Three-dimensional hydrogel scaffold and preparation method and bone repair application thereof

Abstract

The invention relates to the technical field of medical biological materials, in particular to a three-dimensional hydrogel scaffold as well as a preparation method and bone repair application thereof. The three-dimensional hydrogel scaffold is of a stacked three-dimensional ordered structure built by a plurality of hydrogel tubes, each layer comprises a plurality of hydrogel tubes arranged in parallel at intervals, an included angle is formed between every two adjacent layers of hydrogel tubes, and the component of the three-dimensional hydrogel scaffold comprises sodium alginate; in the three-dimensional hydrogel stent, macropores are formed between every two hydrogel tubes, a fully-communicated micro-channel is formed in each hydrogel tube, and the outer surface and the inner surface of each hydrogel tube are each provided with a surface orientation nanofiber structure. The three-dimensional hydrogel scaffold disclosed by the invention can be well applied to the aspect of bone repair, the three-dimensional hydrogel scaffold is successfully constructed by utilizing a 3D printing, non-uniform cross-linking and stretching drying combined technology, and the preparation process is simple and accurate, easy to operate and beneficial to industrial popularization and application.

Description

Technical Field

[0001] This invention relates to the field of medical biomaterials technology, and in particular to a three-dimensional hydrogel scaffold, its preparation method, and its application in bone repair. Background Technology

[0002] In the field of medical biomaterials technology, three-dimensional hydrogel scaffolds that simultaneously possess macropores, fully interconnected microchannels, and surface-oriented nanofiber structures have important applications, especially in bone repair.

[0003] On the one hand, there are currently two main methods for preparing hydrogel scaffolds with microchannels: one is coaxial 3D printing, which uses 3D printing technology with coaxial needles to directly construct hydrogel scaffolds with microchannels. However, the disadvantage of this method is that the constructed microchannels are not fully interconnected, resulting in poor internal material transport and communication. The second method is the sacrificial template method, which first prints a water-soluble material scaffold, then infuses the scaffold with hydrogel containing living cells. After the hydrogel crosslinks, the original printed scaffold is removed, forming a microchannel (hollow tube) structure. However, the disadvantage of this method is that although the constructed microchannels have good connectivity, the hydrogel scaffold lacks overall structural controllability and does not have an open macroporous structure. In addition, the inventor's previous patent application CN113476664A disclosed a method for preparing a biological scaffold that combines open macropores and fully connected microchannels. This application uses 3D printing to print open macropores on the biological scaffold, and then performs partial cross-linking on the biological scaffold to form a fully connected microchannel structure. The biological scaffold has both open macropores and fully connected microchannels. However, the biological scaffold in this application does not have a surface orientation structure, and its mechanical properties and structural stability are relatively weak, which limits its practical application.

[0004] On the other hand, the main methods for constructing surface-oriented micro- and nano-structures in biological scaffolds are electrospinning and micro- and nano-lithography. However, both of these methods are only suitable for constructing membrane-like or planar materials and cannot be applied to three-dimensional hydrogel scaffolds.

[0005] In summary, constructing three-dimensional hydrogel scaffolds with both open macropores and fully connected microchannels, as well as surface-oriented nanofiber structures, remains a challenge using existing methods, and no such methods have been reported to date. Therefore, existing technologies require further improvement and development. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a three-dimensional hydrogel scaffold, its preparation method and its application in bone repair. The three-dimensional hydrogel scaffold simultaneously has macropores, fully connected microchannels and surface-oriented nanofiber structures, aiming to solve the problem that existing methods cannot construct a three-dimensional hydrogel scaffold that simultaneously has macropores, fully connected microchannels and surface-oriented nanofiber structures.

[0007] The technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides a three-dimensional hydrogel scaffold, wherein the three-dimensional hydrogel scaffold is a layered, three-dimensional ordered structure constructed by a plurality of hydrogel tubes, each layer comprising a plurality of hydrogel tubes arranged in parallel intervals, the hydrogel tubes of adjacent layers forming an angle, and the components of the three-dimensional hydrogel scaffold include sodium alginate.

[0009] In the three-dimensional hydrogel scaffold, there are macropores between each hydrogel tube, fully interconnected microchannels inside each hydrogel tube, and surface-oriented nanofiber structures on the outer and inner surfaces of each hydrogel tube.

[0010] Optionally, the pore size of the macropore is 200μm to 1000μm, the wall thickness of the hydrogel tube is 40μm to 600μm, the diameter of the fully connected microchannel is 50μm to 500μm, and the diameter of the nanofibers in the surface-oriented nanofiber structure is 20nm to 200nm.

[0011] Optionally, the height of the three-dimensional hydrogel scaffold is 1mm to 30mm, the number of layers of the three-dimensional hydrogel scaffold is 2 to 30, and the number of hydrogel tubes in each layer is 2 to 20.

[0012] Optionally, the included angle is greater than 0° and less than or equal to 90°.

