Composite elastic stent synthesized based on collagenous fiber-hydroxyapatite as well as preparation method and application of composite elastic stent

Abstract

The invention relates to the technical field of biomedical materials and regenerative medicine, in particular to a composite elastic stent synthesized based on collagenous fiber-hydroxyapatite as well as a preparation method and application of the composite elastic stent. The composite elastic stent is formed by compounding collagen and hydroxyapatite, the collagen is self-assembled to form a collagen fiber network, and the hydroxyapatite is uniformly dispersed in the collagen fiber network; the preparation method comprises the following steps: mixing an acidic collagen solution and a hydroxyapatite suspension to form printing ink; after ultrasonic and low-speed centrifugal defoaming, performing three-dimensional printing in a carbopol support bath; performing temperature control incubation to form a collagenous fiber network structure; washing away the support bath by using a salt-containing buffer solution; performing cross-linking treatment; the material shows elastic structure characteristics, observable blood absorption and factor agglutination behaviors, shows good repair capability and biocompatibility in an animal bone-cartilage defect model, and is used in the field of tissue engineering such as bone and cartilage injury repair.

Description

Technical Field

[0001] This invention relates to the fields of biomedical materials and regenerative medicine, and in particular to a composite elastic scaffold based on collagen fiber-hydroxyapatite synthesis, its preparation method, and its application. Background Technology

[0002] Collagen is a major component of the natural extracellular matrix. It has good biocompatibility, biodegradability, and a natural fibrous hierarchical structure, and is therefore widely used as the basis for constructing tissue engineering materials.

[0003] However, hydrogels or scaffolds formed solely from collagen typically have limited mechanical properties. Their fiber networks are prone to irreversible deformation under external forces, making it difficult to maintain structural stability under significant compression or cyclic loading. This makes it difficult for natural rubber raw materials to possess both formability and elasticity.

[0004] To improve the mechanical properties of adhesive raw materials, existing technologies often combine collagen with inorganic components (such as hydroxyapatite) in order to enhance support capacity or provide additional functions.

[0005] However, traditional organic / inorganic composite methods often rely on simple mixing, electrostatic adsorption, or physical embedding, lacking precise control over the interfacial structure. Without effective interfacial control, inorganic particles often aggregate uncontrollably within the collagen network, leading to fiber structure damage, a decline in the overall mechanical properties of the material, and difficulty in forming a three-dimensional elastic structure with synergistic deformation capabilities. Furthermore, the adsorption, liquid retention, and enrichment capabilities of collagen raw materials when in contact with biofluids are closely related to their internal porosity, fiber arrangement, and the characteristics of the organic / inorganic interface.

[0006] While existing collagen-based materials can absorb a certain amount of liquid, they generally lack the ability to simultaneously achieve: (1) the formation of a hierarchical structure after collagen fiberization; (2) uniform and stable dispersion of inorganic particles in the network; (3) significant elasticity and rapid shape recovery behavior; and (4) the ability to adsorb and retain blood or protein-containing liquids. These properties depend on the overall synergy of the material from the nano-interface to the macrostructure, and existing technologies lack a preparation strategy that can simultaneously control the self-assembly of collagen fibers, the interfacial composite state, and the stability of three-dimensional forming. To this end, this invention provides a composite elastic scaffold based on collagen fiber-hydroxyapatite synthesis, its preparation method, and its applications. Summary of the Invention

[0007] To address the problem that inorganic particles in existing collagen-based materials often exhibit uncontrolled aggregation within the collagen network, leading to fiber structure damage, decreased overall material mechanical performance, and difficulty in forming a three-dimensional elastic structure with synergistic deformation capabilities, this invention provides a composite elastic scaffold based on collagen fiber-hydroxyapatite synthesis, its preparation method, and applications. Using collagen as the main component, the inorganic phase is uniformly dispersed through interface regulation, exhibiting elastic behavior on a macroscopic scale, while also possessing good liquid adsorption and enrichment capabilities. The construction of this composite elastic scaffold is based on the internal structure design of the material, overcoming the limitations of existing collagen-based materials in terms of mechanical response, interface structure, and liquid adsorption performance.

[0008] The present invention provides a composite elastic scaffold based on collagen fiber-hydroxyapatite synthesis, which is achieved through the following technical solution: A composite elastic scaffold based on collagen fiber-hydroxyapatite synthesis is a three-dimensional porous network structure formed by collagen and hydroxyapatite; collagen self-assembles to form a collagen fiber network, and hydroxyapatite is uniformly dispersed in the collagen fiber network.

[0009] The composite elastic scaffold, after being printed in a support bath, incubated under temperature control, and cross-linked, possesses elastic properties.

[0010] Preferably, the collagen fiber network in the composite elastic scaffold is a multi-level nanostructure network, which facilitates blood absorption and factor aggregation.

