydroxyapatite to polycaprolactone can improve the mechanical properties and surface hydrophilicity of the scaffold, increase the number of active sites, and enhance the bioactivity of the scaffold.

[0044] In this invention, the preferred mass ratio of polycaprolactone to nano-hydroxyapatite is (7~9):(1~3), more preferably (7~9):1; as one embodiment of this invention, the mass ratio of polycaprolactone to nano-hydroxyapatite can be 7:3, 8:2, or 9:1. A mass ratio of polycaprolactone to nano-hydroxyapatite within the above range is beneficial for further improving the bioactivity of the scaffold.

[0045] The magnetically responsive hydrogel composite scaffold provided by this invention can optimize the mechanical properties of the scaffold and match the properties of cancellous bone.

[0046] The present invention also provides a method for preparing the magnetically responsive hydrogel composite scaffold described above, comprising the following steps: Porous PCL scaffolds were fabricated using 3D printing. A magnetically responsive hydrogel precursor is polymerized in the porous PCL scaffold in a static magnetic field to obtain a magnetically responsive hydrogel composite scaffold. The magnetically responsive hydrogel precursor is a solution obtained by mixing the monomer solution and the initiator described in the above technical solution.

[0047] This invention utilizes 3D printing to fabricate porous PCL scaffolds.

[0048] As one embodiment of the present invention, the raw material for preparing the porous PCL scaffold can be a composite material of nano-hydroxyapatite and polycaprolactone (PCL-nHA composite material).

[0049] As one embodiment of the present invention, the nano-hydroxyapatite and polycaprolactone can be dried before use; the present invention does not particularly limit the specific parameters of the drying, as long as the moisture in the raw materials can be removed.

[0050] As one embodiment of the present invention, the preparation method of the PCL-nHA composite material is as follows: polycaprolactone (PCL) is melted at 100°C, nano-hydroxyapatite (n-HA) is added to the molten PCL, stirred evenly, and then vacuum dried for 24 hours to obtain the PCL-nHA composite material, which is then sealed and stored.

[0051] In one embodiment of the present invention, the 3D printing operation may include: constructing a support model, performing layer-by-layer processing using software slicing functionality, and laying the layers alternately along the 0° / 90° direction using a Zig-Zag routing pattern. The present invention does not particularly limit other operations of the 3D printing; conventional operations in the art can be used.

[0052] In one embodiment of the present invention, the 3D printing parameters may include: nozzle diameter 0.4 mm, line spacing 1.0 mm, layer height 0.3 mm, and extrusion speed 0.2 mm / s.2 The printing speed is 1.5 mm / s, and the printing temperature is 100°C. This invention does not impose any particular limitations on other parameters of the 3D printing process; parameters conventional in the field can be used.

[0053] After obtaining the porous PCL scaffold, the present invention polymerizes the magnetically responsive hydrogel precursor in the porous PCL scaffold in a static magnetic field to obtain a magnetically responsive hydrogel composite scaffold.

[0054] In this invention, the magnetically responsive hydrogel precursor is a solution obtained by mixing the monomer solution and the initiator described in the above technical solution.

[0055] As one embodiment of the present invention, the method of polymerizing the magnetically responsive hydrogel precursor in the porous PCL scaffold can be: dripping the magnetically responsive hydrogel precursor onto the porous PCL scaffold, so that the precursor flows into the pores of the porous PCL scaffold.

[0056] In this invention, the polymerization process is preferably the same as the above-described technical solution, and will not be repeated here.

[0057] The present invention also provides the application of the magnetically responsive hydrogel composite scaffold described above in bone injury repair.

[0058] In this invention, the application includes applying a dynamic magnetic field to the magnetically responsive hydrogel composite scaffold. As one embodiment of this invention, the dynamic magnetic field may be applied without a magnetic field for days 1-4 of application, with a 0.2T magnetic field applied for days 5-9, and with a 0.4T magnetic field applied for days 10-14.

[0059] This invention applies a dynamic magnetic field to a magnetically responsive hydrogel composite scaffold, which allows the scaffold's stiffness to adapt to the laws of bone injury repair, thereby more effectively promoting the biological activity of MSCs.