[0013] Optionally, the components of the three-dimensional hydrogel scaffold may further include formulation materials, which are one or more of bacterial cellulose, methacrylamide gelatin, polyvinyl alcohol, hydroxyapatite, bioglass, collagen, gelatin, tricalcium β-phosphate, graphene, and hyaluronic acid.

[0014] A second aspect of the present invention provides a method for preparing a three-dimensional hydrogel scaffold, comprising:

[0015] Step 1: Prepare sodium alginate into a printable biomaterial. Use 3D printing to form several printing lines from the printable biomaterial and print it into a three-dimensional hydrogel scaffold intermediate D1 with large pores. The three-dimensional hydrogel scaffold intermediate D1 is a layered, three-dimensional ordered structure built by several printing lines. Each layer includes multiple printing lines arranged in parallel and spaced intervals. The printing lines of adjacent layers are at an angle to each other, and there are large pores between each printing line.

[0016] Step 2: Immerse the three-dimensional hydrogel scaffold intermediate D1 obtained in Step 1 in a crosslinking agent solution, so that the biomaterial on the surface of each printed line is crosslinked while the internal biomaterial is not crosslinked, to obtain the three-dimensional hydrogel scaffold intermediate D2.

[0017] Step 3: Immerse the three-dimensional hydrogel scaffold intermediate D2 obtained in step 2 in an aqueous solution to remove the uncrosslinked biomaterial inside each printing line. The printing line becomes a hydrogel tube with fully connected microchannels, resulting in a three-dimensional hydrogel scaffold intermediate D3 with macropores and fully connected microchannels.

[0018] Step 4: Immerse the three-dimensional hydrogel scaffold intermediate D3 obtained in Step 3 in the crosslinking agent solution again for complete crosslinking to obtain the three-dimensional hydrogel scaffold intermediate D4.

[0019] Step 5: The three-dimensional hydrogel scaffold intermediate D4 obtained in Step 4 is stretched and dried to form surface-oriented nanofiber structures on the outer and inner surfaces of each hydrogel tube, thus obtaining the final three-dimensional hydrogel scaffold.

[0020] Optionally, in step one, the step of preparing sodium alginate into printable biomaterials specifically includes:

[0021] Sodium alginate, formulation materials, and an aqueous solution are mixed to prepare a printable biomaterial, wherein the mass ratio of sodium alginate, formulation materials, and aqueous solution is 10-20:0-10:80-90, and the weight percentage concentration of sodium alginate in the printable biomaterial is greater than or equal to 10%.

[0022] Optionally, in step two, the crosslinking agent solution is a copper chloride solution or a calcium chloride solution; the concentration of the crosslinking agent solution is 0.1 mol / L to 2 mol / L; and the soaking time is 5 s to 120 s.

[0023] Optionally, in step five, during the stretching and drying process, the stretching ratio of the three-dimensional hydrogel scaffold intermediate D4 is greater than 20%.

[0024] A third aspect of the present invention provides an application of a three-dimensional hydrogel scaffold in bone repair.

[0025] Beneficial Effects: The three-dimensional hydrogel scaffold provided by this invention possesses macropores, fully interconnected microchannels, and surface-oriented nanofiber structures. The macropores facilitate tissue ingrowth and oxygen / nutrient transport, while the fully interconnected microchannels facilitate the transport, exchange, and migration of oxygen / nutrients and cellular substances within the scaffold. Furthermore, these microchannels can promote cell proliferation and migration through signaling pathways, thereby promoting angiogenesis and bone regeneration in vivo. The surface-oriented nanofiber structure helps regulate cell orientation, working in synergy with the fully interconnected microchannels to promote angiogenesis and bone regeneration. Therefore, the three-dimensional hydrogel scaffold of this invention can be well applied to bone repair. Moreover, this invention successfully constructs a three-dimensional hydrogel scaffold using a combined 3D printing, non-uniform cross-linking, and stretching-drying technique. The fabrication process is simple, accurate, and easy to operate, facilitating industrial promotion and application. Attached Figure Description

[0026] Figure 1 A schematic diagram of the structure of the three-dimensional hydrogel scaffold provided by the present invention.

[0027] Figure 2 The flowchart illustrates the fabrication process of the three-dimensional hydrogel scaffold provided by this invention.

[0028] Figure 3 This is a top view of the three-dimensional hydrogel scaffold prepared in Example 1.

[0029] Figure 4 This is a scanning electron microscope image of the oriented nanofiber structure on the outer surface of the three-dimensional hydrogel scaffold prepared in Example 1.

[0030] Figure 5 This is a scanning electron microscope image of the oriented nanofiber structure on the inner surface of the three-dimensional hydrogel scaffold prepared in Example 1.

[0031] Figure 6 Scanning electron microscope image of the surface-oriented microstructure of the three-dimensional hydrogel scaffold prepared in Comparative Example 1.

[0032] Figure 7 This is a scanning electron microscope image of the surface-oriented nanofiber structure of the three-dimensional hydrogel scaffold prepared in Example 2.