[0011] Preferably, the hydroxyapatite has a particle size of 20 nm to 1 μm.

[0012] The composite elastic scaffold in this invention utilizes a Carbopol support bath to maintain the three-dimensional structure of collagen during the printing process. After temperature-controlled incubation, it forms a fibrous network, which is then cross-linked and freeze-dried to obtain the final product.

[0013] The problem of uniform dispersion of hydroxyapatite (HAp) in collagen was successfully solved using a "sodium citrate-hydroxyapatite (HAp) dispersion system". This system constructs an elastic collagen fiber-hydroxyapatite (HAp) three-dimensional scaffold (previous collagen scaffolds were brittle or soft and could not be compressed).

[0014] The composite elastic scaffold of this invention exhibits observable blood adsorption / factor aggregation behavior, forming continuous repair tissue in animal osteochondral models.

[0015] The present invention provides a method for preparing a composite elastic scaffold based on collagen fiber-hydroxyapatite synthesis, which is achieved through the following technical solution: A method for preparing a composite elastic scaffold based on collagen fiber-hydroxyapatite synthesis, comprising the following steps: Step 1: Prepare acidic collagen solution and sodium citrate / hydroxyapatite (HAp) suspension respectively; Step 2: The acidic collagen solution and sodium citrate / hydroxyapatite (HAp) suspension are mixed evenly at a volume ratio of (1-4):1, and then subjected to ultrasonic and centrifugal treatment to obtain printing ink. Step 3: Using the printing ink from Step 2, perform 3D printing in a support bath. The printing material in the support bath is incubated at 35-38℃ / 70-100%RH for 1-5 hours to form a collagen fiber network structure. Step 4: Wash away the support bath with PBS buffer containing NaCl; Step 5: Crosslinking treatment was performed using an alcoholic solution of glutaraldehyde or 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) at a concentration of 0.5±0.1wt%. The crosslinking temperature was 37±0.5℃ and the crosslinking reaction time was 4~12h. Subsequently, the composite elastic scaffold with a three-dimensional porous network structure was obtained by freeze drying.

[0016] Preferably, the acidic collagen solution in step one is prepared as follows: collagen is mixed evenly with a 0.5-5.0M acidic aqueous solution to obtain an acidic collagen aqueous solution with a collagen mass concentration of 10-30 mg / mL; the acidic aqueous solution is at least one of acetic acid aqueous solution, hydrochloric acid aqueous solution, and carbonic acid aqueous solution.

[0017] Preferably, the sodium citrate / hydroxyapatite HAp suspension in step one is prepared as follows: hydroxyapatite with a particle size of 20nm to 1μm and sodium citrate are thoroughly mixed and dispersed in PBS buffer at a mass ratio of (0.1-3.5):1 to obtain a sodium citrate / hydroxyapatite HAp suspension with a hydroxyapatite solid content of 10% to 50%.

[0018] Preferably, the ultrasonic treatment parameters in step two are as follows: ultrasonic frequency 20-40 kHz, ultrasonic time 5-20 min; the centrifugation treatment parameters in step two are as follows: temperature 4±0.5℃, centrifugation speed 1500±500 rap / min, centrifugation 3±1 min.

[0019] Preferably, the preparation method of the support bath in step three is as follows: Carbopol and PB buffer are stirred and dispersed evenly to obtain a PB buffer with a Carbopol concentration of 1-2 w / v%. After stopping stirring, the mixture is allowed to stand for 5-24 hours to allow the Carbopol to swell completely. The pH is adjusted to about 7.0-7.4 to ensure that the system is fully swollen and presents a uniform gel state. Then, the system is centrifuged at 8000±2000 rap / min for 4±1 min at a temperature of 4±0.5℃ and stored at low temperature to obtain the support bath.

[0020] Preferably, the 3D printing parameters in step three are as follows: 27G specification needles are used for 3D printing, the printing line spacing is set to 0.6±0.1mm; the layer height is set to 0.21±0.01mm; and the printing speed is set to 10±2mm / s.

[0021] Preferably, in step three, three-dimensional printing is performed in a support bath using predetermined path parameters and layer height parameters. The printed structure includes, but is not limited to, a cylinder with a diameter of approximately 3 mm and a height of approximately 3 mm, a cylinder with a diameter of approximately 3.5 mm and a height of approximately 3 mm, and a cuboid with a side length of 10 mm and a height of approximately 4 mm. After printing, the support bath ink system is in an unstable state and needs to be incubated in a constant temperature and humidity chamber of 35~38℃ and 70~100% for 1~5 hours to allow the collagen ink to complete fiber assembly and fix its shape.

[0022] Preferably, in step four, the support bath is washed away using a PBS buffer containing NaCl, and a PBS buffer containing 2wt% to 5wt% NaCl is recommended.