[0060] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0061] Example 1 A magnetically responsive hydrogel, prepared by the following method: 4g of acrylamide (AM) and 0.04g of N,N'-methylenebisacrylamide (MBA) were weighed and dissolved in 20mL of deionized water. The solution was stirred at room temperature to obtain an AM-MBA solution. 0.4g of SA was weighed and added to 20mL of deionized water. The solution was stirred at 60℃ to obtain an SA solution. The AM-MBA solution and SA solution were mixed and stirred continuously with a magnetic stirrer for 1h to ensure that all components were fully mixed and homogeneous to obtain an AM-SA solution. 0.63g of 3μm Fe3O4 was added to the AM-SA solution and placed on a mechanical stirrer for 1h. The mixture was then ultrasonically dispersed for 2h to ensure that the Fe3O4 particles were uniformly dispersed to obtain a monomer solution. 0.1225mL of ammonium persulfate (APS) and 0.01225mL of tetramethylethylenediamine (TEMED) were added sequentially to the monomer solution and stirred until homogeneous to obtain a precursor. The precursor was immediately transferred to a predetermined mold and placed in a 0.5T magnetic field for polymerization until gelation was complete, resulting in a magnetically responsive hydrogel, denoted as PAM-SA-2.5wt%.

[0062] Example 2 A magnetically responsive hydrogel, designated PAM-SA-5wt%, was prepared using the same method as in Example 1, except that the amount of Fe3O4 used was 1.26g.

[0063] Comparative Example 1 A magnetically responsive hydrogel, designated PAM-SA-10wt%, was prepared using the same method as in Example 1, except that the amount of Fe3O4 used was 2.52g.

[0064] Comparative Example 2 A magnetically responsive hydrogel, designated PAM-SA-20wt%, was prepared using the same method as in Example 1, except that the amount of Fe3O4 used was 5.04g.

[0065] Comparative Example 3 A hydrogel, designated PAM-SA, was prepared using the same method as in Example 1, except that Fe3O4 was omitted.

[0066] Comparative Example 4 A magnetically responsive hydrogel, designated PAM-Fe3O4, was prepared using the same method as in Example 1, except that SA was omitted.

[0067] Comparative Example 5 A hydrogel, designated PAM-Control, was prepared using the same method as in Example 1, except that SA and Fe3O4 were omitted.

[0068] Example 3 A magnetically responsive hydrogel composite scaffold is prepared by the following method: The dried PCL and n-HA were weighed at a mass ratio of 8:2. The PCL was melted at 100℃, and the n-HA was added to the molten PCL and stirred and mixed. The mixture was then vacuum dried for 24 hours to obtain the PCL / n-HA composite material, which was then sealed and stored. Porous PCL scaffolds were fabricated using the aforementioned PCL-nHA composite material via 3D printing: A porous cuboid bone scaffold model was constructed. After the model was completed, it was layered using the software's slicing function, and layers were alternately laid out along the 0° / 90° direction using a Zig-Zag routing pattern. The printing parameters were as follows: nozzle diameter 0.4 mm, line spacing 1.0 mm, layer height 0.3 mm, and extrusion speed 0.2 mm / s². 2 / s, printing speed 1.5mm / s, printing temperature 100℃; The precursor of Example 2 was dropped onto the porous PCL scaffold and then polymerized in a 0.5T magnetic field to obtain a magnetically responsive hydrogel composite scaffold.

[0069] Example 4 A magnetically responsive hydrogel composite scaffold is prepared using the same method as in Example 4, except that the mass ratio of PCL to n-HA is 9:1.

[0070] Example 5 A magnetically responsive hydrogel composite scaffold is prepared using the same method as in Example 4, except that the mass ratio of PCL to n-HA is 7:3.

[0071] Comparative Example 6 A magnetically responsive hydrogel composite scaffold is prepared using the same method as in Example 4, except that n-HA is omitted.

[0072] Test Example 1 The stability of the magnetically responsive hydrogel precursors of Example 1 and Comparative Example 4 was observed, and the results are as follows: Figure 1 As shown; Figure 1 In the diagram, A represents the state of the precursor at different times, B is a schematic diagram of the interaction mechanism between SA and Fe3O4, and C represents the curing time of the magnetically responsive hydrogel.

