3D printing organic-inorganic composite scaffold with interconnection holes and preparation method and application thereof

Abstract

The application discloses a 3D printing organic-inorganic composite scaffold with excellent interconnection and intercommunication holes and a preparation method and application thereof. The scaffold is prepared by layer-by-layer printing through an extrusion type 3D printing method of composite ink including hydroxyapatite, polyvinyl alcohol, kappa-carrageenan and a dispersing agent. The ink has good printing property, and a bone repair scaffold with an interconnection and intercommunication hole structure can be prepared. The scaffold can not only maintain the original morphology of the scaffold, but also promote cell adhesion and growth, and provide a channel for cells to transport oxygen, nutrients and metabolic products.

Description

Technical Field

[0001] This invention belongs to the field of bone tissue engineering, and specifically relates to a 3D-printed organic-inorganic composite scaffold with interconnected pores, its preparation method, and its application. Background Technology

[0002] Bones are a vital component of the human body, accounting for approximately 14.7% of total body mass. Macroscopically, bone consists of 50-70 wt% minerals, 20-40 wt% organic matter, 5-10 wt% water, and 1-5 wt% lipids. Microscopically, bone comprises an outer layer of compact bone and an inner layer of cancellous bone. The inner cortex and ends contain numerous irregular, sheet-like or linear bone structures called trabeculae. The irregular arrangement of trabeculae forms a honeycomb-like network, giving bone its porous internal structure. The main components of trabeculae are type I collagen fibers with a triple-helical structure and hydroxyapatite (HA). The organic phase (type I collagen fibers) provides flexibility and elasticity, while the inorganic phase (HA) provides rigidity and resistance to mechanical stress. Hydroxyapatite is arranged and tightly bound along the long axis of collagen fibers. This highly ordered microscopic structure gives bones high strength and toughness, and enables bones to protect organs, promote movement, support the body, and store and release lipids and minerals.

[0003] In healthy individuals, the resorption by osteoclasts and the formation of osteoblasts within bone are in a dynamic equilibrium. Osteoblasts are mononuclear bone cells that produce and mineralize the bone matrix, thereby promoting bone formation; osteoblasts differentiate from MSCs into osteocytes. Osteocytes are the most abundant cell type in the bone matrix and play a crucial role in coordinating the activities of osteoblasts and osteoclasts during bone remodeling. Osteoclasts secrete various enzymes to dissolve and absorb bone matrix and bone minerals, degrading minerals and releasing them into the bloodstream as calcium ions. The dynamic properties of bone are mediated by bone formation caused by osteoblasts and bone resorption caused by osteoclasts. This dynamic process contributes to the balance of the bone immune system, known as bone homeostasis. Defective osteoclast activity leads to osteosclerosis. Similarly, hyperactive osteoclast activity leads to osteoporosis. Therefore, imbalance in bone homeostasis is the root cause of osteoporotic fractures.

[0004] In my country, approximately 100 million people suffer from bone health issues to varying degrees, with countless cases involving bone defects caused by injury, infection, tumor resection, and bone deformities. In many cases, bone injuries heal spontaneously through two distinct mechanisms: primary and secondary fracture healing. Secondary fracture healing is the most common form, progressing from fracture hematoma to granulation tissue, then to cartilage callus, hard callus, and finally bone remodeling, ultimately leading to new bone formation. While most bone injuries heal successfully, up to 10% to 20% exhibit abnormal healing, requiring additional treatment intervention. Currently, autologous bone grafting is the primary treatment and clinical gold standard for large bone defects; however, it has limitations, including the potential for secondary injury and a limited amount of bone available for harvest. Although orthopedic biologics are promising, the safety of growth factor-based therapies remains questionable due to dose-related side effects. In addition, the limitations of orthopedic biologics include the lack of spatial control over scaffold structures to anatomically match complex bone defect sites, and the lack of temporal control over combined therapeutic agents to maximize their efficacy.