[0033] Figure 8 This is a scanning electron microscope image of the surface-oriented nanofiber structure of the three-dimensional hydrogel scaffold prepared in Example 3.

[0034] Figure 9 This is a scanning electron microscope image of the surface-oriented nanofiber structure of the three-dimensional hydrogel scaffold prepared in Example 4.

[0035] Figure 10This is a scanning electron microscope image of the surface-oriented nanofiber structure of the three-dimensional hydrogel scaffold prepared in Example 5.

[0036] Figure 11 This is a scanning electron microscope image of the surface-oriented nanofiber structure of the three-dimensional hydrogel scaffold prepared in Example 6.

[0037] Figure 12 This is a scanning electron microscope image of the surface-oriented nanofiber structure of the three-dimensional hydrogel scaffold prepared in Example 7.

[0038] Figure 13 H&E staining image of a three-dimensional hydrogel scaffold repairing a skull defect in a rat. Detailed Implementation

[0039] This invention provides a three-dimensional hydrogel scaffold, its preparation method, and its application in bone repair. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0040] like Figure 1 As shown, this embodiment of the invention provides a three-dimensional hydrogel scaffold, wherein the three-dimensional hydrogel scaffold is a layered, three-dimensional ordered structure constructed by a plurality of hydrogel tubes, each layer including a plurality of hydrogel tubes arranged in parallel intervals, the hydrogel tubes of adjacent layers forming an angle, and the components of the three-dimensional hydrogel scaffold include sodium alginate.

[0041] In the three-dimensional hydrogel scaffold, there are large pores between each hydrogel tube ( Figure 1 The marked 1 indicates a large pore, and each hydrogel tube has fully connected microchannels inside. Figure 1 The number 2 indicates a fully connected microchannel, and each hydrogel tube has a surface-oriented nanofiber structure on both its outer and inner surfaces.

[0042] The macropores in the three-dimensional hydrogel scaffold of this invention are formed by stacked hydrogel tubes arranged at intervals between each layer. For example, two hydrogel tubes in one layer are arranged at intervals, and the gap between these two hydrogel tubes and the hole formed by the hydrogel tubes in the upper and lower layers are the macropores.

[0043] The fully connected microchannels in the three-dimensional hydrogel scaffold of this invention are formed by the microchannels inside the hydrogel tubes. Each hydrogel tube in the three-dimensional hydrogel scaffold is hollow and has microchannels. The microchannels of each hydrogel tube are fully connected in the three-dimensional hydrogel scaffold, which means that each hydrogel tube has fully connected microchannels inside.

[0044] In the three-dimensional hydrogel scaffold of this invention, each hydrogel tube has a surface-oriented nanofiber structure on both its outer and inner surfaces. This surface-oriented nanofiber structure means that the entire wall of the hydrogel tubes in the three-dimensional hydrogel scaffold is composed of oriented nanofibers, significantly improving the overall mechanical properties of the scaffold. Compared to scaffolds without an oriented nanofiber structure, its tensile strength is increased by two times. Furthermore, both the inner and outer surfaces of the hydrogel tubes in the three-dimensional hydrogel scaffold have ordered nanofiber bundle structures, and this oriented nanofiber structure is maintained on the surface even after immersion in PBS or cell culture medium for 28 days. In addition, the surface-oriented nanofiber structure in this embodiment of the invention, compared to the surface micron-sized fiber bundle structure, significantly improves the overall mechanical properties of the scaffold and more effectively promotes angiogenesis and bone regeneration.

[0045] The three-dimensional hydrogel scaffold provided in this invention combines macroporous structures, fully connected microchannels, and surface-oriented nanofiber structures. The macroporous structure facilitates tissue ingrowth and oxygen / nutrient transport; the fully connected microchannel structure facilitates the transport, exchange, and migration of oxygen / nutrients and cellular substances within the scaffold, and can promote cell proliferation and migration through signaling pathways, thereby promoting angiogenesis and bone regeneration in vivo; the surface-oriented nanofiber structure helps regulate cell orientation, and in conjunction with the fully connected microchannel structure, activates the cAMP-RAP1 and MAPK pathways respectively, and jointly promotes the downstream PI3K / Hif-1 pathway, thus coordinating with the fully connected microchannel structure to promote angiogenesis and bone tissue regeneration. Therefore, the three-dimensional hydrogel scaffold of this invention can be well applied to bone repair.

[0046] The main component of the hydrogel scaffold in this invention is sodium alginate, which has good biocompatibility and excellent bone repair effect.

[0047] In one embodiment, the pore size of the macropore is 200 μm to 1000 μm. Preferably, the pore size of the macropore is 200 μm to 800 μm, for example, it can be 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, etc. A pore size within this range is most conducive to bone tissue regeneration.

[0048] In one embodiment, the wall thickness of the hydrogel tube is 40 μm to 600 μm, for example, it can be 40 μm, 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, etc. With the wall thickness of the hydrogel tube within this range, the three-dimensional hydrogel scaffold possesses both high porosity and good structural stability.