[0023] The present invention provides a composite elastic scaffold based on collagen fiber-hydroxyapatite synthesis, which has a biomimetic structure that exhibits stronger elasticity and blood absorption capacity, and can effectively repair osteocartilage damage. That is, the composite elastic scaffold is used in the field of tissue engineering, specifically for osteocartilage damage repair.

[0024] In summary, the present invention has the following advantages: 1. This invention achieves stable shaping of collagen during 3D printing: Natural collagen is prone to collapse and lacks self-support during printing, making it difficult to maintain its structure. This invention uses a Carbopol support bath to stabilize and fix the collagen printing ink during the shaping process; subsequently, temperature-controlled incubation allows the collagen to form a fiber network in situ within the printed structure, thereby obtaining a 3D scaffold with an internal fibrous structure, overcoming the problem of poor shape retention in printing with existing collagen raw materials.

[0025] 2. This invention significantly improves the dispersibility and stability of hydroxyapatite in collagen systems: In traditional collagen / hydroxyapatite (HAp) composites, hydroxyapatite (HAp) tends to agglomerate and distribute unevenly. This invention achieves effective dispersion of hydroxyapatite through a "sodium citrate-hydroxyapatite (HAp) dispersion system," resulting in a uniform distribution within the collagen network. SAXS data (Dm, Rg, S / V) confirms structural characteristics different from Comparative Example 1, avoiding the problem of difficult dispersion control in existing materials.

[0026] 3. This invention achieves three-dimensional elastic properties: Traditional collagen scaffolds are generally fragile and prone to breakage. This invention, through a combined process of printing, fiberization, and cross-linking, enables the final scaffold to have high-fidelity structural features, exhibiting a mechanical response capable of repeated compression cycles and observable energy dissipation behavior, significantly improving structural stability and making it suitable for tissue repair scenarios under stress.

[0027] 4. This invention has the ability to construct a variety of complex three-dimensional structures: relying on the support bath printing system, the printing ink of this invention can form a variety of geometric shapes, including multi-branched vascular structures, hand shapes, and brain-shaped structures with surface textures, realizing the ability to construct three-dimensional structures under complex shape conditions, which is difficult to achieve with existing natural collagen-based hydrogel materials.

[0028] 5. The adjustability of the blood adsorption and factor aggregation behavior of this invention demonstrates good structural adaptability and tissue repair performance in animal osteocartilage injury models: The material of this invention exhibits a different numerical range in the BCI test than Comparative Example 1, showing the adjustability of the material's blood adsorption and factor aggregation behavior, providing verifiable evidence for its application in osteocartilage repair or hemorrhage environments. The illustrated examples show that the material can conform to the defect site in animal models, and MRI and tissue staining results show an observable morphology of continuously repaired tissue, providing experimental support for the application of the material in the field of osteocartilage repair. Attached Figure Description

[0029] Figure 1 The figure shows the shape change of the composite elastic support material in Example 1 under the action of external force and after the removal of external force: The figure shows the change in the appearance of the material from t=0 s to t=1s (after the removal of external force) during the pressure process.

[0030] Figure 2 The figures show different structural models of the composite elastic scaffold material in this invention formed by 3D printing, including various shapes such as hand structure, branching tube structure and brain-shaped structure.

[0031] Figure 3 The microstructure and elemental distribution of the composite elastic scaffold materials in Example 1 and Comparative Example 1 are shown in the following diagrams: (A) The left and middle images are scanning electron microscope (SEM) images of the two sets of materials, showing the morphology of the particle structure or fiber structure on the material surface at different magnifications; the right image is the energy spectrum distribution of Ca, showing the spatial distribution characteristics of the Ca element signal on the material surface; (B) A transmission electron microscope (TEM) image of a single collagen fiber, showing that HAp is uniformly distributed in the field of view.

[0032] Figure 4The structural parameter test results of the composite elastic scaffold materials in Example 1 and Comparative Example 1 are as follows: (A) Small-angle X-ray scattering (SAXS) curves of the two groups of samples; (B) Comparison of surface gauge (Dm) results; (C) Comparison of cluster size (Rg) results; (D) Comparison of relative specific surface area (S / V) results.

[0033] Figure 5 The compression performance test results of the composite elastic support material in Example 1 and Comparative Example 1 are as follows: (A) Compression stress-strain curves and compression modulus values ​​of Example 1 and Comparative Example 1; the compression modulus of Example 1 is about 110 kPa and the modulus of Comparative Example 1 is about 30 kPa; (B) Hysteresis curves and energy dissipation values ​​of the two groups of samples in 1000 cycles of compression test.