[0005] To overcome the limitations of current treatment options, research is increasingly turning to the engineering of 3D-printed bone graft alternatives. 3D printing can create patient-specific bone graft alternatives, customized to fit the site of bone loss. The technology also helps control the microstructure of the scaffold, including pore size, total pore volume, and shape. Although currently lacking clinical evidence and regulatory approval, 3D printing technology can meet individualized patient needs, printing three-dimensional scaffolds with controllable structures and pore sizes to promote cell migration and bone growth.

[0006] Scaffolds are typically developed through various traditional manufacturing methods, including particle leaching, thermal phase separation, solvent casting, electrospinning, melt molding, gas foaming, and freeze drying. However, these techniques have limitations in terms of minimal control over toxic solvent residues, scaffold composition, structure, micropore shape, interconnection, distribution, and pore size. 3D printing, commonly known as additive manufacturing, refers to layer-by-layer stacking supported by computer-aided design (CAD) that helps manufacture high-quality scaffolds. The 3D printing process begins with designing a 3D geometric model using CAD software. The CAD file is then converted into a Standard Triangulation Language (STL) file format, which is the standard input for almost all 3D printing systems. The STL file is imported into the 3D printing software to adjust printing parameters and object orientation, and then the object geometry is sliced ​​at a distance equal to the layer thickness. This information is transferred to the 3D printing system for additive manufacturing of the part. Finally, post-processing of the printed material is performed, such as cleaning the object, removing support material, and surface finishing. Currently, the mainstream 3D printing technologies are extrusion printing, selective laser sintering (SLS), and stereolithography (SLA). Each of these technologies has its own advantages and disadvantages. Considering cost and printing temperature, extrusion printing is typically chosen to create composite scaffolds. Extrusion printing offers advantages such as low equipment cost and a wide range of printing materials. However, its greatest advantage is its ability to load growth factors, cells, and proteins, which can aid in bone healing within the scaffold.

[0007] Existing materials for bone repair can be broadly categorized into three types: metals, inorganic ceramics, and organic materials. Inorganic ceramics are the preferred material for bone repair due to their osteoinductive and osteoconductive properties; however, these single inorganic ceramics suffer from high brittleness, thus failing to provide adequate mechanical support. Organic materials, on the other hand, possess good toughness and elasticity, as well as excellent biocompatibility and degradation properties.

[0008] Currently, there are three main types of inks used for extrusion printing: shear-thinning inks, rapid sol-gel transition inks, and support bath inks. Shear-thinning inks are the most commonly used. Most polymer solutions used in these inks are shear-thinned; the application of shear force and the orientation of amorphous polymer chains reduce the ink's viscosity, allowing it to be extruded from the syringe under certain pressure and rate. However, to function effectively in 3D printing, the material must also possess rapid shear recovery capability to maintain its printed shape. While extrusion 3D printing offers advantages such as a wide range of printable materials, the inherent drawback of insufficient precision in the printed scaffold is unavoidable. Therefore, developing a bio-ink with good printable precision is essential.

[0009] In addition, ideal bone tissue engineering materials need to have the following characteristics: biocompatibility, which can degrade into non-toxic decomposition products after implantation in the body without causing inflammation and immune rejection; biodegradability, which means that the scaffold has controllable degradation, can be degraded by host enzymes or biological processes, and can supplement tissue growth inward while maintaining sufficient support; and bioactivity, including osteoconductivity and osteoinductive properties. Summary of the Invention

[0010] To address the issues of poor printability in current extrusion-based 3D printed bone repair scaffolds, this invention provides an ink suitable for extrusion-based 3D printing, a 3D printed composite scaffold with interconnected pores based on the ink, its preparation method, and its application.

[0011] Specifically, the present invention provides an ink comprising hydroxyapatite, polyvinyl alcohol, κ-carrageenan, and a dispersant.