[0049] In one embodiment, the diameter of the fully connected microchannel is 50 μm to 500 μm, for example, it can be 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, etc. Within this diameter range, the fully connected microchannels in the three-dimensional hydrogel scaffold facilitate the transport and exchange of internal substances while also providing the space required for cell migration and tissue ingrowth.

[0050] In one embodiment, the nanofiber diameter of the surface-oriented nanofiber structure is 20 nm to 200 nm. Within this range, the nanofiber diameter effectively regulates cell morphology, achieving a structural effect that promotes angiogenesis and osteogenic formation. Preferably, the nanofiber diameter of the surface-oriented nanofiber structure is 50 nm to 100 nm, for example, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, etc.

[0051] In one embodiment, the height of the three-dimensional hydrogel scaffold is 1mm to 30mm, the number of layers in the three-dimensional hydrogel scaffold is 2 to 30, and the number of hydrogel tubes in each layer is 2 to 20. The height, number of layers, and number of hydrogel tubes in each layer of the three-dimensional hydrogel scaffold are within the range described above, which basically meets the size requirements for constructing a three-dimensional scaffold for human bone defects.

[0052] In one embodiment, the included angle is greater than 0° and less than or equal to 90°; preferably, the included angle is 90°.

[0053] In one embodiment, the three-dimensional hydrogel scaffold further comprises formulation materials. The addition of formulation materials can further improve the overall mechanical strength and structural stability of the scaffold in solution. For example, a three-dimensional hydrogel scaffold prepared by combining 12.5% ​​sodium alginate with 5% bacterial cellulose and 2.5% methacrylamide gelatin can maintain its fully connected microchannel structure and ordered surface-oriented nanofiber structure even after being immersed in PBS for 28 days.

[0054] In one embodiment, the formulation material is one or more selected from bacterial cellulose, methacrylamide gelatin, polyvinyl alcohol, hydroxyapatite, bioglass, collagen, gelatin, tricalcium β-phosphate, graphene, and hyaluronic acid.

[0055] In one embodiment, the components of the three-dimensional hydrogel scaffold include sodium alginate, bacterial cellulose, and methacrylamide gelatin.

[0056] In one embodiment, the mass ratio of sodium alginate to the formulation material in the three-dimensional hydrogel scaffold is 50–100:0–50. Within this mass ratio range, a three-dimensional hydrogel scaffold simultaneously possessing macroporous, fully permeable microchannel structures and surface-ordered oriented nanofiber structures can be well constructed. Preferably, the mass ratio of sodium alginate to the formulation material in the three-dimensional hydrogel scaffold is 10–20:1–10.

[0057] like Figure 2 As shown, this embodiment of the invention also provides a method for preparing a three-dimensional hydrogel scaffold, comprising:

[0058] Step 1: 3D printing sodium alginate-based scaffold: Sodium alginate is prepared into a printable biomaterial. Using 3D printing, the printable biomaterial is formed into several printing lines and printed into a three-dimensional hydrogel scaffold intermediate D1 with large pores. The three-dimensional hydrogel scaffold intermediate D1 is a layered, three-dimensional ordered structure built by several printing lines. Each layer includes multiple printing lines arranged in parallel and spaced intervals. The printing lines of adjacent layers are at an angle, and there are large pores between each printing line.

[0059] Step 2, Non-uniform surface crosslinking: The three-dimensional hydrogel scaffold intermediate D1 obtained in Step 1 is immersed in a crosslinking agent solution (CuCl2 solution or CaCl2 solution) so that the biomaterial on the surface of each printed line is crosslinked while the internal biomaterial is not crosslinked, thus obtaining the three-dimensional hydrogel scaffold intermediate D2.

[0060] Step 3: Remove the uncrosslinked part inside: Cut off the end of each printing line of the three-dimensional hydrogel scaffold intermediate D2 obtained in step 2, and then soak it in an aqueous solution to remove the uncrosslinked biomaterial inside each printing line. The printing line becomes a hydrogel tube with fully connected microchannels, and a three-dimensional hydrogel scaffold intermediate D3 with macropores and fully connected microchannels is obtained.

[0061] Step 4, complete cross-linking: The three-dimensional hydrogel scaffold intermediate D3 obtained in step 3 is immersed again in the cross-linking agent solution to complete cross-linking, and the three-dimensional hydrogel scaffold intermediate D4 is obtained.

[0062] Step 5, Pre-stretching and drying: The three-dimensional hydrogel scaffold intermediate D4 obtained in Step 4 is stretched and dried to form surface-oriented nanofiber structures on the outer and inner surfaces of each hydrogel tube, thus obtaining the final three-dimensional hydrogel scaffold.