[0073] from Figure 1 As can be seen in Figure A, Fe3O4 undergoes rapid sedimentation in the monomer solution (left bottle), while it remains uniformly dispersed in the AM-SA solution (right bottle), indicating the stabilizing effect of SA on the particles. Figure 1 Figure B shows a schematic diagram of the interaction between SA and Fe3O4: the carboxyl oxygen atom (-COO) of SA - Fe on the Fe3O4 surface 2+ / Fe 3+ Empty orbitals form strong coordination bonds, which enable SA molecules to tightly encapsulate Fe3O4 particles, forming a hydrophilic protective layer and significantly improving particle dispersibility. Figure 1 As can be seen from the C value, Fe3O4 remained stably dispersed during the 5-minute gelation period, eventually forming a uniform magnetically responsive hydrogel.

[0074] Test Example 2 The adsorption properties of the hydrogels in Examples 1-2 and Comparative Examples 1-4 were tested, specifically their porosity, swelling ratio, anti-degradation ability, and ion adsorption capacity.

[0075] The prepared magnetically responsive hydrogel was freeze-dried for 72 hours and swollen in deionized water until saturated. The mass of the hydrogel after freeze-drying and after swelling was weighed, and then the porosity was calculated using the following formula. PR(%) = W2 - W1 / ρV ×100%; Wherein, W1 and W2 represent the mass of the hydrogel after freeze-drying and after swelling, respectively. ρ It is the density of deionized water. V It is the volume of the hydrogel after swelling.

[0076] The swelling rate (SR) of the hydrogel in deionized water was calculated using the differential weight method. The prepared magnetically responsive hydrogel was freeze-dried for 72 h, and the dry weight W1 of the hydrogel was recorded. Then it was soaked in deionized water. Every once in a while, the hydrogel was taken out, excess surface moisture was removed, and the wet weight W2 of the hydrogel was recorded until it no longer changed or the change was small. The swelling rate was calculated according to the following formula. SR(%)=(W2-W1) / W1×100%.

[0077] The degradation performance of hydrogels in a medium containing 10% FBS was studied using the differential gravimetric method, and the mass retention (MR) was used for characterization. After the hydrogels swelled to saturation in a medium containing 10% FBS, they were continued to be immersed in the medium. The mass of the hydrogels was recorded daily for 7 consecutive days, and the mass retention was calculated according to the following formula. MR(%) = W2 / W1 × 100%; Where W2 is the mass of hydrogel recorded each day, and W1 is the mass of hydrogel recorded on the first day.

[0078] The magnetically responsive hydrogel was immersed in deionized water for 24 hours, and the Fe content was measured afterward. 2+ / Fe 3+ The content is calculated as 0.2 g / mL.

[0079] Test results are as follows Figure 2 As shown; Figure 2In the figure, A represents the porosity of the hydrogel, "NS" indicates that the porosity of each sample is not significantly different, B is the swelling kinetics curve of the hydrogel, C is the hydrogel degradation curve, and D is the iron ion release curve.

[0080] from Figure 2 As shown in Figure A, the porosity of the magnetically responsive hydrogel remains around 80%, indicating that the incorporation of Fe3O4 particles did not significantly alter its porosity. This further suggests that the addition of Fe3O4 did not significantly change the basic cross-linking mechanism of the PAM-SA network (covalent cross-linking of PAM and ionic cross-linking of SA). This is because the uniformly dispersed Fe3O4 particles mainly fill the spaces originally occupied by water or the gaps in the network. Although they occupy some space, they do not significantly compress or destroy the original polymer network framework.

[0081] from Figure 2 As can be seen from Figure B, the hydrogel reaches swelling equilibrium in about 8 hours, avoiding the impact of continuous swelling on its performance. As the Fe3O4 particle content increases, the swelling rate of the magnetically responsive hydrogel gradually decreases. This is because Fe3O4 has a high density and does not absorb water. Its addition increases the mass of the hydrogel after freeze-drying, thus leading to a decrease in the swelling rate.

[0082] from Figure 2 As can be seen from Figure C, continuous testing revealed that the hydrogel did not exhibit degradation ability in a culture medium containing 10% FBS, indicating that the hydrogel was relatively stable.

[0083] from Figure 2 As can be seen from D, with the increase of Fe3O4 content, Fe 2+ / Fe 3+ The release gradually increases, and when the Fe3O4 content is 20wt%, Fe 2+ / Fe 3+ The surge in release may be due to the aggregation of Fe3O4 particles at high concentrations, which disrupts the integrity of the hydrogel network, making it easier for the encapsulated ions to be released.