[0034] Figure 6 The results of the coagulation performance tests of the composite elastic stent materials in Examples 1, 2, 3 and Comparative Example 1 are shown: In Figure A, the materials of each example formed different degrees of coagulation morphology after contact with whole blood; Figure B shows the BCI (Blood Clotting Index) values ​​obtained by the materials of the examples and Comparative Example 1 under the same conditions; the BCI values ​​of the four groups of samples are different, and Examples 1, 2 and 3 all showed different BCI values ​​from Comparative Example 1 under the test conditions.

[0035] Figure 7 The graph shows a comparison of the contents of TGF-β1, BMP-2, PDGF, IGF-1 and VEGF detected in the composite elastic stent materials of Example 1 and Comparative Example 1 after blood absorption. The pink bars in the bar chart represent the composite elastic stent material of Example 1, and the blue bars represent the composite elastic stent material of Comparative Example 1.

[0036] Figure 8 Gross, MRI and histological evaluation of bone-cartilage defect repair in different treatment groups: (A) Gross morphology of the defect site after sampling in each treatment group; (B) MRI imaging results of the defect area in each group; (C) Histological sections of the defect tissue stained with H&E, toluidine blue and safranin O-fast green in each group, used to observe the repair of cartilage tissue and matrix.

[0037] Figure 9 This is a diagram illustrating the biocompatibility of the composite elastic scaffold material in Example 1 after implantation in the human body. Detailed Implementation

[0038] To further understand the inventiveness and technical advancements of this invention, the preferred embodiments of this invention will be discussed in detail below with reference to examples and comparative examples.

[0039] Example: A composite elastic scaffold based on collagen fiber-hydroxyapatite synthesis has a three-dimensional porous network structure, which is formed by the combination of collagen and hydroxyapatite. The particle size of hydroxyapatite is 20nm to 1μm.

[0040] The composite elastic scaffold in this invention utilizes a Carbopol support bath to maintain the three-dimensional structure of collagen during the printing process. After temperature-controlled incubation, it forms a fibrous network, which is then cross-linked and freeze-dried to obtain the final product.

[0041] Collagen self-assembles to form a three-dimensional porous network of collagen fibers. This three-dimensional porous network of collagen fibers is a multi-level nanostructure network. Hydroxyapatite is uniformly dispersed in the collagen fiber network, which is conducive to blood absorption and factor aggregation, and can effectively repair osteocartilage damage. That is, the composite elastic scaffold is used in the field of tissue engineering, specifically for osteocartilage damage repair.

[0042] A method for preparing a composite elastic scaffold based on collagen fiber-hydroxyapatite synthesis, comprising the following steps: Step 1: Prepare acidic collagen solution and sodium citrate / hydroxyapatite (HAp) suspension respectively; The preparation method of acidic collagen solution is as follows: Collagen is mixed evenly with 0.5-5.0M acidic aqueous solution, wherein the acidic aqueous solution is at least one of acetic acid aqueous solution, hydrochloric acid aqueous solution, and carbonic acid aqueous solution, to obtain an acidic collagen aqueous solution with a collagen mass concentration of 10-30 mg / mL; The preparation method of sodium citrate / hydroxyapatite HAp suspension is as follows: Hydroxyapatite with a particle size of 20nm to 1μm and sodium citrate are thoroughly mixed and dispersed in PBS buffer at a mass ratio of (0.1-3.5):1 to obtain sodium citrate / hydroxyapatite HAp suspension with a hydroxyapatite solid content of 10% to 50%. Step 2: The acidic collagen solution and sodium citrate / hydroxyapatite (HAp) suspension are mixed evenly at a volume ratio of (1-4):1, and then subjected to ultrasonic and centrifugal treatment to obtain printing ink. The ultrasonic treatment parameters are as follows: ultrasonic frequency 20-40 kHz, ultrasonic time 5-20 min; the centrifugation treatment parameters are as follows: temperature 4±0.5℃, centrifugation speed 1500±500 rap / min, centrifugation 3±1 min. Step 3: Using the printing ink from Step 2, perform 3D printing in a support bath. The printing material in the support bath is incubated at 35-38℃ / 70-100%RH for 1-5 hours to form a collagen fiber network structure. The preparation method of the support bath is as follows: Carbopol and PB buffer are stirred and dispersed evenly to obtain PB buffer with a Carbopol concentration of 1-2 w / v%. Stirring is stopped and the mixture is allowed to stand for 5-24 h to allow the Carbopol to swell completely. The pH is adjusted to about 7.0-7.4 to ensure that the system is fully swollen and presents a uniform gel state. Then, the system is centrifuged at 8000±2000 rap / min for 4±1 min at a temperature of 4±0.5℃ and stored at low temperature to obtain the support bath. The 3D printing parameters are as follows: 27G specification needles are used for 3D printing; the line spacing is set to 0.6±0.1mm; the layer height is set to 0.21±0.01mm; and the printing speed is set to 10±2mm / s. Step 4: Wash the support bath with PBS buffer containing NaCl (2-5 wt%). Step 5: Crosslinking treatment was performed using an alcoholic solution of glutaraldehyde or 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) at a concentration of 0.5±0.1wt%. The crosslinking temperature was 37±0.5℃ and the crosslinking reaction time was 4~12h. Subsequently, the composite elastic scaffold with a three-dimensional porous network structure was obtained by freeze drying.