[0063] This invention successfully prepared a three-dimensional hydrogel scaffold with macroporous structures, fully interconnected microchannels, and surface-oriented nanofibers using a combined 3D printing, non-uniform crosslinking, and stretching-drying technique. First, a macroporous three-dimensional hydrogel scaffold was constructed using 3D printing. Then, fully interconnected microchannels and surface-oriented nanofibers were added to the scaffold. Non-uniform surface crosslinking was performed by immersing the macroporous three-dimensional hydrogel scaffold in a crosslinking agent solution, resulting in a non-uniformly crosslinked three-dimensional hydrogel scaffold intermediate D2, where the biomaterial on the surface of each printed line is crosslinked while the internal biomaterial remains uncrosslinked. By immersing the three-dimensional hydrogel scaffold intermediate D2 in an aqueous solution to remove uncrosslinked biomaterials inside the printing line, the printing line is transformed into a hydrogel tube with fully interconnected microchannels, thus constructing a fully interconnected microchannel and obtaining a three-dimensional hydrogel scaffold intermediate D3 with macropores and fully interconnected microchannels. Then, the three-dimensional hydrogel scaffold intermediate D3 is immersed in a crosslinking agent solution for a second complete crosslinking, resulting in a fully crosslinked three-dimensional hydrogel scaffold intermediate D4. Finally, a surface-oriented nanofiber structure is constructed using stretching and directional drying techniques. The surface-oriented nanofiber structure exhibits an ordered arrangement of nanofiber bundles.

[0064] In step one, in one embodiment, the step of preparing sodium alginate into printable biomaterials specifically includes:

[0065] Sodium alginate, formulation materials, and water-based solutions are mixed to prepare printable biomaterials.

[0066] In one embodiment, the mass ratio of sodium alginate, formulation materials, and water-based solution is 10-20:0-10:80-90.

[0067] In one embodiment, the sodium alginate concentration in the printable biomaterial is greater than or equal to 10% by weight. If the sodium alginate concentration is too low, the printing effect is poor, and the lower layers of the scaffold are prone to deformation when printing multilayer three-dimensional hydrogel scaffolds. Furthermore, low concentrations of sodium alginate do not effectively inhibit subsequent ion crosslinking, causing the printed lines (hydrogel tubes) of the scaffold to be rapidly and completely crosslinked, failing to achieve the effect of uneven crosslinking, and thus preventing the fabrication of structures with fully interconnected microchannels.

[0068] In one embodiment, the sodium alginate concentration in the printable biomaterial is greater than or equal to 10% and less than or equal to 20% by weight.

[0069] In one embodiment, the aqueous solution is one or more of deionized water, phosphate buffer, sodium chloride aqueous solution, and cell culture medium.

[0070] In one embodiment, the 3D printing parameters are set as follows: printing air pressure of 600 kPa and printing speed of 8-10 mm / s.

[0071] In step two, in one embodiment, the crosslinking agent solution is a copper chloride solution or a calcium chloride solution.

[0072] In one embodiment, the concentration of the crosslinking agent solution is 0.1 mol / L to 2 mol / L, and the soaking time is 5 s to 120 s. When forming a fully connected microchannel structure, it is necessary to control the concentration of the crosslinking agent solution and the soaking time of the scaffold within these ranges. This ensures that the scaffold is in a non-uniform crosslinked state, where the biomaterial on the surface of each printed line (hydrogel tube) is crosslinked while the internal biomaterial remains uncrosslinked. Forming this non-uniform crosslinked state is crucial for constructing a fully connected microchannel structure. Furthermore, the concentration of the crosslinking agent solution must be matched with the soaking time; generally, the higher the concentration, the shorter the required soaking time.

[0073] In one embodiment, the three-dimensional hydrogel scaffold intermediate D2 is a three-dimensional hydrogel scaffold with large pores, wherein the biomaterial on the surface of each printed line is cross-linked while the biomaterial inside is not cross-linked.

[0074] In step three, in one embodiment, the three-dimensional hydrogel scaffold intermediate D2 obtained in step two is immersed in an aqueous solution to remove uncrosslinked biomaterial inside each printed line, transforming the printed line into a hydrogel tube with fully interconnected microchannels. This is achieved by immersing the three-dimensional hydrogel scaffold intermediate D2 in an aqueous solution, causing the uncrosslinked hydrogel inside the printed line to swell. Then, through repeated injection and perfusion of deionized water at the open end, all the uncrosslinked hydrogel is removed. In this way, the printed lines in the three-dimensional hydrogel scaffold become hollow structures, transforming the printed lines into hydrogel tubes with microchannels, and the microchannels of the hydrogel tubes are fully interconnected within the three-dimensional hydrogel scaffold.

[0075] In step four, in one embodiment, the operation of fully crosslinking the three-dimensional hydrogel scaffold intermediate D3 obtained in step three is adjusted according to the composition of the scaffold. Generally, the three-dimensional hydrogel scaffold is first immersed again in a crosslinking agent solution to allow sodium alginate to be fully crosslinked. If the three-dimensional hydrogel scaffold also contains photocrosslinkable components, the scaffold also needs to be photocrosslinked. Photocrosslinking can form an interpenetrating hydrogel network with ionically crosslinked sodium alginate, further enhancing its mechanical strength and structural stability in the solution system.