[0012] Research has found that the properties of bio-inks play a crucial role in the fabrication of integrated layer-by-layer constructs and the formation of functional tissues or organs. Physically, bio-inks should exhibit gelation properties after extrusion from the nozzle tip, displaying a solidified filament morphology. Their mechanical strength should be sufficient to support the deposition of upper layers, and their physical structure should possess an interconnected porous structure. Physiologically, bio-inks should provide a suitable microenvironment to support various cellular activities, such as migration, proliferation, differentiation, and specific tissue formation. Furthermore, bio-inks also need excellent printability—the ability to form 3D structures with good fidelity and integrity—and cell viability—the survival rate of cells after printing—which are considered key representative standards of physical and physiological properties, respectively. Based on this, the present invention proposes a bio-ink with the above-mentioned composition, possessing all the aforementioned performance requirements.

[0013] According to an embodiment of the present invention, the mass ratio of the hydroxyapatite to the total mass of κ-carrageenan and polyvinyl alcohol is 6:(3-5), with exemplary ratios of 6:3, 6:4, and 6:5.

[0014] According to an embodiment of the present invention, the mass ratio of κ-carrageenan to polyvinyl alcohol is (1-3):(1-3), with exemplary ratios of 1:3, 1:1, and 3:1.

[0015] According to an embodiment of the present invention, the concentration of polyvinyl alcohol in the ink is 10-15%, exemplarily 10%, 12%, and 15%.

[0016] According to an embodiment of the present invention, the dispersant is water. Specifically, the dispersant is deionized water.

[0017] According to an embodiment of the present invention, the hydroxyapatite is a product obtained by co-precipitation and calcination of a phosphorus source, ammonia, a calcium source, and water. Specifically, the phosphorus source is selected from diammonium hydrogen phosphate, and the calcium source is selected from calcium nitrate tetrahydrate.

[0018] The present invention also provides a method for preparing the above-mentioned ink, the method comprising: mixing hydroxyapatite, polyvinyl alcohol, κ-carrageenan and a dispersant to obtain the ink.

[0019] Specifically, the definition of the dispersant is the same as above.

[0020] According to an embodiment of the present invention, the hydroxyapatite, polyvinyl alcohol, and κ-carrageenan are selected and used in the same proportions as described above.

[0021] The present invention also provides a scaffold comprising hydroxyapatite, polyvinyl alcohol, and κ-carrageenan.

[0022] According to an embodiment of the present invention, the hydroxyapatite, polyvinyl alcohol, and κ-carrageenan are selected and used in the same proportions as described above.

[0023] According to an embodiment of the present invention, the scaffold is prepared by printing the above-mentioned ink layer by layer using an extrusion 3D printing method.

[0024] According to an embodiment of the present invention, the scaffold is a 3D-printed organic-inorganic composite scaffold with interconnected holes.

[0025] According to an embodiment of the present invention, the diameter of the bottom hole of the bracket is 300-500 μm, exemplarily (390.00±42.12) μm; the diameter of the side hole of the bracket is 400-550 μm, exemplarily (474.90±36.00) μm.

[0026] According to an embodiment of the present invention, biosignals and / or growth factors may be attached to the scaffold.

[0027] The scaffold of this invention possesses biocompatibility (specifically, it degrades into non-toxic decomposition products after implantation without triggering inflammation or immune rejection); biodegradability (specifically, the scaffold of this invention exhibits controlled degradation, capable of degradation by host enzymes or biological processes, and can promote tissue inward growth while maintaining sufficient support); and bioactivity, including osteoconductivity and osteoinductive properties. The scaffold of this invention can interact and bind to host tissues and can further contain biological signals and growth factors to stimulate cell inward growth, attachment, and differentiation. Furthermore, the scaffold of this invention has an excellent interconnected porous structure with sufficient porosity, which effectively promotes cell inward growth without compromising mechanical properties. Moreover, the mechanical properties of the scaffold of this invention are matched to human bone, with compressive, elastic, and fatigue strength comparable to host tissues, while maintaining structural integrity in vivo, making it a scaffold with great application potential.