[0043] Example 1: A method for preparing a composite elastic scaffold based on collagen fiber-hydroxyapatite synthesis, the steps of which are as follows: Step 1: Prepare acidic collagen solution and sodium citrate / hydroxyapatite (HAp) suspension respectively; The preparation method of acidic collagen solution is as follows: 800 mg of collagen (purchased from Jiangsu Bochuang Biotechnology Co., Ltd., type I bovine Achilles tendon collagen raw material) is added to 40 mL of 0.5 M acetic acid aqueous solution, and the collagen is fully dissolved under stirring to obtain an acidic collagen solution with a mass concentration of 20 mg / mL. The preparation method of sodium citrate / hydroxyapatite HAp suspension is as follows: 48 mg of hydroxyapatite (HAp, average particle size about 60 nm) and 48 mg of sodium citrate are added to 0.4 mL of PBS solution, stirred at room temperature for 1 h, and then ultrasonically dispersed at 40 kHz for 10 min to fully disperse HAp and obtain sodium citrate / hydroxyapatite HAp suspension. Simultaneously, the support bath was prepared: 1.5g of Carbopol ETD2020 (purchased from Shanghai Changwei Pharmaceutical Excipients Technology Co., Ltd.) was weighed and added to the rapidly stirred PB buffer to prepare a PB buffer with a Carbopol concentration of 1.5w / v%. Stirring was stopped, and the buffer was fully hydrated at room temperature for 12h. The pH was adjusted to 7 with 5M NaOH aqueous solution. The buffer was centrifuged at 8000rad / min for 5min to remove air bubbles and then transferred to a printing container for later use. Step 2: Mix 1.2 mL of acidic collagen solution with 0.4 mL of sodium citrate / hydroxyapatite (HAp) suspension. Stir under mixing conditions until the solution is homogeneous, adjusting the final collagen concentration in the printing ink to approximately 15 mg / mL. Transfer the mixture to a 3 mL syringe and ultrasonically disperse the syringe containing the printing ink at 20 kHz for 5 min. After ultrasonic dispersion, place the syringe in a centrifuge and centrifuge at 2000 rad / min for 3 min to remove air bubbles, making the resulting solution suitable for 3D printing. Step 3: Using the printing ink from Step 2, perform 3D printing in the support bath. The 3D printing is as follows: a 27G specification needle is used for 3D printing, the line spacing is set to 0.6mm, the layer height is set to 0.21mm, and the printing speed is set to 10mm / s. The resulting printed material forms corresponding 3D structures in shapes of different complexities, demonstrating the ability to construct various types of 3D shapes. Figure 2 The printed material in the support bath was placed in a constant temperature and humidity chamber and incubated for 2.0 h under temperature control conditions of 37℃ / 85%RH to form a collagen fiber network structure. Step 4: After incubation, wash the carbomer with PBS solution containing 5% NaCl to remove any residual carbomer components in the support bath. Step 5: The washed material is placed in an ethanol aqueous solution with a concentration of 0.5wt% glutaraldehyde (ethanol to water volume ratio of 1 / 1) for crosslinking treatment. The crosslinking temperature is controlled at a constant temperature of 37℃ and the crosslinking reaction time is 5.0h. After the crosslinking is completed, the material is taken out and freeze-dried to obtain a composite elastic scaffold with a three-dimensional porous network structure.

[0044] See Figure 1 The resulting composite elastic stent recovers its shape within 1 second after being compressed.

[0045] The difference between Example 2 and Example 1 is as follows: In step five, the washed material is placed in an ethanol-water solution of 0.5wt% 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride DMTMM (CAS: 3945-69-5) (ethanol to water volume ratio of 1 / 1) for crosslinking treatment. The crosslinking temperature is controlled at a constant 37°C and the crosslinking reaction time is 12.0 h. After the crosslinking is completed, the material is taken out and freeze-dried to obtain a composite elastic scaffold with a three-dimensional porous network structure. The remaining steps are the same.

[0046] The difference between Example 3 and Example 1 is as follows: In step one, the preparation method of sodium citrate / hydroxyapatite HAp suspension is as follows: 48 mg of hydroxyapatite (HAp, with an average particle size of about 60 nm) and 48 mg of sodium citrate are added to 0.4 mL of PBS solution, stirred at room temperature for 1 h, and then ultrasonically dispersed at 40 kHz for 10 min to fully disperse HAp and obtain sodium citrate / hydroxyapatite HAp suspension. The remaining steps are the same.