[0076] In one embodiment, in the step of immersing the three-dimensional hydrogel scaffold intermediate D3 again in the crosslinking agent solution for complete crosslinking, the immersion time is 60s to 1800s.

[0077] In step five, in one embodiment, the stretching refers to the oriented stretching of each hydrogel tube in the three-dimensional hydrogel scaffold, thereby forming a surface-oriented nanofiber structure on the outer and inner surfaces of each hydrogel tube.

[0078] In one embodiment, during the stretching and drying process, the stretching ratio of the three-dimensional hydrogel scaffold intermediate D4 is greater than 20%. This stretching ratio represents the proportion relative to the original length of each hydrogel tube within the three-dimensional hydrogel scaffold. Furthermore, because each hydrogel tube is stretched individually, resulting in a relatively long and slender tube (high length-to-diameter ratio), the stress is uniform across all parts of each tube, enabling the construction of nanoscale ordered and oriented nanofiber structures on the entire inner and outer surfaces of the three-dimensional hydrogel scaffold. In contrast, processing existing hydrogel films results in insufficiently uniform stress, making it difficult to achieve a nanoscale overall structure.

[0079] In one embodiment, the stretching ratio of the three-dimensional hydrogel scaffold intermediate D4 is 45%-55%. Within this stretching ratio range, the overall mechanical properties of the three-dimensional hydrogel scaffold are nearly twice that of a stretching ratio of 20%, and the entire surface of the scaffold exhibits a highly ordered, oriented nanoscale (100 nm) fibrous structure.

[0080] In this embodiment of the invention, during the stretching and drying process, the oriented shrinkage of the hydrogel fibers forms highly oriented surface-oriented nanofiber structures on both the inner and outer surfaces of the three-dimensional hydrogel scaffold. Compared to thin film materials, the presence of surface-oriented nanofiber structures on both the inner and outer surfaces of the three-dimensional hydrogel scaffold significantly increases the specific surface area of ​​the material while maintaining structural uniformity across the entire surface, thus maximizing the regulation of angiogenesis and osteogenic effects in cells.

[0081] This invention also provides an application of a three-dimensional hydrogel scaffold in bone repair.

[0082] The present invention will be further described below through specific embodiments.

[0083] Example 1

[0084] This embodiment provides a three-dimensional hydrogel scaffold with macropores, fully interconnected microchannels, and surface-oriented nanofiber structures, as well as its preparation method, as detailed below:

[0085] Step 1: Dissolve 1.6g of sodium alginate (Alg, 12.5% ​​by weight), 0.64g of bacterial cellulose (BC, 5% by weight), and 0.32g of methacrylamide gelatin (GelMA, 2.5% by weight, with 0.05% photoinitiator I2959 added) in 10.24g of deionized water to prepare a printable biomaterial. Use a 3D printer (printing pressure 600kPa, printing speed 9mm / s) to form several printing lines from the printable biomaterial and print it into a three-dimensional hydrogel scaffold intermediate D1 with large pores. The three-dimensional hydrogel scaffold intermediate D1 exhibits a four-layer three-dimensional ordered structure built by several printing lines. Each layer includes five printing lines arranged in parallel intervals. The printing lines of adjacent layers are at a 90° angle, and there are large pores between each printing line.

[0086] Step 2: Immerse the three-dimensional hydrogel scaffold intermediate D1 with macropores obtained in Step 1 in a 0.1 mol / L copper chloride (CuCl2) solution for 1 minute to crosslink the biomaterial on the surface of each printed line while the biomaterial inside is not crosslinked, thus obtaining the three-dimensional hydrogel scaffold intermediate D2.

[0087] Step 3: Cut off the ends of each printed hydrogel line of the three-dimensional hydrogel scaffold intermediate D2 obtained in Step 2, and then soak it in deionized water to remove the uncrosslinked biomaterial inside each printed line. Each printed line becomes a hollow structure, and each printed line forms a hydrogel tube with microchannels. The hydrogel tube with microchannels is fully connected in the three-dimensional hydrogel scaffold, that is, the three-dimensional hydrogel scaffold intermediate D3 with macropores and fully connected microchannels is obtained.

[0088] Step 4: The three-dimensional hydrogel scaffold intermediate D3 with macropores and fully interconnected microchannels obtained in Step 3 is immersed again in a 1 mol / L calcium chloride solution for 10 minutes to achieve complete cross-linking, and then irradiated under ultraviolet light for 10 minutes to achieve cross-linking, to obtain a fully cross-linked three-dimensional hydrogel scaffold intermediate D4.

[0089] Step 5: Finally, for the fully cross-linked three-dimensional hydrogel scaffold intermediate D4 with macropores and fully interconnected microchannels obtained in Step 4, fix the end of each hydrogel tube on a stretcher and stretch it by 50% of its original length. Finally, allow it to dry naturally at room temperature. This yields a three-dimensional hydrogel scaffold that combines macropores (pore size 600-800 μm), fully interconnected microchannels (diameter 400 μm), and surface-oriented nanofiber structure (nanofiber diameter 100 nm).