[0084] Test Example 3 The mechanical properties of the magnetically responsive hydrogels of Examples 1-2 and Comparative Examples 1-2 were tested under different magnetic fields: An electronic universal testing machine was used to test the stiffness of the hydrogels under different magnetic field strengths through compression tests. The samples were placed on a base plate of a cylindrical structure (diameter = 15 mm, height = 10 mm) with a magnet, and then compression tests were performed at a speed of 2 mm / min. The test results are as follows: Figures 3-6 As shown in the figure, * indicates statistical differences.

[0085] contrast Figures 3-6It can be seen that under the same magnetic field, the elastic modulus of the hydrogel increases significantly with the increase of Fe3O4 mass fraction. For the same Fe3O4 mass fraction, the elastic modulus also increases with the increase of magnetic field strength from 0T to 0.4T. When the Fe3O4 mass fraction is 5wt%, the elastic modulus increases from 30KPa to 82KPa; when the Fe3O4 mass fraction is 2.5wt%, the elastic modulus increases from 21KPa to 64KPa; when the Fe3O4 mass fraction is 10wt%, the elastic modulus increases from 37KPa to 93KPa; and when the Fe3O4 mass fraction is 20wt%, the elastic modulus increases from 49KPa to 112KPa. This indicates that the matrix stiffness can be controlled in real time, dynamically, and reversibly through an external magnetic field in this system, providing an ideal platform for simulating the dynamic mechanical environment in vivo.

[0086] Test Example 4 The bioactivity of the hydrogels in Examples 1-2 and Comparative Examples 1-3 and Comparative Example 5 was tested as follows: The prepared hydrogels were sterilized by irradiation under a UV lamp for 1 hour; the sterilized hydrogels were added to DMEM-high sugar medium (containing 1% penicillin-streptomycin solution) at a ratio of 0.2 g / mL (material mass / extraction liquid volume); the mixture was placed in a shaker at 37°C and 100 r / min for 72 hours, sterilized by filtering through a 0.22 μm filter membrane, and stored in a sealed container at 4°C. To evaluate the effect of magnetically responsive hydrogels with different Fe3O4 contents on the survival rate of BMSCs, cells were arranged at 3 × 10⁻⁶ cells / cells. 3 / wells were seeded into 96-well plates, and the solution was replaced with the appropriate extraction medium after 12 hours. Cells in the wells were detected using a CCK-8 assay kit on days 1 and 3. Three replicates were set up for each group.

[0087] Cells were loaded at 6 × 10 3 / wells were seeded into 48-well plates, and the solution was replaced with the appropriate extraction medium after 12 hours. Cells in each group were stained using the Calcein / PI cell viability and cytotoxicity assay kit on days 1 and 3, respectively. Live cells showed green fluorescence and dead cells showed red fluorescence in the field of view. Each group was set up with 3 replicates.

[0088] Add an appropriate amount of 0.2 mg / mL sulfosuccinimide 6-(4-azo-2-nitroaniline)hexanoate (sulfo-SANPAH) solution to the surface of the sterilized hydrogel and irradiate with ultraviolet light for 15 min; after thorough washing with HEPES buffer, add an appropriate amount of collagen I to the surface of the hydrogel to firmly cross-link it; then, introduce cells at a rate of 2 × 10⁻⁶ cells / mL. 4 / wells were seeded into 24-well plates, and the cells in the wells were detected on days 1 and 3 using the CCK-8 assay kit and the Calcein / PI cell viability and cytotoxicity assay kit; each group had 3 replicates.

[0089] Test results are as follows Figure 7 As shown. Figure 7 In Figure A, the live / dead cell staining method was used to assess cell viability after co-culturing with hydrogels for 1 and 3 days. In Figure B, the CCK-8 assay was used to assess cell viability after co-culturing with hydrogels for 1 and 3 days. Figure 7 It can be seen that the cell survival rate was less than 80% in the groups containing 10wt% and 20wt% Fe3O4.