[0047] The difference between Comparative Example 1 and Example 1 is as follows: In step one, the preparation method of HAp suspension is as follows: 48 mg of hydroxyapatite is added to 0.4 mL of PBS solution, stirred at room temperature for 1 h, and then ultrasonically dispersed at 40 kHz for 10 min to fully disperse HAp and obtain HAp suspension. The remaining steps are the same.

[0048] A comparison of Example 1 and Comparative Example 1 shows that: in Comparative Example 1, the hydroxyapatite (HAp) is unevenly dispersed in the scaffold, and a large amount of hydroxyapatite (HAp) agglomerates (see Example 1). Figure 3 This results in low strength and poor elasticity of the fabricated scaffold, making the scaffold material easily damaged under low stress (see...). Figure 5 In Example 1, the hydroxyapatite (HAp) was uniformly dispersed in the composite elastic scaffold, resulting in high strength and high elasticity. This result indicates that appropriate sodium citrate can uniformly disperse hydroxyapatite (HAp) in the scaffold, which is key to obtaining a high-strength, high-elasticity collagen fiber structure.

[0049] Performance testing: (1) The collagen fiber / hydroxyapatite composite elastic scaffold prepared in Example 1 and Comparative Example 1 were subjected to SEM, TEM and EDS tests.

[0050] See Figure 3 As can be seen from the SEM image on the left and the magnified view in the middle column, both Example 1 and Comparative Example 1 have fibrous structures. The surface of the composite elastic scaffold material in Example 1 exhibits a porous structure composed of microscale particles, with small pore spacing. In contrast, the surface of the scaffold material in Comparative Example 1 consists of granular or aggregated structures, with a certain degree of non-uniform particle distribution.

[0051] See Figure 3 As shown in the Ca element distribution map (EDS Mapping) on ​​the right side of A, the Ca element signal distribution of the scaffold material in Example 1 is relatively uniform, with the image displaying continuous and dense dot-like signals. In contrast, the Ca element in the scaffold material in Comparative Example 1 exhibits a relatively clustered distribution, with multiple bright spots indicating concentrated Ca signals. The above images demonstrate the observable differences in surface microstructure and elemental distribution between the two types of materials.

[0052] See Figure 3 The white arrow points to the center, which is a single collagen fiber, while the yellow arrow points to a homogeneous, dense layer of nanoparticles. These nanoparticles exist both inside and outside the collagen fiber.

[0053] (2) The collagen fiber / hydroxyapatite composite elastic scaffold prepared in Example 1 and the collagen fiber scaffold prepared in Comparative Example 1 were used to test small angle X-ray scattering (SAXS) to analyze the uniformity of HAp dispersion in the scaffold.

[0054] SAXS test Figure 4 In the scattering curves of Example 1 and Comparative Example 1, both exhibit continuous attenuation across the low to high q value range, but the curve distribution of Example 1 differs slightly from that of Comparative Example 1. Surface gauge (Dm) results show that the Dm value of Example 1 is greater than that of Comparative Example 1; cluster size (Rg) results show that the Rg value of Example 1 is higher than that of Comparative Example 1; and relative surface area (S / V) results show that the S / V value of Example 1 is greater than that of Comparative Example 1. These test data reflect the differences in nanostructure characteristics between Example 1 and Comparative Example 1, including changes in surface gauge, cluster size, and relative surface area.

[0055] (3) The collagen fiber / hydroxyapatite composite elastic scaffold prepared in Example 1 and Comparative Example 1 were used for compression test and cyclic compression test.

[0056] See Figure 5 Figure A shows a comparison of the compressive stress-strain curves and compressive modulus of Example 1 and Comparative Example 1. As can be seen from the curves, both groups of samples exhibit a stress-strain increase trend with increasing applied strain. Specifically, the stress value of Example 1 is generally higher than that of Comparative Example 1 within the test range. The bar chart on the right shows the compressive modulus values ​​of the two groups of samples. The compressive modulus of Example 1 is approximately 110 kPa, while that of Comparative Example 1 is approximately 30 kPa. The two are numerically distinguishable. Figure 5 Figure B shows the stress-strain hysteresis curves of Example 1 and Comparative Example 1 obtained under the same loading conditions after 1000 cycles of compression. The hysteresis loop of Example 1 shows a certain overlap trend in each cycle; the hysteresis loop of Comparative Example 1 also shows a trend of changing with the cycles. There are observable differences in the morphology of the hysteresis curves between the two groups of samples. The bar chart on the right shows the energy dissipation values ​​of the two groups of samples. The energy dissipation of Example 1 is approximately 65 kJ·m⁻³, and the energy dissipation of Comparative Example 1 is approximately 30 kJ·m⁻³.

[0057] The results show that the mechanical response and energy dissipation values ​​of the two groups of samples are different under cyclic compression test conditions.