[0090] Figure 3 This is a top view of the three-dimensional hydrogel scaffold prepared in this embodiment. Figure 4This is a scanning electron microscope image of the oriented nanofiber structure on the outer surface of the three-dimensional hydrogel scaffold prepared in this embodiment. Figure 5 This is a scanning electron microscope image of the oriented nanofiber structure on the inner surface of the three-dimensional hydrogel scaffold prepared in this embodiment.

[0091] Comparative Example 1

[0092] The preparation method is the same as in Example 1, except that the stretching ratio in step five is adjusted to 20% to obtain a three-dimensional hydrogel scaffold with an orientation morphology scale on the surface of the three-dimensional hydrogel scaffold at the micrometer level. Figure 6 This is a scanning electron microscope image of the surface-oriented microstructure of the three-dimensional hydrogel scaffold prepared in Comparative Example 1.

[0093] Example 2

[0094] The preparation method is the same as in Example 1, except that the amount of BC in step one is adjusted to 0.96g and the weight percentage concentration is 7.5%, and GelMA is not used. In step two, the BC is crosslinked with 0.1mol / L calcium chloride (CaCl2) solution for 1 minute. Finally, a three-dimensional hydrogel scaffold with macroporous, fully connected microchannel and surface-oriented nanofiber structure is prepared. Figure 7 This is a scanning electron microscope image of the surface-oriented nanofiber structure of the three-dimensional hydrogel scaffold prepared in this embodiment.

[0095] Example 3

[0096] The preparation method is the same as in Example 1, except that the amount of BC in step one is adjusted to 0.8g with a weight percentage concentration of 6.25%, the amount of GelMA is adjusted to 0.16g with a weight percentage concentration of 1.25%, and in step two, it is crosslinked with 0.1mol / L calcium chloride (CaCl2) solution for 1 minute. Finally, a three-dimensional hydrogel scaffold with macroporous, fully connected microchannel and surface-oriented nanofiber structure is prepared. Figure 8 This is a scanning electron microscope image of the surface-oriented nanofiber structure of the three-dimensional hydrogel scaffold prepared in this embodiment.

[0097] Example 4

[0098] The preparation method is the same as in Example 1, except that in step 2, crosslinking is performed with 0.1 mol / L calcium chloride (CaCl2) solution for 1 minute instead of copper chloride, and a three-dimensional hydrogel scaffold with macropores, fully connected microchannels and surface-oriented nanostructures is finally prepared. Figure 9 This is a scanning electron microscope image of the surface-oriented nanostructure of the three-dimensional hydrogel scaffold prepared in this embodiment.

[0099] Example 5

[0100] The preparation method is the same as in Example 1, except that the amount of BC is adjusted to 0.48g and the weight percentage concentration is 3.75%, the amount of GelMA is adjusted to 0.48g and the weight percentage concentration is 3.75%, and in step two, it is crosslinked with 0.1mol / L calcium chloride (CaCl2) solution for 1 minute. Finally, a three-dimensional hydrogel scaffold with macroporous, fully connected microchannel and surface-oriented nanofiber structure is prepared. Figure 10 This is a scanning electron microscope image of the surface-oriented nanofiber structure of the three-dimensional hydrogel scaffold prepared in this embodiment.

[0101] Example 6

[0102] The preparation method is the same as in Example 1, except that in step one, the amount of BC is adjusted to 0.32g with a weight percentage concentration of 2.5%, the amount of GelMA is adjusted to 0.64g with a weight percentage concentration of 5%, and in step two, it is crosslinked with 0.1mol / L calcium chloride (CaCl2) solution for 1 minute. Finally, a three-dimensional hydrogel scaffold with macroporous, fully connected microchannel and surface-oriented nanofiber structure is prepared. Figure 11 This is a scanning electron microscope image of the surface-oriented nanofiber structure of the three-dimensional hydrogel scaffold prepared in this embodiment.

[0103] Example 7

[0104] The preparation method is the same as in Example 1, except that the amount of GelMA is adjusted to 0.96g and the weight percentage concentration is 7.5%, instead of using BC, and crosslinking is performed with 0.1mol / L calcium chloride (CaCl2) solution for 1 minute in step 2, finally preparing a three-dimensional hydrogel scaffold with macropores, fully connected microchannels and surface-oriented nanofiber structure. Figure 12 This is a scanning electron microscope image of the surface-oriented nanofiber structure of the three-dimensional hydrogel scaffold prepared in this embodiment.

[0105] Example 8

[0106] The three-dimensional hydrogel scaffold with macroporous, fully connected microchannels and surface-oriented nanofiber structure prepared in Example 1 was implanted into the skull defect site (circular defect with a diameter of 5 mm) of 5-6 week old SD rats. After 4 and 12 weeks, the samples were removed, fixed, and subjected to micro-CT scanning analysis and decalcification. The samples were then embedded in paraffin and prepared into sections. The sections were then stained with H&E and Masson staining and photographed for analysis.