[0028] The present invention also provides a method for preparing the above-mentioned scaffold, the method comprising: preparing the scaffold by printing the above-mentioned ink layer by layer using an extrusion 3D printing method.

[0029] According to an embodiment of the present invention, the method specifically includes the following steps:

[0030] (1) Preparation of hydroxyapatite powder;

[0031] (2) Dissolve polyvinyl alcohol powder in water and prepare a polyvinyl alcohol aqueous solution by heating and stirring.

[0032] (3) Disperse hydroxyapatite powder and κ-carrageenan powder in a polyvinyl alcohol aqueous solution and stir to obtain ink;

[0033] (4) Use extrusion 3D printing method to print ink layer by layer.

[0034] According to an embodiment of the present invention, step (1) specifically includes: mixing a calcium source (such as calcium nitrate tetrahydrate), a phosphorus source (such as diammonium hydrogen phosphate), ammonia water and water, and then calcining the mixture using a co-precipitation method to obtain hydroxyapatite powder.

[0035] According to an embodiment of the present invention, the method for preparing the scaffold may further include step (5): drying the printed scaffold until it is completely dry, and then taking it out to obtain a 3D printed bone repair scaffold with an interconnected pore structure.

[0036] Preferably, in step (1), the molar ratio of calcium source (such as calcium nitrate tetrahydrate), phosphorus source (such as diammonium hydrogen phosphate), ammonia water and water is 10:6:8:(800-1200).

[0037] Preferably, in step (1), the calcium source (such as calcium nitrate tetrahydrate) and the phosphorus source (such as diammonium hydrogen phosphate) are first dissolved in water, and then ammonia water is added to it.

[0038] Preferably, in step (1), the temperature of the coprecipitation reaction is 60-80°C, for example 70°C; and the time of the coprecipitation reaction is 1-4 hours, for example 2 hours.

[0039] Preferably, in step (1), the calcination temperature is 700–900°C, exemplarily 700°C, 800°C, or 900°C; and the calcination time is 1–4 hours, exemplarily 2 hours. High-temperature calcination helps remove impurities and obtain well-crystallized, high-purity nano-hydroxyapatite.

[0040] Preferably, in step (2), the concentration of the polyvinyl alcohol aqueous solution is 10-15%, for example 10%, 12%, or 15%.

[0041] Preferably, polyvinyl alcohol powder is dissolved in water and heated to dissolve. Preferably, the heating temperature is 80-100°C, exemplarily 90°C, and the heating time is 1-4 hours, exemplarily 2 hours.

[0042] Preferably, in step (3), the total mass ratio of hydroxyapatite powder, κ-carrageenan powder and polyvinyl alcohol is 6:(3-5), for example 6:3, 6:4, 6:5.

[0043] According to an embodiment of the present invention, the mass ratio of κ-carrageenan to polyvinyl alcohol is (1-3):(1-3), exemplarily 1:3, 1:1, or 3:1.

[0044] Preferably, in step (3), the stirring temperature is room temperature and the stirring time is 3-5 minutes.

[0045] Preferably, in step (4), the infill density of the 3D printed scaffold is 40%-60%; for example, 40%, 50%, and 60%.

[0046] Preferably, in step (5), the drying time is 6-48 hours, for example 24 hours, and the drying temperature is 37°C.

[0047] According to an exemplary embodiment of the present invention, the method for preparing the stent includes the following steps:

[0048] (1) Calcium nitrate tetrahydrate, diammonium hydrogen phosphate, ammonia and water were mixed in a molar ratio of 10:6:8:(800-1200) and hydroxyapatite powder was prepared by coprecipitation.