[0090] Test Example 5 The effect of different magnetic stimuli on osteogenic differentiation of MSCs using the magnetically responsive hydrogel in Example 2 was detected by an ALP activity assay: cells were sputtered at 5 × 10⁻⁶ m³ / h. 4 Cells were seeded in 24-well plates. After cell adhesion, different magnetic stimulation parameters were applied to the experimental groups; no magnetic stimulation was applied to the control group. All groups were replaced with osteogenic induction medium (containing sodium β-glycerophosphate, ascorbic acid, dexamethasone, etc.). Cells were cultured at 37°C in a 5% CO2 incubator. On day 7 of osteogenic induction culture, cells were stained according to the standard ALP staining kit procedure. The area and depth of ALP-positive areas in each group were observed and recorded. The test results are as follows: Figure 8 As shown in the figure, * indicates statistical difference, Control represents the control group without magnetic field, and Dynamic represents the application of dynamic magnetic stimulation (no magnetic field applied on days 1-2, a 0.2T magnetic field applied on days 3-5, and a 0.4T magnetic field applied on days 6-7). From Figure 8 It can be seen that dynamic magnetic stimulation can more effectively promote osteogenic differentiation of cells, and its effect is significantly better than continuous static magnetic stimulation and no magnetic stimulation.

[0091] Test Example 6 The porous PCL scaffolds of Examples 3-5 and Comparative Example 6 were observed using a scanning electron microscope, and SEM images were obtained, as follows: Figures 9-12 As shown. Comparison Figure 9 and Figures 10-12 It can be seen that the addition of n-HA to the raw materials resulted in an uneven surface on the porous PCL scaffold, especially at 30wt% n-HA. Figure 12 The surface of the scaffold shows many obvious protrusions, which, when magnified under a microscope, appear to be aggregated n-HA particles.

[0092] Test Example 7 Quasi-static uniaxial compression tests were performed on the porous PCL scaffolds (rectangular bone scaffold specimens with dimensions of 10mm × 10mm × 5mm) of Examples 3-5 and Comparative Example 6 using a universal testing machine. The compression speed was set to 0.5mm / min, and the compression termination displacement was 3mm. Each group was tested three times. The universal testing machine automatically recorded the load-displacement data for each test specimen and converted it into a stress-strain relationship according to the following formula: σ = F / S; ε = h1 / h0; E=σ / ε; In the above formula, F is the stress load value (N), h1 is the corresponding displacement value (mm); S and h0 are the original cross-sectional areas of the sample (mm²). 2 The original height (mm) is given by the stress-strain curve; σ is the compressive stress (MPa), ε is the strain value, and E is the elastic modulus (MPa). According to the stress-strain curve, the elastic modulus is approximately the slope of the line connecting the two points of 30% and 40% of the yield strength. According to the ISO 844:2007 test standard, the stress value corresponding to 10% strain is taken as the compressive strength. The final mechanical property data are expressed as the average value ± standard deviation of the test results of three sets of parallel specimens.

[0093] Test results are as follows Figure 13 As shown, Figure 13 Diagram A shows the compressive stress, and diagram B shows the elastic modulus. From... Figure 13 It can be seen that the compressive strength of the porous PCL scaffold first increases and then decreases with the increase of n-HA content, and the compressive strength reaches its peak at 20wt%n-HA. Combined with the SEM observation results, the decrease in compressive strength at 30wt%n-HA may be attributed to the agglomeration of n-HA particles disrupting the continuity of the PCL extruded filament structure.

[0094] Test Example 8 The bioactivity of the porous PCL scaffolds in Examples 3-5 and Comparative Example 6 was tested: The porous PCL scaffolds were sterilized by immersing them in 75% ethanol solution for 1 h, and then transferred to PBS buffer solution for 24 h. In accordance with ISO 10993-12 standard, the porous PCL scaffolds and extraction medium were added to DMEM-high glucose medium (containing 1% penicillin-streptomycin solution) at a ratio of 0.2 g / mL (material mass / extraction liquid volume), and extracted at 37 °C and 100 r / min for 72 h. The scaffolds were then sterilized by filtering through a 0.22 μm filter membrane and stored at 4 °C in a sealed container.

[0095] To evaluate the effect of bone scaffolds with different n-HA contents on BMSC cell survival, cells were prepared at a ratio of 3 × 10⁶ cells / year. 3 / wells were seeded into 96-well plates, and the solution was replaced with the appropriate extraction medium after 12 hours. Cells in the wells were detected using a CCK-8 assay kit on days 1 and 3. Three replicates were set up for each group.

[0096] Cells were loaded at 6 × 10 3 / wells were seeded into 48-well plates, and the solution was replaced with the appropriate extraction medium after 12 hours. Cells in each group were stained using the Calcein / PI cell viability and cytotoxicity assay kit on days 1 and 3, respectively. Live cells showed green fluorescence and dead cells showed red fluorescence in the field of view. Each group was set up with 3 replicates.