[0058] (4) The prepared collagen / hydroxyapatite composite elastic fiber scaffold was used to test the blood absorption performance of the material.

[0059] Slowly add 0.1 ml of anticoagulated rabbit whole blood to the printing frame, and incubate at 37°C for 5 min after addition. Rinse the frame with 3.0 ml of PBS and incubate for 5 min. Collect the supernatant in a cuvette / ELISA plate and measure the absorbance at 540 nm, correcting with a deionized water blank. Blood coagulation index BCI (%) = A sample / A control *100%. The lower the BCI, the more blood cells are "trapped" in the clot, and the stronger the coagulation / hemostasis ability.

[0060] See Figure 6 The BCI value of Example 1 is approximately 12%, which indicates the highest degree of blood coagulation.

[0061] The BCI value of Example 2 was approximately 20%, and the BCI value of Example 3 was approximately 35%. The BCI value of Comparative Example 1 was approximately 65%. These results indicate that, under the same testing conditions, materials with different formulations or structures exhibit distinguishable differences in their BCI values. Overall, the blood from the Examples 1 showed a higher degree of agglutination than that of Comparative Example 1.

[0062] See Figure 7 In Example 1 and Comparative Example 1, after adsorbing blood, blood factors such as TGF-β1, BMP-2, PDGF, IGF-1, and VEGF were detected using an ELISA kit. As can be seen from the bar chart, under the same test conditions, the levels of the above-mentioned factors detected in Example 1 were all higher than those in Comparative Example 1. In summary, the detected values ​​of each factor showed a distinguishable difference between the two groups of materials, indicating that the materials of Example 1 retained more factors in the blood adsorbed under these test conditions than those of Comparative Example 1.

[0063] (5) The collagen / hydroxyapatite composite elastic fiber scaffold prepared in Example 1 and Comparative Example 1 were used to test the cartilage repair ability of the materials.

[0064] All animals underwent surgery under general anesthesia. The articular surface was exposed using a common knee approach, and a standardized osteochondral defect was established in the femoral trochlea or medial femoral condyle region by a specialist. The defect was cylindrical, penetrating the cartilage layer and extending into the subchondral bone, creating a model that could not spontaneously and completely repair itself. The defect area was flushed with saline to remove debris. A dried scaffold was implanted into the defect cavity, allowing it to absorb wound blood and adhere closely to the surrounding tissue, maintaining the correct orientation (cartilage layer towards the joint cavity). No significant interference was confirmed during joint flexion and extension. No material was implanted in the control group. Postoperatively, animals received standardized care, analgesia, and observation. Animals were allowed free movement in their cages to achieve a joint environment consistent with physiological weight-bearing. Three months postoperatively, tissue samples were harvested for gross observation, imaging examination, and histological and immunohistochemical analysis. The osteochondral units were fixed, decalcified, embedded, and sectioned using standard methods.

[0065] Five groups were compared: blank (Black), natural collagen-printed scaffold (Col), scaffold prepared in Example 1, scaffold prepared in Comparative Example 1, and allogeneic osteochondral tissue (positive control). Based on macroscopic observation, MRI imaging, and histological staining results, Example 1 showed the most significant and near-normal cartilage regeneration effect in bone-cartilage defect repair. Compared with the blank group, the pure collagen group, and Comparative Example 1, Example 1 not only macroscopically presented a smooth, continuous, and transparent cartilage-like surface (…), but also… Figure 8 A), also appears as a uniform low signal on MRI, close to that of normal cartilage, indicating that its repair tissue is more mature in terms of water content, structural density, and subchondral bone reconstruction. Figure 8 B). Histological examination further confirmed that this group formed a typical hyaline cartilage-like layered structure with regular cell morphology, and showed strong and uniform proteoglycan positive signals in both Toluidine Blue and Safranin O / FastGreen staining, indicating sufficient matrix deposition and high-quality cartilage-like tissue. Figure 8 C). The aforementioned repair advantages may stem from the comprehensive optimization of the scaffold material used in Example 1 in terms of structural design, biocompatibility, and mechanical properties. The osteochondral repair level was scored, and the results are shown in Table 1. Notably, the repair effect of Example 1 was comparable to that of the positive control group, suggesting that this scaffold has potential clinical application value in promoting integrated reconstruction of the bone-cartilage interface.

[0066] To assess the potential impact of materials / treatments on systemic toxicity in experimental animals, routine H&E staining was performed on the heart, liver, lungs, kidneys, and spleen samples taken in Example 1. Figure 9The top row shows low-power fields (×40), and the bottom row shows high-power fields (×200) of the corresponding selected area. Myocardial fibers were observed to be regularly arranged without necrosis or inflammatory cell infiltration; hepatocyte lamellar structure was intact, without fatty degeneration or necrosis; alveolar structure was clear, without obvious inflammation or edema; glomerular and tubular structures were normal, without tubular necrosis or protein casts; the white and red pulp of the spleen were clearly demarcated, and the lymphoid follicle structure was normal. These results suggest that no significant pathological damage occurred in the major organs of the treated group. Overall, the structures of all major organs and tissues were normal, with no obvious toxic or damaging changes, indicating that the tested material has good in vivo biocompatibility.