[0049] (2) Polyvinyl alcohol is mixed with water to prepare a polyvinyl alcohol aqueous solution;

[0050] (3) Mix hydroxyapatite powder, κ-carrageenan powder and polyvinyl alcohol aqueous solution, and stir to prepare printing ink;

[0051] (4) Using extrusion 3D printing technology, print ink with a density of 40%-60% layer by layer to obtain a shaped scaffold;

[0052] (5) The formed scaffold is dried to obtain a scaffold. Specifically, the scaffold has an interconnected porous structure and can be referred to as a hydroxyapatite-polyvinyl alcohol / κ-carrageenan 3D printed bone repair scaffold.

[0053] The present invention also provides the use of the above-described scaffold in the preparation of drugs that promote angiogenesis. For example, its use in the preparation of drugs for bone defect repair (osteoporotic fractures).

[0054] The present invention also provides a method for treating or alleviating bone defects, comprising providing the above-mentioned scaffold to an individual in need of such treatment.

[0055] The present invention also provides a method for treating or alleviating a fracture, comprising providing the aforementioned brace to an individual in need. For example, the fracture may be an osteoporotic fracture.

[0056] The beneficial effects of this invention are:

[0057] The novel composite ink of this invention has good printability and can be used to prepare bone repair scaffolds with interconnected pore structures. The scaffolds of this invention can not only maintain the original morphology of the scaffold, but also these interconnected pores can promote cell adhesion and growth, and provide channels for cells to transport oxygen, nutrients and metabolic products. Attached Figure Description

[0058] Figure 1 In the diagram, A represents a macroscopic schematic of three types of scaffolds prepared when the solid content ratio of κ-carrageenan (κ-CA) to PVA is 1:3. Figure 1 In the figure, B represents a macroscopic schematic diagram of three types of scaffolds prepared when the solid content ratio of κ-carrageenan (κ-CA) to PVA is 1:1. Figure 1 In the figure, C represents a macroscopic schematic diagram of three types of scaffolds prepared when the solid content ratio of κ-carrageenan (κ-CA) to PVA is 3:1. Figure 1 D represents the fidelity (Pr) test results of the brackets printed with nine different ink ratios in Example 1 and the brackets printed with inks in Comparative Example 1.

[0059] Figure 2 The rheological properties of the inks in Example 1 and Comparative Example 1 are as follows: Figure 2 In Figure A, the shear thinning properties of the inks in Example 1 and Comparative Example 1 are tested. Figure 2 B represents the viscoelastic performance test of the inks in Example 1 and Comparative Example 1; Figure 2 The self-healing performance test of the inks in Example 1 and Comparative Example 1 is shown in CF.

[0060] Figure 3 The printing process of the bracket in Example 1 and the aperture diagram of the bracket are shown.

[0061] Figure 4 The graph shows the cell viability assay of the scaffolds in Example 1 and Comparative Example 1. Detailed Implementation

[0062] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0063] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0064] Example 1

[0065] A method for fabricating a 3D-printed organic-inorganic composite scaffold with interconnected holes includes the following steps:

[0066] (1) Dissolve 0.5062 mol Ca(NO3)2·4H2O and 0.3104 mol (NH4)2HPO4 in 500 mL of deionized water, respectively, and then stir magnetically at room temperature until completely dissolved to prepare solutions A and B. Solution B is then introduced into solution A to obtain solution C, which is then gently heated. Ammonia is added to solution C, and the pH of solution C is adjusted to 9.5-10 using a pH meter. Solution C is then heated in a 70 °C water bath and stirred vigorously for 2 h. After the reaction is complete, the resulting white precipitate is filtered and washed three times with deionized water and three times with anhydrous ethanol. The filtered product is then dried at 60 °C for 24 h and sintered at 800 °C for 2 h to remove impurities, finally yielding hydroxyapatite powder.

[0067] (2) Weigh 10g, 12g and 15g of polyvinyl alcohol (PVA, degree of alcoholysis: 98-99% (mol / mol)) powder respectively, dissolve in 100g of deionized water, and heat at 90℃ for 2h to form polyvinyl alcohol aqueous solutions with concentrations of 10%, 12% and 15% respectively.