[0107] Figure 13The image shows the H&E staining of the three-dimensional hydrogel scaffold repairing skull defects in rats, demonstrating that the three-dimensional hydrogel scaffold prepared in this invention, which combines macroporous, fully connected microchannels and surface-oriented nanofiber structures, has a good repair effect on skull defects.

[0108] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A three-dimensional hydrogel scaffold, characterized in that, The three-dimensional hydrogel scaffold is a layered and three-dimensional ordered structure built by a plurality of hydrogel tubes, each layer comprising a plurality of hydrogel tubes arranged in parallel and at intervals, and the hydrogel tubes of adjacent two layers are at an included angle, and the components of the three-dimensional hydrogel scaffold comprise sodium alginate; In the three-dimensional hydrogel scaffold, each hydrogel tube has a large pore, and each hydrogel tube has a fully connected microchannel inside, and the outer surface and the inner surface of each hydrogel tube have a surface-oriented nanofiber structure.

2. The three-dimensional hydrogel scaffold of claim 1, wherein, The pore size of the large pore is 200-1000 μm, the wall thickness of the hydrogel tube is 40-600 μm, the diameter of the fully connected microchannel is 50-500 μm, and the nanofiber diameter of the surface-oriented nanofiber structure is 20-200 nm.

3. The three-dimensional hydrogel scaffold of claim 1, wherein, The height of the three-dimensional hydrogel scaffold is 1-30 mm, the number of layers of the three-dimensional hydrogel scaffold is 2-30, and the number of hydrogel tubes in each layer is 2-20.

4. The three-dimensional hydrogel scaffold of claim 1, wherein, The included angle is greater than 0° and less than or equal to 90°.

5. The three-dimensional hydrogel scaffold of claim 1, wherein, The components of the three-dimensional hydrogel scaffold further comprise a formula material, and the formula material is one or more of bacterial cellulose, methacrylated gelatin, polyvinyl alcohol, hydroxyapatite, bioglass, collagen, gelatin, β-tricalcium phosphate, graphene, and hyaluronic acid.

6. A method of preparing a three-dimensional hydrogel scaffold according to claim 1, wherein, Comprise: Step one, prepare the sodium alginate into a printable biomaterial, and use the 3D printing method to form a plurality of printing lines and print into a three-dimensional hydrogel scaffold intermediate D1 with a large pore; the three-dimensional hydrogel scaffold intermediate D1 is a layered and three-dimensional ordered structure built by a plurality of printing lines, each layer comprising a plurality of printing lines arranged in parallel and at intervals, and the printing lines of adjacent two layers are at an included angle, and each printing line has a large pore; Step two, immerse the three-dimensional hydrogel scaffold intermediate D1 obtained in step one in a crosslinking agent solution, so that the biomaterial on the surface of each printing line is crosslinked and the biomaterial inside is not crosslinked, to obtain a three-dimensional hydrogel scaffold intermediate D2; Step three, immerse the three-dimensional hydrogel scaffold intermediate D2 obtained in step two in a water-based solution to remove the biomaterial inside each printing line, and the printing line becomes a hydrogel tube with a fully connected microchannel, to obtain a three-dimensional hydrogel scaffold intermediate D3 with a large pore and a fully connected microchannel; Step four, immerse the three-dimensional hydrogel scaffold intermediate D3 obtained in step three in a crosslinking agent solution again for complete crosslinking, to obtain a three-dimensional hydrogel scaffold intermediate D4; Step five, stretch and dry the three-dimensional hydrogel scaffold intermediate D4 obtained in step four, so that the outer surface and the inner surface of each hydrogel tube form a surface-oriented nanofiber structure, to obtain a final three-dimensional hydrogel scaffold.

7. The method of claim 6, wherein the three-dimensional hydrogel scaffold is prepared by, In step one, the step of preparing the sodium alginate into a printable biomaterial specifically comprises: A printable biomaterial is prepared by mixing sodium alginate, a formula material and a water-based solution, wherein the mass ratio of the sodium alginate, the formula material and the water-based solution is 10-20:0-10:80-90, and the printable biomaterial has a sodium alginate concentration of 10% or more.

8. The method of claim 6, wherein the three-dimensional hydrogel scaffold is prepared by, In step two, the crosslinking agent solution is a copper chloride solution or a calcium chloride solution; The concentration of the crosslinking agent solution is 0.1-2 mol / L; The soaking time is 5-120 s.

9. The method of claim 6, wherein the three-dimensional hydrogel scaffold is prepared by, In step five, in the stretching and drying process, the stretching ratio of the three-dimensional hydrogel scaffold intermediate D4 is greater than 20%.

10. Use of the three-dimensional hydrogel scaffold of claim 1 in bone repair.

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