[0067] Table 1: Performance Test Results of Collagen-Based Materials It should be noted that this specific embodiment is merely an explanation of the technical solution of the present invention and is not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A composite elastic scaffold based on collagen fiber-hydroxyapatite synthesis, characterized in that: The composite elastic scaffold is a three-dimensional porous network structure formed by collagen and hydroxyapatite; the collagen self-assembles to form a collagen fiber network, and the hydroxyapatite is uniformly dispersed in the collagen fiber network.

2. The composite elastic scaffold based on collagen fiber-hydroxyapatite synthesis according to claim 1, characterized in that: The collagen fiber network in the composite elastic scaffold is a multi-level nanostructure network.

3. The composite elastic scaffold based on collagen fiber-hydroxyapatite synthesis according to claim 1, characterized in that: The hydroxyapatite has a particle size of 20 nm to 1 μm.

4. A method for preparing a composite elastic scaffold based on collagen fiber-hydroxyapatite synthesis according to any one of claims 1-3, characterized in that: Includes the following steps: Step 1: Prepare acidic collagen solution and sodium citrate / hydroxyapatite (HAp) suspension respectively; Step 2: The acidic collagen solution and sodium citrate / hydroxyapatite (HAp) suspension are mixed evenly at a volume ratio of (1-4):1, and then subjected to ultrasonic and centrifugal treatment to obtain printing ink. Step 3: Using the printing ink from Step 2, perform 3D printing in a support bath. The printing material in the support bath is incubated at 35-38℃ / 70-100%RH for 1-5 hours to form a collagen fiber network structure. Step 4: Wash away the support bath with PBS buffer containing NaCl; Step 5: Crosslinking treatment was performed using an alcoholic solution of glutaraldehyde or 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) at a concentration of 0.5±0.1wt%. The crosslinking temperature was 37±0.5℃ and the crosslinking reaction time was 4~12h. Subsequently, the composite elastic scaffold with a three-dimensional porous network structure was obtained by freeze drying.

5. The method for preparing the composite elastic scaffold according to claim 4, characterized in that: The preparation method of the acidic collagen solution in step one is as follows: Collagen is mixed evenly with a 0.5-5.0M acidic aqueous solution to obtain an acidic collagen aqueous solution with a collagen mass concentration of 10-30 mg / mL; the acidic aqueous solution is at least one of acetic acid aqueous solution, hydrochloric acid aqueous solution, and carbonic acid aqueous solution.

6. The method for preparing the composite elastic scaffold according to claim 4, characterized in that: The preparation method of the sodium citrate / hydroxyapatite HAp suspension in step one is as follows: Hydroxyapatite with a particle size of 20nm to 1μm and sodium citrate are thoroughly mixed and dispersed in PBS buffer at a mass ratio of (0.1-3.5):1 to obtain a sodium citrate / hydroxyapatite HAp suspension with a hydroxyapatite solid content of 10% to 50%.

7. The method for preparing the composite elastic scaffold according to claim 4, characterized in that: The ultrasonic treatment parameters are as follows: ultrasonic frequency 20-40 kHz, ultrasonic time 5-20 min; the centrifugation treatment parameters are as follows: temperature 4±0.5℃, centrifugation speed 1500±500 rap / min, centrifugation 3±1 min.

8. The method for preparing the composite elastic scaffold according to claim 4, characterized in that: The preparation method of the support bath in step three is as follows: Carbopol and PB buffer are stirred and dispersed evenly to obtain PB buffer with a Carbopol concentration of 1-2 w / v%. After stopping stirring, the mixture is allowed to stand for 5-24 h to allow the Carbopol to swell completely. The pH is adjusted to about 7.0-7.4 to ensure that the system is fully swollen and presents a uniform gel state. Then, the mixture is centrifuged at 8000±2000 rap / min for 4±1 min at a temperature of 4±0.5℃ and stored at low temperature to obtain the support bath.

9. The method for preparing the composite elastic scaffold according to claim 4, characterized in that: The 3D printing parameters in step three are as follows: 27G specification needles are used for 3D printing, the printing line spacing is set to 0.6±0.1mm, the layer height is set to 0.21±0.01mm, and the printing speed is set to 10±2mm / s.

10. A synthesis method based on collagen fibers and hydroxyapatite according to any one of claims 1-3, characterized in that: Used in the field of tissue engineering, specifically for the repair of osteochondral injuries.

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