[0068] (3) According to the mass ratio of inorganic to organic = 6:4, weigh 6g of hydroxyapatite powder and 1g of κ-carrageenan powder (purchased from Aladdin), dissolve them in a polyvinyl alcohol aqueous solution with a solid content of 3g (the masses of polyvinyl alcohol aqueous solutions with concentrations of 10%, 12%, and 15% are 30g, 25g, and 20g, respectively), stir with a glass rod at room temperature for 3-5 minutes until a uniform composite ink is formed, and name the scaffolds 1:3-10%, 1:3-12%, and 1:3-15% according to the ratio of organic κ-carrageenan to polyvinyl alcohol solid content.

[0069] According to the inorganic:organic ratio of 6:4, 6g of hydroxyapatite powder and 2g of κ-carrageenan powder (purchased from Aladdin) were weighed in sequence and dissolved in a polyvinyl alcohol aqueous solution with a solid content of 2g (the masses of polyvinyl alcohol aqueous solutions with concentrations of 10%, 12%, and 15% were 20g, 16.7g, and 13.3g, respectively). The solution was stirred with a glass rod at room temperature for 3-5 minutes until a uniform composite ink was formed. The scaffolds were named 1:1-10%, 1:1-12%, and 1:1-15% according to the ratio of organic κ-carrageenan to polyvinyl alcohol solid content.

[0070] According to the inorganic:organic ratio of 6:4, 6g of hydroxyapatite powder and 3g of κ-carrageenan powder (purchased from Aladdin) were weighed in sequence and dissolved in a polyvinyl alcohol aqueous solution with a solid content of 1g (the masses of polyvinyl alcohol aqueous solutions with concentrations of 10%, 12%, and 15% were 10g, 8.3g, and 6.7g, respectively). The solution was stirred with a glass rod at room temperature for 3-5 minutes until a uniform composite ink was formed. The scaffolds were named 3:1-10%, 3:1-12%, and 3:1-15% according to the ratio of organic κ-carrageenan to polyvinyl alcohol solid content.

[0071] (4) Cylindrical models with diameters of 15×5mm and 15×30mm were constructed using modeling software, and slicing files were exported using slicing software. The layer thickness was 0.6mm and the infill density was 50%. Then, an extrusion 3D printer was used to print "ink" layer by layer under the control of gcode to obtain a porous scaffold. After printing, a hydroxyapatite-polyvinyl alcohol / κ-carrageenan scaffold was obtained and dried in an oven at 37℃ for 24 hours. The scaffold was named HA+PVA / κ-CA.

[0072] Comparative Example 1

[0073] A method for fabricating a 3D-printed organic-inorganic composite scaffold with interconnected holes includes the following steps:

[0074] (1) To verify the effect of the introduction of κ-carrageenan on the printability of the scaffold, an HA+PVA ink without κ-carrageenan was prepared, wherein the concentration of the PVA aqueous solution was 15%. 6g of the hydroxyapatite powder prepared in step (1) of Example 1 was dispersed in a polyvinyl alcohol aqueous solution with a solid content of 4g (the mass of the 15% polyvinyl alcohol aqueous solution was 26.67g). The solution was stirred with a glass rod at room temperature for 3-5 minutes until a uniform composite ink was formed, named HA+PVA-15%.

[0075] (2) Cylindrical models with diameters of 15×5mm and 15×30mm were constructed using modeling software, and slice files were exported using sliced ​​parts. The layer thickness was 0.6mm, and the infill density was 50%. Then, an extrusion 3D printer was used to print "ink" layer by layer under the control of gcode code to obtain a porous scaffold. After printing, a hydroxyapatite-polyvinyl alcohol scaffold was obtained and dried in an oven at 37℃ for 24 hours. The scaffold was named HA+PVA-15%.