[0097] To observe the morphology of cells on a porous PCL scaffold, 1×10 5 BMSCs were seeded onto sterilized porous PCL scaffolds; samples were taken at 4h, 8h and 12h after cell seeding for DAPI staining and observation.

[0098] Test results are as follows Figure 14 As shown. Figure 14 Figure A shows the live / dead cell staining method used to assess cell viability after co-culturing with porous PCL scaffold extracts for 1 and 3 days. "NS" in the figure indicates no significant difference in cell viability among samples, and "Control" represents the blank group. Figure B shows the CCK-8 assay used to assess cell viability after co-culturing with porous PCL scaffold extracts for 1 and 3 days. Figure 14 It can be seen that the cell survival rate of each group is above 85%. According to the ISO10993-5 standard, none of the porous PCL scaffolds showed obvious cytotoxicity, which meets the basic requirements for bone scaffolds in bone tissue engineering.

[0099] Test Example 9 The effect of different magnetic stimuli on osteogenic differentiation of mesenchymal stem cells was detected using an ALP activity assay in Example 3: cells were sputtered at 5 × 10⁻⁶ ppm. 4 Cells were seeded in 12-well plates and, after cell attachment, different magnetic stimulation parameters were applied to the experimental groups; no magnetic stimulation was applied to the control group. All groups were replaced with osteogenic induction medium (containing sodium β-glycerophosphate, ascorbic acid, dexamethasone, etc.). Cells were cultured at 37°C in a 5% CO2 incubator. On day 14 of osteogenic induction culture, alkaline phosphatase (ALP) was quantitatively detected, and the results are as follows: Figure 15 As shown in the figure, * indicates statistical difference, Control indicates no magnetic field is applied, and Dynamic indicates dynamic magnetic stimulation is applied (no magnetic field is applied on days 1-4, a 0.2T magnetic field is applied on days 5-9, and a 0.4T magnetic field is applied on days 10-14).

[0100] from Figure 15It can be seen that dynamic magnetic stimulation can more effectively promote osteogenic differentiation of cells, and its effect is significantly better than continuous static stimulation and the control group without stimulation.

[0101] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a magnetically responsive hydrogel, comprising the following steps: Acrylamide, crosslinking agent, sodium alginate, iron oxide, and water were mixed to obtain a monomer solution; The mass fraction of iron(III) oxide in the monomer solution is 2-8%; The monomer solution was mixed with an initiator and polymerized in a static magnetic field to obtain a magnetically responsive hydrogel.

2. The preparation method according to claim 1, characterized in that, The mass fraction of sodium alginate in the monomer solution is 0.5-1.5%.

3. The preparation method according to claim 1, characterized in that, The strength of the static magnetic field is 0.1~0.5T.

4. The preparation method according to claim 1, characterized in that, The initiator is persulfate, and the volume ratio of the initiator to the monomer solution is (0.5~1.5):

200.

5. The preparation method according to claim 1, characterized in that, The mass ratio of acrylamide to crosslinking agent is (8~12):(0.05~0.15).

6. The magnetically responsive hydrogel prepared by the preparation method according to any one of claims 1 to 5, characterized in that, The magnetite in the magnetically responsive hydrogel is arranged in an orderly manner.

7. A magnetically responsive hydrogel composite scaffold, characterized in that, It includes a porous PCL scaffold and a magnetically responsive hydrogel filling the pores of the porous PCL scaffold; the magnetically responsive hydrogel is the magnetically responsive hydrogel of claim 6.

8. The magnetically responsive hydrogel composite scaffold according to claim 7, characterized in that, The porous PCL scaffold is made of polycaprolactone and nano-hydroxyapatite, and the mass ratio of polycaprolactone to nano-hydroxyapatite is (7~9):(1~3).

9. The method for preparing the magnetically responsive hydrogel composite scaffold according to claim 7 or 8, characterized in that, Includes the following steps: Porous PCL scaffolds were fabricated using 3D printing. A magnetically responsive hydrogel precursor is polymerized in the porous PCL scaffold in a static magnetic field to obtain a magnetically responsive hydrogel composite scaffold. The magnetically responsive hydrogel precursor is a solution obtained by mixing the monomer solution of claim 1 with an initiator.

10. The application of the magnetically responsive hydrogel composite scaffold according to claim 7 or 8 in bone injury repair, characterized in that, include: A dynamic magnetic field is applied to the magnetically responsive hydrogel composite scaffold.

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