[0076] Experimental Example 1

[0077] To explore the impact of bio-ink formulations on scaffold printability and to optimize the ink ratio for best printability, this invention introduces a semi-quantitative method based on rheological measurements and image analysis to evaluate the printability of bio-inks. Specifically, for rheological testing, a rotational rheometer was used to detect the rheological, viscoelastic, and self-healing properties of the bio-ink at room temperature. Rheological properties were assessed by testing the relationship between ink shear rate and viscosity, with the shear rate range set to 0.1-1000 s⁻¹. -1 Viscoelastic performance testing was conducted on the changes in storage modulus (G') and dissipation modulus (G”) of the ink under a 1Hz frequency scan and a strain range of 1%-1000%. Self-healing performance testing assessed the damage and recovery effects of the ink under repeated oscillatory strain. For semi-quantitative image testing, the pore area and perimeter of the scaffold were measured using the built-in scale of a stereomicroscope, and the scaffold fidelity (Pr) was calculated based on a printable semi-quantitative formula. Results are as follows... Figure 1 As shown.

[0078] In this study, the printability of the scaffolds prepared in Example 1 and Comparative Example 1 was measured based on fidelity (Pr):

[0079] Pr = L 2 / 16A

[0080] Where L represents the perimeter of the square grid formed by the support structure, and A represents the area enclosed. Each set of supports was photographed (n=5), and the supports were observed using an optical microscope to measure the perimeter and area of ​​the grid.

[0081] Figure 1Experimental results show that the printing effects of scaffolds prepared in Example 1 with different ratios of organic κ-carrageenan to polyvinyl alcohol solids vary. The scaffolds prepared with ratios of 1:3-15%, 1:1-12%, and 3:1-10% exhibit the best interconnected pore structure. The Pr value further verifies this result, with a Pr value closer to 1 indicating better printability. Furthermore, the scaffolds prepared in Comparative Example 1 have the same printing effect as those with a ratio of 1:3-10% organic κ-carrageenan to polyvinyl alcohol solids; the grid of the scaffold collapses completely, indicating insufficient ink gelation ability. In contrast, the scaffolds prepared in Example 1 using κ-carrageenan all exhibit a certain pore structure, with the scaffolds prepared with ratios of 1:3-15%, 1:1-12%, and 3:1-10% showing clearly defined interconnected channels and Pr values ​​closest to 1. Therefore, they possess the best printability.

[0082] Experimental Example 2

[0083] The rheological properties of bio-inks are also a key factor in evaluating printability. This invention investigated the relationship between the viscosity and shear rate of bio-inks, revealing pseudoplastic behavior characterized by shear thinning. Further analysis of the ink modulus showed that, across the entire strain range, the loss modulus (G”) of the scaffold prepared in Comparative Example 1 remained higher than the storage modulus (G’), revealing its viscosity-to-elasticity characteristic. Therefore, it was prone to collapse after extrusion, consistent with the Pr value structure. Conversely, at low strain, the scaffolds prepared in Example 1 with organic κ-carrageenan to polyvinyl alcohol solid content ratios of 1:3-15%, 1:1-12%, and 3:1-10% exhibited G’ exceeding G”, indicating that the elasticity of the bio-ink predominated after extrusion, thus facilitating the ink to maintain a stable structure without collapse during post-extrusion.

[0084] The rapid self-healing capabilities of the scaffolds prepared in Example 1 (organic κ-carrageenan to polyvinyl alcohol solids ratios of 1:3-15%, 1:1-12%, and 3:1-10%) and Comparative Example 1 were subsequently evaluated. Cyclic strain testing demonstrated that the bio-ink in Example 1 (organic κ-carrageenan to polyvinyl alcohol solids ratios of 1:3-15%, 1:1-12%, and 3:1-10%) exhibited alternating transitions between G' and G”, implying a reversible gel-to-fluid transition. However, Comparative Example 1 (HA+PVA-15%) lacked the ability to revert to a gel state under low strain, thus lacking printability.

[0085] Experimental Example 3

[0086] Macroscopic printing of the support structure reveals that the supports of Example 1 do not collapse during the printing process, and a very obvious porous structure is visible. Further printing of a tall support structure (using modeling software to construct a Φ15×30mm cylindrical model and exporting slice files using slicing software, with a layer thickness of 0.6mm and an infill density of 50%, then printing "ink" layer by layer using an extrusion 3D printer under the control of gcode) also demonstrates the support structure's excellent printability, and the material does not collapse. More importantly, the side hole structure is particularly prominent. For tall supports, due to the more pronounced longitudinal gravity, side hole collapse is inevitable. However, the printed support structure of this invention exhibits an excellent side hole structure, which is further evident during drying.

[0087] The results in summary indicate that this invention has developed a novel composite ink, and by adjusting its formulation, a scaffold with good printability has been obtained. Moreover, the scaffold prepared by this invention can maintain the original structure and interconnected holes.

[0088] Experiment Example 4

[0089] Three scaffolds with optimal fidelity Pr selected in Experimental Example 1 (the three scaffolds prepared with organic matter κ-carrageenan and polyvinyl alcohol solid content ratios of 1:3-15%, 1:1-12%, and 3:1-10%) and the scaffold in Comparative Example 1 were soaked in culture medium to prepare scaffold extracts. The scaffold extracts were prepared according to ISO 10993-12 2007 standards. The extracts were then used to culture MC3T3-E1 cells, and cell viability was measured using a cell counting kit-8 (CCK-8). The experimental results are as follows. Figure 4 As shown in the figure, the three scaffolds prepared with organic κ-carrageenan and polyvinyl alcohol solid content ratios of 1:3-15%, 1:1-12%, and 3:1-10% all exhibit good cell compatibility, and cell viability increases with time, indicating that the scaffolds of the present invention can promote cell adhesion and growth.

[0090] The exemplary embodiments of the present invention have been described above. However, the scope of protection of this application is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a stent, characterized in that, Includes the following steps: (1) Preparation of hydroxyapatite powder; (2) Dissolve polyvinyl alcohol powder in water and prepare a polyvinyl alcohol aqueous solution by heating and stirring; (3) Disperse hydroxyapatite powder and κ-carrageenan powder in a polyvinyl alcohol aqueous solution and stir to obtain ink; (4) Use extrusion 3D printing method to print ink layer by layer; The mass ratio of the hydroxyapatite to the total mass of κ-carrageenan and polyvinyl alcohol is 6:(3-5). The mass ratio of κ-carrageenan to polyvinyl alcohol is 1:3, 1:1, or 3:

1. Step (1) specifically includes: mixing calcium nitrate tetrahydrate, diammonium hydrogen phosphate, ammonia water and water, and then calcining the mixture using a co-precipitation method to obtain hydroxyapatite powder; The molar ratio of calcium nitrate tetrahydrate, diammonium hydrogen phosphate, ammonia, and water is 10:6:8:(800-1200). The temperature for the coprecipitation reaction is 60~80℃; the reaction time is 1~4h. The calcination temperature is 700~900℃; the calcination time is 1~4h.

2. The preparation method according to claim 1, characterized in that, In step (2), the concentration of the polyvinyl alcohol aqueous solution is 10-15%.

3. The preparation method according to claim 1, characterized in that, In step (4), the infill density of the 3D printed scaffold is 40%-60%.

4. The use of the stent prepared by the preparation method according to any one of claims 1-3 in the preparation of drugs that promote angiogenesis.

5. The application as described in claim 4, characterized in that, The scaffold is used in the preparation of drugs for bone defect repair.

6. The application as described in claim 5, characterized in that, The scaffold is used in the preparation of drugs for osteoporotic fractures.

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